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: Yi Cheng, Chien-Huang Lin, Jing-Yun Chen, Chien-Hua Li, Yu-Tin Liu, Bing-Chang Chen
• Journal: PloS one (PLoS One) Vol. 11 Issue 8 Pg. e0160593 ( 2016) ISSN: 1932-6203 [Electronic] United States
• PMID: 27486656 (Publication Type: Journal Article)

Chemical References

• CCN2 protein, human
• GLI1 protein, human
• GLI2 protein, human
• Kruppel-Like Transcription Factors
• Nuclear Proteins
• Transcription Factor AP-1
• Zinc Finger Protein GLI1
• Zinc Finger Protein Gli2
• Connective Tissue Growth Factor
• Mitogen-Activated Protein Kinase 3
• MAP Kinase Kinase Kinase 1
• MAP3K1 protein, human
• MAP Kinase Kinase 1
• MAP2K1 protein, human

Topics

• Cells, Cultured
• Connective Tissue Growth Factor (genetics, metabolism)
• Fibroblasts (metabolism)
• Humans
• Hypoxia (genetics, metabolism)
• Kruppel-Like Transcription Factors (genetics, metabolism)
• Lung (metabolism)
• MAP Kinase Kinase 1 (metabolism)
• MAP Kinase Kinase Kinase 1 (metabolism)
• MAP Kinase Signaling System (genetics, physiology)
• Mitogen-Activated Protein Kinase 3 (metabolism)
• Nuclear Proteins (genetics, metabolism)
• Primary Cell Culture
• Pulmonary Fibrosis (genetics, metabolism, pathology)
• Transcription Factor AP-1 (metabolism)
• Zinc Finger Protein GLI1 (genetics, metabolism)
• Zinc Finger Protein Gli2