Xue-Ru Wu

Biosketch / Results /

Xue-Ru Wu, M.D.

Professor; Vice Chair of Research
Departments of Urology (Urology) and Pathology

Contact Info

Address
423 E 23 Street
Urology Floor 18 Room 18064S
Veterans Administration
New York, NY 10010

212-951-5429
212-951-5424
Xue-Ru.Wu@nyumc.org

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Education

— Shanghai, Medical Education

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Research Summary

Diseases involving the urinary bladder are of major clinical and social concerns. Bladder cancer is the fifth most common neoplasm and the twelfth leading cause of cancer deaths in the United States. Urinary tract infections are one of most common infectious diseases, accounting for 8-10 million physician''s visits annually. Together, these two diseases cost over 5 billion health care dollars in the clinical management. Despite extensive studies, little is known about the pathogenesis of these bladder disorders.

Our approach to better understand the molecular pathogenesis of bladder diseases is to utilize a group of bladder-specific markers?the uroplakins?that we have recently identified. These proteins, naturally forming two-dimensional crystals, are synthesized by all mammalian bladders studied, represent the major differentiation products of the normal bladder epithelium and are retained by a majority of the human bladder cancers. They are therefore excellent lineage-specific markers for bladder epithelium and for differentiating bladder cancers from cancers from other tissue origins. In addition, we have found that two of the uroplakins, uroplakins Ia and Ib, can serve as the major urothelial receptors for type 1-fimbriated E. coli, which cause more than 85% of the urinary tract infections. The preferential binding between a sub-population of E. coli that are predominant in urinary tract infection and uroplakins provides a molecular explanation for the recently recognized tissue tropism of uropathogenic E. coli. To study the bladder tumorigenesis, we have developed transgenic mouse models by specifically expressing activated oncogenes and mutated tumor suppressor genes in bladder epithelium. Mice harboring simian virus 40 large T oncogene, whose protein product inactivates p53 and retinoblastoma tumor suppressor protein, induced carcinoma in situ which progresses to invasive and metastatic transitional cell carcinomas. Mice harboring an activated H-ras induced bladder epithelial hyperplasia which progresses to superficial papillary tumors. These transgenic models provide strong experimental evidence that bladder cancers develop and progress via two distinctive pathways each of which is caused by unique genetic defects. Ongoing research efforts include the development of novel transgenic and knockout models, and the identification of molecular signatures of cancer progression through high-throughput screening, biochemical and cell biologic approaches.

Research Interests

Molecular Pathogenesis of Urinary Bladder Diseases

Research Keywords

Oncogenes, tumor suppressor genes, bladder cancer, urinary tract infection, transgenic mouse models

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Acrolein- and 4-Aminobiphenyl-DNA adducts in human bladder mucosa and tumor tissue and their mutagenicity in human urothelial cells
Lee, Hyun-Wook; Wang, Hsiang-Tsui; Weng, Mao-Wen; Hu, Yu; Chen, Wei-Sheng; Chou, David; Liu, Yan; Donin, Nicholas; Huang, William C; Lepor, Herbert; Wu, Xue-Ru; Wang, Hailin; Beland, Frederick A; Tang, Moon-Shong
2014 May;:67-67, Oncotarget
Tobacco smoke (TS) is a major cause of human bladder cancer (BC). Two components in TS, 4-aminobiphenyl (4-ABP) and acrolein, which also are environmental contaminants, can cause bladder tumor in rat models. Their role in TS related BC has not been forthcoming. To establish the relationship between acrolein and 4-ABP exposure and BC, we analyzed acrolein-deoxyguanosine (dG) and 4-ABP-DNA adducts in normal human urothelial mucosa (NHUM) and bladder tumor tissues (BTT), and measured their mutagenicity in human urothelial cells. We found that the acrolein-dG levels in NHUM and BTT are 10-30 fold higher than 4-ABP-DNA adduct levels and that the acrolein-dG levels in BTT are 2 fold higher than in NHUM. Both acrolein-dG and 4-ABP-DNA adducts are mutagenic; however, the former are 5 fold more mutagenic than the latter. These two types of DNA adducts induce different mutational signatures and spectra. We found that acrolein inhibits nucleotide excision and base excision repair and induces repair protein degradation in urothelial cells. Since acrolein is abundant in TS, inhaled acrolein is excreted into urine and accumulates in the bladder and because acrolein inhibits DNA repair and acrolein-dG DNA adducts are mutagenic, we propose that acrolein is a major bladder carcinogen in TS.
— id: 1036762, year: 2014, vol: , page: 67, stat: Journal Article,

Progress made in the use of animal models for the study of high-risk, nonmuscle invasive bladder cancer
Lin-Tsai, Opal; Taylor, John A 3rd; Clark, Peter E; Adam, Rosalyn M; Wu, Xue-Ru; DeGraff, David J
2014 Jun;:?-?, Current opinion in urology
PURPOSE OF REVIEW: High-risk, nonmuscle invasive bladder cancer (HR-NMIBC) represents a costly and difficult-to-treat disease, the molecular pathogenesis of which has a limited understanding. Most preclinical models for the study of bladder cancer are more appropriate for the study of advanced disease. However, recent key advances in preclinical animal models places us at an opportune position to better understand HR-NMIBC. RECENT FINDINGS: Discoveries in the basic sciences allow us to better understand tumor biology when building models of bladder cancer. Of note, a key study on urothelial progenitor cells recently highlighted an important role for Sonic hedgehog-positive cells and retinoid signaling that is essential for urothelial development and regeneration. In the translational realm, transgenic mouse models continue to be used, with a recent interest in the role of Wnt/beta-catenin in urothelial carcinomas. Tissue recombination models are also being increasingly utilized to better recreate the tissue microenvironment and better understand stromal-epithelial interactions and the impact of genetic alterations on tissue differentiation. Lastly, the avatar mouse systems, which involve direct xenotransplantation of human tumor specimens into immunocompromised mice, represent an additional approach to study cancer characteristics in a preserved tissue context. SUMMARY: With molecular alterations remaining an unclear area of our understanding of HR-NMIBC, preclinical models of bladder cancer serve as essential tools to discover specific genetic compromises in disease pathogenesis and the therapeutics to treat them.
— id: 1046542, year: 2014, vol: , page: ?, stat: Journal Article,

Chemoprevention of Urothelial Cell Carcinoma Growth and Invasion by the Dual COX-LOX Inhibitor Licofelone in UPII-SV40T Transgenic Mice
Madka, Venkateshwar; Mohammed, Altaf; Li, Qian; Zhang, Yuting; Patlolla, Jagan M R; Biddick, Laura; Lightfoot, Stan; Wu, Xue-Ru; Steele, Vernon; Kopelovich, Levy; Rao, Chinthalapally V
2014 May;:708-716 e99644, Cancer prevention research (Philadelphia, Pa.)
Epidemiologic and clinical data suggest that use of anti-inflammatory agents is associated with reduced risk for bladder cancer. We determined the chemopreventive efficacy of licofelone, a dual COX-lipoxygenase (LOX) inhibitor, in a transgenic UPII-SV40T mouse model of urothelial transitional cell carcinoma (TCC). After genotyping, six-week-old UPII-SV40T mice (n = 30/group) were fed control (AIN-76A) or experimental diets containing 150 or 300 ppm licofelone for 34 weeks. At 40 weeks of age, all mice were euthanized, and urinary bladders were collected to determine urothelial tumor weights and to evaluate histopathology. Results showed that bladders of the transgenic mice fed control diet weighed 3 to 5-fold more than did those of the wild-type mice due to urothelial tumor growth. However, treatment of transgenic mice with licofelone led to a significant, dose-dependent inhibition of the urothelial tumor growth (by 68.6%-80.2%, P < 0.0001 in males; by 36.9%-55.3%, P < 0.0001 in females) compared with the control group. The licofelone diet led to the development of significantly fewer invasive tumors in these transgenic mice. Urothelial tumor progression to invasive TCC was inhibited in both male (up to 50%; P < 0.01) and female mice (41%-44%; P < 0.003). Urothelial tumors of the licofelone-fed mice showed an increase in apoptosis (p53, p21, Bax, and caspase3) with a decrease in proliferation, inflammation, and angiogenesis markers (proliferating cell nuclear antigen, COX-2, 5-LOX, prostaglandin E synthase 1, FLAP, and VEGF). These results suggest that licofelone can serve as potential chemopreventive for bladder TCC. Cancer Prev Res; 7(7); 1-9. (c)2014 AACR.
— id: 1046552, year: 2014, vol: , page: 708, stat: Journal Article,

SNX31: A Novel Sorting Nexin Associated with the Uroplakin-Degrading Multivesicular Bodies in Terminally Differentiated Urothelial Cells
Vieira, Neide; Deng, Fang-Ming; Liang, Feng-Xia; Liao, Yi; Chang, Jennifer; Zhou, Ge; Zheng, Weiyue; Simon, Jean-Pierre; Ding, Mingxiao; Wu, Xue-Ru; Romih, Rok; Kreibich, Gert; Sun, Tung-Tien
2014 ;9(6):e99644-e99644 e99644, PLoS ONE
Uroplakins (UP), a group of integral membrane proteins, are major urothelial differentiation products that form 2D crystals of 16-nm particles (urothelial plaques) covering the apical surface of mammalian bladder urothelium. They contribute to the urothelial barrier function and, one of them, UPIa, serves as the receptor for uropathogenic Escherichia coli. It is therefore important to understand the mechanism by which these surface-associated uroplakins are degraded. While it is known that endocytosed uroplakin plaques are targeted to and line the multivesicular bodies (MVBs), it is unclear how these rigid-looking plaques can go to the highly curved membranes of intraluminal vesicles (ILVs). From a cDNA subtraction library, we identified a highly urothelium-specific sorting nexin, SNX31. SNX31 is expressed, like uroplakins, in terminally differentiated urothelial umbrella cells where it is predominantly associated with MVBs. Apical membrane proteins including uroplakins that are surface biotin-tagged are endocytosed and targeted to the SNX31-positive MVBs. EM localization demonstrated that SNX31 and uroplakins are both associated not only with the limiting membranes of MVBs containing uroplakin plaques, but also with ILVs. SNX31 can bind, on one hand, the PtdIns3P-enriched lipids via its N-terminal PX-domain, and, on the other hand, it binds uroplakins as demonstrated by co-immunoprecipitation and proximity ligation assay, and by its reduced membrane association in uroplakin II-deficient urothelium. The fact that in urothelial umbrella cells MVBs are the only major intracellular organelles enriched in both PtdIns3P and uroplakins may explain SNX31's MVB-specificity in these cells. However, in MDCK and other cultured cells transfected SNX31 can bind to early endosomes possibly via lipids. These data support a model in which SNX31 mediates the endocytic degradation of uroplakins by disassembling/collapsing the MVB-associated uroplakin plaques, thus enabling the uroplakin-containing (but 'softened') membranes to bud and form the ILVs for lysosomal degradation and/or exosome formation.
— id: 1033592, year: 2014, vol: 9, page: e99644, stat: Journal Article,

Loss of p27 upregulates MnSOD in a STAT3-dependent manner, disrupts intracellular redox activity and enhances cell migration
Zhang, Dongyun; Wang, Yulei; Liang, Yuguang; Zhang, Min; Wei, Jinlong; Zheng, Xiao; Li, Fei; Meng, Yan; Zhu, Nina Wu; Li, Jingxia; Wu, Xue-Ru; Huang, Chuanshu
2014 Apr;:2920-2933, Journal of cell science
Cell migration is a dynamic process that is central to a variety of physiological functions as well as disease pathogenesis. The modulation of cell migration by p27 has been reported, but the exact mechanism(s) whereby p27 intersects with downstream effectors that control cell migration have not been elucidated. By systematically comparing p27+/+ MEFs with genetically ablated p27-/- MEFs using wound healing, transwell and time-lapse microscopic analyses, we provide direct evidence demonstrating that p27 inhibits both directional and random cell migration. Identical results were obtained with normal and cancer epithelial cells using complementary knockdown and overexpression approaches. Additional studies revealed that overexpression of manganese superoxide dismutase (MnSOD) and reduced intracellular oxidation played a key role in increased cell migration in p27-deficient cells. Furthermore, we identified signal transducer and activator of transcription 3 (STAT3) as the transcription factor responsible for p27-regulated MnSOD expression which was further mediated by ERKs/ATF1-dependent transactivation of CRE within the stat3 promoter. Collectively, our data strongly indicate that p27 plays a crucially negative role in cell migration by inhibiting MnSOD expression in a STAT-3 dependent manner.
— id: 1055612, year: 2014, vol: , page: 2920, stat: Journal Article,

Tamm-Horsfall Protein Translocates to the Basolateral Domain of Thick Ascending Limbs, Interstitium and Circulation during Recovery from Acute Kidney Injury
El-Achkar, Tarek M; McCracken, Ruth; Liu, Yan; Heitmeier, Monique R; Bourgeois, Soline; Ryerse, Jan; Wu, Xue-Ru
2013 Feb;:F1066-F1075, American journal of physiology. Renal physiology
Tamm-Horsfall protein (THP) is a glycoprotein normally targeted to the apical membrane domain of the kidney's thick ascending limbs (TAL). We previously showed that THP of TAL confers protection to proximal tubules against acute kidney injury (AKI) via a possible cross-talk between the two functionally distinct tubular segments. However, the extent, timing, specificity and functional effects of basolateral translocation of THP during AKI remain unclear. Using an ischemia-reperfusion (IRI) model of murine AKI, we show here that, while THP expression in TAL is down-regulated at the peak of injury, it is significantly upregulated 48 hours after IRI. Confocal immunofluorescence and immunoelectron microscopy reveal a major redirection of THP during recovery from the apical membrane domain of TAL towards the basolateral domain, interstitium and basal compartment of S3 segments. This corresponds with increased THP in the serum but not in the urine. The overall epithelial polarity of TAL cells does not change, as evidenced by correct apical targeting of NKCC2 and basolateral targeting of Na+-K+-ATPase. Compared to the wild-type, THP-/- mice show a significantly delayed renal recovery after IRI, due possibly to reduced suppression by THP of pro-inflammatory cytokines and chemokines such as MCP-1 during recovery. Taken together, our data suggest that THP redistribution in the TAL after AKI is a protein-specific event, and its increased interstitial presence negatively regulates the evolving inflammatory signaling in neighboring proximal tubules, thereby enhancing kidney recovery. The increase of serum THP may be used as a prognostic biomarker for recovery from AKI.
— id: 265582, year: 2013, vol: , page: F1066, stat: Journal Article,

The DNA damage checkpoint precedes activation of ARF in response to escalating oncogenic stress during tumorigenesis
Evangelou, K; Bartkova, J; Kotsinas, A; Pateras, I S; Liontos, M; Velimezi, G; Kosar, M; Liloglou, T; Trougakos, I P; Dyrskjot, L; Andersen, C L; Papaioannou, M; Drosos, Y; Papafotiou, G; Hodny, Z; Sosa-Pineda, B; Wu, X-R; Klinakis, A; Orntoft, T; Lukas, J; Bartek, J; Gorgoulis, V G
2013 Jul;:1485-1497, Cell death & differentiation
Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16INK4A, a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic 'hits', compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload.Cell Death and Differentiation advance online publication, 12 July 2013; doi:10.1038/cdd.2013.76.
— id: 426102, year: 2013, vol: , page: 1485, stat: Journal Article,

Cyclin D1 Downregulation Contributes to Anti-Cancer Effect of Isorhapontigenin (ISO) on Human Bladder Cancer Cells
Fang, Yong; Cao, Zipeng; Hou, Qi; Ma, Chen; Yao, Chunsuo; Li, Jingxia; Wu, Xue-Ru; Huang, Chuanshu
2013 May;:924-92g, Molecular cancer therapeutics
Isorhapontigenin (ISO) is a new derivative of stilbene compound that was isolated from the Chinese herb Gnetum Cleistostachyum, and has been used for treatment of bladder cancers for centuries. In our current studies, we have explored the potential inhibitory effect and molecular mechanisms underlying ISO anti-cancer effects on anchorage-independent growth of human bladder cancer cell lines. We found that ISO showed a significant inhibitory effect on human bladder cancer cell growth and was accompanied with related cell cycle G0/G1 arrest as well as downregulation of Cyclin D1 expression at the transcriptional level in UMUC3 and RT112 cells. Further studies identified that ISO down-regulated Cyclin D1 gene transcription via inhibition of SP1 transactivation. Moreover, ectopic expression of GFP-Cyclin D1 rendered UMUC3 cells resistant to induction of cell cycle G0/G1 arrest and inhibition of cancer cell anchorage-independent growth by ISO treatment. Together, our studies demonstrate that ISO is an active compound that mediates for Gnetum Cleistostachyum's induction of cell cycle G0/G1 arrest and inhibition of cancer cell anchorage-independent growth through down-regulating SP1/Cyclin D1 axis in bladder cancer cells. Our studies provide a novel insight into understanding the anti-cancer activity of the Chinese herb Gnetum Cleistostachyum and its isolate ISO.
— id: 426052, year: 2013, vol: , page: 924, stat: Journal Article,

Decreased Tumorigenesis and Mortality from Bladder Cancer in Mice Lacking Urothelial Androgen Receptor
Hsu, Jong-Wei; Hsu, Iawen; Xu, Defeng; Miyamoto, Hiroshi; Liang, Liang; Wu, Xue-Ru; Shyr, Chih-Rong; Chang, Chawnshang
2013 Mar;:1811-1820, American journal of pathology
Much fewer mice lacking androgen receptor (AR) in the entire body develop bladder cancer (BCa). However, the role of urothelial AR (Uro-AR) in BCa development remains unclear. In the present study, we generated mice that lacked only Uro-AR (Uro-AR-/y) to develop BCa by using the carcinogen BBN [N-butyl-N-(4-hydroxybutyl)-nitrosamine] and found that Uro-AR-/y mice had a lower incidence of BCa and a higher survival rate than did their wild-type (WT; Uro-AR+/y) littermates. In vitro assay also demonstrated that Uro-AR facilitates the neoplastic transformation of normal urothelial cells to carcinoma. IHC staining exhibited less DNA damage, with much higher expression of p53 and its downstream target protein PNCA in Uro-AR-/y than that found in WT urothelium, which suggests that Uro-AR may modulate bladder tumorigenesis through p53-PCNA DNA repair signaling. Indeed, Uro-AR-/y mice with the transgene, simian vacuolating virus 40 T (SV40T), in the urothelium (Uro-SV40T-AR-/y) had a similar incidence of BCa as did their WT littermates (Uro-SV40T-AR+/y), and p53 was inactivated by SV40T in both genotypes. Use of the AR degradation enhancer ASC-J9 led to suppression of bladder tumorigenesis, with few adverse effects in the BBN-induced BCa mouse model. Together, these results provide the first direct in vivo evidence that Uro-AR has an important role in promoting bladder tumorigenesis and BCa progression. Targeting AR with ASC-J9 may provide a novel approach to suppress BCa initiation.
— id: 265562, year: 2013, vol: , page: 1811, stat: Journal Article,

Conformational inactivation induces immunogenicity of the receptor-binding pocket of a bacterial adhesin
Kisiela, Dagmara I; Rodriguez, Victoria B; Tchesnokova, Veronika; Avagyan, Hovhannes; Aprikian, Pavel; Liu, Yan; Wu, Xue-Ru; Thomas, Wendy E; Sokurenko, Evgeni V
2013 Nov;:19089-19094, Proceedings of the National Academy of Sciences of the United States of America
Inhibiting antibodies targeting receptor-binding pockets in proteins is a major focus in the development of vaccines and in antibody-based therapeutic strategies. Here, by using a common mannose-specific fimbrial adhesin of Escherichia coli, FimH, we demonstrate that locking the adhesin in a low-binding conformation induces the production of binding pocket-specific, adhesion-inhibiting antibodies. A di-sulfide bridge was introduced into the conformationally dynamic FimH lectin domain, away from the mannose-binding pocket but rendering it defective with regard to mannose binding. Unlike the native, functionally active lectin domain, the functionally defective domain was potent in inducing inhibitory monoclonal antibodies that blocked FimH-mediated bacterial adhesion to epithelial cells and urinary bladder infection in mice. Inhibition of adhesion involved direct competition between the antibodies and mannose for the binding pocket. Binding pocket-specific inhibitory antibodies also were abundant in polyclonal immune serum raised against the functionally defective lectin domain. The monoclonal antibodies elicited against the binding-defective protein bound to the high-affinity conformation of the adhesin more avidly than to the low-affinity form. However, both soluble mannose and blood plasma more strongly inhibited antibody recognition of the high-affinity FimH conformation than the low-affinity form. We propose that in the functionally active conformation the binding-pocket epitopes are shielded from targeted antibody development by ligand masking and that strong immunogenicity of the binding pocket is unblocked when the adhesive domain is in the nonbinding conformation.
— id: 600702, year: 2013, vol: , page: 19089, stat: Journal Article,

KAVA chalcone, Flavokawain A, inhibits urothelial tumorigenesis in the UPII-SV40T transgenic mouse model
Liu, Zhongbo; Xu, Xia; Li, Xuesen; Liu, Shuman; Simoneau, Anne R; He, Feng; Wu, Xue-Ru; Zi, Xiaolin
2013 Oct;:1365-1375, Cancer prevention research (Philadelphia, Pa.)
Flavokawain A (FKA) is the predominant chalcone identified from the kava plant. We have previously demonstrated that FKA preferentially inhibits the growth of p53 defective bladder cancer cell lines. Here we examined whether FKA could inhibit bladder cancer development and progression in vivo in the UPII-SV40T transgenic model that resembles human urothelial cell carcinoma (UCC) with defects in the p53 and the retinoblastoma (RB) protein pathways. Genotyped UPII-SV40T mice were fed orally with vehicle control (AIN-93M) or FKA (6 g /kg food; 0.6%) for 318 days starting at 28 days of age. More than 64% of the male mice fed with FKA-containing food survived beyond 318 days of age, whereas only about 38% of the male mice fed with vehicle control food survived to that age (p= 0.0383). The mean bladder weights of surviving male transgenic mice with the control diet versus the FKA diet were 234.6 +/- 72.5 versus 96.1+/-69.4 mg (P=0.0002). FKA was excreted primarily through the urinary tract and concentrated in the urine up to 8.4 mumol/L, averaging about 38 times (males) and 15 times (females) more concentrated than in the plasma (P=0.0001). FKA treatment inhibited the occurrence of high-grade papillary UCC, a precursor to invasive urothelial cancer, by 42.1%. A decreased expression of Ki67, survivin and XIAP and increased expression of p27 and DR5 and number of TUNEL-positive apoptotic cells were observed in the urothelial tissue of FKA-fed mice. These results suggest a potential of FKA in preventing the recurrence and progression of non-muscle invasive UCC.
— id: 600712, year: 2013, vol: , page: 1365, stat: Journal Article,

Effect of CpG methylation at different sequence context on acrolein- and BPDE-DNA binding and mutagenesis
Wang, Hsiang-Tsui; Weng, Mao-Wen; Chen, Wen-Chi; Yobin, Michael; Pan, Jishen; Chung, Fung-Lung; Wu, Xue-Ru; Rom, William; Tang, Moon-Shong
2013 Jan;34(1):220-227, Carcinogenesis
Acrolein (Acr), an alpha,beta-unsaturated aldehyde, is abundant in tobacco smoke and cooking and exhaust fumes. Acr induces mutagenic alpha- and gamma- hydroxy-1,N(2)-cyclic propano-deoxyguanosine adducts in normal human bronchial epithelial cells. Our earlier work has found that Acr-induced DNA damage preferentially occurs at lung cancer p53 mutational hotspots that contain CpG sites and that methylation at CpG sites enhances Acr-DNA binding at these sites. Based on these results, we hypothesized that this enhancement of Acr-DNA binding leads to p53 mutational hotspots in lung cancer. In this study, using a shuttle vector supF system, we tested this hypothesis by determining the effect of CpG methylation on Acr-DNA binding and the mutations in human lung fibroblasts. We found that CpG methylation enhances Acr-induced mutations significantly. Although CpG methylation enhances Acr-DNA binging at all CpG sites, it enhances mutations at selective-TCGA-sites. Similarly, we found that CpG methylation enhances benzo(a)pyrene diol epoxide binding at all -CpG- sites. However, the methylated CpG sequences in which benzo(a)pyrene diol epoxide-induced mutations are enhanced are different from the CpG sequences in which Acr-induced mutations are enhanced. CpG methylation greatly increases Acr-induced G to T and G to A mutation frequency to levels similar to these types of mutations found in the CpG sites in the p53 gene in tobacco smoke-related lung cancer. These results indicate that both CpG sequence context and the chemical nature of the carcinogens are crucial factors for determining the effect of CpG methylation on mutagenesis.
— id: 216532, year: 2013, vol: 34, page: 220, stat: Journal Article,

Uromodulin upregulates TRPV5 by impairing caveolin-mediated endocytosis
Wolf, Matthias T F; Wu, Xue-Ru; Huang, Chou-Long
2013 Mar;:130-137, Kidney international
Uromodulin (UMOD) is synthesized in the thick ascending limb and secreted into urine as the most abundant protein. Association studies in humans suggest protective effects of UMOD against calcium-containing kidney stones. Mice carrying mutations of Umod found in human UMOD-associated kidney disease (UAKD) and Umod-deficient mice exhibit hypercalciuria. The mechanism for UMOD regulation of urinary Ca2+ excretion is incompletely understood. We examined if UMOD regulates TRPV5 and TRPV6, channels critical for renal transcellular Ca2+ reabsorption. Coexpression with UMOD increased whole-cell TRPV5 current density in HEK293 cells. In biotinylation studies, UMOD increased TRPV5 cell-surface abundance. Extracellular application of purified UMOD upregulated TRPV5 current density within physiological relevant concentration ranges. UMOD exerted a similar effect on TRPV6. TRPV5 undergoes constitutive caveolin-mediated endocytosis. UMOD had no effect on TRPV5 in a caveolin-1-deficient cell line. Expression of recombinant caveolin-1 in these cells restored the ability of UMOD to upregulate TRPV5. Secretion of UAKD-mutant UMOD was markedly reduced and coexpression of mutant UMOD with TRPV5 failed to increase its current. Immunofluorescent studies demonstrated lower TRPV5 expression in Umod-/- mice compared with wild-type. UMOD upregulates TRPV5 by acting from extracellular and by decreasing endocytosis of TRPV5. The stimulation of Ca2+ reabsorption via TRPV5 by UMOD may contribute to protection against kidney-stone formation.Kidney International advance online publication, 6 March 2013; doi:10.1038/ki.2013.63.
— id: 265572, year: 2013, vol: , page: 130, stat: Journal Article,

Aristolochic acid-associated urothelial cancer in Taiwan
Chen, CH; Dickman, KG; Moriya, M; Zavadil, J; Sidorenko, VS; Edwards, KL; Gnatenko, DV; Wu, L; Turesky, RJ; Wu, XR; Pu, YS; Grollman, AP
2012 Apr;:8241-8246, Proceedings of the National Academy of Sciences of the United States of America
Aristolochic acid, a potent human carcinogen produced by Aristolochia plants, is associated with urothelial carcinoma of the upper urinary tract (UUC). Following metabolic activation, aristolochic acid reacts with DNA to form aristolactam (AL)-DNA adducts. These lesions concentrate in the renal cortex, where they serve as a sensitive and specific biomarker of exposure, and are found also in the urothelium, where they give rise to a unique mutational signature in the TP53 tumor-suppressor gene. Using AL-DNA adducts and TP53 mutation spectra as biomarkers, we conducted a molecular epidemiologic study of UUC in Taiwan, where the incidence of UUC is the highest reported anywhere in the world and where Aristolochia herbal remedies have been used extensively for many years. Our study involves 151 UUC patients, with 25 patients with renal cell carcinomas serving as a control group. The TP53 mutational signature in patients with UUC, dominated by otherwise rare A:T to T:A transversions, is identical to that observed in UUC associated with Balkan endemic nephropathy, an environmental disease. Prominent TP53 mutational hotspots include the adenine bases of (5')AG (acceptor) splice sites located almost exclusively on the nontranscribed strand. A:T to T:A mutations also were detected at activating positions in the FGFR3 and HRAS oncogenes. AL-DNA adducts were present in the renal cortex of 83% of patients with A:T to T:A mutations in TP53, FGFR3, or HRAS. We conclude that exposure to aristolochic acid contributes significantly to the incidence of UUC in Taiwan, a finding with significant implications for global public health.
— id: 164510, year: 2012, vol: , page: 8241, stat: Journal Article,

Loss of the Urothelial Differentiation Marker FOXA1 Is Associated with High Grade, Late Stage Bladder Cancer and Increased Tumor Proliferation
Degraff, David J; Clark, Peter E; Cates, Justin M; Yamashita, Hironobu; Robinson, Victoria L; Yu, Xiuping; Smolkin, Mark E; Chang, Sam S; Cookson, Michael S; Herrick, Mary K; Shariat, Shahrokh F; Steinberg, Gary D; Frierson, Henry F; Wu, Xue-Ru; Theodorescu, Dan; Matusik, Robert J
2012 ;7(5):e36669-e36669 e36669, PLoS ONE
Approximately 50% of patients with muscle-invasive bladder cancer (MIBC) develop metastatic disease, which is almost invariably lethal. However, our understanding of pathways that drive aggressive behavior of MIBC is incomplete. Members of the FOXA subfamily of transcription factors are implicated in normal urogenital development and urologic malignancies. FOXA proteins are implicated in normal urothelial differentiation, but their role in bladder cancer is unknown. We examined FOXA expression in commonly used in vitro models of bladder cancer and in human bladder cancer specimens, and used a novel in vivo tissue recombination system to determine the functional significance of FOXA1 expression in bladder cancer. Logistic regression analysis showed decreased FOXA1 expression is associated with increasing tumor stage (p<0.001), and loss of FOXA1 is associated with high histologic grade (p<0.001). Also, we found that bladder urothelium that has undergone keratinizing squamous metaplasia, a precursor to the development of squamous cell carcinoma (SCC) exhibited loss of FOXA1 expression. Furthermore, 81% of cases of SCC of the bladder were negative for FOXA1 staining compared to only 40% of urothelial cell carcinomas. In addition, we showed that a subpopulation of FOXA1 negative urothelial tumor cells are highly proliferative. Knockdown of FOXA1 in RT4 bladder cancer cells resulted in increased expression of UPK1B, UPK2, UPK3A, and UPK3B, decreased E-cadherin expression and significantly increased cell proliferation, while overexpression of FOXA1 in T24 cells increased E-cadherin expression and significantly decreased cell growth and invasion. In vivo recombination of bladder cancer cells engineered to exhibit reduced FOXA1 expression with embryonic rat bladder mesenchyme and subsequent renal capsule engraftment resulted in enhanced tumor proliferation. These findings provide the first evidence linking loss of FOXA1 expression with histological subtypes of MIBC and urothelial cell proliferation, and suggest an important role for FOXA1 in the malignant phenotype of MIBC.
— id: 167757, year: 2012, vol: 7, page: e36669, stat: Journal Article,

Uromodulin in Kidney Injury: An Instigator, Bystander, or Protector?
El-Achkar TM; Wu XR
2012 Jan 23;:452-461 #, American journal of kidney diseases
Uromodulin, also known as Tamm-Horsfall protein, is a glycoprotein expressed exclusively by renal tubular cells lining the thick ascending limb of the loop of Henle. Although the physiologic functions of this protein remain elusive, significant progress has been made during the last decade that highlights the importance of uromodulin in the pathophysiology of various diseases, such as medullary cystic kidney disease, urinary tract infections, and nephrolithiasis. Meanwhile, there is renewed interest in the role of uromodulin in kidney injury, both acute and chronic. In this article, we review the existing evidence that supports a role for uromodulin in acute kidney injury, chronic kidney disease, and renal inflammation. Contrary to the conventional view of uromodulin as an instigator in kidney injury, new data from uromodulin knockout mice show a protective role for this protein in acute kidney injury, possibly through downregulating interstitial inflammation. In chronic kidney disease, uromodulin excretion, when adjusted for kidney function, is increased; the significance of this is unclear. Although it has been suggested that uromodulin exacerbates progressive kidney injury, we propose that the elevation in uromodulin secretion is instead reactive to injury and reflects an increase of uromodulin in the renal parenchyma, where it slows the injury process
— id: 150549, year: 2012, vol: , page: 452, stat: Journal Article,

The Chinese Herb Isolate Isorhapontigenin Induces Apoptosis in Human Cancer Cells by Down-regulating Overexpression of Antiapoptotic Protein XIAP
Fang, Yong; Yu, Yonghui; Hou, Qi; Zheng, Xiao; Zhang, Min; Zhang, Dongyun; Li, Jingxia; Wu, Xue-Ru; Huang, Chuanshu
2012 Oct;287(42):35234-35243, Journal of biological chemistry
Although the Chinese herb Gnetum cleistostachyum has been used as a remedy for cancers for hundred years, the active compounds and molecular mechanisms underlying its anti-cancer activity have not been explored. Recently a new derivative of stilbene compound, isorhapontigenin (ISO), was isolated from this Chinese herb. In the present study, we examined the potential of ISO in anti-cancer activity and the mechanisms involved in human cancer cell lines. We found that ISO exhibited significant inhibitory effects on human bladder cancer cell growth that was accompanied by marked apoptotic induction as well as down-regulation of the X-linked inhibitor of apoptosis protein (XIAP). Further studies have shown that ISO down-regulation of XIAP protein expression was only observed in endogenous XIAP, but not in constitutionally exogenously expressed XIAP in the same cells, excluding the possibility of ISO regulating XIAP expression at the level of protein degradation. We also identified that ISO down-regulated XIAP gene transcription via inhibition of Sp1 transactivation. There was no significant effect of ISO on apoptosis and colony formation of cells transfected with exogenous HA-tagged XIAP. Collectively, current studies, for the first time to the best of our knowledge, identify ISO as a major active compound for the anti-cancer activity of G. cleistostachyum by down-regulation of XIAP expression and induction of apoptosis through specific targeting of a SP1 pathway, and cast new light on the treatment of the cancer patients with XIAP overexpression.
— id: 180079, year: 2012, vol: 287, page: 35234, stat: Journal Article,

Tissue-specific mutagenesis by N-butyl-N-(4-hydroxybutyl)nitrosamine as the basis for urothelial carcinogenesis
He, Zhiming; Kosinska, Wieslawa; Zhao, Zhong-Lin; Wu, Xue-Ru; Guttenplan, Joseph B
2012 Feb;742(1-2):92-95, Mutation research
Bladder cancer is one of the few cancers that have been linked to carcinogens in the environment and tobacco smoke. Of the carcinogens tested in mouse chemical carcinogenesis models, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) is one that reproducibly causes high-grade, invasive cancers in the urinary bladder, but not in any other tissues. However, the basis for such a high-level tissue-specificity has not been explored. Using mutagenesis in lacI (Big Blue) mice, we show here that BBN is a potent mutagen and it causes high-level of mutagenesis specifically in the epithelial cells (urothelial) of the urinary bladder. After a 2-6-week treatment of 0.05% BBN in the drinking water, mutagenesis in urothelial cells of male and female mice was about two orders of magnitude greater than the spontaneous mutation background. In contrast, mutagenesis in smooth muscle cells of the urinary bladder was about five times lower than in urothelial tissue. No appreciable increase in mutagenesis was observed in kidney, ureter, liver or forestomach. In lacI (Big Blue) rats, BBN mutagenesis was also elevated in urothelial cells, albeit not nearly as profoundly as in mice. This provides a potential explanation as to why rats are less prone than mice to the formation of aggressive form of bladder cancer induced by BBN. Our results suggest that the propensity to BBN-triggered mutagenesis of urothelial cells underlies its heightened susceptibility to this carcinogen and that mutagenesis induced by BBN represents a novel model for initiation of bladder carcinogenesis.
— id: 156491, year: 2012, vol: 742, page: 92, stat: Journal Article,

E3 Ligase Activity of XIAP RING Domain Is Required for XIAP-Mediated Cancer Cell Migration, but Not for Its RhoGDI Binding Activity
Liu, Jinyi; Zhang, Dongyun; Luo, Wenjing; Yu, Jianxiu; Li, Jingxia; Yu, Yonghui; Zhang, Xinhai; Chen, Jingyuan; Wu, Xue-Ru; Huang, Chuanshu
2012 ;7(4):e35682-e35682 e35682, PLoS ONE
Although an increased expression level of XIAP is associated with cancer cell metastasis, the underlying molecular mechanisms remain largely unexplored. To verify the specific structural basis of XIAP for regulation of cancer cell migration, we introduced different XIAP domains into XIAP(-/-) HCT116 cells, and found that reconstitutive expression of full length HA-XIAP and HA-XIAP DeltaBIR, both of which have intact RING domain, restored beta-Actin expression, actin polymerization and cancer cell motility. Whereas introduction of HA-XIAP DeltaRING or H467A mutant, which abolished its E3 ligase function, did not show obvious restoration, demonstrating that E3 ligase activity of XIAP RING domain played a crucial role of XIAP in regulation of cancer cell motility. Moreover, RING domain rather than BIR domain was required for interaction with RhoGDI independent on its E3 ligase activity. To sum up, our present studies found that role of XIAP in regulating cellular motility was uncoupled from its caspase-inhibitory properties, but related to physical interaction between RhoGDI and its RING domain. Although E3 ligase activity of RING domain contributed to cell migration, it was not involved in RhoGDI binding nor its ubiquitinational modification.
— id: 165633, year: 2012, vol: 7, page: e35682, stat: Journal Article,

Tamm-Horsfall protein regulates circulating and renal cytokines by affecting glomerular filtration rate and acting as a urinary cytokine trap
Liu, Y; El-Achkar, TM; Wu, XR
2012 Mar;:16365-16378, Journal of biological chemistry
Although few organ systems play a more important role than the kidneys in cytokine catabolism, the mechanism(s) regulating this pivotal physiological function and how its deficiency affects systemic cytokine homeostasis remain unclear. Here we show that elimination of Tamm-Horsfall protein (THP) expression from mouse kidneys caused a marked elevation of circulating IFN-gamma, IL1-alpha, TNF-alpha, IL6, CXCL1 and IL13. Accompanying this were enlarged spleens with prominent white-pulp macrophage infiltration. Lipopolysaccharide (LPS) exacerbated the increase of serum cytokines without a corresponding increase in their urinary excretion in THP knockout (KO) mice. This, along with the rise of serum cystatin C and the reduced inulin and creatinine clearance from the circulation, suggested that diminished glomerular filtration may contribute to reduced cytokine clearance in THP KO mice both at the baseline and under stress. Unlike wild-type mice where renal and urinary cytokines formed specific in vivo complexes with THP, this trapping effect was absent in THP KO mice, thus explaining why cytokine signaling pathways were activated in renal epithelial cells in such mice. Our study provides new evidence implicating an important role of THP in influencing cytokine clearance and acting as a decoy receptor for urinary cytokines. Based on these and other data, we present a unifying model that underscores the role of THP as a major regulator of renal and systemic immunity.
— id: 162338, year: 2012, vol: , page: 16365, stat: Journal Article,

Molecular and cellular effects of Tamm-Horsfall protein mutations and their rescue by chemical chaperones
Ma L; Liu Y; El-Achkar TM; Wu XR
2012 Jan;287(2):1290-1305, Journal of biological chemistry
Correct folding of a nascent polypeptide in the lumen of endoplasmic reticulum (ER) into a three-dimensional conformation is a crucial step in the stability, intracellular trafficking and targeting to the final destination of a protein. By transiently and stably expressing human-relevant mutants of Tamm-Horsfall protein in polarized MDCK cells, we show here that a cysteine-altering mutation in evolutionally conserved cysteine-rich domain had more severe defects in ER exit, surface translocation and triggered more apoptosis than a cysteine-altering mutation outside the domain. Both mutants were able to specifically bind and trap the wild-type THP and prevent it from exiting the ER and translocating to the cell surface. This explains at least partly why in patients with THP-associated diseases there is a marked urinary reduction of both the mutant and the wild-type THP. Exposure of mutant-expressing cells to low temperature (30(0)C), osmolytes (glycerol, trimethylamine N-oxide and dimethyl sulfoxide) and Ca2+-ATP inhibitor, thapsigargin only slightly relieved ER retention and increased surface targeting of the mutants. In contrast, sodium 4-phenylbutyrate and probenecid, the latter a uricosuric drug used clinically to treat gout, markedly reduced ER retention of the mutants and increased their surface translocation and secretion into the culture media. The rescue of the THP mutants was associated with the restoration of the level and subcellular localization of cytosolic chaperone HSP70. Our results reveal intricate mechanistic details that may underlie THP-associated diseases, and suggest that novel therapeutics enhancing the refolding of THP mutants may be of important value in therapy
— id: 145778, year: 2012, vol: 287, page: 1290, stat: Journal Article,

Effect of carcinogenic acrolein on DNA repair and mutagenic susceptibility
Wang HT; Hu Y; Tong D; Huang J; Gu L; Wu XR; Chung FL; Li GM; Tang MS
2012 Jan 24;:12379-12386 #, Journal of biological chemistry
Acrolein (Acr), a ubiquitous environmental contaminant, is a human carcinogen. Acr can react with DNA to form mutagenic alpha- and gamma-hydroxy-1, N2-cyclic propano-2-deoxyguanosine adducts (alpha-OH-Acr-dG and gamma-OH-Acr-dG). We demonstrate here that Acr-dG adducts can be efficiently repaired by the nucleotide excision repair (NER) pathway in normal human bronchial epithelia (NHBE) and lung fibroblasts (NHLF). However, the same adducts were poorly processed in cell lysates isolated from Acr-treated NHBE and NHLF, suggesting that Acr inhibits NER. In addition, we show that Acr treatment also inhibits base excision repair (BER) and mismatch repair (MMR). While Acr does not change the expression of XPA, XPC, hOGG1, PMS2 or MLH1 genes, it causes a reduction of XPA, XPC, hOGG1, PMS2 and MLH1 proteins; this effect, however, can be neutralized by the proteasome inhibitor, MG132. Acr treatment further enhances both bulky and oxidative DNA damage-induced mutagenesis. These results indicate that Acr not only damages DNA, but can also modify DNA repair proteins and further causes degradation of these modified repair proteins. We propose that these two detrimental effects contribute to Acr mutagenicity and carcinogenicity
— id: 150550, year: 2012, vol: , page: 12379, stat: Journal Article,

RhoGDI SUMOylation at K138 Increases Its Binding Activity to Rho GTPase and its Inhibiting Cancer Cell Motility
Yu, J; Zhang, D; Liu, J; Li, J; Yu, Y; Wu, XR; Huang, C
2012 Mar;:13752-13760, Journal of biological chemistry
The Rho GDP dissociation inhibitor (RhoGDI) can bind to small GTPases and keep them in a biologically inactive state in cytoplasm, through which it affects actin polymerization and cell motility. However, mechanisms underlying how RhoGDI regulates Rho GTPases complex formation/membrane extraction/GTPase dissociation remains largely unexplored. Our previous studies reported that XIAP interacted with RhoGDI via its RING domain and negatively modulated RhoGDI SUMOylation and HCT116 cancer cell migration. Here, we identified that RhoGDI SUMOylation specifically occurred at K138, which was inhibited by XIAP RING domain. We further demonstrated that RhoGDI SUMOylation at K138 was crucial for inhibiting actin polymerization and cytoskeleton formation as well as cancer cell motility. Moreover, SUMO-RhoGDI had a much higher binding affinity to small Rho GTPase compared to un-SUMOylated form of RhoGDI. Taken together, our study demonstrated a novel modification of RhoGDI, SUMOylation at K138, which played a key role in regulating Rho GTPase activation in cancer cells. The physiological regulation of RhoGDI SUMOylation by the RING domain of XIAP may account for XIAP's modulation of cancer cell invasion and metastasis.
— id: 162339, year: 2012, vol: , page: 13752, stat: Journal Article,

Urothelial tumor initiation requires deregulation of multiple signaling pathways: implications in target-based therapies
Zhou, H; Huang, HY; Shapiro, E; Lepor, H; Huang, WC; Mohammadi, M; Mohr, I; Tang, MS; Huang, C; Wu, XR
2012 Feb;:770-780, Carcinogenesis
Although formation of urothelial carcinoma of the bladder (UCB) requires multiple steps and proceeds along divergent pathways, the underlying genetic and molecular determinants for each step and pathway remain undefined. By developing transgenic mice expressing single or combinatorial genetic alterations in urothelium, we demonstrated here that overcoming oncogene-induced compensatory tumor barriers was critical for urothelial tumor initiation. Constitutively active Ha-ras (Ras*) elicited urothelial hyperplasia that was persistent and did not progress to tumors over a 10 months period. This resistance to tumorigenesis coincided with increased expression of p53 and all pRb family proteins. Expression of a Simian virus 40 T antigen (SV40T), which disables p53 and pRb family proteins, in urothelial cells expressing Ras* triggered early-onset, rapidly-growing and high-grade papillary UCB that strongly resembled the human counterpart (pTaG3). Urothelial cells expressing both Ras* and SV40T had defective G(1)/S checkpoint, elevated Ras-GTPase and hyperactivated AKT-mTOR signaling. Inhibition of the AKT-mTOR pathway with rapamycin significantly reduced the size of high-grade papillary UCB but hyperactivated mitogen-activated protein kinase (MAPK). Inhibition of AKT-mTOR, MAPK and STAT3 altogether resulted in much greater tumor reduction and longer survival than did inhibition of AKT-mTOR pathway alone. Our studies provide the first experimental evidence delineating the combinatorial genetic events required for initiating high-grade papillary UCB, a poorly defined and highly challenging clinical entity. Furthermore, they suggest that targeted therapy using a single agent such as rapamycin may not be highly effective in controlling high-grade UCB and that combination therapy employing inhibitors against multiple targets are more likely to achieve desirable therapeutic outcomes.
— id: 162340, year: 2012, vol: , page: 770, stat: Journal Article,

K-Ras and {beta}-catenin mutations cooperate with Fgfr3 mutations in mice to promote tumorigenesis in the skin and lung, but not in the bladder
Ahmad I; Singh LB; Foth M; Morris CA; Taketo MM; Wu XR; Leung HY; Sansom OJ; Iwata T
2011 Jul-Aug;4(4):548-555, Disease models & mechanisms
The human fibroblast growth factor receptor 3 (FGFR3) gene is frequently mutated in superficial urothelial cell carcinoma (UCC). To test the functional significance of FGFR3 activating mutations as a 'driver' of UCC, we targeted the expression of mutated Fgfr3 to the murine urothelium using Cre-loxP recombination driven by the uroplakin II promoter. The introduction of the Fgfr3 mutations resulted in no obvious effect on tumorigenesis up to 18 months of age. Furthermore, even when the Fgfr3 mutations were introduced together with K-Ras or beta-catenin (Ctnnb1) activating mutations, no urothelial dysplasia or UCC was observed. Interestingly, however, owing to a sporadic ectopic Cre recombinase expression in the skin and lung of these mice, Fgfr3 mutation caused papilloma and promoted lung tumorigenesis in cooperation with K-Ras and beta-catenin activation, respectively. These results indicate that activation of FGFR3 can cooperate with other mutations to drive tumorigenesis in a context-dependent manner, and support the hypothesis that activation of FGFR3 signaling contributes to human cancer
— id: 131662, year: 2011, vol: 4, page: 548, stat: Journal Article,

beta-Catenin activation synergizes with PTEN loss to cause bladder cancer formation
Ahmad, I; Morton, J P; Singh, L B; Radulescu, S M; Ridgway, R A; Patel, S; Woodgett, J; Winton, D J; Taketo, M M; Wu, X-R; Leung, H Y; Sansom, O J
2011 Jan 13;30(2):178-189, Oncogene
Although deregulation of the Wnt signalling pathway has been implicated in urothelial cell carcinoma (UCC), the functional significance is unknown. To test its importance, we have targeted expression of an activated form of beta-catenin to the urothelium of transgenic mice using Cre-Lox technology (UroIICRE(+) beta-catenin(exon3/+)). Expression of this activated form of beta-catenin led to the formation of localized hyperproliferative lesions by 3 months, which did not progress to malignancy. These lesions were characterized by a marked increase of the phosphatase and tensin homologue (PTEN) tumour suppressor protein. This appears to be a direct consequence of activating Wnt signalling in the bladder as conditional deletion of the adenomatous polyposis coli (Apc) gene within the adult bladder led rapidly to coincident beta-catenin and PTEN expression. This PTEN expression blocked proliferation. Next, we combined PTEN deficiency with beta-catenin activation and found that this caused papillary UCC. These tumours had increased pAKT signalling and were dependent on mammalian target of rapamycin (mTOR). Importantly, in human UCC, there was a significant correlation between high levels of beta-catenin and pAKT (and low levels of PTEN). Taken together these data show that deregulated Wnt signalling has a critical role in promoting UCC, and suggests that human UCC that have high levels of Wnt and PI3 kinase signalling may be responsive to mTOR inhibition
— id: 132742, year: 2011, vol: 30, page: 178, stat: Journal Article,

Ras mutation cooperates with beta-catenin activation to drive bladder tumourigenesis
Ahmad, I; Patel, R; Liu, Y; Singh, L B; Taketo, M M; Wu, X-R; Leung, H Y; Sansom, O J
2011 ;2:e124-e124, Cell death & disease
Mutations in the Ras family of proteins (predominantly in H-Ras) occur in approximately 40% of urothelial cell carcinoma (UCC). However, relatively little is known about subsequent mutations/pathway alterations that allow tumour progression. Indeed, expressing mutant H-Ras within the mouse bladder does not lead to tumour formation, unless this is expressed at high levels. The Wnt signalling pathway is deregulated in approximately 25% of UCC, so we examined if this correlated with the activation of MAPK signalling in human UCC and found a significant correlation. To test the functional significance of this association we examined the impact of combining Ras mutation (H-Ras(Q61L) or K-Ras(G12D)) with an activating beta-catenin mutation within the mouse bladder using Cre-LoxP technology. Although alone, neither Ras mutation nor beta-catenin activation led to UCC (within 12 months), mice carrying both mutations rapidly developed UCC. Mechanistically this was associated with reduced levels of p21 with dependence on the MAPK signalling pathway. Moreover, tumours from these mice were sensitive to MEK inhibition. Importantly, in human UCC there was a negative correlation between levels of p-ERK and p21 suggesting that p21 accumulation may block tumour progression following Ras mutation. Taken together these data definitively show Ras pathway activation strongly cooperates with Wnt signalling to drive UCC in vivo
— id: 132242, year: 2011, vol: 2, page: e124, stat: Journal Article,

Tamm-Horsfall Protein Deficient Thick Ascending Limbs Promote Injury to Neighboring S3 Segments in an MIP-2 Dependent Mechanism
El-Achkar TM; McCracken R; Rauchman M; Heitmeier MR; Al-Aly Z; Dagher PC; Wu XR
2011 Apr;300(4):F999-1007, American journal of physiology. Renal physiology
Tamm-Horsfall Protein (THP) is a glycoprotein expressed exclusively in thick ascending limbs (TAL) of the kidney. We recently described a novel protective role of THP against acute kidney injury (AKI) via down-regulation of inflammation in the outer medulla (OM). Our current study investigates the mechanistic relationships among the status of THP, inflammation and tubular injury. Using an ischemia-reperfusion model in wild type and THP-/- mice, we demonstrate that it is the S3 proximal segments but not the THP-deficient TAL, that are the main targets of tubular injury during AKI. The injured S3 segments that are surrounded by neutrophils in THP-/- mice have marked over-expression of neutrophil chemo-attractant MIP-2, as compared to wild-type counterparts. Neutralizing MIP-2 antibody rescues S3 segments from injury, decreases neutrophil infiltration and improves kidney function in THP-/- mice. Furthermore, using immunofluorescence volumetric imaging of wild-type mouse kidneys, we show that ischemia alters the intracellular translocation of THP in the TAL cells by partially shifting it from its default apical surface domain to the basolateral domain, the latter being contiguous to the basolateral surface of S3 segments. Concomitant with this is the upregulation, in the basolateral surface of S3 segments, of the scavenger receptor SRB-1, a putative receptor for THP. We conclude that TAL affects the susceptibility of S3 segments to injury at least in part by regulating MIP-2 expression in a THP-dependent manner. Our findings raise the interesting possibility of a direct role of basolaterally-released THP on regulating inflammation in S3 segments
— id: 120836, year: 2011, vol: 300, page: F999, stat: Journal Article,

Organ, species and sex differences in mutagenesis induced by butyl-hydroxybutyl-N-nitrosamine in big blue transgenic rodents as the basis for the strict organ specificity of this environmental carcinogen metabolite
He, Z; Strasberg-Rieber, Z -L Z; Kosinska, W; Wu, X -R; Guttenplan, J B
2011 15 Apr 2011;71(8):-, Cancer research
Although butyl-hydroxybutyl-N-nitrosamine (BBN) is a powerful carcinogen that specifically causes bladder cancer in rodents, the reason for such a high degree of organ specificity is unclear. Mutagenesis induced by BBN in a chromosomally incorporated mutagenesis reporter gene was measured in bladder urothelial cells and smooth muscle cells of mice or rats and compared with mutagenesis in liver, kidney, ureter and forestomach. BBN was administered as a 0.05% solution in water for 2 weeks followed by 4 weeks of water to 6 male and female mice and 4 male rats. All animals were euthanized at 6 weeks. Since mutagenesis in the reporter gene is a surrogate for mutagenesis in general, we hypothesized that mutations also induced in critical growth control genes would contribute to the tumor formation induced by BBN. The mutant fractions in urothelial cells were: 257 +/- 25 mutants / 105 pfu for male mice, 276 +/- 43 for female mice and 54 +/- 6 for rats. In contrast, the mutant fractions in bladder smooth muscle cells were 8-9 fold lower than these values in all animals. In non-bladder tissues such as liver, kidney, ureter and forestomach, the mutant fractions were between 2 and 5 mutants / 105 pfu, a level comparable to the control groups without BBN treatment. Hence mutagenesis correlated extremely well with the organ specificity for cancer induction by BBN. Additionally, mutagenesis level was found to be significantly higher in mice than in rats, consistent with the fact that BBN is a much more potent bladder carcinogen in mice. Finally, no significant difference in mutagenicity of BBN between male and female mice was observed, suggesting that post-initiation processes may be responsible for the gender difference in bladder tumor susceptibility. BBN is a primary metabolite of the environmental carcinogen, dibutylnitrosamine and hence a potential human carcinogen. These results provide molecular explanations to the bewildering organ and species specificity of BBN, and indicate that BBN-induced mutagenesis in the urinary bladder represents a highly appropriate model for initiation of bladder cancer, and combined with other models can help elucidate distinct steps in bladder carcinogenesis
— id: 162905, year: 2011, vol: 71, page: , stat: Journal Article,

The X-linked inhibitor of apoptosis protein (XIAP) mediates cancer cell motility Via RhoGDP dissociation inhibitor (RhoGDI)-dependent regulation of cytoskeleton
Liu J; Zhang D; Luo W; Yu Y; Yu J; Li J; Zhang X; Zhang B; Chen J; Wu XR; Rosas-Acosta G; Huang C
2011 May 6;286(18):15630-15640, Journal of biological chemistry
The X-linked inhibitor of apoptosis protein (XIAP) overexpression has been found to be associated with malignant cancer progression and aggression in individuals with many types of cancers. However, the molecular basis of XIAP in the regulation of cancer cell biological behavior remains largely unknown. In the current study, we found that the deficiency of XIAP expression in human cancer cells by approach of either knockout or knockdown led to a marked reduction of beta-Actin polymerization and cytoskeleton formation. Consistently, cell migration and invasion were also decreased in XIAP-deficient cells in comparison to those in the parental wild type cells. Subsequent studies demonstrated that the regulation of cell motility by XIAP depended on its interaction with RhoGDP dissociation inhibitor (RhoGDI) via XIAP RING domain. Furthermore, XIAP was found to negatively regulate RhoGDI SUMOylation, which might affect its activity in controlling cell motility. Collectively, our studies provide novel insights into the molecular mechanisms by which XIAP regulates cancer invasion, and offer further theoretical basis for setting XIAP as a potential prognostic marker and specific target for treatment of cancers with metastatic property
— id: 131663, year: 2011, vol: 286, page: 15630, stat: Journal Article,

The epithelium-molecular landscaping for an interactive barrier
Chai, Karl; Kitamura, Kenichiro; McCann, Amanda; Wu, Xue-Ru
2010 ;2010:870506-870506, Journal of biomedicine & biotechnology
— id: 132737, year: 2010, vol: 2010, page: 870506, stat: Journal Article,

Comparison of the pathology of interstitial plaque in human ICSF stone patients to NHERF-1 and THP-null mice
Evan, Andrew P; Weinman, Edward J; Wu, Xue-Ru; Lingeman, James E; Worcester, Elaine M; Coe, Fredric L
2010 Dec;38(6):439-452, Urological research
Extensive evidence now supports the role of papillary interstitial deposits-Randall's plaques-in the formation of stones in the idiopathic, calcium oxalate stone former. These plaques begin as deposits of apatite in the basement membranes of the thin limbs of Henle's loop, but can grow to become extensive deposits beneath the epithelium covering the papillary surface. Erosion of this covering epithelium allows deposition of calcium oxalate onto this plaque material, and the transition of mineral type and organic material from plaque to stone has been investigated. The fraction of the papilla surface that is covered with Randall's plaque correlates with stone number in these patients, as well as with urine calcium excretion, and plaque coverage also correlates inversely with urine volume and pH. Two animal models--the NHERF-1 and THP-null mice--have been shown to develop sites of interstitial apatite plaque in the renal papilla. In these animal models, the sites of interstitial plaque in the inner medulla are similar to that found in human idiopathic calcium oxalate stone formers, except that the deposits in the mouse models are not localized solely to the basement membrane of the thin limbs of Henle's loop, as in humans. This may be due to the different morphology of the human versus mouse papillary region. Both mouse models appear to be important to characterize further in order to determine how well they mimic human kidney stone disease
— id: 132736, year: 2010, vol: 38, page: 439, stat: Journal Article,

Progressive renal papillary calcification and ureteral stone formation in mice deficient for Tamm-Horsfall protein
Liu, Yan; Mo, Lan; Goldfarb, David S; Evan, Andrew P; Liang, Fengxia; Khan, Saeed R; Lieske, John C; Wu, Xue-Ru
2010 Sep;299(3):F469-F478, American journal of physiology. Renal physiology
Mammalian urine contains a range of macromolecule proteins that play critical roles in renal stone formation, among which Tamm-Horsfall protein (THP) is by far the most abundant. While THP is a potent inhibitor of crystal aggregation in vitro and its ablation in vivo predisposes one of the two existing mouse models to spontaneous intrarenal calcium crystallization, key controversies remain regarding the role of THP in nephrolithiasis. By carrying out a long-range follow-up of more than 250 THP-null mice and their wild-type controls, we demonstrate here that renal calcification is a highly consistent phenotype of the THP-null mice that is age and partially gene dosage dependent, but is gender and genetic background independent. Renal calcification in THP-null mice is progressive, and by 15 mo over 85% of all the THP-null mice develop spontaneous intrarenal crystals. The crystals consist primarily of calcium phosphate in the form of hydroxyapatite, are located more frequently in the interstitial space of the renal papillae than intratubularly, particularly in older animals, and lack accompanying inflammatory cell infiltration. The interstitial deposits of hydroxyapatite observed in THP-null mice bear strong resemblances to the renal crystals found in human kidneys bearing idiopathic calcium oxalate stones. Compared with 24-h urine from the wild-type mice, that of THP-null mice is supersaturated with brushite (calcium phosphate), a stone precursor, and has reduced urinary excretion of citrate, a stone inhibitor. While less frequent than renal calcinosis, renal pelvic and ureteral stones and hydronephrosis occur in the aged THP-null mice. These results provide direct in vivo evidence indicating that normal THP plays an important role in defending the urinary system against calcification and suggest that reduced expression and/or decreased function of THP could contribute to nephrolithiasis
— id: 138204, year: 2010, vol: 299, page: F469, stat: Journal Article,

Neuropilin-VEGF signaling pathway acts as a key modulator of vascular, lymphatic, and inflammatory cell responses of the bladder to intravesical BCG treatment
Saban, Marcia R; Sferra, Thomas J; Davis, Carole A; Simpson, Cindy; Allen, Arielle; Maier, Julie; Fowler, Ben; Knowlton, Nicholas; Birder, Lori; Wu, Xue-Ru; Saban, Ricardo
2010 Dec;299(6):F1245-F1256, American journal of physiology. Renal physiology
Recent findings indicate that VEGF receptors and coreceptors (neuropilins; NRP) are expressed on nonendothelial cells in human bladder urothelium, in one human bladder cancer cell line (J82), and in the mouse bladder urothelium. In addition, VEGFR1, VEGFR2, NRP1, and NRP2 expressions were upregulated in animal models of chronic bladder inflammation induced by four weekly instillations of protease-activated receptors (PAR)-activating peptides or bacillus Calmette-Guerin (BCG) into the mouse bladder. Here, we used four weekly instillations of BCG as a model for chronic bladder inflammation to further investigate whether VEGF receptors and NRPs play a role in the migration of inflammatory cells and inflammation-induced lymphangiogenesis and angiogenesis. For this purpose, we used neutralizing antibodies that were engineered to specifically block the binding of VEGF to NRP (anti-NRP1(B)) and the binding of semaphorins to NRP (anti-NRP1(A)). C57BL/6 mice received intraperitoneal injections of PBS, anti-NRP1(A)- or anti-NRP1(B)-neutralizing antibodies and then were challenged chronically with intravesical PBS or BCG. At the end of chronic challenge period, a fluorescent internalizable tracer, scVEGF/Cy5.5, was administered to all mice and near-infrared fluorescence images were obtained in vivo and in real time. BCG increased the overall accumulation of scVEGF/Cy5.5 in the urinary bladder urothelium and inflammatory cells. In addition, BCG increased the density of blood and lymphatic vessels concomitantly with an upregulation of NRP2 expression in lymphatic vessels. Treatment of the mice with NRP1-neutralizing antibodies dramatically reduced scVEGF/Cy5.5 uptake, polymorphonuclear (myeloperoxidase-positive cells) and dendritic cell (CD11c-positive cells) infiltration, and decreased the overall density of BCG-induced blood and lymphatic vessels. These results implicate NRPs as critical in vivo regulators of the vascular and inflammatory responses to the intravesical administration of BCG
— id: 132735, year: 2010, vol: 299, page: F1245, stat: Journal Article,

Utility of Uroplakin and PAX-2 Immunohistochemistry in the Diagnosis of Primary Adenocarcinoma of the Urinary Bladder
Ye, H; Wu, X; Hansel, DE; Melamed, J; Epstein, JI
2010 FEB ;23(3):229A-230A, Modern pathology
— id: 109937, year: 2010, vol: 23, page: 229A, stat: Journal Article,

Utility of Uroplakin and PAX-2 Immunohistochemistry in the Diagnosis of Primary Adenocarcinoma of the Urinary Bladder
Ye, H; Wu, X; Hansel, DE; Melamed, J; Epstein, JI
2010 FEB ;90(11):229A-230A, Laboratory investigation
— id: 109956, year: 2010, vol: 90, page: 229A, 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,

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,

Immunohistochemical Panel to Identify the Primary Site of Invasive Micropapillary Carcinoma
Lotan, Tamara L; Ye, Huihui; Melamed, Jonathan; Wu, Xue-Ru; Shih, Ie-Ming; Epstein, Jonathan I
2009 Jul;33(7):1037-1041, American journal of surgical pathology
Invasive micropapillary carcinoma (IMC) is generally an aggressive morphologic variant that has been described in the bladder, lung, breast, salivary gland, gastrointestinal tract, and ovary. Given the morphologic similarities between IMCs arising from different organ systems and the high propensity of this histologic subtype for lymphatic metastasis, it may be necessary to use immunohistochemical (IHC) markers to determine the primary site of an IMC. Few studies have compared the IHC profiles of IMCs originating from different sites. We tested a panel of 11 IHC markers for their ability to distinguish urothelial, lung, breast, and ovarian IMC using a tissue microarray constructed with primary tumor tissue from 47 patients with IMC (13 bladder, 6 lung, 16 breast, and 12 ovarian). For each tumor, correct classification as IMC was verified by reverse polarity MUC1 expression. We found that immunostaining for uroplakin, CK20, TTF-1, estrogen receptor (ER), WT-1 and/or PAX8, and mammaglobin was the best panel for determining the most likely primary site of IMC. The best markers to identify urothelial IMC were uroplakin and CK20, whereas p63, high molecular weight cytokeratin, and thrombomodulin were less sensitive and specific. Lung IMC was uniformly TTF-1 positive. Breast IMC was ER positive, mammaglobin positive, and PAX8/WT-1 negative, while ovarian IMC was ER positive, mammaglobin negative, and PAX8/WT-1 positive. In the metastatic setting, or when IMC occurs without an associated in situ or conventional carcinoma component, staining for uroplakin, CK20, TTF-1, ER and WT-1, and/or PAX8, and mammaglobin is the best panel for accurately classifying the likely primary site of IMC
— id: 95436, year: 2009, vol: 33, page: 1037, stat: Journal Article,

Biology of urothelial tumorigenesis: insights from genetically engineered mice
Wu, Xue-Ru
2009 Dec;28(3-4):281-290, Cancer metastasis reviews
Urothelium, one of the slowest cycling epithelia in the body, embodies a unique biological context for cellular transformation. Introduction of oncogenes into or removing tumor suppressor genes from the urothelial cells or a combination of both using the transgenic and/or knockout mouse approaches has provided useful insights into the molecular mechanisms of urothelial transformation and tumorigenesis. It is becoming increasingly clear that over-activation of the receptor tyrosine kinase (RTK) pathway, as exemplified by the constitutively activated Ha-ras oncogene, is both necessary and sufficient to initiate the low-grade, non-invasive urothelial carcinomas. Dosage of the mutated Ha-ras, but not concurrent inactivation of pro-senescence molecules p16Ink4a and p19Arf, dictates whether and when the low-grade urothelial carcinomas arise. Inactivation of both p53 and pRb, a prevailing paradigm previously proposed for muscle-invasive urothelial tumorigenesis, is found to be necessary but insufficient to initiate this urothelial carcinoma variant. Instead, downregulation in p53/pRb co-deficient urothelial cells of p107, a pRb family member, is associated with the genesis of the muscle-invasive bladder cancers. p53 deficiency also seems to be capable of cooperating with that of PTEN in eliciting invasive urothelial carcinomas. The genetically engineered mice have improved the molecular definition of the divergent pathways of urothelial tumorigenesis and progression, helped delineate the intricate crosstalk among different genetic alterations within a urothelium-specific context, identified new prognostic markers and novel therapeutic targets potentially applicable for clinical intervention, and provided in vivo platforms for testing preventive strategies of bladder cancer
— id: 106275, year: 2009, vol: 28, page: 281, 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,

Tamm-Horsfall protein protects the kidney from ischemic injury by decreasing inflammation and altering TLR4 expression
El-Achkar, Tarek M; Wu, Xue-Ru; Rauchman, Michael; McCracken, Ruth; Kiefer, Susan; Dagher, Pierre C
2008 Aug;295(2):F534-F544, American journal of physiology. Renal physiology
Tamm-Horsfall protein (THP) is a glycoprotein with unclear functions expressed exclusively in thick ascending limbs (TAL) of the kidney. Its role in ischemic acute kidney injury is uncertain, with previous data suggesting a possible negative effect by enhancing cast formation and promoting inflammation. Using a recently characterized THP knockout mouse (THP-/-), we investigated the role of THP in renal ischemia-reperfusion injury (IRI). In wild-type mice (THP+/+), THP expression was increased by injury. THP-/- mice developed more functional and histological renal damage after IRI compared with THP+/+. THP-/- kidneys showed more inflammation and tubular necrosis. Cast formation correlated with the severity of injury and was independent of THP presence. THP absence was associated with a more necrotic, rather than apoptotic, phenotype of cell death. The outer medulla was predominantly affected, where significant interstitial neutrophil infiltration was detected in proximity to injured S3 proximal tubular segments and TAL. This coincided with an enhanced expression of the innate immunity receptor Toll-like receptor 4 (TLR4) in S3 segments of THP-/- compared with THP+/+ mice. Specifically, a basolateral S3 expression of TLR4 was more evident in THP-/- kidneys compared with a more apical distribution in THP+/+. Such basolateral location for TLR4 allows a greater interaction with proinflammatory ligands present in the interstitium during ischemia. In conclusion, we are showing a completely novel role for a very old protein in the setting of renal injury. Our data suggest that THP stabilizes the outer medulla in the face of injury by decreasing inflammation, possibly through an effect on TLR4
— id: 132739, year: 2008, vol: 295, page: F534, stat: Journal Article,

VEGF receptors and neuropilins are expressed in the urothelial and neuronal cells in normal mouse urinary bladder and are upregulated in inflammation
Saban, Marcia R; Backer, Joseph M; Backer, Marina V; Maier, Julie; Fowler, Ben; Davis, Carole A; Simpson, Cindy; Wu, Xue-Ru; Birder, Lori; Freeman, Michael R; Soker, Shay; Hurst, Robert E; Saban, Ricardo
2008 Jul;295(1):F60-F72, American journal of physiology. Renal physiology
Recent evidence supports a role for vascular endothelium growth factor (VEGF) signaling in bladder inflammation. However, it is not clear what bladder cells are targeted by VEGF. Therefore, we determined the nature of cells responding to VEGF in normal and inflamed bladders by tagging such cells in vivo with a targeted fluorescent tracer, scVEGF/Cy, an engineered single-chain VEGF labeled with Cy5.5 dye, which identifies cells with accessible and functionally active VEGF receptors. Inflammation was induced by intravesical instillation of PAR-activating peptides or BCG. In vivo NIRF imaging with intravenously injected scVEGF/Cy revealed accumulation of the tracer in the control mouse bladder and established that inflammation increased the steady-state levels of tracer uptake. Ex vivo colocalization of Cy5.5 dye revealed that in normal and at a higher level in inflamed bladder, accumulation of scVEGF/Cy occurs in both urothelial and ganglial cells, expressing VEGF receptors VEGFR-1 and VEGFR-2, as well as VEGF coreceptors neuropilins (NRP) NRP1 and NRP2. PCR results indicate that the messages for VEGF-Rs and NRPs are present in the bladder mucosa and ChIP/QPCR analysis indicated that inflammation induced upregulation of genes encoding VEGFRs and NRPs. Our results strongly suggest new and blossoming VEGF-driven processes in bladder urothelial cells and ganglia in the course of inflammation. We expect that molecular imaging of the VEGF pathway in the urinary tract by receptor-mediated cell tagging in vivo will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of bladder-targeting drugs and treatments
— id: 132740, year: 2008, vol: 295, page: F60, stat: Journal Article,

Molecular networks discriminating mouse bladder responses to intravesical bacillus Calmette-Guerin (BCG), LPS, and TNF-alpha
Saban, Marcia R; O'Donnell, Michael A; Hurst, Robert E; Wu, Xue-Ru; Simpson, Cindy; Dozmorov, Igor; Davis, Carole; Saban, Ricardo
2008 ;9:4-4, BMC immunology
BACKGROUND: Despite being a mainstay for treating superficial bladder carcinoma and a promising agent for interstitial cystitis, the precise mechanism of Bacillus Calmette-Guerin (BCG) remains poorly understood. It is particularly unclear whether BCG is capable of altering gene expression in the bladder target organ beyond its well-recognized pro-inflammatory effects and how this relates to its therapeutic efficacy. The objective of this study was to determine differentially expressed genes in the mouse bladder following chronic intravesical BCG therapy and to compare the results to non-specific pro inflammatory stimuli (LPS and TNF-alpha). For this purpose, C57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-alpha. Seven days after the last instillation, the urothelium along with the submucosa was removed from detrusor muscle and the RNA was extracted from both layers for cDNA array experiments. Microarray results were normalized by a robust regression analysis and only genes with an expression above a conditional threshold of 0.001 (3SD above background) were selected for analysis. Next, genes presenting a 3-fold ratio in regard to the control group were entered in Ingenuity Pathway Analysis (IPA) for a comparative analysis in order to determine genes specifically regulated by BCG, TNF-alpha, and LPS. In addition, the transcriptome was precipitated with an antibody against RNA polymerase II and real-time polymerase chain reaction assay (Q-PCR) was used to confirm some of the BCG-specific transcripts. RESULTS: Molecular networks of treatment-specific genes generated several hypotheses regarding the mode of action of BCG. BCG-specific genes involved small GTPases and BCG-specific networks overlapped with the following canonical signaling pathways: axonal guidance, B cell receptor, aryl hydrocarbon receptor, IL-6, PPAR, Wnt/beta-catenin, and cAMP. In addition, a specific detrusor network expressed a high degree of overlap with the development of the lymphatic system. Interestingly, TNF-alpha-specific networks overlapped with the following canonical signaling pathways: PPAR, death receptor, and apoptosis. Finally, LPS-specific networks overlapped with the LPS/IL-1 mediated inhibition of RXR. Because NF-kappaB occupied a central position in several networks, we further determined whether this transcription factor was part of the responses to BCG. Electrophoretic mobility shift assays confirmed the participation of NF-kappaB in the mouse bladder responses to BCG. In addition, BCG treatment of a human urothelial cancer cell line (J82) also increased the binding activity of NF-kappaB, as determined by precipitation of the chromatin by a NF-kappaB-p65 antibody and Q-PCR of genes bearing a NF-kappaB consensus sequence. Next, we tested the hypothesis of whether small GTPases such as LRG-47 are involved in the uptake of BCG by the bladder urothelium. CONCLUSION: As expected, BCG treatment induces the transcription of genes belonging to common pro-inflammatory networks. However, BCG also induces unique genes belonging to molecular networks involved in axonal guidance and lymphatic system development within the bladder target organ. In addition, NF-kappaB seems to play a predominant role in the bladder responses to BCG therapy. Finally, in intact urothelium, BCG-GFP internalizes in LRG-47-positive vesicles. These results provide a molecular framework for the further study of the involvement of immune and nervous systems in the bladder responses to BCG therapy
— id: 132741, year: 2008, vol: 9, page: 4, stat: Journal Article,

Urothelial expression of neuropilins and VEGF receptors in control and interstitial cystitis patients
Saban, Ricardo; Saban, Marcia R; Maier, Julie; Fowler, Ben; Tengowski, Mark; Davis, Carole A; Wu, Xue-Ru; Culkin, Daniel J; Hauser, Paul; Backer, Joseph; Hurst, Robert E
2008 Dec;295(6):F1613-F1623, American journal of physiology. Renal physiology
Interstitial cystitis (IC) is a chronic and painful bladder syndrome of unknown cause with no reliable biological marker or effective therapy. Vascular endothelial growth factor (VEGF), which plays a key role in bladder inflammation, is closely associated with the vascular alterations observed in patients with IC. However, our recent findings of VEGF receptors (VEGF-Rs) and VEGF coreceptors on nonendothelial cells in human and mouse urothelium suggest that additional VEGF targets and functions are possible in IC bladders. We report here that VEGF-Rs and coreceptors (neuropilins; NRP) are strongly expressed in both the human bladder urothelium and in the human bladder cancer cell line (J82) and that the expression of NRP2 and VEGF-R1 is significantly downregulated in IC compared with control subjects. In addition, treatment of J82 cells with bacillus Calmette-Guerin (BCG), a novel treatment strategy for IC, upregulates the messages for NRPs and VEGF-Rs. Furthermore, intravesical instillation of an internalizable VEGF fluorescent tracer (scVEGF/Cy5.5) into mouse urinary bladders results in a marked ligand accumulation in the urothelium and bladder parenchyma, indicating that urothelial VEGF-Rs are functionally active and capable of ligand interaction and internalization. Our results suggest that the VEGF pathway is altered in IC, that urinary VEGF may gain access to the bladder wall via these receptors, and that BCG treatment may replenish the missing VEGF-Rs/NRP receptors. Together, these results suggest that levels of NRPs, VEGF-Rs, and VEGF are new putative markers for the diagnosis of IC and that modulating these receptors can be exploited as therapeutic strategies
— id: 132738, year: 2008, vol: 295, page: F1613, stat: Journal Article,

Oncogene-induced replication stress preferentially targets common fragile sites in preneoplastic lesions. A genome-wide study
Tsantoulis, P K; Kotsinas, A; Sfikakis, P P; Evangelou, K; Sideridou, M; Levy, B; Mo, L; Kittas, C; Wu, X-R; Papavassiliou, A G; Gorgoulis, V G
2008 May 22;27(23):3256-3264, Oncogene
Common fragile sites (CFSs) are regions of the genome prone to breakage by replication inhibitors (extrinsic replication stress). Recently, we and others observed that oncogene-induced replication stress (RS) induces DNA damage from the earliest stages of cancer. Our aim was to perform a genome-wide analysis in precancerous and cancerous experimental models to examine whether allelic imbalance occurs within CFSs. Subsequently, CFSs sequence characteristics were assessed. We used a growth-factor-induced human skin hyperplasia and a H-ras-induced mouse hyperplastic urothelium as preneoplastic models, along with an inducible U2OS-CDT1(Tet-ON) cancer cell line model, all bearing established oncogene-induced RS stimuli. Human DNA was analysed with Affymetrix SNP microarrays, while mouse DNA was analysed with Nimblegen array CGH. We studied 56 aphidicolin-type CFSs and 1914 regions of control, nonfragile DNA. Our theoretical in silico analysis spanned 2.16 billion nonoverlapping bases on human chromosomes 1-22. Our results provide direct experimental evidence indicating that genomic alterations were more common within CFSs in epidermal and urothelial preneoplastic lesions as well as in cancer. CFSs were on average less flexible than nonfragile regions, contained more guanine-cytosine (GC) and Alu sequences. Importantly, regions with loss-of-heterozygosity were also less flexible and had a higher Alu percentage.Oncogene advance online publication, 17 December 2007; doi:10.1038/sj.onc.1210989
— id: 75193, year: 2008, vol: 27, page: 3256, stat: Journal Article,

Loss of Urinary Macromolecules in Mice Causes Interstitial and Intratubular Renal Calcification Dependent on the Underlying Conditions
Wu, XR; Lieske, JC; Evan, AP; Sommer, AJ; Liaw, L; Mo, L
2008 NOV 25 ;1049(22):113-119, AIP conference proceedings
Urinary protein macromolecules have long been thought to play a role in influencing the various phases of urolithiasis including nucleation, growth, aggregation of mineral crystals and their subsequent adhesion to the renal epithelial cells. However, compelling evidence regarding their precise role was lacking, due partly to the fact that most prior studies were done it? vitro and results were highly variable depending on the experimental conditions. The advent of genetic engineering technology has made it possible to study urinary protein macromolecules within an in vivo biological system. Indeed, recent studies have begun to shed light on the net effects of loss of one or more macromolecules on the earliest steps of urolithiasis. This paper focuses on the in vivo consequences of inactivating Tamm-Horsfall protein and/or osteopontin, two major urinary glycoproteins, using the knockout approach. The renal phenotypes of both single and double knockout mice under spontaneous or hyperoxaluric conditions will be described. The functional significance of the urinary macromolecules as critical defense factors against renal calcification will also be discussed
— id: 91289, year: 2008, vol: 1049, page: 113, 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,

Decreased DOC-2/DAB2 expression in urothelial carcinoma of the bladder
Karam, Jose A; Shariat, Shahrokh F; Huang, Hong-Ying; Pong, Rey-Chen; Ashfaq, Raheela; Shapiro, Ellen; Lotan, Yair; Sagalowsky, Arthur I; Wu, Xue-Ru; Hsieh, Jer-Tsong
2007 Aug 1;13(15 Pt 1):4400-4406, Clinical cancer research
PURPOSE: DOC-2/DAB2 (differentially expressed in ovarian carcinoma-2/disabled-2), a potential tumor suppressor gene, is underexpressed in several cancers. Little is known about the expression of this gene in urothelial carcinoma of the bladder (UCB). We profiled DOC-2/DAB2 expression in mouse and human normal and neoplastic urothelia. EXPERIMENTAL DESIGN: Immunohistochemical staining for DOC-2/DAB2 was carried out on tissue specimens from two transgenic mouse models with urothelium-specific molecular alterations and on a tissue microarray containing cores from 9 normal controls, 44 patients who underwent transurethral resection of the bladder tumor (TURBT), 195 patients who underwent radical cystectomy for UCB, and 39 lymph nodes with metastatic UCB. RESULTS: Normal mouse urothelium stained uniformly with DOC-2/DAB2. Weaker staining was observed in low-grade, superficial papillary bladder tumors from transgenic mice harboring constitutively active Ha-Ras, whereas carcinoma in situ-like lesions and high-grade bladder tumors from transgenic mice expressing a SV40 T antigen completely lacked DOC-2/DAB2 expression. In human tissues, DOC-2/DAB2 expression was decreased in 11% of normal bladder specimens, 59% of TURBT specimens, 65% of radical cystectomy specimens, and 77% of the metastatic lymph node specimens. Decreased DOC-2/DAB2 expression was associated with advanced pathologic stage (P = 0.023), lymph node metastases (P = 0.050), and lymphovascular invasion (P < 0.001). In univariable, but not in multivariable analysis, decreased DOC-2/DAB2 was associated with an increased probability of bladder cancer recurrence (log-rank test, P = 0.020) and bladder cancer-specific mortality (log-rank test, P = 0.023). CONCLUSIONS: Decreased DOC-2/DAB2 expression seems to occur early in bladder tumorigenesis and becomes more prominent in advanced stages of UCB
— id: 73405, year: 2007, vol: 13, page: 4400, stat: Journal Article,

Renal Calcinosis and Stone Formation in Mice Lacking Osteopontin, Tamm-Horsfall Protein or Both
Mo, Lan; Liaw, Lucy; Evan, Andrew P; Sommer, Andre J; Lieske, John C; Wu, Xue-Ru
2007 Dec;293(6):F1935-F1943, American journal of physiology. Renal physiology
Although often supersaturated with mineral salts such as calcium phosphate and calcium oxalate, normal urine possesses an innate ability to keep them from forming harmful crystals. This inhibitory activity has been attributed to the presence of urinary macromolecules, although controversies abound regarding their role, or lack thereof, in preventing renal mineralization. Here we show that 10% of the mice lacking osteopontin (OPN) and 14.3% of the mice lacking Tamm-Horsfall protein (THP) spontaneously form interstitial deposits of calcium phosphate within the renal papillae, events never seen in wild-type mice. Lack of both proteins causes renal crystallization in 39.3% of the double null mice. Urinalysis revealed elevated concentrations of urine phosphorus and brushite (calcium phosphate) supersaturation in THP-null and OPN/THP-double null mice, suggesting that impaired phosphorus handling may be linked to interstitial papillary calcinosis in THP-, but not in OPN-null mice. In contrast, experimentally induced hyperoxaluria provokes widespread intratubular calcium oxalate crystallization and stone formation in OPN/THP double null mice, while completely sparing the wild-type controls. Whole urine from OPN-, THP-, or double-null mice all possessed a dramatically reduced ability to inhibit the adhesion of calcium oxalate monohydrate crystals to renal epithelial cells. These data establish OPN and THP as powerful and functionally synergistic inhibitors of calcium phosphate and calcium oxalate crystallization in vivo, and suggest that defects in either molecule may contribute to renal calcinosis and stone formation, an exceedingly common condition that afflicts up to 12% males and 5% females. Key words: nephrolithiasis, Tamm-Horsfall protein, kidney stone
— id: 74321, year: 2007, vol: 293, page: F1935, 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,

Repeated BCG treatment of mouse bladder selectively stimulates small GTPases and HLA antigens and inhibits single-spanning uroplakins
Saban, Marcia R; Hellmich, Helen L; Simpson, Cindy; Davis, Carole A; Lang, Mark L; Ihnat, Michael A; O'Donnell, Michael A; Wu, Xue-Ru; Saban, Ricardo
2007 Nov 2;7(1):204-204, BMC cancer
ABSTRACT: Background Despite being a mainstay for treating superficial bladder carcinoma and a promising agent for interstitial cystitis, the precise mechanism of Bacillus Calmette-Guerin (BCG) remains poorly understood. It is particularly unclear whether BCG is capable of altering gene expression beyond its well-recognized pro-inflammatory effects and how this relates to its therapeutic efficacy. The objective of this study was to determine differentially expressed genes in the mouse bladder following repeated intravesical BCG therapy. Methods Mice were transurethrally instilled with BCG or pyrogen-free on days 1, 7, 14, and 21. Seven days after the last instillation, urothelia along with the submucosa was removed and amplified ds-DNA was prepared from control- and BCG-treated bladder mucosa and used to generate suppression subtractive hybridization (SSH). Plasmids from control- and BCG-specific differentially expressed clones and confirmed by Virtual Northern were then purified and the inserts were sequenced and annotated. Finally, chromatin immune precipitation combined with real-time polymerase chain reaction assay (ChIP/Q-PCR) was used to validate SSH-selected transcripts. Results Repeated intravesical BCG treatment induced an up regulation of genes associated with antigen presentation (B2M, HLA-A, HLA-DQA1, HLA-DQB2, HLA-E, HLA-G, IGHG, and IGH) and representatives of two IFN-induced small GTPase families: the GBPs (GBP1, GBP2, and GBP5) and the p47GTPases (IIGTP1, IIGTP2, and TGTP). Genes expressed in saline-treated bladders but down-regulated by BCG included: the single-spanning uroplakins (UPK3a and UPK2), SPRR2G, GSTM5, and RSP 19. Conclusion Here we introduced a hypothesis-generator approach to determine key genes involved in the urothelium/sumbmucosa responses to BCG therapy. Urinary bladder responds to repeated BCG treatment by up-regulating not only antigen presentation-related genes, but also GBPs and p47 small GTPases, both potentially serving to mount a resistance to the replication of the Mycobacterium. It will be of tremendous future interest to determine whether these immune response cascades play a role in the anti-cancer effects exerted by BCG
— id: 75191, year: 2007, vol: 7, page: 204, stat: Journal Article,

Lymphatic vessel density and function in experimental bladder cancer
Saban, Marcia R; Towner, Rheal; Smith, Nataliya; Abbott, Andrew; Neeman, Michal; Davis, Carole A; Simpson, Cindy; Maier, Julie; Memet, Sylvie; Wu, Xue-Ru; Saban, Ricardo
2007 Nov 29;7(1):219-219, BMC cancer
ABSTRACT: The lymphatics form a second circulatory system that drains the extracellular fluid and proteins from the tumor microenvironment, and provides an exclusive environment in which immune cells interact and respond to foreign antigen. Both cancer and inflammation are known to induce lymphangiogenesis. However, little is known about bladder lymphatic vessels and their involvement in cancer formation and progression. METHODS: A double transgenic mouse model was generated by crossing a bladder cancer-induced transgenic, in which SV40 large T antigen was under the control of uroplakin II promoter, with another transgenic mouse harboring a lacZ reporter gene under the control of an NF-kB-responsive promoter (kB-lacZ) exhibiting constitutive activity of b-galactosidase in lymphatic endothelial cells. In this new mouse model (SV40-lacZ), we examined the lymphatic vessel density (LVD) and function (LVF) during bladder cancer progression. LVD was performed in bladder whole mounts and cross-sections by fluorescent immunohistochemistry (IHC) using LYVE-1 antibody. LVF was assessed by real-time in vivo imaging techniques using a contrast agent (biotin-BSA-Gd-DTPA-Cy5.5; Gd-Cy5.5) suitable for both magnetic resonance imaging (MRI) and near infrared fluorescence (NIRF). In addition, IHC of Cy5.5 was used for time-course analysis of co-localization of Gd-Cy5.5 with LYVE-1-positive lymphatics and CD31-positive blood vessels. RESULTS: SV40-lacZ mice develop bladder cancer and permitted visualization of lymphatics. A significant increase in LVD was found concomitantly with bladder cancer progression. Double labeling of the bladder cross-sections with LYVE-1 and Ki-67 antibodies indicated cancer-induced lymphangiogenesis. MRI detected mouse bladder cancer, as early as 4 months, and permitted to follow tumor sizes during cancer progression. Using Gd-Cy5.5 as a contrast agent for MRI-guided lymphangiography, we determined a possible reduction of lymphatic flow within the tumoral area. In addition, NIRF studies of Gd-Cy5.5 confirmed its temporal distribution between CD31-positive blood vessels and LYVE-1 positive lymphatic vessels. CONCLUSIONS: SV40-lacZ mice permit the visualization of lymphatics during bladder cancer progression. Gd-Cy5.5, as a double contrast agent for NIRF and MRI, permits to quantify delivery, transport rates, and volumes of macromolecular fluid flow through the interstitial-lymphatic continuum. Our results open the path for the study of lymphatic activity in vivo and in real time, and support the role of lymphangiogenesis during bladder cancer progression
— id: 75190, year: 2007, vol: 7, page: 219, stat: Journal Article,

Early detection and measurement of urothelial tumors in mice
Johnson, Aimee M; Conover, David L; Huang, Jiaoti; Messing, Edward M; Ning, Ruola; O'Connell, Mary J; Rossi, M Adrian; Sun, Tung-Tien; Wood, Ronald W; Wu, Xue-Ru; Reeder, Jay E
2006 Jun;67(6):1309-1314, Urology
OBJECTIVES: To establish reliable noninvasive in vivo methods to detect, measure, and monitor experimentally induced urothelial tumors in mice. METHODS: UPII-SV40T transgenic mice reliably develop bladder tumors by expression of simian virus 40 large T antigen specifically in bladder urothelium through the use of the uroplakin II promoter. Two wild-type and 10 UPII-SV40T transgenic mice were monitored for microhematuria two to three times weekly using dipstick analysis. A unique flat panel detector-based cone beam computed tomography (FPD-CBCT) system imaged the urinary tracts of anesthetized mice after tail vein injection of an iodinated contrast agent (Omnipaque) that is excreted in urine. Within 10 seconds, the FPD-CBCT system acquired 290 two-dimensional images, which produced three-dimensional volumes with true isotropic resolution (180 microm)3 using a filtered back projection-based modified Feldkamp reconstruction algorithm. Amira, version 3.1.1-1, for MacOSX was used for data analysis and advanced visualization of the three-dimensional reconstructed FPD-CBCT images. RESULTS: Hematuria was present in UPII-SV40T transgenic mice at 32 days of age; the wild-type animals exhibited no hematuria. Filling defects, associated with histologically confirmed tumors, in the bladders of the UPII-SV40T transgenic mice were visualized in the reconstructed FPD-CBCT images 1 to 45 minutes after contrast agent injection. Longitudinal FPD-CBCT imaging sessions showed the tumor position, volume, and growth. CONCLUSIONS: The combination of early detection of hematuria and high-resolution in vivo FPD-CBCT imaging of murine bladder tumors enabled accurate longitudinal assessment of tumor growth and progression in individual animals. This approach could provide an important alternative to serial sacrifice experimental designs, while refining statistical power and reducing animal use
— id: 66475, year: 2006, vol: 67, page: 1309, stat: Journal Article,

Gene deletion in urothelium by specific expression of Cre recombinase
Mo L; Cheng J; Lee EY; Sun TT; Wu XR
2006 ;175(4):1569-, Journal of urology
— id: 62773, year: 2006, vol: 175, page: 1569, 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,

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,

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,

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,

IMMUNOLOCALIZATION OF ESTROGEN RECEPTOR alpha AND beta IN HUMAN FETAL PROSTATE
Shapiro, Ellen; Huang, Hongying; Masch, Rachel J; McFadden, Deborah E; Wilson, E Lynette; Wu, Xue-Ru
2005 Nov;174(5):2051-2053, Journal of urology
PURPOSE:: We examined the immunolocalization of estrogen receptor (ER)alpha and ERbeta in the human fetal prostate. MATERIALS AND METHODS:: Tissue sections from human fetal prostates at 7 to 22 weeks of gestation were stained with antibodies to ERalpha, ERbeta, and cytokeratin 10 and 14. RESULTS:: ERalpha expression was not detected until 15 weeks of gestation with sparse staining in the utricle. By 19 weeks increased ERalpha expression was seen in the luminal cells of the ventral urogenital epithelium (UGE), basal cells of the dorsal UGE, utricle, distal periurethral ducts, peripheral stroma and posterior prostatic duct. K14 was detected in basal cells of the UGE and in several posterior acini. At 22 weeks ERalpha expression was more intense in all of these areas. ERbeta was expressed throughout the UGE, ejaculatory ducts, mullerian ducts and entire stroma at 7 weeks. Intense ERbeta staining was observed in these areas and in the prostatic buds by 8 weeks with persistent intense staining through 22 weeks. CONCLUSIONS:: To our knowledge we report the first immunolocalization of ERalpha in the human fetal prostate and the earliest demonstration of ERbeta expression in the prostate at 7 weeks of gestation. ERbeta expression is intense during ductal morphogenesis, suggesting a role in normal glandular growth and proliferation. The induction of squamous metaplasia in the UGE, distal periurethral ducts and utricle is associated with ERalpha expression in these areas, while the induction of squamous metaplasia in peripheral prostatic acini is associated with peripheral stromal ERalpha expression. This study suggests estrogen signaling pathways in the human fetal prostate via ERalpha that involve epithelial-epithelial and epithelial-stromal interactions
— id: 58459, year: 2005, vol: 174, page: 2051, stat: Journal Article,

IMMUNOLOCALIZATION OF ANDROGEN RECEPTOR AND ESTROGEN RECEPTORS alpha AND beta IN HUMAN FETAL TESTIS AND EPIDIDYMIS
Shapiro, Ellen; Huang, Hongying; Masch, Rachel J; McFadden, Deborah E; Wu, Xue-Ru; Ostrer, Harry
2005 Oct;174(4, Part 2 of 2):1695-8; discussion 1698, Journal of urology
PURPOSE:: Expression and cellular localization of the androgen receptor (AR) and estrogen receptor (ER) isoforms were determined using antibodies specific to these receptors and to specific cell types. MATERIALS AND METHODS:: Gonads and genitourinary structures were removed from 5 human male fetuses 7 to 22 weeks of gestational age. Sections were stained with antibodies to AR, ERalpha and ERbeta, P450 scc and smooth muscle actin. RESULTS:: AR was present in undifferentiated gonadal cells, peritubular myoid cells and in some Leydig and stromal cells at 7 weeks of gestation. The number of AR positive peritubular myoid cells remained constant through 22 weeks of gestation but the number of AR positive stromal cells continued to increase through 22 weeks. ERalpha was apparent by 12 weeks of gestation with perinuclear staining of Leydig cells, peaked at 16 weeks and then diminished. ERbeta was first observed at 7 weeks in undifferentiated gonadal cells. By 12 weeks of gestation ERbeta was apparent in germ cells, PTMC and Leydig cells. In the epididymis AR was expressed in the epithelium and stroma of the efferent ductules and the ductus epididymis by 7 weeks of gestation with increased expression by 12 weeks. A similar pattern of staining was observed for ERbeta. By contrast, staining of ERalpha was observed only in the epithelium of the epididymis from 7 weeks of gestation onward with no apparent ERalpha staining in the tail of the epididymis. CONCLUSIONS:: These findings are compatible with the well-known roles of androgen signaling in sexual differentiation and spermatogenesis in humans. The role of estrogens in the developing human testis and epididymis remains unknown
— id: 57793, year: 2005, vol: 174, page: 1695, stat: Journal Article,

Urothelial tumorigenesis: a tale of divergent pathways
Wu, Xue-Ru
2005 Sep;5(9):713-725, Nature reviews. Cancer
Urothelial carcinoma of the bladder is unique among epithelial carcinomas in its divergent pathways of tumorigenesis. Low-grade papillary tumours rarely become muscle-invasive and they frequently harbour gene mutations that constitutively activate the receptor tyrosine kinase-Ras pathway. By contrast, most high-grade invasive tumours progress to life-threatening metastases and have defects in the p53 and the retinoblastoma protein pathways. Correcting pathway-specific defects represents an attractive strategy for the molecular therapy of urothelial carcinomas
— id: 57725, year: 2005, vol: 5, page: 713, stat: Journal Article,

Variation of high mannose chains of Tamm-Horsfall glycoprotein confers differential binding to type 1-fimbriated Escherichia coli
Cavallone, Daniela; Malagolini, Nadia; Monti, Angela; Wu, Xue-Ru; Serafini-Cessi, Franca
2004 Jan 2;279(1):216-222, Journal of biological chemistry
Tamm-Horsfall glycoprotein (THP), the most abundant protein in mammalian urine, has been implicated in defending the urinary tract against infections by type 1-fimbriated Escherichia coli. Recent experimental evidence indicates that the defensive capability of THP relies on its single high mannose chain, which binds to E. coli FimH lectin and competes with mannosylated uroplakin receptors on the bladder surface. Here we describe several major differences, on both structural and functional levels, between human THP (hTHP) and pig THP (pTHP). pTHP contains a much higher proportion (47%) of Man5GlcNAc2 than does hTHP (8%). FimH-expressing E. coli adhere to monomeric pTHP at an approximately 3-fold higher level than to monomeric hTHP. This suggests that the shorter high mannose chain (Man5GlcNAc2) is a much better binder for FimH than the longer chains (Man6-7GlcNAc2) and that pTHP is a more potent urinary defense factor than hTHP. In addition, unlike hTHP whose polyantennary glycans are exclusively capped by sialic acid and sulfate groups, those of pTHP are also terminated by Galalpha1,3Gal epitope. This is consistent with the fact that the outer medulla of pig kidney expresses the alpha1,3-galactosyltransferase, which is completely absent in human kidney. Finally, pTHP is more resistant to leukocyte elastase hydrolysis than hTHP, thus explaining why pTHP is much less prone to urinary degradation than hTHP. These results demonstrate for the first time that the species variations of the glycomoiety of THP can lead to the differential binding of THP to type 1-fimbriated E. coli and that the differences in high mannose processing may reflect species-specific adaptation of urinary defenses against E. coli infections
— id: 42020, year: 2004, vol: 279, page: 216, 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,

Tamm-Horsfall protein is a critical renal defense factor protecting against calcium oxalate crystal formation
Mo, Lan; Huang, Hong-Ying; Zhu, Xin-Hua; Shapiro, Ellen; Hasty, David L; Wu, Xue-Ru
2004 Sep;66(3):1159-1166, Kidney international
Tamm-Horsfall protein is a critical renal defense factor protecting against calcium oxalate crystal formation. Background. The tubular fluid of the mammalian kidney is often supersaturated with mineral salts, but crystallization rarely occurs under normal conditions. The unique ability of the kidney to avoid harmful crystal formation has long been attributed to the inhibitory activity of the urinary macromolecules, although few in vivo studies have been carried out to examine this hypothesis. Here we examined the role of Tamm-Horsfall protein (THP), the principal urinary protein, in urinary defense against renal calcium crystal formation, using a THP knockout model that we recently developed. Methods. Wild-type and THP knockout mice were examined for the spontaneous formation of renal calcium crystals using von Kossa staining. The susceptibility of these mice to experimentally induced renal crystal formation was evaluated by administering mice with ethylene glycol, a precursor of oxalate, and vitamin D(3), which increases calcium absorption. Renal calcium crystals were visualized by von Kossa stain, dark field microscopy with polarized light and scanning electron microscopy. Results. Inactivating the THP gene in mouse embryonic stem cells results in spontaneous formation of calcium crystals in adult kidneys. Excessive intake of calcium and oxalate, precursors of the most common type of human renal stones, dramatically increases both the frequency and the severity of renal calcium crystal formation in THP-deficient, but not in wild-type mice. Under high calcium/oxalate conditions, the absence of THP triggers a marked, adaptive induction in renal epithelial cells of osteopontin (OPN), a potent inhibitor of bone mineralization and vascular calcification. Thus, OPN may serve as an inducible inhibitor of calcium crystallization, whereas THP can serve as a constitutive and apparently more effective inhibitor. Conclusion. These results provide the first in vivo evidence that THP is a critical urinary defense factor and suggest that its deficiency could be an important contributing factor in human nephrolithiasis, a condition afflicting tens of millions of people in the world annually
— id: 44481, year: 2004, vol: 66, page: 1159, stat: Journal Article,

Ablation of the Tamm-Horsfall protein gene increases susceptibility of mice to bladder colonization by type 1-fimbriated Escherichia coli
Mo, Lan; Zhu, Xin-Hua; Huang, Hong-Ying; Shapiro, Ellen; Hasty, David L; Wu, Xue-Ru
2004 Apr;286(4):F795-F802, American journal of physiology. Renal physiology
The adhesion of uropathogenic Escherichia coli to the urothelial surface of the bladder is a prerequisite for the establishment of bladder infections. This adhesion process relies on E. coli adhesins and their cognate urothelial receptors, and it also is influenced by an intricate array of defense mechanisms of the urinary system. In this study, we examined the in vivo role of Tamm-Horsfall protein (THP), the most abundant urinary protein, in innate urinary defense. We genetically ablated the mouse THP gene and found that THP deficiency predisposes mice to bladder infections by type 1-fimbriated E. coli. Inoculation of too few type 1-fimbriated E. coli to colonize wild-type mice caused significant bladder colonization in THP-knockout mice. In contrast, THP deficiency did not enhance the ability of P-fimbriated E. coli to colonize the bladder. Our results provide the first in vivo evidence indicating that under physiological conditions, the mannosylated THP can serve as an effective soluble 'receptor,' binding to the type 1-fimbriated E. coli and competitively inhibiting them from adhering to the uroplakin Ia receptors present on the urothelial surface. These results suggest that potential THP defects, either quantitative or qualitative, could predispose the urinary bladder to bacterial infections. The generation of THP-deficient mice established the role of THP as a first line of urinary defense and should help elucidate other potential functions of this major protein in urinary tract physiology and diseases
— id: 48190, year: 2004, vol: 286, page: F795, 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,

THE PROSTATIC UTRICLE IS NOT A MULLERIAN DUCT REMNANT: IMMUNOHISTOCHEMICAL EVIDENCE FOR A DISTINCT UROGENITAL SINUS ORIGIN
Shapiro, Ellen; Huang, Hongying; McFadden, Deborah E; Masch, Rachel J; Ng, Eliza; Lepor, Herbert; Wu, Xue-Ru
2004 Oct;172(4, Part 2 Of 2):1753-6; discussion 1756, Journal of urology
PURPOSE:: The embryological origin of the utricle is thought to be a remnant of the fused caudal ends of the mullerian ducts (MDs). Others propose that the urogenital sinus (UGS) contributes either partially or totally to the development of this structure. Using immunohistochemical probes, we provide strong evidence that the utricle is of UGS origin only. MATERIALS AND METHODS:: Human fetal prostates, gestational ages 9 to 24 weeks, were serially cross-sectioned. Representative sections were stained with antibodies to p63 (basal cell marker), vimentin (mesoderm marker), uroplakins (marker for urothelium) Pax-2 (expressed in ductal and mesenchyme of urogenital system including the MDs and wolffian ducts) and Ki67 (proliferation). Apoptosis was detected with the TUNEL assay. RESULTS:: By 9 weeks there was weak expression of p63 in the basal layer of the UGS. At 11 weeks there was increased staining of p63 in the UGS and some p63 staining of the fused MDs, which expressed Pax-2 at this time. At 14 to 15 weeks as the MDs were undergoing apoptosis, there was an ingrowth of uroplakin-expressing UGS epithelium into the periurethral stroma, which formed a plate of p63 positive cells just beneath the UGS that was Ki67 positive. The remaining caudal MD epithelium was p63 negative and expressed vimentin and Pax-2. By 17 weeks the plate of p63 positive cells elongated forming the utricle that remained p63 positive but Pax-2 and vimentin negative. CONCLUSIONS:: We show that the utricle forms as an ingrowth of specialized cells from the dorsal wall of the UGS as the caudal MDs regress
— id: 44930, year: 2004, vol: 172, page: 1753, stat: Journal Article,

New concepts on the development of the vagina
Shapiro, Ellen; Huang, Hongying; Wu, Xue-Ru
2004 ;545(1):173-185, Advances in experimental medicine & biology
— id: 42301, year: 2004, vol: 545, page: 173, 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,

Renal tubule-specific expression and urinary secretion of human hormone: a kidney-based transgenic bioreactor growth
Zhu, Xinhua; Cheng, Jin; Huang, Liwei; Gao, Jin; Zhang, Zhong-Ting; Pak, Joanne; Wu, Xue-Ru
2003 Apr;12(2):155-162, Transgenic research
Tissue-specific expression of human genes and secretion of human proteins into the body fluids in transgenic animals provides an important means of manufacturing large-quantity and high-quality pharmaceuticals. The present study demonstrates using transgenic mice that a 3.0 kb promoter of the mouse Tamm-Horsfall protein (THP, or uromodulin) gene directs the specific expression of human growth hormone (hGH) gene in the kidney followed by the secretion of hGH protein into the urine. hGH expression was detected in renal tubules that actively produce the THP, that is, the ascending limb of Henle's loop and distal convoluted tubules. Up to 500 ng/ml of hGH was detected in the urine, and this level remained constant throughout the 10-month observation period. hGH was also detectable in the stomach epithelium and serum in two of the transgenic lines, suggesting position-dependent effects of the transgene and leakage of hGH from the site of synthesis into the bloodstream, respectively. These results indicate that the 3.0 kb mouse THP promoter is primarily kidney-specific and can be used to convert kidney into a bioreactor in transgenic animals to produce recombinant proteins. Given the capacity of urine production independent of age, sex and lactation, the ease of urinary protein purification, and the potentially distinct machinery for post-translational modifications in the kidney epithelial cells, the kidney-based transgenic bioreactor may offer unique opportunities for producing certain complex pharmaceuticals
— id: 39231, year: 2003, vol: 12, page: 155, stat: Journal Article,

The human prostate expresses sonic hedgehog during fetal development
Barnett, Daniel H; Huang, Hong-Ying; Wu, Xue-Ru; Laciak, Robert; Shapiro, Ellen; Bushman, Wade
2002 Nov;168(5):2206-2210, Journal of urology
PURPOSE: The keynote event of prostate ductal development is the formation of epithelial buds that invade the urogenital sinus mesenchyma. Studies in mice have shown that budding requires the signaling peptide, which is expressed in the epithelium of the prostatic anlagen. We report our characterization of (SHH) expression in the human fetal prostate. MATERIALS AND METHODS: Reverse transcriptase-polymerase chain reaction was performed in fetal prostate RNA isolated at 15.5 and 18 weeks of gestation, respectively. Immunostaining was performed on sections from 7 male fetuses at 9.5 to 34 and in 4 female fetuses at 9 to 18 weeks of gestation. RESULTS: Weak staining for was seen in the prostatic urethra at 9.5 weeks. Intense staining was seen at 11.5 and 13 weeks in the prostatic urothelium and nascent prostatic buds. Staining was slightly diminished at 16.5, further diminished at 18 to 20 and absent at 34 weeks. expression at 15.5 and 18 weeks was confirmed by reverse transcriptase-polymerase chain reaction assay of freshly isolated prostate tissue. Comparative immunostaining in the female showed urothelial staining at 9 and 12 weeks with staining greatest above the entrance of the mullerian ducts. Staining diminished earlier in the female (14 weeks) than in the male and was almost absent at 18 weeks. CONCLUSIONS: expression in the human fetal prostate is contemporaneous with the fetal testosterone surge and with ductal budding of the prostatic urothelium. expression is also present in the female urogenital sinus but in the absence of testosterone it is not associated with ductal budding
— id: 75192, year: 2002, vol: 168, page: 2206, 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,

Isolation of mouse THP gene promoter and demonstration of its kidney-specific activity in transgenic mice
Zhu, Xinhua; Cheng, Jin; Gao, Jing; Lepor, Herbert; Zhang, Zhong-Ting; Pak, Joanne; Wu, Xue-Ru
2002 Apr;282(4):F608-F617, American journal of physiology. Renal physiology
Tamm-Horsfall protein (THP), the most abundant urinary protein synthesized by the kidney epithelial cells, is believed to play important and diverse roles in the urinary system, including renal water balance, immunosuppression, urinary stone formation, and inhibition of bacterial adhesion. In the present study, we describe the isolation of a 9.3-kb, 5'-region of the mouse THP gene and show the highly conserved nature of its proximal 589-bp, 5'-flanking sequence with that in rats, cattle, and humans. We also demonstrate using the transgenic mouse approach that a 3.0-kb, proximal 5'-flanking sequence is sufficient to drive the kidney-specific expression of a heterologous reporter gene. Within the kidney, transgene expression was confined to the renal tubules that endogenously expressed the THP protein, which suggests specific transgene activity in the thick ascending limb of the loop of Henle and early distal convoluted tubules. Our results establish the kidney- and nephron-segment-specific expression of the mouse THP gene. The availability of the mouse THP gene promoter that functions in vivo should facilitate additional studies of the molecular mechanisms of kidney-specific gene regulation and should provide new molecular tools for better understanding renal physiology and disease through nephron-specific gene targeting
— id: 39700, year: 2002, vol: 282, page: F608, 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,

Tamm-Horsfall protein binds to type 1 fimbriated Escherichia coli and prevents E. coli from binding to uroplakin Ia and Ib receptors
Pak J; Pu Y; Zhang ZT; Hasty DL; Wu XR
2001 Mar 30;276(13):9924-9930, Journal of biological chemistry
The adherence of uropathogenic Escherichia coli to the urothelial surface, a critical first step in the pathogenesis of urinary tract infection (UTI), is controlled by three key elements: E. coli adhesins, host receptors, and host defense mechanisms. Although much has been learned about E. coli adhesins and their urothelial receptors, little is known about the role of host defense in the adherence process. Here we show that Tamm-Horsfall protein (THP) is the principal urinary protein that binds specifically to type 1 fimbriated E. coli, the main cause of UTI. The binding was highly specific and saturable and could be inhibited by d-mannose and abolished by endoglycosidase H treatment of THP, suggesting that the binding is mediated by the high-mannose moieties of THP. It is species-conserved, occurring in both human and mouse THPs. In addition, the binding to THP was much greater with an E. coli strain bearing a phenotypic variant of the type 1 fimbrial FimH adhesin characteristic of those prevalent in UTI isolates compared with the one prevalent in isolates from the large intestine of healthy individuals. Finally, a physiological concentration of THP completely abolished the binding of type 1 fimbriated E. coli to uroplakins Ia and Ib, two putative urothelial receptors for type 1 fimbriae. These results establish, on a functional level, that THP contains conserved high-mannose moieties capable of specific interaction with type 1 fimbriae and strongly suggest that this major urinary glycoprotein is a key urinary anti-adherence factor serving to prevent type 1 fimbriated E. coli from binding to the urothelial receptors
— id: 26762, year: 2001, vol: 276, page: 9924, 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,

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,

Terminal glycosylation of bovine uroplakin III, one of the major integral-membrane glycoproteins of mammalian bladder
Malagolini N; Cavallone D; Wu XR; Serafini-Cessi F
2000 Jul 26;1475(3):231-237, Biochimica & biophysica acta
Uroplakin III (UPIII) is one of the major transmembrane glycoproteins exposed at the luminal face of mammalian bladder. We investigated the terminal glycosylation of bovine UPIII in order to ascertain whether it contains the alpha 2,3-sialylated sequence thus potentially serving as a receptor for uropathogenic Escherichia coli expressing type S adhesins. We report the occurrence of sialic acid in alpha 2,3- and alpha 2,6-linkage to galactose in bovine UPIII glycans as evidenced by the sensitivity of UPIII to both Vibrio cholera and Newcastle disease virus neuraminidase and by the colocalization of UPIII antigen and material detected by lectins of Sambucus nigra and Maackia amurensis on the luminal face of the bladder. We also present evidence that UPIII glycans are capped by Gal-alpha 1,3-Gal epitope. Consistently, alpha 2,3- and alpha 2, 6-sialyltransferase, as well as alpha 1,3-galactosyltransferase were found to be present in the cells detached from the luminal side of bovine bladder, which are responsible for the UPIII biosynthesis. The putative role of UPIII sialylated glycans in enhancing the uropathogenicity of E. coli expressing type S adhesins is discussed
— id: 34164, year: 2000, vol: 1475, page: 231, stat: Journal Article,

Uroplakin and androgen receptor expression in the human fetal genital tract: insights into the development of the vagina
Shapiro E; Huang HY; Wu XR
2000 Sep;164(3 Pt 2):1048-1051, Journal of urology
PURPOSE: Although a dual origin of the vagina has been popularized, other theories support a mullerian or wolffian duct origin or various combinations of these structures and the urogenital sinus. Uroplakins are specialized membrane proteins of the urothelial plaque, constituting the asymmetrical unit membrane of the bladder, and represent specific molecular markers of urothelial differentiation. We hypothesize that the epithelium of the dorsal wall of the urogenital sinus is involved in the formation of the sinovaginal bulbs and will express uroplakins. In addition, localization of the androgen receptor and its temporal expression during development may in part explain the varied effects of androgens on the lower female genital tract in congenital adrenal hyperplasia. MATERIALS AND METHODS: Lower genitourinary tracts from 4 human female fetuses (9 to 18 weeks) were serially sagittally sectioned. Representative sections were stained with hematoxylin and eosin, rabbit antibodies against panuroplakin and antibodies to the androgen receptor. RESULTS: At 9 weeks of gestation the urogenital sinus showed evidence of evagination and the formation of the sinovaginal bulbs. The urothelium of the entire urogenital sinus expressed uroplakins including the region of the dorsal wall involved in evagination and formation of the sinovaginal bulbs. The mullerian ducts were in direct contact with the area of urogenital sinus evagination but were not in continuity with the sinus. Androgen receptors were expressed in the epithelium and the stroma of the urogenital sinus, sinovaginal bulbs, and mullerian and wolffian ducts. By 14 weeks androgen receptor expression was almost absent in the urothelium of the urogenital sinus, and the epithelium and surrounding stroma of the lower vagina and mullerian ducts. CONCLUSIONS: The area of evagination of the urogenital sinus expresses uroplakins, is involved in the formation of the sinovaginal bulbs and further substantiates the urogenital sinus origin of the lower vagina. Since testosterone inhibits formation of the lower vagina, the timing of exposure to systemic testosterone in congenital adrenal hyperplasia will determine the phenotypic appearance of the external genitalia and effect of testosterone on the development of the lower genital tract. If exposure to testosterone occurs after 12 weeks only clitoromegaly occurs. Androgen receptor is absent in the urogenital sinus urothelium, vaginal epithelium and mullerian ducts by 14 weeks, suggesting that these tissues become androgen insensitive and vaginal development will proceed normally after that critical time
— id: 11531, year: 2000, vol: 164, page: 1048, 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,

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,

Uroplakin and androgen receptor expression in the human fetal female genital tract: Insights into the development of the vagina in normal females and in congenital adrenal hyperplasia
Shapiro, E; Huang, HY; Wu, XR
1999 SEP ;104(3):859-860, Pediatrics
— id: 53840, year: 1999, vol: 104, page: 859, stat: Journal Article,

Uroplakins as markers of urothelial differentiation
Sun TT; Liang FX; Wu XR
1999 ;462:7-18; discussion 103, Advances in experimental medicine & biology
— id: 11902, year: 1999, vol: 462, page: 7, 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,

New concepts on the histological changes in experimental augmentation cystoplasty: Insights into the development of neoplastic transformation at the enterovesical anastomosis
Gitlin, JS; Wu, XR; Ritchey, ML; Shapiro, E
1998 SEP ;102(3):843-843, Pediatrics
— id: 53771, year: 1998, vol: 102, page: 843, stat: Journal Article,

Variants of the FimH adhesion confer distinct patterns of interaction of E-coli with urinary bladder
Hasty, DL; Wu, XR; Sokurenko, E
1998 NOV ;9(11):501A-501A, Molecular biology of the cell
— id: 53653, year: 1998, vol: 9, page: 501A, stat: Journal Article,

Three dimensional analysis of the 16-nm urothelial plaque particle: Luminal surface exposure, preferential head-to-head interaction, and dynamic plaque/hinge formation
Kachar, B; Liang, F; Lins, U; Ding, M; Wu, XR; Stoffler, D; Acbi, U; Sun, TT
1998 NOV ;9(11):78A-78A, Molecular biology of the cell
— id: 53642, year: 1998, vol: 9, page: 78A, stat: Journal Article,

Pathogenic adaptation of Escherichia coli by natural variation of the FimH adhesin
Sokurenko EV; Chesnokova V; Dykhuizen DE; Ofek I; Wu XR; Krogfelt KA; Struve C; Schembri MA; Hasty DL
1998 Jul 21;95(15):8922-8926, Proceedings of the National Academy of Sciences of the United States of America
Conventional wisdom regarding mechanisms of bacterial pathogenesis holds that pathogens arise by external acquisition of distinct virulence factors, whereas determinants shared by pathogens and commensals are considered to be functionally equivalent and have been ignored as genes that could become adapted specifically for virulence. It is shown here, however, that genetic variation in an originally commensal trait, the FimH lectin of type 1 fimbriae, can change the tropism of Escherichia coli, shifting it toward a urovirulent phenotype. Random point mutations in fimH genes that increase binding of the adhesin to mono-mannose residues, structures abundant in the oligosaccharide moieties of urothelial glycoproteins, confer increased virulence in the mouse urinary tract. These mutant FimH variants, however, are characterized by increased sensitivity to soluble inhibitors bathing the oropharyngeal mucosa, the physiological portal of E. coli. This functional trade-off seems to be detrimental for the intestinal ecology of the urovirulent E. coli. Thus, bacterial virulence can be increased by random functional mutations in a commensal trait that are adaptive for a pathologic environment, even at the cost of reduced physiological fitness in the nonpathologic habitat
— id: 7800, year: 1998, vol: 95, page: 8922, 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,

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,

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,

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,

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,

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,

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,

Rapid communication: PstI, HindIII, and TaqI restriction fragment length polymorphisms in the bovine Uroplakin IB gene
Ryan AM; Womack JE; Lin JH; Yu J; Wu XR; Sun TT
1994 Jul;72(7):1909-1909, Journal of animal science
— id: 34165, year: 1994, vol: 72, page: 1909, stat: Journal Article,

TaqI and BglII RFLPs for the bovine uroplakin III loci UPK3A and UPK3B
Ryan AM; Womack JE; Wu XR; Sun TT
1994 Feb;25(1):64-64, Animal genetics
— id: 34167, year: 1994, vol: 25, page: 64, stat: Journal Article,

Rapid communication: BamHI and TaqI restriction fragment length polymorphisms in the bovine Uroplakin IA gene
Ryan AM; Womack JE; Yu J; Lin JH; Wu XR; Sun TT
1994 Jul;72(7):1908-1908, Journal of animal science
— id: 34166, year: 1994, vol: 72, page: 1908, 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,

[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,

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,

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,

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,