Human Airway Smooth Muscle Growth Regulation

描述（申请人摘要）：本提案的长期目标是 确定细胞和分子信号传导过程， 蛋白偶联受体（GPCRs）调节人气道平滑肌的生长 (ASM)细胞许多炎症和收缩剂已知激活 GPCR在哮喘患者的气道中升高， 其特征在于气道高反应性和增加的ASM质量。私家侦探的 研究证实，尽管大多数GPCR激动剂不能刺激 ASM自身生长，这些激动剂能够显著 增加多肽生长因子的增殖作用。的 GPCR和受体酪氨酸激酶之间协同作用的潜在机制 (RTK)激活未知。这一提议的核心假设是， GPCRS和RTK贡献不同的信号，可以合作激活 p7 Os 6 k和引起HASM生长的协同诱导。这种假设是 广泛的初步数据表明， 用RTK（EGF）和GPCR（凝血酶， 组胺、卡巴胆碱和血栓烷）激动剂。为了验证这个假设，我们 将研究特异性抑制分子/途径的影响， p70 s6 k的潜在上游：1）p70 s6 k活性; 2）p70 s6 k 3）由EGF、GCPR激动剂刺激的ASM生长，或 两者在目标1中，GPCR依赖性跨膜信号传导事件的作用 （特异性异源三聚体G蛋白活化，膜描绘的支架 p70 s6 k活化中的蛋白质形成）将主要使用分子生物学方法进行分析。 抑制异源三聚体G蛋白、抑制蛋白和局灶性 粘附依赖性信号传导。在目标2中，我们将靶向细胞溶质分子， （钙和磷酸肌醇、粘着斑激酶、Src、Akt、Akt和PKC 同种型）以类似的方式检测它们在增加的p70 s6 k activation.在目标3中，p70 s6 k的突变体将在ASM培养物中表达， 分析EGF和GPCR激动剂在细胞增殖中的相对贡献的目的， p70 s6 k的特定氨基酸的磷酸化。在目标4中， 介导GPCR依赖性调节p70 s6 k的分子/途径 在RTK+ GPCR依赖性细胞生长的测定中，活化将被抑制。 总的来说，这些研究将阐明GPCRS作为 生长信号，增强我们对一个重要的生长调节因子的理解。 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.