Department of Internal Medicine III and Institute for Clinical Immunology, University of Erlangen-Nuremberg, , Erlangen, Germany.
Ann Rheum Dis. 2014 Jun;73(6):1232-9. doi: 10.1136/annrheumdis-2012-203194. Epub 2013 May 22.
Activated Wnt signalling with decreased expression of endogenous inhibitors has recently been characterised as a central pathomechanism in systemic sclerosis (SSc). Aberrant epigenetic modifications also contribute to the persistent activation of SSc fibroblasts. We investigated whether increased Wnt signalling and epigenetic changes in SSc are causally linked via promoter hypermethylation-induced silencing of Wnt antagonists.
The methylation status of endogenous Wnt antagonists in leucocytes and fibroblasts was evaluated by methylation-specific PCR. 5-aza-2'-deoxycytidine was used to inhibit DNA methyltransferases (Dnmts) in cultured fibroblasts and in the mouse model of bleomycin-induced skin fibrosis. Activation of Wnt signalling was assessed by analysing Axin2 mRNA levels and by staining for β-catenin.
The promoters of DKK1 and SFRP1 were hypermethylated in fibroblasts and peripheral blood mononuclear cells of patients with SSc. Promoter hypermethylation resulted in impaired transcription and decreased expression of DKK1 and SFRP1 in SSc. Treatment of SSc fibroblasts or bleomycin-challenged mice with 5-aza prevented promoter methylation-induced silencing and increased the expression of both genes to normal levels. Reactivation of DKK1 and SFRP1 transcription by 5-aza inhibited canonical Wnt signalling in vitro and in vivo and effectively ameliorated experimental fibrosis.
We demonstrate that hypermethylation of the promoters of DKK1 and SFRP1 contributes to aberrant Wnt signalling in SSc and that Dnmt inhibition effectively reduces Wnt signalling. These data provide a novel link between epigenetic alterations and increased Wnt signalling in SSc and also have translational implications because Dnmt inhibitors are already approved for clinical use.
最近，下调内源性抑制剂的激活 Wnt 信号被认为是全身性硬皮病（SSc）的中心发病机制。异常的表观遗传修饰也有助于 SSc 成纤维细胞的持续激活。我们研究了 SSc 中增加的 Wnt 信号和表观遗传变化是否通过启动子超甲基化诱导 Wnt 拮抗剂沉默而具有因果关系。
通过甲基化特异性 PCR 评估白细胞和成纤维细胞中内源性 Wnt 拮抗剂的甲基化状态。5-氮杂-2'-脱氧胞苷用于抑制培养的成纤维细胞中和博来霉素诱导的皮肤纤维化小鼠模型中的 DNA 甲基转移酶（Dnmts）。通过分析 Axin2 mRNA 水平和β-连环蛋白染色来评估 Wnt 信号的激活。
SSc 患者的成纤维细胞和外周血单个核细胞中 DKK1 和 SFRP1 的启动子发生超甲基化。启动子超甲基化导致 SSc 中 DKK1 和 SFRP1 的转录受损和表达减少。用 5-氮杂处理 SSc 成纤维细胞或博来霉素处理的小鼠可防止启动子甲基化诱导的沉默，并将这两个基因的表达增加到正常水平。5-氮杂重新激活 DKK1 和 SFRP1 转录可抑制体外和体内的经典 Wnt 信号，并有效改善实验性纤维化。
我们证明 DKK1 和 SFRP1 启动子的超甲基化导致 SSc 中异常的 Wnt 信号，并且 Dnmt 抑制有效降低了 Wnt 信号。这些数据为 SSc 中表观遗传改变与增加的 Wnt 信号之间提供了新的联系，并且因为 Dnmt 抑制剂已经被批准用于临床用途，所以具有转化意义。
