1,25-dihydroxyvitamin D3 increases nuclear vitamin D3 receptors by blocking ubiquitin/proteasome-mediated degradation in human skin
1,25-dihydroxyvitamin D3 increases nuclear vitamin D3 receptors by blocking ubiquitin/proteasome-mediated degradation in human skin
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DOI：
10.1210/me.13.10.1686
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发表时间：
1999-10-01
影响因子：
--
通讯作者：
Voorhees, JJ
中科院分区：
文献类型：
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作者：
Li, XY;Boudjelal, M;Voorhees, JJ
1，25-二羟基维生素D-3（D-3）通过结合并激活调节靶基因转录的核维生素D-3受体（VDR）发挥其作用。我们已经研究了体内人皮肤和培养的人角质形成细胞中VDR水平的调节。定量配体结合分析显示，人皮肤每个细胞表达约220个VDR，其以高亲和力结合D-3 [（解离常数（K-d）= 0.22 nM]。在人皮肤细胞核提取物中，VDR仅与含有维生素D-3反应元件的DNA结合，作为类维生素A X受体的异二聚体。局部应用D-3到人类皮肤的VDR蛋白水平升高2倍，如通过配体结合和DNA结合测定所测量的。相反，D-3类似物卡泊三烯对VDR水平没有影响。局部D-3对VDR mRNA没有影响，表明D-3刺激VDR的合成和/或抑制VDR的降解。为了研究后一种可能性，在存在或不存在D-3的情况下，将重组VDR与皮肤裂解物一起孵育。D-3的存在基本上保护VDR免受人皮肤裂解物的降解。VDR降解被蛋白酶体抑制剂抑制，但不是溶酶体或丝氨酸蛋白酶抑制剂。在培养的角质形成细胞中，D-3或蛋白酶体抑制剂增加VDR蛋白而不影响VDR mRNA水平。在细胞中，VDR被泛素化，这种泛素化被D-3抑制。蛋白酶体抑制剂与D-3的组合增强VDR介导的基因表达，通过诱导培养的角质形成细胞中的维生素D-3 24-羟化酶mRNA来测量。总之，我们的研究结果表明，低VDR水平的维持，部分，通过泛素/蛋白酶体介导的降解和低VDR水平限制D-3信号。D-3对其核受体具有双重积极影响，同时刺激VDR反式激活活性和延缓VDR降解。
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.