Department of Internal Medicine, Hospital U.M. Valdecilla-IFIMAV, University of Cantabria, RETICEF, Santander, Spain.
J Bone Miner Res. 2012 Apr;27(4):926-37. doi: 10.1002/jbmr.1491.
Sclerostin, encoded by the SOST gene, is a potent inhibitor of bone formation, produced by osteocytes, not by osteoblasts, but little is known about the molecular mechanisms controlling its expression. We aimed to test the hypothesis that epigenetic mechanisms, specifically DNA methylation, modulate SOST expression. We found two CpG-rich regions in SOST: region 1, located in the proximal promoter, and region 2, around exon 1. qMSP and pyrosequencing analysis of DNA methylation showed that region 2 was largely methylated in all samples analyzed. In contrast, marked differences were observed in region 1. Whereas the CpG-rich region 1 was hypermethylated in osteoblasts, this region was largely hypomethylated in microdissected human osteocytes. Bone lining cells showed a methylation profile between primary osteoblasts and osteocytes. Whereas SOST expression was detected at very low level or not at all by RT-qPCR in several human osteoblastic and nonosteoblastic cell lines, and human primary osteoblasts under basal conditions, it was dramatically upregulated (up to 1300-fold) by the demethylating agent AzadC. Experiments using reporter vectors demonstrated the functional importance of the region -581/+30 of the SOST gene, which contains the CpG-rich region 1. In vitro methylation of this CpG-island impaired nuclear protein binding and led to a 75 ± 12% inhibition of promoter activity. In addition, BMP-2-induced expression of SOST was markedly enhanced in cells demethylated by AzadC. Overall, these results strongly suggest that DNA methylation is involved in the regulation of SOST expression during osteoblast-osteocyte transition, presumably by preventing the binding of transcription factors to the proximal promoter. To our knowledge, our data provide first ever evidence of the involvement of DNA methylation in the regulation of SOST expression and may help to establish convenient experimental models for further studies of human sclerostin.
骨硬化蛋白（Sclerostin）由 SOST 基因编码，是一种强效的骨形成抑制剂，由骨细胞产生，而非成骨细胞产生，但人们对控制其表达的分子机制知之甚少。我们旨在验证这样一个假说，即表观遗传机制，特别是 DNA 甲基化，调节 SOST 的表达。我们在 SOST 中发现了两个富含 CpG 的区域：区域 1 位于近端启动子，区域 2 位于外显子 1 周围。对 DNA 甲基化的 qMSP 和焦磷酸测序分析表明，所有分析样本中区域 2 都被高度甲基化。相比之下，区域 1 则存在显著差异。富含 CpG 的区域 1 在成骨细胞中呈高甲基化，而在微分离的人骨细胞中则呈低甲基化。骨衬细胞的甲基化谱则处于原代成骨细胞和骨细胞之间。尽管在几种人成骨细胞和非成骨细胞系以及人原代成骨细胞的基础条件下，RT-qPCR 检测到 SOST 的表达水平很低或根本检测不到，但去甲基化剂 AzadC 可使其表达水平显著上调（高达 1300 倍）。使用报告载体的实验表明，SOST 基因-581/+30 区域的功能非常重要，该区域包含富含 CpG 的区域 1。该 CpG 岛的体外甲基化会削弱核蛋白结合，并导致启动子活性下降 75±12%。此外，经 AzadC 去甲基化的细胞中，BMP-2 诱导的 SOST 表达显著增强。总的来说，这些结果强烈表明，DNA 甲基化参与了成骨细胞-骨细胞转化过程中 SOST 表达的调控，可能是通过阻止转录因子与近端启动子结合来实现的。据我们所知，我们的数据首次提供了 DNA 甲基化参与 SOST 表达调控的证据，并可能有助于建立方便的实验模型，以进一步研究人类骨硬化蛋白。
