The human transient receptor potential vanilloid type 6 distal promoter contains multiple vitamin D receptor binding sites that mediate activation by 1,25-dihydroxyvitamin D3 in intestinal cells
The human transient receptor potential vanilloid type 6 distal promoter contains multiple vitamin D receptor binding sites that mediate activation by 1,25-dihydroxyvitamin D3 in intestinal cells
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DOI：
10.1210/me.2006-0031
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发表时间：
2006-06-01
影响因子：
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通讯作者：
Pike, J. Wesley
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文献类型：
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作者：
Meyer, Mark B.;Watanuki, Makoto;Pike, J. Wesley
瞬时受体电位香草样蛋白6 (TRPV6) （ECAC2, CaT1）是肠上皮细胞膜上负责钙进入的主要离子通道。1,25-二羟基维生素D-3 [1,25-(OH)(2)D-3]在转录水平上积极调节其表达。在本报告中，我们确定了1,25-(OH)(2)D-3调控TRPV6的完整机制。基于肠道细胞系中一个7kb TRPV6启动子片段的激素响应性，我们采用染色质免疫沉淀（ChIP）扫描方法在TRPV6基因座内寻找可能的维生素D受体（VDR）和类视黄醇X受体（RXR）调控区域。VDR/RXR结合范围较广，相对于TRPV6转录起始位点在-1.2 ~ -5.5 kb之间。这些结果与计算机分析一致，显示了位于-1.2,-2.1,-3.5,-4.3和-5.5 kb的推定调控元件（VDREs）。尽管进行了ChIP分析，但只有TRPV6基因中含有-2.1和-4.3 kb推定元件的区域将1.25 -(OH)(2)D-3响应转移到异源启动子上。进一步的研究表明，这两个活性区域中的每一个都含有由两个独立的调控元件组成的复合VDREs。当在天然TRPV6启动子内检测时，-2.1-和-4.3- kb区域的VDRE以及-1.2 kb区域的VDRE的突变消除了对1,25-(OH) D-2(3)的所有应答。最后的ChIP分析显示，VDR/RXR异源二聚体与TRPV6基因结合时，伴随着类固醇受体共激活因子1的募集以及组蛋白4乙酰化的广泛变化。这些研究确定了1,25-(OH)(2)D-3调节人肠细胞中TRPV6表达的机制。
Transient receptor potential vanilloid type 6 (TRPV6) (ECAC2, CaT1) is the major ion channel in intestinal epithelial cell membranes responsible for calcium entry. Its expression is actively regulated at the transcriptional level by 1,25-dihydroxyvitamin D-3 [1,25-(OH)(2)D-3]. In this report, we identify mechanisms integral to the regulation of TRPV6 by 1,25-(OH)(2)D-3. Based upon the hormonal responsiveness of a 7-kb TRPV6 promoter fragment in intestinal cell lines, we used a chromatin immunoprecipitation (ChIP) scanning method to search for possible vitamin D receptor (VDR) and retinoid X receptor (RXR) regulatory regions within the TRPV6 locus. VDR/RXR binding was broad, ranging from -1.2 to -5.5 kb relative to the start site of TRPV6 transcription. These results were consistent with an in silico analysis that revealed putative regulatory elements (VDREs) located at -1.2, -2.1, -3.5, -4.3, and -5.5 kb. Despite the ChIP analyses, only regions of the TRPV6 gene that contained putative elements at -2.1 and -4.3 kb transferred 1,25-(OH)(2)D-3 response to a heterologous promoter. Further study revealed that each of these two active regions contained composite VDREs comprised of two separate regulatory elements. Mutagenesis of the VDREs within the -2.1- and -4.3- kb region and the VDRE at -1.2 kb abrogated all response to 1,25-(OH) D-2(3) when examined within the natural TRPV6 promoter. A final ChIP assay revealed that VDR/RXR heterodimer binding to the TRPV6 gene was accompanied by both the recruitment of steroid receptor coactivator 1 as well as a broad change in histone 4 acetylation. These studies identify a mechanism by which 1,25-(OH)(2)D-3 regulates the expression of TRPV6 in human intestinal cells.