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（由申请人提供）：发育中的肺部损伤通常会引起周围肺血管平滑肌细胞（SMC）的增生和肺血管的早熟肌肉化，从而限制肺血流并引起肺动脉高压。已经观察到针对肺CGMP水平升高的药物，例如吸入一氧化氮气体，可降低异常的肺动脉动脉SMC SMC增殖并改善动物模型中受伤的新生儿肺发育。但是，这些疗法的保护作用是不完整的。该赠款建议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。环状GMP主要通过刺激CGMP依赖性蛋白激酶I（PKGI）调节PASMC表型。新兴的证据表明，CGMP刺激的核蛋白和转录因子（例如CREB和ATF-1）调节SMC表型的PKGI核定位和磷酸化。最近，我们观察到CGMP刺激的PKGI蛋白水解产生了COOH末端组成型活性激酶片段PKGI？，它易位到SMC核，对于基因表达的反式激活至关重要。 PKGI蛋白水解裂解位点的研究和底物诱饵的作用表明普洛蛋白转化酶是PKGI的负责？ SMC中的核定位。但是，调节PKGI蛋白水解和PKGI的机制？核定位和PKGI的作用？在调节SMC表型时，未知。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。我们将在以下目的中检验大鼠PASMC中的这一假设。特定的目标1表征了普洛蛋白转化酶在调节PKGI蛋白水解和核PKGI信号传导中的特定作用。我们将使用RNAi识别哪种前蛋白转化酶调节PKGI蛋白水解。我们还将使用原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检查了调节PKGI核定位的机制。高尔基体在PKGI中的PKGI蛋白水解和活性核转运的作用？将评估核PKGI信号传导。特定目标3测试PKGI蛋白水解和核PKGI的功能意义？调节PASMC分化和增殖的隔室和测试PKGI蛋白水解是否缺乏损伤发育中的肺部。本赠款应用中提出的研究将提供有关调节CGMP驱动的SMC表型调制的机制的新信息。他们可能会确定可能在血管损伤中异常异常的途径，并为预防肺血管疾病的疗法提供新的靶标。 公共卫生相关性：小儿肺损伤患者的肺血管疾病与肺动脉高压，心力衰竭以及有时死亡有关。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，这些肌肉细胞分化和增殖在肺血管疾病中受异常调节。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信号传导的疗法如何预防或改善肺损伤儿科患者的肺血管疾病。