PKD Family Kinase Function and Signaling in Lung Fibroblasts

特发性肺纤维化(IPF)是一种慢性、进行性的、通常是致命的不明原因的肺部疾病。 病因以肺成纤维细胞活化和增殖、细胞外过度沉积为特征 基质(ECM)和正常肺结构的破坏。IPF患者的长期存活率较低， 5年存活率仅为20%。从历史上看，抗炎药一直是治疗的主要手段，但 已被证明在减轻疾病的严重程度或进展方面无效。因此，有 迫切需要确定新的可用药靶点以开发有效的治疗新疗法 IPF患者。由于肺成纤维细胞是IPF的主要效应细胞，新型药物的鉴定 控制IPF肺成纤维细胞生物学的分子或途径可能导致更特异和有效的 IPF的治疗性干预。在这项建议中，我们试图确定丝氨酸/苏氨酸蛋白 蛋白激酶D(PKD)家族在IPF成纤维细胞的激活、细胞外基质的产生和表达中起关键作用 促纤维化生长因子受体。PKD家族包括PKD1、PKD2和PKD3。在这里，我们发现了 首次在IPF标志性病变成纤维细胞灶中检测到所有PKD亚型。我们 进一步表明，在原代IPF成纤维细胞中，PKD1和PKD2被结构性地激活，而在对照细胞中不被激活 正常人肺成纤维细胞。此外，PKD1和PKD2还可被促纤维化生长进一步激活 原代IPF成纤维细胞的影响因素。有趣的是，抑制PKD显著下调了 前胶原和纤维连接蛋白对IPF成纤维细胞的mRNA水平无明显影响。鉴于，PKD 抑制显著抑制促纤维化的血小板衍化生长因子的mRNA和蛋白水平 成纤维细胞中的受体(PDGFR)。根据这些新的发现，重新提交的R21提案是 旨在验证PKD家族蛋白激酶可能在IPF成纤维细胞生物学中发挥关键作用的假设， 包括成纤维细胞的激活、细胞外基质的产生和促纤维生长因子受体的表达，其中 可能为阻止IPF进展提供新的可用药靶点。这项重新提交提案的具体目的是 目的：1)研究成纤维细胞激动剂诱导的蛋白酪氨酸激酶家族的激活。 目的：2)研究蛋白酪氨酸激酶家族在肺纤维化中的作用及其机制。 在原代IPF成纤维细胞中产生促纤维化基质；目的3)确定PKD家族激酶的作用 以及促纤维化的PDGF受体在IPF成纤维细胞中的表达机制。我们预计这将是 一项提案将确定PKD家族激酶是涉及肺成纤维细胞生物学的新的和重要的调节因子 在IPF的发病和进展中，因此靶向抑制PKD家族激酶或特定的PKD 异构体可能为IPF患者提供一种新的、有效的治疗干预手段。

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and usually fatal lung disease of unknown etiology, characterized by lung fibroblast activation and proliferation, excessive deposition of extracellular matrix (ECM), and destruction of the normal lung architecture. Long-term survival of IPF patients is poor, with a 5-year survival rate of only 20%. Historically, anti-inflammatory agents have been the mainstay of therapy, but have been proven to be ineffective in reducing either the severity or progression of the disease. Hence, there is a profound need for the identification of novel drugable targets to develop efficacious new therapies for treating IPF patients. Because lung fibroblasts are primary effector cells in IPF, the identification of novel drugable molecules or pathways that control IPF lung fibroblast biology may lead to more specific and efficacious therapeutic intervention in IPF. In this proposal, we seek to determine whether the serine/threonine protein kinase D (PKD) family kinases play critical roles in IPF fibroblast activation, production of ECM, and expression of profibrotic growth factor receptors. PKD family kinases include PKD1, PKD2 and PKD3. Herein, we found for the first time that all the PKD isoforms were readily detected in fibroblastic foci, the hallmark lesions of IPF. We further showed that PKD1 and PKD2 were constitutively activated in primary IPF fibroblasts but not in control normal human lung fibroblasts. Moreover, PKD1 and PKD2 could be further activated by profibrotic growth factors in primary IPF fibroblasts. Interestingly, PKD inhibition markedly downregulated the protein levels of procollagen and fibronectin without significantly affecting their mRNA levels in IPF fibroblasts. Whereas, PKD inhibition markedly suppressed both mRNA and protein levels of profibrotic platelet-derived growth factor receptor-¿ (PDGFR¿) in the fibroblasts. On the basis of these novel findings, this resubmission R21 proposal is designed to test the hypothesis that PKD family kinases may play critical roles in IPF fibroblast biology, including the fibroblast activation, ECM production and expression of profibrotic growth factor receptors, which may offer novel drugable targets for halting IPF progression. The Specific Aims of this resubmission proposal are: Aim 1) To characterize the constitutive and profibrotic agonist-induced activation of PKD family kinases in primary IPF fibroblasts; Aim 2) To determine the roles of PKD family kinases and the mechanisms in the production of profibrotic matrix in primary IPF fibroblasts; Aim 3) To determine the roles of PKD family kinases and the mechanisms in IPF fibroblast expression of profibrotic PDGF receptor-¿. We anticipate that this proposal will identify PKD family kinases as novel and important modulators of lung fibroblast biology involved in the pathogenesis and progression of IPF, therefore targeting to inhibit PKD family kinases or a specific PKD isoform may provide a novel and efficacious therapeutic intervention for IPF patients.