RGS: A Molecular Switch Regulating Irreversible ASM Growth/Contraction in Asthma

RGS：调节哮喘不可逆 ASM 生长/收缩的分子开关

基本信息

批准号：
8120246
负责人：
Reynold Alexander Panettieri
金额：
$ 49.87万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-01 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8120246
关键词：
1-Phosphatidylinositol 3-Kinase Affect Agonist Air Asthma Attenuated Biopsy Bronchoalveolar Lavage Fluid Bronchoconstriction Calcium Cell Degranulation Cells Complementary DNA Contracts Coupling Data Disease Event Fluorescence Functional disorder G-substrate GTP-Binding Protein Regulators GTP-Binding Proteins Goals Growth Growth Factor Guanosine Triphosphate Phosphohydrolases Human Hyperplasia Image Inflammation Laboratories Lung Measurement Measures Mediating Mediator of activation protein Metalcaptase Microscopy Mitogens Modeling Molecular Morbidity - disease rate Muscle Cells Muscle Contraction Myosin Light Chains Obstruction Pathogenesis Pathway interactions Pattern Phosphatidylinositols Phosphorylation Phosphotransferases Process Proto-Oncogene Protein pp60 (c-src)Proto-Oncogene Proteins c-akt RGS Proteins Receptor Activation Role Severities Severity of illness Signal Pathway Signal Transduction Signaling Molecule Slice Small Interfering RNA Smooth Muscle Myocytes Spirometry Stimulation of Cell Proliferation Techniques Testing Time Tissues Transfection Work abstracting airway hyperresponsiveness airway inflammation airway obstruction cell growth cytokine inflammatory marker insight mast cell methacholine mortality muscle form muscle hypertrophy mutant new therapeutic target prevent protein expression protein protein interaction release of sequestered calcium ion into cytoplasm respiratory smooth muscle response therapeutic target

项目摘要

DESCRIPTION (provided by applicant): Airway inflammation and reversible bronchoconstriction characterize asthma; however, in severe disease, irreversible airway obstruction and increases in airway smooth muscle (ASM) mass/growth commonly occur. Growth factors require activation of Src, phosphatidylinositol 3-kinase (PI3K) and S6K1 to stimulate ASM growth. Our new data shows that mitogens increase ASM expression of regulators of G-protein signaling (RGS) proteins which inactivate G proteins and co-localize with phospho-p85 (p-p85) PI3K to facilitate S6K1 activation. Importantly, RGS protein knockdown inhibited ASM growth; mitogens inhibited agonist-induced contraction and decreased cytosolic calcium levels [Ca2+]i. These data suggest that specific RGS proteins serve as molecular switches to promote ASM growth while inhibiting excitation-contraction (E-C) coupling. Our central hypothesis states that RGS proteins promote mitogen-induced ASM proliferation in a PI3K-dependent manner and inhibit agonist-induced contraction by altering E-C coupling. Newly developed techniques by the PI using RGS knockdown of human ASM from normals and those with asthma will define how RGS proteins regulate cell growth. The use of precision cut lung slices (PCLS) and calcium imaging will define how RGS proteins regulate ASM contraction. Fluorescence lifetime imaging microscopy (FLIM) will identify RGS protein- protein interactions in biopsies and ASM cells from subjects with asthma. In Aim 1, levels of RGS protein expression and PI3K activation will be defined in ASM cells and in PCLS. The necessity and sufficiency of RGS proteins to regulate Src, PI3K, S6K1 activation and growth will be defined in ASM transfected with mutant RGS constructs. Using FLIM, alterations in ASM mass, RGS protein expression and p-p85 PI3K in endobronchial biopsies will be assessed. In Aim 2, the necessity and sufficiency of RGS proteins to regulate [Ca2+]i and contraction will be determined. RGS protein effects on agonist-induced PLC activation, myosin light chain phosphorylation, [Ca2+]i levels and RhoA kinase activation will be clarified in ASM transfected with mutant contracts. Our studies will identify the key mechanisms by which RGS proteins regulate ASM growth and contractility while providing insight into therapeutic targets to abrogate ASM hyperplasia and hyperresponsiveness in asthma. (End of Abstract)

描述（由申请人提供）：气道炎症和可逆的支气管收缩表征哮喘；但是，在严重疾病中，通常发生不可逆的气道阻塞和气道平滑肌（ASM）质量/生长的增加。生长因子需要激活SRC，磷脂酰肌醇3-激酶（PI3K）和S6K1以刺激ASM生长。我们的新数据表明，有丝分裂剂会增加G蛋白信号传导（RGS）蛋白的调节剂的ASM表达，该蛋白会使G蛋白失活并与Phosho-P85（P-P85）PI3K共定位以促进S6K1激活。重要的是，RGS蛋白敲低抑制了ASM的生长。有丝分裂原抑制激动剂引起的收缩和胞质钙水平降低[Ca2+] i。这些数据表明，特定的RGS蛋白是分子开关，以促进ASM生长，同时抑制激发 - 收缩（E-C）偶联。我们的中央假设指出，RGS蛋白以PI3K依赖性方式促进有丝分裂原诱导的ASM增殖，并通过改变E-C耦合来抑制激动剂诱导的收缩。 PI使用人类ASM的RGS敲低正常的ASM和哮喘患者的新技术将定义RGS蛋白如何调节细胞生长。精确切割的肺切片（PCL）和钙成像的使用将定义RGS蛋白如何调节ASM收缩。荧光寿命成像显微镜（FLIM）将鉴定活活检中的RGS蛋白质蛋白质相互作用和患有哮喘受试者的ASM细胞中的RGS蛋白质相互作用。在AIM 1中，将在ASM细胞和PCL中定义RGS蛋白表达和PI3K激活的水平。 RGS蛋白的必要性和充分性调节SRC，PI3K，S6K1激活和生长将在用突变体RGS构建体转染的ASM中定义。使用FLIM，将评估ASM质量，RGS蛋白表达和p-P85 PI3K的改变。在AIM 2中，将确定RGS蛋白调节[Ca2+] I和收缩的必要性和充分性。 RGS蛋白对激动剂诱导的PLC激活，肌球蛋白轻链磷酸化，[Ca2+] I水平和RhoA激酶激活的影响将在用突变合同转染的ASM中阐明。我们的研究将确定RGS蛋白调节ASM生长和收缩力的关键机制，同时提供对治疗靶标的见解，以消除ASM增生和哮喘中的高反应性。（抽象的结尾）