Checkpoint Function of PTPalpha in Pathological Fibrogenesis in the Lung

项目概要/摘要 纤维化，即纤维结缔组织的形成，在发育和伤口愈合中至关重要。 然而，当不受限制时，病理性纤维化导致肺和其他组织的进行性纤维化。 导致器官衰竭。肺的弥漫性、进行性纤维化是特发性肺纤维化的标志。 纤维化（IPF）是一种持续进行并最终致命的病症。虽然最近的试验表明 吡非尼酮和尼达尼布可以减缓肺功能下降的速度， 迫切需要不仅减缓纤维化的进展而且解决已建立的纤维化。我们有 发现蛋白酪氨酸磷酸酶-α（PTP α）促进TGF β依赖性纤维化反应， 肺成纤维细胞，代表纤维化途径中的关键检查点。该项目将解决 假设PTP α通过间接控制酪氨酸磷酸化状态促进肺纤维化 TGF β受体（T β R）II的胞质尾区残基，从而增强Smad依赖性纤维化信号， 成纤维细胞我们的实验构建是PTP α放大和抑制来自T β Rs的纤维化信号， ECM在基于整合素的局灶性粘连的背景下，从而增强胶原蛋白和纤连蛋白的产生 导致组织纤维化。使用药理学、生物化学（质谱， 免疫沉淀、重组蛋白的体外分析和磷酸化蛋白质组学分析），和 分子（siRNA基因沉默，RNA Seq）的方法在培养的人类和小鼠成纤维细胞，我们将 确定PTP α如何被募集到TGF β受体复合物中并调节酪氨酸磷酸化， 受体复合物中的T β R和相关分子间接通过Src酪氨酸激酶。然后我们将 确定PTP α对促纤维化基因下游Smad依赖性表达的影响，包括 胶原、纤连蛋白、α-SMA和miR-29。我们将评估PTP α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PTP α是如何被募集到粘连灶的 并将来自机械僵硬的"纤维化" ECM的信号与通过Src、T β Rs和 FAK最终导致纤维化反应。这些研究将采用基于分子和成像的方法 在ECM包被的聚丙烯酰胺水凝胶上生长的成纤维细胞中表达荧光融合蛋白， 改变刚度。然后，我们将在肺纤维化的临床前动物模型中检验我们的假设。我们将 使用Cre驱动的Ptpraf/f小鼠中成纤维细胞特异性PTP α基因缺失的影响， 在三种肺纤维化模型中的成纤维细胞特异性启动子（DERMO1、Col1a1和Col1a2）：（i） 重组TGF-β的腺病毒表达;（ii）单剂量和（iii）多剂量经皮内博莱霉素。 Src激酶的作用将在这些模型中使用基因靶向小鼠和药理学方法进行评估。 抑制剂的最终，这些知识将用于开发小分子或生物方法 选择性靶向这些促纤维化途径以治疗人类肺纤维化。

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.