Mechanisms regulating PKGI proteolysis and modulation of pulmonary SMC phenotype

调节 PKGI 蛋白水解和肺 SMC 表型调节的机制

• 批准号: 7885712
• 负责人: JESSE D ROBERTS
• 金额: $ 43.85万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7885712
• 关键词: Amino Acid Sequence; Animal Model; Antibodies; Applications Grants; Binding; Blood Vessels; Blood flow; Breathing; Bronchopulmonary Dysplasia; Cell Differentiation process; Cell Nucleus; Cell Proliferation; Cell physiology; Cessation of life; Childhood; Cleaved cell; Cyclic AMP Response Element; Cyclic AMP-Responsive DNA-Binding Protein; Cyclic GMP; Distal; Gases; Gene Expression; Golgi Apparatus; Growth; Guanylate Cyclase; Heart failure; Human; Hyperplasia; Hypertension; In Vitro; Infant; Injury; Laboratories; Lead; Lung; Membrane; Molecular; Morbidity - disease rate; Natriuretic Peptides; Newborn Animals; Newborn Infant; Nitric Oxide; Nuclear; Nuclear Localization Signal; Nuclear Protein; Nuclear Proteins; Pathway interactions; Patients; Peptide Hydrolases; Peptide Signal Sequences; Peripheral; Phenotype; Phosphorylation; Phosphotransferases; Play; Proprotein Convertases; Proteins; Proteolysis; Proteolytic Processing; Pulmonary Hypertension; Pulmonary artery structure; Pulmonary vessels; RNA Interference; Rattus; Research Project Grants; Research Proposals; Role; Signal Transduction; Site; Smooth Muscle Myocytes; Testing; Transactivation; Vascular Diseases; activating transcription factor 1; base; cGMP production; cGMP-dependent protein kinase I; congenital heart disorder; enzyme activity; improved; infant outcome; inhaled nitric oxide; inhibitor/antagonist; injured; lung development; lung injury; mortality; mutant; novel; nucleocytoplasmic transport; prevent; protective effect; public health relevance; pup; transcription factor

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.

描述（申请人提供）：发育中的肺损伤常常引起外周肺血管平滑肌细胞（SMC）增生和肺血管早熟肌化，从而限制肺血流并引起肺动脉高压。在动物模型中，已观察到旨在增加肺部 cGMP 水平的药物（例如吸入一氧化氮）可以减少异常的肺动脉 SMC 增殖并改善受损的新生儿肺部发育。然而，这些疗法的保护作用并不完整。该拨款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。新的证据表明，cGMP 刺激的 PKGI 核定位以及核蛋白和转录因子（例如 CREB ​​和 ATF-1）的磷酸化可调节 SMC 表型。最近我们观察到，cGMP 刺激的 PKGI 蛋白水解作用产生 COOH 末端组成型活性激酶片段 PKGI？，该片段易位至 SMC 细胞核，对于基因表达的反式激活至关重要。对 PKGI 蛋白水解裂解位点和底物诱饵作用的研究表明，前蛋白转化酶是 PKGI 的罪魁祸首？ SMC 中的核定位。然而，PKGI蛋白水解和PKGI的调节机制是什么？核定位和PKGI的作用？调节 SMC 表型的作用尚不清楚。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。具体目标 1 描述了前蛋白转化酶在调节 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 蛋白水解中的作用以及 PKGI 中主动核转运的作用？将评估核 PKGI 信号传导中的作用。具体目的3 测试PKGI蛋白水解和核PKGI的功能意义？分区在调节 PASMC 分化和增殖中的作用，并测试 PKGI 蛋白水解作用在受损的发育肺中是否存在缺陷。本拨款申请中提出的研究将提供有关调节 cGMP 驱动的 SMC 表型调节机制的新信息。他们可能会识别血管损伤中可能异常的途径，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：肺损伤儿科患者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，这些细胞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病因的重要机制信息，以及针对调节 cGMP 信号传导的疗法如何预防或改善肺损伤儿科患者的肺血管疾病。