Checkpoint Function of PTPalpha in Pathological Fibrogenesis in the Lung

项目摘要/摘要 纤维化，即纤维结缔组织的形成，在发育和伤口愈合中是必不可少的。 然而，如果不加限制，病理性纤维化会导致肺和其他组织的进行性纤维化。 导致器官衰竭的器官。弥漫性进行性肺纤维化是特发性肺纤维化的特征 纤维化(IPF)，一种无情地进展并最终致命的疾病。虽然最近的试验表明 吡非尼酮和9tedanib可以减缓肺功能下降的速度，这是一种基于机制的新疗法 这不仅需要减缓纤维化的进展，而且迫切需要解决已建立的纤维化。我们有 发现蛋白酪氨酸磷酸酶-α(PtPα)促进转化生长因子β依赖的肝纤维化反应 肺成纤维细胞，代表了纤维化形成途径中的关键检查点。该项目将解决 PtPα通过间接控制酪氨酸磷酸化状态促进肺纤维化的假说 转化生长因子β受体(T-βR)II胞浆尾部残基增强Smad依赖的肝纤维化信号 成纤维细胞。我们的实验构建是ptpα放大并延长来自TβRs和 细胞外基质在基于整合素的局灶性粘连中的作用，从而促进胶原和纤维连接蛋白的产生 导致组织纤维化。使用药理、生化(质谱仪， 免疫沉淀、重组蛋白的体外分析和磷酸蛋白质组分析)，以及 分子(siRNA基因沉默，RNA Seq)在培养的人和小鼠成纤维细胞中的方法，我们将 确定pTPα如何被募集到转化生长因子β受体复合体，并调节酪氨酸磷酸化。 Tβ受体及其相关分子通过src酪氨酸激酶间接进入受体复合体。到时候我们会的 确定ptpα对下游Smad依赖的促纤维化基因表达的影响，包括 胶原蛋白、纤维连接蛋白、α-SMA和miR-29。我们将评估ptpα在肌成纤维细胞中的重要性 分化、增殖和凋亡。然后我们将确定pTPα是如何被募集到焦点粘连上的 并将来自机械僵硬的“纤维化”细胞外基质的信号与通过Src、TβRs传递的信号相结合，以及 FAK最终导致纤维化反应。这些研究将使用分子和基于成像的方法。 在细胞外基质包裹的聚丙烯酰胺水凝胶上培养的成纤维细胞中表达荧光融合蛋白 不同的硬度。然后，我们将在肺纤维化的临床前动物模型中测试我们的假设。我们会 用Cre法检测成纤维细胞特异性α基因缺失对PTPREF/f小鼠的影响 成纤维细胞特异性启动子(DERMO1、COL1a1和COL1a2)在三种肺纤维化模型中的作用：(I) 重组转化生长因子-β的腺病毒表达；(Ii)单剂和(Iii)气管内注射博莱霉素。 在这些模型中，将使用基因靶向的小鼠和药理学方法来评估Src激酶的作用 抑制剂。最终，这些知识将被用于开发小分子或生物学方法。 选择性地靶向这些促纤维化途径来治疗人类的肺纤维化。

PROJECT SUMMARY/ABSTRACT Fibrogenesis, the formation of fibrous connective tissue, is essential in development and wound healing. When unrestrained, however, pathologic fibrogenesis contributes to progressive fibrosis of the lungs and other organs leading to organ failure. Diffuse, progressive fibrosis of the lungs is a hallmark of idiopathic pulmonary fibrosis (IPF), a condition that is relentlessly progressive and ultimately fatal. While recent trials have shown that pirfenidone and nintedanib can slow the rate of decline in lung function, novel mechanism-based therapies that not only slow the progression of fibrosis but resolve established fibrosis are urgently needed. We have discovered that protein tyrosine phosphatase-α (PTPα) promotes TGFβ-dependent fibrogenic responses in lung fibroblasts, representing a key checkpoint in the fibrogenic pathway. This project will address the hypothesis that PTPα promotes fibrosis in the lung by indirectly controlling the phosphorylation state of tyrosine residues in the cytoplasmic tail of TGFβ receptor (TβR)II thus enhancing Smad-dependent fibrogenic signals in fibroblasts. Our experimental construct is that PTPα amplifies and prolongs fibrogenic signals from TβRs and the ECM in the context of integrin-based focal adhesions thus enhancing production of collagen and fibronectin leading to tissue fibrosis. Using a combination of pharmacological, biochemical (mass spec, immunoprecipitation, in vitro analysis of recombinant proteins, and phospho-proteomic analysis), and molecular (siRNA gene silencing, RNA Seq) approaches in cultured human and murine fibroblasts, we will determine how PTPα is recruited to the TGFβ receptor complex and regulates tyrosine phosphorylation of TβRs and associated molecules in the receptor complex indirectly through Src tyrosine kinases. We will then ascertain the effects of PTPα on downstream Smad-dependent expression of profibrotic genes including collagen, fibronectin, α-SMA, and miR-29. We will assess the importance of PTPα in myofibroblast differentiation, proliferation, and apoptosis. We will then determine how PTPα is recruited to focal adhesions and integrates signals from a mechanically stiff `fibrotic' ECM with signals transduced through Src, TβRs, and FAK culminating in fibrogenic responses. These studies will employ molecular and imaging-based approaches with expression of fluorescent fusion proteins in fibroblasts grown on ECM-coated polyacrylamide hydrogels of varying stiffness. We will then test our hypothesis in preclinical animal models of pulmonary fibrosis. We will determine the effect of fibroblast-specific genetic deletion of PTPα in our Ptpraf/f mice using Cre driven by fibroblast-specific promoters (DERMO1, Col1a1, and Col1a2) in three models of pulmonary fibrosis: (i) adenoviral expression of recombinant TGF-β; (ii) single dose and (iii) multiple dose intratracheal bleomycin. The role of Src kinases will be assessed in these models using gene-targeted mice and pharmacological inhibitors. Ultimately, this knowledge will be used to develop small molecule or biological approaches selectively targeting these profibrotic pathways to treat pulmonary fibrosis in humans.