Rho Kinase and Airway Hyperresponsiveness

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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.