Novel Mechanisms of Smooth Muscle Beta2-receptor Regulation Relevant to Asthma

与哮喘相关的平滑肌β2受体调节新机制

• 批准号: 8661247
• 负责人: JEFFREY D HASDAY
• 金额: $ 36.38万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-19 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8661247
• 关键词: Acute; Address; Adrenergic Receptor; Affect; Agonist; Allergens; Allergic; Animal Model; Arrestins; Asthma; Binding; Binding Sites; Biochemical; Bronchial Spasm; Bronchodilation; Cell Line; Cell model; Cells; Chronic Obstructive Airway Disease; Complex; Coupled; Coupling; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Disease; Down-Regulation; Drug Targeting; Drug usage; Elements; Event; Family; Feedback; Functional disorder; G protein coupled receptor kinase; Goals; Grant; Human; In Vitro; Individual; Inflammatory; Interleukin-13; Knowledge; Ligands; Link; Lung diseases; Mediating; Methods; MicroRNAs; Modeling; Molecular; Mus; Muscle function; Mutate; Organ; Ovalbumin; Pathway interactions; Phosphorylation; Phosphorylation Site; Physiological; Physiology; Receptor Activation; Receptor Cell; Receptor Signaling; Regulation; Relaxation; Reporting; Role; Seeds; Series; Shapes; Signal Transduction; Site; Smooth Muscle; Smooth Muscle Myocytes; Specificity; Symptoms; System; Tachyphylaxis; Techniques; Testing; Transgenic Mice; Transgenic Organisms; Untranslated Regions; arrestin 2; base; effective therapy; human GPRK7 protein; improved; in vivo; insight; mRNA Expression; member; mouse model; mutant; novel; phosphoric diester hydrolase; protein expression; receptor; receptor binding; receptor downregulation; receptor expression; receptor function; receptor internalization; respiratory smooth muscle; response

DESCRIPTION (provided by applicant): Obstructive pulmonary diseases such as asthma and COPD are frequently treated with ¿-agonists. These drugs target the ¿2AR expressed on airway smooth muscle and are used for both long term control and rescue from acute bronchospasm. Of particular importance in human pathophysiology and treatment, ¿2AR signaling can be desensitized by b-agonist treatment, as well as the asthmatic inflammatory milieu. The mechanisms of this regulation are poorly understood, particularly in smooth muscle with associated physiologic function. These gaps in our knowledge have impaired our basic understanding of receptor-relaxation coupling and our ability to improve therapy for the treatment of bronchospasm. Mechanisms which will be explored in this grant include regulation by protein kinase A (PKA) and G-protein coupled receptor kinases (GRKs), and the let-7 family of microRNAs (miRNA). Our previous studies in airway smooth muscle point to a significant role for PKA phosphorylation of the receptor that alters GRK interaction with ¿2AR which had not been previously defined. Specific Aim 1 will delineate this mechanism of altered ¿2AR function with studies addressing phosphorylation, 2-arrestin recruitment, receptor internalization and phosphodiesterase (PDE) recruitment. Additionally, we have identified that ¿2AR baseline expression is directly regulated by let-7 miRNA. And, in an apparent feedback loop, let-7 miRNA is regulated by prolonged agonist activation of ¿2AR. In Specific Aim 2, transgenic mice will be generated with targeted expression on airway smooth muscle of WT ¿2AR and a mutant ¿2AR lacking the let-7 seed region in the 3"UTR. These mice will be studied to ascertain the significance of let-7 in establishing ¿2AR expression and its physiologic relevance in regulating bronchodilation. Additional studies will focus on the mechanisms and physiologic relevance of the apparent feedback loop, which may establish a new, and perhaps the most important, mechanism of ¿2AR downregulation by agonist. Loss of ¿2AR function in the asthmatic state has been reported in humans and animal models yet the mechanisms remain unknown, particularly with regard to linking cellular events to physiologic consequences. Evidence suggests several potential mechanisms that may involve PKA and GRK actions at the receptor, or altered let-7 miRNA expression. We have developed several transgenic mice that express mutated ¿2AR so that these potential mechanisms can be explored. In Specific Aim 3 transgenic mice lacking phosphorylation sites for PKA, GRK, PKA/GRK and the let-7 binding domains will be crossed into the inducible IL-13 allergic mouse model with confirmatory studies in the ovalbumin (OVA) allergen induced mouse model. Results from these various transgenic mice will define the specific mechanisms relevant to ¿2AR dysfunction in the asthmatic state. Taken together, these proposed studies will combine molecular, cellular and transgenic approaches to define mechanisms of ¿2AR function and regulation relevant to asthma.

描述（由申请人提供）：阻塞性肺疾病，如哮喘和慢性阻塞性肺病，经常使用激动剂治疗。这些药物靶向气道平滑肌上表达的¿2AR，用于急性支气管痉挛的长期控制和抢救。在人类病理生理和治疗中特别重要的是，ar信号可以通过b激动剂治疗脱敏，以及哮喘炎症环境。这种调节的机制尚不清楚，特别是与平滑肌相关的生理功能。这些知识上的空白损害了我们对受体-松弛耦合的基本理解，也损害了我们改善支气管痉挛治疗的能力。该基金将探索的机制包括蛋白激酶A （PKA）和g蛋白偶联受体激酶（GRKs）以及let-7 microrna家族（miRNA）的调控。我们之前对气道平滑肌的研究表明，PKA磷酸化的受体改变了GRK与¿2AR的相互作用，这在以前没有被定义。特异性目标1将通过研究磷酸化、2-抑制蛋白募集、受体内化和磷酸二酯酶（PDE）募集来描述这种改变的ar功能机制。此外，我们已经确定了let-7 miRNA直接调节¿2AR的基线表达。并且，在一个明显的反馈回路中，let-7 miRNA受持续激活的激动剂调控。在Specific Aim 2中，将在气道平滑肌上靶向表达WT¿2AR和缺乏3”UTR中let-7种子区的突变型¿2AR。将对这些小鼠进行研究，以确定let-7在建立¿2AR表达及其调节支气管扩张的生理相关性中的意义。进一步的研究将集中于表观反馈回路的机制和生理相关性，这可能会建立一个新的，也许是最重要的，激动剂下调¿2AR的机制。在人类和动物模型中已经报道了哮喘状态下ar功能的丧失，但其机制尚不清楚，特别是关于将细胞事件与生理后果联系起来。有证据表明，几种潜在的机制可能涉及PKA和GRK对受体的作用，或改变let-7 miRNA的表达。我们已经开发了几种表达突变的¿2AR的转基因小鼠，以便探索这些潜在的机制。在Specific Aim 3中，缺乏PKA、GRK、PKA/GRK和let-7结合域磷酸化位点的转基因小鼠将被交叉到可诱导的IL-13过敏小鼠模型中，并在卵清蛋白（OVA）过敏原诱导的小鼠模型中进行验证研究。这些不同转基因小鼠的结果将确定哮喘状态下与¿2AR功能障碍相关的具体机制。总之，这些拟议的研究将结合分子、细胞和转基因方法来确定与哮喘相关的ar功能和调控机制。