The role of SOCS3 in the pathogenesis of fibrotic disorders

该研究的目的是表征SOCS蛋白，特别是SOCS3在纤维化条件下的作用，并评估SOCS3的表观遗传抑制是否有助于JAK-STAT信号级联的增强激活，而JAK-STAT信号级联先前已被证明有助于系统性硬化症（SSc）的发病机制。我们的初步结果显示，在SSc患者的皮肤和培养成纤维细胞中，SOCS3的表达明显下降，这可以通过刺激正常成纤维细胞，使TGFβ水平持续升高来模拟。此外，体外通过siRNA或体内通过特异性敲除成纤维细胞来敲除SOCS3诱导成纤维细胞向肌成纤维细胞分化、胶原合成和组织纤维化。为了证实SOCS3对成纤维细胞活化和组织纤维化的抑制作用，我们将在体外进行强制过表达SOCS3和在体内不同小鼠模型中进行成纤维细胞特异性敲除的实验。随后，我们想要表征SOCS3调节成纤维细胞激活的信号级联。为此，我们计划共敲低SOCS3和不同Janus激酶（JAKs）和STAT蛋白，过表达突变激酶并进行共免疫沉淀（CoIP）测定。在我们的初步结果中，我们也可以证明在SSc成纤维细胞和TGFβ刺激的正常成纤维细胞中，SOCS3启动子的甲基化增加。由于DNA甲基化与抑制组蛋白标记一起作用以沉默基因的转录，我们进一步旨在更详细地表征纤维化中SOCS3抑制的调控表观遗传机制。因此，我们将通过染色质免疫沉淀（ChIP）、潜在相关酶和蛋白的表达分析、CoIP分析以及sirna介导的敲低和强制过表达来分析DNA甲基化和组蛋白修饰的相互作用。最后，我们将分析靶向DNA甲基化在不同实验性纤维化模型中的治疗潜力。首先，我们计划评估成纤维细胞特异性敲除Dnmt3a在博来霉素和tgf β riact诱导的纤维化中的结果。此外，我们将在紧皮1 （Tsk1）小鼠模型和慢性移植物抗宿主病（cGvHD）小鼠模型中评估5-aza-2 ' -脱氧胞苷（5-aza，地西他宾）对DNA甲基转移的药理学抑制作用。

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).