Smooth Muscle Cell Protein Serotonylation and Pulmonary Hypertension

描述（由申请人提供）：我们相信我们发现了5-羟色胺（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。这是很重要的，因为这些SMC的功能参与肺动脉高压（PH）和5- HT/5-HT转运蛋白（SERT）系统已与临床和实验PH。从初步数据的SMC蛋白的翻译后修饰的生化过程中，通过转酰胺基（乙酰化）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有关，是一种主要的蛋白质，经历了羟丙基化。通过组织酶转氨酶（TGase）发生的SMC蛋白的这种增强的FN酰化受到PDGF受体和可能与PH相关的其他细胞表面受体的影响。此外，作为体内必然结果，在继发于暴露于缺氧的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发生增强的FN酰化，并且我们已经确定了一些PAH患者的肺和血清中羟丙甲酰基化FN的显著升高。我们建议在本申请中进一步探索肺组织SERT，TGase和蛋白酰化可能在刺激SMC增殖和迁移以及PH发展中的作用，采用各种细胞培养，转染，siRNA，免疫沉淀和免疫印迹技术，并使用正常和转基因啮齿动物模型实验。具体而言，我们将1）更好地确定SERT和TGase在SMC增殖、迁移和由5-HT产生的细胞信号中的作用; 2）确定FN和相关整合素的N-酰基化在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对SMC蛋白/FN N-酰基化的修饰，它们是PH的已知参与者;和4）在发生继发于缺氧的PH或在施用野百合碱后的啮齿动物中、在SERT KO和“敲入”转基因小鼠中、在SERT KO大鼠中和在TGase缺陷的小鼠中，检查肺和肺动脉蛋白/FN羟化的关联。显示SERT过度活跃的SERT“敲入”动物来自范德比尔特大学的国际公认的SERT专家兰迪·布莱克利博士。来自密歇根州立大学的Stephanie Watts博士是SERT的另一位专家，他将合作进行与SERT KO大鼠及其分离的肺动脉SMC相关的研究。我们希望更好地确定SERT，TGase活性和蛋白质羟丙酰化在PH发展中的任何作用，并考虑通过这些途径可能用于治疗这种疾病的可能的新干预策略。

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.