Smooth Muscle Cell Protein Serotonylation and Pulmonary Hypertension

描述(申请人提供)：我们相信我们已经发现了一种新的解释，解释了5-羟色胺(5-羟色胺)促进肺动脉平滑肌细胞(SMC)增殖和迁移的机制。这一点很重要，因为这些SMC功能参与了肺动脉高压(PH)，而5-羟色胺/5-羟色胺转运体(SERT)系统与临床和实验性PH有关。根据初步数据，SMC蛋白通过转酰胺化(5-羟色胺基化)翻译后修饰的生化过程导致SMC的增殖和迁移。纤维连接蛋白(FN)是一种重要的细胞外基质蛋白，与PH有关，是一种主要的5-羟色胺基化蛋白。这种通过组织酶转谷氨酰胺酶(TGase)发生的SMC蛋白的5-羟色胺增强作用，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，作为体内增强的必然结果，在继发于缺氧的PH的小鼠和大鼠的肺组织以及给予野百合碱的大鼠的肺组织中，Fn的5-羟色胺基化显著增加，我们还发现一些PAH患者的肺和血清中5-羟色胺基化的Fn显著升高。在这一应用中，我们建议通过各种细胞培养、转基因、siRNA、免疫沉淀和免疫印迹技术，以及使用正常和转基因啮齿动物模型实验，进一步探讨肺组织SERT、TGase和蛋白5-羟色化在刺激SMC增殖和PH的迁移和发展中可能发挥的作用。具体地说，我们将1)更好地确定SERT和TGase在5-羟色胺诱导的SMC增殖、迁移和细胞信号转导中的作用；2)确定FN及相关整合素的5羟色化在5-羟色胺诱导的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 5羟色化的修饰；以及4)研究缺氧继发高血压或给予野百合碱后肺和肺动脉蛋白/FN 5羟色化的关联。显示SERT过度活跃的SERT“敲入”动物来自范德比尔特大学国际公认的SERT专家兰迪·布莱克利博士。密歇根州立大学的斯蒂芬妮·瓦茨博士是SERT的另一位专家，她将参与与SERT KO大鼠及其隔离的肺动脉SMC相关的研究。我们希望更好地确定SERT、TGase活性和蛋白质5羟色胺在PH发生发展中的作用，并考虑通过这些途径可能用于治疗这种疾病的新的干预策略。 公共卫生相关性：5-羟色胺和5-羟色胺转运体被认为通过引起肺动脉平滑肌细胞的增殖和迁移而参与了肺动脉高压的发展。我们相信，我们已经确定了5-羟色胺通过改变平滑肌细胞蛋白(特别是纤维连接蛋白)、转运体使5-羟色胺内化和一种名为组织转谷氨酰胺酶(称为蛋白质5-羟色胺基化的过程)的酶的作用来实现这一功能的机制。更好地了解5-羟色胺的过程及其与实验性肺动脉高压的关系，可能有助于更好地理解肺动脉高压的发病机制，并找到治疗该疾病的新方法。

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. PUBLIC HEALTH RELEVANCE: Serotonin and the serotonin transporter are thought to participate in the development of pulmonary hypertension by causing proliferation and migration of pulmonary artery smooth muscle cells. We believe we have identified a mechanism by which serotonin does this by its alteration of smooth muscle cell proteins (in particular fibronectin), following cellular internalization of serotonin by the transporter and action of an enzyme called tissue transglutaminase ( a process called serotonylation of protein). A better understanding of the process of serotonylation and the association of it with experimental pulmonary hypertension as proposed in this application may lead to a better understanding of the pathogenesis of pulmonary hypertension and novel treatments for the disease.