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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL054685-04
Application #
2750480
Study Section
Lung Biology and Pathology Study Section (LBPA)
Project Start
1995-08-03
Project End
2000-07-31
Budget Start
1998-08-01
Budget End
1999-07-31
Support Year
4
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Chicago
Department
Pediatrics
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637
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