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Tissue fibrosis caused by a pathological activation of systemic sclerosis (SSc) fibroblasts is a major hallmark of SSc. Transforming growth factor beta (TGFβ)is a major mediator of fibrosis and has been identified as key-player in the pathogenesis of SSc. Alterations in DNA methylation and subsequent changes in gene transcription have been implicated in the pathogenesis of SSc. The aim of the present study was to investigate whether TGFβ induces DNA methylation.
The methylation status of suppressor of cytokine signalling 3 (socs-3) in fibroblasts was evaluated by methylation-specific PCR. The expression of socs-3 was analysed by real-time PCR and immunohistochemistry. The expression of socs-3 was specifically targeted by small interfering RNA (siRNA). Adenoviral overexpression was used to analyse TGFβ-induced hypermethylation in vivo. The anti-fibrotic potential of 5-aza for prevention and treatment of established fibrosis was analysed in the mouse model of bleomycin-induced skin fibrosis.
socs-3 potently regulated the release of collagen in fibroblasts. Knockdown of socs-3 by siRNA in healthy fibroblasts significantly increased mRNA and protein levels of collagen. The expression of socs-3 was strongly reduced in SSc fibroblasts and in the skin of SSc patients. The promoter of socs-3 was heavily hypermethylated in SSc fibroblasts. Incubation with 5-aza reactivated the expression of socs-3 in SSc fibroblasts, but had no effects in control fibroblasts. The hypermethylation of socs-3 in SSc might be mediated by TGFβ. TGFβ-induced hypermethylation of the promoter of socs-3 in healthy fibroblasts similar to SSc fibroblasts and strongly reduced the expression of socs-3. The increased DNA methylation was mediated by TGFβ-dependent induction of the DNA methyltransferase 3a. Coincubation with 5-aza prevented not only the inhibitory effects of TGFβ on the expression of socs-3, but also reduced the TGFβ-induced stimulation of collagen synthesis. Adenoviral overexpression of TGFβ in vivo induced DNA methyltransferase 3a with subsequent hypermethylation of socs-3. 5-aza prevented dermal fibrosis upon bleomycin challenge and induced regression of pre-established fibrosis with a decrease in dermal thickening of 44±3% below pre-treatment levels (p<0.05).
We demonstrate that TGFβ induces silencing of the anti-fibrotic gene socs-3 via DNA methyltransferase 3a-dependent promoter hypermethylation. Inhibition of the TGFβ-induced silencing of socs-3 by 5-aza reduced the stimulatory effects of TGFβ on the collagen synthesis in vitro and exerted potent anti-fibrotic effects in vivo. Thus, we identify inhibitors of DNA methyltransferases as novel anti-fibrotic drugs and provide a novel mechanism for the profibrotic effects of TGFβ.