title: Alteration of integrin-dependent adhesion and signaling in EMT-like MDCK cells established through overexpression of calreticulin.
abstract: Calreticulin (CRT) is a multi-functional Ca(2+) -binding molecular chaperone in the endoplasmic reticulum. We previously reported that kidney epithelial cell-derived Madin-Darby Canine Kidney cells were transformed into mesenchymal-like cells by gene transfection of CRT. In this study, we investigated the altered characteristics of cell adhesion in these epithelial-mesenchymal transition (EMT)-like cells. Several extracellular matrix substrata were tested, and cell adhesion to fibronectin was found to be specifically increased in the CRT-overexpressing cells compared to controls. The expression of integrins was significantly up-regulated in subunits α5 and αV, resulting in an increase in the formation of complexes such as α5β1 and αVβ3. These integrins also contributed to the enhanced binding of fibronectin. In the CRT-overexpressing cells, the phosphorylation of Akt, a downstream target of integrin-linked kinase (ILK), was up-regulated on attachment to fibronectin or collagen IV. Integrin-associated signaling through ILK was also promoted on attachment to fibronectin, suggesting some of the correlation between ILK and Akt in the CRT-overexpressing cells. Furthermore, on treatment with 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester, a membrane-permeable Ca(2+) chelator, the enhanced Akt signaling was suppressed with a concomitant decrease in the formation of complexes between integrins and ILK in the CRT-overexpressing cells. In conclusion, these findings demonstrate that CRT regulates cell-substratum adhesion by modulating integrin-associated signaling through altered Ca(2+) homeostasis in the CRT-overexpressing EMT-like cells, suggesting a novel regulatory role for CRT in EMT.
authors.affiliation: Department of Biochemistry, Wakayama Medical University School of Medicine, Wakayama, Japan. y-ihara@wakayama-med.ac.jp.
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Ihara
authors.forename: Yoko
authors.initials: Y
authors.lastname: Inai
authors.forename: Midori
authors.initials: M
authors.lastname: Ikezaki
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D061207:Calcium Ionophores / Q000494:pharmacology; D037282:Calreticulin / Q000378:metabolism*; D002448:Cell Adhesion; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D004285:Dogs; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D058750:Epithelial-Mesenchymal Transition; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D039081:Integrin alpha5beta1 / Q000378:metabolism*; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D046912:Multiprotein Complexes / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism*; D015398:Signal Transduction; D000099200:Scaffold Protein ILK
chemical_list: D002118:Calcium; D061207:Calcium Ionophores; D037282:Calreticulin; D002614:Chelating Agents; D004533:Egtazic Acid; D005353:Fibronectins; D039081:Integrin alpha5beta1; D039302:Integrin alphaVbeta3; D015759:Ionomycin; D046912:Multiprotein Complexes; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D000099200:Scaffold Protein ILK; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester