Integrin-mediated Regulation of Airway Smooth Muscle

描述(申请人提供)：A9？1整合素在呼吸道平滑肌中高表达。我们培育的只在平滑肌细胞中缺乏这种整合素的小鼠在体内有明显的气道高反应性，来自这些小鼠的肺切片增加了气道狭窄。我们以前已经证明，A9亚单位胞浆结构域直接与精胺/亚精胺乙酰转移酶(SSAT)结合，SSAT是高阶多胺分解代谢的限速步骤，这种结合和多胺分解代谢是A9？1功能的重要调节因子。SSAT的药理稳定性也增强了A9？1依赖的对气道平滑肌收缩的预防作用。在目前的应用中，我们将使用4个平行的实验系统(活体AHR、气管环收缩、肺切片上的气道狭窄和气道平滑肌细胞的缩短)来系统地评价A9？1对多种收缩激动剂和异丙肾上腺素引起的松弛反应的影响。我们将利用A9和SSAT的各种突变和嵌合构建物、SSAT基因敲除小鼠的体内和体外研究、催化失活的SSAT突变体以及遗传和药物抑制剂来彻底研究A9与SSAT的相互作用对这一反应的贡献。肌力的产生依赖于钙依赖的肌动蛋白-肌球蛋白交叉桥联和平行肌动蛋白聚合，我们将系统地评估A9？1在每一条途径上的作用。由于高阶多胺的两个主要胞浆作用是通过KIR通道阻止钾外流和激活脂蛋白激酶PIP5K1？，因此我们将特别关注KIR通道和PIP5K1？的作用。在这个过程中。拟议的研究将检验这样的总体假设，即结扎的A9？1通常通过浓缩SSAT、分解多胺从而抑制钾外流和/或PIP2的产生来抑制气道狭窄，从而减少钙振荡，减少肌球蛋白-肌球蛋白的交叉桥联和/或损害肌动蛋白聚合。这一途径的异常，无论是后天的还是遗传的，都可能导致哮喘等疾病，这些疾病的特征是气道狭窄加剧。 公共卫生相关性： 项目夸大的气道狭窄是哮喘的一个主要特征。我们发现，只有在平滑肌中缺乏特定蛋白质(整合素)的小鼠会夸大呼吸道狭窄。在这项提案中，我们将确定这种整合素通常如何防止呼吸道狭窄，这一过程可能会在哮喘等疾病中受到干扰。

DESCRIPTION (provided by applicant): The a9¿1 integrin is highly expressed in airway smooth muscle. Mice we have generated lacking this integrin only in smooth muscle cells have marked in vivo airway hyperresponsiveness and lung slices from these mice have increased airway narrowing. We have previously shown that the a9 subunit cytoplasmic domain directly binds the enzyme spermine/spermidine acetyltransferase (SSAT), the rate limiting step in catabolism of higher order polyamines, and that this association and polyamine catabolism are important modulators of a9¿1 function. Pharmacologic stabilization of SSAT also augments a9¿1-dependent prevention of airway smooth muscle contraction. In the current application we will systematically evaluate the effects of a9¿1 on responses of airway smooth muscle to multiple contractile agonists and to isoproterenol-induced relaxation using 4 parallel experimental systems (in vivo AHR, tracheal ring contraction, airway narrowing in lung slices and shortening of airway smooth muscle cells). We will utilize a variety of mutant and chimeric constructs of a9 and SSAT, in vivo and in vitro studies with SSAT knockout mice, catalytically inactive SSAT mutants and genetic and pharmacologic inhibitors to thoroughly examine what contribution interaction of a9 with SSAT makes to this response. Force generation in smooth muscle depends on calcium-dependent actin-myosin cross-bridging and parallel actin polymerization, and we will systematically evaluate the effects of a9¿1 on each of these pathways. Because the two major cytosolic effects of higher order polyamines are prevention of potassium efflux through Kir channels and activation of the lipid kinase, PIP5K1?, we will pay special attention to the roles of Kir channels and PIP5K1? in this process. The proposed studies will test the overall hypothesis that ligated a9¿1 normally serves as a brake on airway narrowing by concentrating SSAT, catabolizing polyamines and thus inhibiting potassium efflux and/or PIP2 production, resulting in reduced calcium oscillations, decreased actin-myosin cross-bridging and/or impaired actin polymerization. Abnormalities in this pathway, either acquired or genetic, could contribute to diseases such as asthma that are characterized by enhanced airway narrowing. PUBLIC HEALTH RELEVANCE: Project Exaggerated airway narrowing is a central feature of asthma. We have found that mice lacking a specific protein (an integrin) only in smooth muscle have exaggerated airway narrowing. In this proposal we will determine how this integrin normally prevents airway narrowing, a process that could be perturbed in diseases such as asthma.