MECHANISMS REGULATING AIRWAY MUSCLE GROWTH

DESCRIPTION (Applicant's abstract): The goal of this proposal is to define the molecular signaling process that regulate mitogen-induced growth of human airway smooth muscle (ASM) cells. This question is critical to an understanding of the pathogenesis of asthma, where increased ASM mass is a well documented pathologic finding. The PI's studies establish that activation of the P21ras, phosphotidylinositol 3-kinase (PtdIns 3-kinase) and p70s6k are necessary to stimulate agonist-induced ASM cell growth. The PI proposes to elucidate the upstream regulators and downstream effectors of Ras, PtdIns 3-kinase and p70s6k and establish their interdependence in the transduction of mitogenic signals. Newly-developed techniques for cell transfection and microinjection will enable analysis of both kinase activation and proliferation in both cultures and individual ASM cells. The central hypothesis of the proposal is that p21ras, PtdIns 3-kinase, and p70s6k are essential, interdependent modulators of ASM growth. P21ras is regulated upstream by Src and activates PtdIns 3-kinase, whereas Protein Kinase B (PKB) and phosphotidylinositol-dependent kinase 1 (PDK1) serve critical roles as both downstream effectors of PtdIns 3-kinase and upstream modulators of p70s6k. To test this hypothesis, in Aim 1, Src and Src-dependent Ras activation by agonists will be characterized using in vitro kinase assays. The effects of Src inhibition on agonist-induced DNA synthesis will be determined in cell microinjection with neutralizing Src antibodies, or in cultures transfected with dominant negative (D-N) Src cDNA. Ras activation and DNA synthesis measured in cells transfected with constitutively-active (CA) Src will define whether Src is sufficient to stimulate mitogenesis. In Aim 2, the role of G-beta-gamma subunits, Src, and Ras as mediators of PtdIns 3-kinase activity and PtdIns 3-kinase-dependent growth will be examined in cells transfected with G-beta-gamma sequestrants, CA and D-N Src and Ras. The role of PKB and PDK1 as downstream effectors of PtdIns 3-kinase-dependent growth will be examined in cells transfected with CA or D-N PKB or PDK1. In Aim 3, to determine whether PKB or PDK1 is sufficient or necessary to stimulate p70s6k, p70s6k activation will be measured in cells transfected with CA PKB or PDK1 or in agonist-treated cells transfected with D-N PKB or PDK1. To determine whether PKC-zeta is necessary or sufficient to stimulate p70s6k and cell growth will be determined in cells transfected with D-N PKC-zeta and treated with agonists, and in cells transfected with CA PKC-zeta. These studies will define the mechanism by which agonists induce ASM cell growth such that new issues in the pathogenesis of asthma will be addressed and therapeutic measures to prevent these alterations can be developed.