PKD Family Kinase Function and Signaling in Lung Fibroblasts

特发性肺纤维化（IPF）是一种慢性、进行性和通常致死性的肺部疾病， 病因学，特征为肺成纤维细胞活化和增殖，细胞外 基质（ECM）和正常肺结构的破坏。IPF患者的长期生存率较差， 5-年生存率仅20%。从历史上看，抗炎药一直是治疗的主要药物，但 已被证明在降低疾病的严重程度或进展方面无效。因此， 对鉴定新的可药物化靶点以开发用于治疗癌症的有效新疗法的深刻需求 IPF患者。由于肺成纤维细胞是IPF的主要效应细胞，因此鉴定新的可药用的 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的治疗干预。在这个建议中，我们试图确定丝氨酸/苏氨酸蛋白是否 激酶D（PKD）家族激酶在IPF成纤维细胞活化、ECM产生和表达中起关键作用 促纤维化生长因子受体PKD家族激酶包括PKD 1、PKD 2和PKD 3。在此，我们发现， 首次在成纤维细胞病灶（IPF的标志性病变）中容易检测到所有PKD亚型。我们 进一步表明PKD 1和PKD 2在原代IPF成纤维细胞中组成性激活，但在对照组中没有 正常人肺成纤维细胞。此外，PKD 1和PKD 2可被促纤维化生长进一步激活 原代IPF成纤维细胞中的因子。有趣的是，PKD抑制显著下调了 前胶原和纤连蛋白，而不显著影响其在IPF成纤维细胞中的mRNA水平。然而，PKD 抑制显著抑制促纤维化血小板衍生生长因子的mRNA和蛋白水平 受体（PDGFR）。基于这些新发现，重新提交的R21提案是 旨在检验PKD家族激酶可能在IPF成纤维细胞生物学中发挥关键作用的假设， 包括成纤维细胞活化、ECM产生和促纤维化生长因子受体的表达， 可能为阻止IPF进展提供新的药物靶点。重新提交提案的具体目的 目的：1）表征在大鼠肝纤维化模型中PKD家族激酶的组成性和促纤维化激动剂诱导的激活。 目的2）确定PKD家族激酶在IPF成纤维细胞中的作用及其机制。 原代IPF成纤维细胞中促纤维化基质的产生;目的3）确定PKD家族激酶的作用 以及促纤维化PDGF受体在IPF成纤维细胞中的表达机制。我们预计， 一项提案将确定PKD家族激酶作为肺成纤维细胞生物学的新的重要调节剂， 在IPF的发病机制和进展中，因此靶向抑制PKD家族激酶或特定PKD 同种型可能为IPF患者提供一种新的有效的治疗干预。

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.