Pamela Cowin, Ph.D.

A Systematic Screen for Micro-RNAs Regulating the Canonical Wnt Pathway
Anton, Roman; Chatterjee, Sujash S; Simundza, Julia; Cowin, Pamela; Dasgupta, Ramanuj
2011 ;6(10):e26257-e26257, PLoS ONE
MicroRNAs (miRs) and the canonical Wnt pathway are known to be dysregulated in human cancers and play key roles during cancer initiation and progression. To identify miRs that can modulate the activity of the Wnt pathway we performed a cell-based overexpression screen of 470 miRs in human HEK293 cells. We identified 38 candidate miRs that either activate or repress the canonical Wnt pathway. A literature survey of all verified candidate miRs revealed that the Wnt-repressing miRs tend to be anti-oncomiRs and down-regulated in cancers while Wnt-activating miRs tend to be oncomiRs and upregulated during tumorigenesis. Epistasis-based functional validation of three candidate miRs, miR-1, miR-25 and miR-613, confirmed their inhibitory role in repressing the Wnt pathway and suggest that while miR-25 may function at the level of a-catenin (beta-cat), miR-1 and miR-613 act upstream of beta-cat. Both miR-25 and miR-1 inhibit cell proliferation and viability during selection of human colon cancer cell lines that exhibit dysregulated Wnt signaling. Finally, transduction of miR-1 expressing lentiviruses into primary mammary organoids derived from Conductin-lacZ mice significantly reduced the expression of the Wnt-sensitive beta-gal reporter. In summary, these findings suggest the potential use of Wnt-modulating miRs as diagnostic and therapeutic tools in Wnt-dependent diseases, such as cancer
— id: 140537, year: 2011, vol: 6, page: e26257, stat: Journal Article,

Choreographing Metastasis to the Tune of LTBP
Chandramouli, Anupama; Simundza, Julia; Pinderhughes, Alicia; Cowin, Pamela
2011 Jun;16(2):67-80, Journal of mammary gland biology & neoplasia
Latent Transforming Growth Factor beta (TGFbeta) Binding Proteins (LTBPs) are chaperones and determinants of TGFbeta isoform-specific secretion. They belong to the LTBP/Fibrillin family and form integral components of the fibronectin and microfibrillar extracellular matrix (ECM). LTBPs serve as master regulators of TGFbeta bioavailability, functioning to incorporate and spatially pattern latent TGFbeta at regular intervals within the ECM, and actively participate in integrin-mediated stretch activation of TGFbeta in vivo. In so doing they create a highly patterned sensory system where local changes in ECM tension can be detected and transduced into focal signals. The physiological role of LTBPs in the mammary gland remains largely unstudied, however both loss and gain of LTBP expression is found in breast cancers and breast cancer cell lines. Importantly, elevated LTBP1 levels appear in two gene signatures predictive of enhanced metastatic behavior. LTBP may promote metastasis by providing the bridge between structural and signaling components of the epithelial to mesenchymal transition (EMT)
— id: 132580, year: 2011, vol: 16, page: 67, stat: Journal Article,

A mouse transgenic approach to induce beta-catenin signaling in a temporally controlled manner
Mukherjee, Atish; Soyal, Selma M; Li, Jie; Ying, Yan; Szwarc, Maria M; He, Bin; Kommagani, Ramakrishna; Hodgson, Myles C; Hiremath, Minoti; Cowin, Pamela; Lydon, John P
2011 Aug;20(4):827-840, Transgenic research
Although constitutive murine transgenic models have provided important insights into beta-catenin signaling in tissue morphogenesis and tumorigenesis, these models are unable to express activated beta-catenin in a temporally controlled manner. Therefore, to enable the induction (and subsequent de-induction) of beta-catenin signaling during a predetermined time-period or developmental stage, we have generated and characterized a TETO-DeltaN89beta-catenin responder transgenic mouse. Crossed with the MTB transgenic effector mouse, which targets the expression of the reverse tetracycline transactivator (rtTA) to the mammary epithelium, we demonstrate that the stabilized (and activated) form of beta-catenin (DeltaN89beta-catenin) is expressed only in the presence doxycycline-activated rtTA in the mammary epithelial compartment. Furthermore, we show that transgene-derived DeltaN89beta-catenin elicits significant mammary epithelial proliferation and precocious alveologenesis in the virgin doxycycline-treated MTB/TETO-DeltaN89beta-catenin bitransgenic. Remarkably, deinduction of TETO-DeltaN89beta-catenin transgene expression (through doxycycline withdrawal) results in the reversal of these morphological changes. Importantly, continued activation of the TETO-DeltaN89beta-catenin transgene results in palpable mammary tumors (within 7-9 months) in the doxycycline-treated virgin MTB/TETO-DeltaN89beta-catenin bigenic but not in the same bitransgenic without doxycycline administration. Collectively, these mammary epithelial responses to DeltaN89beta-catenin expression agree with previous reports using conventional transgenesis and therefore confirm that DeltaN89beta-catenin functions as expected in this doxycycline-responsive bigenic system. In sum, our mammary gland studies demonstrate 'proof-of-principle' for using the TETO-DeltaN89beta-catenin transgenic responder to activate (and then de-activate) beta-catenin signaling in any tissue of interest in a spatiotemporal specific fashion
— id: 138283, year: 2011, vol: 20, page: 827, stat: Journal Article,

Distinct function of androgen receptor coactivator ARA70alpha and ARA70beta in mammary gland development, and in breast cancer
Wu, Xinyu; Chen, Fei; Sahin, Aysegul; Albarracin, Constance; Pei, Zhiheng; Zou, Xuanyi; Singh, Baljit; Xu, Ruliang; Daniels, Garrett; Li, Yirong; Wei, Jianjun; Blake, Marvin; Schneider, Robert J; Cowin, Pamela; Lee, Peng
2011 Jul;128(2):391-400, Breast cancer research & treatment
Steroid receptor coactivators are important in regulating the function of the receptors in endocrine organ development and in cancers, including breast. Androgen receptor (AR) coactivator ARA70, was first identified as a gene fused to the ret oncogene and later characterized as an AR coactivator. We previously reported that the full length ARA70alpha functions as a tumor suppressor gene and that ARA70beta functions as an oncogene in prostate cancer. Here we show that both ARA70alpha and ARA70beta function as AR and estrogen receptor (ER) coactivators in breast cancer cells. However, ARA70alpha and ARA70beta serve different functions in mammary gland development and breast cancer tumorigenesis. We observed hypoplastic development of mammary glands in MMTV driven ARA70alpha transgenic mice and overgrowth of mammary glands in ARA70beta transgenic mice at virgin and pregnant stages. We determined that ARA70alpha inhibited cell proliferation, and that ARA70beta promotes proliferation in MCF7 breast cancer cells. These effects were observed in hormone-free media, or in media with androgen or estrogen, though to varying degrees. Additionally, we observed that ARA70beta strongly enhanced the invasive ability of MCF7 breast cancer cells in in vitro Matrigel assays. Significantly, decreased ARA70alpha expression is associated with increased tendency of breast cancer metastasis. In summary, ARA70alpha and ARA70beta have distinct effects in mammary gland development and in the progression of breast cancer
— id: 138162, year: 2011, vol: 128, page: 391, stat: Journal Article,

Molecular mechanisms guiding embryonic mammary gland development
Cowin, Pamela; Wysolmerski, John
2010 Jun 1;2(6):a003251-a003251, Cold Spring Harbor perspectives in biology
The mammary gland is an epidermal appendage that begins to form during embryogenesis, but whose development is only completed during pregnancy. Each mammary gland begins as a budlike invagination of the surface ectoderm, which then gives rise to a simple duct system by birth. Subsequent development occurs during sexual maturation and during pregnancy and lactation. In this review, we outline the distinct stages of embryonic mammary development and discuss the molecular pathways involved in the regulation of morphogenesis at each stage. We also discuss the potential relevance of embryonic breast development to the pathophysiology of breast cancer and highlight questions for future research
— id: 109851, year: 2010, vol: 2, page: a003251, stat: Journal Article,

Key signaling nodes in mammary gland development and cancer: beta-catenin
Incassati, Angela; Chandramouli, Anupama; Eelkema, Rachel; Cowin, Pamela
2010 ;12(6):213-213, Breast cancer research
beta-Catenin plays important roles in mammary development and tumorigenesis through its functions in cell adhesion, signal transduction and regulation of cell-context-specific gene expression. Studies in mice have highlighted the critical role of beta-catenin signaling for stem cell biology at multiple stages of mammary development. Deregulated beta-catenin signaling disturbs stem and progenitor cell dynamics and induces mammary tumors in mice. Recent data showing deregulated beta-catenin signaling in metaplastic and basal-type tumors suggest a similar link to reactivated developmental pathways and human breast cancer. The present review will discuss beta-catenin as a central transducer of numerous signaling pathways and its role in mammary development and breast cancer
— id: 138282, year: 2010, vol: 12, page: 213, stat: Journal Article,

Links between transforming growth factor-beta and canonical Wnt signaling yield new insights into breast cancer susceptibility, suppression and tumor heterogeneity
Incassati, Angela; Pinderhughes, Alicia; Eelkema, Rachel; Cowin, Pamela
2009 ;11(3):103-103, Breast cancer research
In a recent issue of Breast Cancer Research, investigators from the Serra laboratory describe a novel mechanism of transforming growth factor (TGF)-beta tumor suppression. Previously, the authors discovered that stromal TGF-beta signaled through Wnt5a to restrain pubertal ductal elongation and branching. Here, they show that inhibition of stromal TGF-beta signaling or Wnt5a loss leads to increased beta-catenin transcriptional activity and reduced latency in mammary tumor models, with tumors displaying a higher proportion of progenitor cell markers. These findings reveal a novel intersection of two tumor suppressors with a potent oncogenic pathway and highlight the need for further study on the role played by canonical Wnt signaling in breast cancer susceptibility and subtype
— id: 101284, year: 2009, vol: 11, page: 103, stat: Journal Article,

MMTV-Wnt1 and -DeltaN89beta-catenin induce canonical signaling in distinct progenitors and differentially activate Hedgehog signaling within mammary tumors
Teissedre, Brigitte; Pinderhughes, Alicia; Incassati, Angela; Hatsell, Sarah J; Hiremath, Minoti; Cowin, Pamela
2009 ;4(2):e4537-e4537, PLoS ONE
Canonical Wnt/beta-catenin signaling regulates stem/progenitor cells and, when perturbed, induces many human cancers. A significant proportion of human breast cancer is associated with loss of secreted Wnt antagonists and mice expressing MMTV-Wnt1 and MMTV-DeltaN89beta-catenin develop mammary adenocarcinomas. Many studies have assumed these mouse models of breast cancer to be equivalent. Here we show that MMTV-Wnt1 and MMTV-DeltaN89beta-catenin transgenes induce tumors with different phenotypes. Using axin2/conductin reporter genes we show that MMTV-Wnt1 and MMTV-DeltaN89beta-catenin activate canonical Wnt signaling within distinct cell-types. DeltaN89beta-catenin activated signaling within a luminal subpopulation scattered along ducts that exhibited a K18(+)ER(-)PR(-)CD24(high)CD49f(low) profile and progenitor properties. In contrast, MMTV-Wnt1 induced canonical signaling in K14(+) basal cells with CD24/CD49f profiles characteristic of two distinct stem/progenitor cell-types. MMTV-Wnt1 produced additional profound effects on multiple cell-types that correlated with focal activation of the Hedgehog pathway. We document that large melanocytic nevi are a hitherto unreported hallmark of early hyperplastic Wnt1 glands. These nevi formed along the primary mammary ducts and were associated with Hedgehog pathway activity within a subset of melanocytes and surrounding stroma. Hh pathway activity also occurred within tumor-associated stromal and K14(+)/p63(+) subpopulations in a manner correlated with Wnt1 tumor onset. These data show MMTV-Wnt1 and MMTV-DeltaN89beta-catenin induce canonical signaling in distinct progenitors and that Hedgehog pathway activation is linked to melanocytic nevi and mammary tumor onset arising from excess Wnt1 ligand. They further suggest that Hedgehog pathway activation maybe a critical component and useful indicator of breast tumors arising from unopposed Wnt1 ligand
— id: 96250, year: 2009, vol: 4, page: e4537, stat: Journal Article,

Plakoglobin is required for effective intermediate filament anchorage to desmosomes
Acehan, Devrim; Petzold, Christopher; Gumper, Iwona; Sabatini, David D; Muller, Eliane J; Cowin, Pamela; Stokes, David L
2008 Nov;128(11):2665-2675, Journal of investigative dermatology
Desmosomes are adhesive junctions that provide mechanical coupling between cells. Plakoglobin (PG) is a major component of the intracellular plaque that serves to connect transmembrane elements to the cytoskeleton. We have used electron tomography and immunolabeling to investigate the consequences of PG knockout on the molecular architecture of the intracellular plaque in cultured keratinocytes. Although knockout keratinocytes form substantial numbers of desmosome-like junctions and have a relatively normal intercellular distribution of desmosomal cadherins, their cytoplasmic plaques are sparse and anchoring of intermediate filaments is defective. In the knockout, beta-catenin appears to substitute for PG in the clustering of cadherins, but is unable to recruit normal levels of plakophilin-1 and desmoplakin to the plaque. By comparing tomograms of wild type and knockout desmosomes, we have assigned particular densities to desmoplakin and described their interaction with intermediate filaments. Desmoplakin molecules are more extended in wild type than knockout desmosomes, as if intermediate filament connections produced tension within the plaque. On the basis of our observations, we propose a particular assembly sequence, beginning with cadherin clustering within the plasma membrane, followed by recruitment of plakophilin and desmoplakin to the plaque, and ending with anchoring of intermediate filaments, which represents the key to adhesive strength
— id: 93304, year: 2008, vol: 128, page: 2665, stat: Journal Article,

Breast cancer progression: controversies and consensus in the molecular mechanisms of metastasis and EMT
Cowin, Pamela; Welch, Danny R
2007 Sep;12(2-3):99-102, Journal of mammary gland biology & neoplasia
— id: 91971, year: 2007, vol: 12, page: 99, stat: Journal Article,

The pattern of beta-catenin responsiveness within the mammary gland is regulated by progesterone receptor
Hiremath, Minoti; Lydon, John P; Cowin, Pamela
2007 Oct;134(20):3703-3712, Development
Experiments involving beta-catenin loss- and gain-of-function in the mammary gland have decisively demonstrated the role of this protein in normal alveologenesis. However, the relationship between hormonal and beta-catenin signaling has not been investigated. In this study, we demonstrate that activated beta-catenin rescues alveologenesis in progesterone receptor (PR; Pgr)-null mice during pregnancy. Two distinct subsets of mammary cells respond to expression of DeltaN89beta-catenin. Cells at ductal tips are inherently beta-catenin-responsive and form alveoli in the absence of PR. However, PR activity confers beta-catenin responsiveness to progenitor cells along the lateral ductal borders in the virgin gland. Once activated by beta-catenin, responding cells switch on an alveolar differentiation program that is indistinguishable from that observed in pregnancy and is curtailed by PR signaling
— id: 76076, year: 2007, vol: 134, page: 3703, stat: Journal Article,

Gli3-mediated repression of Hedgehog targets is required for normal mammary development
Hatsell, Sarah J; Cowin, Pamela
2006 Sep;133(18):3661-3670, Development
The Hedgehog pathway is vital for the development of many epidermal appendages, but its role in mammary development has been unclear. Here, we show that although Gli2 and Gli3 are expressed during embryonic mammary development, transcriptional reporters of positive Hedgehog signaling are absent. Nevertheless, Gli3(xt/xt) embryos show aberrant early mammary marker expression and lack two pairs of mammary buds, demonstrating that Gli3 is essential for mammary bud formation and preceding patterning events. Misactivation of the Hedgehog pathway by targeted expression of the constitutive activator Gli1, from the Gli2 promoter in Gli3(xt/+) mice, also induces mammary bud loss. Moreover, loss of Gli3 expression induces Gli1 misexpression in mammary mesenchyme. These results establish that the essential function of Gli3 during embryonic mammary development is to repress Hedgehog/Gli1-inducible targets. During postnatal mammary development, Gli2 and Gli3 are expressed in stromal and myoepithelial cells, and Gli3 is also found within the lumenal epithelium. Again, transcriptional reporters of positive Hedgehog signaling are absent from these cell types, yet are expressed robustly within mammary lymphatics. Thus, positive Hedgehog signaling is absent throughout mammary development, distinguishing the mammary gland from other epidermal appendages, such as hair follicles, which require Hedgehog pathway activity
— id: 69584, year: 2006, vol: 133, page: 3661, stat: Journal Article,

Cadherins and catenins in breast cancer
Cowin, Pamela; Rowlands, Tracey M; Hatsell, Sarah J
2005 Oct;17(5):499-508, Current opinion in cell biology
Recent studies show that cadherins and catenins are hormonally regulated and carry out physiological roles during mammary development but have pathological effects when deregulated. E-cadherin expression is irreversibly lost in invasive lobular breast cancer (ILC). Animal models of ILC provide mechanistic insight, confirming that E-cadherin serves as both a tumor suppressor and an invasion suppressor in ILC. Ductal breast cancer involves complex, reversible, epigenetic modulation of multiple cadherins. Transcriptional regulators of E-cadherin have been identified that induce epithelial-to-mesenchymal transitions. Catenins are lost or mislocalized in tumors lacking cadherins. However, beta-catenin signaling is upregulated by numerous pathways in >50% of breast tumors and animal models suggest its oncogenic function in breast relates to its role in mammary progenitor cell expansion
— id: 96251, year: 2005, vol: 17, page: 499, stat: Journal Article,

Beta-catenin induces a population of radio-resistant alveolar stem/progenitors that progress to form hormone-independent breast tumors in mice
Formenti, SC; Hiremath, M; Yang, A; Demaria, S; Cowin, P
2005 ;63(2):2396-2396, International journal of radiation oncology biology physics
— id: 109265, year: 2005, vol: 63, page: 2396, stat: Journal Article,

Bone morphogenetic protein signaling regulates postnatal hair follicle differentiation and cycling
Guha, Udayan; Mecklenburg, Lars; Cowin, Pamela; Kan, Lixin; O'Guin, W Michael; D'Vizio, Dolores; Pestell, Richard G; Paus, Ralf; Kessler, John A
2004 Sep;165(3):729-740, American journal of pathology
Hair follicle morphogenesis and cycling were examined in transgenic mice that overexpress the bone morphogenetic protein (BMP) inhibitor Noggin under the control of the neuron-specific enolase promoter. The Noggin transgene was misexpressed in the proximal portion of the hair follicle, primarily the matrix cells, apart from the usual expression in neurons. Transgene expression appeared only after induction of both the primary (tylotrich) and secondary (nontylotrich) pelage hair follicles had already occurred, thus allowing examination of the role of BMP signaling in follicles that had been induced normally in the presence of BMPs. The overexpression of Noggin in these animals resulted in a dramatic loss of hair postnatally. There was an apparently normal, but shortened period of postnatal hair follicle morphogenesis, followed by premature initiation of hair follicle cycling via entry into the first catagen transformation. This resulted in a complete loss of hair shafts from the nontylotrich hair follicles in these mice while the tylotrich hair follicles were normal. The onset of anagen of the first postnatal hair follicle cycle was also accelerated in the transgenic mice. Our results show that BMP signaling is specifically required for proper proliferation and differentiation during late morphogenesis of nontylotrich hair follicles and that inhibition of this signaling pathway may be one of the triggers for the onset of catagen when the follicles are in anagen and the onset of anagen when the follicles are in telogen. Ectopic sebocyte differentiation was another hallmark of the phenotype of these transgenic mice suggesting that BMP signaling may be an important determinant of lineage selection by common progenitor cells in the skin. BMPs likely promote a hair follicle-type differentiation pathway of keratinocytes while suppressing the sebaceous differentiation pathway of skin epithelium
— id: 44783, year: 2004, vol: 165, page: 729, stat: Journal Article,

Beta-catenin and Cyclin D1: Connecting Development to Breast Cancer
Rowlands, Tracey M; Pechenkina, Irina V; Hatsell, Sarah; Cowin, Pamela
2004 Mar-Apr;3(2):145-148, Cell cycle
Beta-catenin and cyclin D1 have attracted considerable attention due to their proto-oncogenic roles in human cancer. The finding of cyclin D1 as a direct target gene of beta-catenin in colon cancer cells led to the assumption that cyclin D1 upregulation is pivotal to beta-catenin's oncogenicity. Our recent paper shows that this is not the case; cyclin D1 dampens the oncogenicity of activated beta-catenin (MMTV-DN89beta-catenin). The relationships and dependencies of beta-catenin and cyclin D1 point to distinct, essential and sequential roles during alveologenesis. These results support the concept that both beta-catenin's and cyclin D1's actions are more sophisticated than simple acceleration of the cell cycle clock. These proteins are employed at critical junctures involving cell fate decisions that we speculate require specific types of cell cycle to traverse
— id: 41643, year: 2004, vol: 3, page: 145, stat: Journal Article,

Plakoglobin is O-glycosylated close to the N-terminal destruction box
Hatsell, Sarah; Medina, Lillian; Merola, Joe; Haltiwanger, Robert; Cowin, Pamela
2003 Sep 26;278(39):37745-37752, Journal of biological chemistry
Plakoglobin provides a key linkage in protein chains that connect desmosomal and classical cadherins to the cytoskeleton. It is also present in a significant cytosolic pool that has the capacity to impact on canonical Wnt signaling by competing for interaction with partner proteins of beta-catenin. The closely related protein, beta-catenin, is rapidly targeted for proteasomal degradation by phosphorylation of a 'destruction box' within the N-terminal domain. Inhibition of this process forms the basis of Wnt signaling. This destruction box is also found in the N-terminal domain of plakoglobin. We report that plakoglobin is modified by the addition of O-GlcNAc at a single site in close proximity to the destruction box. O-GlcNAc modification has been proposed to counteract phosphorylation, provide protection from proteasomal degradation, mediate signal transduction, silence transcription, and regulate multimolecular protein assembly. This finding has potential implications for understanding the roles of plakoglobin
— id: 38123, year: 2003, vol: 278, page: 37745, stat: Journal Article,

Beta-catenin and Tcfs in mammary development and cancer
Hatsell, Sarah; Rowlands, Tracey; Hiremath, Minoti; Cowin, Pamela
2003 Apr;8(2):145-158, Journal of mammary gland biology & neoplasia
Beta-catenin regulates cell-cell adhesion and transduces signals from many pathways to regulate the transcriptional activities of Tcf/Lef DNA binding factors. Gene ablation and transgenic expression studies strongly support the concept that beta-catenin together with Lef/Tcf factors act as a switch to determine cell fate and promote cell survival and proliferation at several stages during mammary gland development. Mice expressing the negative regulator of Wnt/beta-catenin signaling (K14-Dkk) fail to form mammary buds, and those lacking Lef-1 show an early arrest in this process at stage E13.5. Stabilized deltaN89beta-catenin initiates precocious alveologenesis during pubertal development, and negative regulators of endogenous beta-catenin signaling suppress normal alveologenesis during pregnancy. Stabilized beta-catenin induces hyperplasia and mammary tumors in mice. Each of the beta-catenin-induced phenotypes is accompanied by upregulation of the target genes cyclin D1 and c-myc. Cyclin D1, however, is dispensable for tumor formation and the initiation of alveologenesis but is essential for later alveolar expansion
— id: 44866, year: 2003, vol: 8, page: 145, stat: Journal Article,

Untangling desmosomal knots with electron tomography
He, Wanzhong; Cowin, Pamela; Stokes, David L
2003 Oct 3;302(5642):109-113, Science
Cell adhesion by adherens junctions and desmosomes relies on interactions between cadherin molecules. However, the molecular interfaces that define molecular specificity and that mediate adhesion remain controversial. We used electron tomography of plastic sections from neonatal mouse skin to visualize the organization of desmosomes in situ. The resulting three-dimensional maps reveal individual cadherin molecules forming discrete groups and interacting through their tips. Fitting of an x-ray crystal structure for C-cadherin to these maps is consistent with a flexible intermolecular interface mediated by an exchange of amino-terminal tryptophans. This flexibility suggests a novel mechanism for generating both cis and trans interactions and for propagating these adhesive interactions along the junction
— id: 38124, year: 2003, vol: 302, page: 109, stat: Journal Article,

Evidence that transgenes encoding components of the Wnt signaling pathway preferentially induce mammary cancers from progenitor cells
Li, Yi; Welm, Bryan; Podsypanina, Katrina; Huang, Shixia; Chamorro, Mario; Zhang, Xiaomei; Rowlands, Tracey; Egeblad, Mikala; Cowin, Pam; Werb, Zena; Tan, Lee K; Rosen, Jeffrey M; Varmus, Harold E
2003 Dec 23;100(26):15853-15858, Proceedings of the National Academy of Sciences of the United States of America
Breast cancer is a genetically and clinically heterogeneous disease, and the contributions of different target cells and different oncogenic mutations to this heterogeneity are not well understood. Here we report that mammary tumors induced by components of the Wnt signaling pathway contain heterogeneous cell types and express early developmental markers, in contrast to tumors induced by other signaling elements. Expression of the Wnt-1 protooncogene in mammary glands of transgenic mice expands a population of epithelial cells expressing progenitor cell markers, keratin 6 and Sca-1; subsequent tumors express these markers and contain luminal epithelial and myoepithelial tumor cells that share a secondary mutation, loss of Pten, implying that they arose from a common progenitor. Mammary tumors arising in transgenic mice expressing beta-catenin and c-Myc, downstream components of the canonical Wnt signaling pathway, also contain a significant proportion of myoepithelial cells and cells expressing keratin 6. Progenitor cell markers and myoepithelial cells, however, are lacking in mammary tumors from transgenic mice expressing Neu, H-Ras, or polyoma middle T antigen. These results suggest that mammary stem cells and/or progenitors to mammary luminal epithelial and myoepithelial cells may be the targets for oncogenesis by Wnt-1 signaling elements. Thus, the developmental heterogeneity of different breast cancers is in part a consequence of differential effects of oncogenes on distinct cell types in the breast
— id: 41642, year: 2003, vol: 100, page: 15853, stat: Journal Article,

Mammary gland development requires syndecan-1 to create a beta-catenin/TCF-responsive mammary epithelial subpopulation
Liu, Bob Y; Kim, Young Chul; Leatherberry, Vicki; Cowin, Pam; Alexander, Caroline M
2003 Dec 18;22(58):9243-9253, Oncogene
Mice with a null mutation in the cell surface heparan sulfate (HS) proteoglycan, syndecan-1 (Sdc1), develop almost normally, but resist mammary tumor development in response to Wnt-1. Here, we test the hypothesis that Sdc1 promotes Wnt-1-induced tumor development by interacting with the Wnt cell surface signaling complex. Thus, the response of Sdc1-/- mammary epithelial cells (mecs) to the intracellular, activated Wnt signal transducer, DeltaNbeta-catenin, was assayed both in vitro and in vivo, to test whether beta-catenin/TCF transactivation was Sdc1-independent. Surprisingly, we found that the expression of a canonical Wnt pathway reporter, TOP-FLASH, was reduced by 50% in both unstimulated Sdc1-/- mecs and in stimulated cells responding to Wnt1 or DeltaNbeta-catenin. Tumor development in response to DeltaNbeta-catenin was also significantly delayed on a Sdc1-/- background. Furthermore, the average beta-catenin/TCF transactivation per cell was normal in Sdc1-/- mec cultures, but the number of responsive cells was reduced by 50%. Sdc1-/- mecs show compensatory changes that maintain the number of HS chains, hence these experiments cannot test the coreceptor activity of HS for Wnt signaling. We propose that TCF-dependent transactivational activity is suppressed in 50% of cells in Sdc1-/- glands, and conclude that the major effect of Sdc1 does not map to the activity of the Wnt signaling complex, but to another pathway to create or stabilize the beta-catenin/TCF-responsive tumor precursor cells in mouse mammary gland
— id: 41641, year: 2003, vol: 22, page: 9243, stat: Journal Article,

Molecular cloning of the mouse Ltbp-1 gene reveals tissue specific expression of alternatively spliced forms
Noguera, Irene; Obata, Hiroto; Gualandris, Anna; Cowin, Pamela; Rifkin, Daniel B
2003 Apr 10;308(5642):31-41, Gene
Latent transforming growth factor binding proteins (Ltbp-1, -2, -3 and -4) and fibrillins (Fbn-1 and -2) are structurally related cysteine-rich extracellular matrix proteins that localize to the 10 nm microfibrils. Ltbp-1 is thought to promote the secretion and proper folding of the small latent transforming growth factor beta (TGF-beta) complex (TGF-beta plus its propeptide) and is implicated in sequestering it in the extracellular matrix. Here we report the isolation of the mouse Ltbp-1 complementary DNA (cDNA) and gene. The longer form of the Ltbp-1 cDNA encodes a predicted 1713 amino acid protein containing 18 epidermal growth factor-like repeats, four 8-cysteine domains and several motifs that suggest interactions with alpha(IV)beta(1) and alpha(9)beta(1) integrins. Northern blotting analyses indicate that long and short Ltbp-1 transcripts are widely expressed in adult mouse tissues and most abundantly expressed in heart. Ltbp-1 is a single copy gene that maps to chromosome 17, band E (1-3) and encompasses more than 212 kb. The Ltbp-1 gene contains 34 exons and shows a similar organization to the LTBP-2 gene, suggesting that these genes originated from a common ancestral gene
— id: 38125, year: 2003, vol: 308, page: 31, stat: Journal Article,

Dissecting the roles of beta-catenin and cyclin D1 during mammary development and neoplasia
Rowlands, Tracey M; Pechenkina, Irina V; Hatsell, Sarah J; Pestell, Richard G; Cowin, Pamela
2003 Sep 30;100(20):11400-11405, Proceedings of the National Academy of Sciences of the United States of America
A considerable body of circumstantial data suggests that cyclin D1 is an attractive candidate to mediate the effects of beta-catenin in mammary tissue. To test the functional significance of these correlative findings, we investigated the genetic interaction between transcriptionally active beta-catenin (DeltaN89beta-catenin) and its target gene cyclin D1 in the mouse mammary gland during pubertal development, pregnancy, and tumorigenesis. Our data demonstrate that cyclin D1 is dispensable for the DeltaN89beta-catenin-stimulated initiation of alveologenesis in virgin females, for the de novo induction of alveoli in males, and for the formation of tumors. Indeed, lack of cyclin D1 accentuates and enhances these hyperplastic and tumorigenic DeltaN89beta-catenin phenotypes. Although alveologenesis is initiated by DeltaN89beta-catenin in a cyclin D1-independent fashion, up-regulation of cyclin D1 occurs in DeltaN89beta-catenin mice and its expression remains essential for the completion of alveolar development during the later stages of pregnancy. Thus, alveologenesis is a two-step process, and cyclin D1 activity during late alveologenesis cannot be replaced by the activity of other beta-catenin target genes that successfully drive proliferation at earlier stages
— id: 38126, year: 2003, vol: 100, page: 11400, stat: Journal Article,

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

Deconstructing desmoplakin
Hatsell, S; Cowin, P
2001 DEC ;3(12):E270-E272, Nature cell biology
Mature desmosomes are the main adhesive junctions in epithelia and cardiac muscle. Now new work shows that the desmosomal protein desmoplakin is also essential for the maturation of adherens junctions. Desmoplakin clamps down on the transient zippering courtship of the classical cadherins, promoting the maturation of puncta adherens junctions and cortical actin remodelling, steps that are essential for cell adhesion
— id: 54788, year: 2001, vol: 3, page: E270, stat: Journal Article,

Delta N89 beta-catenin induces precocious development, differentiation, and neoplasia in mammary gland
Imbert A; Eelkema R; Jordan S; Feiner H; Cowin P
2001 Apr 30;153(3):555-568, Journal of cell biology
To investigate the role of beta-catenin in mammary gland development and neoplasia, we expressed a stabilized, transcriptionally active form of beta-catenin lacking the NH(2)-terminal 89 amino acids (Delta N 89 beta-catenin) under the control of the mouse mammary tumor virus long terminal repeat. Our results show that Delta N 89 beta-catenin induces precocious lobuloalveolar development and differentiation in the mammary glands of both male and female mice. Virgin Delta N 89 beta-catenin mammary glands resemble those found in wild-type (wt) pregnant mice and inappropriately express cyclin D1 mRNA. In contrast to wt mammary glands, which resume a virgin appearance after cessation of lactation, transgenic mammary glands involute to a midpregnant status. All transgenic females develop multiple aggressive adenocarcinomas early in life. Surprisingly, the Delta N89 beta-catenin phenotype differs from those elicited by overexpression of Wnt genes in this gland. In particular, Delta N 89 beta-catenin has no effect on ductal side branching. This suggests that Wnt induction of ductal branching involves additional downstream effectors or modulators
— id: 20693, year: 2001, vol: 153, page: 555, stat: Journal Article,

Appearance of Langerhans cells in the epidermis of Tgfb1(-/-) SCID mice: paracrine and autocrine effects of transforming growth factor-beta 1 and -beta 2(1)
Thomas RM; Belsito DV; Huang C; Chen Lz LZ; Ormsby I; Simmons WJ; Cowin P; Shaw J; Doetschman T; Thorbecke GJ
2001 Dec;117(6):1574-1580, Journal of investigative dermatology
A striking immunologic abnormality of normal and SCID Tgfb1(-/-) mice is the total absence of Langerhans cells in their epidermis. Here we show that transfer of Tgfb1(+/-) SCID bone marrow causes, within a few weeks, the appearance of Langerhans cells in the epidermis of gamma-irradiated and unirradiated Tgfb1(-/-) SCID recipients. In addition, local injection of 2 x 10(5) latent transforming growth factor-beta1 cDNA-transduced cloned CD4+ T lymphocytes causes the appearance of Langerhans cells in the ear epidermis of Tgfb1(-/-) SCID mice. This effect is enhanced by antigen-specific activation of these T cells. Injection of recombinant active transforming growth factor-beta 2 into the ear of Tgfb1(-/-) SCID mice also results in the migration of Langerhans cells into the epidermis locally, but no epidermal Langerhans cells are seen after systemic injections of transforming growth factor-beta 2. Our results suggest that transforming growth factor-beta can act in paracrine as well as autocrine fashion to induce the differentiation of precursors into Langerhans cells. Furthermore, these results indicate that the relative roles of different transforming growth factor-beta isoforms in vivo may be influenced by their local availability and/or the regulation of their conversion from latent into active form
— id: 32235, year: 2001, vol: 117, page: 1574, stat: Journal Article,

Plakoglobin suppresses epithelial proliferation and hair growth in vivo
Charpentier E; Lavker RM; Acquista E; Cowin P
2000 Apr 17;149(2):503-520, Journal of cell biology
Plakoglobin regulates cell adhesion by providing a modulatable connection between both classical and desmosomal cadherins and their respective cytoskeletal linker proteins. Both plakoglobin and the related protein beta-catenin are posttranscriptionally upregulated in response to Wnt-1 in cultured cells. Upregulation of beta-catenin has been implicated in potentiating hyperproliferation and tumor formation. To investigate the role of plakoglobin in these functions we expressed a full-length (PG) and an NH(2)-terminally truncated form of plakoglobin (DeltaN80PG) in mouse epidermis and hair follicles, tissues which undergo continuous and easily observed postnatal renewal and remodeling. Expression of these constructs results in stunted hair growth, a phenotype that has also been observed in transgenic mice expressing Wnt3 and Dvl2 (Millar et al. 1999). Hair follicles from PG and DeltaN80PG mice show premature termination of the growth phase (anagen) of the hair cycle, an event that is regulated in part by FGF5 (Hebert et al. 1994). The proliferative rate of the epidermal cells was reduced and apoptotic changes, which are associated with entry into the regressive phase of the hair follicle cycle (catagen), occurred earlier than usual
— id: 11747, year: 2000, vol: 149, page: 503, stat: Journal Article,

Cytoskeletal-membrane interactions and signal transduction
Cowin, Pam; Klymkowsky, Michael
New York : Chapman & Hall ; Austin, Tex. : Landes Bioscience, c1997,
— id: 576, year: 1997, vol: , page: , stat: ,

Cytoskeleton-membrane interactions [published erratum appears in Curr Opin Cell Biol 1996 Apr;8(2):following 244]
Cowin P; Burke B
1996 Feb;8(1):56-65, Current opinion in cell biology
Associations between the cytoskeleton and cellular membranes, both within the cell and at points of cell contact, play a central role in determining cell shape and tissue integrity. During the past few years, it has become clear that many of these cytoskeleton-membrane interactions go far beyond simple mechanical linkages. For example, proteins that act as linker molecules at the adherens junctions and desmosomes in the plasma membrane have newly recognized functions in signal transduction pathways. These functions have profound effects on cell behaviour during development. In addition, within the nucleus, the lamin branch of the intermediate filament protein family appears to have a key role in defining the protein composition of the inner nuclear membrane by means of extensive interactions with integral membrane proteins. The identities of these integral membrane proteins are only now coming to light
— id: 6896, year: 1996, vol: 8, page: 56, stat: Journal Article,

Desmosomal cadherin binding domains of plakoglobin
Witcher LL; Collins R; Puttagunta S; Mechanic SE; Munson M; Gumbiner B; Cowin P
1996 May 3;271(18):10904-10909, Journal of biological chemistry
Plakoglobin is a major component of both desmosomes and adherens junctions. At these sites it binds to the cytoplasmic domains of cadherin cell-cell adhesion proteins and regulates their adhesive and cytoskeletal binding functions. Plakoglobin also forms distinct cytosolic protein complexes that function in pathways of tumor suppression and cell fate determination. Recent studies in Xenopus suggest that cadherins inhibit the signaling functions of plakoglobin presumably by sequestering this protein at the membrane and depleting its cytosolic pool. To understand the reciprocal regulation between desmosomal cadherins (desmoglein and desmocollin) and plakoglobin, we have sought to identify the binding domains involved in the formation of these protein complexes. Plakoglobin comprises 13 central repeats flanked by amino-terminal and carboxyl-terminal domains. Our results show that repeats 1-4 are involved in binding desmoglein-1. In contrast, the interaction of plakoglobin with desmocollin-1a is sensitive to deletion of either end of the central repeat domain. The binding sites for two adherens junction components, alpha-catenin and classical cadherins, overlap these sites. Competition among these proteins for binding sites on plakoglobin may therefore account for the distinct composition of adherens junctions and desmosomes
— id: 7059, year: 1996, vol: 271, page: 10904, stat: Journal Article,

Protein zero, a nervous system adhesion molecule, triggers epithelial reversion in host carcinoma cells
Doyle JP; Stempak JG; Cowin P; Colman DR; D'Urso D
1995 Oct;131(2):465-482, Journal of cell biology
Protein zero (P(o)) is the immunoglobulin gene superfamily glycoprotein that mediates the self-adhesion of the Schwann cell plasma membrane that yields compact myelin. HeLa is a poorly differentiated carcinoma cell line that has lost characteristic morphological features of the cervical epithelium from which it originated. Normally, HeLa cells are not self-adherent. However, when P(o) is artificially expressed in this line, cells rapidly aggregate, and P(o) concentrates specifically at cell-cell contact sites. Rows of desmosomes are generated at these interfaces, the plasma membrane localization of cingulin and ZO-1, proteins that have been shown to be associated with tight junctions, is substantially increased, and cytokeratins coalesce into a cohesive intracellular network. Immunofluorescence patterns for the adherens junction proteins N-cadherin, alpha-catenin, and vinculin, and the desmosomal polypeptides desmoplakin, desmocollin, and desmoglein, are also markedly enhanced at the cell surface. Our data demonstrate that obligatory cell-cell adhesion, which in this case is initially brought about by the homophilic association of P(o) molecules across the intercellular cleft, triggers pronounced augmentation of the normally sluggish or sub-basal cell adhesion program in HeLa cells, culminating in suppression of the transformed state and reversion of the monolayer to an epithelioid phenotype. Furthermore, this response is apparently accompanied by an increase in mRNA and protein levels for desmoplakin and N-cadherin which are normally associated with epithelial junctions. Our conclusions are supported by analyses of ten proteins we examined immunochemically (P(o), cingulin, ZO-1, desmoplakin, desmoglein, desmocollin, N-cadherin, alpha-catenin, vinculin, and cytokeratin-18), and by quantitative polymerase chain reactions to measure relative amounts of desmoplakin and N-cadherin mRNAs. P(o) has no known signaling properties; the dramatic phenotypic changes we observed are highly likely to have developed in direct response to P(o)-induced cell adhesion. More generally, the ability of this 'foreign' membrane adhesion protein to stimulate desmosome and adherens junction formation by augmenting well-studied cadherin-based adhesion mechanisms raises the possibility that perhaps any bona fide cell adhesion molecule, when functionally expressed, can engage common intracellular pathways and trigger reversion of a carcinoma to an epithelial-like phenotype
— id: 16330, year: 1995, vol: 131, page: 465, stat: Journal Article,

Unraveling the cytoplasmic interactions of the cadherin superfamily [see comments]
Cowin P
1994 Nov 8;91(23):10759-10761, Proceedings of the National Academy of Sciences of the United States of America
— id: 6598, year: 1994, vol: 91, page: 10759, stat: Journal Article,

Interactions of the cytoplasmic domain of the desmosomal cadherin Dsg1 with plakoglobin
Mathur M; Goodwin L; Cowin P
1994 May 13;269(19):14075-14080, Journal of biological chemistry
Dsg1 is a 165-kDa glycoprotein component of suprabasal epidermal desmosomes and the prototype of a subset of the cadherin superfamily of cell-cell adhesion proteins known as desmogleins. The adhesive function of classical cadherins is known to be dependent upon their association with cytoplasmic components called catenins. In the case of desmogleins, a single interaction has been described with a protein called plakoglobin that is found in desmosomal plaques, adherens junctions, and the cytosol. Several proteins with homology to plakoglobin have been described that regulate junction assembly and implement morphoregulatory signals. To address the functional significance of plakoglobin-desmoglein interaction, we have mapped the sequences of Dsg1 that are crucial for this association by using blot overlay techniques. By examining the binding of plakoglobin to a deletion series of the Dsg1 cytoplasmic domain expressed as fusion proteins, we have defined a 19-amino acid sequence that is important for association. This region of Dsg1 sequence shows significant similarity to the catenin-binding domain of classical cadherins, suggesting a common mechanism for the association of plakoglobin with desmosomes and adherens junctions
— id: 6448, year: 1994, vol: 269, page: 14075, stat: Journal Article,

Structure of DSG1, the bovine desmosomal cadherin gene encoding the pemphigus foliaceus antigen. Evidence of polymorphism
Puttagunta S; Mathur M; Cowin P
1994 Jan 21;269(3):1949-1955, Journal of biological chemistry
The cadherin superfamily of calcium-dependent cell-cell adhesion and recognition proteins can be categorized into a number of subsets on the basis of the distinct cytoplasmic sequences of their members. Currently these families include classical cadherins, desmogleins, desmocollins, protocadherins, and the products of the Drosophila genes FAT and Dachsous. Dsg1, the prototype of the desmoglein family, is a major component of epidermal desmosomes and the antigenic target of antibodies found in the sera of patients with the blistering disease, pemphigus foliaceus. In this study, we determined the organization of the bovine DSG1 gene. This gene consists of 15 exons distributed over > 37.5 kilobases of genomic DNA. A comparison of DSG1 with genes encoding classical cadherins revealed a striking conservation of exon boundaries in regions encoding the ectodomain and to a more limited extent among those encoding the cytoplasmic domain. Polymorphism was found in a sequence of DSG1 encoding protein proximal to the external face of the plasma membrane. This region is topologically equivalent to a domain of classical cadherins that harbors epitopes recognized by adhesion-disrupting antibodies. We discuss these results with regard to the evolution of the cadherin superfamily and their implications for the definition of pemphigus epitopes
— id: 6489, year: 1994, vol: 269, page: 1949, stat: Journal Article,

Alterations in beta-catenin phosphorylation and plakoglobin expression in human breast cancer cells
Sommers CL; Gelmann EP; Kemler R; Cowin P; Byers SW
1994 Jul 1;54(13):3544-3552, Cancer research
Because the cell adhesion molecule epithelial cadherin (E-cadherin) is absent in many invasive carcinomas, we transfected the E-cadherin gene into E-cadherin-negative, invasive breast cancer cell lines BT549 and HS578t to investigate the role of E-cadherin in invasive behavior. Although the transfected E-cadherin could mediate calcium-dependent aggregation to E-cadherin-transfected L-cells, morphology and invasiveness of the breast cancer cells were not altered. We investigated the strength of the linkage of the transfected E-cadherin to the actin cytoskeleton by examining the Triton X-100 solubility of the transfected E-cadherin. In BT549 and HS578t cells, a large proportion of the transfected E-cadherin was Triton soluble, whereas in E-cadherin-positive MCF-7 cells, Triton-insoluble E-cadherin was apparent at cell-cell borders. Interaction of E-cadherin with the actin cytoskeleton is thought to be mediated by the E-cadherin-binding proteins alpha-catenin, beta-catenin, and plakoglobin. We found normal levels of alpha-catenin and beta-catenin in BT549 and HS578t cells; however, low levels of plakoglobin were expressed in these cells compared to those found in weakly invasive MCF-7 cells. Furthermore, levels of tyrosine phosphorylation of beta-catenin were elevated in E-cadherin-transfected BT549 and HS578t cells compared to MCF-7 cells. We conclude that other factors such as the expression and appropriate posttranslational modification of cadherin-associated proteins must be in place for E-cadherin to be fully functional, i.e., to alter invasiveness. During cancer progression, loss of E-cadherin expression itself or multiple other mechanisms that lead to loss of cell-cell adhesion (mutation, loss of catenin expression, alterations in phosphorylation) may contribute to a more metastatic phenotype
— id: 16331, year: 1994, vol: 54, page: 3544, stat: Journal Article,

Expression of Wnt-1 in PC12 cells results in modulation of plakoglobin and E-cadherin and increased cellular adhesion
Bradley RS; Cowin P; Brown AM
1993 Dec;123(6 Pt 2):1857-1865, Journal of cell biology
The Wnt-1 gene plays an essential role in fetal brain development and encodes a secreted protein whose signaling mechanism is presently unknown. In this report we have investigated intracellular mechanisms by which the Wnt-1 gene induces morphological changes in PC12 pheochromocytoma cells. PC12 cells expressing Wnt-1 show increased steady-state levels of the adhesive junction protein plakoglobin, and an altered distribution of this protein within the cell. This effect appears similar to a modulation of the plakoglobin homolog, Armadillo, that occurs in Drosophila embryos in response to the Wnt-1 homolog, wingless (Riggleman, B., P. Schedl, and E. Wieschaus. 1990. Cell. 63:549-560). In addition, PC12/Wnt-1 cells show elevated expression of E-cadherin and increased calcium-dependent cell-cell adhesion. These results imply evolutionary conservation of cellular responses to Wnt-1/wingless and indicate that in certain cell types Wnt-1 may act to modulate cell adhesion mechanisms
— id: 16332, year: 1993, vol: 123, page: 1857, stat: Journal Article,

Nomenclature of the desmosomal cadherins
Buxton RS; Cowin P; Franke WW; Garrod DR; Green KJ; King IA; Koch PJ; Magee AI; Rees DA; Stanley JR; et al.
1993 May;121(3):481-483, Journal of cell biology
— id: 16333, year: 1993, vol: 121, page: 481, stat: Journal Article,

Components of intercellular adhesive junctions and their role in morphogenesis
Cowin P; Brown A
Molecular basis of morphogenesis New York : Wiley-Liss, 1993,
— id: 3526, year: 1993, vol: , page: 49, stat: Chapter,

Desmocollins form a distinct subset of the cadherin family of cell adhesion molecules
Mechanic S; Raynor K; Hill JE; Cowin P
1991 May 15;88(10):4476-4480, Proceedings of the National Academy of Sciences of the United States of America
The desmosomal adhesive core is formed by four major components: desmoglein (Mr, 165,000), desmocollins I and II (Mr, 120,000 and 110,000, respectively), and a Mr 22,000 protein. Here, we report the cloning and sequencing of cDNAs encoding a bovine desmocollin. The open reading frame found in the longest cDNA, 5 kilobases, contains a region encoding a protein of 839 amino acids. The features of the deduced amino acid sequence imply that the mature 707-amino acid desmocollin is a type I transmembrane protein that is produced by proteolytic cleavage of an 810-amino acid precursor. The ectodomain of desmocollin contains repeats that show extensive sequence similarity to members of the cadherin family of calcium-dependent cell adhesion molecules. A comparison of the amino acid sequences of desmocollin, desmoglein, and the cadherins shows that although these intercellular junctional adhesion molecules share a consensus sequence in their adhesive domains that defines them as a family, several features, including the divergence in the sequence of their cytoplasmic tails, divide them into three distinct subtypes
— id: 14021, year: 1991, vol: 88, page: 4476, stat: Journal Article,

Desmoglein shows extensive homology to the cadherin family of cell adhesion molecules
Goodwin L; Hill JE; Raynor K; Raszi L; Manabe M; Cowin P
1990 Dec 31;173(3):1224-1230, Biochemical & biophysical research communications
Desmoglein is a major adhesive component of the desmosome. It is also at least one of the antigenic targets of pathogenic antibodies circulating in the sera of patients with the blistering disease Pemphigus foliaceus. To examine the molecular basis of desmosomal adhesion and to further our understanding of its disruption in various bullous disorders we have cloned cDNAs encoding four of the extracellular domains of desmoglein. The predicted amino acid sequence of these clones shows extensive homology with the cadherin class of calcium-dependent cell adhesion molecules. Desmoglein represents a novel subtype of this family
— id: 14242, year: 1990, vol: 173, page: 1224, stat: Journal Article,

Molecular cloning and amino acid sequence of human plakoglobin, the common junctional plaque protein
Franke WW; Goldschmidt MD; Zimbelmann R; Mueller HM; Schiller DL; Cowin P
1989 Jun;86(11):4027-4031, Proceedings of the National Academy of Sciences of the United States of America
Plakoglobin is a major cytoplasmic protein that occurs in a soluble and a membrane-associated form and is the only known constituent common to the submembranous plaques of both kinds of adhering junctions, the desmosomes and the intermediate junctions. Using a partial cDNA clone for bovine plakoglobin, we isolated cDNAs encoding human plakoglobin, determined its nucleotide sequence, and deduced the complete amino acid sequence. The polypeptide encoded by the cDNA was synthesized by in vitro transcription and translation and identified by its comigration with authentic plakoglobin in two-dimensional gel electrophoresis. The identity was further confirmed by comparison of the deduced sequence with the directly determined amino acid sequence of two fragments from bovine plakoglobin. Analysis of the plakoglobin sequence showed the protein (744 amino acids; 81,750 Da) to be unrelated to any other known proteins, highly conserved between human and bovine tissues, and characterized by numerous changes between hydrophilic and hydrophobic sections. Only one kind of plakoglobin mRNA (3.4 kilobases) was found in most tissues, but an additional mRNA (3.7 kilobases) was detected in certain human tumor cell lines. This longer mRNA may be represented by a second type of plakoglobin cDNA, which contains an insertion of 297 nucleotides in the 3' non-coding region
— id: 16334, year: 1989, vol: 86, page: 4027, stat: Journal Article,

The desmosomal plaque and the cytoskeleton
Franke WW; Cowin P; Schmelz M; Kapprell HP
1987 ;125(3):26-48, CIBA Foundation symposium
Two major plasma membrane domains are involved in the architectural organization of the cytoskeleton. Both are junctions of the adherens category characterized by the presence of dense plaques associated with the cytoplasmic surface of their membranes. The plaques serve as specific anchorage structures for two different types of cytoplasmic filaments. Intermediate-sized filaments (IF) of several types, i.e. cytokeratin IF in epithelial cells, desmin IF in cardiac myocytes and vimentin IF in arachnoidal cells of meninges, meningiomas and several other cells, attach to the desmosomal plaques, whereas actin-containing microfilaments associate with non-desmosomal adhering junctions such as the zonula adherens, fascia adherens and punctum adherens. The plaques of both kinds of adhering junctions contain a common acidic polypeptide of Mr 83,000 identical to 'band 5 protein' of bovine snout epidermal desmosomes. However, other plaque components are mutually exclusive to one of the two subclasses of adhering junctions. The desmosomal plaque structure, which does not contain vinculin and alpha-actinin, comprises representatives of cytoplasmic, non-membrane-integrated proteins such as desmoplakin(s) and the cytoplasmic portions of transmembrane glycoproteins such as 'band 3 glycoprotein'. The analysis of both categories of junction-associated plaques should provide a basis for understanding the establishment and the dynamics of junction-cytoskeleton interaction
— id: 16337, year: 1987, vol: 125, page: 26, stat: Journal Article,

Immunolocalization of plakoglobin in endothelial junctions: identification as a special type of Zonulae adhaerentes
Franke WW; Kapprell HP; Cowin P
1987 ;59(3):205-218, Biology of the cell
We have characterized the junctions between endothelial cells of diverse blood vessels at the light and electron microscopic level using various antibodies to plakoglobin (polypeptide Mr 83,000) and vinculin. Endothelial cells from fenestrated and non-fenestrated capillaries to large arteries are connected to each other by extended junctions that are coated on their cytoplasmic face by plaques of loosely matted filamentous material that form a continuous belt system along the cell circumference. These plaques are devoid of desmosome-specific proteins such as desmoplakin(s) and desmoglein, but contain plakoglobin. Immunofluorescence microscopic reactions of these regions with vinculin antibodies have also been observed, although they are much weaker and less consistent. This composition, together with their association with actin microfilaments, classifies this extended plaque system as Zonulae adhaerentes. Our results also show that such endothelia may be distinguished from truly epithelial cells by the absence of desmosomes and intermediate filaments of the cytokeratin type. The relationship of the various kinds of adhering junctions and the physiological importance of these junctions are discussed
— id: 16338, year: 1987, vol: 59, page: 205, stat: Journal Article,

Plakoglobin is a component of the filamentous subplasmalemmal coat of lens cells
Franke WW; Kapprell HP; Cowin P
1987 Jun;43(3):301-315, European journal of cell biology
The plasma membranes of the cells of the superficial layer of the eye lens and the lens fibres are in close intercellular contact, leaving an intermembrane space of approximately 20 nm or less throughout their entire length. This plasma membrane is underlaid by a filamentous, cytoplasmic web containing actin, proteins of the spectrin and band 4.1 families, alpha-actinin and vinculin. Using immunofluorescence microscopy and immunoblotting of gel electrophoretically separated proteins, we show that plakoglobin, the plaque protein common to desmosomal and nondesmosomal adhering junctions, is present in lens cells and is also a component of the subplasmalemmal coat of these cells. Plakoglobin also exists in the extended regions of intercellular contacts between cultured lenticular cells where it often colocalizes with vinculin but does not occur in other vinculin-rich plasma membrane regions such as the focal adhesions at the ventral cell surface. Plakoglobin associated with plasma membrane regions can also be identified in various other adhesive cultured cells, but it is not detected in cells and tissues that do not establish firm intercellular junctions such as erythrocytes, platelets, cultured myeloma cells and smooth muscle tissue. We conclude that plakoglobin occurs, at least in lens cells, throughout the entire subplasmalemmal coat, coexisting in this situation not only with vinculin but also with spectrin and 4.1 protein(s). This colocalization infers the presence of a distinct, complex type of membrane-skeleton assembly involving the actin filament-associated junctional plaque elements plakoglobin and vinculin together with actin-associated proteins of the spectrin and band 4.1 protein families
— id: 16336, year: 1987, vol: 43, page: 301, stat: Journal Article,

Biochemical characterization of the soluble form of the junctional plaque protein, plakoglobin, from different cell types
Kapprell HP; Cowin P; Franke WW
1987 Aug 3;166(3):505-517, European journal of biochemistry
A polypeptide of identical molecular mass (Mr 83,000) and charge to desmosomal plakoglobin from bovine snout epidermis was identified in soluble and pelletable fractions from diverse tissues and cells of different mammalian species, including cells and tissues devoid of desmosomes (e.g. endothelial, retinal, lenticular cells, fibroblasts). The protein, however, was not detected in erythrocytes and platelets and in myeloma cells, nor in smooth muscle tissue. In all cells examined, the plakoglobin soluble upon cell lysis in buffers of near-physiological pH and ionic strength (21-31% of the total plakoglobin in the different cell types) was found to exist in a distinct molecular form. On sucrose gradient centrifugation it appeared at about 7 S and gel filtration chromatography revealed a Stokes radius of about 5.0 nm, from which an Mr of about 170,000 was estimated. By using isoelectric focusing under non-denaturing conditions, soluble approximately equal to 7-S plakoglobin had an isoelectric point at about pH 5.3. The plaque-bound and the soluble form of plakoglobin were indistinguishable by electrical charge and molecular mass, regardless of the source, indicating molecular identity. Cross-linking of soluble proteins with cupric 1,10-phenanthroline resulted in the formaton of a cross-linked product of plakoglobin with similar physical properties as the native approximately equal to 7-S particle, which is compatible with the interpretation that the soluble plakoglobin particle is a dimer. While a major proportion of the plakoglobin in the desmosomal plaque was resistant to various extraction procedures, plakoglobin present in the plaques of non-desmosome-containing cells and tissues was readily extractable under low and high salt conditions. This indicates that differences exist in the binding of plakoglobin to desmosomal plaques and the plaques of non-demosomal junctions
— id: 16335, year: 1987, vol: 166, page: 505, stat: Journal Article,

Plakoglobin: a protein common to different kinds of intercellular adhering junctions
Cowin P; Kapprell HP; Franke WW; Tamkun J; Hynes RO
1986 Sep 26;46(7):1063-1073, Cell
We have established, by means of a monoclonal antibody and a cDNA clone, that a desmosomal polypeptide of Mr 83,000 also occurs at the plaques of other types of adhering junctions, including the vinculin-actin-associated intercellular junctions, e.g., the zonula adhaerens of epithelial cells and the endothelial, lens, and Sertoli cell junctions. This is the first component found in common among otherwise biochemically distinct plaque domains. Despite its concentration at these intercellular junctions, it is absent from the respective cell-substratum contact sites. In addition, it appears in a globular soluble 7S form in the cytoplasm. We discuss the significance of this protein, for which the name plakoglobin is proposed, in terms of its interaction with such biochemically diverse membrane domains and their different types of associated cytoskeletal filaments
— id: 16341, year: 1986, vol: 46, page: 1063, stat: Journal Article,

Desmosomal proteins: new markers for identification and classification of tumors
Moll R; Cowin P; Kapprell HP; Franke WW
1986 Jan;54(1):4-25, Laboratory investigation
— id: 16342, year: 1986, vol: 54, page: 4, stat: Journal Article,

A constitutive transmembrane glycoprotein of Mr 165,000 (desmoglein) in epidermal and non-epidermal desmosomes. I. Biochemical identification of the polypeptide
Schmelz M; Duden R; Cowin P; Franke WW
1986 Dec;42(2):177-183, European journal of cell biology
Two murine monoclonal antibodies (DG 3.4 and DG 3.10) raised against a major glycoprotein ('band 3 component') from desmosomes of bovine muzzle epidermis were used in immunoblot experiments following SDS-polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis to identify this or immunologically related proteins in other bovine tissues and cultured cell lines. In all desmosome-bearing cells, i.e. cells also expressing desmoplakins, including representative of stratified, transitional and simple epithelia as well as myocardium, only a single distinct polypeptide of identical Mr value (165,000) and electrical charge was detected. These findings, together with the immunolocalization results reported in the companion paper indicate that this glycoprotein (desmoglein) is a general constituent protein of desmosomes, providing a case of an integral membrane protein co-expressed with non-membranous desmosomal proteins such as the plaque component, desmoplakin I. Our results further suggest that, contrary to previous suggestions, desmoglein is very similar, if not identical in different cells of the same species and does not display significant cell type diversity
— id: 16339, year: 1986, vol: 42, page: 177, stat: Journal Article,

A constitutive transmembrane glycoprotein of Mr 165,000 (desmoglein) in epidermal and non-epidermal desmosomes. II. Immunolocalization and microinjection studies
Schmelz M; Duden R; Cowin P; Franke WW
1986 Dec;42(2):184-199, European journal of cell biology
Using two monoclonal antibodies described in the preceding paper we determined by immunofluorescence microscopy the distribution of an integral membrane protein of the desmosomal domain, the major glycopolypeptide of Mr 165,000 (bovine muzzle epidermal desmosome band 3; desmoglein) in various normal tissues, tumors and cultured cell lines from several mammalian species. This protein was detected in dotted or streak-like arrays along cell boundary structures which were known to contain non-membrane-integrated desmosomal plaque proteins such as desmoplakins. This is true for epithelial, i.e. cytokeratin-expressing cell types, for the desmin-producing myocardiac and Purkinje fiber cells of the heart, and for certain vimentin-containing cells such as arachnoidal and meningiomal cells and dendritic follicular cells of lymph nodes. However, on the basis of both immunoblot and immunocytochemical reactions, the protein is absent from non-desmosomal adhering junctions, including those devoid of desmoplakin but containing another plaque protein, plakoglobin ('band 5 protein'). We have used these antibodies to localize their epitopes with respect to the cell membrane. By immunoelectron microscopy we found that both epitopes are located in the desmosomal plaques, and this was confirmed by microinjection of purified antibodies into living cultured cells which resulted in labelling of the plaques. From these findings, taken together with previous analyses and localizations of the carbohydrate moieties of this glycoprotein, we conclude that desmoglein is a transmembrane glycoprotein which projects into--and contributes to--the desmosomal plaque structure. This glycoprotein represents a general component of true desmosomes and it is coexpressed with obligatory desmosome-specific plaque proteins such as desmoplakin I. The potential value of this glycoprotein as a desmosomal and cell type marker in histology and tumor diagnosis is discussed
— id: 16340, year: 1986, vol: 42, page: 184, stat: Journal Article,

The complement of desmosomal plaque proteins in different cell types
Cowin P; Kapprell HP; Franke WW
1985 Oct;101(4):1442-1454, Journal of cell biology
Desmosomal plaque proteins have been identified in immunoblotting and immunolocalization experiments on a wide range of cell types from several species, using a panel of monoclonal murine antibodies to desmoplakins I and II and a guinea pig antiserum to desmosomal band 5 protein. Specifically, we have taken advantage of the fact that certain antibodies react with both desmoplakins I and II, whereas others react only with desmoplakin I, indicating that desmoplakin I contains unique regions not present on the closely related desmoplakin II. While some of these antibodies recognize epitopes conserved between chick and man, others display a narrow species specificity. The results show that proteins whose size, charge, and biochemical behavior are very similar to those of desmoplakin I and band 5 protein of cow snout epidermis are present in all desmosomes examined. These include examples of simple and pseudostratified epithelia and myocardial tissue, in addition to those of stratified epithelia. In contrast, in immunoblotting experiments, we have detected desmoplakin II only among cells of stratified and pseudostratified epithelial tissues. This suggests that the desmosomal plaque structure varies in its complement of polypeptides in a cell-type specific manner. We conclude that the obligatory desmosomal plaque proteins, desmoplakin I and band 5 protein, are expressed in a coordinate fashion but independently from other differentiation programs of expression such as those specific for either epithelial or cardiac cells
— id: 16344, year: 1985, vol: 101, page: 1442, stat: Journal Article,

Maintenance of desmosomes in mouse hepatocytes after drug-induced rearrangement of cytokeratin filament material. Demonstration of independence of desmosomes and intermediate-sized filaments
Denk H; Lackinger E; Cowin P; Franke WW
1985 Nov;161(1):161-171, Experimental cell research
The distribution of desmosomes and cytokeratin filaments (tonofilaments) in hepatocytes of normal mice and those intoxicated with griseofulvin was studied by immunofluorescence microscopy. Treatment with griseofulvin over prolonged periods of time resulted in the dissociation of cytokeratin filaments from the plasma membrane and the inclusions of cytokeratin material in typical cytoplasmic aggregates, i.e. 'Mallory bodies'. However, such hepatocytes still displayed typical desmosomal arrays, including rather regularly spaced desmosomes along the bile canaliculi. These observations show that, in this tissue, desmosomes are able to maintain their characteristic positions along the plasma membrane after disconnection of the intermediate filament cytoskeleton. This indicates that maintenance of desmosomal integrity and position is independent of desmosome anchorage to tonofilaments. The results are discussed in relation to current concepts of desmosome formation and turnover
— id: 16343, year: 1985, vol: 161, page: 161, stat: Journal Article,

Biochemical characterization of desmosomal proteins isolated from bovine muzzle epidermis: amino acid and carbohydrate composition
Kapprell HP; Cowin P; Franke WW; Ponstingl H; Opferkuch HJ
1985 Mar;36(2):217-229, European journal of cell biology
The seven major desmosomal polypeptides from isolated bovine muzzle desmosomes ranging from Mr 75 000 to 250 000 were separated by gel electrophoresis, isolated and characterized with respect to their amino acid composition and sugar content. The two largest polypeptides (bands 1 and 2), i.e. desmoplakins I and II, are similar in their amino acid composition, confirming our previous immunological and biochemical data, and display a relatively high glycine content. In contrast, the other two cytoplasmic components also believed to be associated with the desmosomal plaque, i.e. polypeptides of bands 5 (Mr 83 000) and 6 (Mr 75 000), differ significantly in their amino acid composition from the desmoplakins and from each other. All four candidate polypeptides for plaque association, i.e. bands 1, 2, 5, and 6, show no significant glycosylation. The glycoproteins 4a and 4b (Mr 115 000 and 130 000) are similar in their amino acid composition, peptide analysis and immunological reactivity. Both are relatively rich in mannose and galactose but also contain sialic acid. Our determinations also indicate that the two polypeptides differ significantly in their N-acetylglucosamine and mannose content. Most, if not all, of the sugar residues are associated with a water-soluble fragment of Mr 15 500 obtained after limited digestion with V8 protease. The glycopolypeptides obtained in band 3 (Mr 164 000-175 000) are distinct from the glycopolypeptides 4a and 4b in amino acid composition, sugar content, isoelectric pH values, certain antigenic determinants and in their pattern of cleavage products obtained by treatment with proteases or cyanogen bromide. The results identify polypeptides of bands 3, 4a and 4b as glycosylated with characteristic sugar compositions. It is suggested that the major glycoproteins (bands 3, 4a, 4b) of the desmosome are integral membrane components arranged in a special way conferring resistance to detergent treatment. The possible roles of these glycoproteins in cell recognition and in adhesive functions of the desmosome are discussed
— id: 16345, year: 1985, vol: 36, page: 217, stat: Journal Article,

Distribution of desmosomal components in the tissues of vertebrates, studied by fluorescent antibody staining
Cowin P; Mattey D; Garrod D
1984 Mar;66(2):119-132, Journal of cell science
In previous work we used immunofluorescent staining with specific antibodies to study the distribution of five desmosomal antigens in the epithelia of different vertebrate animals. We showed that all five antigens were present in all epithelia studied in human, bovine, rat, guinea pig, chick and frog (Rana pipiens) tissues. It was concluded that desmosomes are highly conserved structures. This paper extends those studies: by including three other species, a lizard (Lacerta viridis), the axolotl (Ambystoma mexicanum) and the trout (Salmo trutta), and by looking at several tissues in more detail. The principal results are as follows. The epidermis of all species down to the frog stain with equal intensity for all desmosomal antigens. In the epidermis of axolotl and trout, staining for desmosomal plaque constituents is present, but staining for the desmosomal glycoproteins is greatly reduced or absent. Within mammalian species as well as chick, lizard and frog, staining for the 115 X 10(3) and 100 X 10(3) molecular weight desmosomal glycoproteins is less intense in non-epidermal tissues than in the epidermis, while staining for desmosomal plaque constituents and for the 150 X 10(3) molecular weight glycoprotein is undiminished. It is possible, therefore, that slight differences exist between certain glycoproteins of epidermis and non-epidermal epithelia. The hearts of lower vertebrates (lizard, frog, axolotl and trout) stain only for individual desmosomal plaque antigens. The pillar cells of trout gill stain, adjacent to their collagenous columns, for one desmosomal plaque antigen. There is a fibrous cytoplasmic mat in this position but no desmosomes. Thus one of the desmosomal antigens may have a function outside the desmosome
— id: 16347, year: 1984, vol: 66, page: 119, stat: Journal Article,

Identification of desmosomal surface components (desmocollins) and inhibition of desmosome formation by specific Fab'
Cowin P; Mattey D; Garrod D
1984 Aug;70(2):41-60, Journal of cell science
Specific antibodies against the components of desmosomes, the adhesive junctions of epithelial cells, have been used to determine which components are located on the cell surface. Three criteria have been used: fluorescent antibody staining, immuno-gold labelling and electron microscopy, and quantitative measurements of antibody binding using [125I]protein A. When these techniques were applied to living Madin-Darby bovine kidney (MDBK) cells, antibodies against only two desmosomal components, glycoproteins of approximately 115 X 10(3) Mr and 100 X 10(3) Mr, bound to the cell surface. Antibodies against all other components, the 230 and 205 X 10(3) Mr proteins (desmoplakins), the 150 X 10(3) Mr glycoprotein and the 82 and 86 X 10(3) Mr proteins reacted in fluorescent antibody staining only after cells had been fixed and made permeable. MDBK cells were cultured in the presence of univalent fragments (Fab') of anti-desmosomal antibodies for periods from 24 h to 72 h. After these times cells were fixed, made permeable, and stained with anti-desmoplakin antibody to assay for desmosome formation. Fab' derived from anti-100 X 10(3) Mr protein specifically inhibited desmosome formation, whereas Fab's from anti-desmoplakin, anti-150 X 10(3) Mr and anti-82 and 86 X 10(3) Mr proteins were without effect. We conclude that the 100 X 10(3) Mr and the immunologically related 115 X 10(3) Mr components are located on the cell surface and are directly involved in cell-cell adhesion. We have named them desmocollins to denote that they are involved in the adhesive function of desmosomes. The modulation of desmocollin distribution during monolayer formation and establishment of epithelial polarity has also been studied. Fluorescent and immuno-gold labelling using Fab' or IgG at 4 degrees C revealed that desmocollins were initially evenly dispersed over the cell surface. Staining with IgG at 37 degrees C caused the desmocollins to 'patch' but not to 'cap'. With the establishment of confluency, desmocollins were gradually removed from the upper surfaces of the cells (or masked and rendered inaccessible to antibody) being confined to the lateral and probably basal regions of the cells. Treatment of confluent monolayers with 3 mM-EGTA rendered the desmocollins stainable, probably by causing their release from lateral constraint. Desmocollin staining at the cell surface was not appreciably reduced during 5 h of EGTA treatment, suggesting that desmocollins, unlike desmosomal plaques, may not be internalized after junction breakdown
— id: 16346, year: 1984, vol: 70, page: 41, stat: Journal Article,

Antibodies to epithelial desmosomes show wide tissue and species cross-reactivity
Cowin P; Garrod DR
1983 Mar 10;302(5904):148-150, Nature
Many workers regard cell adhesion as a highly specific phenomenon, believing that different molecular mechanisms are involved in the adhesion of cells of different tissues and different species. We believe that the evidence from cell behaviour is against this view and that cells share common adhesion mechanisms (for reviews see refs 1, 2); however, molecular evidence is lacking. As an approach to providing such evidence we have begun to study desmosomes, the cell-surface organelles responsible for strong intercellular adhesion in epithelia. We have raised antisera against each of five high-molecular weight (MW) desmosomal components. Having determined the specificity of our antisera by immunoblotting, we show here that each gives a staining pattern corresponding to the distribution of desmosomes in a range of tissues from different vertebrate species, demonstrating that desmosomal components are widely shared and highly conserved
— id: 16348, year: 1983, vol: 302, page: 148, stat: Journal Article,

The cytoskeleton and substratum adhesion in chick embryonic corneal epithelial cells
Billig D; Nicol A; McGinty R; Cowin P; Morgan J; Garrod D
1982 Oct;57(5904):51-71, Journal of cell science
— id: 16349, year: 1982, vol: 57, page: 51, stat: Journal Article,

Antitubulin antibody in healthy adults and patients with infectious mononucleosis and its relationship to smooth muscle antibody (SMA)
Mead GM; Cowin P; Whitehouse JM
1980 Feb;39(2):328-336, Clinical & experimental immunology
Antibody to tubulin in man has been studied using a specific radioimmunoassay, affinity chromatography radioimmunoassay but markedly increased levels were noted in patients with infectious mononucleosis where the antibody was predominantly IgM in type. This finding was confirmed on fluorescence microscopy. Affinity chromatography purified antibody produced characteristic microtubular staining of fixed 3T3 cells, but in addition, produced weak staining of cryostat sections of rat tissue, similar in distribution to that of smooth muscle antibody. Our studies indicate that the IgM smooth muscle antibody found in infectious mononucleosis by IF techniques is at least in part due to an antitubulin antibody
— id: 16350, year: 1980, vol: 39, page: 328, stat: Journal Article,

Measurement of antibodies to tubulin by radioimmunoassay
Mead GM; Cowin P; Whitehouse JM
1979 ;28(3-4):243-253, Journal of immunological methods
A solid-phase double antibody radioimmunoassay capable of measuring antibody to tubulin, the principal component of microtubules, is described. This assay is simple, combining sensitivity with specificity and also allowing determination of antibody subclasses
— id: 16351, year: 1979, vol: 28, page: 243, stat: Journal Article,