Smooth Muscle Cell Protein Serotonylation and Pulmonary Hypertension

平滑肌细胞蛋白血清素化与肺动脉高压

• 批准号: 8690956
• 负责人: Barry Fanburg
• 金额: $ 46.81万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8690956
• 关键词: Active Biological Transport; Amines; Animal Model; Animals; BMP2 gene; BMPR2 gene; Biochemical Process; Cell Culture Techniques; Cell Proliferation; Cell Surface Receptors; Cell physiology; Clinical; Data; Development; Disease; Enzymes; Exposure to; Extracellular Matrix Proteins; Fibronectins; Human; Hypoxia; Immunoblotting; Immunoprecipitation; Integrins; Intervention; Knock-in Mouse; Knowledge; Lead; Lung; MAP Kinase Gene; Michigan; Mitogen-Activated Protein Kinase Inhibitor; Modification; Molecular; Monocrotaline; Mus; PDGFRB gene; Participant; Pathogenesis; Pathway interactions; Patients; Platelet-Derived Growth Factor; Platelet-Derived Growth Factor Receptor; Post-Translational Protein Processing; Process; Proteins; Pulmonary Hypertension; Pulmonary artery structure; Rattus; Regulation; Rodent; Rodent Model; Role; Secondary to; Serotonin; Serum; Signal Pathway; Signal Transduction; Small Interfering RNA; Smooth Muscle Myocytes; Structure of parenchyma of lung; System; Techniques; Testing; Tissues; Transfection; Transgenic Mice; Transgenic Organisms; Transglutaminases; Universities; Vascular remodeling; cell growth; cell motility; hemodynamics; in vivo; inhibitor/antagonist; migration; mutant; novel; novel therapeutic intervention; public health relevance; receptor; research study; response; rho; serotonin transporter; transglutaminase 2; uptake

DESCRIPTION (provided by applicant): We believe we have discovered a novel explanation for the mechanism by which serotonin (5- HT) produces pulmonary artery smooth muscle cell (SMC) proliferation and migration. This is important since these SMC functions participate in pulmonary hypertension (PH) and the 5- HT/5-HT transporter (SERT) system has been associated with clinical and experimental PH. From preliminary data the biochemical process of posttranslational modification of SMC protein through transamidation (serotonylation) leads to SMC proliferation and migration. Fibronectin (FN), an important extracellular matrix protein that has been associated with PH, is a major protein undergoing serotonylation. This enhanced serotonylation of SMC protein, occurring through the tissue enzyme transglutaminase (TGase), is influenced by the PDGF receptor and possibly other cell surface receptors that have been associated with PH. Furthermore, as an in vivo corollary enhanced serotonylation of FN occurs in lung tissue of mice and rats developing PH secondary to exposure to hypoxia and in rats given monocrotaline, and we have identified marked elevation of serotonylated FN in lungs and sera of some patients with PAH. We propose in this application to explore further the roles that lung tissue SERT, TGase and protein serotonylation may have in stimulation of SMC proliferation and migration and development of PH with a variety of cell culture, transfection, siRNA, immunoprecipitation and immunoblotting techniques and with the use of normal and transgenic rodent model experiments. Specifically, we will 1) better define the roles of SERT and TGase in SMC proliferation, migration and cell signaling produced by 5-HT; 2) determine the roles of serotonylation of FN and related integrins in 5-HT induced SMC proliferation and migration; 3) assess modification of SMC protein/FN serotonylation by hypoxia, PDGF/PDGFR and BMP/BMPR, known participants in PH; and 4) examine the association of lung and pulmonary artery protein/FN serotonylation in rodents developing PH secondary to hypoxia or following administration of monocrotaline, in SERT KO and "knock in" transgenic mice, in SERT KO rats and in mice deficient in TGase. The SERT "knock-in" animals that show overactivity of SERT are from Dr. Randy Blakely, an internationally recognized expert in SERT, at Vanderbilt University. Dr. Stephanie Watts from Michigan State University, another expert in SERT, will collaborate in studies related to SERT KO rats and their isolated pulmonary artery SMC's. We hope to better define any role of SERT, TGase activity and protein serotonylation in the development of PH and to consider possible new interventional strategies through these pathways that might be used for treating this disease.

描述（由申请人提供）：我们相信我们已经发现了对血清素（5-HT）引起肺动脉平滑肌细胞（SMC）增殖和迁移的机制的新解释。这很重要，因为这些 SMC 功能参与肺动脉高压 (PH)，并且 5-HT/5-HT 转运蛋白 (SERT) 系统与临床和实验性 PH 相关。从初步数据来看，SMC 蛋白通过转酰胺基作用（血清素化）进行翻译后修饰的生化过程会导致 SMC 增殖和迁移。纤连蛋白 (FN) 是一种重要的细胞外基质蛋白，与 PH 相关，是一种经历血清素化的主要蛋白。通过组织酶转谷氨酰胺酶 (TGase) 发生的 SMC 蛋白血清素化增强受到 PDGF 受体以及可能与 PH 相关的其他细胞表面受体的影响。此外，由于在因暴露于缺氧而继发PH的小鼠和大鼠的肺组织中以及在给予野百合碱的大鼠中，FN的体内推论增强的血清素化发生在肺组织中，并且我们已经发现一些PAH患者的肺和血清中血清素化的FN显着升高。在本申请中，我们建议通过各种细胞培养、转染、siRNA、免疫沉淀和免疫印迹技术以及使用使用正常和转基因啮齿动物模型实验。具体来说，我们将1）更好地定义SERT和TGase在SMC增殖、迁移和5-HT产生的细胞信号传导中的作用； 2)确定FN和相关整合素的血清素化在5-HT诱导的SMC增殖和迁移中的作用； 3) 评估缺氧、PDGF/PDGFR 和 BMP/BMPR（已知 PH 参与者）对 SMC 蛋白/FN 血清素化的修饰； 4) 检查因缺氧或给予野百合碱后继发PH的啮齿类动物、SERT KO和“敲入”转基因小鼠、SERT KO大鼠和缺乏TGase的小鼠中肺和肺动脉蛋白/FN血清素化的关联。表现出 SERT 过度活跃的 SERT“敲入”动物来自范德比尔特大学国际公认的 SERT 专家 Randy Blakely 博士。另一位 SERT 专家、密歇根州立大学的 Stephanie Watts 博士将合作开展与 SERT KO 大鼠及其离体肺动脉 SMC 相关的研究。我们希望更好地定义 SERT、TGase 活性和蛋白质血清素化在 PH 发展中的作用，并考虑通过这些途径可能用于治疗这种疾病的新干预策略。