Epigenetic silencing of CYP24 in tumor-derived endothelial cells contributes to selective growth inhibition by calcitriol
Epigenetic silencing of CYP24 in tumor-derived endothelial cells contributes to selective growth inhibition by calcitriol
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DOI：
10.1074/jbc.m608894200
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发表时间：
2007-03-23
影响因子：
4.8
通讯作者：
Trump, Donald L.
中科院分区：
文献类型：
--
作者：
Chung, Ivy;Karpf, Adam R.;Trump, Donald L.
骨化三醇（1，25-二羟基胆钙化醇）是维生素D的最活跃形式，与基质胶衍生的内皮细胞（MDEC）相比，对肿瘤衍生的内皮细胞（TDEC）具有选择性抗增殖作用。虽然这两种细胞类型都有一个完整的维生素D受体信号传导轴，这项研究表明，在治疗与骨化三醇，24-羟化酶（CYP 24）mRNA，蛋白质和酶活性显着诱导MDEC在时间依赖性的方式，但不是在TDEC。此外，用CYP 24小干扰RNA治疗MDEC恢复了对骨化三醇的敏感性。为了研究TDEC中缺乏CYP 24诱导，我们检测了这两种细胞类型中CYP 24基因启动子区域的甲基化模式。我们鉴定了位于5'端的两个假定的CpG岛区域。使用甲基化特异性PCR和亚硫酸氢盐测序，我们确定这些CpG岛在TDEC中是高甲基化的，但在MDEC中不是。这些数据可以解释招聘维生素D受体的启动子区域在MDEC，但不是TDEC，染色质免疫沉淀分析显示。用DNA甲基转移酶抑制剂5-氮杂-2 '-脱氧胞苷治疗TDEC恢复了骨化三醇介导的CYP 24诱导，这导致对骨化三醇生长抑制作用的敏感性丧失。在从其他肿瘤分离的内皮细胞中也观察到CYP 24启动子高甲基化，但在从正常小鼠组织分离的内皮细胞中未观察到。这些观察结果表明，CYP 24启动子的甲基化状态在分离自不同微环境（肿瘤与正常）的内皮细胞中不同，并且CYP 24的甲基化沉默有助于内皮细胞中选择性骨化三醇介导的生长抑制。
Calcitriol (1,25-dihydroxycholecalciferol), the most active form of vitamin D, has selective anti-proliferative effects on tumor-derived endothelial cells (TDEC) compared with Matrigel-derived endothelial cells (MDEC). Although both cell types have an intact vitamin D receptor-signaling axis, this study demonstrates that upon treatment with calcitriol, 24-hydroxylase (CYP24) mRNA, protein and enzymatic activity were markedly induced in MDEC in a time-dependent manner but not in TDEC. Furthermore, treatment of MDEC with a CYP24 small interfering RNA restored sensitivity to calcitriol. To investigate the lack of CYP24 induction in TDEC, we examined methylation patterns in the promoter regions of the CYP24 gene in these two cell types. We identified two putative CpG island regions located at the 5' end. Using methylation-specific PCR and bisulfite sequencing, we determined that these CpG islands were hypermethylated in TDEC but not in MDEC. These data may explain the recruitment of vitamin D receptor to the promoter region in MDEC but not TDEC, as revealed by chromatin immunoprecipitation analyses. Treatment of TDEC with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine restored calcitriol-mediated induction of CYP24, which led to loss of sensitivity to calcitriol growth inhibitory effects. CYP24 promoter hypermethylation was also observed in endothelial cells isolated from other tumors but not in endothelial cells isolated from normal mouse tissues. These observations indicate that the methylation status of the CYP24 promoter differs in endothelial cells isolated from different microenvironments (tumor versus normal) and that methylation silencing of CYP24 contributes to selective calcitriol-mediated growth inhibition in endothelial cells.