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

描述(申请人提供)：阻塞性肺疾病，如哮喘和慢性阻塞性肺病，经常使用非激动剂治疗。这些药物针对的是气道平滑肌上表达的2AR，用于长期控制和抢救急性支气管痉挛。在人类病理生理学和治疗中特别重要的是，2AR信号可以被b-激动剂治疗以及哮喘的炎症环境脱敏。这一调节机制尚不清楚，特别是在具有相关生理功能的平滑肌中。我们知识中的这些差距损害了我们对受体-松弛偶联的基本理解，以及我们改进治疗支气管痉挛的能力。这项资助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)的调节，以及let-7家族的microRNAs(MiRNA)。我们之前对气道平滑肌的研究指出，PKA对受体的磷酸化具有重要作用，它改变了GRK与2AR的相互作用，这是以前没有定义的。具体目标1将通过研究磷酸化、2-arrestin募集、受体内化和磷酸二酯酶(PDE)募集来描述2AR功能改变的机制。此外，我们还发现2AR的基线表达直接受let-7 miRNA的调节。而且，在一个明显的反馈环中，let-7 miRNA受到2AR激动剂长期激活的调节。在特定的目的2中，将产生靶向表达WT？2AR的转基因小鼠和在3“非编码区缺失let-7种子区的突变体？2AR。将对这些小鼠进行研究，以确定let-7在建立2AR表达方面的意义及其在调节支气管扩张方面的生理相关性。其他研究将集中在表观反馈环的机制和生理相关性上，这可能建立一种新的，也可能是最重要的，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的2AR功能丧失，但其机制仍不清楚，特别是关于将细胞事件与生理后果联系起来的机制。有证据表明，一些潜在的机制可能涉及到受体上的PKA和GRK作用，或者是let-7miRNA表达的改变。我们已经开发了几只表达突变的2AR的转基因小鼠，以便探索这些潜在的机制。针对特定目的，将3只缺乏PKA、GRK、PKA/GRK磷酸化位点和let-7结合域的转基因小鼠交叉进入诱导性IL-13过敏小鼠模型，并在卵清蛋白(OVA)变应原诱导小鼠模型中进行确证研究。来自这些不同转基因小鼠的结果将确定与哮喘状态下的2AR功能障碍相关的特定机制。综上所述，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功能和调节机制。

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.