1,25-dihydroxyvitamin D3 increases nuclear vitamin D3 receptors by blocking ubiquitin/proteasome-mediated degradation in human skin

1,25-Dihydroxyvitamin D-3 (D-3) exerts its effects by binding to and activating nuclear vitamin D-3 receptors (VDRs) that regulate transcription of target genes. We have investigated regulation of VDR levels in human skin in vivo and in cultured human keratinocytes. Quantitative ligand-binding analysis revealed that human skin expressed approximately 220 VDRs per cell, which bound D-3 with high affinity [(dissociation constant (K-d) = 0.22 nM]. In human skin nuclear extracts, VDR exclusively bound to DNA containing vitamin D-3 response elements as heterodimers with retinoid X receptors. Topical application of D-3 to human skin elevated VDR protein levels 2-fold, as measured by both ligand-binding and DNA-binding assays. In contrast, the D-3 analog calcipotriene had no effect on VDR levels. Topical D-3 had no effect on VDR mRNA, indicating that D-3 either stimulated synthesis and/or inhibited degradation of VDRs. To investigate this latter possibility, recombinant VDRs were incubated with skin lysates in the presence or absence of D-3. The presence of D-3 substantially protected VDRs against degradation by human skin lysates. VDR degradation was inhibited by proteasome inhibitors, but not lysosome or serine protease inhibitors. In cultured keratinocytes, D-3 or proteasome inhibitors increased VDR protein without affecting VDR mRNA levels. In cells, VDR was ubiquitinated and this ubiquitination was inhibited by D-3. Proteasome inhibitors in combination with D-3 enhanced VDR-mediated gene expression, as measured by induction of vitamin D-3 24-hydroxylase mRNA in cultured keratinocytes. Taken together, our findings indicate that low VDR levels are maintained, in part, through ubiquitin/proteasome-mediated degradation and that low VDR levels limit D-3 signaling. D-3 exerts dual positive influences on its nuclear receptor, simultaneously stimulating VDR transactivation activity and retarding VDR degradation.