title: The role of RhoA and cytoskeleton in myofibroblast transformation in hyperoxic lung fibrosis.
abstract: 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.
authors.affiliation: Department of Pharmacology & Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA; Department of Respiratory Medicine, The First People's Hospital of Yichang, Yichang, China; The People's Hospital, China Three Gorges University, Yichang, Hubei Province, China.
authors.forename: Jixiang
authors.initials: J
authors.lastname: Ni
authors.affiliation: Department of Cell Biology & Anatomy, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Dong
authors.affiliation: Department of Pharmacology & Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA.
authors.forename: Weihong
authors.initials: W
authors.lastname: Han
authors.affiliation: Department of Pharmacology & Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Kondrikov
authors.affiliation: Department of Pharmacology & Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA; Department of Medicine, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA; Vascular Biology Center, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA; Center for Biotechnology & Genomic Medicine, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA. Electronic address: ysu@gru.edu.
authors.forename: Yunchao
authors.initials: Y
authors.lastname: Su
mesh_terms: D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt / Q000494:pharmacology; D000199:Actins / Q000235:genetics; D021581:Active Transport, Cell Nucleus; D000818:Animals; D003094:Collagen / Q000096:biosynthesis; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000502:physiology*; D047630:Depsipeptides / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D018496:Hyperoxia / Q000150:complications*; D008297:Male; D051379:Mice; D058628:Myofibroblasts / Q000473:pathology*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000502:physiology; D011401:Promoter Regions, Genetic; D011658:Pulmonary Fibrosis / Q000209:etiology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015534:Trans-Activators / Q000502:physiology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: D000199:Actins; D047630:Depsipeptides; D016166:Free Radical Scavengers; C511173:Mrtfa protein, mouse; D017382:Reactive Oxygen Species; D015534:Trans-Activators; C544013:alpha-smooth muscle actin, mouse; C057531:jasplakinolide; D015638:Cytochalasin D; D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; D003094:Collagen; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C422943:Nox4 protein, mouse; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein
keywords: Collagen; Fibroblasts; Free radicals; Lung; MRTF-A; Oxygen toxicity; Reactive oxygen species