performed and analyzed experiments

performed and analyzed experiments. enteropathy, which is usually associated with mutations of AMG-1694 the gene (9). Although Lei (8) reported a certain degree of embryonic lethality, the reasons for these obvious discrepancies in phenotypes remain unknown. Furthermore, molecular mechanisms responsible for the observed congenital tufting enteropathy phenotypes were deviating. Guerra (7) proposed a role for adherens junctions with a mislocalization of E-cadherin and -catenin in AMG-1694 the developing intestine (7), whereas Lei (8) excluded the involvement of E-cadherin and -catenin, which were properly located, and they claimed a function for mEpcam in the recruitment of claudins to tight junctions. A role for Epcam in the formation of functional adherens junctions via E-cadherin was further described during epiboly processes in the developing zebrafish embryo and in embryonic development of (10, 11). Similar to reports by Nagao (6), depletion of Epcam in was lethal, suggesting an essential role for Epcam in embryonic development (11). Work by Z?ller and co-workers (12) further revealed a physical conversation of Epcam with Claudin 7 and a regulatory role in the formation of metastases from rat carcinoma cells. A comparable beneficial effect of Epcam on invasion and migration was observed in (11, 13) and human breast malignancy cell lines (14, 15). In contrast, IFN-alphaA loss of Epcam during epithelial-to-mesenchymal transition (EMT) in circulating and disseminating tumor cells (16,C18) and in zebrafish was reported (19). Knockdown of EPCAM in esophageal carcinoma cells induced a mesenchymal phenotype along with increased migration and invasion (16) and conformed with a dynamic expression AMG-1694 of EPCAM during tumor progression (20). Besides this complex and intricate role in cell adhesion and tissue integrity, HEPCAM was associated early on with a proliferative state of epithelia, especially in carcinomas (21, 22). This involvement in the regulation of proliferation and progression through the cell cycle was analyzed in-depth over the last decade. HEPCAM regulated proliferation of breast malignancy cell lines (14), fibroblasts, and human embryonic kidney cells, in which it induced the transcription of the proto-oncogene c-MYC (23). To induce cell cycle progression, HEPCAM undergoes regulated intramembrane proteolysis (RIP), which includes a series of consecutive proteolytic cleavages of receptors within lipid bilayers (24, 25). The regulated feature is usually conducted by sheddases within the extracellular domain of substrates, generating a C-terminal fragment (CTF), which is a substrate for -secretase. Commonly, -secretase cleaves CTFs at two distinct ?- and -sites to produce A-like and intracellular fragments (ICD). To date, numerous membrane proteins have been identified as targets of RIP, including prominent molecules such as amyloid precursor protein (APP) and NOTCH receptors (26, 27). RIP of substrates has two major functions in that it can initiate signaling through ICDs of receptors and, additionally, result in degradation of substrates (28). Pathologic conditions, such as Alzheimer disease, result from abnormal processing of APP with formation of the disease promoting the A fragment known to induce neurodegenerative plaques (27). RIP of EPCAM results in shedding of the extracellular domain name HEPEX and in -secretase-dependent release of the intracellular signaling domain name HEPICD (29). Through interactions with the scaffolding protein FHL2 and -catenin, HEPICD can translocate into the nucleus and bind to regulatory element of target genes, including cyclin D1 (29, 30). Exact amino acid sequences involved in cleavage have been mapped for murine Epcam (31), but they remain unidentified for the therapeutic target HEPCAM. In this work, we have investigated regulated cleavage ofHEPCAM at the single amino acid level and then resolved its implication in cell adhesion. We demonstrate a broad cleavage pattern of EpCAM with numerous extra- and intracellular products. However, inhibition of cleavage did not affect adhesion of HEPCAM-expressing cells. Through the use of knock-out and knockdown cell lines, we demonstrate that HEPCAM has no detectable effect on cell-matrix or cell-cell adhesion in the context of the carcinoma cells used herein. Thus, a general role of HEPCAM as an active cell adhesion molecule in carcinoma cells appears either lacking or context- and cell type-dependent. Experimental Procedures Cell Lines Human embryonic kidney cells (HEK) 293 (German Collection of Microorganisms and Cell Cultures, DSMZ number ACC305), human colon carcinoma cell line.