Stretch augments TGF-β1 expression through RhoA/ROCK1/2, PTK, and PI3K in airway smooth muscle cells

Junaith Mohamed, Aladin M. Boriek

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24 Citations (Scopus)

Abstract

Transforming growth factor-β1 (TGF-β1) 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-β1 gene expression in human airway smooth muscle cells (HASMCs) remain elusive. Here, we show that mechanical stretch of HASMCs augmented TGF-β1 expression through a de novo RNA synthesis mechanism. Luciferase reporter assays revealed that stretch-induced TGF-β1 expression was mediated through the enhanced activation of TGF-β1 promoter. Interestingly, selective inhibitors of PTK, PI3K, or MEK1/2 attenuated TGF-β1 expression through blocking ERK1/2 phosphorylation and TGF-β1 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-β1 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-β1 expression through repressing the DNA binding activity of AP-1. Site-directed mutagenesis demonstrated that two AP-1 binding sites in the TGF-β1 promoter region are responsible for stretch-induced TGF-β1 expression. Overall, in HASMCs, mechanical stretch plays an important role in TGF-β1 gene upregulation through a stretch-induced signaling pathway, which could be a potential therapeutic intervention for TGF-β1-induced pathogenesis in asthma.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume299
Issue number3
DOIs
StatePublished - Sep 1 2010
Externally publishedYes

Fingerprint

Transforming Growth Factors
Phosphatidylinositol 3-Kinases
Smooth Muscle Myocytes
Transcription Factor AP-1
GTP Phosphohydrolases
Asthma
Phosphorylation
DNA
Transferases
Site-Directed Mutagenesis
Guanosine Triphosphate
Luciferases
Genetic Promoter Regions
Oligonucleotides
Small Interfering RNA
Transfection
Up-Regulation
Binding Sites
RNA

All Science Journal Classification (ASJC) codes

  • Cell Biology
  • Physiology
  • Medicine(all)
  • Physiology (medical)
  • Pulmonary and Respiratory Medicine

Cite this

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title = "Stretch augments TGF-β1 expression through RhoA/ROCK1/2, PTK, and PI3K in airway smooth muscle cells",
abstract = "Transforming growth factor-β1 (TGF-β1) 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-β1 gene expression in human airway smooth muscle cells (HASMCs) remain elusive. Here, we show that mechanical stretch of HASMCs augmented TGF-β1 expression through a de novo RNA synthesis mechanism. Luciferase reporter assays revealed that stretch-induced TGF-β1 expression was mediated through the enhanced activation of TGF-β1 promoter. Interestingly, selective inhibitors of PTK, PI3K, or MEK1/2 attenuated TGF-β1 expression through blocking ERK1/2 phosphorylation and TGF-β1 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-β1 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-β1 expression through repressing the DNA binding activity of AP-1. Site-directed mutagenesis demonstrated that two AP-1 binding sites in the TGF-β1 promoter region are responsible for stretch-induced TGF-β1 expression. Overall, in HASMCs, mechanical stretch plays an important role in TGF-β1 gene upregulation through a stretch-induced signaling pathway, which could be a potential therapeutic intervention for TGF-β1-induced pathogenesis in asthma.",
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AB - Transforming growth factor-β1 (TGF-β1) 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-β1 gene expression in human airway smooth muscle cells (HASMCs) remain elusive. Here, we show that mechanical stretch of HASMCs augmented TGF-β1 expression through a de novo RNA synthesis mechanism. Luciferase reporter assays revealed that stretch-induced TGF-β1 expression was mediated through the enhanced activation of TGF-β1 promoter. Interestingly, selective inhibitors of PTK, PI3K, or MEK1/2 attenuated TGF-β1 expression through blocking ERK1/2 phosphorylation and TGF-β1 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-β1 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-β1 expression through repressing the DNA binding activity of AP-1. Site-directed mutagenesis demonstrated that two AP-1 binding sites in the TGF-β1 promoter region are responsible for stretch-induced TGF-β1 expression. Overall, in HASMCs, mechanical stretch plays an important role in TGF-β1 gene upregulation through a stretch-induced signaling pathway, which could be a potential therapeutic intervention for TGF-β1-induced pathogenesis in asthma.

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