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Reactive oxygen species-dependent RhoA activation mediates collagen synthesis in hyperoxic lung fibrosis.

ABSTRACT: Lung fibrosis is an ultimate consequence of pulmonary oxygen toxicity in human and animal models. Excessive production and deposition of extracellular matrix proteins, e.g., collagen-I, is the most important feature of pulmonary fibrosis in hyperoxia-induced lung injury. In this study, we investigated the roles of RhoA and reactive oxygen species (ROS) in collagen-I synthesis in hyperoxic lung fibroblasts and in a mouse model of oxygen toxicity. Exposure of human lung fibroblasts to hyperoxia resulted in RhoA activation and an increase in collagen-I synthesis and cell proliferation. Inhibition of RhoA by C3 transferase CT-04, dominant-negative RhoA mutant T19N, or RhoA siRNA prevented hyperoxia-induced collagen-I synthesis. The constitutively active RhoA mutant Q63L mimicked the effect of hyperoxia on collagen-I expression. Moreover, the Rho kinase inhibitor Y27632 inhibited collagen-I synthesis in hyperoxic lung fibroblasts and fibrosis in mouse lungs after oxygen toxicity. Furthermore, the ROS scavenger tiron attenuated hyperoxia-induced increases in RhoA activation and collagen-I synthesis in lung fibroblasts and mouse lungs after oxygen toxicity. More importantly, we found that hyperoxia induced separation of guanine nucleotide dissociation inhibitor (GDI) from RhoA in lung fibroblasts and mouse lungs. Further, tiron prevented the separation of GDI from RhoA in hyperoxic lung fibroblasts and mouse lungs with oxygen toxicity. Together, these results indicate that ROS-induced separation of GDI from RhoA leads to RhoA activation with oxygen toxicity. ROS-dependent RhoA activation is responsible for the increase in collagen-I synthesis in hyperoxic lung fibroblasts and mouse lungs.

SUBMITTER: Kondrikov D

PROVIDER: S-EPMC3097427 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Reactive oxygen species-dependent RhoA activation mediates collagen synthesis in hyperoxic lung fibrosis.

Kondrikov Dmitry D Caldwell Ruth B RB Dong Zheng Z Su Yunchao Y

Free radical biology & medicine 20110323 11

Lung fibrosis is an ultimate consequence of pulmonary oxygen toxicity in human and animal models. Excessive production and deposition of extracellular matrix proteins, e.g., collagen-I, is the most important feature of pulmonary fibrosis in hyperoxia-induced lung injury. In this study, we investigated the roles of RhoA and reactive oxygen species (ROS) in collagen-I synthesis in hyperoxic lung fibroblasts and in a mouse model of oxygen toxicity. Exposure of human lung fibroblasts to hyperoxia re ...[more]

PMID: 21439370

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Project description:Myofibroblast transformation is a key process in the pathogenesis of lung fibrosis. We have previously reported that hyperoxia induces RhoA activation in HFL-1 lung fibroblasts and RhoA mediates collagen synthesis in hyperoxic lung fibrosis. In this study, we investigated the role of RhoA and actin cytoskeleton in hyperoxia-induced myofibroblast transformation. Exposure of HFL-1 lung fibroblasts to hyperoxia stimulated actin filament formation, shift of G-actin to F-actin, nuclear colocalization of myocardin-related transcription factor-A (MRTF-A), recruitment of MRTF-A to the α-smooth muscle actin (α-SMA) gene promoter, myofibroblast transformation, and collagen-I synthesis. Inhibition of RhoA by C3 transferase CT-04 or dominant-negative RhoA mutant T19N, and inhibition of ROCK by Y27632, prevented myofibroblast transformation and collagen-I synthesis. Moreover, inhibition of RhoA by CT-04 prevented hyperoxia-induced actin filament formation, shift of G-actin to F-actin, and nuclear colocalization of MRTF-A. In addition, disrupting actin filaments with cytochalasin D or scavenging reactive oxygen species (ROS) with tiron attenuated actin filament formation, nuclear colocalization of MRTF-A, myofibroblast transformation, and collagen-I synthesis. Furthermore, overexpression of constitutively active RhoA mutant Q63L or stabilization of actin filaments recapitulated the effects of hyperoxia on the actin cytoskeleton and nuclear colocalization of MRTF-A, myofibroblast transformation, and collagen-I synthesis. Interestingly, knocking down MRTF-A prevented hyperoxia-induced increase in the recruitment of MRTF-A to the serum response factor transcriptional complex on the α-SMA gene promoter, myofibroblast transformation, and collagen-I synthesis. Finally, Y27632 and tiron attenuated hyperoxia-induced increases in α-SMA and collagen-I in mouse lungs. Together, these results indicate that the actin cytoskeletal reorganization due to the ROS/RhoA-ROCK pathway mediates myofibroblast transformation and collagen synthesis in lung fibrosis of oxygen toxicity. MRTF-A contributes to the regulatory effect of the actin cytoskeleton on myofibroblast transformation during hyperoxia.

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Reactive oxygen species-dependent RhoA activation mediates collagen synthesis in hyperoxic lung fibrosis.

Publications

Reactive oxygen species-dependent RhoA activation mediates collagen synthesis in hyperoxic lung fibrosis.

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