Inactivation of the calcium sensing receptor inhibits E-cadherin-mediated cell-cell adhesion and calcium-induced differentiation in human epidermal keratinocytes

Abstract

Extracellular Ca2+ (Ca2+o) is a critical regulator that promotes differentiation in epidermal keratinocytes. The calcium sensing receptor (CaR) is essential for mediating Ca2+ signaling during Ca2+o-induced differentiation. Inactivation of the endogenous CaR-encoding gene CASR by adenoviral expression of a CaR antisense cDNA inhibited the Ca2+o-induced increase in intracellular free calcium (Ca2+i) and expression of terminal differentiation genes, while promoting apoptosis. Ca2+o also instigates E-cadherin-mediated cell-cell adhesion, which plays a critical role in orchestrating cellular signals mediating cell survival and differentiation. Raising Ca2+o concentration ([Ca 2+]o) from 0.03 to 2 mM rapidly induced the co-localization of α-, β-, and p120-catenin with E-cadherin in the intercellular adherens junctions (AJs). To assess whether CaR is required for the Ca2+o-induced activation of E-cadherin signaling, we examined the impact of CaR inactivation on AJ formation. Decreased CaR expression suppressed the Ca2+o-induced AJ formation, membrane translocation, and the complex formation of E-cadherin, catenins, and the phosphatidylinositol 3-kinase (PI3K), although the expression of these proteins was not affected. The assembly of the E-cadherin-catenin-PI3K complex was sensitive to the pharmacologic inhibition of Src family tyrosine kinases but was not affected by inhibition of Ca2+o-induced rise in Ca2+i. Inhibition of CaR expression blocked the Ca 2+o-induced tyrosine phosphorylation of β-, γ-, and p120-catenin, PI3K, and the tyrosine kinase Fyn and the association of Fyn with E-cadherin and PI3K. Our results indicate that the CaR regulates cell survival and Ca2+o-induced differentiation in keratinocytes at least in part by activating the E-cadherin/PI3K pathway through a Src family tyrosine kinase-mediated signaling.