Rho Kinase and Airway Hyperresponsiveness

描述(由申请人提供)：Rho激酶或岩石，通过对肌动蛋白细胞骨架的影响来调节平滑肌、嗜酸性粒细胞和淋巴细胞的功能。因此，ROCK通路可能是哮喘治疗的一个重要且相对未被探索的靶点。存在两种岩石异构体，ROCK1和ROCK2，它们有不同的表达，可能受到不同的调控，而且它们针对的一些底物也不同。我们产生了ROCK1(ROCK1)或ROCK2(ROCK2)杂合子缺陷小鼠，它们的ROCK1或ROCK2分别减少了50%。初步数据显示，卵蛋白(OVA)致敏和攻击小鼠的肺部ROCK激活，并显示OVA诱导的ROCK2与野生型(WT)小鼠的AHR显著减少，尽管这两个品系的Th2细胞因子表达相似。相比之下，OVA诱导的Th2细胞因子表达和AHR在ROCK1小鼠中几乎被取消。与ROCK1小鼠相比，ROCK2小鼠的平滑肌细胞和组织的收缩和增殖能力降低。因此，我们的假设是，岩石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。为了验证这一假设，在目标1中，我们将评估(在WT、ROCK1、ROCK2和ROCK1/2小鼠中)OVA致敏和挑战后ROCK在肺部和胸部淋巴结中的表达和活性的时间进程和位置。我们还将测量ROCK信号通路上游的RhoA和RhoGEF分子，以及ROCK靶标MBS、CPI-17和Ef1a的磷酸化。将评估OVA增敏和挑战对呼吸道反应性、肺部炎症、Th2细胞因子表达和气道重塑的影响。在目标2中，我们将评估ROCKS在OVA攻击过程中对T淋巴细胞的作用。将ROCK充足小鼠的T淋巴细胞过继转移到ROCK1和ROCK2小鼠体内。我们还将在体内和体外检测ROCK1或ROCK2不足对OVA刺激的淋巴细胞增殖的影响。在目标3中，我们将利用培育条件性ROCK1和ROCK2基因敲除小鼠产生的平滑肌特异性ROCK1和ROCK2基因敲除来评估在OVA攻击过程中岩石在平滑肌中的作用。转基因小鼠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。我们的初步数据表明，这些小鼠是存活的，发育正常。我们还将测试这一假设，即ROCK2是平滑肌细胞骨架重塑和增殖所必需的，并且这些事件需要ROCK2介导的Ef1a、MBS和CPI-17的磷酸化。了解岩石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。 与公共健康相关：Rho激酶或岩石，通过对肌动蛋白细胞骨架的影响来调节平滑肌、嗜酸性粒细胞和淋巴细胞的功能。因此，ROCK通路可能是哮喘治疗的一个重要且相对未被探索的靶点。了解岩石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.