Profibrotic Mechanisms of the TRPV4-PI3K-gamma Protein Complex

摘要 特发性肺纤维化（IPF）是一种致命的疾病，迫切需要药物治疗。为了 为了成功地改善肺纤维化，更好地理解纤维化发病机制是至关重要的。 肌成纤维细胞是关键的纤维化效应细胞。人们早就知道， 周围组织/基质的机械性质，沿着活性TGF-β，是 肌成纤维细胞分化尽管整合素和其他受体参与细胞-基质相互作用， 驱动肌成纤维细胞分化的特异性机械传感器仍然是难以捉摸的。我们已经鉴定出TRPV 4 作为驱动肺中肌成纤维细胞分化和纤维化的关键机械传感器。TRPV 4是一种 瞬时受体电位中牵张激活的质膜阳离子通道，香草素家族 （TRPV4）。此外，TRPV 4在基质硬度水平驱动肌成纤维细胞分化和纤维化， 与肺部疾病直接生物学和临床相关。我们已经证明了TRPV 4的重要性 与肌成纤维细胞分化和实验性肺纤维化和人IPF的关系。我们的小说出版了， 初步数据进一步表明，TRPV 4的促纤维化作用取决于其与纤维化的生物化学联系。 细胞溶质PI 3 K γ通过PI 3 K γ独特的氨基末端非催化结构域，随后是 蛋白质复合物的质膜。基于这些数据，我们提出了一个新的假设， TGFβ通过诱导TRPV 4-PI 3 K γ促进肌成纤维细胞分化和肺纤维化 蛋白质复合物转移到质膜。三个协调一致的具体目标， 损失函数实验设计将确定关键的TRPV 4-PI 3 K γ途径相互作用， 肌成纤维细胞分化和纤维化。他们将利用计算机模拟和体外功能设计的增益和损失 以及在结构水平、细胞水平和已建立的实验鼠水平上的体内系统 模型，以及人类疾病。AIM 1将精确地定义TRPV 4和TRPV 5之间的氨基酸相互作用位点。 而PI 3 K γ、AIM 2则决定了TGFβ诱导TRPV 4-PI 3 K γ转位的机制 复合物的PM，在那里他们的行为，以驱动肌成纤维细胞分化和AIM 3将测试的概念， PI 3 K γ的氨基端结构域是体内肺纤维化的必要和充分驱动。所有关键 这些发现将在正常和IPF患者来源的细胞和组织中得到验证。完成后，我们将有一个 详细而全面地了解TRPV 4-PI 3 K γ轴 介导肺纤维化。作为几种小分子，PI 3 K γ和TRPV 4的选择性抑制剂， 在不同的发展阶段，从这个“概念验证”研究中获得的知识可以迅速转化为 肺纤维化疾病的新治疗方法。

ABSTRACT Idiopathic pulmonary fibrosis (IPF) is a fatal disorder with urgent need for a medical cure. In order to successfully ameliorate pulmonary fibrosis, a better understanding of fibrotic pathogenesis is vital. Myofibroblasts are key fibrosis-effector cells. It has long been known that the response to changes in the mechanical properties of the surrounding tissue/matrix, along with active TGF-β, are critical drivers of myofibroblast differentiation. Although integrins and other receptors participate in cell-matrix interactions, the specific mechanosensor driving myofibroblast differentiation has remained elusive. We have identified TRPV4 as the critical mechanosensor for driving both myofibroblast differentiation and fibrosis in the lung. TRPV4 is a stretch-activatable, plasma membrane cation channel in the transient receptor potential, vanilloid family (TRPV4). Moreover, TRPV4 drives myofibroblast differentiation and fibrosis at levels of matrix stiffness that are directly biologically and clinically relevant to pulmonary disease. We have demonstrated TRPV4’s importance to myofibroblast differentiation and experimental pulmonary fibrosis and human IPF. Our novel published and preliminary data further suggests that TRPV4’s pro-fibrotic actions depend on its biochemical association with cytosolic PI3Kγ via PI3kγ’s unique aminoterminal, non-catalytic domain, followed by the translocation of the protein complex to the plasma membrane. Based on this data, we have formulated the novel hypothesis that the TGFβ drives myofibroblast differentiation and pulmonary fibrosis by inducing the TRPV4-PI3Kγ protein complex to translocate to the plasma membrane. Three coordinated specific aims with gain and loss function experimental designs will determine the key TRPV4-PI3Kγ pathway interactions that drive myofibroblast differentiation and fibrosis. They will utilize gain and loss of function design in in silico, in vitro and in vivo systems at the structural level, cellular level, and at the levels of established experimental murine models, and in human disease. AIM 1 will precisely define the amino acid(s) interacting sites between TRPV4 and PI3Kγ, AIM 2 will determine the mechanism whereby TGFβ induces the translocation of TRPV4-PI3Kγ complexes to the PM where they act to drive myofibroblast differentiation and AIM 3 will test the concept that the aminoterminal domain of PI3Kγ is necessary and sufficient to drive pulmonary fibrosis in vivo. All key findings will be validated in normal and IPF patient-derived cells and tissue. When complete, we will have a detailed and comprehensive understanding of the precise mechanism by which the TRPV4-PI3Kγ axis mediates pulmonary fibrosis. As several small molecule, selective inhibitors of both PI3Kγ and TRPV4 are in various phases of development, the knowledge gained from this “proof of concept” study could rapidly translate into novel therapeutic approaches for pulmonary fibrotic disorders.