TY - JOUR
T1 - The role of RhoA and cytoskeleton in myofibroblast transformation in hyperoxic lung fibrosis
AU - Ni, Jixiang
AU - Dong, Zheng
AU - Han, Weihong
AU - Kondrikov, Dmitry
AU - Su, Yunchao
N1 - Funding Information:
This work was supported by NIH Grant HL088261 and HL115078 (to Y.S.), Flight Attendants Medical Research Institute Grants 072104 and 113018 (to Y.S.), and National Natural Science Foundation of China Grant NSFC 81200052 (to J.N.).
PY - 2013
Y1 - 2013
N2 - 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.
AB - 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.
KW - Collagen
KW - Fibroblasts
KW - Free radicals
KW - Lung
KW - MRTF-A
KW - Oxygen toxicity
KW - Reactive oxygen species
UR - http://www.scopus.com/inward/record.url?scp=84878829007&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84878829007&partnerID=8YFLogxK
U2 - 10.1016/j.freeradbiomed.2013.03.012
DO - 10.1016/j.freeradbiomed.2013.03.012
M3 - Article
AN - SCOPUS:84878829007
SN - 0891-5849
VL - 61
SP - 26
EP - 39
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
ER -