Tung-Tien Sun, Ph.D.

Effects of shear stress on the number and function of endothelial progenitor cells adhered to specific matrices
Xiao L; Wang G; Jiang T; Tang C; Wu X; Sun T
2011 Mar 23;:0 L-0 L, Journal of applied biomaterials & biomechanics : JABB
Purpose: The aim of this study was to screen specific adherent matrix for endothelial progenitor cells (EPCs), which can be used for antibody capturing stents. Methods: In this study, the adhesion of EPCs on different matrices containing three different antibodies, VEGFR-2, CD34, CD133, was observed under shear stress in a flow chamber. Nitric oxide (NO) release, cell proliferation and the retention rate of EPCs, were measured separately. Results: The results demonstrated that shear stress within a certain range can promote proliferation and NO secretion of EPCs. Under the same shear stress, the EPCs showed stronger adhesion on matrix-containing CD133 antibody than on the other matrices. Conclusions: CD133 antibody has the potential application for EPCs capture
— id: 132743, year: 2011, vol: , page: 0 L, stat: Journal Article,

Overexpression of NGF in mouse urothelium leads to neuronal hyperinnervation, pelvic sensitivity, and changes in urinary bladder function
Schnegelsberg, Birthe; Sun, Tung-Tien; Cain, Gary; Bhattacharya, Anindya; Nunn, Philip A; Ford, Anthony P D W; Vizzard, Margaret A; Cockayne, Debra A
2010 Mar;298(3):R534-R547, American journal of physiology. Regulatory, integrative, & comparative physiology
NGF has been suggested to play a role in urinary bladder dysfunction by mediating inflammation, as well as morphological and functional changes, in sensory and sympathetic neurons innervating the urinary bladder. To further explore the role of NGF in bladder sensory function, we generated a transgenic mouse model of chronic NGF overexpression in the bladder using the urothelium-specific uroplakin II (UPII) promoter. NGF mRNA and protein were expressed at higher levels in the bladders of NGF-overexpressing (NGF-OE) transgenic mice compared with wild-type littermate controls from postnatal day 7 through 12-16 wk of age. Overexpression of NGF led to urinary bladder enlargement characterized by marked nerve fiber hyperplasia in the submucosa and detrusor smooth muscle and elevated numbers of tissue mast cells. There was a marked increase in the density of CGRP- and substance P-positive C-fiber sensory afferents, neurofilament 200-positive myelinated sensory afferents, and tyrosine hydroxylase-positive sympathetic nerve fibers in the suburothelial nerve plexus. CGRP-positive ganglia were also present in the urinary bladders of transgenic mice. Transgenic mice had reduced urinary bladder capacity and an increase in the number and amplitude of nonvoiding bladder contractions under baseline conditions in conscious open-voiding cystometry. These changes in urinary bladder function were further associated with an increased referred somatic pelvic hypersensitivity. Thus, chronic urothelial NGF overexpression in transgenic mice leads to neuronal proliferation, focal increases in urinary bladder mast cells, increased urinary bladder reflex activity, and pelvic hypersensitivity. NGF-overexpressing mice may, therefore, provide a useful transgenic model for exploring the role of NGF in urinary bladder dysfunction
— id: 115888, year: 2010, vol: 298, page: R534, stat: Journal Article,

Location of corneal epithelial stem cells
Sun, Tung-Tien; Tseng, Scheffer C; Lavker, Robert M
2010 Feb 25;463(7284):E10-E11, Nature
Arising from: Majo, F., Rochat, A., Nicolas, M., Jaoude, G. A. & Barrandon, Y. 456, 250-254 (2008).; Majo et al. replyThe longstanding concept that corneal epithelial stem cells reside mainly in the limbus is supported by the absence of major corneal epithelial differentiation markers, that is, K3 and K12 keratins, in limbal basal cells (these markers are expressed, however, in corneal basal cells, thus distinguishing the mode of keratin expression in corneal epithelium from that of all other stratified epithelia), the centripetal migration of corneal epithelial cells, the exclusive location of slow-cycling cells in the limbal basal layer, the superior in vitro proliferative potential of limbal epithelial cells, and the transplanted limbal cells' ability to reconstitute corneal epithelium in vivo (reviewed in refs 1-4). Moreover, previous data indicate that corneal and conjunctival epithelia represent two separate cell lineages (reviewed in refs 1-4). Majo et al. suggested, however, that corneal and conjunctival epithelia are equipotent, and that identical oligopotent stem cells are present throughout the corneal, limbal and conjunctival epithelia. We point out here that these suggestions are inconsistent with many known growth, differentiation and cell migration properties of the anterior ocular epithelia
— id: 107392, year: 2010, vol: 463, page: E10, stat: Journal Article,

Temporally and spatially controllable gene expression and knockout in mouse urothelium
Zhou, Haiping; Liu, Yan; He, Feng; Mo, Lan; Sun, Tung-Tien; Wu, Xue-Ru
2010 Aug;299(2):F387-F395, American journal of physiology. Renal physiology
Urothelium that lines almost the entire urinary tract performs important functions and is prone to assaults by urinary microbials, metabolites, and carcinogens. To improve our understanding of urothelial physiology and disease pathogenesis, we sought to develop two novel transgenic systems, one that would allow inducible and urothelium-specific gene expression, and another that would allow inducible and urothelium-specific knockout. Toward this end, we combined the ability of the mouse uroplakin II promoter (mUPII) to drive urothelium-specific gene expression with a versatile tetracycline-mediated inducible system. We found that, when constructed under the control of mUPII, only a modified, reverse tetracycline trans-activator (rtTA-M2), but not its original version (rtTA), could efficiently trans-activate reporter gene expression in mouse urothelium on doxycycline (Dox) induction. The mUPII/rtTA-M2-inducible system retained its strict urothelial specificity, had no background activity in the absence of Dox, and responded rapidly to Dox administration. Using a reporter gene whose expression was secondarily controlled by histone remodeling, we were able to identify, colocalize with 5-bromo-2-deoxyuridine incorporation, and semiquantify newly divided urothelial cells. Finally, we established that, when combined with a Cre recombinase under the control of the tetracycline operon, the mUPII-driven rtTA-M2 could inducibly inactivate any gene of interest in mouse urothelium. The establishment of these two new transgenic mouse systems enables the manipulation of gene expression and/or inactivation in adult mouse urothelium at any given time, thus minimizing potential compensatory effects due to gene overexpression or loss and allowing more accurate modeling of urothelial diseases than previously reported constitutive systems
— id: 138180, year: 2010, vol: 299, page: F387, stat: Journal Article,

Alterations in bladder function associated with urothelial defects in uroplakin II and IIIa knockout mice
Aboushwareb, Tamer; Zhou, Ge; Deng, Fang-Ming; Turner, Chanda; Andersson, Karl-Erik; Tar, Moses; Zhao, Weixin; Melman, Arnold; D'Agostino, Ralph Jr; Sun, Tung-Tien; Christ, George J
2009 ;28(8):1028-1033, Neurourology & urodynamics
AIMS: The effects of deleting genes encoding uroplakins II (UPII) and III (UPIIIa) on mouse bladder physiology/dysfunction were studied in male and female wild type and knockout (KO) mice. METHODS: UPII, UPIIIa, and WT mice were catheterized using previously described techniques. Continuous cystometry was conducted in conscious, freely moving animals. Bladder strips were harvested after animal sacrifice and pharmacological studies and EFS were conducted in an organ chamber. Histological studies were also carried on with H&E staining to identify differences among the three mouse types. RESULTS: These studies have revealed numerous alterations, some of which were apparently gender-specific. Nonvoiding contractions were common in both UPII and UPIIIa KO mice, although more severe in the former. In particular, the increased bladder capacity, micturition pressure and demonstrable nonvoiding contractions observed in the male UPII KO's, were reminiscent of an obstruction-like syndrome accompanied by evidence of emerging bladder decompensation, as reflected by an increased residual volume. Pharmacological studies revealed a modest, gender-specific reduction in sensitivity of isolated detrusor strips from UPII KO female mice to carbachol-induced contractions. A similar reduction was observed in UPIIIa KO female mice. Histological investigation showed urothelial hyperplasia in both UPII KO and UPIIIa KO mice, although again, apparently more severe in the former. CONCLUSIONS: These results confirm and extend previous work to indicate that urothelial defects due to uroplakin deficiency are associated with significant alterations in bladder function and further highlight the importance of the urothelium to bladder physiology/dysfunction
— id: 115886, year: 2009, vol: 28, page: 1028, stat: Journal Article,

Intron evolution: testing hypotheses of intron evolution using the phylogenomics of tetraspanins
Garcia-Espana, Antonio; Mares, Roso; Sun, Tung-Tien; Desalle, Rob
2009 ;4(3):e4680-e4680, PLoS ONE
BACKGROUND: Although large scale informatics studies on introns can be useful in making broad inferences concerning patterns of intron gain and loss, more specific questions about intron evolution at a finer scale can be addressed using a gene family where structure and function are well known. Genome wide surveys of tetraspanins from a broad array of organisms with fully sequenced genomes are an excellent means to understand specifics of intron evolution. Our approach incorporated several new fully sequenced genomes that cover the major lineages of the animal kingdom as well as plants, protists and fungi. The analysis of exon/intron gene structure in such an evolutionary broad set of genomes allowed us to identify ancestral intron structure in tetraspanins throughout the eukaryotic tree of life. METHODOLOGY/PRINCIPAL FINDINGS: We performed a phylogenomic analysis of the intron/exon structure of the tetraspanin protein family. In addition, to the already characterized tetraspanin introns numbered 1 through 6 found in animals, three additional ancient, phase 0 introns we call 4a, 4b and 4c were found. These three novel introns in combination with the ancestral introns 1 to 6, define three basic tetraspanin gene structures which have been conserved throughout the animal kingdom. Our phylogenomic approach also allows the estimation of the time at which the introns of the 33 human tetraspanin paralogs appeared, which in many cases coincides with the concomitant acquisition of new introns. On the other hand, we observed that new introns (introns other than 1-6, 4a, b and c) were not randomly inserted into the tetraspanin gene structure. The region of tetraspanin genes corresponding to the small extracellular loop (SEL) accounts for only 10.5% of the total sequence length but had 46% of the new animal intron insertions. CONCLUSIONS/SIGNIFICANCE: Our results indicate that tests of intron evolution are strengthened by the phylogenomic approach with specific gene families like tetraspanins. These tests add to our understanding of genomic innovation coupled to major evolutionary divergence events, functional constraints and the timing of the appearance of evolutionary novelty
— id: 98997, year: 2009, vol: 4, page: e4680, stat: Journal Article,

Involvement of vps33a in the fusion of uroplakin-degrading multivesicular bodies with lysosomes
Guo, Xuemei; Tu, Liyu; Gumper, Iwona; Plesken, Heide; Novak, Edward K; Chintala, Sreenivasulu; Swank, Richard T; Pastores, Gregory; Torres, Paola; Izumi, Tetsuro; Sun, Tung-Tien; Sabatini, David D; Kreibich, Gert
2009 Sep;10(9):1350-1361, Traffic
The apical surface of the terminally differentiated mouse bladder urothelium is largely covered by urothelial plaques, consisting of hexagonally packed 16-nm uroplakin particles. These plaques are delivered to the cell surface by fusiform vesicles (FVs) that are the most abundant cytoplasmic organelles. We have analyzed the functional involvement of several proteins in the apical delivery and endocytic degradation of uroplakin proteins. Although FVs have an acidified lumen and Rab27b, which localizes to these organelles, is known to be involved in the targeting of lysosome-related organelles (LROs), FVs are CD63 negative and are therefore not typical LROs. Vps33a is a Sec1-related protein that plays a role in vesicular transport to the lysosomal compartment. A point mutation in mouse Vps33a (Buff mouse) causes albinism and bleeding (Hermansky-Pudlak syndrome) because of abnormalities in the trafficking of melanosomes and platelets. These Buff mice showed a novel phenotype observed in urothelial umbrella cells, where the uroplakin-delivering FVs were almost completely replaced by Rab27b-negative multivesicular bodies (MVBs) involved in uroplakin degradation. MVB accumulation leads to an increase in the amounts of uroplakins, Lysosomal-associated membrane protein (LAMP)-1/2, and the activities of beta-hexosaminidase and beta-glucocerebrosidase. These results suggest that FVs can be regarded as specialized secretory granules that deliver crystalline arrays of uroplakins to the cell surface, and that the Vps33a mutation interferes with the fusion of MVBs with mature lysosomes thus blocking uroplakin degradation
— id: 101636, year: 2009, vol: 10, page: 1350, stat: Journal Article,

Deficiency of pRb family proteins and p53 in invasive urothelial tumorigenesis
He, Feng; Mo, Lan; Zheng, Xiao-Yong; Hu, Changkun; Lepor, Herbert; Lee, Eva Y-H P; Sun, Tung-Tien; Wu, Xue-Ru
2009 Dec 15;69(24):9413-9421, Cancer research
Defects in pRb tumor suppressor pathway occur in approximately 50% of the deadly muscle-invasive urothelial carcinomas in humans and urothelial carcinoma is the most prevalent epithelial cancer in long-term survivors of hereditary retinoblastomas caused by loss-of-function RB1 mutations. Here, we show that conditional inactivation of both RB1 alleles in mouse urothelium failed to accelerate urothelial proliferation. Instead, it profoundly activated the p53 pathway, leading to extensive apoptosis, and selectively induced pRb family member p107. Thus, pRb loss triggered multiple fail-safe mechanisms whereby urothelial cells evade tumorigenesis. Additional loss of p53 in pRb-deficient urothelial cells removed these p53-dependent tumor barriers, resulting in late-onset hyperplasia, umbrella cell nuclear atypia, and rare-occurring low-grade, superficial papillary bladder tumors, without eliciting invasive carcinomas. Importantly, mice deficient in both pRb and p53, but not those deficient in either protein alone, were highly susceptible to subthreshold carcinogen exposure and developed invasive urothelial carcinomas that strongly resembled the human counterparts. The invasive lesions had a marked reduction of p107 but not p130 of the pRb family. Our data provide compelling evidence, indicating that urothelium, one of the slowest cycling epithelia, is remarkably resistant to transformation by pRb or p53 deficiency; that concurrent loss of these two tumor suppressors is necessary but insufficient to initiate urothelial tumorigenesis along the invasive pathway; that p107 may play a critical role in suppressing invasive urothelial tumor formation; and that replacing/restoring the function of pRb, p107, or p53 could be explored as a potential therapeutic strategy to block urothelial tumor progression
— id: 105925, year: 2009, vol: 69, page: 9413, stat: Journal Article,

Bacteria-induced uroplakin signaling mediates bladder response to infection
Thumbikat, Praveen; Berry, Ruth E; Zhou, Ge; Billips, Benjamin K; Yaggie, Ryan E; Zaichuk, Tetiana; Sun, Tung-Tien; Schaeffer, Anthony J; Klumpp, David J
2009 May;5(5):e1000415-e1000415, PLoS pathogens
Urinary tract infections are the second most common infectious disease in humans and are predominantly caused by uropathogenic E. coli (UPEC). A majority of UPEC isolates express the type 1 pilus adhesin, FimH, and cell culture and murine studies demonstrate that FimH is involved in invasion and apoptosis of urothelial cells. FimH initiates bladder pathology by binding to the uroplakin receptor complex, but the subsequent events mediating pathogenesis have not been fully characterized. We report a hitherto undiscovered signaling role for the UPIIIa protein, the only major uroplakin with a potential cytoplasmic signaling domain, in bacterial invasion and apoptosis. In response to FimH adhesin binding, the UPIIIa cytoplasmic tail undergoes phosphorylation on a specific threonine residue by casein kinase II, followed by an elevation of intracellular calcium. Pharmacological inhibition of these signaling events abrogates bacterial invasion and urothelial apoptosis in vitro and in vivo. Our studies suggest that bacteria-induced UPIIIa signaling is a critical mediator of bladder responses to insult by uropathogenic E. coli
— id: 100510, year: 2009, vol: 5, page: e1000415, stat: Journal Article,

Uropathogenic E. coli adhesin-induced host cell receptor conformational changes: implications in transmembrane signaling transduction
Wang, Huaibin; Min, Guangwei; Glockshuber, Rudi; Sun, Tung-Tien; Kong, Xiang-Peng
2009 Sep 18;392(2):352-361, Journal of molecular biology
Urinary tract infection is the second most common infectious disease and is caused predominantly by type 1-fimbriated uropathogenic Escherichia coli (UPEC). UPEC initiates infection by attaching to uroplakin (UP) Ia, its urothelial surface receptor, via the FimH adhesins capping the distal end of its fimbriae. UP Ia, together with UP Ib, UP II, and UP IIIa, forms a 16-nm receptor complex that is assembled into hexagonally packed, two-dimensional crystals (urothelial plaques) covering >90% of the urothelial apical surface. Recent studies indicate that FimH is the invasin of UPEC as its attachment to the urothelial surface can induce cellular signaling events including calcium elevation and the phosphorylation of the UP IIIa cytoplasmic tail, leading to cytoskeletal rearrangements and bacterial invasion. However, it remains unknown how the binding of FimH to the UP receptor triggers a signal that can be transmitted through the highly impermeable urothelial apical membrane. We show here by cryo-electron microscopy that FimH binding to the extracellular domain of UP Ia induces global conformational changes in the entire UP receptor complex, including a coordinated movement of the tightly bundled transmembrane helices. This movement of the transmembrane helix bundles can cause a corresponding lateral translocation of the UP cytoplasmic tails, which can be sufficient to trigger downstream signaling events. Our results suggest a novel pathogen-induced transmembrane signal transduction mechanism that plays a key role in the initial stages of UPEC invasion and receptor-mediated bacterial invasion in general
— id: 101952, year: 2009, vol: 392, page: 352, stat: Journal Article,

Uroplakins in urothelial biology, function, and disease
Wu, Xue-Ru; Kong, Xiang-Peng; Pellicer, Angel; Kreibich, Gert; Sun, Tung-Tien
2009 Jun;75(11):1153-1165, Kidney international
Urothelium covers the inner surfaces of the renal pelvis, ureter, bladder, and prostatic urethra. Although morphologically similar, the urothelia in these anatomic locations differ in their embryonic origin and lineages of cellular differentiation, as reflected in their different uroplakin content, expandability during micturition, and susceptibility to chemical carcinogens. Previously thought to be an inert tissue forming a passive barrier between the urine and blood, urothelia have recently been shown to have a secretory activity that actively modifies urine composition. Urothelial cells express a number of ion channels, receptors, and ligands, enabling them to receive and send signals and communicate with adjoining cells and their broader environment. The urothelial surface bears specific receptors that not only allow uropathogenic E. coli to attach to and invade the bladder mucosa, but also provide a route by which the bacteria ascend through the ureters to the kidney to cause pyelonephritis. Genetic ablation of one or more uroplakin genes in mice causes severe retrograde vesicoureteral reflux, hydronephrosis, and renal failure, conditions that mirror certain human congenital diseases. Clearly, abnormalities of the lower urinary tract can impact the upper tract, and vice versa, through the urothelial connection. In this review, we highlight recent advances in the field of urothelial biology by focusing on the uroplakins, a group of urothelium-specific and differentiation-dependent integral membrane proteins. We discuss these proteins' biochemistry, structure, assembly, intracellular trafficking, and their emerging roles in urothelial biology, function, and pathological processes. We also call attention to important areas where greater investigative efforts are warranted.Kidney International (2009) 75, 1153-1165; doi:10.1038/ki.2009.73; published online 1 April 2009
— id: 98907, year: 2009, vol: 75, page: 1153, stat: Journal Article,

Appearance of new tetraspanin genes during vertebrate evolution
Garcia-Espana, Antonio; Chung, Pei-Jung; Sarkar, Indra Neil; Stiner, Eric; Sun, Tung-Tien; Desalle, Rob
2008 Apr;91(4):326-334, Genomics
A detailed phylogenetic analysis of tetraspanins from 10 fully sequenced metazoan genomes and several fungal and protist genomes gives insight into their evolutionary origins and organization. Our analysis suggests that the superfamily can be divided into four large families. These four families-the CD family, CD63 family, uroplakin family, and RDS family-are further classified as consisting of several ortholog groups. The clustering of several ortholog groups together, such as the CD9/Tsp2/CD81 cluster, suggests functional relatedness of those ortholog groups. The fact that our studies are based on whole genome analysis enabled us to estimate not only the phylogenetic relationships among the tetraspanins, but also the first appearance in the tree of life of certain tetraspanin ortholog groups. Taken together, our data suggest that the tetraspanins are derived from a single (or a few) ancestral gene(s) through sequence divergence, rather than convergence, and that the majority of tetraspanins found in the human genome are vertebrate (21 instances), tetrapod (4 instances), or mammalian (6 instances) inventions
— id: 115884, year: 2008, vol: 91, page: 326, stat: Journal Article,

Voiding pattern analysis as a surrogate for cystometric evaluation in uroplakin II knockout mice
Hodges, Steve J; Zhou, Ge; Deng, Fang-Ming; Aboushwareb, Tamer; Turner, Chanda; Andersson, Karl-Erik; Santago, Pete; Case, Doug; Sun, Tung-Tien; Christ, George J
2008 May;179(5):2046-2051, Journal of urology
PURPOSE: Previous study has shown that the absence of uroplakin II can cause urinary tract dysfunction, including vesicoureteral reflux and renal abnormalities, as well as micturition pattern changes. We developed a simple surrogate measure of bladder function using ultraviolet visualization of urinary voiding patterns in a uroplakin II knockout mouse animal model. MATERIALS AND METHODS: Three male and 3 female WT mice, and 3 male and 3 female uroplakin II knockout mice were evaluated by cystometric analysis and voiding pattern markings. Voiding pattern markings were graded by independent observers on a scale of 1 to 5 according to the degree of dispersion of voided urine. Statistical analysis was then used to correlate voiding dispersion grades with cystometric parameters in the same mice. RESULTS: The degree of dispersion of voiding pattern markings correlated with several measures of bladder function. Specifically the Pearson correlation coefficients for the observed voiding patterns highly correlated with baseline pressure, threshold pressure and intermicturition pressure measurements made during conscious cystometry in these mice (p <0.05). CONCLUSIONS: Ultraviolet visualization of urinary voiding patterns of mice correlated well with certain measures of standard cystometric evaluations. As such, this method provides a simple, noninvasive method of evaluating mouse bladder function. Implementation of this methodology, which can potentially be automated for high throughput analysis, can accelerate the development of novel therapy for certain important aspects of bladder disease/dysfunction
— id: 115885, year: 2008, vol: 179, page: 2046, stat: Journal Article,

Assembly of a membrane receptor complex: roles of the uroplakin II prosequence in regulating uroplakin bacterial receptor oligomerization
Hu, Chih-Chi Andrew; Bachmann, Thomas; Zhou, Ge; Liang, Feng-Xia; Ghiso, Jorge; Kreibich, Gert; Sun, Tung-Tien
2008 Sep 1;414(2):195-203, Biochemical journal
The apical surface of the mammalian urothelium is almost completely covered by two-dimensional protein crystals (known as urothelial plaques) of hexagonally packed 16 nm particles consisting of two UP (uroplakin) heterodimers, i.e. UPs Ia/II and Ib/III pairs. UPs are functionally important as they contribute to the urothelial permeability barrier function, and UPIa may serve as the receptor for the uropathogenic Escherichia coli that causes over 90% of urinary tract infections. We study here how the UP proteins are assembled and targeted to the urothelial apical surface, paying special attention to the roles of the prosequence of UPII in UP oligomerization. We show that (i) the formation of the UPIa/UPII heterodimer, necessary for ER (endoplasmic reticulum) exit, requires disulfide formation in the prosequence domain of proUPII (the immature form of UPII still containing its prosequence); (ii) differentiation-dependent N-glycosylation of the prosequence leads to UP stabilization; (iii) a failure to form tetramers in cultured urothelial cells, in part due to altered glycosylation of the prosequence, may block two-dimensional crystal formation; and (iv) the prosequence of UPII remains attached to the mature protein complex on the urothelial apical surface even after it has been cleaved by the trans-Golgi-network-associated furin. Our results indicate that proper secondary modifications of the prosequence of UPII play important roles in regulating the oligomerization and function of the UP protein complex
— id: 81088, year: 2008, vol: 414, page: 195, stat: Journal Article,

Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting
Luque-Garcia, Jose L; Zhou, Ge; Spellman, Daniel S; Sun, Tung-Tien; Neubert, Thomas A
2008 Feb;7(2):308-314, Molecular & cellular proteomics
We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion
— id: 76651, year: 2008, vol: 7, page: 308, stat: Journal Article,

Tailbud-derived mesenchyme promotes urinary tract segmentation via BMP4 signaling
Brenner-Anantharam, Andrea; Cebrian, Cristina; Guillaume, Richard; Hurtado, Romulo; Sun, Tung-Tien; Herzlinger, Doris
2007 May;134(10):1967-1975, Development
Urinary tract morphogenesis requires the sub-division of the ureteric bud (UB) into the intra-renal collecting system and ureter, two tissues with unique structural and functional properties. In this report we investigate the cellular and molecular mechanisms that mediate their differentiation. Fate mapping experiments in the developing chick indicate that the UB is surrounded by two distinct mesenchymal populations: nephrogenic mesenchyme derived from the intermediate mesoderm and tailbud-derived mesoderm, which is selectively associated with the domain of the UB that differentiates into the ureter. Functional experiments utilizing murine metanephric kidney explants show that BMP4, a paracrine factor secreted by tailbud-derived mesenchyme, is required for ureter morphogenesis. Conversely, ectopic BMP4 signaling is sufficient to induce ureter morphogenesis in domains of the UB normally fated to differentiate into the intra-renal collecting system. Collectively, these results indicate that the border between the kidney and ureter forms where mesenchymal tissues originating in two different areas of the early embryo meet. These data raise the possibility that the susceptibility of this junction to congenital defects in humans, such as ureteral-pelvic obstructions, may be related to the complex morphogenetic movements that are required to integrate cells from these different lineages into a single functional structure
— id: 71590, year: 2007, vol: 134, page: 1967, stat: Journal Article,

The histone deacetylase inhibitor belinostat (PXD101) suppresses bladder cancer cell growth in vitro and in vivo
Buckley, Michael T; Yoon, Joanne; Yee, Herman; Chiriboga, Luis; Liebes, Leonard; Ara, Gulshan; Qian, Xiaozhong; Bajorin, Dean F; Sun, Tung-Tien; Wu, Xue-Ru; Osman, Iman
2007 Oct 12;5(1):49-49, Journal of translational medicine
ABSTRACT: BACKGROUND: Treatment options for patients with recurrent superficial bladder cancer are limited, necessitating aggressive exploration of new treatment strategies that effectively prevent recurrence and progression to invasive disease. We assessed the effects of belinostat (previously PXD101), a novel histone deacetylase inhibitor, on a panel of human bladder cancer cell lines representing superficial and invasive disease, and on a transgenic mouse model of superficial bladder cancer. METHODS: Growth inhibition and cell cycle distribution effect of belinostat on 5637, T24, J82, and RT4 urothelial lines were assessed. Ha-ras transgenic mice with established superficial bladder cancer were randomized to receive either belinostat or vehicle alone, and assessed for bladder weight, hematuria, gene expression profiling, and immunohistochemistry (IHC). RESULTS: Belinostat had a significant linear dose-dependent growth inhibition on all cell lines (IC50 range of 1.0-10.0 micromolar). The 5637 cell line, which was derived from a superficial papillary tumor, was the most sensitive to treatment. Belinostat (100 mg/kg, intraperitoneal, 5 days each week for 3 weeks) treated mice had less bladder weight (p<0.05), and no hematuria compared with 6/10 control mice that developed at least one episode. IHC of bladder tumors showed less cell proliferation and a higher expression of p21WAF1 in the belinostat-treated mice. Gene expression profile analysis revealed 56 genes significantly different in the treated group; these included the upregulation of p21WAF1, induction of core histone deacetylase (HDAC), and cell communication genes. CONCLUSION: Our data demonstrate that belinostat inhibits bladder cancer and supports the clinical evaluation of belinostat for the treatment of patients with superficial bladder cancer
— id: 74320, year: 2007, vol: 5, page: 49, stat: Journal Article,

Persistent uroplakin expression in advanced urothelial carcinomas: implications in urothelial tumor progression and clinical outcome
Huang, Hong-Ying; Shariat, Shahrokh F; Sun, Tung-Tien; Lepor, Herbert; Shapiro, Ellen; Hsieh, Jer-Tsong; Ashfaq, Raheela; Lotan, Yair; Wu, Xue-Ru
2007 Nov;38(11):1703-1713, Human pathology
As the terminal differentiation products of human urothelium, uroplakins (UPs) would be expected to diminish during urothelial tumorigenesis. Surprisingly, recent studies found UPs to be retained even by well-advanced urothelial carcinomas, suggesting that the loss of UPs does not strictly parallel urothelial transformation. Little is known, however, about whether the status of UPs is associated with a particular pathologic parameter, the tumor's biological behavior, or patient outcome. Here we assessed UP expression by immunohistochemistry on tissue arrays from 285 patients with bladder urothelial carcinomas or nontumor conditions. UPs were expressed in all 9 normal urothelial specimens, 63 of 74 (85%) patients with non-muscle-invasive urothelial carcinomas on transurethral resection, 104 of 202 (51.5%) patients who underwent radical cystectomy for advanced urothelial carcinomas, and 33 of 50 (66%) lymph node metastases. Normally associated with urothelial apical surface, UPs were localized aberrantly in tumors, including microluminal, basal-laminal, cytoplasmic, or uniform patterns. In non-muscle-invasive diseases, there was no association between UP expression and disease recurrence, progression, or mortality. In contrast, in invasive diseases, absent UP expression was significantly associated with advanced pathologic stage, lymph node metastases, disease recurrence, and bladder cancer-specific mortality (P = .042, P = .035, P = .023, and P = .022, respectively) in univariate analyses. Furthermore, UP status was independent of key cell-cycle regulators, including p53, pRb, p27, and cyclin D1, thus excluding a functional link between these 2 groups of proteins. Our data demonstrate for the first time that persistent UP expression is associated with a favorable clinical outcome and that UPs may be used as adjunct markers for predicting the prognoses of patients with invasive and metastatic bladder carcinomas. Our results also suggest that UP-positive and -negative carcinomas have different clonal origins or may be derived from different cancer stem cells
— id: 73404, year: 2007, vol: 38, page: 1703, stat: Journal Article,

Compositional differences between infant and adult human corneal basement membranes
Kabosova, Andrea; Azar, Dimitri T; Bannikov, Gregory A; Campbell, Kevin P; Durbeej, Madeleine; Ghohestani, Reza F; Jones, Jonathan C R; Kenney, M Cristina; Koch, Manuel; Ninomiya, Yoshifumi; Patton, Bruce L; Paulsson, Mats; Sado, Yoshikazu; Sage, E Helene; Sasaki, Takako; Sorokin, Lydia M; Steiner-Champliaud, Marie-France; Sun, Tung-Tien; Sundarraj, Nirmala; Timpl, Rupert; Virtanen, Ismo; Ljubimov, Alexander V
2007 Nov;48(11):4989-4999, Investigative ophthalmology & visual science. IOVS
PURPOSE: Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development. METHODS: Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components. RESULTS: Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from alpha1-alpha2 to alpha3-alpha4 chains after 3 years of life; in the adult, alpha1-alpha2 chains were retained only in the limbal BM. Laminin alpha2 and beta2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, beta2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin gamma3 chain. In the infant DM, type IV collagen alpha1-alpha6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years. CONCLUSIONS: The distribution of laminin gamma3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation
— id: 115883, year: 2007, vol: 48, page: 4989, stat: Journal Article,

Hyperactivation of Ha-ras oncogene, but not Ink4a/Arf deficiency, triggers bladder tumorigenesis
Mo, Lan; Zheng, Xiaoyong; Huang, Hong-Ying; Shapiro, Ellen; Lepor, Herbert; Cordon-Cardo, Carlos; Sun, Tung-Tien; Wu, Xue-Ru
2007 Feb 1;117(2):314-325, Journal of clinical investigation
Although ras is a potent mitogenic oncogene, its tumorigenicity depends on cellular context and cooperative events. Here we show that low-level expression of a constitutively active Ha-ras in mouse urothelium induces simple urothelial hyperplasia that is resistant to progression to full-fledged bladder tumors even in the absence of Ink4a/Arf. In stark contrast, doubling of the gene dosage of the activated Ha-ras triggered early-onset, rapidly growing, and 100% penetrant tumors throughout the urinary tract. Tumor initiation required superseding a rate-limiting step between simple and nodular hyperplasia, the latter of which is marked by the emergence of mesenchymal components and the coactivation of AKT and STAT pathways as well as PTEN inactivation. These results indicate that overactivation of Ha-ras is both necessary and sufficient to induce bladder tumors along a low-grade, noninvasive papillary pathway, and they shed light on the recent findings that ras activation, via point mutation, overexpression, or intensified signaling from FGF receptor 3, occurs in 70%-90% of these tumors in humans. Our results highlight the critical importance of the dosage/strength of Ha-ras activation in dictating its tumorigenicity - a mechanism of oncogene activation not fully appreciated to date. Finally, our results have clinical implications, as inhibiting ras and/or its downstream effectors, such as AKT and STAT3/5, could provide alternative means to treat low-grade, superficial papillary bladder tumors, the most common tumor in the urinary system
— id: 70641, year: 2007, vol: 117, page: 314, stat: Journal Article,

Origin of the tetraspanin uroplakins and their co-evolution with associated proteins: implications for uroplakin structure and function
Garcia-Espana, Antonio; Chung, Pei-Jung; Zhao, Xiaoqian; Lee, Andy; Pellicer, Angel; Yu, Jun; Sun, Tung-Tien; Desalle, Rob
2006 Nov;41(2):355-367, Molecular phylogenetics & evolution
Genome level information coupled with phylogenetic analysis of specific genes and gene families allow for a better understanding of the structure and function of their protein products. In this study, we examine the mammalian uroplakins (UPs) Ia and Ib, members of the tetraspanin superfamily, that interact with uroplakins UPII and UPIIIa/IIIb, respectively, using a phylogenetic approach of these genes from whole genome sequences. These proteins interact to form urothelial plaques that play a central role in the permeability barrier function of the apical urothelial surface of the urinary bladder. Since these plaques are found exclusively in mammalian urothelium, it is enigmatic that UP-like genomic sequences were recently found in lower vertebrates without a typical urothelium. We have cloned full-length UP-related cDNAs from frog (Xenopus laevis), chicken (Gallus gallus), and zebrafish (Danio rerio), and combined these data with sequence information from their orthologs in all the available fully sequenced and annotated animal genomes. Phylogenetic analyses of all the available uroplakin sequences, and an understanding of their distribution in several animal taxa, suggest that: (i) the UPIa/UPIb and UPII/UPIII genes evolved by gene duplication in the common ancestor of vertebrates; (ii) uroplakins can be lost in different combinations in vertebrate lineages; and (iii) there is a strong co-evolutionary relationship between UPIa and UPIb and their partners UPII and UPIIIa/IIIb, respectively. The co-evolution of the tetraspanin UPs and their associated proteins may fine-tune the structure and function of uroplakin complexes enabling them to perform diverse species- and tissue-specific functions. The structure and function of uroplakins, which are also expressed in Xenopus kidney, oocytes and fat body, are much more versatile than hitherto appreciated
— id: 115882, year: 2006, vol: 41, page: 355, stat: Journal Article,

Use of nitrocellulose membranes for protein characterization by matrix-assisted laser desorption/ionization mass spectrometry
Luque-Garcia, Jose L; Zhou, Ge; Sun, Tung-Tien; Neubert, Thomas A
2006 Jul 15;78(14):5102-5108, Analytical chemistry
We present an improved method for MALDI-MS analysis of proteins that have been electroblotted onto a nitrocellulose (NC) membrane. With this approach, electroblotted proteins can be analyzed directly for intact molecular weight determination or after on-membrane digestion by dissolution of the nitrocellulose in MALDI matrix solution containing 70% acetonitrile and 30% methanol. This solution helps maintain solubility of proteins and peptides while dissolving the NC membrane, which is dissolved by 100% acetone in other protocols. On-membrane tryptic digestion using this method requires half the time of in-gel digestion and results in fewer missed cleavages and better protein coverage. For the membrane proteins studied, bovine uroplakins II and III, the protein coverage was almost twice that provided by conventional in-gel digestion, and the transmembrane domains of both uroplakins were detected only after on-membrane digestion. We also demonstrated the compatibility with MALDI-MS of a new dye, MemCode, which is specifically designed for staining NC membrane-immobilized proteins and is faster and more sensitive than Ponceau-S. Our improved on-membrane digestion protocol greatly improves the study of soluble and, particularly strikingly, integral membrane proteins by mass spectrometry
— id: 71579, year: 2006, vol: 78, page: 5102, stat: Journal Article,

Structural basis for tetraspanin functions as revealed by the cryo-EM structure of uroplakin complexes at 6-A resolution
Min, Guangwei; Wang, Huaibin; Sun, Tung-Tien; Kong, Xiang-Peng
2006 Jun 19;173(6):975-983, Journal of cell biology
Tetraspanin uroplakins (UPs) Ia and Ib, together with their single-spanning transmembrane protein partners UP II and IIIa, form a unique crystalline 2D array of 16-nm particles covering almost the entire urothelial surface. A 6 A-resolution cryo-EM structure of the UP particle revealed that the UP tetraspanins have a rod-shaped structure consisting of four closely packed transmembrane helices that extend into the extracellular loops, capped by a disulfide-stabilized head domain. The UP tetraspanins form the primary complexes with their partners through tight interactions of the transmembrane domains as well as the extracellular domains, so that the head domains of their tall partners can bridge each other at the top of the heterotetramer. The secondary interactions between the primary complexes and the tertiary interaction between the 16-nm particles contribute to the formation of the UP tetraspanin network. The rod-shaped tetraspanin structure allows it to serve as stable pilings in the lipid sea, ideal for docking partner proteins to form structural/signaling networks
— id: 67387, year: 2006, vol: 173, page: 975, stat: Journal Article,

Novel blood biomarkers of human urinary bladder cancer
Osman, Iman; Bajorin, Dean F; Sun, Tung-Tien; Zhong, Hong; Douglas, Diah; Scattergood, Joseph; Zheng, Run; Han, Mark; Marshall, K Wayne; Liew, Choong-Chin
2006 Jun 1;12(11 Pt 1):3374-3380, Clinical cancer research
PURPOSE: Recent data indicate that cDNA microarray gene expression profile of blood cells can reflect disease states and thus have diagnostic value. We tested the hypothesis that blood cell gene expression can differentiate between bladder cancer and other genitourinary cancers as well as between bladder cancer and healthy controls. EXPERIMENTAL DESIGN: We used Affymetrix U133 Plus 2.0 GeneChip (Affymetrix, Santa Clara, CA) to profile circulating blood total RNA from 35 patients diagnosed with one of three types of genitourinary cancer [bladder cancer (n = 16), testicular cancer (n = 10), and renal cell carcinoma (n = 9)] and compared their cDNA profiles with those of 10 healthy subjects. We then verified the expression levels of selected genes from the Affymetrix results in a larger number of bladder cancer patients (n = 40) and healthy controls (n = 27). RESULTS: Blood gene expression profiles distinguished bladder cancer patients from healthy controls and from testicular and renal cancer patients. Differential expression of a combined set of seven gene transcripts (insulin-like growth factor-binding protein 7, sorting nexin 16, chondroitin sulfate proteoglycan 6, and cathepsin D, chromodomain helicase DNA-binding protein 2, nell-like 2, and tumor necrosis factor receptor superfamily member 7) was able to discriminate bladder cancer from control samples with a sensitivity of 83% (95% confidence interval, 67-93%) and a specificity of 93% (95% confidence interval, 76-99%). CONCLUSION: We have shown that the gene expression profile of circulating blood cells can distinguish bladder cancer from other types of genitourinary cancer and healthy controls and can be used to identify novel blood markers for bladder cancer
— id: 68525, year: 2006, vol: 12, page: 3374, stat: Journal Article,

EEDA: A protein associated with an early stage of stratified epithelial differentiation
Sun, Lijie; Ryan, David G; Zhou, Mingyuan; Sun, Tung-Tien; Lavker, Robert M
2006 Jan;206(1):103-111, Journal of cellular physiology
Using suppressive subtractive hybridization, we have identified a novel gene, which we named early epithelial differentiation associated (EEDA), which is uniquely associated with an early stage of stratified epithelial differentiation. In epidermis, esophageal epithelium, and tongue epithelium, EEDA mRNA, and antigen was abundant in suprabasal cells, but was barely detectable in more differentiated cells. Consistent with the limbal location of corneal epithelial stem cells, EEDA was expressed in basal corneal epithelial cells that are out of the stem cell compartment, as well as the suprabasal corneal epithelial cells. The strongest EEDA expression occurred in suprabasal precortical cells of mouse, bovine, and human anagen follicles. Developmental studies showed that the appearance of EEDA in embryonic mouse epidermis (E 15.5) coincided with morphological keratinization. Interestingly, EEDA expression is turned off when epithelia were perturbed by wounding and by cultivation under both low and high Ca(2+) conditions. Our results indicate that EEDA is involved in the early stages of normal epithelial differentiation, and that EEDA is important for the 'normal' differentiation pathway in a wide range of stratified epithelia. (c) 2005 Wiley-Liss, Inc
— id: 59000, year: 2006, vol: 206, page: 103, stat: Journal Article,

Altered phenotype of cultured urothelial and other stratified epithelial cells: implications for wound healing
Sun, Tung-Tien
2006 Jul;291(1):F9-21, American journal of physiology. Renal physiology
The differentiation of cultured stratified epithelial cells can deviate significantly from that of normal epithelium, leading to suggestions that cultured cells undergo abnormal differentiation, or a truncated differentiation. Thus cultured epidermal and corneal epithelial cells stop synthesizing their tissue-specific keratin pair K1/K10 and K3/K12, respectively. The replacement of these keratins in the suprabasal compartment by K6/K16 keratins that are made by all stratified squamous epithelia during hyperplasia rules out a truncated differentiation. Importantly, the keratin pattern of in vivo corneal epithelium undergoing wound repair mimics that of cultured rabbit corneal epithelial cells. Although cultured urothelial cells continue to synthesize uroplakins, which normally form two-dimensional crystalline urothelial plaques covering almost the entire apical urothelial surface, these proteins do not assemble into crystals in cultured cells. Cultured epithelial cells can, however, rapidly regain normal differentiation on the removal of mitogenic stimuli, the use of a suitable extracellular matrix, or the transplantation of the cells to an in vivo, nonmitogenic environment. These data suggest that cultured epithelial cells adopt altered differentiation patterns mimicking in vivo regenerating or hyperplastic epithelia. Blocking the synthesis of tissue-specific differentiation products, such as the K1 and K10 keratins designed to form extensive disulfide cross-links in cornified cells, or the assembly of uroplakin plaques allows epithelial cells to better migrate and proliferate, activities that are of overriding importance during wound repair. Cultured urothelial and other stratified epithelial cells provide excellent models for studying the regulation of the synthesis and assembly of differentiation products, a key cellular process during epithelial wound repair
— id: 111747, year: 2006, vol: 291, page: F9, stat: Journal Article,

Integrity of all four transmembrane domains of the tetraspanin uroplakin Ib is required for its exit from the ER
Tu, Liyu; Kong, Xiang-Peng; Sun, Tung-Tien; Kreibich, Gert
2006 Dec 15;119(Pt 24):5077-5086, Journal of cell science
The surface of the mammalian urinary bladder is covered by a crystalline, asymmetric unit membrane (AUM) structure that contains the four major uroplakins (UPs): Ia, Ib, II and IIIa. UPIa and UPIb belong to the family of tetraspanins. Although UPIa and UPIb are structurally conserved, only UPIb could exit from the endoplasmic reticulum (ER) and reach the cell surface when expressed alone in 293T cells. Modifications of the large extracellular loop of UPIb, such as mutation of the N-glycosylation site or the cysteines involved in the formation of three disulfide bridges, or exchanging the large luminal loop of UPIb with that of UPIa did not affect the ability of UPIb to reach the cell surface. However, modifications of any of the four transmembrane domains of UPIb led to ER retention, suggesting that the proper formation of helical bundles consisting of the tetraspanin transmembrane domains is a prerequisite for UPIb to exit from the ER. Results of sedimentation analysis suggested that aggregate formation is a mechanism for ER retention
— id: 71581, year: 2006, vol: 119, page: 5077, stat: Journal Article,

Distinct glycan structures of uroplakins Ia and Ib: structural basis for the selective binding of FimH adhesin to uroplakin Ia
Xie, Bo; Zhou, Ge; Chan, Shiu-Yung; Shapiro, Ellen; Kong, Xiang-Peng; Wu, Xue-Ru; Sun, Tung-Tien; Costello, Catherine E
2006 May 26;281(21):14644-14653, Journal of biological chemistry
Although it has been shown that mouse uroplakin (UP) Ia, a major glycoprotein of urothelial apical surface, can serve as the receptor for the FimH lectin adhesin of type 1-fimbriated Escherichia coli, the organism that causes a great majority of urinary tract infections, the glycan structure of this native receptor was unknown. Using a sensitive approach that combines in-gel glycosidase and protease digestions, permethylation of released glycans, and mass spectrometry, we have elucidated for the first time the native glycoform structures of the mouse UPIa receptor and those of its non-binding homolog, UPIb, and have determined the glycosylation site occupancy. UPIa presents a high level of terminally exposed mannose residues (located on Man(6)GlcNAc(2) to Man(9)GlcNAc(2)) that are capable of specifically interacting with FimH. We have shown that this property is conserved not only in the mouse uroplakins but also in cattle and, even more importantly, in human UPIa, thus establishing the concept that UPIa is a major urothelial receptor in humans and other mammals for the mannose-specific FimH variant. In contrast, our results indicate that most terminally exposed glycans of mouse UPIb are non-mannose residues, thus explaining the failure of FimH to bind to this UPIb. In cattle, on the other hand, complex carbohydrates constituted only about 20% of the UPIb N-linked glycans. Human UPIa contained exclusively high mannose glycans, and human UPIb contained only complex glycans. The drastically different carbohydrate processing of the UPIa and UPIb proteins, two closely related members of the tetraspanin family, may reflect differences in their folding and masking due to their interactions with their associated proteins, UPII and UPIIIa, respectively. Results from this study shed light on the molecular pathogenesis of urinary tract infections and may aid in the design of glyco-mimetic inhibitors for preventing and treating this disease
— id: 66476, year: 2006, vol: 281, page: 14644, stat: Journal Article,

Expression of an olfactomedin-related gene in rat hair follicular papilla cells
Cao, Qiong; Yu, Dawen; Lee, Andy; Kasai, Yuko; Tychsen, Birte; Paus, Ralf; Freedberg, Irwin M; Sun, Tung-Tien
2005 Jul;125(1):24-33, Journal of investigative dermatology
Follicular papilla (FP) cells, but not their closely related dermal fibroblasts, can maintain hair growth suggesting cell type-specific molecular signals. To define the molecular differences between these two cell types, we generated a subtraction complementary DNA (cDNA) library highly enriched in FP-specific cDNA. Differential screening identified FP-1 as the most abundant cDNA sequence in this subtraction library. FP-1 message RNA is highly abundant in cultured rat vibrissa FP cells, can be detected at very low levels in the stomach and the ovary, and is undetectable in cultured dermal fibroblasts and in 16 rat non-follicular tissues. The full-length, 2.3 kb FP-1 cDNA encodes a protein of 549 amino acids harboring a signal peptide, collagen triple helix repeats, and an olfactomedin-like domain. Monospecific rabbit antibodies to FP-1 recognize in cultured FP cells a single approximately 72 kDa glycoprotein with a approximately 60 kDa protein core. FP-1 protein is expressed in vivo in a hair cycle-dependent manner, as it can be detected in FP during anagen, but not in catagen and telogen phases of the hair cycle. FP-1 is presumably a highly specific extracellular matrix protein synthesized by FP cells and may be involved in the organization of FP during certain phases of normal or pathological hair growth
— id: 56394, year: 2005, vol: 125, page: 24, stat: Journal Article,

Differential expression of cell cycle regulators in phenotypic variants of transgenically induced bladder tumors: implications for tumor behavior
Garcia-Espana, Antonio; Salazar, Edgard; Sun, Tung-Tien; Wu, Xue-Ru; Pellicer, Angel
2005 Feb 15;65(4):1150-1157, Cancer research
Proteins controlling cell growth, differentiation, apoptosis, and oncogenic stress are often deregulated in tumor cells. However, whether such deregulations affect tumor behavior remains poorly understood in many tumor types. We recently showed that the urothelium-specific expression of activated H-ras and SV40 T antigen in transgenic mice produced two distinctive types of tumors strongly resembling the human superficial papillary tumors and carcinoma in situ of the bladder, respectively. Here we assessed the expression of a key set of cell cycle regulators in these mouse tumors and in a new transgenic line expressing a cyclin D1 oncogene in the urothelium. We found that urothelia of the wild-type and cyclin D1 transgenic mice exhibited a profile of cell cycle regulators found in quiescent (G(0)) cells, indicating that urothelium overexpressing the cyclin D1 (an 8-fold increase) is reminiscent of normal urothelium and remains slow-cycling. Low-grade superficial papillary tumors induced by activated H-ras had no detectable Rb family proteins (Rb, p107, and p130) and late cell cycle cyclins and kinases (cyclin A, E, and CDK1), but had increased level of p16, p53, and MDM2. These data suggest that the inactivation of the Rb pathway plays an important role in H-ras-induced superficial papillary tumors and that oncogenic H-ras can induce a compensatory activation of alternative tumor suppressor pathways. In contrast, carcinoma in situ of the bladder induced by SV40 T antigen had increased expression of cell cycle regulators mainly active in post-G(1) phases. The fact that phenotypically different bladder tumors exhibit different patterns of cell cycle regulators may explain why these tumors have different propensity to progress to invasive tumors. Our results indicate that the transgenic mouse models can be used not only for studying tumorigenesis but also for evaluating therapeutic strategies that target specific cell cycle regulators
— id: 48701, year: 2005, vol: 65, page: 1150, stat: Journal Article,

Assembly of Urothelial Plaques: Tetraspanin Function in Membrane Protein Trafficking
Hu, Chih-Chi Andrew; Liang, Feng-Xia; Zhou, Ge; Tu, Liyu; Tang, Chih-Hang Anthony; Zhou, Jessica; Kreibich, Gert; Sun, Tung-Tien
2005 Sep;16(9):3937-3950, Molecular biology of the cell
Monitoring Editor: Jeffrey Brodsky The apical surface of mammalian urothelium is covered by 16-nm protein particles packed hexagonally to form 2D crystals of asymmetric unit membranes (AUM) that contribute to the remarkable permeability barrier function of the urinary bladder. We have shown previously that bovine AUMs contain four major integral membrane proteins, i.e., uroplakins Ia, Ib, II and IIIa, and that UPIa and Ib (both tetraspanins) form heterodimers with UPII and IIIa, respectively. Using a panel of antibodies recognizing different conformational states of uroplakins, we demonstrate that the UPIa-dependent, furin-mediated cleavage of the prosequence of UPII leads to global conformational changes in mature UPII, and that UPIb also induces conformational changes in its partner UPIIIa. We further demonstrate that tetraspanins CD9, CD81 and CD82 can stabilize their partner protein CD4. These results indicate that tetraspanin uroplakins, and some other tetraspanin proteins, can induce conformational changes leading to the ER-exit, stabilization and cell surface expression of their associated, single-transmembrane-domained partner proteins, and thus can function as 'maturation-facilitators.' We propose a model of AUM assembly in which conformational changes in integral membrane proteins induced by uroplakin interactions, differentiation-dependent glycosylation and the removal of the prosequence of UPII play roles in regulating the assembly of uroplakins to form AUM
— id: 56078, year: 2005, vol: 16, page: 3937, stat: Journal Article,

De novo Uroplakin IIIa heterozygous mutations cause human renal adysplasia leading to severe kidney failure
Jenkins, Dagan; Bitner-Glindzicz, Maria; Malcolm, Sue; Hu, Chih-Chi A; Allison, Jennifer; Winyard, Paul J D; Gullett, Ambrose M; Thomas, David F M; Belk, Rachel A; Feather, Sally A; Sun, Tung-Tien; Woolf, Adrian S
2005 Jul;16(7):2141-2149, Journal of the American Society of Nephrology
Human renal adysplasia usually occurs sporadically, and bilateral disease is the most common cause of childhood end-stage renal failure, a condition that is lethal without intervention using dialysis or transplantation. De novo heterozygous mutations in Uroplakin IIIa (UPIIIa) are reported in four of 17 children with kidney failure caused by renal adysplasia in the absence of an overt urinary tract obstruction. One girl and one boy in unrelated kindreds had a missense mutation at a CpG dinucleotide in the cytoplasmic domain of UPIIIa (Pro273Leu), both of whom had severe vesicoureteric reflux, and the girl had persistent cloaca; two other patients had de novo mutations in the 3' UTR (963 T-->G; 1003 T-->C), and they had renal adysplasia in the absence of any other anomaly. The mutations were absent in all sets of parents and in siblings, none of whom had radiologic evidence of renal adysplasia, and mutations were absent in two panels of 192 ethnically matched control chromosomes. UPIIIa was expressed in nascent urothelia in ureter and renal pelvis of human embryos, and it is suggested that perturbed urothelial differentiation may generate human kidney malformations, perhaps by altering differentiation of adjacent smooth muscle cells such that the metanephros is exposed to a functional obstruction of urine flow. With advances in renal replacement therapy, children with renal failure, who would otherwise have died, are surviving to adulthood. Therefore, although the mechanisms of action of the UPIIIa mutations have yet to be determined, these findings have important implications regarding genetic counseling of affected individuals who reach reproductive age
— id: 59001, year: 2005, vol: 16, page: 2141, stat: Journal Article,

Cellular basis of urothelial squamous metaplasia: roles of lineage heterogeneity and cell replacement
Liang, Feng-Xia; Bosland, Maarten C; Huang, Hongying; Romih, Rok; Baptiste, Solange; Deng, Fang-Ming; Wu, Xue-Ru; Shapiro, Ellen; Sun, Tung-Tien
2005 Dec 5;171(5):835-844, Journal of cell biology
Although the epithelial lining of much of the mammalian urinary tract is known simply as the urothelium, this epithelium can be divided into at least three lineages of renal pelvis/ureter, bladder/trigone, and proximal urethra based on their embryonic origin, uroplakin content, keratin expression pattern, in vitro growth potential, and propensity to keratinize during vitamin A deficiency. Moreover, these cells remain phenotypically distinct even after they have been serially passaged under identical culture conditions, thus ruling out local mesenchymal influence as the sole cause of their in vivo differences. During vitamin A deficiency, mouse urothelium form multiple keratinized foci in proximal urethra probably originating from scattered K14-positive basal cells, and the keratinized epithelium expands horizontally to replace the surrounding normal urothelium. These data suggest that the urothelium consists of multiple cell lineages, that trigone urothelium is closely related to the urothelium covering the rest of the bladder, and that lineage heterogeneity coupled with cell migration/replacement form the cellular basis for urothelial squamous metaplasia
— id: 59934, year: 2005, vol: 171, page: 835, stat: Journal Article,

Interpreting epithelial cancer biology in the context of stem cells: tumor properties and therapeutic implications
Miller, Stanley J; Lavker, Robert M; Sun, Tung-Tien
2005 Sep 25;1756(1):25-52, Biochimica & biophysica acta
Over 90% of all human neoplasia is derived from epithelia. Significant progress has been made in the identification of stem cells of many epithelia. In general, epithelial stem cells lack differentiation markers, have superior in vivo and in vitro proliferative potential, form clusters in association with a specialized mesenchymal environment (the 'niche'), are located in well-protected and nourished sites, and are slow-cycling and thus can be experimentally identified as 'label-retaining cells'. Stem cells may divide symmetrically giving rise to two identical stem cell progeny. Any stem cells in the niche, which defines the size of the stem cell pool, may be randomly expelled from the niche due to population pressure (the stochastic model). Alternatively, a stem cell may divide asymmetrically yielding one stem cell and one non-stem cell that is destined to exit from the stem cell niche (asymmetric division model). Stem cells separated from their niche lose their stemness, although such a loss may be reversible, becoming 'transit-amplifying cells' that are rapidly proliferating but have a more limited proliferative potential, and can give rise to terminally differentiated cells. The identification of the stem cell subpopulation in a normal epithelium leads to a better understanding of many previously enigmatic properties of an epithelium including the preferential sites of carcinoma formation, as exemplified by the almost exclusive association of corneal epithelial carcinoma with the limbus, the corneal epithelial stem cell zone. Being long-term residents in an epithelium, stem cells are uniquely susceptible to the accumulation of multiple, oncogenic changes giving rise to tumors. The application of the stem cell concept can explain many important carcinoma features including the clonal origin and heterogeneity of tumors, the occasional formation of tumors from the transit amplifying cells or progenitor cells, the formation of precancerous 'patches' and 'fields', the mesenchymal influence on carcinoma formation and behavior, and the plasticity of tumor cells. While the concept of cancer stem cells is extremely useful and it is generally assumed that such cells are derived from normal stem cells, more work is needed to identify and characterize epithelial cancer stem cells, to address their precise relationship with normal stem cells, to study their markers and their proliferative and differentiation properties and to design new therapies that can overcome their unusual resistance to chemotherapy and other conventional tumor modalities
— id: 58999, year: 2005, vol: 1756, page: 25, stat: Journal Article,

Gene Deletion in Urothelium by Specific Expression of Cre Recombinase
Mo, Lan; Cheng, Jin; Lee, Eva Y-H P; Sun, Tung-Tien; Wu, Xue-Ru
2005 Apr 19;289(3):F562-F568, American journal of physiology. Renal physiology
Urothelium that lines almost the entire urinary tract acts as a permeability barrier and is involved in the pathogenesis of major urinary diseases including urothelial carcinoma, urinary tract infection and interstitial cystitis. However, investigation of urothelial biology and diseases has been hampered by the lack of tissue-specific approaches. To address this deficiency, we sought to develop a urothelium-specific knockout system using the Cre/loxP strategy. Transgenic mouse lines were generated in which a 3.6-kb mouse uroplakin II (UPII) promoter was used to drive the expression of Cre recombinase (Cre). Among the multiple tissues analyzed, Cre was found to be expressed exclusively in the urothelia of the transgenic mice. Crossing a UPII-Cre transgenic line with a ROSA26-LacZ reporter line, in which LacZ expression depends on Cre-mediated deletion of a floxed 'stop' sequence, led to LacZ expression only in the urothelium. Gene recombination was also observed when the UPII-Cre line was crossed to an independent line in which a part of the p53 gene was flanked by the loxP sequences (floxed p53). Truncation of the p53 gene and mRNA were observed exclusively in the urothelia of double transgenic mice harboring both the UPII-Cre transgene and the floxed p53 allele. These results demonstrate for the first time the feasibility and potentially wide applicability of the UPII-Cre transgenic mice to inactivate any genes of interest in the urothelium
— id: 51116, year: 2005, vol: 289, page: F562, stat: Journal Article,

Urothelial umbrella cells of human ureter are heterogeneous with respect to their uroplakin composition: different degrees of urothelial maturity in ureter and bladder?
Riedel, Ina; Liang, Feng-Xia; Deng, Fang-Ming; Tu, Liyu; Kreibich, Gert; Wu, Xue-Ru; Sun, Tung-Tien; Hergt, Michaela; Moll, Roland
2005 Mar;84(2-3):393-405, European journal of cell biology
Urothelial umbrella cells are characterized by apical, rigid membrane plaques, which contain four major uroplakin proteins (UP Ia, Ib, II and III) forming UPIa/UPII and UPIb/UPIII pairs. These integral membrane proteins are thought to play an important role in maintaining the physical integrity and the permeability barrier function of the urothelium. We asked whether the four uroplakins always coexpress in the entire human lower urinary tract. We stained immunohistochemically (ABC-peroxidase method) paraffin sections of normal human ureter (n = 18) and urinary bladder (n = 10) using rabbit antibodies against UPIa, UPIb, UPII and UPIII; a recently raised mouse monoclonal antibody (MAb), AU1, and two new MAbs, AU2 and AU3, all against UPIII; and mouse MAbs against umbrella cell-associated cytokeratins CK18 and CK20. Immunoblotting showed that AU1, AU2 and AU3 antibodies all recognized the N-terminal extracellular domain of bovine UPIII. By immunohistochemistry, we found that in 15/18 cases of human ureter, but in only 2/10 cases of bladder, groups of normal-looking, CK18-positive umbrella cells lacked both UPIII and UPIb immunostaining. The UPIb/UPIII-negative cells showed either normal or reduced amounts of UPIa and UPII staining. These data were confirmed by double immunofluorescence microscopy. The distribution of the UPIb/UPIII-negative umbrella cells was not correlated with localized urothelial proliferation (Ki-67 staining) or with the distribution pattern of CK20. Similar heterogeneities were observed in bovine but not in mouse ureter. We provide the first evidence that urothelial umbrella cells are heterogeneous as some normal-looking umbrella cells can possess only one, instead of two, uroplakin pairs. This heterogeneity seems more prominent in the urothelium of human ureter than that of bladder. This finding may indicate that ureter urothelium is intrinsically different from bladder urothelium. Alternatively, a single lineage of urothelium may exhibit different phenotypes resulting from extrinsic modulations due to distinct mesenchymal influence and different degrees of pressure and stretch in bladder versus ureter. Additional studies are needed to distinguish these two possibilities and to elucidate the physiological and pathological significance of the observed urothelial and uroplakin heterogeneity
— id: 51032, year: 2005, vol: 84, page: 393, stat: Journal Article,

A survivin gene signature predicts aggressive tumor behavior
Salz, Whitney; Eisenberg, Dan; Plescia, Janet; Garlick, David S; Weiss, Robert M; Wu, Xue-Ru; Sun, Tung-Tien; Altieri, Dario C
2005 May 1;65(9):3531-3534, Cancer research
Gene signatures that predict aggressive tumor behavior at the earliest stages of disease, ideally before overt tissue abnormalities, are urgently needed. To search for such genes, we generated a transgenic model of survivin, an essential regulator of cell division and apoptosis overexpressed in cancer. Transgenic expression of survivin in the urinary bladder did not cause histologic abnormalities of the urothelium. However, microarray analysis revealed that survivin-expressing bladders exhibited profound changes in gene expression profile affecting extracellular matrix and inflammatory genes. Following exposure to a bladder carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (OH-BBN), survivin transgenic animals exhibited accelerated tumor progression, preferential incidence of tumors as compared with premalignant lesions, and dramatically abbreviated survival. Conversely, transgenic expression of a survivin Thr(34)-->Ala dominant-negative mutant did not cause changes in gene expression or accelerated tumor progression after OH-BBN treatment. Therefore, survivin expression induces global transcriptional changes in the tissue microenvironment that may promote tumorigenesis. Detection of survivin or its associated gene signature may provide an early biomarker of aggressive tumor behavior before the appearance of tissue abnormalities
— id: 51752, year: 2005, vol: 65, page: 3531, stat: Journal Article,

p53 deficiency provokes urothelial proliferation and synergizes with activated Ha-ras in promoting urothelial tumorigenesis
Gao, Jing; Huang, Hong-Ying; Pak, Joanne; Cheng, Jin; Zhang, Zhong-Ting; Shapiro, Ellen; Pellicer, Angel; Sun, Tung-Tien; Wu, Xue-Ru
2004 Jan 22;23(3):687-696, Oncogene
Mutation and deletion of the p53 tumor suppressor gene are arguably the most prevalent among the multiple genetic alterations found in human bladder cancer, but these p53 defects are primarily associated with the advanced diseases, and their roles in bladder tumor initiation and in synergizing with oncogenes in tumor progression have yet to be defined. Using the mouse uroplakin II gene promoter, we have targeted into urothelium of transgenic mice a dominant-negative mutant of p53 that lacks the DNA-binding domain but retains the tetramerization domain. Urothelium-expressed p53 mutant binds to and stabilizes the endogenous wild-type p53, induces nuclear abnormality, hyperplasia and occasionally dysplasia, without eliciting frank carcinomas. Concurrent expression of the p53 mutant with an activated Ha-ras, the latter of which alone induces urothelial hyperplasia, fails to accelerate tumor formation. In contrast, the expression of the activated Ha-ras in the absence of p53, as accomplished by crossing the activated Ha-ras transgenic mice with the p53 knockout mice, results in early-onset bladder tumors that are either low-grade superficial papillary or high grade in nature. These results provide the first in vivo experimental evidence that p53 deficiency predisposes the urothelium to hyperproliferation, but is insufficient for bladder tumorigenesis; that the mere reduction of p53 dosage, as produced in transgenic mice expressing the dominant-negative p53 or in heterozygous p53 knockouts, is incapable of synergizing with Ha-ras to induce bladder tumors; and that the complete loss of p53 is a prerequisite for collaborating with activated Ha-ras to promote bladder tumorigenesis
— id: 42019, year: 2004, vol: 23, page: 687, stat: Journal Article,

Lack of major involvement of human uroplakin genes in vesicoureteral reflux: Implications for disease heterogeneity
Jiang, Songshan; Gitlin, Jordan; Deng, Fang-Ming; Liang, Feng-Xia; Lee, Andy; Atala, Anthony; Bauer, Stuart B; Ehrlich, Garth D; Feather, Sally A; Goldberg, Judith D; Goodship, Judith A; Goodship, Timothy H J; Hermanns, Monika; Hu, Fen Ze; Jones, Katrin E; Malcolm, Sue; Mendelsohn, Cathy; Preston, Robert A; Retik, Alan B; Schneck, Francis X; Wright, Victoria; Ye, Xiang Y; Woolf, Adrian S; Wu, Xue-Ru; Ostrer, Harry; Shapiro, Ellen; Yu, Jun; Sun, Tung-Tien
2004 Jul;66(1):10-19, Kidney international
Lack of major involvement of human uroplakin genes in vesicoureteral reflux: Implications for disease heterogeneity. Background. Primary vesicoureteral reflux (VUR) is a hereditary disorder characterized by the retrograde flow of urine into the ureters and kidneys. It affects about 1% of the young children and is thus one of the most common hereditary diseases. Its associated nephropathy is an important cause of end-stage renal failure in children and adults. Recent studies indicate that genetic ablation of mouse uroplakin (UP) III gene, which encodes a 47 kD urothelial-specific integral membrane protein forming urothelial plaques, causes VUR and hydronephrosis. Methods. To begin to determine whether mutations in UP genes might play a role in human VUR, we genotyped all four UP genes in 76 patients with radiologically proven primary VUR by polymerase chain reaction (PCR) amplification and sequencing of all their exons plus 50 to 150 bp of flanking intronic sequences. Results. Eighteen single nucleotide polymorphisms (SNPs) were identified, seven of which were missense, with no truncation or frame shift mutations. Since healthy relatives of the VUR probands are not reliable negative controls for VUR, we used a population of 90 race-matched, healthy individuals, unrelated to the VUR patients, as controls to perform an association study. Most of the SNPs were not found to be significantly associated with VUR. However, SNP1 of UP Ia gene affecting a C to T conversion and an Ala7Val change, and SNP7 of UP III affecting a C to G conversion and a Pro154Ala change, were marginally associated with VUR (both P= 0.08). Studies of additional cases yielded a second set of data that, in combination with the first set, confirmed a weak association of UP III SNP7 in VUR (P= 0.036 adjusted for both subsets of cases vs. controls). Conclusion. Such a weak association and the lack of families with simple dominant Mendelian inheritance suggest that missense changes of uroplakin genes cannot play a dominant role in causing VUR in humans, although they may be weak risk factors contributing to a complex polygenic disease. The fact that no truncation or frame shift mutations have been found in any of the VUR patients, coupled with our recent finding that some breeding pairs of UP III knockout mice yield litters that show not only VUR, but also severe hydronephrosis and neonatal death, raises the possibility that major uroplakin mutations could be embryonically or postnatally lethal in humans
— id: 43158, year: 2004, vol: 66, page: 10, stat: Journal Article,

Roles of uroplakins in plaque formation, umbrella cell enlargement, and urinary tract diseases
Kong, Xiang-Tian; Deng, Fang-Ming; Hu, Ping; Liang, Feng-Xia; Zhou, Ge; Auerbach, Anna B; Genieser, Nancy; Nelson, Peter K; Robbins, Edith S; Shapiro, Ellen; Kachar, Bechara; Sun, Tung-Tien
2004 Dec 20;167(6):1195-1204, Journal of cell biology
The apical surface of mouse urothelium is covered by two-dimensional crystals (plaques) of uroplakin (UP) particles. To study uroplakin function, we ablated the mouse UPII gene. A comparison of the phenotypes of UPII- and UPIII-deficient mice yielded new insights into the mechanism of plaque formation and some fundamental features of urothelial differentiation. Although UPIII knockout yielded small plaques, UPII knockout abolished plaque formation, indicating that both uroplakin heterodimers (UPIa/II and UPIb/III or IIIb) are required for plaque assembly. Both knockouts had elevated UPIb gene expression, suggesting that this is a general response to defective plaque assembly. Both knockouts also had small superficial cells, suggesting that continued fusion of uroplakin-delivering vesicles with the apical surface may contribute to umbrella cell enlargement. Both knockouts experienced vesicoureteral reflux, hydronephrosis, renal dysfunction, and, in the offspring of some breeding pairs, renal failure and neonatal death. These results highlight the functional importance of uroplakins and establish uroplakin defects as a possible cause of major urinary tract anomalies and death
— id: 48112, year: 2004, vol: 167, page: 1195, stat: Journal Article,

Corneal epithelial stem cells at the limbus: looking at some old problems from a new angle
Lavker, Robert M; Tseng, Scheffer C G; Sun, Tung-Tien
2004 Mar;78(3):433-446, Experimental eye research
Corneal epithelium is traditionally thought to be a self-sufficient, self-renewing tissue implying that its stem cells are located in its basal cell layer. Recent studies indicate however that corneal epithelial stem cells reside in the basal layer of peripheral cornea in the limbal zone, and that corneal and conjunctival epithelia represent distinct cell lineages. These ideas are supported by the unique limbal/corneal expression pattern of the K3 keratin marker for corneal-type differentiation; the restriction of the slow-cycling (label-retaining) cells in the limbus; the distinct keratin expression patterns of corneal and conjunctival epithelial cells even when they are provided with identical in vivo and in vitro growth environments; and the limbal cells' superior ability as compared with central corneal epithelial cells in undergoing in vitro proliferation and in reconstituting in vivo an intact corneal epithelium. The realization that corneal epithelial stem cells reside in the limbal zone provides explanations for several paradoxical properties of corneal epithelium including its 'mature-looking' basal cells, the preponderance of tumor formation in the limbal zone, and the centripetal cellular migration. The limbal stem cell concept has led to a better understanding of the strategies of corneal epithelial repair, to a new classification of various anterior surface epithelial diseases, to the use of limbal stem cells for the reconstruction of corneal epithelium damaged or lost as a consequence of trauma or disease ('limbal stem cell transplantation'), and to the rejection of the traditional notion of 'conjunctival transdifferentiation'. The fact that corneal epithelial stem cells reside outside of the cornea proper suggests that studying corneal epithelium per se without taking into account its limbal zone will yield partial pictures. Future studies need to address the signals that constitute the limbal stem cell niche, the mechanism by which amniotic membrane facilitates limbal stem cell transplantation and ex vivo expansion, and the lineage flexibility of limbal stem cells
— id: 49560, year: 2004, vol: 78, page: 433, stat: Journal Article,

Detection of circulating cancer cells expressing uroplakins and epidermal growth factor receptor in bladder cancer patients
Osman, Iman; Kang, Melissa; Lee, Andy; Deng, Fang-Ming; Polsky, David; Mikhail, Maryann; Chang, Caroline; David, Dexter A; Mitra, Nandita; Wu, Xue-Ru; Sun, Tung-Tien; Bajorin, Dean F
2004 Oct 10;111(6):934-939, International journal of cancer
Our purpose was to determine the clinical relevance of the detection of circulating tumor cells (CTCs) expressing urothelial and epithelial markers in bladder cancer patients. Sixty-two patients who presented to Memorial Sloan-Kettering Cancer Center between July 2000 and September 2001 were studied. Peripheral blood was tested by nested RT-PCR assay for uroplakins (UPs) Ia, Ib, II and III as well as for epidermal growth factor receptor (EGFR). We determined the sensitivity and specificity of each individual marker and the combinations of UPIa/UPII and UPIb/UPIII. The latter strategy was based on our data, which showed that UPIa and UPIb form heterodimers with UPII and UPIII, respectively. Forty patients had clinically advanced bladder cancer and 22 had no evidence of disease at the time of assay. Eight of the 22 patients recurred during the follow-up period. All 8 patients were positive at presentation for UPIa/UPII. The combination of UPIa/UPII provided the best sensitivity (75%) of detecting CTCs, with a specificity of 50%. The combination of UPIb/UPIII was the most specific (79%) but had modest sensitivity (31%). Detection of EGFR-positive cells alone and in combination with UPs was inferior to that for UPIa/UPII. Combinations of urothelial markers are superior to single urothelial or epithelial markers in detecting CTCs in bladder cancer patients. Further efforts are under way to confirm the potential predictive value of these markers in a prospectively designed study of a larger cohort of patients.
— id: 44185, year: 2004, vol: 111, page: 934, stat: Journal Article,

Excessive trust in authorities and its influence on experimental design
Sun, Tung-Tien
2004 Jul;5(7):577-581, Nature reviews. Molecular cell biology
— id: 43629, year: 2004, vol: 5, page: 577, stat: Journal Article,

Corneal epithelial stem cells: past, present, and future
Sun, Tung-Tien; Lavker, Robert M
2004 Sep;9(3):202-207, Journal of investigative dermatology symposium proceedings
Corneal epithelium is a self-renewing tissue. Recent studies indicate that corneal epithelial stem cells reside preferentially in the basal layer of peripheral cornea in the limbal zone, rather than uniformly in the entire corneal epithelium. This idea is supported by a unique limbal/corneal expression pattern of the K3 keratin marker for corneal-type differentiation; the preferential distribution of the slow-cycling (label-retaining) cells in the limbus; the superior proliferative capacity of limbal cells as compared with central corneal epithelial cells in vitro and in vivo; and the ability of limbal basal cells to rescue/reconstitute severely damaged or completely depleted corneal epithelium upon transplantation. The limbal/stem cell concept provides explanations for several paradoxical properties of corneal epithelium including the predominance of tumor formation in the limbal zone, the centripetal migration of peripheral corneal cells toward the central cornea, and the 'mature-looking' phenotype of the corneal basal cells. The limbal stem cell concept has led to a better understanding of the strategies that a stratified squamous epithelium uses in repair, to a new classification of various anterior surface epithelial diseases, to a repudiation of the classical idea of 'conjunctival transdifferentiation', and to a new surgical procedure called limbal stem cell transplantation
— id: 48100, year: 2004, vol: 9, page: 202, stat: Journal Article,

Expression profiles of tyrosine kinases in cultured follicular papilla cells versus dermal fibroblasts
Yu, Dawen; Cao, Qiong; He, Zhijun; Sun, Tung-Tien
2004 Aug;123(2):283-290, Journal of investigative dermatology
Tyrosine kinases play crucial roles in cell differentiation and proliferation. Using degenerative primed PCR followed by differential display, we analyzed the tyrosine kinase expression profiles of cultured rat follicular papilla (FP) cells versus dermal fibroblasts. We showed that c-met, cdc2, and tec were preferentially expressed in cultured FP cells, whereas alpha-platelet-derived growth factor receptor (alpha-PDGFR) was preferentially expressed in cultured fibroblasts. The cell type specificity of these tyrosine kinases was confirmed by semi-quantitative RT-PCR using both rat and human cultured cells. Consistent with these results, hepatocyte growth factor preferentially stimulated the growth of rat FP cells, whereas PDGF-AA preferentially stimulated rat fibroblasts. High concentrations of some these kinases are also found in the follicular matrix keratinocytes as revealed by in situ hybridization. The expression of specific tyrosine kinases in FP and matrix cells may play roles in regulating hair growth and cycling
— id: 44186, year: 2004, vol: 123, page: 283, stat: Journal Article,

Rab27b is associated with fusiform vesicles and may be involved in targeting uroplakins to urothelial apical membranes
Chen, Yanru; Guo, Xuemei; Deng, Fang-Ming; Liang, Feng-Xia; Sun, Wenyu; Ren, Mindong; Izumi, Tetsuro; Sabatini, David D; Sun, Tung-Tien; Kreibich, Gert
2003 Nov 25;100(24):14012-14017, Proceedings of the National Academy of Sciences of the United States of America
The terminally differentiated umbrella cells of bladder epithelium contain unique cytoplasmic organelles, the fusiform vesicles, which deliver preassembled crystalline arrays of uroplakin proteins to the apical cell surface of urothelial umbrella cells. We have investigated the possible role of Rab proteins in this delivery process, and found Rab27b to be expressed at an extraordinary high level (0.1% of total protein) in urothelium, whereas Rab27b levels were greatly reduced (to <5% of normal urothelium) in cultured urothelial cells, which synthesized only small amounts of uroplakins and failed to form fusiform vesicles. Immuno-electron microscopy showed that Rab27b was associated with the cytoplasmic face of the fusiform vesicles, but not with that of the apical plasma membrane. The association of Rab27b with fusiform vesicles and its differentiation-dependent expression suggest that this Rab protein plays a role in regulating the delivery of fusiform vesicles to the apical plasma membrane of umbrella cells
— id: 42018, year: 2003, vol: 100, page: 14012, stat: Journal Article,

Allelic loss of p53 gene is associated with genesis and maintenance, but not invasion, of mouse carcinoma in situ of the bladder
Cheng, Jin; Huang, Hongying; Pak, Joanne; Shapiro, Ellen; Sun, Tung-Tien; Cordon-Cardo, Carlos; Waldman, Frederic M; Wu, Xue-Ru
2003 Jan 1;63(1):179-185, Cancer research
Carcinoma in situ (CIS) of the bladder has recently been proposed to be a heterogeneous group of diseases with varied histogenesis and biological behavior. In this study, we describe the sequential steps of CIS development and progression in a transgenic mouse model expressing low levels of the SV40 large T antigen. We found that CIS in transgenic mice arose from urothelial dysplasia, that CIS could persist for an extended period of time without invasion, and that the majority of CIS eventually evolved into high-grade, superficial, papillary tumors before a small fraction of them advanced to invasion/metastasis. A genome-wide search of chromosomal imbalances by comparative genomic hybridization revealed that 9 of 11 (82%) of CIS had losses on chromosome 11. Southern blotting demonstrated the allelic loss of the p53 gene, which resides on mouse chromosome 11, in four comparative genomic hybridization-tested tumors and 10 of 11 (91%) additional CIS examined. Consistent with the reduced p53 gene dosage because of the allelic loss and the functional inactivation of p53 protein of the remaining allele by SV40T antigen, there was a dramatic decrease in CIS of Mdm-2, a major p53 target. In contrast, the level of p21, another p53 target, was largely unaltered, suggesting that p21 expression can be regulated by p53-independent mechanisms. These results delineate the early stages of bladder tumorigenesis and suggest that the loss of a p53-bearing chromosome is an early event in bladder tumorigenesis and is crucial for the genesis and the maintenance, but not the progression, of bladder CIS. On the basis of our current and previous transgenic studies, we have proposed an integrated pathway progression model of bladder cancer
— id: 34168, year: 2003, vol: 63, page: 179, stat: Journal Article,

Epithelial stem cells: the eye provides a vision
Lavker, R M; Sun, T-T
2003 Nov;17(8):937-942, Eye
Epithelial stem cells play a central role in tissue homeostasis, wound repair, and carcinogenesis. Corneal epithelial stem cells have been demonstrated to reside in the limbal epithelium, while the fornical zone of the conjunctiva appears to be a predominant site of conjunctival epithelial stem cells. Stem cells of the corneal and conjunctival epithelia, as well as the hair follicle and interfollicular epidermis share important features: they are capable of self renewal; they are relatively quiescent (slow-cycling); they can be induced to proliferate; and they are multipotent. Its becoming apparent that a certain degree of flexibility exists between corneal and hair follicle keratinocytes
— id: 42017, year: 2003, vol: 17, page: 937, stat: Journal Article,

Hair follicle stem cells
Lavker, Robert M; Sun, Tung-Tien; Oshima, Hideo; Barrandon, Yann; Akiyama, Masashi; Ferraris, Corinne; Chevalier, Genevieve; Favier, Bertrand; Jahoda, Colin A B; Dhouailly, Danielle; Panteleyev, Andrei A; Christiano, Angela M
2003 Jun;8(1):28-38, Journal of investigative dermatology symposium proceedings
The workshop on Hair Follicle Stem Cells brought together investigators who have used a variety of approaches to try to understand the biology of follicular epithelial stem cells, and the role that these cells play in regulating the hair cycle. One of the main concepts to emerge from this workshop is that follicular epithelial stem cells are multipotent, capable of giving rise not only to all the cell types of the hair, but also to the epidermis and the sebaceous gland. Furthermore, such multipotent stem cells may represent the ultimate epidermal stem cell. Another example of epithelial stem cell and transit amplifying cell plasticity, was the demonstration that adult corneal epithelium, under the influence of embryonic skin dermis could form an epidermis as well as hair follicles. With regards to the location of follicular epithelial stem cells, immunohistochemical and ultrastructural data was presented, indicating that cells with stem cell attributes were localized to the prominent bulge region of developing human fetal hair follicles. Finally, a new notion was put forth concerning the roles that the bulge-located stem cells and the hair germ cells played with respect to the hair cycle
— id: 49562, year: 2003, vol: 8, page: 28, stat: Journal Article,

Structural basis of urothelial permeability barrier function as revealed by Cryo-EM studies of the 16 nm uroplakin particle
Min, Guangwei; Zhou, Ge; Schapira, Matthieu; Sun, Tung-Tien; Kong, Xiang-Peng
2003 Oct 15;116(Pt 20):4087-4094, Journal of cell science
The apical surface of terminally differentiated mammalian urothelial umbrella cells is covered by numerous plaques consisting of two-dimensional (2D) crystals of hexagonally packed 16 nm uroplakin particles, and functions as a remarkable permeability barrier. To determine the structural basis of this barrier function, we generated, by electron cryo microscopy, a projection map of the isolated mouse urothelial plaques at 7 A and a 3D structure at 10 A resolution. Our results indicate that each 16 nm particle has a central 6 nm lipid-filled 'hole' surrounded by 6 inverted U-shaped subunits, each consisting of an inner and an outer subdomain connected via a distal joint. The transmembrane portion of each subdomain can fit about 5 helices. This finding, coupled with our STEM and EM data, suggests that uroplakin pairs Ia/II and Ib/III are associated with the inner and outer subdomains, respectively. Since the inner subdomains interconnect to form a ring, which can potentially segregate the lipids of the central hole from those outside, the 2D crystalline uroplakin network may impose an organized state and a severely restricted freedom of movement on the lipid components, thus reducing membrane fluidity and contributing to the barrier function of urothelial plaques. Our finding that distinct uroplakin substructures are in contact with the cytoplasmic and exoplasmic leaflets of the plaque suggests that the two leaflets may have different lipid composition and contribute asymmetrically to the barrier function. We propose that the crystalline lattice structure of uroplakin, through its interactions with specialized lipids, plays a major role in the remarkable permeability barrier function of urothelial apical surface. Our results also have implications for the transmembrane signal transduction in urothelial cells as induced by the binding of uropathogenic E. coli to its uroplakin receptor
— id: 39072, year: 2003, vol: 116, page: 4087, stat: Journal Article,

Inverse expression of uroplakins and inducible nitric oxide synthase in the urothelium of patients with bladder outlet obstruction
Romih, R; Korosec, P; Jezernik, K; Sedmak, B; Trsinar, B; Deng, FM; Liang, FX; Sun, TT
2003 APR ;91(6):507-512, BJU international
OBJECTIVE To assess the expression and distribution of uroplakins, protein subunits of the asymmetric unit membrane (AUM), and inducible nitric-oxide synthase (iNOS) in the urinary bladder urothelium of patients with bladder outlet obstruction (BOO) caused by benign prostatic hyperplasia (BPH). PATIENTS AND METHODS Urinary bladder urothelium samples from 15 men (mean age 69 years) with BOO secondary to BPH were processed for light and electron immunocytochemistry. Uroplakins and iNOS were detected, and areas of apical surface covered with AUM were compared with those of iNOS-positive urothelial cells. RESULTS Areas of superficial urothelial cells with no AUM were found in all obstructed bladder samples. The immuno-electron microscopy showed that the uroplakin-positive cells had the characteristic appearance of terminally differentiated umbrella cells, whereas cells from the uroplakin-negative regions were undifferentiated, typically showing microvilli on their apical surface. iNOS was not detected in areas with continuous AUM staining, but was readily detected in the uroplakin-negative areas. There was an inverse correlation between the intensity of uroplakin and iNOS staining. CONCLUSIONS In patients with BOO associated with BPH, some superficial urothelial cells lacked the AUM, suggesting focal compromise of the blood-urine permeability barrier. In such relatively undifferentiated urothelial zones there was an accompanying increase in the expression of iNOS, which marks perturbed urothelial differentiation and may modulate bladder response to the outlet obstruction
— id: 34140, year: 2003, vol: 91, page: 507, stat: Journal Article,

Rab27b association with melanosomes: dominant negative mutants disrupt melanosomal movement
Chen, Yanru; Samaraweera, Preminda; Sun, Tung-Tien; Kreibich, Gert; Orlow, Seth J
2002 Jun;118(6):933-940, Journal of investigative dermatology
The movement of melanosomes from post-Golgi compartments to the periphery of melanocytes is known to be regulated by factors including myosin Va and at least one Rab protein, Rab27a. Mutations in the genes encoding either protein in the mouse result in a hypopigmented phenotype mimicking the human disease Griscelli syndrome. Rab27b and Rab27a share 72% identity and they belong to the same melanocyte/platelet subfamily of Rab proteins. Rab27a orchestrates the transport of melanosomes by recruitment of the actin motor, myosin Va, onto melanosomes. By contrast, the function of Rab27b has remained elusive. In this study, we found that Rab27b mRNA is present in melanocytes and demonstrated the intrinsic GTPase activity of Rab27b protein. We explored the function of Rab27b by overexpression of two dominant negative mutants as well as the wild-type Rab27b in melan-a melanocytes. Green-fluorescent-protein-tagged Rab27b colocalizes with the melanosome marker tyrosinase-related protein 1 and with myosin Va at the cell periphery, whereas Rab27b mutants do not decorate melanosomes, and melanosomes in these mutant transfected cells redistribute from cell periphery to the perinuclear region. Furthermore, transient overexpression of the dominant negative forms of Rab27b caused diminution in both numbers and length of dendrites of melan-a cells. Our results suggest that Rab27b may regulate the outward movement of melanosomes and the formation or maintenance of dendritic extensions in melanocytes
— id: 32487, year: 2002, vol: 118, page: 933, stat: Journal Article,

Overexpression of epidermal growth factor receptor in urothelium elicits urothelial hyperplasia and promotes bladder tumor growth
Cheng, Jin; Huang, Hongying; Zhang, Zhong-Ting; Shapiro, Ellen; Pellicer, Angel; Sun, Tung-Tien; Wu, Xue-Ru
2002 Jul 15;62(14):4157-4163, Cancer research
Although urothelium is constantly bathed in high concentrations of epidermal growth factor (EGF) and most urothelial carcinomas overexpress EGF receptor (EGFr), relatively little is known about the role of EGFr signaling pathway in urothelial growth and transformation. In the present study, we used the uroplakin II gene promoter to drive the urothelial overexpression of EGFr in transgenic mice. Three transgenic lines were established, all expressing a higher level of the EGFr mRNA and protein in the urothelium than the nontransgenic controls. The overexpressed EGFr was functionally active because it was autophosphorylated, and its downstream mitogen-activated protein kinases were highly activated. Phenotypically, the urinary bladders of all transgenic lines developed simple urothelial hyperplasia that was strongly positive for proliferative cell nuclear antigen and weakly positive for bromodeoxyuridine incorporation. When coexpressed with the activated Ha-ras oncogene in double transgenic mice, EGFr had no apparent tumor-enhancing effects over the urothelial hyperplastic phenotype induced by Ha-ras oncogene. However, when coexpressed with the SV40 large T antigen, EGFr accelerated tumor growth and converted the carcinoma in situ of the SV40T mice into high-grade bladder carcinomas, without triggering tumor invasion. Our studies indicate that urothelial overexpression of EGFr can induce urothelial proliferation but not frank carcinoma formation. Our results also suggest that, whereas EGFr and Ha-ras, both of which act in the same signal transduction cascade, stimulated urothelial hyperplasia, they were not synergistic in urothelial tumorigenesis, and EGFr overexpression can cooperate with p53 and pRB dysfunction (as occurring in SV40T transgenic mice) to promote bladder tumor growth
— id: 32471, year: 2002, vol: 62, page: 4157, stat: Journal Article,

Uroplakin IIIb, a urothelial differentiation marker, dimerizes with uroplakin Ib as an early step of urothelial plaque assembly
Deng, Fang-Ming; Liang, Feng-Xia; Tu, Liyu; Resing, Katheryn A; Hu, Ping; Supino, Mark; Hu, Chih-Chi Andrew; Zhou, Ge; Ding, Mingxiao; Kreibich, Gert; Sun, Tung-Tien
2002 Nov 25;159(4):685-694, Journal of cell biology
Urothelial plaques consist of four major uroplakins (Ia, Ib, II, and III) that form two-dimensional crystals covering the apical surface of urothelium, and provide unique opportunities for studying membrane protein assembly. Here, we describe a novel 35-kD urothelial plaque-associated glycoprotein that is closely related to uroplakin III: they have a similar overall type 1 transmembrane topology; their amino acid sequences are 34% identical; they share an extracellular juxtamembrane stretch of 19 amino acids; their exit from the ER requires their forming a heterodimer with uroplakin Ib, but not with any other uroplakins; and UPIII-knockout leads to p35 up-regulation, possibly as a compensatory mechanism. Interestingly, p35 contains a stretch of 80 amino acid residues homologous to a hypothetical human DNA mismatch repair enzyme-related protein. Human p35 gene is mapped to chromosome 7q11.23 near the telomeric duplicated region of Williams-Beuren syndrome, a developmental disorder affecting multiple organs including the urinary tract. These results indicate that p35 (uroplakin IIIb) is a urothelial differentiation product structurally and functionally related to uroplakin III, and that p35-UPIb interaction in the ER is an important early step in urothelial plaque assembly
— id: 33060, year: 2002, vol: 159, page: 685, stat: Journal Article,

Role of membrane proteins in permeability barrier function: uroplakin ablation elevates urothelial permeability
Hu, Ping; Meyers, Susan; Liang, Feng-Xia; Deng, Fang-Ming; Kachar, Bechara; Zeidel, Mark L; Sun, Tung-Tien
2002 Dec;283(6):F1200-F1207, American journal of physiology. Renal physiology
Although water, small nonelectrolytes, and gases are freely permeable through most biological membranes, apical membranes of certain barrier epithelia exhibit extremely low permeabilities to these substances. The role of integral membrane proteins in this barrier function has been unclear. To study this problem, we have ablated the mouse gene encoding uroplakin III (UPIII), one of the major protein subunits in urothelial apical membranes, and measured the permeabilities of these membranes. Ablation of the UPIII gene greatly diminishes the amounts of uroplakins on the apical urothelial membrane (Hu P, Deng FM, Liang FX, Hu CM, Auerbach AB, Shapiro E, Wu XR, Kachar B, and Sun TT. J Cell Biol 151: 961-972, 2000). Our results indicate that normal mouse urothelium exhibits high transepithelial resistance and low urea and water permeabilities. The UPIII-deficient urothelium exhibits a normal transepithelial resistance (normal 2,024 +/- 122, knockout 2,322 +/- 114 Omega. cm(2); P > 0.5). However, the UPIII-deficient apical membrane has a significantly elevated water permeability (normal 0.91 +/- 0.06, knockout 1.83 +/- 0.14 cm/s x 10(-5); P < 0.05). The urea permeability of the UPIII-deficient membrane also increased, although to a lesser extent (normal 2.22 +/- 0.24, knockout 2.93 +/- 0.31 cm/s x 10(-6); P = 0.12). These results indicate that reduced targeting of uroplakins to the apical membrane does not significantly alter the tight junctional barrier but does double the water permeability. We provide the first demonstration that integral membrane proteins contribute to the apical membrane permeability barrier function of urothelium
— id: 39551, year: 2002, vol: 283, page: F1200, stat: Journal Article,

Localization of uroplakin Ia, the urothelial receptor for bacterial adhesin FimH, on the six inner domains of the 16 nm urothelial plaque particle
Min, Guangwei; Stolz, Martin; Zhou, Ge; Liang, Fengxia; Sebbel, Peter; Stoffler, Daniel; Glockshuber, Rudi; Sun, Tung-Tien; Aebi, Ueli; Kong, Xiang-Peng
2002 Apr 12;317(5):697-706, Journal of molecular biology
The binding of uropathogenic Escherichia coli to the urothelial surface is a critical initial event for establishing urinary tract infection, because it prevents the bacteria from being removed by micturition and it triggers bacterial invasion as well as host cell defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium and its urothelial receptor, uroplakin Ia (UPIa). To localize the UPIa receptor on the 16 nm particles that form two-dimensional crystals of asymmetric unit membrane (AUM) covering >90 % of the apical urothelial surface, we constructed a 15 A resolution 3-D model of the mouse 16 nm AUM particle by negative staining and electron crystallography. Similar to previous lower-resolution models of bovine and pig AUM particles, the mouse 16 nm AUM particle consists of six inner and six outer domains that are interconnected to form a twisted ribbon-like structure. Treatment of urothelial plaques with 0.02-0.1 % (v/v) Triton X-100 allowed the stain to penetrate into the membrane, revealing parts of the uroplakin transmembrane moiety with an overall diameter of 14 nm, which was much bigger than the 11 nm value determined earlier by quick-freeze deep-etch. Atomic force microscopy of native, unfixed mouse and bovine urothelial plaques confirmed the overall structure of the luminal 16 nm AUM particle that was raised by 6.5 nm above the luminal membrane surface and, in addition, revealed a circular, 0.5 nm high, cytoplasmic protrusion of approximately 14 nm diameter. Finally, a difference map calculated from the mouse urothelial plaque images collected in the presence and absence of recombinant bacterial FimH/FimC complex revealed the selective binding of FimH to the six inner domains of the 16 nm AUM particle. These results indicate that the 16 nm AUM particle is anchored by a approximately 14 nm diameter transmembrane stalk, and suggest that bacterial binding to UPIa that resides within the six inner domains of the 16 nm AUM particle may preferentially trigger transmembrane signaling involved in bacterial invasion and host cell defense
— id: 59002, year: 2002, vol: 317, page: 697, stat: Journal Article,

Proximal location of mouse prostate epithelial stem cells: a model of prostatic homeostasis
Tsujimura, Akira; Koikawa, Yasuhiro; Salm, Sarah; Takao, Tetsuya; Coetzee, Sandra; Moscatelli, David; Shapiro, Ellen; Lepor, Herbert; Sun, Tung-Tien; Wilson, E Lynette
2002 Jun 24;157(7):1257-1265, Journal of cell biology
Stem cells are believed to regulate normal prostatic homeostasis and to play a role in the etiology of prostate cancer and benign prostatic hyperplasia. We show here that the proximal region of mouse prostatic ducts is enriched in a subpopulation of epithelial cells that exhibit three important attributes of epithelial stem cells: they are slow cycling, possess a high in vitro proliferative potential, and can reconstitute highly branched glandular ductal structures in collagen gels. We propose a model of prostatic homeostasis in which mouse prostatic epithelial stem cells are concentrated in the proximal region of prostatic ducts while the transit-amplifying cells occupy the distal region of the ducts. This model can account for many biological differences between cells of the proximal and distal regions, and has implications for prostatic disease formation
— id: 32485, year: 2002, vol: 157, page: 1257, stat: Journal Article,

Specific heterodimer formation is a prerequisite for uroplakins to exit from the endoplasmic reticulum
Tu, Liyu; Sun, Tung-Tien; Kreibich, Gert
2002 Dec;13(12):4221-4230, Molecular biology of the cell
Much of the lower urinary tract, including the bladder, is lined by a stratified urothelium forming a highly differentiated, superficial umbrella cell layer. The apical plasma membrane as well as abundant cytoplasmic fusiform vesicles of the umbrella cells is covered by two-dimensional crystals that are formed by four membrane proteins named uroplakins (UPs) Ia, Ib, II, and III. UPs are synthesized on membrane-bound polysomes, and after several co- and posttranslational modifications they assemble into planar crystals in a post-Golgi vesicular compartment. Distension of the bladder may cause fusiform vesicles to fuse with the apical plasma membrane. We have investigated the early stages of uroplakin assembly by expressing the four uroplakins in 293T cells. Transfection experiments showed that, when expressed individually, only UPIb can exit from the endoplasmic reticulum (ER) and move to the plasma membrane, whereas UPII and UPIII reach the plasma membrane only when they form heterodimeric complexes with UPIa and UPIb, respectively. Heterodimer formation in the ER was confirmed by pulse-chase experiment followed by coimmunoprecipitation. Our results indicate that the initial building blocks for the assembly of crystalline uroplakin plaques are heterodimeric uroplakin complexes that form in the ER
— id: 34613, year: 2002, vol: 13, page: 4221, stat: Journal Article,

Going undercover
Cowin P; Sun TT
2001 ;107:287-289, Cell
— id: 32401, year: 2001, vol: 107, page: 287, stat: Journal Article,

Urothelial function reconsidered: a new role in urinary protein secretion
Deng F; Ding M; Lavker RM; Sun T
2001 Jun;57(6 Suppl 1):117-117, Urology
— id: 20642, year: 2001, vol: 57, page: 117, stat: Journal Article,

Urothelial function reconsidered: A role in urinary protein secretion
Deng FM; Ding M; Lavker RM; Sun TT
2001 Jan 2;98(1):154-159, Proceedings of the National Academy of Sciences of the United States of America
Mammalian bladder epithelium functions as an effective permeability barrier. We demonstrate here that this epithelium can also function as a secretory tissue directly involved in modifying urinary protein composition. Our data indicate that normal bovine urothelium synthesizes, as its major differentiation products, two well-known proteases: tissue-type plasminogen activator and urokinase, as well as a serine protease inhibitor, PP5. Moreover, we demonstrate that the urothelium secretes these proteins in a polarized fashion into the urine via a cAMP- and calcium-regulated pathway. Urinary plasminogen activators of ruminants are therefore urothelium derived rather then kidney derived as in some other species; this heterogeneity may have evolved in response to different physiological or dietary factors. In conjunction with our recent finding that transgenic mouse urothelium can secrete ectopically expressed human growth hormone into the urine, our data establish that normal mammalian urothelium can function not only as a permeability barrier but also as a secretor of urinary proteins that can play physiological or pathological roles in the urinary tract
— id: 16510, year: 2001, vol: 98, page: 154, stat: Journal Article,

Ablation of uroplakin III gene results in small urothelial plaques, urothelial leakage, and vesicoureteral reflux
Hu P; Deng F; Liang F; Hu C; Auerbach A; Shapiro E; Wu X; Kachar B; Sun T
2001 Jun;57(6 Suppl 1):117-117, Urology
— id: 21194, year: 2001, vol: 57, page: 117, stat: Journal Article,

Organization of uroplakin subunits: transmembrane topology, pair formation and plaque composition
Liang FX; Riedel I; Deng FM; Zhou G; Xu C; Wu XR; Kong XP; Moll R; Sun TT
2001 Apr 1;355(Pt 1):13-18, Biochemical journal
The apical surfaces of urothelial cells are almost entirely covered with plaques consisting of crystalline, hexagonal arrays of 16 nm uroplakin particles. Although all four uroplakins, when SDS-denatured, can be digested by chymotrypsin, most uroplakin domains in native urothelial plaques are resistant to the enzyme, suggesting a tightly packed structure. The only exception is the C-terminal, cytoplasmic tail of UPIII (UPIII) which is highly susceptible to proteolysis, suggesting a loose configuration. When uroplakins are solubilized with 2% octylglucoside and fractionated with ion exchangers, UPIa and UPII were bound as a complex by a cation exchanger, whereas UPIb and UPIII were bound by an anion exchanger. This result is consistent with the fact that UPIa and UPIb are cross-linked to UPII and UPIII, respectively, and suggests that the four uroplakins form two pairs consisting of UPIa/II and UPIb/III. Immunogold labelling using a new mouse monoclonal antibody, AU1, revealed that UPIII is present in all urothelial plaques, indicating that the two uroplakin pairs are not segregated into two different types of urothelial plaque and that all plaques must have a similar uroplakin composition. Taken together, these results indicate that uroplakins form a tightly packed structure, that the four uroplakins interact specifically forming two pairs, and that both uroplakin pairs are required for normal urothelial plaque formation
— id: 21231, year: 2001, vol: 355, page: 13, stat: Journal Article,

Brenner tumors but not transitional cell carcinomas of the ovary show urothelial differentiation: immunohistochemical staining of urothelial markers, including cytokeratins and uroplakins
Riedel I; Czernobilsky B; Lifschitz-Mercer B; Roth LM; Wu XR; Sun TT; Moll R
2001 Feb;438(2):181-191, Virchows archive
To determine whether Brenner tumors and transitional cell carcinomas (TCCs) of the ovary are urothelial in type, the immunoprofiles of 14 Brenner tumors, including three malignant examples, and eight ovarian TCCs were compared with those of Walthard nests, urothelium, 12 urinary bladder TCCs and 17 ovarian adenocarcinomas (serous, endometrioid, mucinous, and undifferentiated type). The immunohistochemical stains used included those for cytokeratins CKs 5/6, CK7, CK8, CK13, and CK20, vimentin, CA125, and the specific urothelial differentiation marker uroplakin III. CK7 and CK8 were broadly expressed in most tumors of ovary and bladder examined, while vimentin was focally present in some ovarian TCCs and adenocarcinomas. As in normal and neoplastic bladder urothelium, urothelial markers, including uroplakin III, CK13, and CK20, were detected in the epithelial nests of Brenner tumors. Brenner tumor cells also expressed uroplakins Ia and II. CA125 was observed focally in some Brenner tumors. In contrast, TCCs of the ovary and Walthard nests lacked uroplakins and were essentially negative for CK20 and CK13 but quite strongly expressed CA125. This immunophenotype closely resembled that found in ovarian adenocarcinomas. Thus, it appears that the only true urothelial-type ovarian neoplasm is the Brenner tumor, whereas ovarian TCC most likely represents a poorly differentiated adenocarcinoma with a morphologic transitional cell pattern. These results may explain the controversies as expressed in the recent literature concerning TCC of the ovary and establish its place among the ovarian adenocarcinomas of mullerian type
— id: 26905, year: 2001, vol: 438, page: 181, stat: Journal Article,

Uroplakin as a marker for typing metastatic transitional cell carcinoma on fine-needle aspiration specimens
Xu X; Sun TT; Gupta PK; Zhang P; Nasuti JF
2001 Jun 25;93(3):216-221, Cancer
BACKGROUND: Immunohistological markers specific for a single type of epithelium are rare. Recently, urothelium tissue-specific genes were cloned. The genes encoded a family of transmembrane proteins, uroplakins, that are expressed only in urothelial mucosa. Using uroplakin antibodies on paraffin-embedded tissue, a previous study demonstrated positive staining in 66% of metastatic transitional cell carcinoma (TCC) cases and negative staining in all other tumors (including breast, ovarian, lung, and gastrointestinal carcinomas) tested. The current study addresses the diagnostic value of uroplakins in conventional fine-needle aspiration (FNA) material in establishing a diagnosis of metastatic TCC. METHODS: Representative slides from 27 FNA cases of metastatic TCC and 52 non-TCC carcinomas were collected. The avidin-biotin-peroxidase method was utilized, using polyclonal antiuroplakin as the primary antibody on 95% ethanol-fixed, Papanicoloau-stained direct smears. RESULTS: Twenty-five of 27 metastatic TCC cases (93%) were found to stain positively for uroplakin with a superficial membrane/microluminal staining pattern. A few cells with diffuse membranous staining also were noted in 48% of the positive metastatic TCC cases. The superficial membrane/microluminal staining pattern was not observed in any of the non-TCC carcinomas. However, approximately 6% of these cases (3 of 52 cases) did show rare tumor cells with diffuse membranous staining. CONCLUSIONS: The application of uroplakin antibodies to 95% ethanol-fixed FNA direct smears has improved the sensitivity of the antibody for metastatic TCC while maintaining a specificity comparable to that of paraffin-embedded tissue. The authors believe that these antibodies have diagnostic potential in cytopathology in the evaluation of metastatic TCC
— id: 26904, year: 2001, vol: 93, page: 216, stat: Journal Article,

Osteopontin gene is expressed in the dermal papilla of pelage follicles in a hair-cycle-dependent manner
Yu DW; Yang T; Sonoda T; Gong Y; Cao Q; Gaffney K; Jensen PJ; Freedberg IM; Lavker RM; Sun TT
2001 Dec;117(6):1554-1558, Journal of investigative dermatology
Hair follicle formation and maintenance involve intimate interactions between follicular epithelial cells and a group of specialized mesenchymal cells known as the dermal papilla. Using the random primer polymerase chain reaction, we have identified an approximately 1.4 kb osteopontin mRNA that is present in large quantities in cultured rat vibrissa dermal papilla cells but undetectable in cultured rat skin fibroblasts. In situ hybridization showed that the osteopontin gene is expressed in dermal papilla cells of pelage follicles during catagen but not in anagen or telogen. As an acidic glycosylated RGD-containing extracellular matrix protein, osteopontin can function both as a cell attachment protein and as a soluble cytokine playing roles in signaling, cell migration, tissue survival, anti-inflammation, and T-cell-mediated cellular immunity. Our results indicate that the comparison of the mRNA of cultured dermal papilla cells and fibroblasts can lead to the identification of not only anagen-specific genes (e.g., nexin 1), but also a catagen-specific gene. We have thus provided evidence that specific genes are turned on during catagen, which is therefore not simply a passive 'degenerative' phase. The functional role of osteopontin in catagen is unclear but it may promote the formation of a tightly aggregated dermal papilla, and/or protect the dermal papilla cells from apoptosis induced by cytokines or hypoxia during catagen
— id: 25706, year: 2001, vol: 117, page: 1554, stat: Journal Article,

Role of Ha-ras activation in superficial papillary pathway of urothelial tumor formation
Zhang ZT; Pak J; Huang HY; Shapiro E; Sun TT; Pellicer A; Wu XR
2001 Apr 12;20(16):1973-1980, Oncogene
Urothelial tumors develop along two distinctive phenotypic pathways (superficial papillary non-invasive tumors versus flat carcinoma in situ lesions), with markedly different biological behavior and prognosis. Although multiple genetic alterations have been identified in human bladder cancer, their cause-effect relationship with the two pathways has not been firmly established. Using a urothelium-specific promoter of the uroplakin II gene, we showed earlier in transgenic mice that the urothelial expression of SV40T antigen, which inactivates p53 and pRb, induced carcinoma in situ and invasive and metastatic bladder cancer. In striking contrast, we demonstrate here that the urothelial expression of an activated Ha-ras in transgenic mice caused urothelial hyperplasia and superficial papillary non-invasive bladder tumors. These results provide strong, direct experimental evidence that the two phenotypical pathways of bladder tumorigenesis are caused by distinctive genetic defects. Our results indicate that Ha-ras activation can induce urothelial proliferation in vivo; and that urothelial hyperplasia is a precursor of low-grade, superficial papillary bladder tumors. Our transgenic models provide unique opportunities to study the detailed molecular events underlying different types of bladder neoplasms, and can serve as useful preclinical models for evaluating the in vivo efficacy of preventive and therapeutic agents that act on various signaling pathways in bladder cancer
— id: 20658, year: 2001, vol: 20, page: 1973, stat: Journal Article,

Uroplakin Ia is the urothelial receptor for uropathogenic Escherichia coli: evidence from in vitro FimH binding
Zhou G; Mo WJ; Sebbel P; Min G; Neubert TA; Glockshuber R; Wu XR; Sun TT; Kong XP
2001 Nov;114(Pt 22):4095-4103, Journal of cell science
The binding of uropathogenic Escherichia coli to the urothelial surface is a crucial initial event for establishing urinary tract infection because it allows the bacteria to gain a foothold on the urothelial surface, thus preventing them from being removed by micturition. In addition, it triggers bacterial invasion as well as host urothelial defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium, a filamentous attachment apparatus, and its urothelial receptor. We have prepared a biotinylated, recombinant FimH-FimC adhesin:chaperone complex and used it to identify its mouse urothelial receptor. The FimH-FimC complex binds specifically to a single 24 kDa major mouse urothelial plaque protein, which we identified as uroplakin Ia by mass spectrometry, cDNA cloning and immunoreactivity. The terminal mannosyl moieties on Asn-169 of uroplakin Ia are responsible for FimH as well as concanavalin A binding. Although FimH binds to uroplakin Ia with only moderate strength (K(d) approximately 100 nM between pH 4 and 9), the binding between multiple fimbriae of a bacterium and the crystalline array of polymerized uroplakin receptors should achieve high avidity and stable bacterial attachment. The FimH-FimC complex binds preferentially to the mouse urothelial umbrella cells in a pattern similar to uroplakin staining. Our results indicate that the structurally related uroplakins Ia and Ib are glycosylated differently, that uroplakin Ia serves as the urothelial receptor for the type 1-fimbriated E. coli, and that the binding of uropathogenic bacteria to uroplakin Ia may play a key role in mediating the urothelial responses to bacterial attachment
— id: 26903, year: 2001, vol: 114, page: 4095, stat: Journal Article,

Ablation of uroplakin III gene results in small urothelial plaques, urothelial leakage, and vesicoureteral reflux
Hu P; Deng FM; Liang FX; Hu CM; Auerbach AB; Shapiro E; Wu XR; Kachar B; Sun TT
2000 Nov 27;151(5):961-972, Journal of cell biology
Urothelium synthesizes a group of integral membrane proteins called uroplakins, which form two-dimensional crystals (urothelial plaques) covering >90% of the apical urothelial surface. We show that the ablation of the mouse uroplakin III (UPIII) gene leads to overexpression, defective glycosylation, and abnormal targeting of uroplakin Ib, the presumed partner of UPIII. The UPIII-depleted urothelium features small plaques, becomes leaky, and has enlarged ureteral orifices resulting in the back flow of urine, hydronephrosis, and altered renal function indicators. Thus, UPIII is an integral subunit of the urothelial plaque and contributes to the permeability barrier function of the urothelium, and UPIII deficiency can lead to global anomalies in the urinary tract. The ablation of a single urothelial-specific gene can therefore cause primary vesicoureteral reflux (VUR), a hereditary disease affecting approximately 1% of pregnancies and representing a leading cause of renal failure in infants. The fact that VUR caused by UPIII deletion seems distinct from that caused by the deletion of angiotensin receptor II gene suggests the existence of VUR subtypes. Mutations in multiple gene, including some that are urothelial specific, may therefore cause different subtypes of primary reflux. Studies of VUR in animal models caused by well-defined genetic defects should lead to improved molecular classification, prenatal diagnosis, and therapy of this important hereditary problem
— id: 26906, year: 2000, vol: 151, page: 961, stat: Journal Article,

Serpins in the human hair follicle
Jensen PJ; Yang T; Yu DW; Baker MS; Risse B; Sun TT; Lavker RM
2000 May;114(5):917-922, Journal of investigative dermatology
Proteinases and their inhibitors are very likely to function as mediators or regulators of the hair growth cycle. Very little information is currently available, however, regarding the specific inhibitors present in human hair follicles at defined stages of their growth cycle. In this study we have analyzed two proteinase inhibitors, plasminogen activator inhibitor type 2 and protease nexin 1, in human hair follicles using in situ hybridization and/or immunohistochemistry. Protease nexin 1 mRNA was found only in the mesenchymal population of the hair follicle, i.e., the follicular papilla cells, during the anagen but not the catagen phase. In contrast, plasminogen activator inhibitor type 2 was localized to several epithelial populations in the follicle: the more differentiated cells of the infundibulum; the companion layer in anagen follicles; and the single layer of outer root sheath cells directly abutting the club hair in telogen follicles. At least some of the plasminogen activator inhibitor type 2 in human follicles appears to be in the relaxed form, as evidenced by strong staining with an antibody that is specific for this form of the inhibitor. This suggests that plasminogen activator inhibitor type 2 interacts with and is cleaved by an endogenous follicular proteinase and supports a constitutive role for this inhibitor in human follicular epithelia
— id: 16514, year: 2000, vol: 114, page: 917, stat: Journal Article,

Epidermal stem cells: properties, markers, and location
Lavker RM; Sun TT
2000 Dec 5;97(25):13473-13475, Proceedings of the National Academy of Sciences of the United States of America
— id: 16511, year: 2000, vol: 97, page: 13473, stat: Journal Article,

Generation of active TGF-beta by prostatic cell cocultures using novel basal and luminal prostatic epithelial cell lines
Salm SN; Koikawa Y; Ogilvie V; Tsujimura A; Coetzee S; Moscatelli D; Moore E; Lepor H; Shapiro E; Sun TT; Wilson EL
2000 Jul;184(1):70-79, Journal of cellular physiology
Two prostatic epithelial lines, one of basal origin and one of luminal origin, were established from the dorsolateral prostates of p53 null mice. The cell lines are nontumorigenic when inoculated subcutaneously under the renal capsule or intraprostatically in syngeneic mice. The luminal cell line (PE-L-1) expresses cytokeratins 8 and 18 and the basal cell line (PE-B-1) expresses cytokeratins 5 and 14. The basal cells require serum for growth, whereas the luminal cells grow only in serum-free medium. Both cell lines require the presence of growth factors for optimal growth in culture, with EGF and FGF-2 having the greatest effect on the growth rate. Both lines express androgen receptor (AR) mRNA and protein. Androgen stimulates growth of the basal cell line, indicating that the ARs are functional, whereas growth of the luminal cells is unaffected by androgens. The luminal line is significantly inhibited by exogenous TGF-beta and produces low levels of endogenous TGF-beta. In contrast, the basal cell line produces significant amounts of TGF-beta and its growth is not influenced by this cytokine. Coculture of luminal cells with prostatic smooth muscle cells results in the generation of increased levels of biologically active TGF-beta, indicating a paracrine mechanism of TGF-beta activation that may be involved in the maintenance of normal prostatic function. To our knowledge this is the first report describing both basal and luminal prostatic cell lines from a single inbred animal species and the first indication that prostatic epithelial and stromal cells interact to generate the biologically active form of TGF-beta. These lines will provide an important model for determining basal/luminal interactions in both in vitro and in vivo assays.
— id: 11685, year: 2000, vol: 184, page: 70, stat: Journal Article,

Transforming growth factor-beta is an autocrine mitogen for a novel androgen-responsive murine prostatic smooth muscle cell line, PSMC1
Salm SN; Koikawa Y; Ogilvie V; Tsujimura A; Coetzee S; Moscatelli D; Moore E; Lepor H; Shapiro E; Sun TT; Wilson EL
2000 Dec;185(3):416-424, Journal of cellular physiology
A prostatic smooth muscle cell line (PSMC1) was established from the dorsolateral prostate of p53 null mice. The cell line is nontumorigenic when inoculated subcutaneously, under the renal capsule or intraprostatically in syngeneic mice. These cells express alpha-smooth muscle actin (alpha-SMA), indicating their smooth muscle origin, and TGF-beta significantly enhances expression of alpha-SMA. The cells express both androgen receptor (AR) mRNA and protein, and respond mitogenically to physiological concentrations of androgens. PSMC1 cells produce significant amounts of TGF-beta, which stimulates growth by an autocrine mechanism. Dihydrotestosterone (DHT) increases proliferation of PSMC1 cells by promoting TGF-beta secretion. Considering the significant inhibitory effect of TGF-beta on prostatic epithelial cells and its stimulatory effect on the PSMC1 cells, we postulate that TGF-beta produced by prostatic smooth muscle cells may have a paracrine effect on the prostatic epithelium. We also postulate that TGF-beta may be involved in the etiology of benign prostatic hyperplasia (BPH) by stimulating excessive stromal proliferation. Line PSMC1 is the first reported androgen-responsive murine smooth muscle cell line. It will be useful for in vivo and in vitro experiments to study the mechanisms of androgen action on prostatic stroma and for delineating the interactions that occur between prostatic smooth muscle and epithelium that may lead to prostatic diseases such as BPH
— id: 26907, year: 2000, vol: 185, page: 416, stat: Journal Article,

CLED: a calcium-linked protein associated with early epithelial differentiation
Sun L; Sun TT; Lavker RM
2000 Aug 25;259(1):96-106, Experimental cell research
Although it has been well established that Ca(2+) plays a key role in triggering keratinocyte differentiation, relatively little is known about the molecules that mediate this signaling process. By analyzing a bovine corneal epithelial subtraction cDNA library, we have identified a novel gene that we named CLED (calcium-linked epithelial differentiation), which encodes a messenger RNA present in all stratified squamous epithelia, hair follicle, the bladder transitional epithelium, and small intestinal epithelium. The deduced amino acid sequence of CLED, based on a bovine partial cDNA and its full-length, human and mouse homologues that have been described only as ESTs, contains 2 EF-hand Ca(2+)-binding domains, a myristoylation motif, and several potential protein kinase phosphorylation sites; the CLED protein is therefore related to the S100 protein family. In all stratified squamous epithelia, the CLED message is associated with the intermediate cell layers. Similar CLED association with cells that are above the proliferative compartment but below the terminally differentiated compartment is seen in hair follicle, bladder, and small intestinal epithelia. The only exception is corneal epithelium, where CLED is expressed in both basal and intermediate cells. The presence of CLED in corneal epithelial basal cells, but not in the adjacent limbal basal (stem) cells, provides additional, strong evidence for the unique lateral heterogeneity of the limbal/corneal epithelium. These results suggest that CLED, via Ca(2+)-related mechanisms, may play a role in the epithelial cell's commitment to undergo early differentiation, and that its down-regulation is required before the cells can undergo the final stages of terminal differentiation
— id: 16513, year: 2000, vol: 259, page: 96, stat: Journal Article,

Involvement of follicular stem cells in forming not only the follicle but also the epidermis
Taylor G; Lehrer MS; Jensen PJ; Sun TT; Lavker RM
2000 Aug 18;102(4):451-461, Cell
The location of follicular and epidermal stem cells in mammalian skin is a crucial issue in cutaneous biology. We demonstrate that hair follicular stem cells, located in the bulge region, can give rise to several cell types of the hair follicle as well as upper follicular cells. Moreover, we devised a double-label technique to show that upper follicular keratinocytes emigrate into the epidermis in normal newborn mouse skin, and in adult mouse skin in response to a penetrating wound. These findings indicate that the hair follicle represents a major repository of keratinocyte stem cells in mouse skin, and that follicular bulge stem cells are potentially bipotent as they can give rise to not only the hair follicle, but also the epidermis
— id: 16512, year: 2000, vol: 102, page: 451, stat: Journal Article,

New concepts of histological changes in experimental augmentation cystoplasty: insights into the development of neoplastic transformation at the enterovesical and gastrovesical anastomosis
Gitlin JS; Wu XR; Sun TT; Ritchey ML; Shapiro E
1999 Sep;162(3 Pt 2):1096-1100, Journal of urology
PURPOSE: To our knowledge the pathogenesis of malignancy associated with ileal cystoplasty, ureterosigmoidostomy and ileal conduits is currently unknown. To gain further insights into the mechanism of neoplastic transformation we studied histological changes in a canine augmentation cystoplasty model. MATERIALS AND METHODS: Enterocystoplasty and gastrocystoplasty were performed using a 5 to 7 cm. patch of ileum in 8 dogs and gastric antrum in 6. Specimens were harvested 4 months postoperatively. Representative 3 microm sections of the enterovesical and gastrovesical junctions were stained with hematoxylin and eosin. Uroplakin expression was assessed using an indirect peroxidase method subjected to double staining with alcian blue and periodic acid-Schiffreagent. RESULTS: The bladder portion of the augmentation cystoplasty had 3 to 4 stratified cell layers covered with a distinctive umbrella cell layer. Strong uroplakin staining was visible in all cell layers except the basal layer. At the enterovesical and gastrovesical junctions 6 to 10 layers of hyperplastic, urothelial appearing cells covered the glandular epithelium of the ileal and gastric segments. These cells expressed uroplakins. At this junction zone there was a marked decrease of underlying enteric glands, which had atrophied in proportion to the degree of urothelial hyperplasia. Double staining of uroplakin stained sections with alcian blue and periodic acid-Schiff reagent revealed mucosubstances in hyperplastic urothelial cells covering the enteral segments, indicating that the cells co-expressed uroplakins and mucins. CONCLUSIONS: Histological changes in this experimental canine model of augmentation cystoplasty indicated that the overgrowth of hyperplastic transitional epithelium develops at the enterovesical and gastrovesical junctions. These cells express not only uroplakins, but also mucosubstances. Our results suggest that the migrated hyperplastic urothelial cells have undergone changes characteristic of the enteric and gastric epithelium, which may have important implications in the pathogenesis of malignancy in bladder augmentations
— id: 11968, year: 1999, vol: 162, page: 1096, stat: Journal Article,

Spiny keratoderma--a demonstration of hair keratin and hair type keratinization
Hashimoto K; Toi Y; Horton S; Sun TT
1999 Jan;26(1):25-30, Journal of cutaneous pathology
Six cases of spiny keratoderma were analyzed with hair specific antikeratin antibodies (AE13, AE14) and by electron microscopy. The keratotic column exhibited a different keratin birefringence and the underlying viable epidermis was less eosinophilic than the surrounding epidermis. AE13, which is specific for hair cortex, was positive in the lower column and variably positive in the viable epidermis, often beyond the columnar lesion. AE14 was negative in the lesion. Electron microscopy demonstrated features of keratinization of normal hair cortex, i.e. by the accretion of keratin filaments without production of keratohyalin or trichohyalin granules. Cementsomes (lamellar granules) and marginal bands were not produced as they are not formed in normal cortical keratinization. It was suggested that spiny keratoderma represents an ectopic hair formation of palms and soles
— id: 7341, year: 1999, vol: 26, page: 25, stat: Journal Article,

Three-dimensional analysis of the 16 nm urothelial plaque particle: luminal surface exposure, preferential head-to-head interaction, and hinge formation
Kachar B; Liang F; Lins U; Ding M; Wu XR; Stoffler D; Aebi U; Sun TT
1999 Jan 15;285(2):595-608, Journal of molecular biology
The luminal surface of mouse urothelium in contact with the urine is almost entirely covered with plaques consisting of uroplakin-containing particles that form p6 hexagonal crystals with a center-to-center distance of 16 nm. A combination of quick-freeze/deep-etch images and our previous negative staining data indicate that the head domain of the uroplakin particle, which is exposed without an extensive glycocalyx shield, interacts closely with the head domains of the neighboring particles, while the membrane-embedded tail domains are farther apart; and that urothelial particles and plaques are not rigid structures as they can change their configuration in response to mechanical perturbations. Based on these data, we have constructed three-dimensional models depicting the structural organization of urothelial particles and plaques. Our models suggest that the head-to-head interaction may play a key role in determining the shape and size of the urothelial plaques. These models can explain many properties of urothelial plaques including their unique shape, detergent-insolubility, and morphological changes during vesicle maturation.
— id: 7961, year: 1999, vol: 285, page: 595, stat: Journal Article,

Detection of circulating uroplakin-positive cells in patients with transitional cell carcinoma of the bladder
Li SM; Zhang ZT; Chan S; McLenan O; Dixon C; Taneja S; Lepor H; Sun TT; Wu XR
1999 Sep;162(3 Pt 1):931-935, Journal of urology
PURPOSE: Although transitional cell carcinoma of the bladder (TCC) metastasizes frequently with devastating consequences, no marker has been available to monitor this process. Uroplakins are a group of specific markers for normal urothelium and are continuously expressed by the majority of TCCs. Detection of uroplakin-positive cells in the circulation would be a strong indication of hematogenous dissemination of tumor cells in patients with TCC. MATERIALS AND METHODS: Total RNAs were extracted from peripheral blood of 60 patients with TCC (50 non-metastatic and 10 metastatic) and 10 healthy controls, reverse-transcribed and subjected to polymerase chain reaction amplification (RT-PCR) using oligonucleotide primers of human uroplakin II gene. A uroplakin-expressing human bladder cancer cell line (RT4) was used as a positive control to establish the sensitivity of the RT-PCR assay. RESULTS: We showed that the PCR-amplification of the mRNA encoding uroplakin II (UPII), a 15-kDa urothelium-specific marker, constitutes a highly sensitive and specific assay for detecting 100% of transitional cell carcinoma tissue, and that this assay can detect a single bladder cancer cell in a 5-ml. blood sample. UPII mRNA was detected in the blood samples of 2 patients with metastatic bladder cancer without chemotherapy and 1 out of 8 such patients with chemotherapy, but not in those of 50 non-metastatic patients or normal controls. CONCLUSIONS: Uroplakin II is a highly specific marker for human TCC and the detection of uroplakin II in the peripheral blood is associated with metastatic spread of bladder cancer cells. The specific and sensitive detection of uroplakin II provides a useful adjunct for detecting bladder cancer metastasis, staging, and monitoring chemotherapeutic response
— id: 6182, year: 1999, vol: 162, page: 931, stat: Journal Article,

Urothelial hinge as a highly specialized membrane: detergent-insolubility, urohingin association, and in vitro formation
Liang F; Kachar B; Ding M; Zhai Z; Wu XR; Sun TT
1999 Jul;65(1):59-69, Differentiation
Urothelial surface is covered by numerous plaques (consisting of asymmetric unit membranes or AUM) that are interconnected by ordinary looking hinge membranes. We describe an improved method for purifying bovine urothelial plaques using 2% sarkosyl and 25 mM NaOH to remove contaminating membrane and peripheral proteins selectively. Highly purified plaques interconnected by intact hinge areas were obtained, indicating that the hinges are as detergent-insoluble as the plaques. These plaque/hinge preparations contained uroplakins, an as yet uncharacterized 18-kDa plaque-associated protein, plus an 85-kDa glycoprotein that is known to be hinge-associated in situ. Examination of the isolated, in vitro-resealed bovine AUM vesicles by quick-freeze deep-etch showed that each AUM particle consists of a 16-nm, luminally exposed 'head' anchored to the lipid bilayer via a 9-mm transmembranous 'tail', and that an AUM plaque can break forming several smaller plaques separated by newly formed particle-free, hinge-like areas. These data lend support to our recently proposed three-dimensional model of mouse urothelial plaques. In addition, our findings suggest that urothelial plaques are dynamic structures that can rearrange giving rise to new plaques with intervening hinges; that the entire urothelial apical surface (both plaque and hinge areas) is highly specialized; and that these two membrane domains may be equally important in fulfilling some of the urothelial functions
— id: 6177, year: 1999, vol: 65, page: 59, stat: Journal Article,

Comparison of uroplakin expression during urothelial carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in rats and mice
Ogawa K; St John M; Luiza de Oliveira M; Arnold L; Shirai T; Sun TT; Cohen SM
1999 Nov-Dec;27(6):645-651, Toxicology pathology
The expression of uroplakins, the tissue-specific and differentiation-dependent membrane proteins of the urothelium, was analyzed immunohistochemically in N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-treated rats and mice during bladder carcinogenesis. Male Fischer 344 rats were treated with 0.05% BBN in the drinking water for 10 wk and were euthanatized at week 20 of the experiment. BBN was administered to male B6D2F, mice; it was either provided at a rate of 0.05% in the drinking water (for 26 wk) or 5 mg BBN was administered by intragastric gavage twice weekly for 10 wk, followed by 20 wk without treatment. In rats, BBN-induced, noninvasive, low-grade, papillary, transitional cell carcinoma (TCC) showed decreased uroplakin-staining of cells lining the lumen but showed increased expression in some nonluminal cells. In mice, nonpapillary, high-grade dysplasia, carcinoma in situ, and invasive carcinoma were induced. There was a marked decrease in the number of uroplakin-positive cells lining the lumen and in nonluminal cells. This occurred in normal-appearing urothelium in BBN-treated mice and in dysplasic urothelium, in carcinoma in situ, and in invasive TCC. The percentage of uroplakin-positive nonluminal cells was higher in control mice than in rats, but it was lower in the mouse than in the rat after BBN treatment. Uroplakin expression was disorderly and focal in BBN-treated urothelium in both species. These results indicate that BBN treatment changed the expression of uroplakins during bladder carcinogenesis, with differences in rats and mice being related to degree of tumor differentiation
— id: 26908, year: 1999, vol: 27, page: 645, stat: Journal Article,

Identification of a cytosolic NADP+-dependent isocitrate dehydrogenase that is preferentially expressed in bovine corneal epithelium. A corneal epithelial crystallin
Sun L; Sun TT; Lavker RM
1999 Jun 11;274(24):17334-17341, Journal of biological chemistry
Recently, metabolic enzymes have been observed in both the lens and corneal epithelium at levels greatly exceeding what is necessary for normal metabolic functions. These proteins have been termed taxon-specific crystallins and are thought to play a role in maintaining tissue transparency. We report here that cytosolic NADP+-dependent isocitrate dehydrogenase (ICDH) represents a new corneal crystallin. Using suppression subtractive hybridization, we identified a gene (with a deduced amino acid sequence that showed 94% identity to rat cytosolic NADP+-dependent ICDH) that is preferentially expressed in bovine corneal epithelium. Northern blots established that its mRNA level in the corneal epithelium was 31-, 39-, 133-, 230-, and 929-fold more than in the liver, bladder epithelium, stomach epithelium, brain, and heart, respectively. This mRNA was detected primarily in corneal epithelial basal cells by in situ hybridization. SDS-polyacrylamide gel electrophoresis, two-dimensional gel analysis, and Western blotting showed that this protein was overexpressed in the corneal epithelium, constituting approximately 13% of the total soluble bovine corneal epithelial proteins. Enzyme assays showed a corresponding overabundance of this protein in bovine corneal epithelium. Taken together, these data indicate that bovine cytosolic ICDH fulfills the criteria for a corneal epithelial crystallin and may be involved in maintaining corneal epithelial transparency
— id: 16517, year: 1999, vol: 274, page: 17334, stat: Journal Article,

Uroplakins as markers of urothelial differentiation
Sun TT; Liang FX; Wu XR
1999 ;462:7-18, Advances in experimental medicine & biology
— id: 11902, year: 1999, vol: 462, page: 7, stat: Journal Article,

Signal joint formation is inhibited in murine scid preB cells and fibroblasts in substrates with homopolymeric coding ends
Sun, T; Ezekiel, U R; Erskine, L; Agulo, R; Bozek, G; Roth, D; Storb, U
1999 Jun;36(8):551-558, Molecular immunology
During B and T lymphocyte development, immunoglobulin and T cell receptor genes are assembled from the germline V, (D) and J gene segments (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Adv. Immunol. 56, 27-150). These DNA rearrangements, responsible for immune system diversity, are mediated by a site specific recombination machinery via recognition signal sequences (RSSs) composed of conserved heptamers and nonamers separated by spacers of 12 or 23 nucleotides (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Adv. Immunol. 56, 27-150). Recombination occurs only between a RSS with a 12mer spacer and a RSS with a 23mer spacer (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Adv. Immunol. 56, 27-150). RAG1 and RAG2 proteins cleave precisely at the RSS-coding sequence border leading to flush signal ends and coding ends with a hairpin structure (Eastman, M., Leu, T., Schatz, D., 1996. Initiation of V(D)J recombination in vitro obeying the 12/23 rule. Nature 380, 85-88; Roth, D.B., Menetski, J.P., Nakajima, P.B., Bosma, M.J., Gellert, M., 1992. V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes. Cell 983-991: Roth, D.B., Zhu, C., Gellert. M., 1993. Characterization of broken DNA molecules associated with V(D)J recombination. Proc. Natl. Acad. Sci. USA 90, 10,788-10,792; van Gent, D., McBlane, J.. Sadofsky, M., Hesse, J., Gellert, M., 1995. Initiation of V(D)J recombination in a cell-free system. Cell 81, 925-934). Signal ends join, forming a signal joint. The hairpin coding ends are opened by a yet unknown endonuclease, and are further processed to form the coding joint (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Ad. Immunol. 56, 27-150.) The murine scid mutation has been shown to affect coding joints, but much less signal joint formation. In this study we demonstrate that the murine scid mutation inhibits correct signal joint formation when both coding ends contain homopolymeric sequences. We suggest that this finding may be due to the function of the SCID protein as an assembly component in V(D)J recombination
— id: 111486, year: 1999, vol: 36, page: 551, stat: Journal Article,

Primary uroepithelial cultures. A model system to analyze umbrella cell barrier function
Truschel ST; Ruiz WG; Shulman T; Pilewski J; Sun TT; Zeidel ML; Apodaca G
1999 May 21;274(21):15020-15029, Journal of biological chemistry
Despite almost 25 years of effort, the development of a highly differentiated and functionally equivalent cell culture model of uroepithelial cells has eluded investigators. We have developed a primary cell culture model of rabbit uroepithelium that consists of an underlying cell layer that interacts with a collagen substratum, an intermediate cell layer, and an upper cell layer of large (25-100 micrometer) superficial cells. When examined at the ultrastructural level, the superficial cells formed junctional complexes and had an asymmetric unit membrane, a hallmark of terminal differentiation in bladder umbrella cells. These cultured 'umbrella' cells expressed uroplakins and a 27-kDa uroepithelial specific antigen that assembled into detergent-resistant asymmetric unit membrane particles. The cultures had low diffusive permeabilities for water (2.8 x 10(-4) cm/s) and urea (3.0 x 10(-7) cm/s) and high transepithelial resistance (>8000 Omega cm2) was achieved when 1 mM CaCl2 was included in the culture medium. The cell cultures expressed an amiloride-sensitive sodium transport pathway and increases in apical membrane capacitance were observed when the cultures were osmotically stretched. The described primary rabbit cell culture model mimics many of the characteristics of uroepithelium found in vivo and should serve as a useful tool to explore normal uroepithelial function as well as dysfunction as a result of disease
— id: 26909, year: 1999, vol: 274, page: 15020, stat: Journal Article,

Urothelium-specific expression of an oncogene in transgenic mice induced the formation of carcinoma in situ and invasive transitional cell carcinoma
Zhang ZT; Pak J; Shapiro E; Sun TT; Wu XR
1999 Jul 15;59(14):3512-3517, Cancer research
Although many genetic alterations are known to be associated with human transitional cell carcinoma (TCC) of the urinary bladder, relatively little is known about the roles of these molecular defects, singular or in combination, in bladder tumorigenesis. We have developed a transgenic mouse model of bladder tumorigenesis using a 3.6-kb promoter of uroplakin II gene to drive the urotheliums-specific expression of oncogenes. In this study, we demonstrate that transgenic mice bearing a low copy number of SV40T transgene developed bladder carcinoma in situ (CIS), whereas those bearing high copies developed CIS as well as invasive and metastatic TCCs. These results indicate that the SV40T inactivation of p53 and retinoblastoma gene products, defects frequently found in human bladder CIS and invasive TCCs, can cause the aggressive form of TCC. Our results also provide experimental proof that CIS is a precursor of invasive TCCs, thus supporting the concept of two distinct pathways of bladder tumorigenesis (papillary versus CIS/invasive TCC). This transgenic system can be used for the systematic dissection of the roles of individual or combinations of specific molecular events in bladder tumorigenesis
— id: 11977, year: 1999, vol: 59, page: 3512, stat: Journal Article,

The bladder as a bioreactor: urothelium production and secretion of growth hormone into urine [see comments]
Kerr DE; Liang F; Bondioli KR; Zhao H; Kreibich G; Wall RJ; Sun TT
1998 Jan;16(1):75-79, Nature biotechnology
Uroplakin genes are expressed in a bladder-specific and differentiation-dependent fashion. Using a 3.6-kb promoter of mouse uroplakin II gene, we have generated transgenic mice that express human growth hormone (hGH) in their bladder epithelium, resulting in its secretion into the urine at 100-500 ng/ml. The levels of urine hGH concentration remain constant for longer than 8 months. hGH is present as aggregates mostly in the uroplakin-delivering cytoplasmic vesicles that are targeted to fuse with the apical surface. Using the bladder as a bioreactor offers unique advantages, including the utility of all animals throughout their lives. Using urine, which contains little protein and lipid, as a starting material facilitates recombinant protein purification
— id: 8368, year: 1998, vol: 16, page: 75, stat: Journal Article,

Phorbol ester preferentially stimulates mouse fornical conjunctival and limbal epithelial cells to proliferate in vivo
Lavker RM; Wei ZG; Sun TT
1998 Feb;39(2):301-307, Investigative ophthalmology & visual science. IOVS
PURPOSE: The authors investigated whether fornical epithelium displays a differential in vivo response to acute and chronic stimulation when compared with bulbar and palpebral epithelia. METHODS: To induce an increase in epithelial proliferation, 0.5% phorbol myristate (TPA) was topically applied in petrolatum daily to both eyes of SENCAR mice for 12 days. Control mice (three per group) received petrolatum only. After 6, 12, 18, and 24 hours (acute) and 2, 3, 4, 5, 7, 9, and 12 days (chronic) of TPA treatment, mice (three per group) were administered intraperitoneally 0.1 ml 40 microCi [3H]thymidine ([3H]TdR) 1 hour before they were killed. Conjunctival epithelium was fixed and processed for autoradiography, and the labeling index (LI; number of [3H]TdR-labeled nuclei per 1000 basal keratinocytes) was determined for each of the epithelial zones. RESULTS: Under normal situations, the LI was lowest in fornical epithelium (1.9 +/- 0.5) compared with bulbar (4.4 +/- 0.9) and palpebral (5.5 +/- 0.5) epithelia. Within 24 hours of TPA treatment, a 12-fold increase in fornical basal cell labeling was noted compared with a 2.5- and 5-fold increase in bulbar and palpebral basal cell labeling, respectively. Fornical epithelium maintained a significantly greater proliferative response (4.5-fold increase) during chronic stimulation than either bulbar or palpebral epithelia (0.5- and 1.5-fold increase, respectively). CONCLUSIONS: The more vigorous response of the fornical epithelium to acute and chronic stimulation is strong evidence that this epithelium has a greater proliferative capacity than the other two epithelia, which is consistent with the authors' hypothesis that conjunctival epithelial stem cells are primarily located in the fornical region
— id: 16524, year: 1998, vol: 39, page: 301, stat: Journal Article,

Strategies of epithelial repair: modulation of stem cell and transit amplifying cell proliferation
Lehrer MS; Sun TT; Lavker RM
1998 Oct;111(Pt 19):2867-2875, Journal of cell science
Using double labeling techniques, we studied the replication of corneal epithelial stem cells that reside exclusively in the limbal zone, and their progeny transit amplifying cells. We show that corneal epithelial stem cells can be induced to enter DNA synthesis by wounding and by TPA. We demonstrate the existence of a hierarchy of TA cells; those of peripheral cornea undergo at least two rounds of DNA synthesis before they become post-mitotic, whereas those of central cornea are capable of only one round of division. However, the cell cycle time of these TA cells can be shortened and the number of times these TA cells can replicate is increased in response to wounding. These results thus demonstrate three strategies of epithelial repair: (i) stem cell replication, (ii) the unleashing of additional rounds of cell proliferation that remain as an untapped reserve under normal circumstances, and (iii) enhancement of TA cell proliferation via a shortening of the cycling time
— id: 16520, year: 1998, vol: 111, page: 2867, stat: Journal Article,

Extracellular matrix changes in human corneas after radial keratotomy
Ljubimov AV; Alba SA; Burgeson RE; Ninomiya Y; Sado Y; Sun TT; Nesburn AB; Kenney MC; Maguen E
1998 Sep;67(3):265-272, Experimental eye research
Extracellular matrix and basement membrane alterations were identified in human corneas after radial keratotomy. Ten normal and five radial keratotomy autopsy corneas (two at 6 months post surgery, and three at 3 years post surgery) were studied by immunofluorescence with antibodies to 28 extracellular matrix and basement membrane components. Outside of radial keratotomy scars, all studied components had a normal distribution. Of stromal extracellular matrix, only type III collagen accumulated around the scars. The basement membrane around epithelial plugs had a normal composition except for type IV collagen. Its alpha1-alpha2 chains, normally present only in the limbal basement membrane, appeared around all plugs. alpha3 and alpha4 chains were very weak or absent in these areas, contrary to nonscarred areas. This basement membrane pattern was similar to the normal limbal but not to the central corneal pattern. Keratin 3 also had a limbal-like, suprabasal expression in the plug epithelium. The stroma around the scars accumulated tenascin-C, fibrillin-1, types VIII and XIV collagen, all of which were absent from normal corneal basement membrane and extracellular matrix. Only tenascin-C showed less staining in anterior scars 3 years post surgery than 6 months post surgery, but still persisted in posterior scars. Incomplete scar healing was evident even 3 years post radial keratotomy. It was manifested by the accumulation of abnormal extracellular matrix in the anterior and posterior scars and by the limbal-like pattern of type IV collagen isoforms in the basement membrane around epithelial plugs
— id: 26910, year: 1998, vol: 67, page: 265, stat: Journal Article,

Autologous transplantation of urothelium into demucosalized gastrointestinal segments: evidence for epithelialization and differentiation of in vitro expanded and transplanted urothelial cells
Schaefer BM; Lorenz C; Back W; Moll R; Sun TT; Schober C; Waag KL; Kramer MD
1998 Jan;159(1):284-290, Journal of urology
PURPOSE: Our study established a technique for in vitro expansion and subsequent transplantation of autologous urothelial cells into vascularized seromuscular segments from stomach and colon in sheep. The proof of proliferation and differentiation of the transplanted urothelium in the absence of resident urothelium is considered to be a prerequisite for use of this technique in bladder augmentation. MATERIALS AND METHODS: Autologous sheep urothelial cells were expanded in vitro and grown on collagen membranes for sheet grafting. Using a vital stain, viability and confluency status of the urothelial graft were determined before transplantation into demucosalized segments isolated from the sheep stomach and colon gastrointestinal pouches. The gastrointestinal segments were sewn up and remained in the abdomen as small pouches stiched to the abdominal wall. Take and differentiation of transplanted cells within the pouch were assessed two and three weeks later using histological and immunohistological means. RESULTS: Urothelial cells grew well on collagen membranes. A confluency status > 40% and co-culturing with 3T3 feeder cells favored successful transplantation. Two weeks after transplantation a multilayered urothelial-like epithelium was found to line the lumen of the pouch. The epithelium was characterized by a distinct urothelium-typical distribution of basal and luminal keratins and the expression of the umbrella cell-specific marker uroplakin III. Moreover, the epithelium had an underlying basal lamina which focally contained collagen type IV. CONCLUSIONS: The data indicate that in vitro expanded urothelial cells are capable of epithelializing demucosalized gastrointestinal segments forming a genuine, differentiated 'neo' urothelium
— id: 26911, year: 1998, vol: 159, page: 284, stat: Journal Article,

Uroplakin II gene is expressed in transitional cell carcinoma but not in bilharzial bladder squamous cell carcinoma: alternative pathways of bladder epithelial differentiation and tumor formation [published erratum appears in Cancer Res 1998 Jul 1;58(13):2904]
Wu RL; Osman I; Wu XR; Lu ML; Zhang ZF; Liang FX; Hamza R; Scher H; Cordon-Cardo C; Sun TT
1998 Mar 15;58(6):1291-1297, Cancer research
Uroplakins (UPs) are integral membrane proteins that are synthesized as the major differentiation products of mammalian urothelium. We have cloned the human UP-II gene and localized it on chromosome 11q23. A survey of 50 transitional cell carcinomas (TCCs) revealed a UP-II polymorphism but no tumor-specific mutations. Immunohistochemical staining using rabbit antisera against a synthetic peptide of UP-II and against total UPs showed UP reactivity in 39.5% (17 of 43 cases) of conventional TCCs, 12.8% (5 of 39) of bilharzial-related TCCs, and 2.7% (1 of 36) of bilharzial-related squamous cell carcinomas (SCCs). The finding that fewer bilharzial TCCs express UPs than conventional TCCs (12.8 versus 40%) raised the possibility that the former are heterogeneous, expressing SCC features to varying degrees. Our data strongly support the hypothesis that urothelium can undergo at least three pathways of differentiation: (a) urothelium-type pathway; (b) epidermis-type pathway; and (c) glandular-type pathway, characterized by the production of UPs, K1/K10 keratins, and secreted glycoproteins, respectively. Vitamin A deficiency and mesenchymal factors may play a role in determining the relative contributions of these pathways to urothelial differentiation as well as to the formation of TCC, SCC, and adenocarcinoma, or a mixture thereof
— id: 7863, year: 1998, vol: 58, page: 1291, stat: Journal Article,

Regulation of K3 keratin gene transcription by Sp1 and AP-2 in differentiating rabbit corneal epithelial cells
Chen TT; Wu RL; Castro-Munozledo F; Sun TT
1997 Jun;17(6):3056-3064, Molecular & cellular biology
Rabbit corneal epithelial cells cultured in the presence of 3T3 feeder cells undergo biochemical differentiation, as evidenced by their initial expression of K5 and K14 keratins characteristic of basal keratinocytes, followed by the subsequent expression of K3 and K12 keratin markers of corneal epithelial differentiation. Previous data established that mutations of an Sp1 site in a DNA element, E, that contains overlapping Sp1 and AP-2 motifs reduce K3 gene promoter activity by 70% in transfection assays. We show here that Sp1 activates while AP-2 represses the K3 promoter. Although undifferentiated corneal epithelial basal cells express equal amounts of Sp1 and AP-2 DNA-binding activities, the differentiated cells down-regulate their Sp1 activity slightly but their AP-2 activity drastically, thus resulting in a six- to sevenfold increase in the Sp1/AP-2 ratio. This change coincides with the activation and suppression of the differentiation-related K3 gene and the basal cell-related K14 keratin gene, respectively. In addition, we show that polyamines, which are present in a high concentration in proliferating basal keratinocytes, can inhibit the binding of Sp1 to its cognate binding motif but not that of AP-2. These results suggest that the relatively low Sp1/AP-2 ratio as well as the polyamine-mediated inhibition of Sp1 binding to the E motif may account, in part, for the suppression of the K3 gene in corneal epithelial basal cells, while the elevated Sp1/AP-2 ratio may be involved in activating the K3 gene in differentiated corneal epithelial cells. Coupled with the previous demonstration that AP-2 activates the K14 gene in basal cells, the switch of the Sp1/AP-2 ratio during corneal epithelial differentiation may play a role in the reciprocal expression of the K3 and K14 genes in the basal and suprabasal cell layers
— id: 8367, year: 1997, vol: 17, page: 3056, stat: Journal Article,

Clonal analysis of the in vivo differentiation potential of keratinocytes
Wei ZG; Lin T; Sun TT; Lavker RM
1997 Mar;38(3):753-761, Investigative ophthalmology & visual science. IOVS
PURPOSE: This study investigated the in vivo differentiation of conjunctival keratinocytes. METHODS: Keratinocytes from the fornical region of the conjunctival epithelium were isolated and plated at low density (5 x 102 per 100-mm dish) in Dulbecco's minimum essential medium containing 20% fetal bovine serum in the presence of mitomycin C-treated 3T3 feeder cells. At this density, only single, isolated cells were attached after overnight culture. Eight days later, small, well-isolated colonies separated from one another by the feeder cells were detached as a sheet from the dish and were injected subcutaneously into the flanks of BALB/c athymic mice through an 18-gauge needle. Within a day, a small firm nodule appeared at the site of injection. At different time points, the animals were killed, and the nodules were excised for morphologic, histogeometric, and cell kinetic analyses. RESULTS: Each implanted colony derived from a single cell gave rise to a single epithelial cyst lined with a reconstituted stratified epithelium. Goblet-like cells loaded with periodic acid-Schiff-positive cytoplasmic granules began to appear singularly in some of the cysts by day 8 postimplantation and were observed in approximately 85% of the cysts by day 14. CONCLUSIONS: Because the cysts formed were derived from clonal populations of epithelial cells and the majority of cysts had a mixed keratinocyte-goblet cell phenotype, these results suggest strongly the existence of a bipotent precursor cell in conjunctival epithelium that can give rise to both goblet and nongoblet cells. This system can be used to study factors that can influence the commitment of pluripotent epithelial stem cells to divergent pathways of differentiation
— id: 16527, year: 1997, vol: 38, page: 753, stat: Journal Article,

Expression of keratohyalin-trichohyalin hybrid granules in molluscum contagiosum
Manabe M; Yaguchi H; Butt KI; O'Guin WM; Sun TT; Ogawa H
1996 Feb;35(2):106-108, International journal of dermatology
BACKGROUND: Recently, in the filiform papillae epithelium of mouse dorsal tongue, we showed the presence of hybrid granules in which filaggrin and trichohyalin were both present, but physically segregated. Further, trichohyalin was also detected in scattered granular cells of a number of hyperplastic skin diseases. METHODS: The epidermis infected with molluscum contagiosum virus (MCV) was studied by conventional electron microscopy in conjunction with light and electron-microscopic immunohistochemistry, using both antifilaggrin and antitrichohyalin antibodies as probes. RESULTS: We found that the granular cells of MCV-infected epidermis contained both filaggrin and trichohyalin. Subsequent electron-microscopic examination showed that the granular cells contained morphologically heterogeneous granules that appeared to be composed of discrete areas of distinct electron densities. Double-labeling, using antibodies to filaggrin and trichohyalin, clearly indicated that filaggrin and trichohyalin were both present in the hybrid granules and that the electron-dense regions contained trichohyalin while the more electron-lucent regions contained filaggrin. CONCLUSIONS: The expression of trichohyalin was a common feature observed in the epidermis from a heterogenous group of hyperplastic conditions, including MCV infection. This finding has led us to speculate that trichohyalin may be specifically or preferentially involved in interacting with the hyperproliferation-related keratin pair (K6/K16), whereas the function of filaggrin is more closely linked to the skin-type keratin pair (K1/K10) that are normal keratins found in the differentiated epidermis
— id: 16671, year: 1996, vol: 35, page: 106, stat: Journal Article,

Analysis of differentiation-associated proteins in rat bladder carcinogenesis
Ogawa K; Sun TT; Cohen SM
1996 May;17(5):961-965, Carcinogenesis
Uroplakins are the major integral membrane proteins synthesized in terminally differentiated, superficial urothelial cells. Alteration of cell differentiation during rat urinary bladder carcinogenesis was analyzed immunohistochemically for the expression of uroplakins. Expression of uroplakins was compared in N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide (FANFT)-, uracil-, sodium saccharin- or sodium ascorbate-induced urothelial simple hyperplasia, papillary-nodular hyperplasia, papilloma and carcinoma. In controls, uroplakins were located only in superficial cells, especially the luminal surface membrane. In FANFT-induced hyperplasia, including simple hyperplasia, intermediate cells also stained and the staining pattern was disorderly and intermittent. In uracil-induced simple hyperplasia, intermediate cells were stained but in an orderly fashion. In sodium saccharin- or sodium ascorbate-induced simple hyperplasia, superficial cells were swollen but alterations were not observed in the staining pattern. In carcinoma induced by FANFT and uracil, uroplakin expression was very disorderly and focal, usually with no expression on surface cells. It appears that disorderly differentiation is an index of bladder malignancy and is an early event in FANFT-induced lesions but a late event in uracil-, sodium saccharin- and sodium ascorbate-induced lesions
— id: 26912, year: 1996, vol: 17, page: 961, stat: Journal Article,

Epithelial growth and differentiation: an overview
Sun TT
1996 ;23(1):1-2, Molecular biology reports
— id: 12666, year: 1996, vol: 23, page: 1, stat: Journal Article,

Formation of asymmetric unit membrane during urothelial differentiation
Sun TT; Zhao H; Provet J; Aebi U; Wu XR
1996 ;23(1):3-11, Molecular biology reports
Mammalian urothelium undergoes unique membrane specialization during terminal differentiation making numerous rigid-looking membrane plaques (0.3-0.5 micron diameter) that cover the apical cell surface. The outer leaflet of these membrane plaques is almost twice as thick as the inner leaflet hence the name asymmetric unit membrane (AUM). Ultrastructural studies established that the outer leaflet of AUM is composed of 16 nm particles forming two dimensional crystals, and that each particle forms a 'twisted ribbon' structure. We showed recently that highly purified bovine AUMs contain four major integral membrane proteins: uroplakins Ia (27 kD), Ib (28 kD), II (15 kD) and III (47 kD). Studies of the protease sensitivity of the different subdomains of uroplakins and other considerations suggest that UPIa and UPIb have 4 transmembrane domains, while UPII and UPIII have only one transmembrane domain. Chemical crosslinking studies showed that UPIa and UPIb, which share 39% amino acid sequence, are topologically adjacent to UPII and UPIII, respectively, thus raising the possibility that there exist two biochemically distinct AUM particles, i.e., those containing UPIa/UPII vs. UPIb/UPIII. Bovine urothelial cells grown in the presence of 3T3 feeder cells undergo clonal growth forming stratified colonies capable of synthesizing and processing all known uroplakins. Transgenic mouse studies showed that a 3.6 kb 5'-flanking sequence of mouse uroplakin II gene can drive the expression of bacterial LacZ gene to express in the urothelium. Further studies on the biosynthesis, assembly and targeting of uroplakins will offer unique opportunities for better understanding the structure and function of AUM as well as the biology of mammalian urothelium
— id: 12665, year: 1996, vol: 23, page: 3, stat: Journal Article,

Rabbit conjunctival and corneal epithelial cells belong to two separate lineages
Wei ZG; Sun TT; Lavker RM
1996 Mar;37(4):523-533, Investigative ophthalmology & visual science. IOVS
PURPOSE: This study investigated rabbit conjunctival and corneal epithelial cells to determine if they belong to two separate lineages. METHODS: Rabbit corneal, limbal, and conjunctival epithelial cells were isolated and grown in Dulbecco's minimum essential media and 20% fetal bovine serum in the presence of mitomycin-treated 3T3 feeder cells. After reaching 80% confluence, 3T3 feeder cells and any contaminating fibroblasts were removed, and epithelial cells were resuspended in fresh Dulbecco's minimum essential media. Aliquots containing 5x10(6) cells were placed subcutaneously into the flanks of athymic mice, which subsequently formed small nodules. At 2, 4, 6, 8, 14, 21, and 28 days, athymic mice were killed and the nodules (epithelial cyst) were excised for light and transmission electron microscopic examination and histochemical and cell kinetic analyses. RESULTS: Within 2 days after injection of single-cell suspensions, cells aggregated to form cysts lined with a stratified squamous epithelium, the structure of which resembled the original in vivo donor sites by 8 days. Limbal- and corneal-derived cysts were comprised only of glycogen-rich stratified epithelial cells. In contrast, only cysts arising from cultured conjunctival cells contained periodic acid-Schiff-positive cells with a goblet cell structure interspersed among stratified epithelial cells. Furthermore, cystic epithelium of conjunctival origin did not accumulate glycogen. CONCLUSIONS: To determine whether distinct phenotypes are caused by intrinsic divergence or by environmental modulation, the behavior of cells can be monitored in an identical in vivo growth environment. The athymic mouse provides such a permissive growth environment for cultured corneal, limbal, and conjunctival epithelial cells. All these cells reproduced their in vivo phenotype when placed in the athymic mouse. Thus, these findings provide the strongest evidence to date that the corneal-limbal lineage is distinct from the conjunctival lineage. These data also support the idea that the progenitor of goblet cells does not reside in the corneal-limbal epithelial compartment
— id: 16530, year: 1996, vol: 37, page: 523, stat: Journal Article,

In vitro binding of type 1-fimbriated Escherichia coli to uroplakins Ia and Ib: relation to urinary tract infections
Wu XR; Sun TT; Medina JJ
1996 Sep 3;93(18):9630-9635, Proceedings of the National Academy of Sciences of the United States of America
Urinary tract infections, caused mainly by Escherichia coli, are among the most common infectious diseases. Most isolates of the uropathogenic E.coli can express type 1 and P fimbriae containing adhesins that recognize cell receptors. While P fimbriae recognize kidney glycolipid receptors and are involved in peyelonephritis, the urothelial for type 1 fimbriae were not identified. We show that type 1-fimbriated E. coli recognize uroplakins Ia and Ib, two major glycoproteins of urothelial apical plaques. Anchorage of E. coli to urothelial surface via type 1 fimbriae-uroplakin I interactions may play a role in its bladder colonization and eventual ascent through the ureters, against urine flow, to invade the kidneys
— id: 8388, year: 1996, vol: 93, page: 9630, stat: Journal Article,

Characterization of hair follicle bulge in human fetal skin: the human fetal bulge is a pool of undifferentiated keratinocytes
Akiyama M; Dale BA; Sun TT; Holbrook KA
1995 Dec;105(6):844-850, Journal of investigative dermatology
It has been suggested that the bulge of the hair follicle contains a pool of follicular stem cells that may serve as a target site of graft-versus-host disease and as a source of cells with carcinogenic potential. The bulge is prominent in the developing follicle although it is a subtle swelling in the adult follicle. In this paper, we studied the bulge in human fetal skin specimens. Ultrastructurally, the bulge cells, especially the interior cells, have abundant free ribosomes and glycogen particles, but almost no cytoplasmic organelles indicative of differentiation. Immunostaining with several specific anti-keratin antibodies demonstrated that the bulge cells express keratins of both stratified and simple epithelia. Melanocytes and Merkel cells, defined by immunohistochemical and ultrastructural criteria, are seen among bulge cells. Laser confocal microscopy revealed that primitive smooth muscle cells attached directly to the bulge initially at the mid-bulbous hair peg, the stage when the bulge is most prominent. K-laminin and type VII collagen are strongly expressed in the dermoepidermal junction of the bulge and between the matrix area of the bulb and the dermal papilla. Thus, the bulge of human hair follicle is not only an attachment site for arrector pili muscle, but also a pool of keratinocytes that are relatively undifferentiated
— id: 26913, year: 1995, vol: 105, page: 844, stat: Journal Article,

A NOVEL NUCLEAR-PROTEIN OF RABBIT CORNEAL KERATINOCYTES THAT BINDS TO THE PROMOTER OF RABBIT K3 KERATIN GENE
CHEN, TT; WU, RL; SUN, TT
1995 MAR 15 ;36(4):S609-S609, Investigative ophthalmology & visual science. IOVS
— id: 87340, year: 1995, vol: 36, page: S609, stat: Journal Article,

Hair follicle stem cells: present concepts
Lavker RM; Sun TT
1995 May;104(5 Suppl):38S-39S, Journal of investigative dermatology
— id: 16532, year: 1995, vol: 104, page: 38S, stat: Journal Article,

A tissue-specific promoter that can drive a foreign gene to express in the suprabasal urothelial cells of transgenic mice
Lin JH; Zhao H; Sun TT
1995 Jan 31;92(3):679-683, Proceedings of the National Academy of Sciences of the United States of America
Uroplakins are a group of integral membrane proteins that are synthesized as the major differentiation products of urothelium. The luminal portions of these proteins form 12-nm protein particles arranged in a two-dimensional crystalline array. The expression of uroplakin genes is bladder specific and differentiation dependent; little is known, however, about their molecular regulation. Here we describe the cloning of mouse uroplakin II gene and demonstrate, in transgenic mouse experiments, that a 3.6-kb 5'-flanking sequence of this gene can drive a bacterial lacZ (reporter) gene to express in the suprabasal cell layers of the urothelium. The transgene was not expressed in any tested (nonurothelial) epithelial and other tissues (except hypothalamus). These results suggest that most of the cis elements that confer the bladder-specific and differentiation-dependent expression of mouse uroplakin II gene must reside in the 3.6-kb sequence. The availability of a promoter capable of delivering a foreign molecule to the differentiated cell layers of bladder epithelium opens avenues for studying normal and pathological urothelial differentiation in transgenic mice
— id: 8454, year: 1995, vol: 92, page: 679, stat: Journal Article,

Human corneal basement membrane heterogeneity: topographical differences in the expression of type IV collagen and laminin isoforms
Ljubimov AV; Burgeson RE; Butkowski RJ; Michael AF; Sun TT; Kenney MC
1995 Apr;72(4):461-473, Laboratory investigation
BACKGROUND: The corneal epithelium converges at the peripheral zone (limbus) with the conjunctival epithelium, forming a continuous sheet with phenotypically distinct regions--central, limbal, and conjunctival. The epithelial basement membrane (EBM) is important for corneal functions and cell adhesion, but its regional composition is poorly understood. Current literature is controversial as to the occurrence of type IV collagen in the cornea. The aim of this study was to investigate in detail corneal basement membrane (BM) composition and correlate it with the differentiation state of contributing cells. EXPERIMENTAL DESIGN: Adult human corneas (N = 8) were cryosectioned and analyzed by immunofluorescence with antibodies to 15 BM components and to keratin 3, a marker of corneal epithelial differentiation. RESULTS: A novel type of spatial heterogeneity ('horizontal') in the EBM composition was found between the central cornea, limbus, and conjunctiva. Central EBM had type IV collagen alpha 3-alpha 5 chains, whereas limbal and conjunctival EBM contained alpha 1-alpha 2 chains and also laminin alpha 2 and beta 2 chains. Limbal EBM in addition had alpha 5(IV) chain. Laminin-1 (alpha 1 beta 1 gamma 1), laminin-5 (alpha 3 beta 3 gamma 2), perlecan, fibronectin, entactin/nidogen, and type VII collagen were seen in the entire EBM. Another novel type of BM heterogeneity ('vertical') was typical for the corneal Descemet's membrane: its stromal face had alpha 1(IV) and alpha 2(IV) chains and fibronectin, whereas alpha 3(IV)-alpha 5(IV) chains, entactin/nidogen, laminin-1, and perlecan were present on the endothelial face. CONCLUSIONS: Type IV collagen controversy is the result of the shifts of isoforms in the limbus and conjunctiva. These shifts and the appearance of additional laminins in the limbus may be related to the differentiation state of corneal cells contributing to the EBM formation. Novel types of BM heterogeneity in the human cornea are described: regional (horizontal) in the EBM and vertical in the Descemet's membrane. The first one may be a common feature of converging complex epithelia, whereas the second one may be another unique property of the Descemet's membrane
— id: 26917, year: 1995, vol: 72, page: 461, stat: Journal Article,

Ectopic expression of a bacterial lacZ gene in the limbic system of transgenic mice
Meyer-Puttlitz B; Lin JH; Sun TT; Margolis RK
1995 Aug 21;6(12):1674-1678, Neuroreport
In three independent lines of transgenic mice, a 3.6 kb 5'-flanking sequence of the uroplakin II gene consistently drives the ectopic expression of a bacterial lacZ reporter gene in brain, in addition to its specific expression in the suprabasal layers of the urothelium. The ectopic expression in brain is especially noteworthy insofar as it is confined to structures comprising the limbic system. These findings provide additional evidence that the cells forming such functional systems share specific biochemical properties, and also indicate that this promoter may be useful as a tool for studying the effects of overexpression of proteins in anatomically and functionally defined central nervous system pathways
— id: 26915, year: 1995, vol: 6, page: 1674, stat: Journal Article,

Uroplakins, specific membrane proteins of urothelial umbrella cells, as histological markers of metastatic transitional cell carcinomas
Moll R; Wu XR; Lin JH; Sun TT
1995 Nov;147(5):1383-1397, American journal of pathology
Uroplakins (UPs) Ia, Ib, II, and III, transmembrane proteins constituting the asymmetrical unit membrane of urothelial umbrella cells, are the first specific urothelial differentiation markers described. We investigated the presence and localization patterns of UPs in various human carcinomas by applying immunohistochemistry (avidin-biotin-peroxidase complex method), using rabbit antibodies against UPs II and III, to paraffin sections. Positive reactions for UP III (sometimes very focal) were noted in 14 of the 16 papillary noninvasive transitional cell carcinomas (TCCs) (88%), 29 of the 55 invasive TCCs (53%), and 23 of the 35 TCC metastases (66%). Different localization patterns of UPs could be distinguished, including superficial membrane staining like that found in normal umbrella cells (in papillary carcinoma), luminal (microluminal) membrane staining (in papillary and invasive carcinoma), and, against expectations, peripheral membrane staining (in invasive carcinoma). Non-TCC carcinomas of various origins (n = 177) were consistently negative for UPs. The presence of UPs in metastatic TCCs represents a prime example of even advanced tumor progression being compatible with the (focal) expression of highly specialized differentiation repertoires. Although of only medium-grade sensitivity, UPs do seem to be highly specific urothelial lineage markers, thus operating up interesting histodiagnostic possibilities in cases of carcinoma metastases of uncertain origin
— id: 26914, year: 1995, vol: 147, page: 1383, stat: Journal Article,

Cutaneous ultrastructural features of the flaky skin (fsn) mouse mutation
Morita K; Hogan ME; Nanney LB; King LE Jr; Manabe M; Sun TT; Sundberg JP
1995 Jun;22(6):385-395, Journal of dermatology
An autosomal recessive genetic disease with clinical and histopathological skin features resembling human psoriasis vulgaris occurs naturally in flaky skin mice (fsn/fsn). Affected mice are normal at birth, except for a hypochromic anemia. Subsequently, they develop hyperkeratotic plaques and acanthosis with elongation of rete ridges. Scanning electron microscopic examination revealed a greatly thickened epidermis, a sparsity of hairs and scale accumulations on the epidermal surface. Hair shafts had conspicuous pits, striations, and exophytic protrusions. Nails were bent at a 90 degrees angle with surface irregularities and accumulations of scale at the nail base. Transmission electron microscopic examination showed increased epidermal thickness, mitochondrial aberrations, and intraepidermal invasion by neutrophils. Keratohyalin abnormalities were detected using immunocytochemical staining for profilaggrin. At the dermal-epidermal junction, numerous macrophages and mast cells were seen in close proximity to focal dissolutions of the basement membrane. A high density of collagen fibers and cellular infiltrates were evident in the papillary dermis. This constellation of ultrastructural aberrations is typically found in psoriasis vulgaris and supports the theory that the flaky skin mouse mutation is a naturally occurring analog to one variety of human psoriasis vulgaris
— id: 26916, year: 1995, vol: 22, page: 385, stat: Journal Article,

Towards the molecular architecture of the asymmetric unit membrane of the mammalian urinary bladder epithelium: a closed "twisted ribbon" structure
Walz T; Haner M; Wu XR; Henn C; Engel A; Sun TT; Aebi U
1995 May 19;248(5):887-900, Journal of molecular biology
The asymmetric unit membrane (AUM) forms numerous plaques covering the apical surface of mammalian urinary bladder epithelium. These plaques contain four major integral membrane proteins called uroplakins Ia, Ib, II and III, which form particles arranged in a well-ordered hexagonal lattice with p6 symmetry and a lattice constant of 16.5 nm. Bovine AUM plaques negatively stained with anionic sodium silicotungstate revealed structural detail to 3.1 nm resolution. Correlation averaging resolved each particle into 12 stain-excluding domains arranged in two concentric rings (inner ring radius (rm) = 3.7 nm, outer ring radius (rout) = 6.6 nm), each with six domains which were rotated by roughly 30 degrees relative to each other. Negative staining with cationic uranyl formate increased the resolution to 2.2 nm and unveiled distinct connections between adjacent AUM particles. These connections may provide a molecular basis for the observed insolubility of the plaques in many detergents. Examination of the luminal face of freeze-dried/unidirectionally metal-shadowed AUM plaques established a left-handed vorticity of the 16 nm protein particles, whereas the cytoplasmic face exhibited no significant surface corrugations. Three-dimensional reconstruction from sodium silicotungstate-stained specimens revealed the AUM particles to be built of six 'V-shaped' subunits anchored upright in the membrane. The mass density distribution within uranyl formate-stained AUM particles was similar except that the inner tip of each V was bridged to the outer tip of an adjacent V, so that the 16 nm AUM particle appeared as a continuous, 'twisted ribbon' embracing a central cavity. Finally, mass measurements of unstained/freeze-dried plaques by scanning transmission electron microscopy yielded a total mass of 1,120 kDa per membrane-bound AUM particle. By imposing constraints on the possible uroplakin stoichiometries within AUM plaques, these data provide a first glimpse of the molecular architecture of the 16 nm particles constituting the plaques
— id: 8380, year: 1995, vol: 248, page: 887, stat: Journal Article,

Label-retaining cells are preferentially located in fornical epithelium: implications on conjunctival epithelial homeostasis
Wei ZG; Cotsarelis G; Sun TT; Lavker RM
1995 Jan;36(1):236-246, Investigative ophthalmology & visual science. IOVS
PURPOSE. To determine the cell kinetic properties of epithelial cells from various zones of the conjunctiva. METHODS. The morphology and cell kinetics of bulbar, fornical, and palpebral conjunctival epithelium were studied in neonatal and adult SENCAR mice. To examine the proliferative rate of the conjunctival epithelium, a single administration of tritiated thymidine (3H-TdR) was used to detect cells in 'S' phase. Proliferative rates were also assessed by determining mitotic activity after an intraperitoneal injection of colchicine to arrest cells in mitosis. To detect slow-cycling cells, mice received 3H-TdR continuously for 1 week. After a 4-week chase, animals were sacrificed and eyes were surgically removed. All tissues were immediately fixed in formalin and processed for histology and autoradiography. RESULTS. Slow-cycling cells, detected as label-retaining cells (LRCs), were identified in bulbar, fornical, and palpebral epithelia, as well as in limbal epithelium. The greatest number of LRCs was found in fornical epithelium. In addition, we found a number of label-retaining goblet cells. This cell population was shown to incorporate 3H-TdR after a single pulse administration, and mitotic figures were seen in goblet cells after colchicine treatment, indicating that conjunctival goblet cells have proliferative capabilities. CONCLUSIONS. These findings are consistent with earlier in vitro data that the fornical epithelium may be a zone enriched in conjunctival epithelial stem cells. This has important implications in conjunctival epithelial development and is relevant in wound repair. Furthermore, the concept that goblet cells are slow-cycling cells with proliferative capabilities provides new insights into the area of conjunctival homeostasis
— id: 16535, year: 1995, vol: 36, page: 236, stat: Journal Article,

Selective interactions of UPIa and UPIb, two members of the transmembrane 4 superfamily, with distinct single transmembrane-domained proteins in differentiated urothelial cells
Wu XR; Medina JJ; Sun TT
1995 Dec 15;270(50):29752-29759, Journal of biological chemistry
The transmembrane 4 (TM4) superfamily contains many important leukocyte differentiation-related surface proteins including CD9, CD37, CD53, and CD81; tumor-associated antigens including CD63/ME491, CO-029, and SAS; and a newly identified metastasis suppressor gene R2. Relatively little is known, however, about the structure and aggregation state of these four transmembrane-domained proteins. The asymmetrical unit membrane (AUM), believed to play a major role in stabilizing the apical surface of mammalian urothelium thus preventing it from rupturing during bladder distention, contains two TM4 members, the uroplakins (UPs) Ia and Ib. In association with two other (single transmembrane-domained) membrane proteins, UPII and UPIII, UPIa and UPIb form 16-nm particles that naturally form two-dimensional crystalline arrays, thus providing unique opportunities for studying membrane structure and function. To better understand how these proteins interact to form the 16-nm particles, we analyzed their nearest neighbor relationship by chemical cross-linking. We show here that UPIa and UPIb, which share 39% of their amino acid sequence, are cross-linked to UPII and UPIII, respectively. We also show that UPIa has a propensity to oligomerize, forming complexes that are stable in SDS, and that UPII can be readily cross-linked to form homodimers. The formation of UPII homodimers is sensitive, however, to octyl glucoside that can solubilize the AUMs. These data suggest that there exist two types of 16-nm AUM particles that contain UPIa/UPII or UPIb/UPIII, and support a model in which the UPIa and UPII occupy the inner and outer domains, respectively, of the UPIa/UPII particle. This model can account for the apparent 'redundancy' of the uroplakins, as the structurally related UPIa and UPIb, by interacting with different partners, may play different roles in AUM formation. The model also suggests that AUM plaques with different uroplakin compositions may differ in their assembly, and in their abilities to interact with an underlying cytoskeleton. Our data indicate that two closely related TM4 proteins, UPIa and UPIb, can be present in the same cell, interacting with distinct partners. AUM thus provides an excellent model system for studying the targeting, processing, and assembly of TM4 proteins
— id: 6976, year: 1995, vol: 270, page: 29752, stat: Journal Article,

AN IN-VIVO ASSAY FOR STUDYING THE FUNCTIONAL IMPORTANCE OF A KERATINOCYTE-SPECIFIC PROMOTER
WU, RL; CHEN, TT; CASTROMUNOZLEDO, F; SUN, TT
1995 MAR 15 ;36(4):S144-S144, Investigative ophthalmology & visual science. IOVS
— id: 87329, year: 1995, vol: 36, page: S144, stat: Journal Article,

Message of nexin 1, a serine protease inhibitor, is accumulated in the follicular papilla during anagen of the hair cycle
Yu DW; Yang T; Sonoda T; Gaffney K; Jensen PJ; Dooley T; Ledbetter S; Freedberg IM; Lavker R; Sun TT
1995 Dec;108(Pt 12):3867-3874, Journal of cell science
A group of specialized mesenchymal cells located at the root of the mammalian hair follicle, known as the follicular or dermal papillary cells, are involved in regulating the hair cycle, during which keratinocytes of the lower follicle undergo proliferation, degeneration and regrowth. Using the arbitrarily primed-PCR approach, we have identified a 1.3 kb messenger RNA that is present in large quantities in cultured rat follicular papillary cells, but not in skin fibroblasts. This mRNA encodes nexin 1, a potent protease inhibitor that can inactivate several growth-modulating serine proteases including thrombin, urokinase and tissue plasminogen activator. In situ hybridization showed that nexin 1 message is accumulated in the follicular papilla cells of anagen follicles, but is undetectable in keratinocytes or other skin mesenchymal cells. In addition, nexin 1 message level varies widely among several immortalized rat vibrissa papillary cell lines, and these levels correlate well with the reported abilities of these cell lines to support in vivo follicular reconstitution. These results suggest a possible role of nexin 1 in regulating hair follicular growth
— id: 12706, year: 1995, vol: 108, page: 3867, stat: Journal Article,

Permeability properties of the mammalian bladder apical membrane
Chang A; Hammond TG; Sun TT; Zeidel ML
1994 Nov;267(5 Pt 1):C1483-C1492, American journal of physiology. Cell physiology
The luminal surface of mammalian bladder is exposed to urine with a composition widely different from that of plasma that bathes the basolateral surface of epithelium. Therefore we predict that the bladder permeability barrier, which is likely located in the apical membrane (AM), will exhibit low permeabilities to water, urea, NH3, H+, and small nonelectrolytes. AM surface area increases as the bladder fills with urine and decreases during emptying, a process that involves cyclical endocytosis and reinsertion of membrane from a pool of AM endosomes (AME). Rigid-appearing plaques composed of three proteins, uroplakins, have been identified and occupy 70-90% of AM surface area. To determine permeability properties of the AM permeability barrier, we purified AME and measured their permeabilities. Rabbit urinary bladders were removed, and their apical surface was exposed to carboxyfluorescein (CF) or horseradish peroxidase (HRP). Exposure to hypotonic and then isotonic basolateral solutions induced endocytosis of luminal CF or HRP into AME. Electron microscopy of bladders after this treatment revealed HRP entrapped within AME bordered by plaques. AME were purified by differential and sucrose-gradient centrifugation, and CF-containing AME were purified 17.0 +/- 3-fold (SD) with respect to homogenate. Analysis of purified AME by flow cytometry showed that > 95% of vesicles contained CF entrapped from luminal solution and were selectively labeled with anti-uroplakin antibody. AME osmotic water permeability averaged 2.3 +/- 0.66 x 10(-4) cm/s and exhibited a high activation energy, indicating that AM contains no water channels. Permeability to urea and NH3 averaged 7.8 +/- 3.7 x 10(-7) and 1.5 +/- 0.3 x 10(-3) cm/s, respectively, which are exceptionally low and similar to permeabilities of other water-tight membranes, including toad urinary bladder and gastric mucosa. AME behaved as a single population in all permeability studies, which will permit future characterization of protein and lipid structure responsible for these unique permeability properties
— id: 26918, year: 1994, vol: 267, page: C1483, stat: Journal Article,

Precursor sequence, processing, and urothelium-specific expression of a major 15-kDa protein subunit of asymmetric unit membrane
Lin JH; Wu XR; Kreibich G; Sun TT
1994 Jan 21;269(3):1775-1784, Journal of biological chemistry
The asymmetric unit membrane (AUM) is a highly specialized biomembrane elaborated by terminally differentiated urothelial cells. It contains quasi-crystalline arrays of 12-nm protein particles each of which is composed of six dumbbell-shaped subdomains. In this paper we describe the precursor sequence, processing and in vitro membrane insertion properties of bovine uroplakin II (UPII), a 15-kDa major protein component of AUM. The cDNA-deduced amino acid sequence revealed that UPII is synthesized as a precursor protein containing a cleavable signal peptide of approximately 26 amino acids, a long pro-sequence of approximately 59 residues harboring three potential N-glycosylation sites, and the mature polypeptide of 100 residues. In vitro translation of UPII mRNA demonstrated that UPII is indeed first synthesized as a 19-kDa precursor, which loses its signal peptide upon insertion into added microsomes; this process is accompanied by the acquisition of high mannose-type oligosaccharides giving rise to a 28-kDa precursor which is completely protected from the digestion by exogenous proteases. These results, together with the presence of a stretch of 25 hydrophobic amino acids at the C terminus, suggest that UPII protein is anchored to the lipid bilayer via its C-terminal membrane-spanning domain with its major N-terminal domain exposed luminally. The formation of the 15-kDa mature UPII requires the removal of the pro-sequence by a furin-like endoprotease. Since only mature UPII devoid of this pro-sequence can interact with 27-kDa uroplakin I, the proteolytic processing of UPII precursor may play an important role in regulating the assembly of AUM. Finally, we showed that genomic sequences cross-hybridizing with bovine UPII cDNA are present in many mammals suggesting that UPII performs a highly conserved function in the terminally differentiated cells of mammalian urinary bladder epithelium
— id: 8249, year: 1994, vol: 269, page: 1775, stat: Journal Article,

Cells within the bulge region of mouse hair follicle transiently proliferate during early anagen: heterogeneity and functional differences of various hair cycles
Wilson C; Cotsarelis G; Wei ZG; Fryer E; Margolis-Fryer J; Ostead M; Tokarek R; Sun TT; Lavker RM
1994 Jan;55(2):127-136, Differentiation
Based on cell kinetic, morphological and several biological considerations, we have recently proposed that hair follicle stem cells reside in the bulge area of the upper follicle. We predicted that during early anagen the normally slow-cycling bulge stem cells may be activated by the abutting dermal papilla cells to undergo transient proliferation giving rise to keratinocytes of the lower follicle. In the present work, we performed tritiated thymidine-labeling of DNA-synthesizing cells and colcemid-arrest of mitotic figures on the skins of 20-23 and 75-80 day old SENCAR mice, when the follicles entered the anagen phase of the 2nd and 3rd hair cycles. The results clearly indicate that the normally slow-cycling bulge cells indeed undergo transient proliferation during early anagen. Similar results were obtained when the telogen follicles are experimentally induced to enter the 3rd hair cycle by plucking and by topical applications of phorbol ester or tretinoin. These results support the notion that bulge cells are follicular stem cells, and that transient proliferation of these cells is a critical feature of early anagen. However, the long duration of the 2nd telogen (> 30 days in mouse) suggests that a new anagen phase does not automatically result from the physical proximity of dermal papilla to the bulge cells, and that another 'factor' is required for the initiation of the 3rd anagen. The tremendous difference in the durations of the first and second telogen (lasting for 2-3 days and > 50 days, respectively) suggests that follicles can exist in a non-cycling state that may be conceptually equivalent to the G0 state of the cell cycle. Our results also underscore the fact that the first hair cycle is distinct from all the subsequent hair cycles in their cellular origin and morphological sequence, and thus should be regarded as a neogenic event
— id: 16537, year: 1994, vol: 55, page: 127, stat: Journal Article,

Functional importance of an Sp1- and an NFkB-related nuclear protein in a keratinocyte-specific promoter of rabbit K3 keratin gene
Wu RL; Chen TT; Sun TT
1994 Nov 11;269(45):28450-28459, Journal of biological chemistry
We have shown previously that a 300-base pair (bp) 5' upstream sequence of rabbit keratin K3 gene (RK3) can function as a keratinocyte-specific promoter in transient transfection assays. Electrophoretic mobility shift assays using various overlapping and mutated oligonucleotides established that corneal keratinocyte nuclear proteins bound in vitro to two sites (B and E). Immunosupershift and UV cross-linking established that the keratinocyte nuclear binding protein of site B (5'-GGGGCTTTCC-3', -262 to -253 bp) was NFkB consisting of the p65 and p50 subunits. The E site contained an unusual GC-rich motif (5'-CCGCCCCCTG-3', -203 to -194 bp) whose sequence deviated from the Sp1 consensus in 4 out of 10 positions; this site bound an Sp 1-related keratinocyte nuclear protein. Mutagenesis of the NFkB, GC motif, and both sites abolished 20, 50, and 75%, respectively, of the promoter activity in transfected keratinocytes. The NFkB-like keratinocyte nuclear protein was barely detectable in kidney epithelial cells, HeLa, and fibroblasts. The Sp1-related nuclear protein was abundant in keratinocytes and simple epithelial cells, but was much less abundant in fibroblasts. These results indicate that NFkB is present in significant quantities in keratinocyte nuclei and that the tissue restriction of the NFkB- and Sp1-related proteins, in combination with other factors, may contribute to the keratinocyte specificity of RK3 promoter
— id: 12864, year: 1994, vol: 269, page: 28450, stat: Journal Article,

Mammalian uroplakins. A group of highly conserved urothelial differentiation-related membrane proteins
Wu XR; Lin JH; Walz T; Haner M; Yu J; Aebi U; Sun TT
1994 May 6;269(18):13716-13724, Journal of biological chemistry
The asymmetric unit membrane (AUM) forms the apical plaques of mammalian urothelium and is believed to play a role in strengthening the urothelial apical surface thus preventing the cells from rupturing during bladder distention. We have shown previously that purified bovine AUMs contain four major integral membrane proteins: the uroplakins Ia (27 kDa), Ib (28 kDa), II (15 kDa), and III (47 kDa). This contradicts some previous reports indicating that some of these proteins are absent in AUMs of several species. Using an improved procedure, we isolated AUMs from, in addition to cattle, eight mammalian species (human, monkey, sheep, pig, dog, rabbit, rat, and mouse). The AUMs of these species appear morphologically similar bearing crystalline patches of 12-nm protein particles with a center-to-center spacing of 16.5 nm. Using antibodies raised against synthetic oligopeptides or individual bovine uroplakins, we established by immunoblotting that the four uroplakins are present in AUMs of all these species. The DNA-deduced amino acid sequences of bovine and mouse uroplakin II revealed 83% identity. These results indicate that uroplakins Ia, Ib, II, and III are the major protein components of probably all mammalian urothelial plaques, and that the sequence and three-dimensional structure of uroplakin molecules are highly conserved during mammalian evolution
— id: 12966, year: 1994, vol: 269, page: 13716, stat: Journal Article,

Uroplakins Ia and Ib, two major differentiation products of bladder epithelium, belong to a family of four transmembrane domain (4TM) proteins
Yu J; Lin JH; Wu XR; Sun TT
1994 Apr;125(1):171-182, Journal of cell biology
The mammalian bladder epithelium elaborates, as a terminal differentiation product, a specialized plasma membrane called asymmetric unit membrane (AUM) which is believed to play a role in strengthening and stabilizing the urothelial apical surface through its interactions with an underlying cytoskeleton. Previous studies indicate that the outer leaflet of AUM is composed of crystalline patches of 12-nm protein particles, and that bovine AUMs contain three major proteins: the 27- to 28-kD uroplakin I, the 15-kD uroplakin II and the 47-kD uroplakin III. As a step towards elucidating the AUM structure and function, we have cloned the cDNAs of bovine uroplakin I (UPI). Our results established the existence of two isoforms of bovine uroplakin I: a 27-kD uroplakin Ia and a 28-kD uroplakin Ib. These two glycoproteins are closely related with 39% identity in their amino acid sequences. Hydropathy plot revealed that both have four potential transmembrane domains (TMDs) with connecting loops of similar length. Proteolytic digestion of UPIa inserted in vitro into microsomal vesicles suggested that its two main hydrophilic loops are exposed to the luminal space, possibly involved in interacting with the luminal domains of other uroplakins to form the 12-nm protein particles. The larger loop connecting TMD3 and TMD4 of both UPIa and UPIb contains six highly conserved cysteine residues; at least one centrally located cysteine doublet in UPIa is involved in forming intramolecular disulfide bridges. The sequences of UPIa and UPIb (the latter is almost identical to a hypothetical, TGF beta-inducible, TI-1 protein of mink lung epithelial cells) are homologous to members of a recently described family all possessing four transmembrane domains (the '4TM family'); members of this family include many important leukocyte differentiation markers such as CD9, CD37, CD53, and CD63. The tissue-specific and differentiation-dependent expression as well as the naturally occurring crystalline state of uroplakin I molecules make them uniquely suitable, as prototype members of the 4TM family, for studying the structure and function of these integral membrane proteins
— id: 6560, year: 1994, vol: 125, page: 171, stat: Journal Article,

Hair follicle stem cells: their location, role in hair cycle, and involvement in skin tumor formation
Lavker RM; Miller S; Wilson C; Cotsarelis G; Wei ZG; Yang JS; Sun TT
1993 Jul;101(1 Suppl):16S-26S, Journal of investigative dermatology
— id: 16540, year: 1993, vol: 101, page: 16S, stat: Journal Article,

Epithelial stem cells, hair follicles, and tumor formation
Lavker RM; Miller SJ; Sun TT
1993 ;128(3):31-43, Recent results in cancer research = Fortschritte der Krebsforschung = Progres dans les recherches sur le cancer
— id: 16545, year: 1993, vol: 128, page: 31, stat: Journal Article,

Cornea-specific expression of K12 keratin during mouse development
Liu CY; Zhu G; Westerhausen-Larson A; Converse R; Kao CW; Sun TT; Kao WW
1993 Nov;12(11):963-974, Current eye research
The full-length cDNA of mouse K12 keratin was characterized by sequencing overlapping cDNA clones isolated from a mouse cornea cDNA library. Using Northern blot hybridization, the radio-labeled cDNA hybridized to a 1.9 kb mRNA from adult cornea, but not from other mouse tissues including snout, esophagus, tongue, and skin. During mouse development, corneas do not express K12 mRNA until 4 days postnatal when the epithelium begins to stratify as judged by Northern blot and in situ hybridization. In situ hybridization with 3H-labeled cDNA probe and immunohistochemical studies with antibodies against a synthetic oligo-peptide deduced from rabbit K12 cDNA demonstrate that this mouse K12 keratin is expressed in all cell layers of adult corneal epithelium, and the suprabasal layers, but not the basal layer of the limbal epithelium. Epidermal growth factor (EGF) has been shown to promote epithelium stratification of cultured chicken and human corneas in vitro. To examine whether EGF can promote K12 expression, EGF was administered to neonatal mice. The results indicate that EGF retards K12 expression by corneal epithelial cells, even though it promotes corneal epithelial stratification during mouse development. Taken together, our results demonstrate that the expression of K12 keratin is cornea-specific, differentiation-dependent, and developmentally regulated
— id: 26919, year: 1993, vol: 12, page: 963, stat: Journal Article,

Hair follicles, stem cells, and skin cancer
Miller SJ; Sun TT; Lavker RM
1993 Mar;100(3):288S-294S, Journal of investigative dermatology
— id: 16544, year: 1993, vol: 100, page: 288S, stat: Journal Article,

Mouse skin is particularly susceptible to tumor initiation during early anagen of the hair cycle: possible involvement of hair follicle stem cells
Miller SJ; Wei ZG; Wilson C; Dzubow L; Sun TT; Lavker RM
1993 Oct;101(4):591-594, Journal of investigative dermatology
Stem cells are believed to be a necessary target of chemical carcinogens. Based on autoradiographic, ultrastructural, and biologic criteria, we have recently proposed that hair follicle stem cells reside not in the bulb, but in the upper outer root sheath in an area called the bulge. Proliferating cells have been shown to be more susceptible to tumor initiation, and we have recently demonstrated that cells in the bulge undergo transient proliferation during early anagen. Therefore, we theorized that mouse skin should be particularly susceptible to carcinogen application during early anagen phase. In this paper, we show that early anagen Swiss and Sencar mouse skin is indeed particularly susceptible to one- and two-stage chemical carcinogenesis, resulting in tumor yields one to five times those obtained with telogen-timed carcinogen application. Our findings implicate a possible involvement of the bulge cells as precursors to some of the skin cancers, and support the concept that these are stem cells. These observations also raise important questions about the cellular origins and biologic behavior of chemically induced murine skin tumors
— id: 16539, year: 1993, vol: 101, page: 591, stat: Journal Article,

[Uroplakin III, a specific membrane protein of urothelial umbrella cells, as a histological markers for metastatic transitional cell carcinomas]
Moll, R; Laufer, J; Wu, X R; Sun, T T
1993 ;77:260-265, Verhandlungen der Deutschen Gesellschaft fur Pathologie
We have investigated, by immunohistochemical staining of various paraffin-embedded carcinoma sections, the tissue-specific expression of uroplakin III--a recently characterized constituent glycoprotein (47 kD) of the asymmetrical unit membrane which forms plaques on apical surface of urothelial umbrella cells. The apical membrane pattern of normal urothelial umbrella cells was in part maintained in papillary transitional cell carcinomas (TCCs). In addition, in both papillary and invasive TCCs, variously sized lumina exhibited positive membrane staining of uroplakin III. In some cases, basal cell membrane staining was seen. Positive reactions (which sometimes were very focal) were noted in 16/18 papillary non-invasive TCCs (89%), 21/37 invasive TCCs (57%) and 12/15 TCC metastases (80%). Non-TCC carcinomas of different origin (n = 63) were consistently negative. These results show that uroplakin III may serve as a useful marker for TCCs, revealing specific urothelial differentiation features to be expressed in such tumors even after metastasis. This marker, while of only intermediate sensitivity, is highly specific, thus opening interesting histodiagnostic possibilities in the case of unclear carcinoma metastases
— id: 132744, year: 1993, vol: 77, page: 260, stat: Journal Article,

Suprabasal change and subsequent formation of disulfide-stabilized homo- and hetero-dimers of keratins during esophageal epithelial differentiation
Pang YY; Schermer A; Yu J; Sun TT
1993 Mar;104(Pt 3):727-740, Journal of cell science
Rabbit esophageal epithelium, a parakeratinized stratified epithelium, synthesizes as one of its major differentiation products a keratin pair consisting of a basic K4 (59 kDa) and an acidic K13 (41 kDa) keratin. Although immunohistochemical staining data suggest that in esophageal epithelia of some other species these two keratins are suprabasally located, antigenic masking of the epitopes in the basal cells has not been ruled out. Using several well-characterized monoclonal antibodies including AE8, which specifically recognizes K13, coupled with biochemical analysis of keratins of basal and suprabasal cells isolated from confluent rabbit esophageal epithelial culture, we have obtained direct evidence that K4 and K13 keratins are largely absent in the undifferentiated basal cells, but are present in large amounts in suprabasal cells. We also show that in the cornified cell layers that are formed during the terminal stage of esophageal epithelial differentiation, K4 and K13 keratins become disulfide-crosslinked to form three different dimers. Two of them (110 kDa and 100 kDa) are heterodimers and consist of equimolar amounts of K4 and K13; they presumably represent isomers crosslinked via different cysteine residues. The third dimer (90 kDa) was found to be a homodimer of the acidic K13 keratin. Trypsinization experiment established that at least some of the disulfide crosslinks in the K4/K13 heterodimer must involve cysteine residues residing in the trypsin-resistant rod domains of keratins. Air-oxidation of in vitro reconstituted filaments reproduced the two heterodimers, which most likely involve the crosslinking between type I and type II keratins of different coiled coils. The formation of these disulfide-crosslinked keratin dimers, instead of higher molecular mass oligomers or polymers as occurring in the epidermis and hair, may contribute to the formation of cornified cells with a physical stability and rigidity that are optimal for esophageal function. Our data also suggest that interactions involved in the formation of homodimers, thought to be metastable and unimportant during the initial step of filament assembly (i.e. tetramer formation), may actually play an important role in stabilizing a higher order structure in mature keratin filaments
— id: 13244, year: 1993, vol: 104, page: 727, stat: Journal Article,

Chromosomal localization of uroplakin genes of cattle and mice
Ryan AM; Womack JE; Yu J; Lin JH; Wu XR; Sun TT; Clarke V; D'Eustachio P
1993 Nov;4(11):656-661, Mammalian genome
The asymmetric unit membrane (AUM) of the apical surface of mammalian urinary bladder epithelium contains several major integral membrane proteins, including uroplakins IA and IB (both 27 kDa), II (15 kDa), and III (47 kDa). These proteins are synthesized only in terminally differentiated bladder epithelial cells. They are encoded by separate genes and, except for uroplakins IA and IB, appear to be unrelated in their amino acid sequences. The genes encoding these uroplakins were mapped to chromosomes of cattle through their segregation in a panel of bovine x rodent somatic cell hybrids. Genes for uroplakins IA, IB, and II were mapped to bovine (BTA) Chromosomes (Chrs) 18 (UPK1A), 1 (UPK1B), and 15 (UPK2), respectively. Two bovine genomic DNA sequences reactive with a uroplakin III cDNA probe were identified and mapped to BTA 6 (UPK3A) and 5 (UPK3B). We have also mapped genes for uroplakins IA and II in mice, to the proximal regions of mouse Chr 7 (Upk1a) and 9 (Upk2), respectively, by analyzing the inheritance of restriction fragment length variants in recombinant inbred mouse strains. These assignments are consistent with linkage relationships known to be conserved between cattle and mice. The mouse genes for uroplakins IB and III were not mapped because the mouse genomic DNA fragments reactive with each probe were invariant among the inbred strains tested. Although the stoichiometry of AUM proteins is nearly constant, the fact that the uroplakin genes are unlinked indicates that their expression must be independently regulated. Our results also suggest likely positions for two human uroplakin genes and should facilitate further analysis of their possible involvement in disease
— id: 17236, year: 1993, vol: 4, page: 656, stat: Journal Article,

In vitro growth and differentiation of rabbit bulbar, fornix, and palpebral conjunctival epithelia. Implications on conjunctival epithelial transdifferentiation and stem cells
Wei ZG; Wu RL; Lavker RM; Sun TT
1993 Apr;34(5):1814-1828, Investigative ophthalmology & visual science. IOVS
PURPOSE. The anterior surface of the eye is covered by several physically contiguous but histologically distinguishable epithelial overlying the cornea, limbus, bulbar conjunctiva, fornix conjunctiva, and palpebral conjunctiva. It is important to determine whether the different phenotypes of these epithelia are the result of intrinsic divergence, extrinsic modulation, or a combination of both. Based on keratin expression and cell kinetic criteria, the authors previously suggested that corneal epithelial stem cells may actually reside in the limbal basal layer. METHODS. In this article, the relationship between the corneal-limbal epithelial cells and conjunctival epithelial cells was analyzed by comparing their growth and differentiation properties in an identical cell culture environment. RESULTS. Using Dispase instead of trypsin to dissociate the cells, the authors were able to grow all five rabbit ocular surface epithelia in the presence of 3T3 feeder cells. They found that corneal and limbal cells synthesize identical keratins, including large amounts of the K3 and K12 markers of corneal-type differentiation. By contrast, all three conjunctival epithelia shared another keratin pattern, with large amounts of simple epithelial keratins but only minute amounts of K3/K12 keratins. CONCLUSIONS. This observation, coupled with previous findings that the 'transdifferentiation' of conjunctival epithelial cells to corneal epithelium appears to be both incomplete and reversible, provides strong evidence that (1) the limbal-corneal epithelial cells form a lineage distinct from the conjunctival lineage and (2) conjunctival transdifferentiation actually represents a process of environmental modulation. In addition, of the three types of conjunctival epithelial cells, fornix cells were found to have a much greater proliferative potential than bulbar and palpebral cells. This observation, coupled with recent finding that fornix is enriched in slow-cycling (label-retaining) cells, raises the possibility that conjunctival epithelial stem cells may preferentially reside in the fornix
— id: 16543, year: 1993, vol: 34, page: 1814, stat: Journal Article,

A 300 bp 5'-upstream sequence of a differentiation-dependent rabbit K3 keratin gene can serve as a keratinocyte-specific promoter
Wu RL; Galvin S; Wu SK; Xu C; Blumenberg M; Sun TT
1993 Jun;105(Pt 2):303-316, Journal of cell science
Keratinocytes of the suprabasal compartment of many stratified epithelia synthesize as a major differentiation product a keratin pair, consisting of an acidic and a basic keratin, which accounts for 10-20% of the newly synthesized proteins. While genes of several differentiation-related keratins have been cloned and studied, relatively little is known about the molecular basis underlying their tissue-specific and differentiation-dependent expression. We have chosen to study, as a prototype of these genes, the gene of K3 keratin, which has the unique property of being expressed in the majority of corneal epithelial basal cells but suprabasally in peripheral cornea, the site of corneal epithelial stem cells. Using a monoclonal antibody, AE5, specific for K3 keratin, and a fragment of human K3 gene as probes, we have isolated several cDNA and genomic clones of rabbit K3 keratin. One genomic clone has been sequenced and characterized, and the identity of its coding sequence with that of cDNAs indicates that it corresponds to the single, functional rabbit K3 gene. Transfection assays showed that its 3.6 kb 5'-upstream sequence can drive a chloramphenicol acetyl transferase (CAT) reporter gene to express in cultured corneal and esophageal epithelial cells, but not in mesothelial and kidney epithelial cells or fibroblasts, all of rabbit origin. Serial deletion experiments narrowed this keratinocyte-specific promoter to within -300 bp upstream of the transcription initiation site. Its activity is not regulated by the coding or 3'-noncoding sequences that have been tested so far. This 300 bp 5'-upstream sequence of K3 keratin gene, which can function in vitro as a keratinocyte-specific promoter, contains two clusters of partially overlapping motifs, one with an NFkB consensus sequence and another with a GC box. The combinatorial effects of these multiple motifs and their cognate binding proteins may play an important role in regulating the expression of this tissue-restricted and differentiation-dependent keratin gene
— id: 6557, year: 1993, vol: 105, page: 303, stat: Journal Article,

Molecular cloning of a 47 kDa tissue-specific and differentiation-dependent urothelial cell surface glycoprotein
Wu XR; Sun TT
1993 Sep;106(Pt 1):31-43, Journal of cell science
Despite the fact that bladder epithelium has many interesting biological features and is a frequent site of carcinoma formation, relatively little is known about its biochemical differentiation. We have shown recently that a 47 kDa glycoprotein, uroplakin III (UPIII), in conjunction with uroplakins I (27 kDa) and II (15 kDa), forms the asymmetric unit membrane (AUM)--a highly specialized biomembrane characteristic of the apical surface of bladder epithelium. Deglycosylation and cDNA sequencing revealed that UPIII contains up to 20 kDa of N-linked sugars attached to a core protein of 28.9 kDa. The presence of an N-terminal signal peptide sequence and a single transmembrane domain located near the C terminus, plus the N-terminal location of all the potential N-glycosylation sites, points to a type I (N-exo/C-cyto) configuration. Thus the mass of the extracellular domain (20 kDa plus up to 20 kDa of sugar) of UPIII greatly exceeds that of its intracellular domain (5 kDa). Such an asymmetrical mass distribution, a feature shared by the other two major uroplakins, provides a molecular explanation as to why the luminal leaflet of AUM is almost twice as thick as the cytoplasmic one. The fact that of the three major proteins of AUM only UPIII has a significant cytoplasmic domain suggests that this molecule may play an important role in AUM-cytoskeleton interaction in terminally differentiated urothelial cells
— id: 13082, year: 1993, vol: 106, page: 31, stat: Journal Article,

Upper human hair follicle contains a subpopulation of keratinocytes with superior in vitro proliferative potential
Yang JS; Lavker RM; Sun TT
1993 Nov;101(5):652-659, Journal of investigative dermatology
We and others have shown previously that corneal keratinocyte stem cells can proliferate in vitro better than their progeny cells. In this paper, we applied this approach to the identification of hair follicular stem cells. When human scalp hair follicles were placed in explant culture, the bulge area yielded best outgrowths. In another experiment, we isolated different subpopulations of human follicular keratinocytes by micro-dissection, dispersed them by trypsin/EDTA into single cells, and grew them in the presence of 3T3 feeder cells. The keratinocytes were then subcultured under identical conditions to compare their in vitro life span. Our results indicate that the life span of keratinocytes of the upper follicle (containing mainly the isthmus area) > sebaceous gland > lower follicle (between the bulge and bulb) > bulb (containing the matrix cells). The cultured upper follicular keratinocytes tend to be small and relatively uniform in size. The poor in vitro growth of matrix cells may reflect their non-stem cell nature and/or special growth requirement(s) satisfied in vivo by the neighboring dermal papilla cells. Unexpectedly, we found that the upper follicular keratinocytes grow even better than epidermal keratinocytes. The existence of a subpopulation of keratinocytes with an in vitro growth potential superior than other known keratinocytes of the skin supports the hypothesis that follicular stem cells reside in the upper follicle. Our data also raise the possibility that putative follicular stem cells are involved not only in forming the follicle, but also in the long-term maintenance of the epidermis. Finally, we discuss the possibility that keratinocyte stem cells, as defined by their in vivo slow-cycling nature, are absent in culture
— id: 6559, year: 1993, vol: 101, page: 652, stat: Journal Article,

Characterization of a keratinocyte-specific extracellular epitope of desmoglein. Implications for desmoglein heterogeneity and function
Loomis CA; Kolega J; Manabe M; Sun TT
1992 Aug 15;267(23):16676-16684, Journal of biological chemistry
Despite the presumed importance of desmoglein, a 160-kDa glycoprotein, in desmosome formation and its possible involvement in certain blistering skin diseases, the precise location and function of this protein have not yet been firmly established. We describe here the characterization of a new monoclonal antibody, AE23, against an extracellular epitope of desmoglein. Both the AE23 epitope and another epitope, defined by the previously characterized DG3.4 antibody, reside on a 160-kDa human epidermal desmoglein as evidenced by their identical solubility profile, their coexistence in a 130-kDa desmoglein degradative product, their coadsorption by an AE23 immunoaffinity column, and the identical changes in the two antigens' electrophoretic mobility after air oxidation and deglycosylation. The AE23 epitope is resistant to various endoglycosidases, suggesting that sugar moieties are not involved. Characterization of several proteolytic fragments of this epidermal desmoglein enabled us to map the DG3.4 epitope to a 96-kDa intracellular domain and the AE23 epitope to an extracellular domain flanked by the plasma membrane and the distal N-glycosylation site(s). However, these two epitopes do not always coexist on the same desmoglein molecule. For example, tissue surveys showed that although the DG3.4 epitope is present in the desmogleins of all epithelial cell types, the AE23 epitope is limited to normal keratinocytes. Moreover, electron microscopic localization data indicate that whereas the DG3.4 epitope is detected in the submembranous plaques of desmosomes, the AE23 epitope is present in the intercellular space of both desmosomal and nondesmosomal areas. These results raise the possibility that there exist several biochemically closely related isoforms of desmoglein, one (AE23+/DG3.4+) restricted to epidermal desmosomes, one (AE23+/DG3.4-) uniformly distributed along the keratinocyte cell surface, and another (AE23-/DG3.4+) present in desmosomes of simple epithelia and basal cells of cultured keratinocytes. The uniform distribution of at least one desmoglein-related antigen in the intercellular space of keratinocytes coupled with the realization that different isoforms of desmogleins form a subfamily of cadherins suggest that desmoglein(s) may play a more general role in keratinocyte adhesion than previously appreciated
— id: 13478, year: 1992, vol: 267, page: 16676, stat: Journal Article,

Interaction of trichohyalin with intermediate filaments: three immunologically defined stages of trichohyalin maturation
O'Guin WM; Sun TT; Manabe M
1992 Jan;98(1):24-32, Journal of investigative dermatology
'Trichohyalin' is a 220-kD protein found in trichohyalin granules that are present as major differentiation products in the medulla and inner root sheath cells of human hair follicles. It was unclear whether this protein served as an intermediate filament precursor in the inner root sheath or as an intermediate-filament-associated (matrix) protein. We have produced a panel of monoclonal antibodies (AE15-17) to this protein and used them to trace its fate during inner root sheath differentiation. These studies have allowed us to define three immunologically distinct forms of this trichohyalin protein. They are 1) the AE15-positive form, which is found throughout all trichohyalin granules; 2) the AE16-positive form, which is localized as discrete punctae on the surface of trichohyalin granules; and 3) the AE17-positive, intermediate-filament-bound form, which associates with the inner root sheath filaments with a regular, 400-nm periodicity. From these results we suggest that the 220-kD trichohyalin protein is an intermediate-filament-associated protein that may play a role in the lateral aggregation, precise alignment, and stabilization of inner root sheath filament bundles
— id: 13728, year: 1992, vol: 98, page: 24, stat: Journal Article,

Identification of an 85-100 kDa glycoprotein as a cell surface marker for an advanced stage of urothelial differentiation: association with the inter-plaque ('hinge') area
Yu J; Manabe M; Sun TT
1992 Jan;1(1):4-12, Epithelial cell biology
Although bladder cancers account for almost 5% of all human cancer deaths, little is known about the biochemistry of urothelial differentiation. We have recently identified three major protein subunits ('uroplakins') of asymmetric unit membranes (AUM), which form rigid-looking plaques covering up to 70% of the apical surface of urothelial superficial (umbrella) cells. The ordinary-looking plasma membranes that interconnect these plaques are believed to be functionally specialized, serving as flexible but durable 'hinges'. Whether these hinge membranes are biochemically unique is unknown. Using a new monoclonal antibody (AE32) we have identified an 85-100 kDa glycoprotein (UGP85) which appears to be urothelium-specific. In both normal urothelium and cultured urothelial colonies this cell surface protein is associated mainly with superficial cells, suggesting that its expression is differentiation dependent. Results from in vitro translation experiments indicated that this glycoprotein contains a core polypeptide of about 55 kDa. Using immunogold localization techniques, we showed that in cultured urothelial colonies--which are known to lack mature AUM plaques--UGP85 is distributed relatively uniformly on the apical surface of some differentiated cells. However, in superficial cells of normal urothelium UGP85 is mainly associated with the hinge areas. These results raise the possibility that UGP85 is a plasma membrane component which can be excluded, to varying extents, from the plaque region as 12 nm protein particles are assembled into a tightly packed paracrystalline AUM structure. The identification of UGP85 provides the first evidence that the hinge areas interconnecting the urothelial plaques are biochemically distinguishable from the plasma membranes of the relatively undifferentiated urothelial cells of the lower cell layers.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 13803, year: 1992, vol: 1, page: 4, stat: Journal Article,

Expression of K12 keratin in alkali-burned rabbit corneas
Zhu G; Ishizaki M; Haseba T; Wu RL; Sun TT; Kao WW
1992 Sep;11(9):875-887, Current eye research
The healing of alkali-injured corneas is characterized by the persistence of polymorphonuclear leukocytes (PMN) in tissues and recurrent corneal epithelial defects. It has been suggested that the proteolytic enzymes secreted by PMN may account in part for the recurrent epithelial defects in the alkali-burned corneas. Cytoplasmic keratins, which form intracellular intermediate filaments, participate in the formation of hemidesmosomes and play a key role in the focal adhesion of epithelial cells to the basement membranes. The K3/K12 keratin pair is a major constituent of differentiated and stratified corneal epithelium. We have recently cloned the cDNA encoding the rabbit K12 keratin. In the present study we examined the expression of K12 keratin during the healing of alkali-burned rabbit corneas by slot-blot and in situ hybridization. Our results indicate that in normal cornea K12 keratin is equally expressed in all cell layers of stratified corneal epithelium and suprabasal layers of limbal epithelium, but not in bulbar conjunctival and other epithelia, i.e., lens, iris, and retinal pigment epithelium. The basal cells of the detached regenerating epithelium of the injured cornea express a very low level of K12 keratin. These observations are consistent with the notion that defective expression of K3/K12 keratins may play a role in the abnormal attachment of the regenerating epithelium to the basement membrane
— id: 26920, year: 1992, vol: 11, page: 875, stat: Journal Article,

Bilateral tubal ectopic pregnancy after in vitro fertilization and embryo transfer
Chang JC; Lin YC; Sun TT
1991 Oct;8(5):292-296, Journal of in vitro fertilization & embryo transfer
— id: 26922, year: 1991, vol: 8, page: 292, stat: Journal Article,

Stem cells of pelage, vibrissae, and eyelash follicles: the hair cycle and tumor formation
Lavker RM; Cotsarelis G; Wei ZG; Sun TT
1991 Dec 26;642(4):214-224, Annals of the New York Academy of Sciences
— id: 16548, year: 1991, vol: 642, page: 214, stat: Journal Article,

Relative proliferative rates of limbal and corneal epithelia. Implications of corneal epithelial migration, circadian rhythm, and suprabasally located DNA-synthesizing keratinocytes
Lavker RM; Dong G; Cheng SZ; Kudoh K; Cotsarelis G; Sun TT
1991 May;32(6):1864-1875, Investigative ophthalmology & visual science. IOVS
An important element of the recently proposed limbal stem cell model is that corneal epithelial cells migrate centripetally. The driving force for this migration is unknown, although it has been suggested that limbal epithelium, proliferates at a higher rate than central corneal epithelium, thus creating a population pressure toward the central cornea. This hypothesis was tested by measuring the relative proliferative rates of limbal and central corneal epithelia using 3H-thymidine autoradiographic techniques. The results indicate that, in both the New Zealand white rabbit and SENCAR mouse, the labeling index (LI) of limbal epithelium is actually lower than that of central corneal epithelium. This difference in LI persists throughout the circadian rhythm cycle. These results suggest that population pressure per se cannot be responsible for the centripetal migration of corneal epithelium and raise the possibility that preferential desquamation of central corneal epithelium may 'draw' peripheral cells toward the central cornea. In both epithelia, the LI peak precedes the mitotic index (MI) peak during circadian cycle by 4-6 hr. These data therefore are in close agreement with earlier results on several nonocular stratified epithelia but contradict an earlier suggestion that the LI and MI peaks of corneal epithelium coincide. Finally, although most of the 3H-thymidine incorporating cells in central cornea may appear to be suprabasally located, they are only partially displaced into the suprabasal compartment. In most cases, such cells are still connected with the basement membrane through a thin stalk of cytoplasm. Since corneal epithelium rests on an exceptionally flat and rigid substratum, an increase in cellular volume in DNA-synthesizing cells may not be tolerated well in an already crowded basal layer. This may explain why an unusually large proportion of DNA-synthesizing cells are expelled preferentially into either a 'second tier basal layer' or into the suprabasal compartment
— id: 16555, year: 1991, vol: 32, page: 1864, stat: Journal Article,

Interaction of filaggrin with keratin filaments during advanced stages of normal human epidermal differentiation and in ichthyosis vulgaris
Manabe M; Sanchez M; Sun TT; Dale BA
1991 Sep;48(1):43-50, Differentiation
Filaggrin is a histidine-rich, basic protein whose name was first proposed based on its ability to aggregate intermediate filaments in vitro. Based on this in vitro observation, it has generally been assumed that filaggrin functions in vivo as a matrix protein which causes keratin filaments to become densely packed in the terminally differentiated cornified cells. Inconsistent with this view however, is the well-known observation that keratin aggregation appears to proceed normally in the affected epidermis of ichthyosis vulgaris patients despite a greatly reduced quantity of filaggrin. To address this issue, we used immuno-electron microscopy to localize filaggrin and its cross-reactive precursor, profilaggrin, in human and mouse epidermis, as well as in ichthyosis vulgaris epidermis. We found that the localization of filaggrin in lower cornified cells correlates precisely with the formation of aggregated keratin filaments, and the disappearance of filaggrin in upper cornified cells correlates precisely with the loosening of keratin filaments. Furthermore, we showed that, even in ichthyosis vulgaris, small amounts of filaggrin/profilaggrin are present as electron-dense deposits associated with keratin filaments in the granular cells, and that the localization of this small amount of antigen again correlates with the aggregation state of keratin filaments. These data strongly suggest that filaggrin is indeed involved in filament aggregation in vivo
— id: 13928, year: 1991, vol: 48, page: 43, stat: Journal Article,

Hair follicular stem cells: the bulge-activation hypothesis
Sun TT; Cotsarelis G; Lavker RM
1991 May;96(5):77S-78S, Journal of investigative dermatology
— id: 14030, year: 1991, vol: 96, page: 77S, stat: Journal Article,

Interaction between dermal papilla and bulge: the rhino mouse mutation as a model system
Sundberg JP; Roop DR; Dunstan R; Lavker R; Sun TT
1991 Dec 26;642(1):496-499, Annals of the New York Academy of Sciences
— id: 26921, year: 1991, vol: 642, page: 496, stat: Journal Article,

Regional heterogeneity in human corneal and limbal epithelia: an immunohistochemical evaluation
Wiley L; SundarRaj N; Sun TT; Thoft RA
1991 Mar;32(3):594-602, Investigative ophthalmology & visual science. IOVS
The authors studied the distribution of specific keratins within the superior, inferior, medial, and lateral regions of human limbus and cornea to determine whether the limbal epithelium exhibits regional heterogeneity in its microstructure. A corneal epithelial basic keratin (K3), recognized by monoclonal antibody AE5, was immunohistochemically undetectable in the basal layers of the limbus in these four regions, but was seen in all layers in the central cornea. The pattern of immunostaining with another monoclonal antibody, AE1, which recognizes several acidic keratins, was complementary to AE5 staining in that AE1 recognized a similar heterogeneity in the limbal epithelial cells. AE1 immunoreacted with the basal cells of the limbus, but not those of the central corneal epithelium. Limbal characteristics, as defined by AE1-positive and AE5-negative staining, extended deeply into peripheral cornea in the superior and inferior regions, but to a lesser extent in the lateral and medial regions. The broader regions of epithelium with limbal characteristics in the superior and inferior regions raises the possibility that these regions play an important role in corneal epithelial maintenance and wound healing
— id: 26923, year: 1991, vol: 32, page: 594, stat: Journal Article,

Transient expression of mouse hair keratins in transfected HeLa cells: interactions between "hard" and "soft" keratins
Yu DW; Pang SY; Checkla DM; Freedberg IM; Sun TT; Bertolino AP
1991 Aug;97(2):354-363, Journal of investigative dermatology
Although it has been shown previously that an acidic (type I) 'soft' keratin can interact with many basic (type II) 'soft' keratins to form 10-nm intermediate filaments, it has been unclear whether 'soft' keratins are compatible with the 'hard' keratins typically found in hair and nail. To address this issue and to generate more structural information about hard keratins, we have isolated and sequenced a cDNA clone that encodes a mouse hair basic keratin (b4). Our sequence data revealed new information regarding the structural conservation of hard keratins as a group, being significantly different from soft keratins. Using expression vectors containing appropriate cDNA inserts, we studied the expression of this basic (b4) as well as an acidic (a1) mouse hair keratin in HeLa cells. The expression of these alien hair keratins in the transfected cells was surveyed using a panel of monoclonal and polyclonal antibodies. Our results indicated that the basic and acidic hair keratin readily incorporated into the existing endogenous soft keratin network of HeLa cells. Overproduction of hair keratin, however, occasionally led to the formation of cytoplasmic aggregates containing both hard and soft keratins. These data suggest that although small amounts of newly synthesized hair keratins can incorporate into the 'scaffolding' of the preformed soft keratin filament network, possibly through dynamic subunit exchange, overproduction of hard keratins can lead to the partial collapse of the soft keratin network. These observations, along with the deduced amino acid sequence data, support and extend the concept that hard and soft keratins, although closely related, are divergent enough to justify their being divided into two separate subgroups
— id: 13959, year: 1991, vol: 97, page: 354, stat: Journal Article,

Appearance of the keratin pair K3/K12 during embryonic and adult corneal epithelial differentiation in the chick and in the rabbit
Chaloin-Dufau C; Sun TT; Dhouailly D
1990 Dec 1;32(2):97-108, Cell differentiation & development
Sequential expression of the K3/K12 keratin pair was studied during epithelial corneal differentiation, using monoclonal antibodies coupled with electrophoretic analysis. In the chick embryo, K12 appears on day 12, while K3 is present at least from day 11. By contrast, in the rabbit embryo, the expression of K12 (at 17 days) precedes that of K3 (at 21 days). In the adult rabbit, K3 is expressed without K12 in part of the limbus. Thus, within the corneal-type keratin pair, either the acidic or the basic partner appears first, according to the developmental stage or the species
— id: 26924, year: 1990, vol: 32, page: 97, stat: Journal Article,

Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis
Cotsarelis G; Sun TT; Lavker RM
1990 Jun 29;61(7):1329-1337, Cell
Inconsistent with the view that hair follicle stem cells reside in the matrix area of the hair bulb, we found that label-retaining cells exist exclusively in the bulge area of the mouse hair follicle. The bulge consists of a subpopulation of outer root sheath cells located in the midportion of the follicle at the arrector pili muscle attachment site. Keratinocytes in the bulge area are relatively undifferentiated ultrastructurally. They are normally slow cycling, but can be stimulated to proliferate transiently by TPA. Located in a well-protected and nourished environment, these cells mark the lower end of the 'permanent' portion of the follicle. Our findings, plus a reevaluation of the literature, suggest that follicular stem cells reside in the bulge region, instead of the lower bulb. This new view provides insights into hair cycle control and the possible involvement of hair follicle stem cells in skin carcinogenesis
— id: 16559, year: 1990, vol: 61, page: 1329, stat: Journal Article,

Assessing the differentiation state of cultured bovine urothelial cells: elevated synthesis of stratification-related K5 and K6 keratins and persistent expression of uroplakin I
Surya B; Yu J; Manabe M; Sun TT
1990 Nov;97(Pt 3):419-432, Journal of cell science
Although significant progress has recently been made in culturing mammalian urothelial cells, relatively little is known about their biochemical differentiation. In this paper, we assessed the differentiation state of cultured bovine urothelial cells by analyzing their keratins and a cell surface marker, uroplakin I. Urothelial cells were serially cultured either in a serum-free medium, or in a serum-containing medium in the presence of 3T3 feeder cells, with similar results. Despite their stratified appearance, both normal urothelium and cultured urothelial cells synthesize mainly K8, K18 and K19, keratins that are typically seen in simple epithelia. However, cultured urothelial cells synthesize a greatly increased amount of K5 and K6 keratins, which are usually expressed by stratified epithelia but present only in trace amounts in normal urothelium. These data indicate that, as far as keratin synthesis is concerned, cultured urothelial cells undergo an altered pattern of differentiation towards a more 'stratified phenotype'; this unusual finding has interesting implications for urothelial evolution. In the meantime, many superficial cells in cultured urothelial colonies make uroplakin I, a 27 x 10(3) Mr protein subunit of the asymmetrical unit membrane (AUM) characteristic of urothelial (superficial) umbrella cells. These results indicate that cultured urothelial cells undergo, at least in part, AUM biogenesis. Cultured urothelial cells thus provide a useful experimental model system for studying certain early steps of AUM formation
— id: 14310, year: 1990, vol: 97, page: 419, stat: Journal Article,

Comparison of limbal and conjunctival autograft transplantation in corneal surface reconstruction in rabbits
Tsai RJ; Sun TT; Tseng SC
1990 Apr;97(4):446-455, Ophthalmology
Destruction of corneal surface was created in one eye of 24 rabbits by n-heptanol corneal epithelial debridement and surgical removal of limbal zone. One month later, the animals were equally subdivided into three groups of eight for limbal transplantation, conjunctival transplantation, and control without transplantation. During a 6-month postoperative follow-up, all corneas in the control group showed progressive vascularization and conjunctivalization. All corneas with limbal transplantation showed progressive decrease of vascularity, verified by fluorescein angiography. In contrast, all but one of the eight corneas of conjunctival transplantation showed progressive vascularization (P = 0.01). More important, the resultant epithelia showed corneal phenotype in limbal transplantation, but remained conjunctival in conjunctival transplantation, as verified by monoclonal antibodies AM-3, APSM-1, and AE-5. These results support the concept of the limbal location of corneal epithelial stem cells, and indicate that complete destruction of the limbal zone resulted in corneal vascularization and conjunctivalization, and that limbal transplantation has a better efficiency than conjunctival transplantation in restoring such destroyed corneal surface
— id: 26926, year: 1990, vol: 97, page: 446, stat: Journal Article,

MICROCYSTIC ADNEXAL CARCINOMA - AN IMMUNOHISTOCHEMICAL COMPARISON WITH OTHER CUTANEOUS APPENDAGE TUMORS
Wick, MR; Cooper, PH; Swanson, PE; Kaye, VN; Sun, TT
1990 Feb;126(2):189-194, Archives of dermatology
— id: 32112, year: 1990, vol: 126, page: 189, stat: Journal Article,

Large scale purification and immunolocalization of bovine uroplakins I, II, and III. Molecular markers of urothelial differentiation
Wu XR; Manabe M; Yu J; Sun TT
1990 Nov 5;265(31):19170-19179, Journal of biological chemistry
The differentiation of mammalian urothelium culminates in the formation of asymmetrical unit membrane (AUM). Using gradient centrifugation and detergent wash, we purified milligram quantities of AUMs which, interestingly, contained three major proteins (15, 27, and 47 kDa) that appeared to be identical to the three immunoaffinity purified, putatively AUM-associated proteins that we described earlier (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell Biol., 111, 1207-1216). Peptide mapping and immunoblotting established that these three proteins were distinct molecules. Using monospecific antibodies to these three proteins, we showed that they were all restricted to the superficial urothelial cells and were AUM-associated. The 27- and 15-kDa proteins were detected exclusively on the luminal side of mature, apical AUMs. In contrast, epitopes of the 47-kDa protein were detected on both sides of apical AUMs suggesting a transmembranous configuration. These results (i) provide the strongest evidence thus far that AUM contains three major proteins (the 27-kDa uroplakin I, 15-kDa uroplakin II, and 47-kDa uroplakin III) which form an extremely insoluble complex, (ii) suggest that uroplakin II, like uroplakin I (Yu, J., Manabe, M., Wu, X.-R., Xu, C., Surya, B., and Sun, T.-T. (1990) J. Cell. Biol. 111, 1207-1216), translocates from one side of the membrane to another during AUM maturation, (iii) indicate that uroplakin III may play a different structural role than uroplakins I and II in AUM formation, and (iv) establish the three uroplakins as markers for an advanced stage of urothelial differentiation
— id: 14277, year: 1990, vol: 265, page: 19170, stat: Journal Article,

Uroplakin I: a 27-kD protein associated with the asymmetric unit membrane of mammalian urothelium
Yu J; Manabe M; Wu XR; Xu C; Surya B; Sun TT
1990 Sep;111(3):1207-1216, Journal of cell biology
The luminal surface of mammalian urothelium is covered with numerous plaques (also known as the asymmetric unit membrane or AUM) composed of semi-crystalline, hexagonal arrays of 12-nm protein particles. Despite the presumed importance of these plaques in stabilizing the urothelial surface during bladder distention, relatively little is known about their protein composition. Using a mouse mAb, AE31, we have identified a 27-kD protein that is urothelium-specific and is differentially expressed in superficial umbrella cells. This protein (pI approximately 5.8) partitions into the detergent phase during Triton X-114 phase separation. Pulse-chase experiments using cultured bovine urothelial cells showed that this protein is synthesized as a 32-kD precursor that is processed through a 30-kD intermediate, to the mature 27-kD form. In cytoplasmic vesicles containing immature AUM, the AE31 epitope is detected in patches on the cytoplasmic side, but in mature, apical AUM it is detected exclusively on the luminal side. This suggests an unusual translocation of the AE31 epitope during AUM maturation; more data are required, however, to substantiate this interpretation. Immunoaffinity purification of the 27-kD protein results in the copurification in approximately molar ratio of a 15-kD protein, as well as a small and variable amount of a 47-kD protein. Immunoblotting data indicate that these three proteins are immunologically distinguishable. This copurified 15-kD protein is relative basic (pI approximately 8.0). Like the 27-kD protein, it is urothelium-specific and is present mainly in the umbrella cells. Together, our data indicate that a 27-kD protein is urothelial plaque-associated (uroplakin I). Based on complex formation data, we provisionally name the 15-kD protein uroplakin II; additional data will be required to determine whether this and the 47-kD protein are integral parts of AUM. The identification of these AUM-associated and -related proteins, plus the availability of a culture system capable of synthesizing and processing some of these molecules, offer new opportunities for studying the detailed structure, assembly, and function of asymmetrical unit membrane
— id: 26925, year: 1990, vol: 111, page: 1207, stat: Journal Article,

Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells
Cotsarelis G; Cheng SZ; Dong G; Sun TT; Lavker RM
1989 Apr 21;57(2):201-209, Cell
Despite the obvious importance of epithelial stem cells in tissue homeostasis and tumorigenesis, little is known about their specific location or biological characteristics. Using 3H-thymidine labeling, we have identified a subpopulation of corneal epithelial basal cells, located in the peripheral cornea in a region called limbus, that are normally slow cycling, but can be stimulated to proliferate in response to wounding and to a tumor promotor, TPA. No such cells can be detected in the central corneal epithelium, suggesting that corneal epithelial stem cells are located in the limbus. A comparison of various types of epithelial stem cells revealed a common set of features, including their preferred location, pigment protection, and growth properties, which presumably play a crucial role in epithelial stem cell function
— id: 16562, year: 1989, vol: 57, page: 201, stat: Journal Article,

Expression of hair-related keratins in a soft epithelium: subpopulations of human and mouse dorsal tongue keratinocytes express keratin markers for hair-, skin- and esophageal-types of differentiation
Dhouailly D; Xu C; Manabe M; Schermer A; Sun TT
1989 Mar;181(1):141-158, Experimental cell research
The dorsal surfaces of mammalian tongues are covered with numerous projections known as filiform papillae whose morphology varies in different species. Using a panel of monoclonal antibodies to keratins as probes, we have established that, in both human and mouse, the interpapillary epithelia express mainly the 'esophageal-type' keratins, while the papillary epithelia express 'skin-type' keratins as well as some keratins reacting with a monoclonal antibody (AE13) to hair keratins. The AE13-reactive proteins of the mouse were found to be very similar to those of authentic mouse hair keratins. However, the corresponding protein of human tongue appears to be different from all known human keratins. This protein has a MW of 51K; it is relatively acidic; it is sulfhydryl-rich, as revealed by iodoacetic acid-induced charge and apparent size shift; it shares an epitope with all the known acidic human hair keratins; and it is associated with keratin fibrils in vivo. This protein may therefore be regarded as a novel type I 'hard' keratin. These data establish that mammalian dorsal tongue epithelia can be divided into at least three compartments that undergo mainly 'esophageal-', 'skin-' and 'hair'-types of differentiation. Different keratin filaments, e.g., those of the esophageal- and hair-types, exhibit strikingly different degrees of lateral aggregation, which can potentially account for the different physical strength and rigidity of various cellular compartments. Our data also suggest the possibility that variations in papillary structure in human and mouse may arise from different spatial arrangements of specific keratinocytes, and/or from the expression of specialized hair-related keratins
— id: 10719, year: 1989, vol: 181, page: 141, stat: Journal Article,

The major pathways of keratinocyte differentiation as defined by keratin expression: an overview
Galvin S; Loomis C; Manabe M; Dhouailly D; Sun TT
1989 ;4:277-299, Advances in dermatology
— id: 10852, year: 1989, vol: 4, page: 277, stat: Journal Article,

Basement membrane heterogeneity and variation in corneal epithelial differentiation
Kolega J; Manabe M; Sun TT
1989 Oct;42(1):54-63, Differentiation
We have previously shown that the expression of a major 64-Kda keratin (K3) in corneal epithelium is site-related. It is found suprabasally in limbal epithelium, but uniformly (basal cells included) in central corneal epithelium. In the present study, we used a panel of antibodies against various components of corneal epithelial basement membrane to investigate a possible correlation between basement membrane heterogeneity and differential (basal vs. suprabasal) K3 keratin expression. One of these antibodies, AE27, stains human conjunctival basement membrane weakly, limbal basement membrane heterogeneously, and central corneal basement membrane strongly. Basal cells resting on basement membrane that stains strongly with AE27 tend to stain with monoclonal antibody AE5, which recognizes keratin K3. Basal cells on basement membrane staining weakly with AE27 tend not to stain with AE5. No such correlation exists between AE5 staining and type IV collagen, which is detectable immunohistochemically in conjunctival and limbal basement membrane, but not in corneal basement membrane overlying Bowman's layer. These results suggest that basement membrane of human corneal/conjunctival epithelium can be divided into at least three domains: the conjunctival basement membrane (type IV collagen-positive, AE27-weak), the limbal basement membrane (type IV collagen-positive, AE27-strong), and corneal basement membrane (type IV collagen-negative, AE27-strong). The results also raise the possibility that basement membrane heterogeneity may play a functional role in regulating keratin expression and other aspects of differentiation of corneal epithelium; more experiments are needed to test this hypothesis
— id: 10470, year: 1989, vol: 42, page: 54, stat: Journal Article,

Expression of keratin 5 as a distinctive feature of epithelial and biphasic mesotheliomas. An immunohistochemical study using monoclonal antibody AE14
Moll R; Dhouailly D; Sun TT
1989 ;58(2):129-145, Virchows archive
In previous biochemical analyses, keratin 5 (Mr 58,000) has been detected in most mesotheliomas with epithelial component but not in pulmonary adenocarcinomas (Blobel et al., Am J Pathol 121: 235-247, 1985). In the present study, we have characterized a monoclonal antibody, AE14, as being selectively specific for keratin 5 (apart from the reactivity with certain hair proteins) as shown by immunoblotting of gel-electrophoretically separated proteins from various tissues. Immunohistochemical screening of a variety of normal human tissues, using immunoperoxidase microscopy on cryostat sections, revealed the binding of this antibody to the basal, immature cells of stratified squamous epithelia, to basal cells of pseudostratified epithelia, to some myoepithelial cells, thymic reticulum cells, certain pancreatic duct cells, as well as a variable subpopulation of mesothelial cells of the pleura and the peritoneum. In 12/13 epithelial and biphasic mesotheliomas of the pleura, heterogeneous but extended staining with antibody AE14 was seen whereas 21 pulmonary adenocarcinomas were negative or, in six of these cases, showed staining of only a few cells. Among carcinomas from other sites, colonic adenocarcinomas and renal cell carcinomas were negative whereas limited staining was found in some pancreatic adenocarcinomas. It is suggested that antibody AE14 may be useful, as a defined polypeptide-specific reagent, in the histologic distinction between mesotheliomas and most adenocarcinomas. Furthermore, the expression patterns of keratin 5 as detected by antibody AE14 in various normal and malignant epithelial tissues are discussed, particularly their relation to processes of squamous metaplasia and their indication of phenotypic tumor heterogeneity
— id: 26927, year: 1989, vol: 58, page: 129, stat: Journal Article,

EXPRESSION OF KERATIN-5 AS A DISTINCTIVE FEATURE OF EPITHELIAL AND BIPHASIC MESOTHELIOMAS - AN IMMUNOHISTOCHEMICAL STUDY USING MONOCLONAL-ANTIBODY AE14
Moll, R; Dhouailly, D; Sun, TT
1989 Dec;58(2):129-145, Virchows archiv B. Cell pathology including molecular pathology
— id: 31764, year: 1989, vol: 58, page: 129, stat: Journal Article,

Association of a basic 25K protein with membrane coating granules of human epidermis
O'Guin WM; Manabe M; Sun TT
1989 Nov;109(5):2313-2321, Journal of cell biology
Keratinocytes of the upper granular layers contain unique round-to-oval granules, 100-500 nm in diameter, in their peripheral cytoplasm. These granules (known as membrane coating granules [MCG], or lamellar granules) fuse with the apical cell surface of uppermost granular cells and discharge their contents into the intercellular space, where they are believed to play a role in establishing the permeability barrier of the epidermis and possibly in regulating the orderly desquamation of terminally differentiated keratinocytes. Using two monoclonal antibodies originally prepared against hair follicle antigens, we have identified a 25K epidermal protein in association with both MCG-like granules in the peripheral cytoplasm of granular cells as well as MCG-derived intercellular material. This protein is relatively basic (pI greater than 8), largely aqueous soluble, methionine deficient, and is relatively abundant in epidermis (comprising up to approximately 0.1% of soluble proteins). Its distribution is restricted to the granular layer of keratinized (cornified) stratified squamous epithelia. The identification of this protein component opens new avenues for studying the molecular mechanisms underlying the establishment of permeability barrier and/or regulation of desquamation
— id: 10436, year: 1989, vol: 109, page: 2313, stat: Journal Article,

Transient synthesis of K6 and K16 keratins in regenerating rabbit corneal epithelium: keratin markers for an alternative pathway of keratinocyte differentiation
Schermer A; Jester JV; Hardy C; Milano D; Sun TT
1989 Dec;42(2):103-110, Differentiation
Cultured rabbit corneal epithelial cells undergo three distinct stages of growth and differentiation characterized by the sequential appearance of K5/K14 keratin markers for basal keratinocytes, K6/K16 keratin markers for 'hyperproliferative' keratinocytes, and K3/K12 keratin markers for corneal-type differentiation. Analyses of [35S]methionine-labeled, newly synthesized keratins revealed that K6/K16 are synthesized only briefly when the cells undergo exponential growth, and their synthesis is suppressed when the cells reach confluence and switch to synthesizing K3/K12. Transient synthesis of K6/K16 was also observed in vivo during corneal epithelial regeneration. Although K6/K16 expression in general correlates well with cellular growth, drug-induced inhibition of corneal epithelial growth and related data on human epidermal keratinocytes indicate that these two events are dissociable. These results establish clearly for the first time a reciprocal relationship, on a protein level, between the synthesis of K6/K16 and a differentiation-related keratin pair, K3/K12. Such a relationship strongly suggests a competitive mechanism controlling the synthesis of these two major classes of keratins in the suprabasal compartment. Our results also indicate that although hyperproliferation is usually accompanied by K6/K16 expression, the reverse is not always true. Taken together, the data suggest that K6/K16 are synthesized, perhaps by default, as an alternative suprabasal keratin pair under conditions that are nonpermissive for keratinocytes to express their normal, differentiation-related keratin pairs
— id: 10422, year: 1989, vol: 42, page: 103, stat: Journal Article,

Patterns of keratin expression define distinct pathways of epithelial development and differentiation
O'Guin WM; Galvin S; Schermer A; Sun TT
1987 ;22:97-125, Current topics in developmental biology
— id: 11434, year: 1987, vol: 22, page: 97, stat: Journal Article,

Suprabasal expression of a 64-kilodalton keratin (no. 3) in developing human corneal epithelium
Rodrigues M; Ben-Zvi A; Krachmer J; Schermer A; Sun TT
1987 ;34(1):60-67, Differentiation
We have previously shown that a basic 64-kilodalton (no. 3 in the catalog of Moll et al.) and an acidic 55-kilodalton (no. 12) keratin are characteristic of suprabasal cell layers in cultured rabbit corneal epithelial colonies, and therefore may be regarded as markers for an advanced stage of corneal epithelial differentiation. Moreover, using an AE5 mouse monoclonal antibody, we showed that the 64-kilodalton keratin marker is expressed suprabasally in limbal epithelium but uniformly (basal layer included) in central corneal epithelium, suggesting that corneal basal cells are in a more differentiated state than limbal basal cells. In conjunction with previous data implicating the centripetal migration of corneal epithelial cells, our data support a model of corneal epithelial maturation in which corneal epithelial stem cells are located in the limbus, the transitional zone between the cornea and conjunctiva. In the present study, we analyzed the expression of the 64-kilodalton keratin in developing human corneal epithelium by immunohistochemical staining. At 8 weeks of gestation, the presumptive corneal epithelium is composed of a single layer of cuboidal cells with an overlying periderm; neither of these cell layers is AE5 positive. At 12-13 weeks of gestation, some superficial cells of the three- to four-layered epithelium become AE5 positive, providing the earliest sign of overt corneal epithelial differentiation. At 36 weeks, although the epithelium is morphologically mature (four to six layers), AE5 produces a suprabasal staining pattern, this being in contrast to the adult epithelium which exhibits uniform staining.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 26928, year: 1987, vol: 34, page: 60, stat: Journal Article,

Differentiation-dependent expression of keratins in human oral epithelia
Clausen H; Vedtofte P; Moe D; Dabelsteen E; Sun TT; Dale B
1986 Mar;86(3):249-254, Journal of investigative dermatology
The polypeptide composition of epithelial keratins varies with the state of differentiation. The epithelia lining the human oral cavity show regional variations in their histology. In the present study, paired samples of nonkeratinized buccal epithelium and keratinized hard palate epithelium were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoblots with monoclonal antibodies AE1, AE2, and AE3, and results were correlated with immunofluorescence staining of tissue sections of the same samples. Keratins from hard palate (Mr 67K, 63-65K, 58K, 56.5K, 56K, 50K, 48K) and epidermis (Mr 67K, 63-65K, 58K, 56.5K, 50K) were similar to each other but distinctly different from those of buccal epithelium (major bands of Mr 52K and 59K, minor bands of 50K and 58K). The immunoblot analysis further indicated the similarity of hard palate and epidermal keratins, in contrast to those of buccal epithelium. Each oral tissue expressed keratins of the type I (AE1, acidic) subfamily and type II (AE3, basic) subfamily. In tissue sections, the predominant staining pattern for nonkeratinized buccal epithelium was: AE1, positive in the basal layer; AE2, negative; AE3, positive in all layers. In contrast, the staining pattern for keratinized palatal epithelium was: AE1 and AE2, positive in the suprabasal layers; AE3, positive in all layers. Strong suprabasal AE1 staining in palate may be related to the presence of the 48K keratin. Some buccal samples showed an alternate staining pattern of spotty suprabasal staining with AE1 and AE2 which was correlated with the expression of the 56.5K and 63-67K keratins, as well as filaggrin. These results suggest differentiation-specific expression of the keratins and show immunologically detectable variation in the apparently normal differentiation pattern of nonkeratinized buccal epithelium
— id: 26933, year: 1986, vol: 86, page: 249, stat: Journal Article,

Monoclonal antibody analysis of bovine epithelial keratins. Specific pairs as defined by coexpression
Cooper D; Sun TT
1986 Apr 5;261(10):4646-4654, Journal of biological chemistry
We have characterized the keratin proteins of various bovine epithelial tissues by one- and two-dimensional gel electrophoresis, coupled with the immunoblot technique using AE1, AE2, AE3, AE5, CA20, BE14, and 6.11 monoclonal antikeratin antibodies. The results indicate that all known bovine keratins can be divided into two subfamilies. The 'acidic' (Type I) subfamily consists of 41-, 43-, 45-, 46-, 50-, 54-, 56-, and 56.5-kDa keratins, all of which have a pI of less than 5.6, and most of them are recognized by our AE1 antibody, whereas the 'neutral-to-basic' (Type II) subfamily consists of 55-, 57-, 58-, 62-65-, 66-, and 67-kDa keratins, all of which have a pI of greater than 6.0 and are recognized by our AE3 antibody. Tissue distribution data and cell culture studies show that, within the two subfamilies, keratins with similar 'size ranks' form a 'pair' as defined by frequent co-expression. Furthermore, within most 'keratin pairs,' the basic keratin is larger than the acidic one by 8-10 kDa. These results provide further support for the concepts of 'keratin subfamilies' and keratin pairs and are consistent with the possibility that the acidic and basic members of at least some keratin pairs may interact specifically during in vivo tonofilament assembly and/or function. Immunoblotting data derived from the use of several monospecific antibodies show that although the size, charge, and pattern of expression of most bovine keratins are similar to those of the human counterparts, there are important exceptions to this rule
— id: 26932, year: 1986, vol: 261, page: 4646, stat: Journal Article,

The role of keratin subfamilies and keratin pairs in the formation of human epidermal intermediate filaments
Eichner R; Sun TT; Aebi U
1986 May;102(5):1767-1777, Journal of cell biology
The four major keratins of normal human epidermis (molecular mass 50, 56.5, 58, and 65-67 kD) can be subdivided on the basis of charge into two subfamilies (acidic 50-kD and 56.5-kD keratins vs. relatively basic 58-kD and 65-67-kD keratins) or subdivided on the basis of co-expression into two 'pairs' (50-kD/58-kD keratin pair synthesized by basal cells vs. 56.5-kD/65-67-kD keratin pair expressed in suprabasal cells). Acidic and basic subfamilies were separated by ion exchange chromatography in 8.5 M urea and tested for their ability to reassemble into 10-nm filaments in vitro. The two keratins in either subfamily did not reassemble into 10-nm filaments unless combined with members of the other subfamily. While electron microscopy of acidic and basic keratins equilibrated in 4.5 M urea showed that keratins within each subfamily formed distinct oligomeric structures, possibly representing precursors in filament assembly, chemical cross-linking followed by gel analysis revealed dimers and larger oligomers only when subfamilies were combined. In addition, among the four major keratins, the acidic 50-kD and basic 58-kD keratins showed preferential association even in 8.5 M urea, enabling us to isolate a 50-kD/58-kD keratin complex by gel filtration. This isolated 50-kD/58-kD keratin pair readily formed 10-nm filaments in vitro. These results demonstrate that in tissues containing multiple keratins, two keratins are sufficient for filament assembly, but one keratin from each subfamily is required. More importantly, these data provide the first evidence for the structural significance of specific co-expressed acidic/basic keratin pairs in the formation of epithelial 10-nm filaments
— id: 26931, year: 1986, vol: 102, page: 1767, stat: Journal Article,

Acidic and basic hair/nail ("hard") keratins: their colocalization in upper cortical and cuticle cells of the human hair follicle and their relationship to "soft" keratins
Lynch MH; O'Guin WM; Hardy C; Mak L; Sun TT
1986 Dec;103(6 Pt 2):2593-2606, Journal of cell biology
Although numerous hair proteins have been studied biochemically and many have been sequenced, relatively little is known about their in situ distribution and differential expression in the hair follicle. To study this problem, we have prepared several mouse monoclonal antibodies that recognize different classes of human hair proteins. Our AE14 antibody recognizes a group of 10-25K hair proteins which most likely corresponds to the high sulfur proteins, our AE12 and AE13 antibodies define a doublet of 44K/46K proteins which are relatively acidic and correspond to the type I low sulfur keratins, and our previously described AE3 antibody recognizes a triplet of 56K/59K/60K proteins which are relatively basic and correspond to the type II low sulfur keratins. Using these and other immunological probes, we demonstrate the following. The acidic 44K/46K and basic 56-60K hair keratins appear coordinately in upper corticle and cuticle cells. The 10-25K, AE14-reactive antigens are expressed only later in more matured corticle cells that are in the upper elongation zone, but these antigens are absent from cuticle cells. The 10-nm filaments of the inner root sheath cells fail to react with any of our monoclonal antibodies and are therefore immunologically distinguishable from the cortex and cuticle filaments. Nail plate contains 10-20% soft keratins in addition to large amounts of hair keratins; these soft keratins have been identified as the 50K/58K and 48K/56K keratin pairs. Taken together, these results suggest that the precursor cells of hair cortex and nail plate share a major pathway of epithelial differentiation, and that the acidic 44K/46K and basic 56-60K hard keratins represent a co-expressed keratin pair which can serve as a marker for hair/nail-type epithelial differentiation
— id: 16674, year: 1986, vol: 103, page: 2593, stat: Journal Article,

Differentiation-dependent changes in the solubility of a 195-kD protein in human epidermal keratinocytes
Ma AS; Sun TT
1986 Jul;103(1):41-48, Journal of cell biology
We have prepared a monoclonal antibody, AE11, that recognizes specifically a 195-kD protein (pI 5.4) of human keratinocytes. This antigen constitutes approximately 0.01-0.1% of total protein in keratinocytes of skin, esophagus, and cornea, and is readily detectable in these cells by immunofluorescent staining and immunoblotting. However, it is barely detectable in MCF mammary carcinoma cells and HeLa cells, and is undetectable in nonepithelial cell types. Results from serial extraction experiments have shown that this protein exists in two distinct pools: a Tris-soluble, and a Tris-insoluble but urea- or SDS-soluble one. The distribution of the 195-kD protein between these two pools appears to be differentiation-related, since relatively undifferentiated cells selected by a low-calcium medium contain primarily the soluble form, while highly differentiated cells contain mainly the insoluble form. Data from immunofluorescent staining and trypsin-sensitivity experiments suggest that the soluble form is cytoplasmic, whereas the insoluble form is submembranously located at the cell periphery of upper, differentiated cells. The insoluble, cell peripheral form of the 195-kD antigen increases progressively during epidermal differentiation; its insolubility appears to be related to the formation of disulfide-bond(s). These results indicate that the 195-kD protein, which has recently been suggested to be involved in cornified envelope formation (Simon, M., and H. Green, 1985, Cell, 36:827-834), undergoes significant changes in its solubility characteristics and intracellular location during keratinocyte maturation
— id: 26929, year: 1986, vol: 103, page: 41, stat: Journal Article,

Clinical, electron microscopic, and monoclonal antibody studies of intraocular epithelial downgrowth
Rodrigues MM; Krachmer JH; Sun TT
1986 ;84(4):146-169, Transactions of the American Ophthalmological Society
Epithelial downgrowth developed in three patients following cataract extraction and keratoplasty. Light and electron microscopy of the downgrowth tissue disclosed stratified squamous epithelium, but could not determine whether they were derived from conjunctival or corneal epithelium. The epithelial downgrowth contained cells that were connected laterally by desmosomal junctions and displayed well-defined basement membranes. Surface epithelial cells exhibited myriad microvillous processes scant to moderate mitochondria. In one case, prominent hemidesmosomal junctions were present. Immunohistochemical staining with monoclonal antikeratin antibodies revealed immunoreactivity with AE1, AE3, and AE11 in all cases, but with a cornea-specific antibody, AE5, in only one case. We concluded that in this last case, the epithelial downgrowth appears to have originated from the corneal epithelium
— id: 26934, year: 1986, vol: 84, page: 146, stat: Journal Article,

Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells
Schermer A; Galvin S; Sun TT
1986 Jul;103(1):49-62, Journal of cell biology
In this paper we present keratin expression data that lend strong support to a model of corneal epithelial maturation in which the stem cells are located in the limbus, the transitional zone between cornea and conjunctiva. Using a new monoclonal antibody, AE5, which is highly specific for a 64,000-mol-wt corneal keratin, designated RK3, we demonstrate that this keratin is localized in all cell layers of rabbit corneal epithelium, but only in the suprabasal layers of the limbal epithelium. Analysis of cultured corneal keratinocytes showed that they express sequentially three major keratin pairs. Early cultures consisting of a monolayer of 'basal' cells express mainly the 50/58K keratins, exponentially growing cells synthesize additional 48/56K keratins, and postconfluent, heavily stratified cultures begin to express the 55/64K corneal keratins. Cell separation experiments showed that basal cells isolated from postconfluent cultures contain predominantly the 50/58K pair, whereas suprabasal cells contain additional 55/64K and 48/56K pairs. Basal cells of the older, postconfluent cultures, however, can become AE5 positive, indicating that suprabasal location is not a prerequisite for the expression of the 64K keratin. Taken together, these results suggest that the acidic 55K and basic 64K keratins represent markers for an advanced stage of corneal epithelial differentiation. The fact that epithelial basal cells of central cornea but not those of the limbus possess the 64K keratin therefore indicates that corneal basal cells are in a more differentiated state than limbal basal cells. These findings, coupled with the known centripetal migration of corneal epithelial cells, strongly suggest that corneal epithelial stem cells are located in the limbus, and that corneal basal cells correspond to 'transient amplifying cells' in the scheme of 'stem cells----transient amplifying cells----terminally differentiated cells.'
— id: 26930, year: 1986, vol: 103, page: 49, stat: Journal Article,

Microcirculation in chronic viral hepatitis: clinical observation and treatment with traditional Chinese medicine
Zhang, Q B; Wu, X H; Sun, T; Jin, H M; Zhai, W R; Xu, Z Y; Dai, Z Y
1986 Aug;99(8):660-664, Chinese medical journal
— id: 145780, year: 1986, vol: 99, page: 660, stat: Journal Article,

Classification of human epithelia and their neoplasms using monoclonal antibodies to keratins: strategies, applications, and limitations
Cooper D; Schermer A; Sun TT
1985 Mar;52(3):243-256, Laboratory investigation
— id: 26937, year: 1985, vol: 52, page: 243, stat: Journal Article,

Expression of epidermal keratins and filaggrin during human fetal skin development
Dale BA; Holbrook KA; Kimball JR; Hoff M; Sun TT
1985 Oct;101(4):1257-1269, Journal of cell biology
The major structural proteins of epithelia, the keratins, and the keratin filament-associated protein, filaggrin, were analyzed in more than 50 samples of human embryonic and fetal skin by one-dimensional SDS PAGE and immunoblotting with monoclonal and polyclonal antibodies. Companion samples were examined by immunohistochemistry and electron microscopy. Based on structural characteristics of the epidermis, four periods of human epidermal development were identified. The first is the embryonic period (before 9 wk estimated gestational age), and the others are within the fetal period: stratification (9-14 wk), follicular keratinization (14-24 wk), and interfollicular keratinization (beginning at approximately 24 wk). Keratin proteins of both the acidic (AE1-reactive, type I) and the basic (AE3-reactive, type II) subfamilies were present throughout development. Keratin intermediate filaments were recognized in the tissue by electron microscopy and immunohistochemical staining. Keratins of 50 and 58 kD were present in the epidermis at all ages studied (8 wk to birth), and those of 56.5 and 67 kD were expressed at the time of stratification and increased in abundance as development proceeded. 40- and 52-kD keratins were present early in development but disappeared with keratinization. Immunohistochemical staining suggested the presence of keratins of 50 and 58 kD in basal cells, 56.5 and 67 kD in intermediate cells, and 40 and 52 kD in the periderm as well as in the basal cells between the time of stratification and birth. Filaggrin was first detected biochemically at approximately 15 wk and was localized immunohistochemically in the keratinizing cells that surround hair follicles. It was identified 8-10 wk later in the granular and cornified cell layers of keratinized interfollicular epidermis. These results demonstrate the following. An intimate relationship exists between expression of structural proteins and morphologic changes during development of the epidermis. The order of expression of individual keratins is consistent with the known expression of keratins in simple vs. stratified vs. keratinized epithelia. Expression of keratins typical of stratified epithelia (50 and 58 kD) precedes stratification, and expression of keratins typical of keratinization (56.5 and 67 kD) precedes keratinization, which suggests that their expression marks the tissue commitment to those processes. Because only keratins that have been demonstrated in various adult tissues are expressed during fetal development, we conclude that there are no 'fetal' keratins per se.(ABSTRACT TRUNCATED AT 400 WORDS)
— id: 26935, year: 1985, vol: 101, page: 1257, stat: Journal Article,

Change in epithelial keratin expression during healing of rabbit corneal wounds
Jester JV; Rodrigues MM; Sun TT
1985 Jun;26(6):828-837, Investigative ophthalmology & visual science. IOVS
Corneal epithelial wound healing following full-thickness trephination and transcorneal freeze injury was studied by electron microscopy and immunofluorescent microscopy using monoclonal antibodies AE1, AE2, and AE3 to human epithelial keratin. Wounds were evaluated at various time intervals between 4 hr and 2 mo after injury. By scanning and transmission electron microscopy, epithelial migration was evident 4 hr after injury and was characterized by thinning of the epithelium and extension of filopodial processes. AE1 monoclonal antibody, which stains specifically the superficial cells of normal corneal epithelium, reacted to cells at the leading edge of the migrating epithelium. By 24 hr, all cells migrating over the wound displayed positive fluorescence with AE1 while the epithelium over the undamaged cornea exhibited normal fluorescence limited to the superficial epithelial cells. In full-thickness corneal wounds, reepithelialization was complete by 1-2 wk; however, all epithelial cells covering the wound remained positive for the AE1 antikeratin antibody. By 2 mo, the AE1 fluorescence returned to normal. In transcorneal freeze injuries, reepithelialization was complete by 4 to 7 days after injury, with all cells overlying the wound reacting with the AE1 antibody. By 2 wk after freeze injury, all epithelial cells appeared to express a normal AE1 staining pattern. No change was noted in the fluorescent distribution of either AE2 antibody, which did not react with the corneal epithelium, or AE3, which reacts with all corneal epithelial cells. These results suggest that healing of corneal epithelial wounds involves changes in keratin expression of the corneal epithelium
— id: 26936, year: 1985, vol: 26, page: 828, stat: Journal Article,

Monoclonal antibody studies of mammalian epithelial keratins: a review
Sun TT; Tseng SC; Huang AJ; Cooper D; Schermer A; Lynch MH; Weiss R; Eichner R
1985 ;455(4):307-329, Annals of the New York Academy of Sciences
— id: 26938, year: 1985, vol: 455, page: 307, stat: Journal Article,

Antikeratin antibodies in tumor diagnosis. Distinction between seminoma and embryonal carcinoma
Battifora H; Sheibani K; Tubbs RR; Kopinski MI; Sun TT
1984 Sep 1;54(5):843-848, Cancer
The authors investigated the presence and distribution of keratin in germ cell tumors using a rabbit-anti-keratin antiserum and a monoclonal antikeratin antibody--which is specific for keratin classes of 40, 50, and 56.5 kdaltons--by various immunohistochemical methods on frozen sections, alcohol-fixed, and formalin-fixed paraffin-embedded tissues. Thirty-four germ cell tumors were studied. These were the following: 18 seminomas, 10 embryonal carcinomas, 2 teratocarcinomas, 3 yolk sac tumors and 1 choriocarcinoma. All seminomas, including four poorly differentiated (so-called anaplastic seminomas), gave negative results, regardless of the method employed. Embryonal carcinoma, the epithelial component of the teratocarcinoma, the yolk sac tumors, and choriocarcinoma were at least focally positive for keratin. The monoclonal antibody provided a cleaner background and stronger staining than the rabbit-anti-total-human-epidermal-keratin antibody. Best results were obtained from fresh-frozen sections or alcohol-fixed, paraffin-embedded materials. Formalin-fixed, nonseminomatous tumors, when predigested with trypsin and incubated overnight with primary antibody, gave no false-negative results but staining was often focal. The authors' results agree with the reported absence of detectable keratin in primordial germ cells of the normal testis, and with prevailing concepts of the histogenesis of germ cell tumors. These results indicate that the presence or absence of keratin by immunocytochemical methods can be helpful in distinguishing seminoma from embryonal carcinoma
— id: 26942, year: 1984, vol: 54, page: 843, stat: Journal Article,

The use of aIF, AE1, and AE3 monoclonal antibodies for the identification and classification of mammalian epithelial keratins
Cooper D; Schermer A; Pruss R; Sun TT
1984 ;28(1):30-35, Differentiation
Recent data have indicated that specific keratin molecules can provide useful markers for studying different types and stages of epithelial differentiation. To utilize these protein markers, however, it is important to establish the keratin nature of the molecules and identify unambiguously the individual keratin species. In this paper, we show that this can be done relatively easily by one- and two-dimensional gel electrophoresis combined with immunoblotting using three monoclonal antibodies (aIF, AE1, and AE3). The aIF antibody has previously been shown to crossreact with all classes of intermediate-filament proteins. Using one- and two-dimensional immunoblotting, we establish that this antibody recognizes all known epithelial keratins of human and rabbit, although the reaction is relatively strong for the larger, basic keratins and is relatively weak for some of the smaller, acidic keratins. In contrast, AE1 and AE3 monoclonal antibodies have previously been shown to be highly specific for the acidic and basic subfamilies of the keratins, respectively. The combined use of the broadly reacting aIF antibody and the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies should facilitate the immunological definition, identification, and classification of mammalian epithelial keratins
— id: 26948, year: 1984, vol: 28, page: 30, stat: Journal Article,

Classification of epidermal keratins according to their immunoreactivity, isoelectric point, and mode of expression
Eichner R; Bonitz P; Sun TT
1984 Apr;98(4):1388-1396, Journal of cell biology
Human epidermal keratinocytes express under various growth conditions a total of at least nine keratins that can be divided into two subfamilies. Subfamily A comprises 40-, 46-, 48-, 50-/50'-, and 56.5-kilodalton (kd) keratins which are relatively acidic (pI less than 5.5) and, with the exception of 46-kd keratin, are recognized by AE1 monoclonal antibody. Subfamily B comprises 52-, 56-, 58-, and 65-67-kd keratins which are relatively basic (pI greater than 6) and are recognized by AE3 monoclonal antibody. Within each keratin subfamily, there is a constant member (50-/50'- and 58-kd keratins of the subfamilies A and B, respectively) that is always expressed. The other seven keratins of both subfamilies are variable members whose expression depends upon the cellular differentiated state, which is in turn modulated by the growth environment. The 56.5-kd keratin (subfamily A) and the 65-67-kd keratins (subfamily B) are coordinately expressed during keratinization. In contrast, the 40-, 46-, and 48-kd keratins (subfamily A) and the 52- and 56-kd keratins (subfamily B) are characteristic of cultured epidermal cells forming nonkeratinized colonies. These results demonstrate that human epidermal keratins can be classified according to their reactivity with monoclonal antikeratin antibodies, isoelectric point, and mode of expression. The classification of keratins into various subgroups may have important implications for the mechanisms of epidermal differentiation, the evolution of keratin heterogeneity, and the use of keratin markers for tumor diagnosis
— id: 26945, year: 1984, vol: 98, page: 1388, stat: Journal Article,

Differential staining of cytoid bodies and skin-limited amyloids with monoclonal anti-keratin antibodies
Eto H; Hashimoto K; Kobayashi H; Fukaya T; Matsumoto M; Sun TT
1984 Sep;116(3):473-481, American journal of pathology
The authors have used 5 different monoclonal antikeratin antibodies to study the antigenic profiles of cytoid bodies and skin-limited amyloids. Monoclonal antibodies AE1 (which stains the basal cell layer in normal human epidermis), AE2 (suprabasal layers), AE3 (whole epidermis), EKH4 (lower 2-3 layers), and EKH1 (recognizes all classes of intermediate filaments) were used to stain frozen skin sections by the indirect immunofluorescent or indirect immunoperoxidase technique. Cytoid bodies in lichen planus (LP) and discoid lupus erythematosus (DLE) were strongly stained with AE1, AE3, EKH4, and EKH1 antibodies but were negative with AE2. In contrast, amyloids in lichen amyloidosus and macular amyloidosis were stained strongly with EKH4 but only weakly or not at all with AE1, AE2, AE3, and EKH1. Amyloid associated with epithelial tumors showed closer immunologic profiles to cytoid body. These findings suggest that epidermal keratins are the major precursor substance of skin-limited amyloids as well as cytoid bodies in LP and DLE. Sequential changes in antigenic profiles from basal cells to amyloids through cytoid bodies further suggest that cytoid bodies may represent one of the precursor substances of skin-limited amyloids
— id: 26941, year: 1984, vol: 116, page: 473, stat: Journal Article,

Simple epithelial nature of some simian virus-40-transformed human epidermal keratinocytes
Hronis TS; Steinberg ML; Defendi V; Sun TT
1984 Dec;44(12 Pt 1):5797-5804, Cancer research
Previous studies have indicated that some Simian-virus-40-transformed human epidermal keratinocytes (SV40-HE) undergo significant changes in their growth and differentiated properties. To better understand the significance of these changes, we have characterized the keratins of SV40-HE cells by one- and two-dimensional immunoblot analysis using the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies. The results indicate that our SV40-HE cells have lost the Mr 58,000 (No. 5), Mr 56,000 (No. 6), Mr 50,000 (No. 14/15), Mr 48,000 (No. 16), and Mr 46,000 (No. 17) keratins that are expressed by cultured normal human keratinocytes. Instead, these cells express mainly Mr 52,000 (No. 8), Mr 45,000 (No. 18), and Mr 40,000 (No. 19) keratins, a set highly characteristic of simple epithelial cells. Furthermore, our SV40-HE cells have ceased to express involucrin, another marker for keratinocytes, and have a greatly diminished ability to undergo in vitro stratification. These results suggest that epidermal cells can sometimes lose their keratinocyte features as a consequence of viral transformation. This finding may have important implications regarding the mechanisms of epithelial differentiation and tumorigenesis and in the use of keratinocyte markers for tumor diagnosis
— id: 21438, year: 1984, vol: 44, page: 5797, stat: Journal Article,

Specific keratins as molecular markers for neoplasms with a stratified epithelial origin
Nelson WG; Battifora H; Santana H; Sun TT
1984 Apr;44(4):1600-1603, Cancer research
The expression of specific keratin polypeptides in human neoplasms was investigated by the immunoblot technique using monoclonal anti-keratin antibodies. Mr 50,000 and 58,000 keratins, recognized by AE1 and AE3 antibodies, respectively, were detected only in carcinomas of stratified epithelial origin, but not in carcinomas derived from simple epithelia. No keratin was detected in nonepithelial tumors including melanoma, lymphoma, neurofibroma, and sarcoma. The results suggest that the Mr 50,000 and 58,000 keratins provide useful molecular markers for identifying neoplasms of stratified squamous epithelial origin
— id: 26946, year: 1984, vol: 44, page: 1600, stat: Journal Article,

Immunohistochemical study of nasopharyngeal carcinoma using monoclonal keratin antibodies
Shi SR; Goodman ML; Bhan AK; Pilch BZ; Chen LB; Sun TT
1984 Oct;117(1):53-63, American journal of pathology
Nasopharyngeal carcinoma (NPC) provides a unique opportunity to evaluate distinctive epidemiologic features and a possible etiologic relationship with Epstein-Barr virus (EBV) in human malignancy. The lack of a uniformly accepted pathologic classification for NPC has limited the application of this data, although the World Health Organization (WHO) developed a classification that may solve this problem. Monoclonal keratin antibodies were used for staining of NPC for evaluation of its assistance in diagnosis and classification. In the present immunohistochemical study, monoclonal keratin antibodies, designated AE1, AE2, and AE3, and a polyclonal keratin antibody (RAK) were used for study of the presence of keratin in 121 cases of NPC obtained from China and the United States. AE1 monoclonal antibody, which recognizes keratin protein classes 56.5K, 50K, and 40K, was shown to be the most sensitive and specific for NPC tumor cells among the keratin antibodies studied. In addition, some different keratin expression patterns could be identified between different kinds of epithelium and different tumor groups, with possible relevance to the histogenesis of the histologic subtypes of NPC
— id: 26940, year: 1984, vol: 117, page: 53, stat: Journal Article,

Cell culture of mammalian thymic epithelial cells: growth, structural, and antigenic properties
Sun TT; Bonitz P; Burns WH
1984 Jan;83(1):1-13, Cellular immunology
The thymus plays an important role in the maturation and differentiation of T lymphocytes. Many of its functions have been attributed to its epithelial component. Past in vitro studies of putative thymic epithelial cells have been hampered by the inability to produce well-characterized cultures of these cells. Using lethally irradiated 3T3 cells as a feeder layer, we have succeeded in growing virtually pure cultures of thymic epithelial (TE) cells from rabbits, mice, and humans. Antikeratin staining provides an unambiguous criterion for positive identification of the epithelial cells. These cells were found to lack Ia-like or theta-like antigens. The ability to culture large quantities of mammalian TE cells should allow for their detailed functional characterization
— id: 26947, year: 1984, vol: 83, page: 1, stat: Journal Article,

Expression of specific keratin markers by rabbit corneal, conjunctival, and esophageal epithelia during vitamin A deficiency
Tseng SC; Hatchell D; Tierney N; Huang AJ; Sun TT
1984 Dec;99(6):2279-2286, Journal of cell biology
Using an in vivo rabbit model system, we have studied the morphological and biochemical changes in corneal, conjunctival, and esophageal epithelia during vitamin A deficiency. Light and electron microscopy showed that the three epithelia undergo different degrees of morphological keratinization. Corneal and conjunctival epithelia became heavily keratinized, forming multiple layers of superficial, anucleated cornified cells. In contrast, esophageal epithelium underwent only minor morphological changes. To correlate morphological alterations with the expression of specific keratin molecules, we have analyzed the keratins from these epithelia by the immunoblot technique using the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies. The results indicate that during vitamin A deficiency, all three epithelia express an AE1-reactive, acidic 56.5-kd keratin and an AE3-reactive, basic 65-67-kd keratin. Furthermore, the expression of these two keratins correlated roughly with the degree of morphological keratinization. AE2 antibody (specific for the 56.5- and 65-67-kd keratins) stained keratinized corneal epithelial sections suprabasally, as in the epidermis, suggesting that these two keratins are expressed mainly during advanced stages of keratinization. These two keratins have previously been suggested to represent markers for epidermal keratinization. Our present data indicate that they can also be expressed by other stratified epithelia during vitamin A deficiency-induced keratinization, and suggest the possibility that they may play a role in the formation of the densely packed tonofilament bundles in cornified cells of keratinized tissues
— id: 26939, year: 1984, vol: 99, page: 2279, stat: Journal Article,

Possible mechanisms for the loss of goblet cells in mucin-deficient disorders
Tseng SC; Hirst LW; Maumenee AE; Kenyon KR; Sun TT; Green WR
1984 Jun;91(6):545-552, Ophthalmology
By studying the pathological changes in human conjunctival flaps and the conjunctival transdifferentiation in rabbits, we have identified and correlated two pathological processes with the loss of goblet cells: (1) loss of vascularization, and (2) intense inflammation. Loss of vascularization may be correlated with the loss of goblet cells in the chronic cicatricial stage of various mucin-deficient disorders, whereas inflammation may be responsible for their absence in the acute inflammatory stage. The exact interrelationship between these two processes remains unknown. The loss of goblet cells appears to be an early sign of a spectrum of squamous metaplasia, an abnormality of epithelial differentiation. We therefore speculate that loss of vascularization and intense inflammation may have an important effect on epithelial differentiation
— id: 26943, year: 1984, vol: 91, page: 545, stat: Journal Article,

Monoclonal antibody analysis of keratin expression in epidermal diseases: a 48- and 56-kdalton keratin as molecular markers for hyperproliferative keratinocytes
Weiss RA; Eichner R; Sun TT
1984 Apr;98(4):1397-1406, Journal of cell biology
The polypeptide composition of epidermal keratin varies in disease. To better understand the biological meaning of these variations, we have analyzed keratins from a number of human epidermal diseases by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The results reveal a continuous spectrum of keratin expression ranging from one closely resembling the normal in vivo pattern to one almost identical to cultured epidermal keratinocytes. Specifically, a 50-kilodalton (kd) (AE1-positive) and a 58-kd (AE3-positive) keratin are present in all diseases, supporting the concept that they represent 'permanent' markers for keratinocytes. A 56.5-kd (AE1) and a 65-67-kd (AE3) keratin, previously shown to be markers for keratinization, are expressed only by lesions retaining a keratinized morphology. A 48-kd (AE1) and a 56-kd (AE3) keratin are present in all hyperproliferative (para- or nonkeratinized) disorders, but not in normal abdominal epidermis or in ichthyosis vulgaris which is a nonhyperproliferative disease. These two keratins have previously been found in various nonepidermal keratinocytes undergoing hyperproliferation, suggesting that these keratins are not epidermis-specific and may represent markers for hyperproliferative keratinocytes in general. In various epidermal diseases, there is a reciprocal expression of the (keratin) markers for hyperproliferation and keratinization, supporting the mutual exclusiveness of the two cellular events. Moreover, our results indicate that, as far as keratin expression is concerned, cultured human epidermal cells resemble and thus may be regarded as a model for epidermal hyperplasia. Finally, the apparent lack of any major, disease-specific keratin changes in the epidermal disorders studied so far implies that keratin abnormalities probably represent the consequence, rather than the cause, of these diseases
— id: 26944, year: 1984, vol: 98, page: 1397, stat: Journal Article,

The fibrillar substructure of keratin filaments unraveled
Aebi U; Fowler WE; Rew P; Sun TT
1983 Oct;97(4):1131-1143, Journal of cell biology
We show that intermediate-sized filaments reconstituted from human epidermal keratins appear unraveled in the presence of phosphate ions. In such unraveling filaments, up to four '4.5-nm protofibrils' can be distinguished, which are helically twisted around each other in a right-handed sense. Lowering the pH of phosphate-containing preparations causes the unraveling filaments to further dissociate into '2-nm protofilaments.' In addition, we find that reconstitution of keratin extracts in the presence of small amounts of trypsin yields paracrystalline arrays of 4.5-nm protofibrils with a prominent 5.4-nm axial repeat. Limited proteolysis of intact filaments immobilized on an electron microscope grid also unveils the presence of 4.5-nm protofibrils within the filament with the same 5.4-nm axial repeat. These results, together with other published data, are consistent with a 10-nm filament model based on three distinct levels of helical organization: (a) the 2-nm protofilament, consisting of multi-chain extended alpha-helical segments coiled around each other; (b) the 4.5-nm protofibril, being a multi-stranded helix of protofilaments; and (c) the 10-nm filament, being a four-stranded helix of protofibrils
— id: 26949, year: 1983, vol: 97, page: 1131, stat: Journal Article,

Epidermal stem cells
Lavker RM; Sun TT
1983 Jul;81(1 Suppl):121s-127s, Journal of investigative dermatology
Stem cells are by definition present in all self-renewing tissues and are believed to play a central role in cell growth and differentiation. Existing evidence suggests that a subpopulation of epidermal basal keratinocytes represents stem cells; however, these cells have never been positively identified. In this paper we review evidence that in monkey palm epidermis there exist two morphologically distinct subpopulations of basal keratinocytes that are spatially segregated. One population, located in the shallow rete ridges, is characterized by a cytoplasm filled with tonofilaments and a highly convoluted ('serrated') dermal-epidermal junction; these cells may play a role in anchoring the epidermis to the dermis. In contrast, the other population, located at the tips of deep rete ridges, is characterized by a 'primitive' cytoplasm containing abundant melanosomes and a relatively flattened ('nonserrated') dermal-epidermal junction. Tritiated thymidine labeling experiments suggest that the nonserrated basal keratinocytes are slow-cycling; however, a highly proliferative population of keratinocytes can be identified immediately above these basal cells. These findings are consistent with the concept that the nonserrated basal keratinocytes may represent stem cells that give rise to suprabasally located, transient amplifying cells before undergoing terminal differentiation. Monkey palm epidermis provides a model system for further studies of primate epidermal stem cells
— id: 16580, year: 1983, vol: 81, page: 121s, stat: Journal Article,

Rapid modulation of keratinocyte differentiation by the external environment
Lavker RM; Sun TT
1983 Apr;80(4):228-237, Journal of investigative dermatology
The ultrastructural differentiation of epidermal keratinocytes cultured in the presence of 3T3 feeder cells is significantly different from that of the epidermis in vivo. Several markers of keratinization, including keratohyaline granules (KGs), membrane-coating granules (MCGs), and an enucleated stratum corneum are essentially absent from cultured cells. When cultured rabbit epidermal cells were trypsinized and injected subcutaneously into athymic mice, the cells reaggregated and formed cysts lined with a stratified squamous epithelium morphologically resembling the in vivo epidermis. In this paper, we examined the differentiation of the injected cells by electron microscopy. Within 24 h after injection, MCGs and KGs appeared in the reaggregated epidermal cells. Horny cells were noted within 48 h. Since basement membrane formation was not completed until much later (between 4-9 days), a direct contact between epidermal cells and a continuous basal lamina structure was not required for the formation of various keratinization markers. Glycogen and lipid droplets, which were abundant in the early (10-48 h) cystic epithelia, gradually disappeared from the basal through the granular layers during days 2-9. By 16 days, the ultrastructure of the cystic epithelium appeared similar to the in vivo rabbit epidermis including the formation of 'rabbit-type' KGs, MCGs, and normal enucleated horny cells. These results provided further evidence that the differentiation of epidermal cells can be modulated significantly and reversibly by the external environment. Moreover, the expression of certain morphologic markers of keratinization (KGs and MCGs) can be modulated rapidly by the growth environment
— id: 16581, year: 1983, vol: 80, page: 228, stat: Journal Article,

The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies
Nelson WG; Sun TT
1983 Jul;97(1):244-251, Journal of cell biology
The keratins are a highly heterogeneous group of proteins that form intermediate filaments in a wide variety of epithelial cells. These proteins can be divided into at least seven major classes according to their molecular weight and their immunological reactivity with monoclonal antibodies. Tissue-distribution studies have revealed a correlation between the expression of specific keratin classes and different morphological features of in vivo epithelial differentiation (simple vs. stratified; keratinized vs. nonkeratinized). Specifically, a 50,000- and a 58,000-dalton keratin class were found in all stratified epithelia but not in simple epithelia, and a 56,500- and a 65-67,000-dalton keratin class were found only in keratinized epidermis. To determine whether these keratin classes can serve as markers for identifying epithelial cells in culture, we analyzed cytoskeletal proteins from various cultured human cells by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The 56,500- and 65-67,000-dalton keratins were not expressed in any cultured epithelial cells examined so far, reflecting the fact that none of them underwent morphological keratinization. The 50,000- and 58,000-dalton keratin classes were detected in all cultured cells that originated from stratified squamous epithelia, but not in cells that originated from simple epithelia. Furthermore, human epidermal cells growing as a monolayer in low calcium medium continued to express the 50,000- and 58,000-dalton keratin classes. These findings suggest that the 50,000- and 58,000-dalton keratin classes may be regarded as 'permanent' markers for stratified squamous epithelial cells (keratinocytes), and that the expression of these keratin markers does not depend on the process of cellular stratification. The selective expression of the 50,000- and 58,000-dalton keratin classes, which are synthesized in large quantities on a per cell basis, may explain the high keratin content of cultured keratinocytes
— id: 26951, year: 1983, vol: 97, page: 244, stat: Journal Article,

Keratin classes: molecular markers for different types of epithelial differentiation
Sun TT; Eichner R; Nelson WG; Tseng SC; Weiss RA; Jarvinen M; Woodcock-Mitchell J
1983 Jul;81(1 Suppl):109s-115s, Journal of investigative dermatology
Keratins are a group of water-insoluble proteins (molecular weight range 40-70 K) that form 10-nm tonofilaments in a wide variety of epithelial cells. The subunit composition of the keratin filaments varies with cell type, period of embryonic development, stage of histologic differentiation, cellular growth environment, and disease state. To better understand the functional significance of individual keratin species, we have generated three monoclonal antikeratin antibodies to different subsets of keratins and used these antibodies to localize specific keratins in normal human epidermis by a combination of immunohistochemical and biochemical techniques. The results indicate that the 50 K and 58 K keratins are present in all cell layers including the relatively undifferentiated basal layer, whereas the 56.5 K and 65-67 K keratins are associated only with the more differentiated cells above the basal layer. In a separate series of experiments, we used the monoclonal antibodies to survey the keratins expressed by various nonepidermal epithelia. The data show that keratins can be divided into at least seven major classes according to their immunologic reactivity and size. Among the keratin classes, the 50 K and 58 K classes appear to be characteristic of all stratified squamous epithelia, whereas the 56.5 K and 65-67 K classes are unique to the keratinized epidermis. These findings suggest that specific keratin classes, as defined by monoclonal antibodies, may serve as useful markers for different types of epithelial differentiation (simple versus stratified, keratinized versus nonkeratinized)
— id: 26950, year: 1983, vol: 81, page: 109s, stat: Journal Article,

Keratin expression during normal epidermal differentiation
Sun TT; Eichner R; Nelson WG; Vidrich A; Woodcock-Mitchell J
1983 ;11(4):277-291, Current problems in dermatology
The four major epidermal keratins (65-67K, 58K, 56K, and 50K) have been localized in various cell layers of normal human epidermis. Guinea pig antisera and mouse monoclonal antibodies were prepared against human epidermal keratins and were characterized with respect to their specificity to individual keratin polypeptides by the immunoblot technique. These antibodies were used to stain vertical frozen sections of skin, and to identify keratins extracted from serial, horizontal skin sections. The results indicate that: (1) a 65-67K keratin component is limited to the suprabasal layers, (2) a 58K keratin is present throughout the epidermis, (3) a 56K keratin appears to be made only in cells above the basal layer, possibly in the upper spinous or granular layer, and (4) a 50K keratin is present in all living layers but is largely eliminated during stratum corneum formation. The 65-67K and 56K keratins, which are characteristic of suprabasal, terminally differentiated keratinocytes, may be regarded as molecular markers of keratinization
— id: 26952, year: 1983, vol: 11, page: 277, stat: Journal Article,

The use of monoclonal antibody to keratin in human epidermal disease: alterations in immunohistochemical staining pattern
Weiss RA; Guillet GY; Freedberg IM; Farmer ER; Small EA; Weiss MM; Sun TT
1983 Sep;81(3):224-230, Journal of investigative dermatology
A monoclonal antikeratin antibody, designated AEl, was used to stain frozen sections of normal and abnormal human skin by the immunofluorescence and peroxidase-antiperoxidase techniques. In normal human epidermis and ichthyosis vulgaris, a nonproliferative epidermal disease, this antibody selectively stained epidermal basal cells. Very different staining patterns were observed in various other epidermal diseases. A suprabasal staining pattern was observed in psoriasis (16 cases), verruca (9), seborrheic keratosis (5), actinic keratosis (2), as well as the epidermis adjacent to certain epidermal neoplasms (4). Basal cell carcinoma (7) showed weak, homogeneous staining. In contrast, a disorganized pattern consisting of cells with various staining intensities was observed in Bowen's disease (2) and squamous cell carcinoma (4). Although the biochemical basis for these altered staining patterns remains to be elucidated, these results provide further evidence that epidermal keratin expression can be affected by various disease states. Moreover, our data suggest that a common alteration in keratin expression, as defined by the suprabasal AEl staining pattern, exists in psoriasis and a number of other benign hyperproliferative epidermal diseases
— id: 16381, year: 1983, vol: 81, page: 224, stat: Journal Article,

Heterogeneity in epidermal basal keratinocytes: morphological and functional correlations
Lavker RM; Sun TT
1982 Mar 5;215(4537):1239-1241, Science
Two structurally distinct populations of basal keratinocytes, nonserrated and serrated, were observed in cynomolgus monkey and human palm epidermis. Anatomical location, fine structural features, and kinetic properties suggest that nonserrated cells represent a stem cell population and that serrated cells help anchor the epidermis to the dermis
— id: 16584, year: 1982, vol: 215, page: 1239, stat: Journal Article,

Correlation of specific keratins with different types of epithelial differentiation: monoclonal antibody studies
Tseng SC; Jarvinen MJ; Nelson WG; Huang JW; Woodcock-Mitchell J; Sun TT
1982 Sep;30(2):361-372, Cell
We have prepared three monoclonal antibodies against human epidermal keratins. These antibodies were highly specific for keratins and, in combination, recognized all major epidermal keratins of several mammalian species. We have used these antibodies to study the tissue distribution of epidermis-related keratins. In various mammalian epithelia, the antibodies recognized seven classes of keratins defined by their immunological reactivity and size. The 40, 46 and 52 kilodalton (kd) keratin classes were present in almost all epithelia; the 50 kd and 58 kd keratin classes were detected in all stratified squamous epithelia, but not in any simple epithelia; and the 56 kd and 65-67 kd keratin classes were unique to keratinized epidermis. Thus the expression of specific keratin classes appeared to correlate with different types of epithelial differentiation (simple versus stratified; keratinized versus nonkeratinized)
— id: 26954, year: 1982, vol: 30, page: 361, stat: Journal Article,

Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies
Woodcock-Mitchell J; Eichner R; Nelson WG; Sun TT
1982 Nov;95(2 Pt 1):580-588, Journal of cell biology
Three monoclonal antibodies (AE1, AE2, and AE3) were prepared against human epidermal keratins and used to study keratin expression during normal epidermal differentiation. Immunofluorescence staining data suggested that the antibodies were specific for keratin-type intermediate filaments. The reactivity of these antibodies to individual human epidermal keratin polypeptides (65-67, 58, 56, and 50 kdaltons) was determined by the immunoblot technique. AE1 reacted with 56 and 50 kdalton keratins, AE2 with 65-67 and 56-kdalton keratins, and AE3 with 65-67 and 58 kdalton keratins. Thus all major epidermal keratins were recognized by at least one of the monoclonal antibodies. Moreover, common antigenic determinants were present in subsets of epidermal keratins. To correlate the expression of specific keratins with different stages of in vivo epidermal differentiation, the antibodies were used for immunohistochemical staining of frozen skin sections. AE1 reacted with epidermal basal cells, AE2 with cells above the basal layer, and AE3 with the entire epidermis. The observation that AE1 and AE2 antibodies (which recognized a common 56 kdalton keratin) stained mutually exclusive parts of the epidermis suggested that certain keratin antigens must be masked in situ. This was shown to be the case by direct analysis of keratins extracted from serial, horizontal skin sections using the immunoblot technique. The results from these immunohistochemical and biochemical approaches suggested that: (a) the 65- to 67-kdalton keratins were present only in cells above the basal layer, (b) the 58-kdalton keratin was detected throughout the entire epidermis including the basal layer, (c) the 56-kdalton keratin was absent in the basal layer and first appeared probably in the upper spinous layer, and (d) the 50-kdalton keratin was the only other major keratin detected in the basal layer and was normally eliminated during s. corneum formation. The 56 and 65-67-kdalton keratins, which are characteristic of epidermal cells undergoing terminal differentiation, may be regarded as molecular markers for keratinization
— id: 26953, year: 1982, vol: 95, page: 580, stat: Journal Article,

Identification of mouse mammary epithelial cells by immunofluorescence with rabbit and guinea pig antikeratin antisera
Asch BB; Burstein NA; Vidrich A; Sun TT
1981 Sep;78(9):5643-5647, Proceedings of the National Academy of Sciences of the United States of America
Few markers are available to identify the three types of mammary epithelial cells--ductal, alveolar, and myoepithelial--especially in pathological conditions and in cell cultures. We have used antisera to human keratins in immunofluorescence to facilitate the identification of the three mouse mammary epithelial cell types. In frozen tissue sections and primary cell cultures, a rabbit antikeratin antiserum specifically stained cytoplasmic filaments in all three types of epithelial cells. A guinea pig antiserum against the same keratin preparation, however, reacted preferentially with filaments in myoepithelial cells and readily detected this cell type in normal, dysplastic, and malignant mammary tissues and cell cultures. Neither antisera reacted with fibroblasts or any other mesenchymal cells. The combined use of the two antikeratin antisera thereby permits rapid surveys of tissue sections and cultures for the localization of not only all epithelial cells but also the subpopulation of myoepithelial cells. Moreover, when mammary cultures established from late-pregnant or lactating mice were stained simultaneously with guinea pig antikeratin and rabbit anticasein antisera, three populations of epithelial cells were mutually exclusive: those stained by anticasein antiserum, those stained by guinea pig antikeratin antiserum, and those stained by neither, consistent with properties of alveolar, myoepithelial, and ductal cells, respectively. These antisera thus offer a tool for studying different epithelial cell types during mammary development, tumorigenesis, and malignant progression
— id: 26956, year: 1981, vol: 78, page: 5643, stat: Journal Article,

Posterior polymorphous dystrophy of the cornea: cell culture studies
Rodrigues MM; Newsome DA; Krachmer JH; Sun TT
1981 Nov;33(5):535-544, Experimental eye research
— id: 26955, year: 1981, vol: 33, page: 535, stat: Journal Article,

Expression of keratin and vimentin intermediate filaments in rabbit bladder epithelial cells at different stages of benzo[a]pyrene-induced neoplastic progression
Summerhayes IC; Cheng YS; Sun TT; Chen LB
1981 Jul;90(1):63-69, Journal of cell biology
Rabbit bladder epithelium, grown on collagen gels and exposed to the chemical carcinogen benzo[a]pyrene, produced nontumorigenic altered foci as well as tumorigenic epithelial cell lines during 120-180 d in culture. Immunofluorescence studies revealed extensive keratin filaments in both primary epithelial cells and benzo[a]pyrene-induced altered epithelial foci but showed no detectable vimentin filaments. The absence of vimentin expression in these cells was confirmed by two-dimensional gel electrophoresis. In contrast, immunofluorescence staining of the cloned benzo[a]pyrene-transformed rabbit bladder epithelial cell line, RBC-1, revealed a reduction in filamentous keratin concomitant with the expression of vimentin filaments. The epithelial nature of this cell line was established by the observation that cells injected into nude mice formed well-differentiated adenocarcinomas. Frozen sections of such tumors showed strong staining with antikeratins antibodies, but no detectable staining with antivimentin antibodies. These results demonstrated a differential expression of intermediate filament type in cells at different stages of neoplastic progression and in cells maintained in different growth environments. It is apparent that the expression of intermediate filaments throughout neoplastic progression is best studied by use of an in vivo model system in parallel with culture studies
— id: 26957, year: 1981, vol: 90, page: 63, stat: Journal Article,

Keratin filaments of corneal epithelial cells
Sun TT; Vidrich A
1981 ;21(1):55-63, Vision research
— id: 26958, year: 1981, vol: 21, page: 55, stat: Journal Article,

The use of antikeratin antiserum as a diagnostic tool: thymoma versus lymphoma
Battifora H; Sun TT; Bahu RM; Rao S
1980 Nov;11(6):635-641, Human pathology
Indirect immunofluorescence staining in two thymomas, one case of thymic hyperplasia, 10 malignant lymphomas and three seminomas was done with an antibody prepared against keratins from human epidermis. Staining was observed only in the epithelial cells of the thymomas and thymic hyperplasia and correlated well with electron microscopic studies. Immunofluorescence staining of thymic tumors with antikeratin antibody provides a simple, specific, and sensitive method for distinguishing thymoma from lymphoma and seminoma. The method may also prove to be useful in other instances in the distinction between epithelial and nonepithelial tumors
— id: 26959, year: 1980, vol: 11, page: 635, stat: Journal Article,

Intrinsic and extrinsic regulation of the differentiation of skin, corneal and esophageal epithelial cells
Doran TI; Vidrich A; Sun TT
1980 Nov;22(1 Pt 1):17-25, Cell
— id: 26960, year: 1980, vol: 22, page: 17, stat: Journal Article,

Human epithelial cells cultured from urine: growth properties and keratin staining
Felix JS; Sun TT; Littlefield JW
1980 Oct;16(10):866-874, In vitro
Epithelial cells can be cultured from the urine of newborn infants, providing a simple, noninvasive biopsy method. We established such cultures by standard techniques from 44% of uncontaminated specimens obtained from newborn infants up to 1 week of age. There was an average of three colonies per milliliter of urine. Many cultures accomplished 15 to 25 population doublings in as many as five subcultures and yielded total potential culture sizes of 10(4) to 6 x 10(8) cells. Plating efficiency was high at each passage. The cultures displayed two morphologically distinct epithelial cell types. Immunofluorescent staining of keratin fibers in most of these cells further identified them as epithelial
— id: 26961, year: 1980, vol: 16, page: 866, stat: Journal Article,

Epithelialization of the corneal endothelium in posterior polymorphous dystrophy
Rodrigues MM; Sun TT; Krachmer J; Newsome D
1980 Jul;19(7):832-835, Investigative ophthalmology & visual science. IOVS
The unusual cell type present on the posterior corneal surface of posterior polymorphous dystrophy patients has been characterized. In addition to microvilli and desmosomes, these cells contain abundant 10 nm filaments which by immunofluorescent staining were shown to consist of keratin proteins, a marker for epithelial cells
— id: 26962, year: 1980, vol: 19, page: 832, stat: Journal Article,

Keratin cytoskeletons in epithelial cells of internal organs
Sun TT; Shih C; Green H
1979 Jun;76(6):2813-2817, Proceedings of the National Academy of Sciences of the United States of America
— id: 26964, year: 1979, vol: 76, page: 2813, stat: Journal Article,

Immunofluorescent staining of keratin fibers in cultured cells
Sun TT; Green H
1978 Jul;14(3):469-476, Cell
Antibody prepared against a group of keratins purified from human stratum corneum was used to identify cells containing keratins by immunofluorescence. In sectioned tissue and in culture, keratinocytes of skin and other stratified squamous epithelia-whether human, rabbit of mouse-stained strongly, indicating homologous amino acid sequences in the keratins of these species. In all cases, the antibody revealed a dense cytoplasmic network of discrete fibers probably consisting of aggregated (tono-) filaments. The pattern of staining was not affected by cytochalasin B or colcemid. No keratins were detected in cultured cells of mesenchymal origin (3T3, NIL, BHK, human diploid fibroblasts) or in connective tissues, indicating that the 100 A filaments of fibroblasts are not related to the keratins. Keratinocytes at all stages of differentiation, including basal cells, stained brightly and therefore contained abundant keratins
— id: 26965, year: 1978, vol: 14, page: 469, stat: Journal Article,

Keratin filaments of cultured human epidermal cells. Formation of intermolecular disulfide bonds during terminal differentiation
Sun TT; Green H
1978 Mar 25;253(6):2053-2060, Journal of biological chemistry
Human epidermal cells grown in culture synthesize abundant keratins. These keratins are similar to those of stratum corneum of human epidermal callus in their insolubility in dilute aqueous buffers, their molecular weight range of 40,000 to 60,000, their immunolgical reactivity, and their ability to assemble into 80 A tonofilaments in vitro; but there are differences in the molecular weights of some of the proteins, the number of components, and their charge heterogeneity, related at least in part to phosphorylation. About 30% of all the proteins of living cultured keratinocytes consists of keratins, compared with over 85% of stratum corneum. All the keratins of human stratum corneum were found to be cross-linked by intermolecular disulfide bonds while most keratins of the living cells were not. As the cells mature in Methocel-stabilized suspension culture, their keratins become increasingly disulfide cross-linked. When uncross-linked tonofilaments of living keratinocytes are dissolved in 8 M urea and the filaments reconstituted in vitro their keratins become disulfide cross-linked under aerobic conditions and consequently insoluble in solutions of 8 M urea or sodium dodecyl sulfate. The results indicate that the uncross-linked state of the keratins in living cells is due to the reducing intracellular environment and not to a precursor state related to the primary structure of the proteins. The disulfide cross-links stabilizing the keratin filaments must be distinguished from the epsilon-(gamma-glutamyl)lysine cross-links stabilizing the cornified cell envelope
— id: 26966, year: 1978, vol: 253, page: 2053, stat: Journal Article,

Control of a cell surface major glycoprotein by epidermal growth factor
Chen LB; Gudor RC; Sun TT; Chen AB; Mosesson MW
1977 Aug 19;197(4305):776-778, Science
When the serum concentration of the culture medium is below 0.7 per-cent, 3T3 mouse cells lose most of their large external transformation sensitive (LETS) protein at the cell surface, Subsequent addition of epidermal growth factor results in the reappearance of massive fibrillar LETS protein networks on the surface of conluent 3T3 cells. Thirteen other hormones tested do not have this effect. It appears that epidermal growth factor can control the expression of LETS protein
— id: 26968, year: 1977, vol: 197, page: 776, stat: Journal Article,

Properties of an epithelial cell type in culture: the epidermal keratinocyte and its dependence on products of the fibroblast
Green H; Rheinwald JG; Sun TT
1977 ;17(2):493-500, Progress in clinical & biological research
Keratinocytes of stratified squamous epithelium can be grown serially in culture and retain the various markers typical of their form of differentiation. In order to form colonies at each transfer, the keratinocytes must be suitably supported by fibroblasts. Established keratinocyte lines of teratomal origin show this dependence, as do diploid strains of finite culture life derived from human skin. For at least some keratinocyte lines, this requirement can be satisfied by soluble products elaborated by the fibroblasts. It is suggested that epithelial cells in general may not be independent cell types and that their poor cultivability may be due to failure to provide suitable fibroblast support. The existence of a number of established lines of epithelial origin that can grow without such support and of lines of fibroblastic origin which cannot support keratinocytes suggests that both epithelial dependence and the fibroblast supporting function can sometimes be lost in established cell lines
— id: 26970, year: 1977, vol: 17, page: 493, stat: Journal Article,

Cultured epithelial cells of cornea, conjunctiva and skin: absence of marked intrinsic divergence of their differentiated states
Sun TT; Green H
1977 Oct 6;269(5628):489-493, Nature
Keratinocytes of three different epithelia grown in cell culture express a large number of differentiation markers with either no differences or relatively small differences, depending on the species. Much of the distinctive phenotype of these epithelia in vivo must be due to external modulation and relatively little, at least in the case of the human, to permanent intrinsic divergence during development
— id: 26967, year: 1977, vol: 269, page: 489, stat: Journal Article,

Thrombin potentiates the mitogenic response of cultured fibroblasts to serum and other growth promoting agents
Zetter BR; Sun TT; Chen LB; Buchanan JM
1977 Aug;92(2):233-239, Journal of cellular physiology
— id: 26969, year: 1977, vol: 92, page: 233, stat: Journal Article,

Differentiation of the epidermal keratinocyte in cell culture: formation of the cornified envelope
Sun TT; Green H
1976 Dec;9(4 Pt 1):511-521, Cell
Human epidermal keratinocytes grow from single cells into stratified colonies. Cells in the upper layers of the colonies lose their ability to divide and begin terminal differentiation. In this process, there develops a cornified cell envelope that remains insoluble after heating in solutions of sodium dodecylsulfate and beta-mercaptoethanol. The insolubility of the cornified envelope depends upon proteins, since after treatment with proteolytic enzymes, the envelope becomes soluble in the detergent. Cells with cornified envelopes can be identified under the light microscope either in living colonies or following fixation and silver nitrate staining. Keratinocytes of the basal layer move in a characteristic way, but cornified cells do not move at all and form an immobile upper layer in the colonies. Keratinocytes disaggregated from growing colonies are of differing size and density, and can be separated on isopycnic gradients of Ficoll. The DNA-synthesizing cells are small (mean diameter 14 mum). The nonmultiplying cells are large and have a protein content proportionate to their size. Their final diameter may exceed 30 microns (volume increase greater than 10 fold). Cornified envelopes are found in some of the large cells but in none of the small. In growing colonies, usually 5-10% of the cells have cornified envelopes. The fraction is reduced in colonies growing in the presence of epidermal growth factor. Strain XB, an established keratinocyte line of mouse teratomal origin, also forms cornified envelopes, but the kinetics of the process are different, indicating that the program of terminal differentiation is not initiated at corresponding times in the two cell types
— id: 26971, year: 1976, vol: 9, page: 511, stat: Journal Article,

The protein topography of the E. coli 30S ribosomal subunit: a preliminary model E. coli/30S subunit/ribosome/spatial arrangement of proteins
Sun TT; Heimark RL; Traut R R
1975 Jan 31;6(1):33-41, Molecular & cellular biochemistry
A three dimensional model is presented which shows the apatial arrangement of 20 of the 21 proteins of the 30S ribosomal subunit of Escherichia coli. The model fulfills several purposes: (a) It summarizes currently available structural and functional data on ribosomal proteins; (b) It suggests an interesting correlation between stoichiometry and function. Functional proteins are both clustered and fractional; (c) It can be evaluated in relationships or point out critical experiments by which it can be tested
— id: 26972, year: 1975, vol: 6, page: 33, stat: Journal Article,

Topography of ribosomal proteins of the Escherichia coli 30S subunit as studied with the reversible cross-linking reagent methyl 4-mercaptobutyrimidate
Sun TT; Bollen A; Kahan L; Traut RR
1974 May 21;13(11):2334-2340, Biochemistry
— id: 26974, year: 1974, vol: 13, page: 2334, stat: Journal Article,

Protein-protein proximity in the association of ribosomal subunits of Escherichia coli: crosslinking of 30 S protein S16 to 50 S proteins by glutaraldehyde or formaldehyde
Sun TT; Traut RR; Kahan L
1974 Aug 15;87(3):509-522, Journal of molecular biology
— id: 26973, year: 1974, vol: 87, page: 509, stat: Journal Article,

The functional and structural homology of ribosomal protein S7 of E. coli strains K and MRE600
Sun TT; Traut RR
1973 Mar 27;122(1):1-9, Molecular & general genetics
— id: 26976, year: 1973, vol: 122, page: 1, stat: Journal Article,

Methyl 4-mercaptobutyrimidate as a cleavable cross-linking reagent and its application to the Escherichia coli 30S ribosome
Traut RR; Bollen A; Sun TT; Hershey JW; Sundberg J; Pierce LR
1973 Aug 14;12(17):3266-3273, Biochemistry
— id: 26975, year: 1973, vol: 12, page: 3266, stat: Journal Article,

Similarity in the size and number of ribosomal proteins from different prokaryotes
Sun TT; Bickle TA; Traut RR
1972 Aug;111(2):474-480, Journal of bacteriology
— id: 26977, year: 1972, vol: 111, page: 474, stat: Journal Article,