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Phophatidylinositol-3 Kinase/Mammalian Target of Rapamycin/p70^S6K Regulates Contractile Protein Accumulation in Airway Myocyte Differentiation

Departments of Medicine and Pediatrics, University of Chicago, Chicago, Illinois; Department of Physiology, and Section of Respiratory Diseases (Asthma/COPD Research Centre), University of Manitoba, and Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada; and Departments of Pediatrics and Communicable Diseases, Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan

Address correspondence to: Andrew J. Halayko, Ph.D., University of Manitoba, Section of Respiratory Diseases, RS321-810 Sherbrook Street, Winnipeg, MB, R3A 1R8 Canada. E-mail: ahalayk{at}cc.umanitoba.ca

Increased airway smooth muscle in airway remodeling results from myocyte proliferation and hypertrophy. Skeletal and vascular smooth muscle hypertrophy is induced by phosphatidylinositide-3 kinase (PI(3) kinase) via mammalian target of rapamycin (mTOR) and p70S6 kinase (p70^S6K). We tested the hypothesis that this pathway regulates contractile protein accumulation in cultured canine airway myocytes acquiring an elongated contractile phenotype in serum-free culture. In vitro assays revealed a sustained activation of PI(3) kinase and p70^S6K during serum deprivation up to 12 d, with concomitant accumulation of SM22 and smooth muscle myosin heavy chain (smMHC) proteins. Immunocytochemistry revealed that activation of PI3K/mTOR/p70^S6K occurred almost exclusively in myocytes that acquire the contractile phenotype. Inhibition of PI(3) kinase or mTOR with LY294002 or rapamycin blocked p70^S6K activation, prevented formation of large elongated contractile phenotype myocytes, and blocked accumulation of SM22 and smMHC. Inhibition of MEK had no effect. Steady-state mRNA abundance for SM22 and smMHC was unaffected by blocking p70^S6K activation. These studies provide primary evidence that PI(3) kinase and mTOR activate p70^S6K in airway myocytes leading to the accumulation of contractile apparatus proteins, differentiation, and growth of large, elongated contractile phenotype airway smooth muscle cells.

Abbreviations: Dulbecco's modified Eagle's medium, DMEM • glycogen synthase kinase-3ß, GSK-3ß • insulin-like growth factor-1, IGF-1 • mammalian target of rapamycin, mTOR • 3-phosphoinositide–dependent protein kinase, PDK1 • phosphatidylinositol-3 kinase, PI(3) kinase • smooth muscle myosin heavy chain, smMHC

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