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Mechanical Strain Inhibits Airway Smooth Muscle Gene Transcription via Protein Kinase C Signaling

Department of Physiology, and Asthma/COPD Research Centre, Department of Internal Medicine, Section of Respiratory Diseases, and Department of Oral Biology, University of Manitoba, Winnipeg; and Biology of Breathing Research Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada

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

Mechanical strain affects airway myocyte phenotype, cytoskeletal architecture, proliferation, and contractile function. We hypothesized that (i) short-term mechanical strain modulates transcription of smooth muscle–specific gene promoters for SM22 and smooth muscle myosin heavy chain (smMHC); and (ii) strain-induced change is mediated by altered actin polymerization in association with activation of protein kinase C (PKC). Primary cultured canine tracheal myocytes were transiently transfected with luciferase reporter plasmids harboring a murine SM22, human smMHC, or artificial serum response factor (SRF)-specific gene promoter and then subjected to cyclic strain for 48 h. This strain protocol significantly reduced transcriptional activity of SM22 and smMHC promoters and an artificial SRF-dependent promoter by 55 ± 5.9%, 57 ± 6.4%, and 75 ± 7.9%, respectively, with concomitant reduction in F/G actin ratio by 31 ± 8%. PKC inhibitors, GF109203X or Gö6976, significantly attenuated these affects. Similar to strain, strain-independent activation of PKC inhibited SM22, smMHC, and SRF-dependent promoter activity by 61 ± 4%, 66 ± 5%, and 28 ± 15%, respectively, and reduced the F/G actin ratio by 30 ± 5%. Gel shift assay revealed that PKC activation led to decreased binding of the required transcription factor, SRF, to CArG elements in the SM22 promoter. These data suggest a previously unknown role for PKC isoforms in mechanosensitive signaling in airway myocytes that is associated with coordinated regulation of actin cytoskeletal dynamics and smooth muscle–specific gene transcription.

Abbreviations: airway smooth muscle, ASM • Dulbecco's modified Eagle's medium, DMEM • deoxyribonuclease, Dnase • electrophoretic mobility shift assay, EMSA • filamentous actin, F-actin • fetal bovine serum, FBS • globular actin, G-actin • nonessential amino acids, NEAA • o-nitrophenyl-ß-D-galactoside, ONPG • phosphate-buffered saline, PBS • protein kinase C, PKC • phorbol-12-myristate-13-acetate, PMA • smooth muscle myosin heavy chain, smMHC • serum response factor, SRF • Tris Borate EDTA, TBE

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