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

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Highlights

  • Pulmonary fibrosis involves the excess collagen deposition.

  • CTGF plays an important role in lung fibrosis.

  • CTGF activates Rac1/MLK3/JNK/AP-1 to induce collagen I expression in lung fibroblasts.

  • CTGF-induced α-SMA expression was inhibited by procollagen I siRNA.

  • Discovery of the signaling pathway of CTGF in collagen I expression may help elucidate the role of CTGF in lung fibrosis.

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.

Abbreviations

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

Keywords

Connective tissue growth factor
Collagen I
Signal transduction
Lung fibrosis
Fibroblasts

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These authors contributed equally to this work.