Role of Phosporylated SOX9 in Systemic Sclerosis Pathogenesis

系统性硬化症(SSC)是一种全身性炎症性自身免疫性疾病 由于皮肤和多个内脏的进行性纤维化和严重的 微血管系统。在系统性肺炎中，SSc的特定病例死亡率最高 自身免疫性疾病，目前，还没有有效的疾病修正治疗的SSc。 因此，迫切需要开发有效的治疗方法来治疗这种疾病 疾病。 尽管导致进行性纤维增生性疾病的详细机制 SSC中的过程尚未完全阐明最近的研究，包括我们的一些 实验室已经确定了可能参与这种复合体的新的分子途径 进程。 最近的研究表明，软骨细胞特异性转录因子SOX9是 出人意料地参与了包括瘢痕疙瘩和肝纤维化在内的各种纤维化疾病。在我们的 初步结果我们发现正常人皮肤成纤维细胞结构性表达 磷酸化的Ser181 SOX9(PSOX9)，即在SSC成纤维细胞中pSOX9的水平增加， 转化生长因子-β对pSOX9水平有明显的刺激作用。 因此，本应用程序的总体目标是探索pSOX9在 激活的肌成纤维细胞的产生，这些细胞最终要对严重的 SSC的成纤维细胞增殖过程，并探讨其分子机制。这个 这项提案的首要目标是：证明pSOX9的促纤维化作用 并确定参与纤维化过程的pSOX9的分子靶点。这个 将实现以下具体目标： 特异性目的1：研究特异性抑制SOX9磷酸化对细胞周期的影响 体外激活的SSc真皮成纤维细胞促纤维化表型。 特定目标2：鉴定表达发生变化的促纤维化基因 诱导人真皮成纤维细胞SOX9磷酸化所致 使用腺病毒和慢病毒转导SOX9特异性激酶。 特定目标3：在活体内证实和验证pSOX9的促纤维化作用 缺失S181磷酸化位点的新型CRISPR-Cas9转基因小鼠 在SOX9中，它们与小鼠杂交后表现出严重的纤维化表型 通过结构性激活转化生长因子-β信号通路。

Systemic Sclerosis (SSc) is a systemic inflammatory autoimmune disease characterized by progressive fibrosis of skin and multiple internal organs and severe alterations in the microvasculature. SSc has the highest case-specific mortality among the systemic autoimmune diseases and currently, there is no effective disease-modifying therapy for SSc. Therefore, there is an urgent unmet need to develop effective therapeutic approaches for the disease. Although the detailed mechanisms responsible for the progressive fibroproliferative process in SSc have not been fully elucidated recent studies, including some from our laboratories, have identified novel molecular pathways that may participate in this complex process. Recent studies have shown that SOX9, a chondrocyte-specific transcription factor, is unexpectedly involved in various fibrotic diseases including keloids and liver fibrosis. In our Preliminary Results we found that normal human dermal fibroblasts constitutively express phoshorylated Ser181 SOX9 (pSOX9), that pSOX9 levels are increased in SSc fibroblasts, and that TGF-β causes a potent stimulation of pSOX9 levels. Therefore, the overall goal of this application is to explore the role of pSOX9 in the generation of activated myofibroblasts, the cells ultimately responsible for the severe fibroproliferative process in SSc, and to examine the molecular mechanisms involved. The overarching objective of this proposal will be to: Demonstrate the profibrotic role of pSOX9 and to identify the molecular targets of pSOX9 involved in the fibrotic process. The following Specific Aims will be pursued: Specific Aim 1: Study the effects of specific inhibition of phosphorylation of SOX9 on the activated profibrotic phenotype of SSc dermal fibroblasts in vitro. Specific Aim 2: Identification of profibrotic genes displaying changes in their expression caused by the induced phosphorylation of SOX9 in normal human dermal fibroblasts employing adenoviral and lentiviral transduction of SOX9-specific kinases. Specific Aim 3: Confirm and validate the profibrotic effects of pSOX9 in vivo employing a novel strain of CRISPR-Cas9 genetically modified mice that lack the S181 phosphorylation site in SOX9 following their intercrossing with mice displaying a severe fibrotic phenotype induced by constitutively activated TGF-β signaling pathway.