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The importance of the extracellular calcium-sensing receptor (CaR) in the stringent control of extracellular Ca(2+) concentration is well established. However, the presence of CaR in tissues not directly involved in regulating mineral ion homeostasis such as the epidermis suggests a role for CaR in other cellular functions. Although extracellular Ca(2+) regulates the differentiation of epidermal keratinocytes, the role of CaR in this process in the epidermis is not fully understood. In this study we showed using in situ hybridization and immunohistochemistry that CaR is expressed in suprabasal keratinocytes of the mammalian epidermis. We then evaluated the changes in epidermal keratinocyte morphology and differentiation in Casr(-/-) mice lacking the full-length CaR. These mice show increased expression of an alternatively spliced form of CaR which lacks acute Ca(2+)-signaling properties. The absence of the full-length CaR in the epidermis resulted in ultrastructural changes (abnormal keratohyalin granule formation and precocious lamellar body secretion) in the terminally differentiated granular keratinocytes. Furthermore, the expression of both mRNA and protein for the calcium inducible keratinocyte differentiation markers, filaggrin and loricrin, were down-regulated in the epidermis of Casr(-/-) mice, whereas the number of proliferating cells were increased even though the calcium gradient within the epidermis was enhanced. Our results demonstrate that the epidermal expression of the full-length CaR is required for the normal terminal differentiation of keratinocytes., (Copyright 2002 Wiley-Liss, Inc.)

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.