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Extracellular Ca(2+) (Ca(2+)(o)) is a critical regulator that promotes differentiation in epidermal keratinocytes. The calcium sensing receptor (CaR) is essential for mediating Ca(2+) signaling during Ca(2+)(o)-induced differentiation. Inactivation of the endogenous CaR-encoding gene CASR by adenoviral expression of a CaR antisense cDNA inhibited the Ca(2+)(o)-induced increase in intracellular free calcium (Ca(2+)(i)) and expression of terminal differentiation genes, while promoting apoptosis. Ca(2+)(o) also instigates E-cadherin-mediated cell-cell adhesion, which plays a critical role in orchestrating cellular signals mediating cell survival and differentiation. Raising Ca(2+)(o) concentration ([Ca(2+)](o)) from 0.03 to 2 mm rapidly induced the co-localization of alpha-, beta-, and p120-catenin with E-cadherin in the intercellular adherens junctions (AJs). To assess whether CaR is required for the Ca(2+)(o)-induced activation of E-cadherin signaling, we examined the impact of CaR inactivation on AJ formation. Decreased CaR expression suppressed the Ca(2+)(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 Ca(2+)(o)-induced rise in Ca(2+)(i). Inhibition of CaR expression blocked the Ca(2+)(o)-induced tyrosine phosphorylation of beta-, gamma-, 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 Ca(2+)(o)-induced differentiation in keratinocytes at least in part by activating the E-cadherin/PI3K pathway through a Src family tyrosine kinase-mediated signaling.

In cultured keratinocytes, the acute increase of the extracellular calcium concentration above 0.03 mM leads to a rapid increase in intracellular calcium concentration ([Ca(2+)]i) and inositol trisphosphate production and, subsequently, to the expression of differentiation-related genes. Previous studies demonstrated that human keratinocytes express the full-length extracellular calcium-sensing receptor (CaR) and an alternatively spliced variant lacking exon 5 and suggested their involvement in calcium regulation of keratinocyte differentiation. To understand the role of the CaR, we transfected keratinocytes with an antisense human CaR cDNA construct and examined its impact on calcium signaling and calcium-induced differentiation. The antisense CaR cDNA significantly reduced the protein level of endogenous CaRs. These cells displayed a marked reduction in the rise in [Ca(2+)]i in response to extracellular calcium or to NPS R-467, a CaR activator, whereas the ATP-evoked rise in [Ca(2+)]i was not affected. Calcium-induced inhibition of cell proliferation and calcium-stimulated expression of the differentiation markers involucrin and transglutaminase were also blocked by the antisense CaR cDNA. When cotransfected with luciferase reporter vectors containing either the involucrin or transglutaminase promoter, the antisense CaR cDNA suppressed the calcium-stimulated promoter activities. These results indicate that CaR is required for mediating calcium signaling and calcium-induced differentiation in keratinocytes.

We have recently reported that human keratinocytes express both the full-length calcium sensing receptor (CaR) and an alternatively spliced form lacking exon 5, which were suggested to be involved in calcium induced keratinocyte differentiation. To understand further the role of these CaRs, we analyzed the structure of mouse CaRs, and investigated their role using a mouse model in which only the full-length CaR was disrupted. Our results show that both the full-length and the alternatively spliced variant lacking exon 5 encoding 77 amino acids of the extracellular domain were expressed in mouse epidermis. The deletion of the full-length CaR increased the production of the alternatively spliced form of CaR in mutant mice. The keratinocytes derived from these mutant mice did not respond to extracellular calcium, suggesting that the full-length CaR is required to mediate calcium signaling in the keratinocytes. The loss of the full-length CaR altered the morphologic appearance of the epidermis and resulted in a reduction of the mRNA and protein levels of the keratinocyte differentiation marker, loricrin. These results indicate that CaR is important in epidermal differentiation, and that the alternatively spliced form does not fully compensate for loss of the full-length CaR.

We have recently reported the presence of the calcium sensing receptor (CaR) in keratinocytes and suggested that it signaled calcium-induced differentiation of these cells. cDNA clones encoding the full-length CaR were isolated from human keratinocytes. In addition, an alternatively spliced form that lacks exon 5, encoding a portion of the extracellular domain, also was found. The in frame deletion of 231 nucleotides of exon 5 resulted in the loss of function of the CaR as measured by calcium-stimulated production of inositol phosphates when transfected into HEK293 cells or keratinocytes. This variant produced a smaller CaR protein with an altered glycosylation pattern compared with the full-length CaR. Coexpression of the spliced variant with the full-length CaR reduced the function of the full-length CaR. The full-length CaR was expressed in undifferentiated keratinocytes consistent with their greater response to elevated extracellular calcium in terms of increased intracellular free calcium and production of inositol phosphates. The full-length CaR decreased as the keratinocytes differentiated with an increase in the ratio of the spliced variant to the full-length form. The relative proportions of these two forms of CaR may regulate the calcium responsiveness of keratinocytes during their differentiation.

In human hepatoma cell line Hep3B/T2, the human heat-shock-inducible gene hsp70 could be induced by insulin. The dose-dependent insulin effect correlates very well with the dissociation constant of the insulin receptor, indicating that the insulin effect is mediated by the insulin receptor. The expression of hsp70 gene was neither significantly induced by growth factors of epidermal and platelet-derived growth factors, nor by tumor promoter, calcium ionophore, cAMP, and glucocorticoids. These results indicate that the induction of expression of hsp70 gene by insulin is very specific and not cell cycle-related. Furthermore, the insulin-induced expression of hsp70 gene is transient, occurring specifically from 4 to 8 h after insulin addition.