Bone and Joint Research Group, Centre for Human Development Stem Cells and Regeneration, Institute of Developmental Science, University of Southampton Medical School, Southampton, UK.
Bone and Joint Research Group, Centre for Human Development Stem Cells and Regeneration, Institute of Developmental Science, University of Southampton Medical School, Southampton, UK; Department of Orthopaedic Surgery, Tohoku University Hospital, Sendai, Japan.
Osteoarthritis Cartilage. 2016 Nov;24(11):1951-1960. doi: 10.1016/j.joca.2016.06.002. Epub 2016 Jun 13.
To examine the methylation profile of the nuclear factor (NF)-κB enhancer region at -5.8 kb of inducible nitric oxide synthase (iNOS) and the subsequent role in the induction of osteoarthritis (OA) via cell cycle regulation.
Percentage methylation was determined by pyrosequencing, gene expression by qRT-PCR and cell proliferation was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Transient transfections were induced to determine the effect of the NF-κB enhancer region on cell proliferation and the influence of DNA methylation.
In vitro de-methylation with 5-aza-dC showed decreased levels of DNA methylation at CpG sites localised at -5.8 kb, which correlated with higher levels of iNOS expression. In vitro methylation of the NF-κB enhancer region at -5.8 kb increased the percentage of cells at G0/G1 cell cycle phase. Loss of methylation within this region correlated with, enhanced proliferation and increased number of cells at G2/M phase. OA chondrocytes demonstrated up-regulation of the G0/G1 cell cycle progression markers Cyclin D1 and CDK6 in contrast to control cells. We demonstrate the loss of methylation that occurs at specific CpG sites localised at the -5.8 kb NF-κB enhancer region of the iNOS gene in OA chondrocytes permits the binding of this transcription factor activating the expression of iNOS. This results in subsequent altered cell cycle regulation, altered proliferative phenotype and transmission of the pathogenic phenotype to daughter cells.
This study indicates that inhibition of cell cycle progression by iNOS enhancer hypermethylation is capable of reducing pro-inflammatory responses via down-regulation of NF-κB with important therapeutic implications in OA.
研究诱导型一氧化氮合酶（iNOS）-5.8kb 核因子（NF）-κB 增强子区域的甲基化谱，并通过细胞周期调控在骨关节炎（OA）诱导中的后续作用。
通过焦磷酸测序确定甲基化百分比，qRT-PCR 检测基因表达，3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）测定细胞增殖。诱导瞬时转染以确定 NF-κB 增强子区域对细胞增殖的影响以及 DNA 甲基化的影响。
体外去甲基化 5-氮杂-2'-脱氧胞苷（5-aza-dC）显示 CpG 位点在-5.8kb 处的 DNA 甲基化水平降低，与 iNOS 表达水平升高相关。体外在-5.8kb 处 NF-κB 增强子区域的甲基化增加了 G0/G1 细胞周期阶段的细胞百分比。该区域内的甲基化丢失与增强的增殖和 G2/M 期细胞数量增加相关。OA 软骨细胞表现出 G0/G1 细胞周期进展标志物细胞周期蛋白 D1 和 CDK6 的上调，与对照细胞相反。我们证明了在 OA 软骨细胞中，iNOS 基因-5.8kb NF-κB 增强子区域内特定 CpG 位点发生的甲基化丢失允许该转录因子结合，激活 iNOS 的表达。这导致随后的细胞周期调控改变、增殖表型改变以及致病表型传递给子细胞。
本研究表明，iNOS 增强子过度甲基化抑制细胞周期进展能够通过下调 NF-κB 减少促炎反应，这在 OA 中具有重要的治疗意义。
