Connective tissue growth factor induces collagen I expression in human lung fibroblasts through the Rac1/MLK3/JNK/AP-1 pathway

Biochim Biophys Acta. 2013 Dec;1833(12):2823-2833. doi: 10.1016/j.bbamcr.2013.07.016. Epub 2013 Jul 29.

Abstract

Connective tissue growth factor (CTGF) plays an important role in lung fibrosis. In this study, we investigated the role of Rac1, mixed-lineage kinase 3 (MLK3), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CTGF-induced collagen I expression in human lung fibroblasts. CTGF caused concentration- and time-dependent increases in collagen I expression. CTGF-induced collagen I expression was inhibited by the dominant negative mutant (DN) of Rac1 (RacN17), MLK3DN, MLK3 inhibitor (K252a), JNK1DN, JNK2DN, a JNK inhibitor (SP600125), and an AP-1 inhibitor (curcumin). Treatment of cells with CTGF caused activation of Rac1, MLK3, JNK, and AP-1. The CTGF-induced increase in MLK3 phosphorylation was inhibited by RacN17. Treatment with RacN17 and the MLK3DN inhibited CTGF-induced JNK phosphorylation. CTGF caused increases in c-Jun phosphorylation and the recruitment of c-Jun and c-Fos to the collagen I promoter. Furthermore, stimulation of cells with the CTGF resulted in increases in AP-1-luciferase activity; this effect was inhibited by Rac1N17, MLK3DN, JNK1DN, and JNK2DN. Moreover, CTGF-induced α-smooth muscle actin (α-SMA) expression was inhibited by the procollagen I small interfering RNA (siRNA). These results suggest for the first time that CTGF acting through Rac1 activates the MLK3/JNK signaling pathway, which in turn initiates AP-1 activation and recruitment of c-Jun and c-Fos to the collagen I promoter and ultimately induces collagen I expression in human lung fibroblasts.

Keywords: AP-1; ASK1; CCAAT/enhancer-binding protein β; CTGF; ChIP; Collagen I; Connective tissue growth factor; ECM; ERK; FCS; Fibroblasts; IL-6; JAK; JNK; Janus kinase; Lung fibrosis; MAPK kinase kinase; MAPKKK; MAPKs; MEM; MLB; MLK3; Minimum Essential Medium; NEAAs; PBD; STAT; Signal transduction; TGF-β; WT; activator protein-1; apoptosis signal-regulating kinase 1; c-Jun N-terminal kinase; c/EBPβ; chromatin immunoprecipitation; connective tissue growth factor; extracellular matrix; extracellular signal-regulated kinase; fetal calf serum; interleukin-6; magnesium lysis buffer; mitogen-activated protein kinases; mixed-lineage kinase 3; non-essential amino acids; p21-binding domain; siRNA; signal transducer and activator of transcription; small interfering RNA; transforming growth factor-β; wild-type; α-SMA; α-smooth muscle actin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism
  • Cell Line
  • Collagen Type I / metabolism*
  • Connective Tissue Growth Factor / pharmacology*
  • Enzyme Activation / drug effects
  • Fibroblasts / drug effects
  • Fibroblasts / enzymology*
  • Humans
  • Lung / cytology*
  • MAP Kinase Kinase Kinases / metabolism*
  • MAP Kinase Signaling System / drug effects
  • Mitogen-Activated Protein Kinase Kinase Kinase 11
  • Models, Biological
  • Phosphorylation / drug effects
  • Transcription Factor AP-1 / metabolism*
  • rac1 GTP-Binding Protein / metabolism*

Substances

  • ACTA2 protein, human
  • Actins
  • Collagen Type I
  • Transcription Factor AP-1
  • Connective Tissue Growth Factor
  • MAP Kinase Kinase Kinases
  • rac1 GTP-Binding Protein