Background/Purpose: Prolonged endogenous activation of fibroblasts is a major hallmark of fibrosing disorders such as Systemic Sclerosis (SSc). It results in tissue fibrosis, organ failure and a high morbidity and mortality. Trimethylation of H3 at lysine residue K27 (H3K27me3) is an epigenetic mark that mediates gene repression. H3K27me3 was recently identified as an important negative regulator of fibroblast activation [1]. Demethylation of H3K27me3 is mediated by JMJD3. JMJD3 inhibitors are currently being tested as therapeutic strategies in malignant diseases. The aim of this study is to characterize the role of JMJD3 in fibroblast activation and to explore JMJD3 as a potential drug target in SSc.

Methods:   The expression of JMJD3 was analyzed by qPCR, IF and Western blot in patients with SSc, healthy controls and in experimental fibrosis. siRNA mediated knockdown and pharmacologic inhibition by GSKJ4 were used to target JMJD3. In vivo, the effects of JMJD3-inhibiton were analyzed in the mouse model of bleomycin-induced dermal fibrosis, in the model of Topoisomerase-I-induced (TopoI) fibrosis and in a mouse model with adenoviral overexpression of a constitutively active transforming growth factor beta (TGF-β) receptor type 1 (TBR^act). Signaling pathways regulated by JMJD3 were analyzed in vitro and in vivo using qPCR, Western Blot and reporter assays.

Results: The expression of JMJD3 was increased in the skin of SSc patients as compared to healthy volunteers. Accumulation of JMJD3 was also observed in experimental models of SSc. TGF-β potently induced the expression of JMJD3 in vitro and in vivo. Pharmacologic inhibition and siRNA mediated knockdown of JMJD3 strongly upregulated the levels of H3K27me3 in vitro and in vivo. Inhibition of JMJD3 reverted the activated fibroblast phenotype in SSc fibroblasts and persistently decreased the expression of contractile fibers and of α-smooth muscle actin. Secretion of collagen was reduced upon inhibition of JMJD3. JMJD3- inhibition also resulted in a significant down-regulation of the profibrotic transcription factor Fra-2 in vitro and in vivo. Overexpression of Fra-2 in JMJD3-knockdown fibroblasts restored the profibrotic effect of JMJD3. In vivo, inhibition of JMJD3 ameliorated fibrosis in bleomycin-, TopoI- and TBR^act-induced experimental fibrosiswith reduced dermal thickening, hydroxyproline content and myofibroblast differentiation. 

Conclusion: We present first evidence that histone methylation by JMJD3 contributes to the activated phenotype of SSc fibroblasts. JMJD3 is upregulated by TGF-β in SSc. Inhibition of JMJD3 prevents the aberrant collagen release and ameliorates the activated fibroblast phenotype in SSc fibroblasts. In vivo inhibition of JMJD3 has potent anti-fibrotic effects in several preclinical models of SSc. Reference: 1. Kramer, M., et al., Inhibition of H3K27 histone trimethylation activates fibroblasts and induces fibrosis. Ann Rheum Dis, 2013. 72(4): p. 614-20.  

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/jumonji-domain-containing-protein-3-jmjd3-as-a-novel-epigenetic-mechanism-of-fibroblast-activation-by-regulation-of-fra-2/