Smooth Muscle Cell Protein Serotonylation and Pulmonary Hypertension

描述（由申请人提供）：我们相信我们已经发现了5-羟色胺（5- HT）产生肺动脉平滑肌细胞（SMC）增殖和迁移的新机制。这一点很重要，因为这些SMC功能参与肺动脉高压（PH）和5-HT /5-HT转运体（SERT）系统与临床和实验PH有关。从初步数据来看，SMC蛋白翻译后修饰的生化过程通过转酰胺（血清素化）导致SMC增殖和迁移。纤维连接蛋白（FN）是一种与PH相关的重要细胞外基质蛋白，是一种主要的血清素化蛋白。通过组织酶转谷氨酰胺酶（TGase）发生的SMC蛋白的5 -羟色胺化增强受到PDGF受体和可能与PH相关的其他细胞表面受体的影响。此外，由于暴露于缺氧而发生PH的小鼠和大鼠的肺组织中，以及服用了单缬氨酸的大鼠中，FN的5 -羟色胺化增强发生在体内。并且我们已经在一些PAH患者的肺和血清中发现了血清素化FN的显著升高。本研究拟通过多种细胞培养、转染、siRNA、免疫沉淀和免疫印迹技术，以及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 -羟色胺化在刺激SMC增殖、PH迁移和发育中的作用。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5-羟色胺化FN及相关整合素在5-羟色胺诱导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血清素化的影响；4)研究在缺氧或给药后发生PH的啮齿动物、SERT KO和“敲入”转基因小鼠、SERT KO大鼠和TGase缺乏小鼠中肺和肺动脉蛋白/FN血清素化的关系。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SERT专家Randy Blakely博士。密歇根州立大学的Stephanie Watts博士是SERT的另一位专家，她将合作进行SERT KO大鼠及其孤立肺动脉SMC的相关研究。我们希望更好地确定SERT、TGase活性和蛋白5 -羟色胺化在PH发展中的作用，并通过这些途径考虑可能用于治疗这种疾病的新干预策略。

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.