Huang Yu-Chun, Hung Wen-Chun
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, China.
J Cell Physiol. 2006 Nov;209(2):363-9. doi: 10.1002/jcp.20741.
Upregulation of p27Kip1 protein in 1,25-dihydroxyvitamin D3-treated cancer cells is mediated via enhancement of gene transcription and reduction of protein degradation. 1,25-dihydroxyvitamin D3 inhibits the expression of p45Skp2, the F-box protein which is implicated in p27Kip1 degradation, to reduce turnover of p27Kip1 protein. In this study, we elucidate the underlying mechanism by which 1,25-dihydroxyvitamin D3 inhibits p45Skp2 in human LNCaP prostate cancer cells. Western blot and RT-PCR analysis suggest that 1,25-dihydroxyvitamin D3 suppresses p45Skp2 via transcriptional repression. Promoter activity assays indicate that 1,25-dihydroxyvitamin D3 directly inhibits p45Skp2 promoter activity. Deletion analysis shows that 1,25-dihydroxyvitamin D3 response element is localized at -447/-291 bp region from the translational start site of the p45Skp2 promoter. Mutation analysis suggests that two Sp1 sites localized at -386/-380 and -309/-294 bp region are required for transcriptional repression. Chromatin immunoprecipitation (CHIP) assay demonstrates that VDR indirectly binds to these Sp1 sites in vivo and this binding is increased after 1,25-dihydroxyvitamin D3 treatment. Re-CHIP assay suggests that VDR and Sp1 form a complex to bind to the Sp1 sites. DNA affinity precipitation assay (DAPA) shows that histone deacetylase 1 (HDAC1) is recruited to the Sp1 sites after 1,25-dihydroxyvitamin D3 stimulation. Re-CHIP assay verifies that binding of Sp1 and HDAC1 to p45Skp2 promoter is enhanced after 1,25-dihydroxyvitamin D3 treatment. HDAC inhibitor trichostatin A (TSA) reverses the inhibition of p45Skp2 promoter activity by 1,25-dihydroxyvitamin D3. Collectively, our results suggest that 1,25-dihydroxyvitamin D3 induces the formation of VDR/Sp1 complex and acts via a Sp1- and HDAC1-depedent pathway to inhibit p45Skp2 transcription.
1,25 - 二羟维生素D3处理的癌细胞中p27Kip1蛋白的上调是通过增强基因转录和减少蛋白质降解介导的。1,25 - 二羟维生素D3抑制p45Skp2的表达，p45Skp2是一种与p27Kip1降解有关的F - box蛋白，从而减少p27Kip1蛋白的周转。在本研究中，我们阐明了1,25 - 二羟维生素D3在人LNCaP前列腺癌细胞中抑制p45Skp2的潜在机制。蛋白质印迹和逆转录 - 聚合酶链反应分析表明，1,25 - 二羟维生素D3通过转录抑制来抑制p45Skp2。启动子活性测定表明，1,25 - 二羟维生素D3直接抑制p45Skp2启动子活性。缺失分析表明，1,25 - 二羟维生素D3反应元件位于p45Skp2启动子翻译起始位点的 - 447 / - 291 bp区域。突变分析表明，位于 - 386 / - 380和 - 309 / - 294 bp区域的两个Sp1位点是转录抑制所必需的。染色质免疫沉淀（CHIP）测定表明，维生素D受体（VDR）在体内间接结合这些Sp1位点，并且在1,25 - 二羟维生素D3处理后这种结合增加。再免疫沉淀测定表明，VDR和Sp1形成复合物以结合Sp1位点。DNA亲和沉淀测定（DAPA）表明，1,25 - 二羟维生素D3刺激后组蛋白去乙酰化酶1（HDAC1）被招募到Sp1位点。再免疫沉淀测定证实，1,25 - 二羟维生素D3处理后Sp1和HDAC1与p45Skp2启动子的结合增强。组蛋白去乙酰化酶抑制剂曲古抑菌素A（TSA）逆转了1,25 - 二羟维生素D3对p45Skp2启动子活性的抑制。总体而言，我们的结果表明，1,25 - 二羟维生素D3诱导VDR / Sp1复合物的形成，并通过依赖Sp1和HDAC1的途径抑制p45Skp2转录。
