DNA demethylation at specific CpG sites in the IL1B promoter in response to inflammatory cytokines in human articular chondrocytes.
DNA demethylation at specific CpG sites in the IL1B promoter in response to inflammatory cytokines in human articular chondrocytes.
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DOI：
10.1002/art.24882
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发表时间：
2009-11
影响因子：
--
通讯作者：
Roach, Helmtrud I.
中科院分区：
文献类型：
--
作者：
Hashimoto, Ko;Oreffo, Richard O. C.;Gibson, Marc B.;Goldring, Mary B.;Roach, Helmtrud I.
确定在用炎性细胞因子长期处理人关节软骨细胞后，编码白细胞介素-1 β（IL-1β）的基因启动子区的DNA甲基化状态的变化是否可解释IL-1B mRNA的表达。将IL-1β或TNFα与制瘤素M（OSM）或5-氮杂-脱氧胞苷（5-aza-dC）组合，每周两次加入正常人关节软骨细胞的原代培养物中，持续4-5周，这些细胞得自股骨颈骨折患者。通过基于SybrGreen的qRT-PCR对从培养前后的相同样品提取的基因组DNA和总RNA测定MMP 13、IL 1B TNFA和DNMT 1的表达。亚硫酸氢盐修饰用于鉴定IL 1B启动子中的哪些CpG位点在表达和非表达细胞之间显示差异甲基化。在关键CpG位点（-299bp）甲基化的细胞百分比通过依赖于甲基化敏感性限制性内切酶和实时PCR的方法进行定量。采用酶联免疫吸附试验（ELISA）检测培养液中IL-1β的分泌。健康软骨细胞不表达IL 1B mRNA，但5-aza-dC使其水平增加5倍，TNFα/OSM使其水平增加100- 1000倍。5-aza-dC可降低CpG甲基化水平，IL-1β可显著降低CpG甲基化水平，TNFα/OSM可完全消除CpG甲基化。在细胞因子处理的软骨细胞中，mRNA被翻译成蛋白质。这些新的发现表明，炎性细胞因子可以改变关键CpG位点的DNA甲基化状态，导致人关节软骨细胞中IL 1B的长期诱导。
To determine whether changes in the DNA methylation status in the promoter region of the gene encoding interleukin-1β (IL-1β) account for expression of IL1B mRNA after long-term treatment of human articular chondrocytes with inflammatory cytokines. IL-1β or TNFα combined with oncostatin M (OSM), or 5-aza-deoxycytidine (5-aza- dC) were added twice weekly for 4–5 weeks to primary cultures of normal human articular chondrocytes, obtained from patients with a femoral neck fracture. Expression of MMP13, IL1B TNFA and DNMT1 were determined by SybrGreen-based qRT-PCR on genomic DNA and total RNA extracted from the same sample before and after culture. Bisulfite modification was used to identify which CpG sites in the IL1B promoter showed differential methylation between expressing and non-expressing cells. The percentages of cells that were methylated at that critical CpG site (−299bp) were quantified by a method that depended on methylation-sensitive restriction enzymes and real-time PCR. Secretion of IL-1β was assessed by enzyme-linked immunoabsorbent assay (ELISA) of the culture media. Healthy chondrocytes did not express IL1B mRNA, but the levels were increased 5-fold by 5-aza-dC and 100- to 1000-fold by or TNFα/OSM. The % CpG methylation was decreased by 5-aza-dC, but reduced considerably more by IL-1β and almost abolished by TNFα/OSM. The mRNA was translated into protein in cytokine-treated chondrocytes. These novel findings indicate that inflammatory cytokines can change DNA methylation status at key CpG sites, resulting in long-term induction of IL1B in human articular chondrocytes.
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