Rho Kinase and Airway Hyperresponsiveness

描述（由申请方提供）：Rho激酶或ROCK通过对肌动蛋白细胞骨架的作用调节平滑肌、嗜酸性粒细胞和淋巴细胞功能。因此，ROCK通路可能是一个重要的，仍然相对未开发的治疗哮喘的目标。存在两种ROCK同种型，ROCK 1和ROCK 2，它们差异表达，可能受到差异调节，并且在它们靶向的一些底物中不同。我们产生了杂合子ROCK 1（ROCK 1）或ROCK 2（ROCK 2）缺陷小鼠，其ROCK 1或ROCK 2分别减少50%。初步数据表明，在卵清蛋白（OVA）致敏和激发小鼠的肺中ROCK活化，并显示出与野生型（WT）小鼠相比，ROCK 2小鼠中OVA诱导的AHR显著降低，尽管两种品系中Th 2细胞因子表达相似。与此相反，在ROCK 1小鼠中，OVA诱导的Th 2细胞因子表达和AHR几乎被消除。与ROCK 1小鼠相比，ROCK 2小鼠的平滑肌细胞和组织的收缩性和增殖能力降低。因此，我们假设ROCK在哮喘的发病机制中至关重要，但在哮喘效应细胞中具有不同的作用。为了检验这一假设，在目标1中，我们将评估（在WT、ROCK 1、ROCK 2和ROCK 1/2小鼠中）在OVA致敏和激发后肺和胸淋巴结中ROCK表达和活性的时程和位点。我们还将测量RhoA和RhoGEF，ROCK信号通路上游的分子，以及MBS，CPI-17和Ef 1a的磷酸化，ROCK的靶点。将评估OVA致敏和激发对气道反应性、肺部炎症、Th 2细胞因子表达和气道重塑的影响。在目标2中，我们将评估ROCK在OVA攻击期间T淋巴细胞中的作用。将来自ROCK充足小鼠的T淋巴细胞连续转移到ROCK 1和ROCK 2小鼠中。我们还将在体内和体外研究ROCK 1或ROCK 2不足对OVA刺激的淋巴细胞增殖的影响。在目标3中，我们将使用通过将条件性ROCK 1和ROCK 2敲除小鼠（floxed小鼠）培育成在平滑肌特异性肌球蛋白重链启动子控制下表达Cre重组酶的转基因小鼠而产生的平滑肌特异性ROCK 1和ROCK 2敲除，评估ROCKs在OVA激发期间在平滑肌中的作用。我们的初步数据表明，这些小鼠是可行的，发育正常。我们还将检验ROCK 2是平滑肌细胞骨架重塑和增殖所必需的假设，以及这些事件需要ROCK 2介导的Ef 1a、MBS和CPI-17磷酸化。了解ROCK在哮喘小鼠模型中的作用可能会导致预防这种疾病的新策略。 公共卫生相关性：Rho激酶或ROCK通过影响肌动蛋白细胞骨架调节平滑肌、嗜酸性粒细胞和淋巴细胞功能。因此，ROCK通路可能是一个重要的，仍然相对未开发的治疗哮喘的目标。了解ROCK在哮喘小鼠模型中的作用可能会导致预防这种疾病的新策略。

DESCRIPTION (provided by applicant): The Rho kinases or ROCKs, regulate smooth muscle, eosinophil, and lymphocyte function via effects on the actin cytoskeleton. Thus, the ROCK pathway may be an important and still relatively unexplored therapeutic target in asthma. Two ROCK isoforms exist, ROCK1 and ROCK2, that are differentially expressed, may be differentially regulated, and differ in some of the substrates they target. We generated heterozygous ROCK1 (ROCK1) or ROCK2 (ROCK2) deficient mice that have a 50% reduction in ROCK1 or ROCK2 respectively. Preliminary data indicate ROCK activation in lungs of ovalbumin (OVA) sensitized and challenged mice and show a marked reduction in OVA-induced AHR in ROCK2 vs wildtype (WT) mice despite similar Th2 cytokine expression in the two strains. In contrast, OVA-induced Th2 cytokine expression and AHR were virtually abolished in ROCK1 mice. Smooth muscle cells and tissues from ROCK2 mice had reduced contractility and proliferative capacity compared to ROCK1 mice. Thus, it is our hypothesis that ROCKs are critically important in the pathogenesis of asthma but have different roles in asthma effector cells. To test this hypothesis, in aim 1 we will assess (in WT, ROCK1, ROCK2, and ROCK1/2 mice) the time course and locus of ROCK expression and activity in the lungs and thoracic lymph nodes following OVA sensitization and challenge. We will also measure RhoA and RhoGEFs, molecules upstream in the ROCK signaling pathway, as well as phosphorylation of MBS, CPI-17, and Ef1a, targets of ROCK. Effects of OVA sensitization and challenge on airway responsiveness, pulmonary inflammation, Th2 cytokine expression, and airway remodeling will be assessed. In aim 2, we will assess the role of ROCKs in T lymphocytes during OVA challenge. Adoptive transfer of T lymphocytes derived from ROCK sufficient mice into ROCK1 and ROCK2 mice will be performed. We will also examine the impact of ROCK1 or ROCK2 insufficiency on OVA- stimulated lymphocyte proliferation, both in vivo and in vitro. In aim 3, we will assess the role of ROCKs in smooth muscle during OVA challenge using smooth muscle specific ROCK1 and ROCK2 knockdowns generated by breeding conditional ROCK1 and ROCK2 knockout mice (floxed mice) to transgenic mice expressing Cre recombinase under control of the smooth muscle-specific myosin heavy chain promoter. Our preliminary data indicate that these mice are viable and develop normally. We will also test the hypothesis that ROCK2 is required for smooth muscle cytoskeletal remodeling, and proliferation, and that these events require ROCK2 mediated phosphorylation of Ef1a, MBS, and CPI-17. Understanding the role of ROCKs in mouse models of asthma could lead to new strategies for preventing this disease. PUBLIC HEALTH RELEVANCE: The Rho kinases or ROCKs, regulate smooth muscle, eosinophil, and lymphocyte function via effects on the actin cytoskeleton. Thus, the ROCK pathway may be an important and still relatively unexplored therapeutic target in asthma. Understanding the role of ROCKs in mouse models of asthma could lead to new strategies for preventing this disease.