Mechanisms regulating PKGI proteolysis and modulation of pulmonary SMC phenotype

描述（由申请人提供）：发育中的肺损伤通常会导致外周肺血管平滑肌细胞（SMC）增生和肺血管过早肌化，从而限制肺血流并引起肺动脉高压。在动物模型中，已经观察到针对增加肺cGMP水平的药剂，例如吸入的一氧化氮气体，减少异常的肺动脉SMC增殖并改善损伤的新生儿肺发育。然而，这些疗法的保护作用是不完全的。这项资助计划的长期目标是进一步研究cGMP调节肺动脉平滑肌细胞（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）调节PASMC表型。新出现的证据表明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蛋白水解产生了一个COOH末端组成型活性激酶片段PKGI？，其易位至SMC的细胞核并且对于基因表达的反式激活是关键的。PKGI蛋白水解切割位点和底物诱饵效应的研究表明，前蛋白转化酶负责PKGI？SMC的核定位。然而，调节PKGI蛋白水解和PKGI？核定位和PKGI的作用？在调节SMC表型中的作用是未知的。我们的中心假设是，PKGI蛋白水解和PKGI？核定位是核PKGI信号传导和SMC分化和增殖的关键决定因素。我们将在大鼠PASMC中测试这一假设，目的如下。特异性目的1表征前蛋白转化酶在调节PKGI蛋白水解和核PKGI信号传导中的特异性作用。我们将使用RNAi来鉴定哪些前蛋白转化酶调节PKGI蛋白水解。我们还将使用前蛋白转化酶抑制剂来测试这些蛋白酶如何调节PASMC分化和增殖。具体目标2检查调节PKGI？核定位的机制。高尔基体在PKGI蛋白水解和PKGI核转运中的作用？在核PKGI信号传导中的作用。具体目标3测试PKGI蛋白水解和核PKGI的功能意义？本发明的目的是研究在调节PASMC分化和增殖中的区室化，并测试PKGI蛋白水解在受损的发育中的肺中是否缺乏。这项研究将为cGMP驱动的SMC表型调控机制提供新的信息。它们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。 公共卫生相关性：肺损伤患儿的肺血管疾病与肺动脉高压、心力衰竭、有时甚至死亡有关。该研究项目旨在了解cGMP信号如何调节肺血管平滑肌细胞分化和增殖，这在肺血管疾病中受到异常调节。该项目的结果将提供有关肺血管疾病原因的重要机制信息，以及针对调节cGMP信号传导的治疗如何预防或改善肺损伤儿科患者的肺血管疾病。

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.