title: New role and molecular mechanism of Gadd45a in hepatic ﬁbrosis.
abstract: OBJECTIVE
  To investigate the role of Gadd45a in hepatic ﬁbrosis and the transforming growth factor (TGF)-β/Smad signaling pathway.
  
  
  METHODS
  Wild-type male BALB/c mice were treated with CCl4 to induce a model of chronic liver injury. Hepatic stellate cells (HSCs) were isolated from the liver of BALB/c mice and were treated with small interfering RNAs (siRNAs) targeting Gadd45a or the pcDNA3.1-Gadd45a recombinant plasmid. Cellular α-smooth muscle actin (α-SMA), β-actin, type I collagen, phospho-Smad2, phospho-Smad3, Smad2, Smad3, and Smad4 were detected by Western blots. The mRNA levels of α-SMA, β-actin, and type I collagen were determined by quantitative real-time (qRT)-PCR analyses. Reactive oxygen species production was monitored by ﬂow cytometry using 2,7-dichlorodihydroﬂuorescein diacetate. Gadd45a, Gadd45b, anti-Gadd45g, type I collagen, and SMA local expression in liver tissue were measured by histologic and immunohistochemical analyses.
  
  
  RESULTS
  Significant downregulation of Gadd45a, but not Gadd45b or Gadd45g, accompanied by activation of the TGF-β/Smad signaling pathways was detected in fibrotic liver tissues of mice and isolated HSCs with chronic liver injury induced by CCl4 treatment. Overexpression of Gadd45a reduced the expression of extracellular matrix proteins and α-SMA in HSCs, whereas transient knockdown of Gadd45a with siRNA reversed this process. Gadd45a inhibited the activity of a plasminogen activator inhibitor-1 promoter construct and (CAGA)9 MLP-Luc, an artificial Smad3/4-specific reporter, as well as reduced the phosphorylation and nuclear translocation of Smad3. Gadd45a showed protective effects by scavenging reactive oxygen species and upregulating antioxidant enzymes.
  
  
  CONCLUSIONS
  Gadd45a may counteract hepatic ﬁbrosis by regulating the activation of HSCs via the inhibition of TGF-β/Smad signaling.
authors.affiliation: Liang Hong, Qing-Feng Sun, Yang-He Wu, Hui Zhang, Rong-Quan Fu, Fu-Jing Cai, Qing-Qing Zhou, Ke Zhou, Qing-Wei Du, Dong Zhang, Shuang Xu, Ji-Guang Ding, Department of Infectious Diseases, the Third Affiliated Hospital to Wenzhou Medical University, Wenzhou 325200, Zhejiang Province, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Hong
authors.affiliation: Liang Hong, Qing-Feng Sun, Yang-He Wu, Hui Zhang, Rong-Quan Fu, Fu-Jing Cai, Qing-Qing Zhou, Ke Zhou, Qing-Wei Du, Dong Zhang, Shuang Xu, Ji-Guang Ding, Department of Infectious Diseases, the Third Affiliated Hospital to Wenzhou Medical University, Wenzhou 325200, Zhejiang Province, China.
authors.forename: Qing-Feng
authors.initials: QF
authors.lastname: Sun
authors.affiliation: Liang Hong, Qing-Feng Sun, Yang-He Wu, Hui Zhang, Rong-Quan Fu, Fu-Jing Cai, Qing-Qing Zhou, Ke Zhou, Qing-Wei Du, Dong Zhang, Shuang Xu, Ji-Guang Ding, Department of Infectious Diseases, the Third Affiliated Hospital to Wenzhou Medical University, Wenzhou 325200, Zhejiang Province, China.
authors.forename: Ting-Yan
authors.initials: TY
authors.lastname: Xu
authors.affiliation: Liang Hong, Qing-Feng Sun, Yang-He Wu, Hui Zhang, Rong-Quan Fu, Fu-Jing Cai, Qing-Qing Zhou, Ke Zhou, Qing-Wei Du, Dong Zhang, Shuang Xu, Ji-Guang Ding, Department of Infectious Diseases, the Third Affiliated Hospital to Wenzhou Medical University, Wenzhou 325200, Zhejiang Province, China.
authors.forename: Yang-He
authors.initials: YH
authors.lastname: Wu
authors.affiliation: Liang Hong, Qing-Feng Sun, Yang-He Wu, Hui Zhang, Rong-Quan Fu, Fu-Jing Cai, Qing-Qing Zhou, Ke Zhou, Qing-Wei Du, Dong Zhang, Shuang Xu, Ji-Guang Ding, Department of Infectious Diseases, the Third Affiliated Hospital to Wenzhou Medical University, Wenzhou 325200, Zhejiang Province, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Liang Hong, Qing-Feng Sun, Yang-He Wu, Hui Zhang, Rong-Quan Fu, Fu-Jing Cai, Qing-Qing Zhou, Ke Zhou, Qing-Wei Du, Dong Zhang, Shuang Xu, Ji-Guang Ding, Department of Infectious Diseases, the Third Affiliated Hospital to Wenzhou Medical University, Wenzhou 325200, Zhejiang Province, China.
authors.forename: Rong-Quan
authors.initials: RQ
authors.lastname: Fu
authors.affiliation: Liang Hong, Qing-Feng Sun, Yang-He Wu, Hui Zhang, Rong-Quan Fu, Fu-Jing Cai, Qing-Qing Zhou, Ke Zhou, Qing-Wei Du, Dong Zhang, Shuang Xu, Ji-Guang Ding, Department of Infectious Diseases, the Third Affiliated Hospital to Wenzhou Medical University, Wenzhou 325200, Zhejiang Province, China.
authors.forename: Fu-Jing
authors.initials: FJ
authors.lastname: Cai
authors.affiliation: Liang Hong, Qing-Feng Sun, Yang-He Wu, Hui Zhang, Rong-Quan Fu, Fu-Jing Cai, Qing-Qing Zhou, Ke Zhou, Qing-Wei Du, Dong Zhang, Shuang Xu, Ji-Guang Ding, Department of Infectious Diseases, the Third Affiliated Hospital to Wenzhou Medical University, Wenzhou 325200, Zhejiang Province, China.
authors.forename: Qing-Qing
authors.initials: QQ
authors.lastname: Zhou
authors.affiliation: Liang Hong, Qing-Feng Sun, Yang-He Wu, Hui Zhang, Rong-Quan Fu, Fu-Jing Cai, Qing-Qing Zhou, Ke Zhou, Qing-Wei Du, Dong Zhang, Shuang Xu, Ji-Guang Ding, Department of Infectious Diseases, the Third Affiliated Hospital to Wenzhou Medical University, Wenzhou 325200, Zhejiang Province, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Zhou
authors.affiliation: Liang Hong, Qing-Feng Sun, Yang-He Wu, Hui Zhang, Rong-Quan Fu, Fu-Jing Cai, Qing-Qing Zhou, Ke Zhou, Qing-Wei Du, Dong Zhang, Shuang Xu, Ji-Guang Ding, Department of Infectious Diseases, the Third Affiliated Hospital to Wenzhou Medical University, Wenzhou 325200, Zhejiang Province, China.
authors.forename: Qing-Wei
authors.initials: QW
authors.lastname: Du
authors.affiliation: Liang Hong, Qing-Feng Sun, Yang-He Wu, Hui Zhang, Rong-Quan Fu, Fu-Jing Cai, Qing-Qing Zhou, Ke Zhou, Qing-Wei Du, Dong Zhang, Shuang Xu, Ji-Guang Ding, Department of Infectious Diseases, the Third Affiliated Hospital to Wenzhou Medical University, Wenzhou 325200, Zhejiang Province, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Liang Hong, Qing-Feng Sun, Yang-He Wu, Hui Zhang, Rong-Quan Fu, Fu-Jing Cai, Qing-Qing Zhou, Ke Zhou, Qing-Wei Du, Dong Zhang, Shuang Xu, Ji-Guang Ding, Department of Infectious Diseases, the Third Affiliated Hospital to Wenzhou Medical University, Wenzhou 325200, Zhejiang Province, China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Xu
authors.affiliation: Liang Hong, Qing-Feng Sun, Yang-He Wu, Hui Zhang, Rong-Quan Fu, Fu-Jing Cai, Qing-Qing Zhou, Ke Zhou, Qing-Wei Du, Dong Zhang, Shuang Xu, Ji-Guang Ding, Department of Infectious Diseases, the Third Affiliated Hospital to Wenzhou Medical University, Wenzhou 325200, Zhejiang Province, China.
authors.forename: Ji-Guang
authors.initials: JG
authors.lastname: Ding
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002251:Carbon Tetrachloride; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D055166:Hepatic Stellate Cells / Q000378:metabolism* / Q000473:pathology; D008099:Liver / Q000378:metabolism* / Q000473:pathology; D008106:Liver Cirrhosis, Experimental / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism* / Q000517:prevention & control*; D008297:Male; D008807:Mice, Inbred BALB C; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D051900:Smad3 Protein / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism; D000097642:GADD45 Proteins
chemical_list: D000199:Actins; D002251:Carbon Tetrachloride; D018797:Cell Cycle Proteins; D024042:Collagen Type I; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; C499072:Gadd45a protein, mouse; D000097642:GADD45 Proteins; C497791:Smad3 protein, mouse; C544013:alpha-smooth muscle actin, mouse
keywords: Antioxidant; Gadd45a; Hepatic stellate cells; Hepatic ﬁbrosis; Transforming growth factor-β/Smad signaling