Evidence for regulated dimerization of cell-cell adhesion molecule (C-CAM) in epithelial cells.
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ABSTRACT:
SUBMITTER: Hunter I
PROVIDER: S-EPMC1218006 | biostudies-other | 1996 Dec
REPOSITORIES: biostudies-other
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Project description:Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-)stem cells, and which plays an important role in the regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM) is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at ?-, ?-, ?-, and ?-sites to generate soluble ectodomains, soluble A?-like-, and intracellular fragments termed mEpEX, mEp-?, and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the ?- and ?-sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF) are further processed to soluble A?-like fragments mEp-? and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the ?-secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified in silico in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent ?-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, ?-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation.
| S-EPMC3756971 | biostudies-literature
Project description:Human epithelial cell adhesion molecule (HEPCAM) is a tumor-associated antigen frequently expressed in carcinomas, which promotes proliferation after regulated intramembrane proteolysis. Here, we describe extracellular shedding of HEPCAM at two ?-sites through a disintegrin and metalloprotease (ADAM) and at one ?-site through BACE1. Transmembrane cleavage by ?-secretase occurs at three ?-sites to generate extracellular A?-like fragments and at two ?-sites to release human EPCAM intracellular domain HEPICD, which is efficiently degraded by the proteasome. Mapping of cleavage sites onto three-dimensional structures of HEPEX cis-dimer predicted conditional availability of ?- and ?-sites. Endocytosis of HEPCAM warrants acidification in cytoplasmic vesicles to dissociate protein cis-dimers required for cleavage by BACE1 at low pH values. Intramembrane cleavage sites are accessible and not part of the structurally important transmembrane helix dimer crossing region. Surprisingly, neither chemical inhibition of cleavage nor cellular knock-out of HEPCAM using CRISPR-Cas9 technology impacted the adhesion of carcinoma cell lines. Hence, a direct function of HEPCAM as an adhesion molecule in carcinoma cells is not supported and appears to be questionable.
| S-EPMC4591836 | biostudies-literature
Project description:In the last few decades, the epithelial cell adhesion molecule (EpCAM) has received increased attention as the main membrane marker used in many enrichment technologies to isolate circulating tumor cells (CTCs). Although there has been a great deal of progress in the implementation of EpCAM-based CTC detection technologies in medical settings, several issues continue to limit their clinical utility. The biology of EpCAM and its role are not completely understood but evidence suggests that the expression of this epithelial cell-surface protein is crucial for metastasis-competent CTCs and may not be lost completely during the epithelial-to-mesenchymal transition. In this review, we summarize the most significant advantages and disadvantages of using EpCAM as a marker for CTC enrichment and its potential biological role in the metastatic cascade.
| S-EPMC7464831 | biostudies-literature
Project description:Cell-CAM 105, a glycoprotein that is involved in recognition and adhesion between isolated rat hepatocytes in vitro, was purified to homogeneity by a combination of immunoaffinity chromatography, gel-exclusion chromatography and ion-exchange chromatography. Electrophoretic, compositional and enzymic analyses were performed and the glycoprotein was shown to consist of two peptide chains, of apparent Mr 110,000 and 105,000 respectively, that are glycosylated to similar extents. Carbohydrate analyses demonstrated the presence of sialic acid, galactose, mannose, fucose and glucosamine, but no galactosamine, indicating that only N-linked oligosaccharides occurred. The total content of carbohydrate amounted to 33%. Peptide mapping indicated that the two peptide chains were structurally very similar. After incubation of cultured hepatocytes with [32P]Pi, phosphorylated cell-CAM 105 could be isolated. Both peptide chains were labelled and phospho-amino-acid analysis demonstrated that serine residues had become phosphorylated. A significant feature of cell-CAM 105 was a susceptibility to autolytic degradation that was difficult to inhibit. The major degradation products had apparent Mr 90,000 and 70,000, respectively. The effect of purified cell-CAM 105 on cell-cell adhesion of re-aggregating hepatocytes was studied. A significant inhibition was observed, indicating that the protein is directly involved in intercellular adhesion of these cells.
| S-EPMC1146876 | biostudies-other
Project description:Epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of human cancers including lung cancer, and its contribution to increased proliferation through upregulation of cell cycle accelerators such as cyclins A and E has been well established in breast and gastric cancers. Nevertheless, very little is known about its role in supporting the survival of cancer cells. In addition, the functional role of EpCAM in the pathogenesis of lung cancer remains to be explored. In this study, we show that RNAi-mediated knockdown of EpCAM suppresses proliferation and clonogenic growth of three EpCAM-expressing lung cancer cell lines (H3255, H358, and HCC827), but does not induce cell cycle arrest in any of these. In addition, EpCAM knockdown inhibits invasion in the highly invasive H358 but not in less invasive H3255 cells in a Transwell assay. Of note, the EpCAM knockdown induces massive apoptosis in the three cell lines as well as in another EpCAM-expressing lung cancer cell line, HCC2279, but to a much lesser extent in a cdk4/hTERT immortalized normal human bronchial epithelial cell line, HBEC4, suggesting that EpCAM could be a therapeutic target for lung cancer. Finally, EpCAM knockdown partially restores contact inhibition in HCC827, in association with p27(Kip1) upregulation. These results indicate that EpCAM could contribute substantially to the pathogenesis of lung cancer, especially cancer cell survival, and suggest that EpCAM targeted therapy for lung cancer may have potential.
| S-EPMC3381954 | biostudies-literature
Project description:Oxidative damage to renal tubular epithelial cells is a fundamental pathogenic mechanism implicated in both acute kidney injury and chronic kidney diseases. Because epithelial cell survival influences the outcome of acute kidney injury and chronic kidney diseases, identifying its molecular regulators could provide new insight into pathobiology and possible new therapeutic strategies for these diseases. We have identified transmembrane and immunoglobulin domain-containing 1 (TMIGD1) as a novel adhesion molecule, which is highly conserved in humans and other species. TMIGD1 is expressed in renal tubular epithelial cells and promotes cell survival. The extracellular domain of TMIGD1 contains two putative immunoglobulin domains and mediates self-dimerization. Our data suggest that TMIGD1 regulates transepithelial electric resistance and permeability of renal epithelial cells. TMIGD1 controls cell migration, cell morphology, and protects renal epithelial cells from oxidative- and nutrient-deprivation-induced cell injury. Hydrogen peroxide-induced oxidative cell injury downregulates TMIGD1 expression and targets it for ubiquitination. Moreover, TMIGD1 expression is significantly affected in both acute kidney injury and in deoxy-corticosterone acetate and sodium chloride (deoxy-corticosterone acetate salt)-induced chronic hypertensive kidney disease mouse models. Taken together, we have identified TMIGD1 as a novel cell adhesion molecule expressed in kidney epithelial cells that protects kidney epithelial cells from oxidative cell injury to promote cell survival.
| S-EPMC4607757 | biostudies-literature
Project description:BackgroundThe epithelial cell adhesion molecule (EpCAM) is a type I transmembrane and glycosylated protein, which is overexpressed in many neoplasms. However, EpCAM has no known ligand partners and the mechanisms by which it functions are not fully understood.AimThis study was performed to discover novel partners of EpCAM, which may provide a better understanding of its functions.MethodsThe membrane fraction of the ERα+ noninvasive breast cancer cell line ZR-75-1 and MCF-7 was extracted and followed by co-immunoprecipitation of EpCAM using C-10, a mouse monoclonal antibody raised against amino acids 24-93 of the EpCAM molecule. As a negative control, MDA-MB-231 and Hs578T were used since they express a negligible amount of EpCAM and are known as EpCAM-/low ERα-/low invasive and tumorigenic breast cancer cell lines.ResultsAnnexin A2 (ANXA2) was found to be selectively and differentially co-immunoprecipitated with EpCAM in the ERα+ breast cancer cells MCF-7 and ZR-75-1. ANXA2 is a multifunctional protein and known to act as a co-receptor for tissue plasminogen activator (tPA) on the surface of endothelial and cancer cells, thereby affecting fibrinolytic activity and neoangiogenesis as well as invasive and metastatic properties. In this study, the association between EpCAM and ANXA2 was found to affect the activity of tPA.ConclusionThis study concludes that ANXA2 co-localizes with EpCAM at the plasma membrane, and the co-localization may have functional implications. Data suggest that EpCAM supports ANXA2 to function as a co-receptor for the tPA, and that EpCAM has a regulatory function on the expression and subcellular localization of ANXA2.
| S-EPMC9124509 | biostudies-literature
Project description:Junctional adhesion molecule-A (JAM-A) is a transmembrane component of tight junctions that has been proposed to play a role in regulating epithelial cell adhesion and migration, yet mechanistic structure-function studies are lacking. Although biochemical and structural studies indicate that JAM-A forms cis-homodimers, the functional significance of dimerization is unclear. Here, we report the effects of cis-dimerization-defective JAM-A mutants on epithelial cell migration and adhesion. Overexpression of dimerization-defective JAM-A mutants in 293T cells inhibited cell spreading and migration across permeable filters. Similar inhibition was observed with using dimerization-blocking antibodies. Analyses of cells expressing the JAM-A dimerization-defective mutant proteins revealed diminished beta1 integrin protein but not mRNA levels. Further analyses of beta1 protein localization and expression after disruption of JAM-A dimerization suggested that internalization of beta1 integrin precedes degradation. A functional link between JAM-A and beta1 integrin was confirmed by restoration of cell migration to control levels after overexpression of beta1 integrin in JAM-A dimerization-defective cells. Last, we show that the functional effects of JAM dimerization require its carboxy-terminal postsynaptic density 95/disc-large/zonula occludins-1 binding motif. These results suggest that dimerization of JAM-A regulates cell migration and adhesion through indirect mechanisms involving posttranscriptional control of beta1 integrin levels.
| S-EPMC2366836 | biostudies-other
Project description:UnlabelledTherapies that target cancer stem cells (CSCs) hold promise in eliminating cancer burden. However, normal stem cells are likely to be targeted owing to their similarities to CSCs. It is established that epithelial cell adhesion molecule (EpCAM) is a biomarker for normal hepatic stem cells (HpSCs), and EpCAM(+) AFP(+) hepatocellular carcinoma (HCC) cells have enriched hepatic CSCs. We sought to determine whether specific microRNAs (miRNAs) exist in hepatic CSCs that are not expressed in normal HpSCs. We performed a pair-wise comparison of the miRNA transcriptome of EpCAM(+) and corresponding EpCAM(-) cells isolated from two primary HCC specimens, as well as from two fetal livers and three healthy adult liver donors by small RNA deep sequencing. We found that miR-150, miR-155, and miR-223 were preferentially highly expressed in EpCAM(+) HCC cells, which was further validated. Their gene surrogates, identified using miRNA and messenger RNA profiling in a cohort of 292 HCC patients, were associated with patient prognosis. We further demonstrated that miR-155 was highly expressed in EpCAM(+) HCC cells, compared to corresponding EpCAM(-) HCC cells, fetal livers with enriched normal hepatic progenitors, and normal adult livers with enriched mature hepatocytes. Suppressing miR-155 resulted in a decreased EpCAM(+) fraction in HCC cells and reduced HCC cell colony formation, migration, and invasion in vitro. The reduced levels of identified miR-155 targets predicted the shortened overall survival and time to recurrence of HCC patients.ConclusionmiR-155 is highly elevated in EpCAM(+) HCC cells and might serve as a molecular target to eradicate the EpCAM(+) CSC population in human HCCs.
| S-EPMC4549211 | biostudies-literature
Project description:Neuron-glia cell adhesion molecule (Ng-CAM) mediates cell adhesion between neurons homophilically and between neurons and glia heterophilically; it also promotes neurite outgrowth. In the chick brain, Ng-CAM is detected as glycoproteins of 190 and 210 kD (Ng-CAM200) with posttranslational cleavage products of 135 kD (F135, which contains most of the extracellular region) and 80 kD (F80, which includes the transmembrane and the cytoplasmic domains). To examine the functions of each of these components, we have expressed Ng-CAM200, F135, and F80 in murine L cells, and F135 and F80 as GST fusion proteins in the pGEX vector in bacteria. Appropriately transfected L cells expressed each of these proteins on their surfaces; F135 was also found in the media of cells transfected with Ng-CAM200 and F135. In addition to binding homophilically, cells transfected with Ng-CAM200 and F135 bound heterophilically to untransfected L cells, suggesting that there is a ligand for Ng-CAM on fibroblasts that may be related to the glial ligand. Detailed studies using the transfected cells and the fusion proteins indicated that both the homophilic and the heterophilic binding activities of Ng-CAM are localized in the F135 fragment of the molecule. The results also indicated that proteolytic cleavage of Ng-CAM200 is not required either for its expression on the cell surface or for cell adhesion and that there is an "anchor" for F135 on L cells (and presumably on neurons). In contrast to the cell binding results, the F80 but not the F135 fusion protein enhanced the outgrowth of neurites from dorsal root ganglion cells; this activity was associated with the FnIII repeats of F80. The observations that a protein corresponding to F135 contains the cell aggregation sites whereas one corresponding to the F80 has the ability to promote neurite outgrowth suggest that proteolytic cleavage may be an important event in regulating these Ng-CAM activities during embryonic development and neural regeneration.
| S-EPMC2120543 | biostudies-other