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1,25(OH)2D regulates a number of cellular events which contribute to its ability to stimulate differentiation of the keratinocyte. 1,25(OH)2D raises the intracellular calcium (Cai) level in part by increasing the expression of the calcium receptor (CaR). This sensitizes the cell to extracellular calcium, triggering the signaling pathway coupled to the CaR, which results in a rise in Cai. 1,25(OH)2D induces the family of phospholipases C (PLC). These enzymes mediate the hydrolysis of phosphatidyl inositol bisphosphate (PIP2) to form inositol tris phosphate (IP3) and diacylglycerol (DG), which stimulate calcium release from intracellular stores and activate protein kinases C (PKC), respectively. The CaR and other G protein coupled receptors signal through PLC-beta, whereas tyrosine kinase growth factor receptors such as the EGF receptor signal through PLC-gamma. Calcium and PKC regulate the expression of genes in part by controlling the levels and activity of AP-1 transcription factors. 1,25(OH)2D also directly induces structural genes such as involucrin, a substrate for transglutaminase, which crosslinks it to other substrates to form the cornified envelope. 1,25(OH)2D regulates gene expression by activating the vitamin D receptor (VDR), a transcription factor, which, in combination with the retinoid X receptor (RXR) or retinoid A receptor (RAR), binds to its vitamin D response elements (VDRE) in the promoters of genes whose expression it regulates. The VDR also binds to one of two coactivator complexes, Mediator/DRIP (VDR interacting proteins) or p160/SRC (steroid hormone receptor complex), complexes which link the VDR to the RNA polymerase complex. We have recently discovered that the binding of VDR to these complexes is sequential. Binding to Mediator/DRIP occurs in the undifferentiated keratinocyte, but as the cell differentiates, DRIP(205) (the key protein of the DRIP complex binding to the VDR) levels fall, and p160/SRC binding takes over. We hypothesize that this sequential replacement of Mediator/DRIP by p160/SRC is critical for differentiation. Squamous cell carcinomas (SCC) fail to respond to the prodifferentiating actions of 1,25(OH)2D. These cells have normal levels of VDR and normal binding of VDR to VDREs. However, they fail to down-regulate DRIP(205) such that the p160/SRC complex fails to bind to VDR. This lack of sequential binding of these coactivator complexes to the VDR, we believe, maintains the cell in a state of continued proliferation and blocks the ability of 1,25(OH)2D to induce the expression of genes required for the differentiation process.

Involucrin is a major protein of the cornified envelope of keratinocytes that provides much of the structural integrity of skin. Its expression is stimulated by a number of agents including calcium and 1,25-dihydroxy-vitamin D3 that promote the differentiation process in keratinocytes. Within the distal regulatory region of the involucrin promoter lies an AP-1 site and an element homologous to other vitamin D response elements. In previous studies mutation of the AP-1 site was found to reduce basal activity and block calcium stimulation of the involucrin promoter, whereas the vitamin D response element was not critical for calcium regulation. In this study both elements proved to be important for 1,25-dihydroxyvitamin D3 stimulation of the involucrin promoter. Mutation of the AP-1 site reduced basal activity and blocked 1,25-dihydroxyvitamin D3 stimulation of the involucrin promoter. In contrast, mutation of the vitamin D response element did not reduce basal expression of the involucrin promoter or prevent calcium stimulation of involucrin gene expression, but blocked 1,25-dihydroxyvitamin D3 stimulation. The vitamin D response element from the involucrin gene bound the vitamin D receptor and the retinoid X receptor, but not the retinoic acid receptor, in a specific manner. We conclude that the AP-1 site and the vitamin D response element in the involucrin promoter play important roles in mediating the action of 1,25-dihydroxyvitamin D3 on involucrin expression, but the vitamin D response element provides specificity for the 1,25-dihydroxyvitamin D3 response lacking at the AP-1 site. [ABSTRACT FROM AUTHOR]

The active vitamin D metabolite, 1,25-dihydroxyvitamin D, acting through the vitamin D receptor, regulates the expression of genes in a variety of vitamin D-responsive tissues, including the epidermis. To investigate the role of the vitamin D receptor in mediating epidermal differentiation, we examined the histomorphology and expression of differentiation markers in the epidermis of vitamin D receptor knockout mice generated by gene targeting. The homozygous knockout mouse displayed a phenotype that closely resembles vitamin D-dependent rickets type II in humans, including the development of rickets and alopecia. Hair loss developed by 3 mo after birth and gradually led to nearly total hair loss by 8 mo. Histologic analysis of the skin of homozygous knockout mice revealed dilation of the hair follicles with the formation of dermal cysts starting at the age of 3 wk. These cysts increased in size and number with age. Epidermal differentiation markers, including involucrin, profilaggrin, and loricrin, detected by immunostaining and in situ hybridization, showed decreased expression levels in homozygous knockout mice from birth until 3 wk, preceding the morphologic changes observed in the hair follicles. Keratin 10 levels, however, were not reduced. At the ultrastructural level, homozygous knockout mice showed increased numbers of small dense granules in the granular layer with few or no surrounding keratin bundles and a loss of keratohyalin granules. Thus, both the interfollicular epidermis and the hair follicle appear to require the vitamin D receptor for normal differentiation. The temporal abnormalities between the two processes reflect the apparent lack of requirement for the vitamin D receptor during the anagen phase of the first (developmental) hair cycle, but with earlier effects on the terminal differentiation of the interfollicular epidermis. [ABSTRACT FROM AUTHOR]