Background/Purpose:  Tissue fibrosis caused by pathological activation of fibroblasts is a major hallmark of systemic sclerosis (SSc). Although fibroblast activation is initially driven by external factors, prolonged activation can render fibroblasts independent of external stimuli. Accumulating evidence suggests that epigenetic alterations play a central role to establish the persistently activated phenotype of fibroblasts. In the present study, we tested the hypothesis that epigenetic silencing of SOCS expression may contribute to the aberrant activation of JAK2 / STAT3 signaling in SSc, and that re-establishment of the endogenous, SOCS-dependent control of JAK / STAT signaling may prevent aberrant fibroblast activation and ameliorate tissue fibrosis.

Methods:  DNA methylation was evaluated by methylation-specific PCR and MeDIP assays. 5-aza-2’-deoxycytidine (5-aza) was used to inhibit DNA methyltransferases (DNMTs). Knockdown analyses were done by nucleofection of siRNA in vitro and by fibroblast-specific knockout mice in vivo.

Results:  SOCS3 was strongly downregulated in skin of SSc patients compared to healthy individuals. Chronically increased levels of TGFβ induced an SSc-like phenotype and reduced the levels of SOCS3 in normal fibroblasts to a level comparable with SSc fibroblasts. These findings suggested that epigenetic mechanisms may account for the reduced expression of SOCS3. Indeed, methylation analyses demonstrated a prominent promoter hypermethylation of SOCS3 in SSc fibroblasts and in normal fibroblasts exposed to persistently high levels of TGFβ. Mechanistically, chronically increased levels of TGFβ promoter hypermethylation of the SOCS3 promoter by induction of DNMT3A. Pharmacological inhibition of DNMTs or selective knockdown of DNMT3A restored the normal expression of SOCS3 and reduced fibroblast activation and collagen release. Knockdown of SOCS3 in normal dermal fibroblasts induced an SSc-like phenotype with increased activation of JAK2-STAT3 signaling, enhanced expression of myofibroblast markers and increased collagen release. Fibroblast-specific knockout of SOCS3 promoted JAK2-STAT3 signaling and aggravated tissue fibrosis in bleomycin- and TGFβRI^act-induced dermal fibrosis. Vice versa, forced overexpression of SOCS3 inhibited TGFβ-induced JAK2-STAT3 signaling and reduced TGFβ-mediated fibroblast activation. Overepxression of SOCS3 also ameliorated the endogenous activation of SSc fibroblasts. Moreover, treatment with DNMT inhibitors or fibroblast-specific knockdown of DNMT3A re-activated the expression of SOCS3, reduced JAK2-STAT3 signaling and exerted potent antifibrotic effects in bleomycin- and TGFβRI^act-induced dermal fibrosis.

Conclusion:  We demonstrate that chronic activation of TGFβ signaling perturbs the epigenetic control of STAT signaling by DNMT3A-induced silencing of SOCS3 expression in SSc. Re-establishment of the endogenous regulation of STAT signaling, either by forced expression of SOCS3 or by pharmacologic inhibition of DNMTs, prevents aberrant STAT3 signaling, inhibits TGFβ-induced fibroblast activation and collagen release and ameliorates experimental fibrosis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/tgf-%ce%b2-induced-epigenetic-silencing-of-socs3-facilitates-stat3-signaling-to-promote-fibroblast-activation-and-tissue-fibrosis-in-systemic-sclerosis/