Vitamin D regulated keratinocyte differentiation: role of coactivators

1,25 Dihydroxyvitamin D (1,25(OH)(2)D) regulates the differentiation of keratinocytes. 1,25(OH)(2)D raises intracellular free calcium (Cai) as a necessary early step toward stimulating differentiation. 1,25(OH)(2)D induces the calcium sensing receptor (CaR) in keratinocytes and enhances the calcium response of these cells. Activation of the CaR by calcium increases intracellular free calcium by a mechanism involving phospholipase C (PLC) cleavage of phosphatidylinositolbisphosphate into inositoltrisphosphate (IP(3)) and diacylglycerol (DG). 1,25(OH)(2)D induces the family of PLCs. PLC-gamma1 has a DR6 VDRE in its promoter which binds and is activated by VDR/RAR rather than VDR/RXR. The involucrin gene, which encodes a critical component of the cornified envelope, contains a DR3 VDRE in its promoter that acts in conjunction with a nearby AP-1 site. The sequential regulation of these genes is critical for the differentiation process. In undifferentiated keratinocytes, the VDR binds preferentially to the DRIP complex of coactivators. However, with differentiation DRIP 205 is no longer produced, and the VDR switches partners to the SRC family (SRC2 and 3). These studies suggest that at least part of the sequential activation of genes required during keratinocyte differentiation is regulated by the change (availability) of these different coactivator complexes.     

Calcium and 1,25(OH)2D: interacting drivers of epidermal differentiation

Both calcium and 1,25(OH)(2)D promote the differentiation of keratinocytes in vitro. The autocrine or paracrine production of 1,25(OH)(2)D by keratinocytes combined with the critical role of the epidermal calcium gradient in regulating keratinocyte differentiation in vivo suggest the physiologic importance of this interaction. The interactions occur at a number of levels. Calcium and 1,25(OH)(2)D synergistically induce involucrin, a protein critical for cornified envelope formation. The involucrin promoter contains an AP-1 site essential for calcium and 1,25(OH)(2)D induction and an adjacent VDRE essential for 1,25(OH)(2)D but not calcium induction. Calcium regulates coactivator complexes that bind to the Vitamin D receptor (VDR). Nuclear extracts from cells grown in low calcium contain an abundance of DRIP(205), whereas calcium induced differentiation leads to reduced DRIP(205) and increased SRC 3 which replaces DRIP in its binding to the VDR. In vivo models support the importance of 1,25(OH)(2)D-calcium interactions in epidermal differentiation. The epidermis of 1alphaOHase null mice fails to form a normal calcium gradient, has reduced expression of proteins critical for barrier function, and shows little recovery of the permeability barrier when disrupted. Thus in vivo and in vitro, calcium and 1,25(OH)(2)D interact at multiple levels to regulate epidermal differentiation.     

Vitamin D and skin cancer: a problem in gene regulation

The skin is the major source of Vitamin D(3) (cholecalciferol), and ultraviolet light (UV) is critical for its formation. Keratinocytes, the major cell in the epidermis, can further convert Vitamin D(3) to its hormonal form, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] (calcitriol). 1,25(OH)(2)D(3) in turn stimulates the differentiation of keratinocytes, raising the hope that 1,25(OH)(2)D(3) may prevent the development of malignancies in these cells. Skin cancers (squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and melanomas) are the most common cancers afflicting humans. UV exposure is linked to the incidence of these cancers-UV is thus good and bad for epidermal health. Our focus is on the mechanisms by which 1,25(OH)(2)D(3) regulates the differentiation of keratinocytes, and how this regulation breaks down in transformed cells. Skin cancers produce 1,25(OH)(2)D(3), contain ample amounts of the Vitamin D receptor (VDR), and respond to 1,25(OH)(2)D(3) with respect to induction of the 24-hydroxylase, but fail to differentiate in response to 1,25(OH)(2)D(3). Why not? The explanation may lie in the overexpression of the DRIP complex, which by interfering with the normal transition from DRIP to SRC as coactivators of the VDR during differentiation, block the induction of genes required for 1,25(OH)(2)D(3)-induced differentiation.     

Discrete roles for peroxisome proliferator-activated receptor gamma and retinoid X receptor in recruiting nuclear receptor coactivators

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a major role in adipogenesis. PPARgamma binds to DNA as a heterodimer with retinoid X receptor (RXR), and PPARgamma-RXR can be activated by ligands specific for either receptor; the presence of both ligands can result in a cooperative effect on the transactivation of target genes. How these ligands mediate transactivation, however, remains unclear. PPARgamma is known to interact with both the p160/SRC-1 family of coactivators and the distinct, multisubunit coactivator complex called DRIP. A single DRIP subunit, DRIP205 (TRAP220, PBP), binds directly to PPARgamma. Here we report that PPARgamma and RXR selectively interacted with DRIP205 and p160 proteins in a ligand-dependent manner. At physiological concentrations, RXR-specific ligands only induced p160 binding to RXR, and PPARgamma-specific ligands exclusively recruited DRIP205 but not p160 coactivators to PPARgamma. This selectivity was not observed in interaction assays off DNA, implying that the specificity of coactivator binding in response to ligand is strongly influenced by the allosteric effects of DNA-bound heterodimers. These coactivator-selective effects were also observed in transient-transfection assays in the presence of overexpressed p160 or DRIP coactivators. The results suggest that the cooperative effects of PPARgamma- and RXR-specific ligands may occur at the level of selective coactivator recruitment.