Published ahead of print on May 18, 2005, doi:10.1165/rcmb.2004-0411OC Am. J. Respir. Cell Mol. Biol., Volume 33, Number 2, August 2005, 195-202 A more recent version of this article appeared on August 1, 2005
Submitted on December 22, 2004 4E-BP Phosphorylation and eIF4E Release are Required for Airway Smooth Muscle HypertrophyLimei Zhou1,1 Department of Pediatrics and Communicable Diseases and Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA, 2 Department of Pediatrics, University of Chicago, Chicago, IL, USA, 3 Department of Cell and Developmental Biology and Department of Medicine, University of Michigan, Ann Arbor, MI, USA * To whom correspondence should be addressed. E-mail: mhershen{at}umich.edu.
The molecular mechanisms of airway smooth muscle hypertrophy, a feature of severe asthma, are poorly understood. We previously established a conditionally-immortalized human bronchial smooth muscle cell line with a temperature-sensitive SV40 large T antigen. Temperature shift and loss of large T cause G1-phase cell cycle arrest that is accompanied by increased airway smooth muscle cell size. In the present study, we hypothesized that phosphorylation of eukaryotic initiation factor-4E (eIF4E)-binding protein (4E-BP), which subsequently releases eIF4E and initiates cap-dependent mRNA translation, was required for airway smooth muscle hypertrophy. Treatment of cells with chemical inhibitors of PI 3-kinase and mTOR blocked protein synthesis and cell growth while decreasing the phosphorylation of 4E-BP and increasing the binding of 4E-BP to eIF4E, consistent with the notion that 4E-BP1 phosphorylation and eIF4E function are required for hypertrophy. To test this directly, we infected cells with a retrovirus encoding a phosphorylation site mutant of 4E-BP1 (AA-4E-BP-1) that dominantly inhibits eIF4E. Upon temperature shift, cells infected with AA-4E-BP-1, but not empty vector, failed to undergo hypertrophic growth. We conclude that phosphorylation of 4E-BP, eIF4E release and cap-dependent protein synthesis are required for hypertrophy of human airway smooth muscle cells.
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