Mechanisms regulating PKGI proteolysis and modulation of pulmonary SMC phenotype

描述（申请人提供）：发育中的肺损伤常引起肺外周血管平滑肌细胞（SMC）增生和肺血管肌肉化早熟，从而限制肺血流，引起肺动脉高压。在动物模型中，已观察到旨在增加肺cGMP水平的药物，如吸入一氧化氮气体，可减少异常肺动脉SMC增殖并改善受损新生儿肺发育。然而，这些疗法的保护作用是不完整的。该资助提案的长期目标是进一步表征cGMP调节肺动脉SMC （PASMC）分化和增殖的分子机制。环GMP主要通过刺激cgmp依赖性蛋白激酶I （PKGI）来调节PASMC表型。越来越多的证据表明，cgmp刺激的PKGI核定位和核蛋白和转录因子（如CREB和ATF-1）的磷酸化调节SMC表型。最近，我们观察到cgmp刺激的PKGI蛋白水解产生cooh末端构成活性的激酶片段PKGI？，它易位到SMC核，对基因表达的反激活至关重要。对PKGI蛋白水解裂解位点和底物诱饵效应的研究表明，蛋白转化酶是PKGI？SMC中的核定位。然而，PKGI蛋白水解和PKGI？核定位与PKGI的作用？在调节SMC表型中的作用尚不清楚。我们的中心假设是PKGI蛋白水解和PKGI？核定位是核PKGI信号和SMC分化和增殖的关键决定因素。我们将在大鼠PASMC中验证这一假设。特异性目标1描述了蛋白转化酶在调节PKGI蛋白水解和核PKGI信号传导中的具体作用。我们将使用RNAi来确定哪些蛋白转化酶调节PKGI蛋白水解。我们还将使用蛋白转化酶抑制剂来测试这些蛋白酶如何调节PASMC的分化和增殖。特异性目的2研究了PKGI核定位的调控机制。高尔基体在PKGI蛋白水解和PKGI主动核转运中的作用？在核PKGI信号中的作用将被评估。特异性目的3检验PKGI蛋白水解和核PKGI的功能意义。区隔调节PASMC的分化和增殖，并检测PKGI蛋白水解是否在损伤的发育中的肺中缺乏。本资助申请中提出的研究将提供有关cgmp驱动的SMC表型调节机制的新信息。它们有可能识别血管损伤中可能异常的通路，并为预防肺血管疾病的治疗提供新的靶点。

DESCRIPTION (provided by applicant): Injury of the developing lung often causes hyperplasia of peripheral pulmonary vascular smooth muscle cells (SMC) and precocious muscularization of pulmonary vessels, thereby restricting lung blood flow and causing pulmonary artery hypertension. Agents directed at increasing lung cGMP levels, such as inhaled nitric oxide gas, have been observed to decrease abnormal pulmonary artery SMC proliferation and improve injured newborn lung development in animal models. However, the protective effect of these therapies is incomplete. The long-term objective of this grant proposal is to further characterize the molecular mechanisms through which cGMP regulates pulmonary artery SMC (PASMC) differentiation and proliferation. Cyclic GMP modulates PASMC phenotype primarily by stimulating cGMP-dependent protein kinase I (PKGI). Emerging evidence suggests that cGMP-stimulated PKGI nuclear localization and phosphorylation of nuclear proteins and transcription factors, such as CREB and ATF-1, regulate SMC phenotype. Recently we observed that cGMP-stimulated PKGI proteolysis generates a COOH-terminal constitutively active kinase fragment, PKGI?, that translocates to the nucleus of SMC and is critical for the transactivation of gene expression. Studies of the PKGI proteolysis cleavage site and the effect of substrate decoys suggest that proprotein convertases are responsible for PKGI? nuclear localization in SMC. However, the mechanisms regulating PKGI proteolysis and PKGI? nuclear localization and the role of PKGI? in modulating SMC phenotype are unknown. Our central hypothesis is that PKGI proteolysis and PKGI? nuclear localization are key determinants of nuclear PKGI signaling and SMC differentiation and proliferation. We will test this hypothesis in rat PASMC in the following aims. Specific aim 1 characterizes the specific role of proprotein convertases in regulating PKGI proteolysis and nuclear PKGI signaling. We will use RNAi to identify which proprotein convertase(s) regulate PKGI proteolysis. We will also use proprotein convertase inhibitors to test how these proteases regulate PASMC differentiation and proliferation. Specific aim 2 examines the mechanisms that regulate the nuclear localization of PKGI?. The role of the Golgi apparatus in PKGI proteolysis and of active nuclear transport in PKGI? in nuclear PKGI signaling will be evaluated. Specific aim 3 tests the functional significance of PKGI proteolysis and nuclear PKGI? compartmentation in regulating PASMC differentiation and proliferation and tests whether PKGI proteolysis is deficient in the injured developing lung. The studies proposed in this grant application will provide novel information about the mechanisms regulating cGMP-driven modulation of SMC phenotype. They are likely to identify pathways that may be abnormal in vascular injury and to provide new targets for therapies directed at preventing pulmonary vascular disease. PUBLIC HEALTH RELEVANCE: Pulmonary vascular disease in pediatric patients with lung injury is associated with pulmonary hypertension, heart failure, and sometimes death. This research project is directed at understanding how cGMP signaling regulates lung vascular smooth muscle cell differentiation and proliferation, which are abnormally regulated in pulmonary vascular disease. The results from this project will provide important mechanistic information about the causes of pulmonary vascular disease and how therapies directed at modulating cGMP signaling might prevent or improve pulmonary vascular disease in pediatric patients with lung injury.