Increased airway smooth muscle mass is thought to contribute to the airway hyperresponsiveness observed in patients with asthma and with bronchopulmonary dysplasia (BPD), a chronic airways disease of prematurely-born infants. The potential importance of abnormal airway smooth muscle cell proliferation in the pathogenesis of airways disease highlights the need for a precise understanding of the early events involved in airway smooth muscle mitogenesis. The major objective of this application is to clarify the roles of mitogen-activated protein (MAP) kinase and other signaling intermediates (ras, raf-1 and MEK-1) in airway myocyte proliferation. To achieve this overall goal, we propose the following Specific Aims: (l) Identify the individual roles of ras, raf-1 and MEK-1 in the activation of MAP kinase in bovine tracheal myocytes. Measurements of ras, raf-1 and MEK-1 activation, as well as that of transfectant p42mapk activity after co-transfection with either dominant- negative or constitutively-active forms of either ras, raf-1 or MEK-1, will be performed to test the hypotheses that (i) MAP kinase activation may be elicited via both ras-dependent and ras-independent pathways; (ii) MAP kinase activation may be elicited via both raf-dependent and raf- independent pathways; and (iii) activation of MEK-l is both required and sufficient for p42mapk activation. (2) Determine the precise roles of MEK-1 and MAP kinase in bovine tracheal myocyte mitogenesis. Immunolocalization of MAP kinase, as well as stable transfections of bovine tracheal myocytes with inducible mutants of MEK- l, the activation of which directly precedes and is sufficient for activation of p42maPk, will be performed to test the hypotheses that: (i) mitogenesis is associated with nuclear translocation of MAP kinase; and (ii) sustained activation of MEK-1 and MAP kinase is sufficient for bovine tracheal myocyte cell cycle traversal. (3) Determine interventions which enhance or inhibit MAP kinase activation, and as a consequence, cell cycle traversal. The effects of H2O2, phosphatase inhibitors and agents which mimic or elicit intracellular cAMP accumulation on MAP kinase-related signaling intermediates and cell cycle traversal will be assessed, both by direct measurement of enzymatic activities and by measuring the activity of transfectant p42mapk after co-transfection with dominant negative mutants of either ras, raf-1, or MEK-1. These experiments will test the hypotheses that: (i) hydrogen peroxide (H2O2) and phosphatase inhibitors enhance growth factor-induced MAP kinase activation and cell cycle traversal, H2O2 via successive activation of protein kinase C, raf- 1 and MEK-1; and (ii) cAMP may attenuate MAP kinase activation and cell cycle traversal by the inhibition of raf-1, via stimulation of protein kinase A. Insights from this work may shed light on parallel mechanisms that may operate in asthma and BPD, and lead to therapeutic interventions.
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