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)：发育中的肺损伤通常会导致周围肺血管平滑肌细胞(SMC)的增殖和肺血管的早熟肌化，从而限制肺血流，导致肺动脉高压。在动物模型中，针对增加肺cGMP水平的药物，如吸入一氧化氮气体，已被观察到可减少异常的肺动脉SMC增殖，改善受损新生肺的发育。然而，这些疗法的保护作用并不完全。这项赠款提案的长期目标是进一步确定cGMP调节肺动脉SMC(PASMC)分化和增殖的分子机制。环状GMP主要通过刺激cGMP依赖的蛋白激酶I(PKGI)来调节PASMC的表型。新的证据表明，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的表型。最近，我们观察到cGMP刺激的PKGI蛋白分解产生一个COOH末端的结构性活性蛋白激酶片段PKGI？，该片段移位到SMC的核中，对基因表达的反式激活起关键作用。对PKGI蛋白水解酶裂解位点和底物诱骗作用的研究表明，原蛋白转换酶对PKGI？SMC中的核定位。然而，PKGI蛋白水解酶和PKGI？核定位和PKGI的作用？在调控中，SMC的表型尚不清楚。我们的中心假设是PKGI蛋白分解和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中验证这一假说，目的如下。特定目的1表征原蛋白转换酶在调节PKGI蛋白分解和核PKGI信号转导中的特殊作用。我们将使用RNA干扰来确定哪个原蛋白转换酶(S)调节PKGI蛋白降解。我们还将使用原蛋白转换酶抑制剂来测试这些酶是如何调控PASMC分化和增殖的。特定目的2研究了调节PKGI？核定位的机制。高尔基体在PKGI蛋白分解中的作用和PKGI中主动的核运输？在核领域，将对PKGI信号进行评估。特异性目标3检测PKGI蛋白降解和核PKGI？的功能意义。分隔法调节PASMC的分化和增殖，并测试在受损的发育中的肺是否缺乏PKGI蛋白分解。这项拨款申请中提出的研究将为调控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：儿童肺血管疾病合并肺损伤与肺高压、心力衰竭相关，有时甚至死亡。本研究旨在了解cGMP信号如何调节肺血管平滑肌细胞的分化和增殖，这些细胞在肺血管疾病中受到异常调控。该项目的结果将为肺血管疾病的病因以及针对调节cGMP信号的治疗如何预防或改善肺损伤儿童患者的肺血管疾病提供重要的机制信息。