title: Induction of Connective Tissue Growth Factor Expression by Hypoxia in Human Lung Fibroblasts via the MEKK1/MEK1/ERK1/GLI-1/GLI-2 and AP-1 Pathways.
abstract: Several reports have indicated that hypoxia, GLI, and connective tissue growth factor (CTGF) contribute to pulmonary fibrosis in idiopathic pulmonary fibrosis. We investigated the participation of mitogen-activated protein kinase kinase (MEK) kinase 1 (MEKK1)/MEK1/ERK1/GLI-1/2 and activator protein-1 (AP-1) signaling in hypoxia-induced CTGF expression in human lung fibroblasts. Hypoxia time-dependently increased CTGF expression, which was attenuated by the small interfering RNA (siRNA) of GLI-1 (GLI-1 siRNA) and GLI-2 (GLI-2 siRNA) in both human lung fibroblast cell line (WI-38) and primary human lung fibroblasts (NHLFs). Moreover, GLI-1 siRNA and GLI-2 siRNA attenuated hypoxia-induced CTGF-luciferase activity, and the treatment of cells with hypoxia induced GLI-1 and GLI-2 translocation. Furthermore, hypoxia-induced CTGF expression was reduced by an MEK inhibitor (PD98059), MEK1 siRNA, ERK inhibitor (U0126), ERK1 siRNA, and MEKK1 siRNA. Both PD98059 and U0126 significantly attenuated hypoxia-induced CTGF-luciferase activity. Hypoxia time-dependently increased MEKK1, ERK, and p38 MAPK phosphorylation. Moreover, SB203580 (a p38 MAPK inhibitor) also apparently inhibited hypoxia-induced CTGF expression. The treatment of cells with hypoxia induced ERK, GLI-1, or GLI-2 complex formation. Hypoxia-induced GLI-1 and GLI-2 translocation into the nucleus was significantly attenuated by U0126. In addition, hypoxia-induced ERK Tyr204 phosphorylation was impeded by MEKK1 siRNA. Moreover, hypoxia-induced CTGF-luciferase activity was attenuated by cells transfected with AP-1 site mutation in a CTGF construct. Exposure to hypoxia caused a time-dependent phosphorylation of c-Jun, but not of c-Fos. Chromatin immunoprecipitation (ChIP) revealed that hypoxia induced the recruitment of c-Jun, GLI-1, and GLI-2 to the AP-1 promoter region of CTGF. Hypoxia-treated cells exhibited an increase in α-smooth muscle actin (α-SMA) and collagen production, which was blocked by GLI-1 siRNA and GLI-2 siRNA. Overall, these data implied that the MEKK1/MEK1/ERK1/GLI-1/GLI-2, and AP-1 pathways mediated hypoxia-induced CTGF expression in human lung fibroblasts. Furthermore, GLI-1 and GLI-2 found to be involved in hypoxia-induced α-SMA and collagen expression.
authors.affiliation: School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Yi
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: Gradual Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Chien-Huang
authors.initials: CH
authors.lastname: Lin
authors.affiliation: Gradual Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Jing-Yun
authors.initials: JY
authors.lastname: Chen
authors.affiliation: Gradual Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Chien-Hua
authors.initials: CH
authors.lastname: Li
authors.affiliation: School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.
authors.forename: Yu-Tin
authors.initials: YT
authors.lastname: Liu
authors.affiliation: School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan.|Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Bing-Chang
authors.initials: BC
authors.lastname: Chen
mesh_terms: D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D000860:Hypoxia / Q000235:genetics* / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D008168:Lung / Q000378:metabolism*; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D048728:MAP Kinase Kinase Kinase 1 / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000235:genetics / Q000502:physiology*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D011658:Pulmonary Fibrosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D000071676:Zinc Finger Protein GLI1 / Q000235:genetics / Q000378:metabolism; D000075746:Zinc Finger Protein Gli2
chemical_list: C526317:CCN2 protein, human; C400551:GLI1 protein, human; C082593:GLI2 protein, human; D051741:Kruppel-Like Transcription Factors; D009687:Nuclear Proteins; D018808:Transcription Factor AP-1; D000071676:Zinc Finger Protein GLI1; D000075746:Zinc Finger Protein Gli2; D055513:Connective Tissue Growth Factor; D048052:Mitogen-Activated Protein Kinase 3; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human