The role of SOCS3 in the pathogenesis of fibrotic disorders

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The aim of the proposed study is to characterize the role of SOCS proteins, in particular of SOCS3, in fibrotic conditions and to evaluate whether epigenetic repression of SOCS3 contributes to the enhanced activation of JAK-STAT signaling cascades, which have been shown previously to contribute to the pathogenesis of systemic sclerosis (SSc).Our preliminary results show a strong decrease in SOCS3 expression in skin as well as in cultured fibroblasts of SSc patients, which could be mimicked by stimulation of normal fibroblasts with persistently increased levels of TGFβ. In addition, knockdown of SOCS3 either in vitro by siRNA or in vivo by fibroblast-specific knockout induced fibroblast-to-myofibroblast-differentiation, collagen synthesis and tissue fibrosis. To confirm the inhibitory role of SOCS3 on fibroblast activation and tissue fibrosis, we will perform experiments with forced overexpression of SOCS3 in vitro and with fibroblast-specific knockout in different mouse models in vivo. Subsequently, we want to characterize the signaling cascade by which SOCS3 regulates fibroblast activation. To this end, we plan to co-knockdown SOCS3 and the different Janus kinases (JAKs) and STAT proteins, to overexpress mutated kinases and to perform co-immunoprecipitation (CoIP) assays.In our preliminary results, we could also demonstrate an increased methylation of the SOCS3 promoter both in SSc fibroblasts and in normal fibroblasts stimulated with TGFβ. As DNA methylation acts in conjunction with repressing histone marks to silence the transcription of genes, we further aim to characterize the regulatory epigenetic mechanisms underlying the repression of SOCS3 in fibrosis in more detail. Therefore, we will analyze the interaction of DNA methylation and histone modifications by chromatin immunoprecipitation (ChIP), expression analyses of the potentially involved enzymes and proteins, CoIP assays as well as siRNA-mediated knockdown and forced overexpression.Finally, we will analyze the therapeutic potential of targeting DNA methylation in different models of experimental fibrosis. At first, we plan to evaluate the outcome of fibroblast-specific knockout of Dnmt3a in bleomycin- and TGFβRIact-induced fibrosis. In addition, we will evaluate pharmacologic inhibition of DNA methyltranferses by 5-aza-2’-deoxycytidine (5-aza, Decitabine) in the mouse model of tight-skin 1 (Tsk1) mice and in chronic graft-versus-host-disease (cGvHD).