Excess airway smooth muscle cell proliferation is thought to contribute to airflow obstruction in patients with asthma. The signaling mechanisms underlying cell proliferation are not completely understood. We have obtained preliminary data indicating that the 21 kD GTPase Rac1 is sufficient to induce transcriptional activation of two G1 cyclins, cyclin D1 and cyclin A, in bovine tracheal myocytes. Our studies also suggest that the effects of Rac1 are mediated by the intracellular generation of reactive oxygen intermediates. Finally, Rac1-induced cyclin D1 expression appears to be regulated by phosphatidylinositol 3- kinase (PI 3-kinase) and independent of ERK activation. To further characterize this newly-identified Rac1 signaling pathway, we propose the following Specific Aims. 1. Determine the precise role of Rho family proteins in bovine tracheal myocyte G1 cyclin expression. We will measure PDGF-induced Rac, cdc42 and Rho activities by assessment of GTP loading and protein translocation. We will determine the requirement and sufficiency of Rho family GTPases for the transcriptional activity of two inducible G1 cyclins, cyclin D1 and cyclin A. To accomplish this, cells will transiently co-transfected with cDNAs encoding dominant-negative and constitutively-active forms of Rho family proteins, in combination with plasmids encoding the human cyclin D1 and cyclin D1 and cyclin A promoters subcloned into luciferase reporter genes. 2: Characterize the downstream signaling intermediates and promoter elements required for Rac1-induced G1 cyclin expression. We will measure intracellular H2O2 concentrations by fluorescence imaging. We will also measure cyclin expression. We will measure intracellular H202 concentrations by fluorescence imaging. We will also measure the effects of antioxidants (ebselen), N-acetyl cystine and catalase) and NADPH oxidase inhibitors (diphenylene iodonium and N-terminal fragment of p67/phox) on Racl-induced cyclin D1 and cyclin A promoter activities. The requirement of MAP kinases will be determined using specific inhibitors )PD98059, SB203580, dominant-negative mutants). Cis-acting DNA sequences required for Rac1-induced cyclin D1 and A promoter activities will be identified using a series of deletion mutants. The importance of the Rac1 pathway for PDGF-induced cell cycle progression will be assessed by examining the effects of relevant inhibitors on G1 cyclin promoter activity, protein-DNA binding and protein abundance. 3. Define the upstream activators of the Rac1 signaling pathway in bovine tracheal myocytes. We will measure PI 3-kinase activity by extraction and separation of phosphoinositol-containing lipids. We will also measure the effects of specific inhibitors and activators of PI 3- kinase and Ras (wortmannin, LY294002, dominant negative and constitutively-active mutants) on Rac1 GTP loading, intracellular H2O2 generation, G1 cyclin promoter activity and protein binding, and G1 cyclin protein abundance. These studies may shed light on parallel mechanisms that may operate in asthma, and lead to therapeutic interventions.
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