Integrin-mediated Regulation of Airway Smooth Muscle

描述（由申请人提供）：α 9 <$1整联蛋白在气道平滑肌中高度表达。我们只在平滑肌细胞中缺乏这种整合素的小鼠具有明显的体内气道高反应性，这些小鼠的肺切片具有增加的气道狭窄。我们以前已经表明，α 9亚基胞质结构域直接结合酶精胺/亚精胺乙酰转移酶（SSAT），在高级多胺的催化速率限制步骤，这种协会和多胺催化剂是重要的调节剂α 9 <$1功能。SSAT的药理学稳定作用也增强了α 9 β 1依赖性对气道平滑肌收缩的预防作用.在本申请中，我们将使用4个平行的实验系统（体内AHR、气管环收缩、肺切片中的气道狭窄和气道平滑肌细胞缩短）系统地评价α 9 <$1对气道平滑肌对多种收缩激动剂和异丙肾上腺素诱导的舒张的反应的影响。我们将利用α 9和SSAT的各种突变体和嵌合构建体，用SSAT敲除小鼠、催化失活的SSAT突变体和遗传和药理学抑制剂进行体内和体外研究，以彻底检查α 9与SSAT的相互作用对这种应答的贡献。平滑肌中的力产生依赖于钙依赖性肌动蛋白-肌球蛋白交联和平行肌动蛋白聚合，我们将系统地评估a9 <$1对这些途径的影响。由于高级多胺的两个主要胞质效应是阻止钾通过Kir通道流出和激活脂质激酶PIP 5 K1？，我们将特别关注Kir通道和PIP 5 K1的作用。在这个过程中。拟议的研究将测试总体假设，即结扎的α 9 <$1通常通过浓缩SSAT、分解代谢多胺并因此抑制钾流出和/或PIP 2产生而对气道狭窄起制动作用，从而导致钙波动减少、肌动蛋白-肌球蛋白交联减少和/或肌动蛋白聚合受损。这一途径的异常，无论是后天的还是遗传的，都可能导致哮喘等以气道狭窄加剧为特征的疾病。 公共卫生相关性： 哮喘的主要症状之一是气道狭窄。我们发现，只有在平滑肌中缺乏特定蛋白质（整合素）的小鼠具有过度的气道狭窄。在这项提案中，我们将确定这种整合素通常如何防止气道狭窄，这是一个可能在哮喘等疾病中受到干扰的过程。

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.