Human Airway Smooth Muscle Growth Regulation

描述(申请人摘要)：这项建议的长期目标是 确定G通过的细胞和分子信号传递过程 蛋白偶联受体(GPCRs)对人气道平滑肌生长的调控作用 (ASM)细胞。已知的多种炎症和收缩药剂可激活 哮喘患者的呼吸道gpcr升高。 以呼吸道高反应性和ASM质量增加为特征。私家侦探S 研究表明，尽管大多数GPCR激动剂无法刺激 ASM自身的生长，这些激动剂能够显著 增强多肽生长因子的增殖作用。这个 GPCR和受体酪氨酸激酶之间协同作用的机制 (RTK)激活未知。这项提议的中心假设是 GPCRs和RTK提供了不同的信号，可以协同激活 P7Os6k，并协同诱导HASM生长。这一假设是 得到大量初步数据的支持，这些数据表明 表皮生长因子和凝血酶原激活剂对ASM细胞p70S6K的激活作用 组胺、卡巴胆碱和血栓烷)激动剂。为了检验这一假设，我们 将研究特定抑制分子/途径的效果 P70S6K的潜在上游：1)p70S6K活性；2)p70S6K 3)被EGF、GCPR激动剂刺激的ASM生长； 两者都有。在目标1中，依赖于gpcr的跨膜信号事件的作用 (特定的异三聚体G蛋白活化，膜描绘的支架 在p70S6K激活中的形成)将主要用分子分析 抑制异源三聚体G蛋白、Arrestin和Focus的生物学技术 黏附依赖信号转导。在目标2中，我们将以胞浆分子为目标 (钙和磷脂酰肌醇、粘着斑激酶、Src、Akt、Akt和PKC 异构体)以类似的方式检测它们在增加的p70S6K中的作用 激活。在目标3中，p70S6K的突变体将在ASM培养中表达 分析EGF和GPCR型激动剂的相对贡献 P70S6K特定氨基酸的磷酸化。在目标4中， 介导gpcr依赖的p70S6K调控的分子/途径 在依赖RTK+GPCRs的细胞生长试验中，激活将被抑制。 总而言之，这些研究将阐明GPCRs作为 增长信号，提高我们对增长的一个重要认识--调控 分子，并确定哮喘治疗管理的潜在靶点。

DESCRIPTION (Applicant's Abstract): The long-range goal of this proposal is to identify the cellular and molecular signaling processes by which G protein-coupled receptors (GPCRs) modulate growth of human airway smooth muscle (ASM) cells. Numerous inflammatory and contractile agents known to activate GPCRs are elevated in the airways of patients with asthma, a disease characterized by airway hyperreactivity and increased ASM mass. The P.I.'s studies establish that despite the inability of most GPCR agonists to stimulate ASM growth by themselves, these agonists are capable of significantly increasing the proliferative effect of polypeptide growth factors. The mechanisms underlying this synergy between GPCR and receptor tyrosine kinase (RTK) activation are unknown. The central hypothesis of this proposal is that GPCRS and RTKs contribute distinct signals that can cooperate to activate p7Os6k and cause a synergistic induction of HASM growth. This hypothesis is supported by extensive preliminary data that demonstrate a prolonged, increased activation of p70s6k in ASM cells stimulated by RTK (EGF) and GPCR (thrombin, histamine, carbachol, and thromboxane) agonists. To test this hypothesis, we will examine the effects of specifically inhibiting molecules/pathways potentially upstream of p70s6k on: 1) p70s6k activity; 2) p70s6k phosphorylation; and 3) ASM growth that is stimulated by EGF, GCPR agonists, or both. In Aim 1, the role of GPCR-dependent transmembrane signaling events (specific heterotrimeric G protein activation, membrane-delineated scaffold formation) in p70s6k activation will be analyzed primarily using molecular biology techniques to inhibit heterotrimeric G protein-, arrestin-, and focal adhesion-dependent signaling. In Aim 2, we will target cytosolic molecules (calcium and phosphoinositides, focal adhesion kinases, Src, Akt, Akt, and PKC isoforms) in a similar manner to examine their roles in increased p70s6k activation. In Aim 3, mutants of p70s6k will be expressed in ASM cultures for the purpose of analyzing the relative contributions of EGF and GPCR agonists in the phosphorylation of specific amino acids of p70s6k. In Aim 4, molecules/pathways shown to mediate the GPCR-dependent modulation p70s6k activation will be inhibited in assays of RTK+GPCR-dependent cell growth. Collectively these studies will clarify the role of GPCRS as transducers of growth signaling, enhance our understanding of an important growth-regulatory molecule, and identify potential targets for therapeutic management of asthma.