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1,25-Dihydroxyvitamin D₃ Increases Nuclear Vitamin D₃ Receptors by Blocking Ubiquitin/Proteasome-Mediated Degradation in Human Skin

Department of Dermatology University of Michigan Medical School Ann Arbor, Michigan 48109

1,25-Dihydroxyvitamin D₃ (D₃) exerts its effects by binding to and activating nuclear vitamin D₃ 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 D₃ with high affinity [(dissociation constant (K_d) = 0.22 nM]. In human skin nuclear extracts, VDR exclusively bound to DNA containing vitamin D₃ response elements as heterodimers with retinoid X receptors. Topical application of D₃ to human skin elevated VDR protein levels 2-fold, as measured by both ligand-binding and DNA-binding assays. In contrast, the D₃ analog calcipotriene had no effect on VDR levels. Topical D₃ had no effect on VDR mRNA, indicating that D₃ 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 D₃. The presence of D₃ 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, D₃ or proteasome inhibitors increased VDR protein without affecting VDR mRNA levels. In cells, VDR was ubiquitinated and this ubiquitination was inhibited by D₃. Proteasome inhibitors in combination with D₃ enhanced VDR-mediated gene expression, as measured by induction of vitamin D₃ 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 D₃ signaling. D₃ exerts dual positive influences on its nuclear receptor, simultaneously stimulating VDR transactivation activity and retarding VDR degradation.

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