Desai R K, van Wijnen A J, Stein J L, Stein G S, Lian J B
Department of Endocrinology, University of Massachusetts Medical Center, Worcester 01655, USA.
Endocrinology. 1995 Dec;136(12):5685-93. doi: 10.1210/endo.136.12.7588324.
The 1,25-dihydroxyvitamin D3 (vitamin D) receptor (VDR) is a key trans-activating protein that mediates calcium regulation as well as cellular proliferation and differentiation. Phosphorylation of the VDR contributes significantly to its functional activity, but the specific mechanisms that mediate this regulation are not well understood. Phosphorylation may influence DNA binding, ligand binding, and protein-protein interactions, including heterodimerization and/or transactivation functions. We used a protein kinase C inhibitor, staurosporine (ST), and an inhibitor of serine-threonine phosphatases, okadaic acid (OA), to elucidate the contribution of VDR phosphorylation to vitamin D-mediated transcription of the osteocalcin (OC) gene. Vitamin D-induced transcription was assayed in transfected ROS 17/2.8 osteosarcoma cells using chloraminphenicol acetyltransferase constructs containing the vitamin D-responsive element (VDRE) at its native locus in the rat OC promoter as well as fused to a heterologous promoter. Both ST and OA inhibit VDRE-mediated and vitamin D-dependent enhancement of OC gene transcription as well as OC biosynthesis, as assessed by RIAs. Results from gel mobility shift and Western blot analyses using nuclear proteins from ROS 17/2.8 cells show that binding of the VDR-retinoid-X receptor heterodimer complex to the OC VDRE is not inhibited in the presence of ST. In contrast, OA does inhibit the formation of complexes interacting with both the OC and osteopontin VDREs; immunoprecipitation studies using 32P-labeled ROS 17/2.8 cells reveal that OA treatment result in ligand-independent hyperphosphorylation of the VDR. Our results suggest that two distinct phosphorylation events modulate rat VDR function. One event is related to transactivation, and the other is also critical to the VDRE-binding activity of VDR-retinoid X receptor-DNA complexes with consequential effects on transactivation.
1,25 - 二羟基维生素D3（维生素D）受体（VDR）是一种关键的反式激活蛋白，介导钙调节以及细胞增殖和分化。VDR的磷酸化对其功能活性有显著贡献，但介导这种调节的具体机制尚不清楚。磷酸化可能影响DNA结合、配体结合以及蛋白质 - 蛋白质相互作用，包括异源二聚化和/或反式激活功能。我们使用蛋白激酶C抑制剂星形孢菌素（ST）和丝氨酸 - 苏氨酸磷酸酶抑制剂冈田酸（OA），以阐明VDR磷酸化对维生素D介导的骨钙素（OC）基因转录的作用。在转染的ROS 17/2.8骨肉瘤细胞中，使用氯霉素乙酰转移酶构建体检测维生素D诱导的转录，该构建体在大鼠OC启动子的天然位点含有维生素D反应元件（VDRE），以及与异源启动子融合。通过放射免疫分析评估，ST和OA均抑制VDRE介导的以及维生素D依赖性的OC基因转录增强和OC生物合成。使用来自ROS 17/2.8细胞的核蛋白进行的凝胶迁移率变动分析和蛋白质印迹分析结果表明，在ST存在下，VDR - 视黄酸X受体异源二聚体复合物与OC VDRE的结合未受抑制。相反，OA确实抑制了与OC和骨桥蛋白VDRE相互作用的复合物的形成；使用32P标记的ROS 17/2.8细胞进行的免疫沉淀研究表明，OA处理导致VDR的配体非依赖性过度磷酸化。我们的结果表明，两种不同的磷酸化事件调节大鼠VDR功能。一个事件与反式激活有关，另一个对VDR - 视黄酸X受体 - DNA复合物的VDRE结合活性也至关重要，从而对反式激活产生影响。
