Stretch augments TGF-beta1 expression through RhoA/ROCK1/2, PTK, and PI3K in airway smooth muscle cells.
Transforming growth factor-beta1 (TGF-beta1) expression in smooth muscle cells may play an important role in the pathogenesis of asthma. However, mechanisms that are involved in the regulation of TGF-beta1 gene expression in human airway smooth muscle cells (HASMCs) remain elusive. Here, we show that mechanical stretch of HASMCs augmented TGF-beta1 expression through a de novo RNA synthesis mechanism. Luciferase reporter assays revealed that stretch-induced TGF-beta1 expression was mediated through the enhanced activation of TGF-beta1 promoter. Interestingly, selective inhibitors of PTK, PI3K, or MEK1/2 attenuated TGF-beta1 expression through blocking ERK1/2 phosphorylation and TGF-beta1 promoter activity in response to stretch. In addition, stretch rapidly and transiently augmented GTP-bound RhoA and Rac1 but not Cdc42 GTPase. Either blockade of RhoA GTPase using C3 transferase, ROCK1/2 using Y27632, or knockdown of endogenous RhoA using RhoA siRNA attenuated stretch-induced TGF-beta1 expression through the inhibition of ERK1/2 phosphorylation. Moreover, stretch augmented DNA binding activity of AP-1 in a time-dependent manner. Either treatment of HASMCs with the inhibitors of RhoA, ROCK1/2, PTK, PI3K, MEK1/2, or AP-1 or transfection of HASMCs with AP-1 decoy oligonucleotide attenuated stretch-induced TGF-beta1 expression through repressing the DNA binding activity of AP-1. Site-directed mutagenesis demonstrated that two AP-1 binding sites in the TGF-beta1 promoter region are responsible for stretch-induced TGF-beta1 expression. Overall, in HASMCs, mechanical stretch plays an important role in TGF-beta1 gene upregulation through a stretch-induced signaling pathway, which could be a potential therapeutic intervention for TGF-beta1-induced pathogenesis in asthma.
Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA.
Junaith Shaik
JS
Mohamed
Aladin M
AM
Boriek
D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012137:Respiratory System / Q000166:cytology / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013314:Stress, Mechanical; D018808:Transcription Factor AP-1 / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D053773:Transforming Growth Factor beta1; D004247:DNA; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; C515851:ROCK1 protein, human; C515852:ROCK2 protein, human; D054460:rho-Associated Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein