Role of Phosporylated SOX9 in Systemic Sclerosis Pathogenesis

磷酸化 SOX9 在系统性硬化症发病机制中的作用

• 批准号: 9452015
• 负责人: Sonsoles Piera-velazquez
• 金额: $ 17.16万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-15 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9452015
• 关键词: Affect; Autoimmune Diseases; CRISPR/Cas technology; Cells; Chondrocytes; Cicatrix; Collagen; Complex; Dermal; Development; Disease; Extracellular Matrix; Fibroblasts; Fibronectins; Fibrosis; Generations; Genes; Goals; Human; In Vitro; Inflammatory; Keloid; Laboratories; Liver Fibrosis; Mediating; Mesenchymal; Molecular; Molecular Target; Mus; Myofibroblast; Names; Organ; Pathogenesis; Pathway interactions; Phenotype; Phosphorylation; Phosphorylation Inhibition; Phosphorylation Site; Phosphotransferases; Population; Process; Production; Research Project Grants; Role; Skin; Smooth Muscle Actin Staining Method; Systemic Scleroderma; Systemic Therapy; TGF Beta Signaling Pathway; Tissues; Transforming Growth Factor beta; Treatment Efficacy; Vascular Diseases; effective therapy; in vivo; mortality; novel; skin fibrosis; systemic autoimmune disease; transcription factor

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.

系统性硬化症（SSc）是一种全身性炎症性自身免疫性疾病， 皮肤和多个内脏器官的进行性纤维化， 微脉管系统SSc在全身性疾病中具有最高的病例特异性死亡率。 自身免疫性疾病，并且目前，对于SSc没有有效的疾病修饰疗法。 因此，迫切需要开发有效的治疗方法来治疗糖尿病。 疾病 虽然进行性纤维增生的详细机制 最近的研究还没有完全阐明SSc的过程，包括我们的一些研究。 实验室，已经确定了新的分子途径，可能参与这一复杂的 过程 最近的研究表明，软骨细胞特异性转录因子SOX 9， 出乎意料地涉及各种纤维化疾病，包括瘢痕疙瘩和肝纤维化。在我们 初步结果我们发现正常人皮肤成纤维细胞组成性表达 磷酸化Ser181 SOX 9（pSOX 9），pSOX 9水平在SSc成纤维细胞中增加， 并且TGF-β引起pSOX 9水平的有效刺激。 因此，本申请的总体目标是探索pSOX 9在细胞凋亡中的作用。 产生活化的肌成纤维细胞，这些细胞最终负责严重的 纤维增生过程中的SSc，并检查所涉及的分子机制。的 本提案的总体目标是：证明pSOX 9的促纤维化作用 并鉴定参与纤维化过程的pSOX 9的分子靶点。的 将实现以下具体目标： 具体目的1：研究特异性抑制SOX 9磷酸化对细胞凋亡的影响。 体外SSc真皮成纤维细胞的活化促纤维化表型。 具体目的2：鉴定表现出表达变化的促纤维化基因 由正常人皮肤成纤维细胞中SOX 9的诱导磷酸化引起 使用腺病毒和慢病毒转导SOX 9特异性激酶。 具体目的3：采用免疫组织化学方法确认和验证pSOX 9在体内的促纤维化作用。 缺乏S181磷酸化位点的CRISPR-Cas9基因修饰小鼠的新品系 在SOX 9中，在它们与表现出严重纤维化表型的小鼠杂交后， 通过组成性激活的TGF-β信号通路。