Department of Internal Medicine, Hospital U.M. Valdecilla-IFIMAV, University of Cantabria, RETICEF, Santander, Spain.
Epigenetics. 2012 Jan 1;7(1):83-91. doi: 10.4161/epi.7.1.18753.
Osteoblasts are specialized cells that form new bone and also indirectly influence bone resorption by producing factors that modulate osteoclast differentiation. Although the methylation of CpG islands plays an important role in the regulation of gene expression, there is still scanty information about its role in human bone. The aim of this study was to investigate the influence of CpG methylation on the transcriptional levels of two osteoblast-derived critical factors in the regulation of osteoclastogenesis: the receptor activator of nuclear factor NF-κB ligand (RANKL) and its soluble decoy receptor osteoprotegerin (OPG). Quantitative methylation specific PCR (qMSP) and pyrosequencing analysis in various cell types showed that the methylation of regulatory regions of these genes, in the vicinity of the transcription start sites, repressed gene transcription, whereas an active transcription was associated with low levels of methylation. In addition, treatment with the DNA demethylating agent 5-azadeoxycitidine promoted a 170-fold induction of RANKL and a 20-fold induction of OPG mRNA expression in HEK-293 cells, which showed hypermethylation of the CpG islands and barely expressed RANKL and OPG transcripts at baseline. Transcriptional levels of both genes were also explored in bone tissue samples from patients with hip fractures and hip osteoarthritis. Although RANKL transcript abundance and the RANKL:OPG transcript ratio were significantly higher in patients with fractures than in those with osteoarthritis (RANKL: 0.76 ± 0.23 vs. 0.24 ± 0.08, p = 0.012; RANKL/OPG: 7.66 ± 2.49 vs. 0.92 ± 0.21, p = 0.002), there was no evidence for differential methylation across patient groups. In conclusion, the association between DNA methylation and the repression of RANKL and OPG expression strongly suggests that methylation-dependent mechanisms influence the transcription of these genes, which play a critical role in osteoclastogenesis. However, other mechanisms appear to be involved in the increased RANKL/OPG ratio of patients with osteoporotic fractures.
成骨细胞是专门形成新骨的细胞，也通过产生调节破骨细胞分化的因子间接影响骨吸收。虽然 CpG 岛的甲基化在基因表达调控中起着重要作用，但关于其在人类骨骼中的作用的信息仍然很少。本研究旨在探讨 CpG 甲基化对两个成骨细胞衍生的关键因子在破骨细胞生成调节中的转录水平的影响：核因子 κB 受体激活剂配体（RANKL）及其可溶性诱饵受体骨保护素（OPG）。各种细胞类型的定量甲基化特异性 PCR（qMSP）和焦磷酸测序分析表明，这些基因转录起始位点附近的调控区的甲基化抑制了基因转录，而活跃的转录与低甲基化水平相关。此外，用 DNA 去甲基化剂 5-氮杂脱氧胞苷处理可使 HEK-293 细胞中 RANKL 的诱导增加 170 倍，OPG mRNA 表达增加 20 倍，这些细胞的 CpG 岛呈高甲基化状态，基线时几乎不表达 RANKL 和 OPG 转录本。还在髋部骨折和髋部骨关节炎患者的骨组织样本中研究了这两个基因的转录水平。虽然骨折患者的 RANKL 转录物丰度和 RANKL:OPG 转录物比值明显高于骨关节炎患者（RANKL：0.76±0.23 与 0.24±0.08，p=0.012；RANKL/OPG：7.66±2.49 与 0.92±0.21，p=0.002），但两组患者之间没有证据表明存在差异甲基化。总之，DNA 甲基化与 RANKL 和 OPG 表达抑制之间的关联强烈表明，甲基化依赖机制影响这些基因的转录，这些基因在破骨细胞生成中起着关键作用。然而，在骨质疏松性骨折患者中，RANKL/OPG 比值的增加似乎涉及其他机制。
