Abstract
CXCL12 (stromal cell-derived factor-1, SDF-1) is a potent chemokine for homing of CXCR4+ fibrocytes to injury sites of lung tissue, which contributes to pulmonary fibrosis. Overexpression of connective tissue growth factor (CTGF) plays a critical role in pulmonary fibrosis. In this study, we investigated the roles of Rac1, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CXCL12-induced CTGF expression in human lung fibroblasts. CXCL12 caused concentration- and time-dependent increases in CTGF expression and CTGF-luciferase activity. CXCL12-induced CTGF expression was inhibited by a CXCR4 antagonist (AMD3100), small interfering RNA of CXCR4 (CXCR4 siRNA), a dominant negative mutant of Rac1 (RacN17), a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor (PD98059), a JNK inhibitor (SP600125), a p21-activated kinase inhibitor (PAK18), c-Jun siRNA, and an AP-1 inhibitor (curcumin). Treatment of cells with CXCL12 caused activations of Rac1, Rho, ERK, and c-Jun. The CXCL12-induced increase in ERK phosphorylation was inhibited by RacN17. Treatment of cells with PD98059 and SP600125 both inhibited CXCL12-induced c-Jun phosphorylation. CXCL12 caused the recruitment of c-Jun and c-Fos binding to the CTGF promoter. Furthermore, CXCL12 induced an increase in α-smooth muscle actin (α-SMA) expression, a myofibroblastic phenotype, and actin stress fiber formation. CXCL12-induced actin stress fiber formation and α-SMA expression were respectively inhibited by AMD3100 and CTGF siRNA. Taken together, our results suggest that CXCL12, acting through CXCR4, activates the Rac/ERK and JNK signaling pathways, which in turn initiates c-Jun phosphorylation, and recruits c-Jun and c-Fos to the CTGF promoter and ultimately induces CTGF expression in human lung fibroblasts. Moreover, overexpression of CTGF mediates CXCL12-induced α-SMA expression.
Highlights
Idiopathic pulmonary fibrosis (IPF) is caused by chronic lung inflammation in response to unknown etiologic agents, leading to tissue destruction, fibroblast overgrowth, myofibroblast formation, and extracellular matrix (ECM) protein deposition, that result in severe respiratory insufficiency [1,2]
CXCL12 was first described as a factor produced by bone marrow stromal cells and is a potent chemoattractant for fibrocytes that contributes to pulmonary fibrosis [11,12]
CXCL12 induces connective tissue growth factor (CTGF) expression A previous study indicated that CXCL12 is a potent chemoattractant for fibrocytes that contributes to pulmonary fibrosis [11,12]
Summary
Idiopathic pulmonary fibrosis (IPF) is caused by chronic lung inflammation in response to unknown etiologic agents, leading to tissue destruction, fibroblast overgrowth, myofibroblast formation, and extracellular matrix (ECM) protein deposition, that result in severe respiratory insufficiency [1,2]. Resident fibroblasts are major regulator cells of ECM protein expression in connective tissues and are recruited to wound sites by the release of inflammatory mediators such as transforming growth factor-b (TGF-b), interleukin (IL)-8/ CXCL8, and connective tissue growth factor (CTGF) [6,7,8]. Fibroblasts express no or only low levels of the CTGF, it is overexpressed during wound repair by fibrotic mediators such as TGF-b, thrombin, and endothelin-1 (ET-1) that contribute to the pulmonary fibrosis [5,8,9]. CXCL12 is a ligand of the chemokine receptor, CXCR4, and plays an important role in pulmonary fibrosis [17]. The roles of CXCL12 in regulating CTGF expression in lung fibroblasts and in fibroblast differentiation are currently unclear
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