Control of the SOST bone enhancer by PTH using MEF2 transcription factors
Control of the SOST bone enhancer by PTH using MEF2 transcription factors
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DOI：
10.1359/jbmr.070804
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发表时间：
2007-12-01
影响因子：
6.2
通讯作者：
Keller, Hansjoerg
中科院分区：
文献类型：
--
作者：
Leupin, Olivier;Kramer, Ina;Keller, Hansjoerg
骨细胞衍生的骨形成抑制剂sclerostin在成人骨中的表达需要远距离增强子。我们发现，MEF 2转录因子控制这个增强子和介导的抑制硬化蛋白的表达PTH.Introduction：由SOST基因编码的硬化蛋白是骨形成的关键调节。SOST表达的缺乏是进行性骨过度生长疾病硬化症和货车Buchem病的原因。我们以前已经确定了一个遥远的增强子内的52-kb的货车Buchem病缺失下游的SOST基因，是必不可少的，其在成人骨的表达。此外，我们和其他人已经报道了SOST表达被PTH抑制。本研究的目的是确定参与SOST骨增强子活性和介导PTH responsibility.Materials和方法的转录因子：通过荧光素酶报告基因分析研究了SOST增强子和启动子的调控。通过足迹分析和功能突变分析，使用表现出内源性SOST表达的成骨细胞样UMR-106细胞的瞬时转染绘制转录因子结合位点。通过序列分析预测特异性转录因子结合，并通过凝胶阻滞试验和抗体诱导的超位移显示。采用原位杂交、定量RT-PCR（qPCR）和免疫组织化学方法检测心肌细胞增强因子2（MEF 2）的表达。MEF 2s在SOST表达中的作用通过报告基因分析和siRNA介导的RNA knockdown.Results进行了评估：PTH完全抑制了SOST骨增强子的转录活性，但不影响SOST启动子。在骨增强剂中鉴定了MEF 2反应元件。它对转录激活、结合MEF 2转录因子和介导的PTH应答是必不可少的。通过qPCR、原位杂交和免疫组织化学显示MEF 2在骨中的表达。MEF 2和sclerostin共定位于骨细胞中。增强子活性通过MEF 2C过表达刺激，并通过显性负性MEF 2C突变体的共表达抑制。最后，siRNA介导的MEF 2A，C和D敲低抑制内源性SOST表达在UMR-106，成骨细胞样cells.Conclusions：这些数据强烈表明，SOST表达在成人骨的骨细胞及其抑制PTH介导的MEF 2A，C和D转录因子控制的SOST骨增强子。因此，MEF 2与成人骨量的调节有关。
Expression of the osteocyte-derived bone formation inhibitor sclerostin in adult bone requires a distant enhancer. We show that MEF2 transcription factors control this enhancer and mediate inhibition of sclerostin expression by PTH.Introduction: Sclerostin encoded by the SOST gene is a key regulator of bone formation. Lack of SOST expression is the cause for the progressive bone overgrowth disorders sclerosteosis and Van Buchem disease. We have previously identified a distant enhancer within the 52-kb Van Buchem disease deletion downstream of the SOST gene that is essential for its expression in adult bone. Furthermore, we and others have reported that SOST expression is suppressed by PTH. The aim of this study was to identify transcription factors involved in SOST bone enhancer activity and mediating PTH responsiveness.Materials and Methods: Regulation of the SOST enhancer and promoter was studied by luciferase reporter gene assays. Transcription factor binding sites were mapped by footprint analysis and functional mutation analyses using transient transfections of osteoblast-like UMR-106 cells that exhibit endogenous SOST expression. Specific transcription factor binding was predicted by sequence analysis and shown by gel retardation assays and antibody-induced supershifts. Expression of myocyte enhancer factors 2 (MEF2) was detected by in situ hybridization, quantitative RT-PCR (qPCR), and immunohistochemistry. The role of MEF2s in SOST expression was assessed by reporter gene assays and siRNA-mediated RNA knockdown.Results: PTH completely suppressed the transcriptional activity of the SOST bone enhancer but did not affect the SOST promoter. A MEF2 response element was identified in the bone enhancer. It was essential for transcriptional activation, bound MEF2 transcription factors, and mediated PTH responsiveness. Expression of MEF2s in bone was shown by qPCR, in situ hybridization, and immunohistochemistry. MEF2s and sclerostin co-localized in osteocytes. Enhancer activity was stimulated by MEF2C overexpression and inhibited by co-expression of a dominant negative MEF2C mutant. Finally, siRNA-mediated knockdown of MEF2A, C, and D suppressed endogenous SOST expression in UMR-106,osteoblast-like cells.Conclusions: These data strongly suggest that SOST expression in osteocytes of adult bone and its inhibition by PTH is mediated by MEF2A, C, and D transcription factors controlling the SOST bone enhancer. Hence, MEF2s are implicated in the regulation of adult bone mass.