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: 8235019
• 负责人: JESSE D ROBERTS
• 金额: $ 43.8万
• 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/8235019
• 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 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; pup; transcription factor

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.

发育中的肺损伤常引起外周肺血管平滑肌增生 平滑肌细胞（SMC）和肺血管的过早肌肉化，从而限制肺血流， 导致肺动脉高压。针对增加肺cGMP水平的药剂，例如吸入的 一氧化氮气体，已经观察到减少异常肺动脉SMC增殖和改善 在动物模型中损伤新生儿肺发育。然而，这些疗法的保护作用是 不完整 这项资助计划的长期目标是通过以下方法进一步表征分子机制： cGMP调节肺动脉平滑肌细胞（PASMC）的分化和增殖。环GMP 主要通过刺激cGMP依赖性蛋白激酶I（PKGI）调节PASMC表型。新兴 有证据表明cGMP刺激的PKGI核定位和核蛋白磷酸化 转录因子CREB、ATF-1等调节SMC表型。最近我们观察到， cGMP刺激的PKGI蛋白水解产生一个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。的研究 PKGI蛋白水解切割位点和底物诱饵的作用表明，前蛋白转化酶是 负责SMC中PKGI的核定位。然而，调节PKGI蛋白水解和 PKGI <$核定位和PKGI <$在调节SMC表型中的作用尚不清楚。我们的中央 假设PKGI蛋白水解和PKGI核定位是核PKGI的关键决定因素 信号传导和SMC分化和增殖。我们将在以下大鼠PASMC中检验这一假设 目标。特异性目的1表征了前蛋白转化酶在调节PKGI蛋白水解中的特异性作用 和核PKGI信号传导。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检查调节核定位的机制 关于PKGI高尔基体在PKGI蛋白水解中的作用和PKGI中主动核转运的作用 将评价核PKGI信号传导。具体目标3测试PKGI蛋白水解的功能意义 和细胞核PKGI区室化在调节PASMC分化和增殖中的作用，并测试是否 PKGI蛋白水解在受损的发育中的肺中是缺乏的。 这项资助申请中提出的研究将提供有关机制的新信息 调节cGMP驱动的SMC表型调节。他们很可能会识别出 本发明的目的是提供用于预防肺血管损伤的治疗的新靶点， 疾病