Aberrant activation of fibroblasts underlies the pathogenesis of fibrotic diseases such systemic sclerosis (SSc). New findings published in The Journal of Clinical Investigation support the contribution of epigenetic imprinting for maintaining the persistently activated pathological phenotype of fibroblasts in SSc.

Credit: Springer Nature Limited

“In this study, we link two core mechanisms of fibrosis: epigenetics and TGFβ–STAT3 signalling,” reports corresponding author Jörg Distler. Suppressor of cytokine signalling 3 (SOCS3) is a negative regulator of TGFβ–STAT3 signalling that is normally induced as part of a negative-feedback response. “We demonstrate that persistent activation of TGFβ signalling induces the expression of DNA methyltransferases DNMT3A and DNMT1 to silence the expression of SOCS3,” explains Distler. “This mechanism further amplifies the stimulatory effects of TGFβ on STAT3 signalling and amplifies TGFβ-induced fibroblast activation and tissue fibrosis.”

Indeed, the expression of SOCS3 was downregulated in the skin and in skin fibroblasts of patients with SSc compared with that of healthy individuals, and expression was also downregulated in the skin of mice with bleomycin-induced fibrosis or fibrosis caused by constitutive activation of TGFβ receptor type I (TBR1act mice). Treatment of fibroblasts from healthy individuals with the DNMT inhibitor 5-azacitidine (5-aza) prevented TGFβ-mediated repression of SOCS3 expression following prolonged stimulation in vitro.

Methylation-specific PCR and methylated DNA immunoprecipitation analysis suggested that TGFβ signalling induces the expression of DNMT3A and DNMT1 via the canonical TGFβ–SMAD pathway to promote hypermethylation of the SOCS3 promoter and silence SOCS3 expression.

Manipulating the expression of SOCS3 in fibroblasts in vitro using siRNA-mediated knockdown, incubation with 5-aza or forced overexpression of SOCS3 showed that SOCS3 limits TGFβ-dependent fibroblast activation (as measured by TGFβ-mediated expression of COL1A1 and COL12A2 mRNA, expression of α-smooth muscle actin, release of collagen protein and formation of stress fibres).

Notably, in vitro and in vivo findings confirmed that SOCS3 inhibits JAK2–STAT3 signalling, and that 5-aza can restore SOCS3-mediated regulation of JAK–STAT signalling.

Fibroblast-specific deletion of SOCS3 in mice exacerbated bleomycin-induced or TBR1act-induced skin fibrosis. By contrast, treatment with 5-aza or fibroblast-specific deletion of DNMT3A ameliorated fibrosis in both models. Notably, 5-aza treatment not only prevented fibrosis, but also induced regression of pre-established bleomycin-induced skin fibrosis.

“We are currently evaluating options to transfer these findings from bench to bedside and are discussing options to run a clinical trial with DNMT inhibitors such as 5-aza,” says Distler.