Enhancement of VDR-mediated transcription by phosphorylation: Correlation with increased interaction between the VDR and DRIP205, a subunit of the VDR-interacting protein coactivator complex
Enhancement of VDR-mediated transcription by phosphorylation: Correlation with increased interaction between the VDR and DRIP205, a subunit of the VDR-interacting protein coactivator complex
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DOI：
10.1210/me.16.2.301
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发表时间：
2002-02-01
影响因子：
--
通讯作者：
Christakos, S
中科院分区：
文献类型：
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作者：
Barletta, F;Freedman, LP;Christakos, S
当用大鼠24-羟化酶[24（OH）ase]启动子转染UMR-106成骨细胞、LLCPK 1肾细胞和VDR转染的COS-7细胞时，（-1，367/+74）或小鼠骨桥蛋白（OPN）启动子（-777/+79），我们发现蛋白磷酸酶抑制剂冈田酸（OA）可显著增强对1，25-（OH）（2）D-3 [1，25-（OH）（2）D-3]的反应2 - 5倍。使用含有近端24（OH）ase维生素D反应元件（VDRE）或OPN VDRE的合成报告基因，也观察到CIA对1，25-（OH）（2）D-3诱导的转录的增强，表明VDRE足以介导该效应。OA还增强了UMR成骨细胞中1，25-（OH）（2）D-3诱导的24（OH）ase和OPN mRNA水平。OA的作用不是由于VDR的上调或VDR-RXR与VDRE相互作用的增加。为了确定磷酸化是否通过调节与蛋白质伴侣的相互作用来调节VDR介导的转录，我们使用谷胱甘肽-S-转移酶下拉测定法研究了磷酸化对VDR和DRIP205（维生素D受体相互作用蛋白（DRIP）共激活因子复合物的亚基）之间蛋白质-蛋白质相互作用的影响。与功能性研究相似，一致发现OA处理增强VDR与DRIP 205的相互作用，比单独存在1，25-（OH）（2）D-3时观察到的相互作用高3 - 4倍。此外，还对活化功能-2缺陷型VDR突变体L417 S进行了研究，该突变体不能响应1，25-（OH）（2）D-3刺激转录或与DRIP205相互作用。然而，在OA的存在下，突变体VDR能够激活24（OH）酶和OPN转录并募集DRIP205，这表明OA处理可能导致激活功能-2缺陷突变体的构象变化，从而产生与DRIP205的活性相互作用表面。综上所述，这些发现表明，VDR和共激活因子（如DRIP205）之间的相互作用增加可能是将细胞外信号与维生素D作用偶联的主要机制。
When UMR-106 osteoblastic cells, LLCPK1 kidney cells, and VDR transfected COS-7 cells were transfected with the rat 24-hydroxylase [24(OH)ase] promoter (-1,367/+74) or the mouse osteopontin (OPN) promoter (-777/+79), we found that the response to 1,25dihydroxyvitamin D-3 [1,25-(OH)(2)D-3] could be significantly enhanced 2- to 5-fold by the protein phosphatase inhibitor, okadaic acid (OA). Enhancement of 1,25-(OH)(2)D-3-induced transcription by CIA was also observed using a synthetic reporter gene containing either the proximal 24(OH)ase vitamin D response element (VDRE) or the OPN VDRE, suggesting that the VDRE is sufficient to mediate this effect. OA also enhanced the 1,25-(OH)(2)D-3-induced levels of 24(OH)ase and OPN mRNA in UMR osteoblastic cells. The effect of OA was not due to an up-regulation of VDR or to an increase in VDR-RXR interaction with the VDRE. To determine whether phosphorylation regulates VDR-mediated transcription by modulating interactions with protein partners, we examined the effect of phosphorylation on the protein-protein interaction between VDR and DRIP205, a subunit of the vitamin D receptor-interacting protein (DRIP) coactivator complex, using glutathione-S-transferase pull-down assays. Similar to the functional studies, OA treatment was consistently found to enhance the interaction of VDR with DRIP205 3- to 4-fold above the interaction observed in the presence of 1,25-(OH)(2)D-3 alone. In addition, studies were done with the activation function-2 defective VDR mutant, L417S, which is unable to stimulate transcription in response to 1,25-(OH)(2)D-3 or to interact with DRIP205. However, in the presence of OA, the mutant VDR was able to activate 24(OH)ase and OPN transcription and to recruit DRIP205, suggesting that OA treatment may result in a conformational change in the activation function-2 defective mutant that creates an active interaction surface with DRIP205. Taken together, these findings suggest that increased interaction between VDR and coactivators such as DRIP205 may be a major mechanism that couples extracellular signals to vitamin D action.