1,25-dihydroxyvitamin D-3 increases nuclear vitamin D-3 receptors by blocking ubiquitin/proteasome-mediated degradation in human skin

1,25-Dihydroxyvitamin D-3 (D-3) exerts its effects by binding to and activa ting nuclear vitamin D-3 receptors (VDRs) that regulate transcription of ta rget genes. We have investigated regulation of VDR levels in human skin in vivo and in cultured human keratinocytes. Quantitative ligand-binding analy sis revealed that human skin expressed approximately 220 VDRs per cell, whi ch bound D-3 with high affinity [(dissociation constant (K-d) = 0.22 nM]. I n human skin nuclear extracts, VDR exclusively bound to DNA containing vita min D-3 response elements as heterodimers with retinoid X receptors. Topica l application of D-3 to human skin elevated VDR protein levels 2-fold, as m easured 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 effec t on VDR mRNA, indicating that D-3 either stimulated synthesis and/or inhib ited degradation of VDRs. To investigate this latter possibility, recombina nt 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 hu man skin lysates. VDR degradation was inhibited by proteasome inhibitors, b ut not lysosome or serine protease inhibitors. In cultured keratinocytes, D -3 or proteasome inhibitors increased VDR protein without affecting VDR mRN A levels. In cells, VDR was ubiquitinated and this ubiquitination was inhib ited by D-3. Proteasome inhibitors in combination with D-3 enhanced VDR-med iated gene expression, as measured by induction of vitamin D-3 24-hydroxyla se mRNA in cultured keratinocytes. Taken together, our findings indicate th at low VDR levels are maintained, in part, through ubiquitin/proteasome-med iated degradation and that low VDR levels limit D-3 signaling. D-3 exerts d ual positive influences on its nuclear receptor, simultaneously stimulating VDR transactivation activity and retarding VDR degradation.