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: 8452162
• 负责人: JESSE D ROBERTS
• 金额: $ 41.64万
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8452162
• 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.

发育中肺的损伤通常会引起周围肺平滑肌的增生 细胞（SMC）和肺动脉壁的早熟肌肉化，从而限制肺血流和 引起肺动脉高压。指向肺CGMP水平增加的代理，例如继承 已经观察到一氧化氮气体可降低异常的肺动脉SMC增殖并改善 动物模型中的新生儿肺发育受伤。但是，这些疗法的受保护作用是 不完整。 该赠款提案的长期目标是通过 CGMP调节肺动脉SMC（PASMC）分化和增殖。循环GMP 通过刺激CGMP依赖性蛋白激酶I（PKGI）来调节PASMC表型。新兴 有证据表明，CGMP刺激的PKGI核定位和核蛋白的磷酸化 和转录因子（例如CREB和ATF-1）调节SMC表型。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，，PKGI， 这将易位到SMC的核，对于基因表达的反式激活至关重要。研究 PKGI蛋白水解裂解位点和底物诱饵的作用表明提谋蛋白转化酶是 负责SMC中的核定位。但是，调查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。我们的中心 假设是PKGI蛋白水解和核定位是核PKGI的关键决定者 信号传导和SMC分化和增殖。我们将在下面的大鼠PASMC中检验该假设 目标。具体目标1表征了促蛋白转化酶在调节PKGI蛋白水解中的特定作用 和核PKGI信号传导。我们将使用RNAi识别哪种促蛋白转化酶调节PKGI 蛋白水解。我们还将使用促蛋白转化酶抑制剂测试这些蛋白酶如何调节PASMC 分化和增殖。特定的目标2检查调节核定位的机制 pkgi。高尔基体在PKGI蛋白水解和PKGI核转运中的作用 将评估核PKGI信号传导。特定目标3测试PKGI蛋白水解的功能意义 和核pkgi¿在确定PASMC分化和增殖方面的隔室，并测试是否测试 PKGI蛋白水解缺乏受伤的发育中的肺。 本赠款应用中提出的研究将提供有关机制的新信息 调节CGMP驱动的SMC表型调制。他们可能会确定可能是 血管损伤异常，为预防肺血管的疗法提供新靶标 疾病。

项目成果

Transglutaminase 2: a new player in bronchopulmonary dysplasia?

转谷氨酰胺酶 2：支气管肺发育不良的新参与者？

• DOI: 10.1183/09031936.00075713
• 发表时间: 2014
• 期刊: The European respiratory journal
• 作者: Witsch,ThiloJ;Niess,Gero;Sakkas,Elpidoforos;Likhoshvay,Tatyana;Becker,Simone;Herold,Susanne;Mayer,Konstantin;Vadász,István;RobertsJr,JesseD;Seeger,Werner;Morty,RoryE
• 通讯作者: Morty,RoryE

The Golgi apparatus regulates cGMP-dependent protein kinase I compartmentation and proteolysis.

• DOI: 10.1152/ajpcell.00199.2014
• 发表时间: 2015-06
• 期刊: American journal of physiology. Cell physiology
• 作者: S. Kato;Jingsi Chen;Katherine H Cornog;Huili Zhang;Jesse D. Roberts

Deregulation of the lysyl hydroxylase matrix cross-linking system in experimental and clinical bronchopulmonary dysplasia.

• DOI: 10.1152/ajplung.00109.2013
• 发表时间: 2014-02
• 期刊: American journal of physiology. Lung cellular and molecular physiology
• 作者: T. Witsch;P. Turowski;E. Sakkas;Gero Niess;S. Becker;S. Herold;K. Mayer;I. Vadász;Jesse D. Roberts-Jess

cGMP-dependent protein kinase I gamma encodes a nuclear localization signal that regulates nuclear compartmentation and function.

cGMP 依赖性蛋白激酶 I gamma 编码调节核区室和功能的核定位信号。

• DOI: 10.1016/j.cellsig.2014.08.004
• 发表时间: 2014
• 期刊: Cellular signalling
• 影响因子: 4.8
• 作者: Chen,Jingsi;RobertsJr,JesseD
• 通讯作者: RobertsJr,JesseD