We did not expect to see extensive co-localization of Dsg2 and Cav-1 since desmogleins are predominantly found in desmosomes and Cav-1 in lipid rafts. S3. Cav-1competing peptides induce loss of keratinocyte adhesion. A431 cells were incubated with 5 M AP or AP-Cav-1 in serum-free medium for 2 hr. Cell sheets were subjected to dispase-based keratinocyte dissociation assay showing more fragmentation after treatment with AP-Cav-1 peptides, as compared to control untreated or treated with AP alone. Shown are representative results from three separate experiments. NIHMS310516-supplement-3.jpg (78K) GUID:?F42984B7-CFEE-4C59-80FB-F10671D839C2 Abstract Desmoglein 2 (Dsg2) is a desmosomal cadherin that is aberrantly expressed in human skin carcinomas. In addition to its well-known role in mediating intercellular desmosomal adhesion, Dsg2 regulates mitogenic signaling that may promote cancer development and progression. However, the mechanisms by which Dsg2 activates these signaling pathways and the relative contribution of its signaling and adhesion functions in tumor progression are poorly understood. In this study we show that Dsg2 associates with caveolin-1 (Cav-1), the major protein of specialized membrane microdomains called caveolae, which functions in both membrane protein turnover and intracellular signaling. Sequence analysis revealed that Dsg2 contains a putative Cav-1 binding motif. A permeable competing peptide resembling the Cav-1 scaffolding domain bound to Dsg2, disrupted normal Dsg2 staining and interfered with the integrity of epithelial sheets in skin tumors from transgenic mice overexpressing Dsg2. Collectively, these data are consistent with the possibility that accumulation of truncated Dsg2 protein interferes with desmosome assembly and/or maintenance to disrupt cell-cell adhesion. Furthermore, the association of Dsg2 with Cav-1 may provide a mechanism for regulating mitogenic signaling and modulating DZNep the cell surface presentation of an important adhesion molecule, both of which could contribute to malignant transformation and tumor progression. null mice revealed that Dsg2 contributes to embryonic stem cell proliferation, particularly in the inner cell mass of the developing blastocyst (Eshkind et al., 2002). Dsg2 is aberrantly expressed in select epithelial malignancies, including squamous cell carcinomas (Biedermann et al., 2005; Harada et al., 1996; Kurzen et al., 2003). Similarly, genetic profiling of prostate cancer cell lines showed increased expression of Dsg2 in a metastatic cell line, as compared to its non-metastatic DZNep syngeneic precursor cell (Trojan et al., 2005). Dsg2 expression is also upregulated in squamous cell carcinoma (SCC) cell lines in comparison to cultured keratinocytes (Denning et al., 1998; Harada et al., 1996; Sch?fer et al., 1994). We recently showed that Dsg2 is highly expressed in malignant skin carcinomas, including squamous cell carcinomas, basal cell carcinomas, sweat and sebaceous gland carcinomas and adenocarcinomas (Brennan and Mahoney, 2009). Collectively, these results support a role for Dsg2 in epithelial cell growth, survival and malignant transformation. However, the mechanisms by which Dsg2 activates these signaling pathways and promotes tumor formation are unknown. Caveolins are a family of hairpin-like, DZNep palmitoylated, integral membrane proteins that oligomerize and bind to cholesterols and sphingolipids to form specialized areas of the membrane distinct from the clathrin-coated pits. The caveolins form flask-shaped invaginations of 50-100 nm in diameter called caveolae (Severs, 1988). There are three caveolin isoforms: Cav-1 ( and ), Cav-2 and Cav-3. While Cav-1 and Cav-2 are ubiquitously expressed, Cav-3 expression is predominantly restricted to muscle cells (Scherer et al., 1995; Tang et al., 1996). Caveolins and caveolae have been implicated as regulators of key cellular functions, including cholesterol transport and homeostasis (Fielding and Fielding, 1995; Smart et al., 1996), endocytosis and endocytic vesicle trafficking (Schnitzer and Oh, 1996), cell adhesion and apoptosis (Kurzchalia and Parton, 1999; Lisanti et al., 1994; Okamoto, 1998; Okamoto et al., 1998; Shaul and Anderson, 1998). Specific cell signals can be also transmitted through a spatially controlled organization of cell receptors into the caveolae. Indeed, the epidermal growth factor (EGF) receptor has been shown to stimulate the phosphorylation of Cav-1, thus enhancing caveolae assembly (Orlichenko et al., 2006; Severs, 1988; Simons and Toomre, 2000; Singer and Nicolson, 1972). Furthermore, Cav-1 is essential for integrin-mediated activation of PI3-K/AKT (Sedding et al., 2005). Conversely, overexpression of Cav-1 abrogates anchorage-independent cell survival (Engelman et al., 1997), and TSPAN2 suppresses cell growth (Lee et al., 1998). Additionally, Cav-1 binds to and inhibits kinases involved in mitogenic signaling pathways. Cav-1 expression can modulate Wnt/-catenin/Lef-1 signaling by regulating the intracellular localization of -catenin (Galbiati et al., 2000). Consistent with these findings, mounting evidence suggests that diseases associated with deregulated signaling pathways often result from aberrant expression or localization of Cav-1. In cancer, the role for Cav-1 is complex, as it serves both as a modulator of tumor suppression as well as oncogenesis. Mutations in the gene have been linked to human breast cancer, suggesting that loss of Cav-1 function plays a significant role in tumor initiation (Chen et al.,.
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