Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by ET_AR-dependent JNK/AP-1 pathway

Abstract

Endothelin-1 (ET-1) acts as a key mediator of vasoconstriction and tissue repair. Overproduction of connective tissue growth factor (CTGF) underlies the development of lung fibrosis. ET-1 induces expression of matrix-associated genes in lung fibroblasts, however, little is known about the signaling pathway of CTGF expression caused by ET-1. In this study, we found that ET-1 caused concentration- and time-dependently increases in CTGF expression in human embryonic lung fibroblast cell line (WI-38). ET-1-induced CTGF expression was inhibited by BQ123 (ET_AR antagonist), but not BQ788 (ET_BR antagonist). Moreover, ET-1-induced CTGF expression was significantly reduced by JNK inhibitor (SP600125), the dominant-negative mutants of JNK1/2 (JNK1/2 DN), and AP-1 inhibitor (curcumin). ET-1 induced phosphorylations of JNK and c-Jun in time-dependent manners. AP-1 luciferase activity was concentration-dependently increased by ET-1, and this effect was attenuated by SP600125. We also found that ET-1-induced CTGF expression was most controlled by the AP-1 binding region of CTGF promoter. ET-1-indiced CTGF luciferase activity was predominately controlled by the sequence −747 to −408 bp upstream of the transcription start site on the human CTGF promoter. Furthermore, ET-1 caused the formation of AP-1-specific DNA-protein complex and the recruitment of c-Jun to the CTGF promoter. Moreover, we found that ET-1 induced α-smooth muscle actin (α-SMA) expression, which was inhibited by BQ123, SP600125, curcumin, and anti-CTGF antibody. These results suggest that ET-1 stimulates expressions of CTGF and α-SMA through ET_AR/JNK/AP-1 signaling pathway, and CTGF is required for ET-1-induced α-SMA expression in human lung fibroblasts.

Introduction

Endothelin-1 (ET-1), first identified for its potent vasoconstrictor activity, appears now as a mediator of organ fibrosis, such as skin and lung [1]. ET-1 is a 21 amino acid peptide and released from the endothelium, epithelium, and vascular smooth muscle cells, and orchestrates a variety of effects, such as tissue repair [2]. Clinical evidence showed that elevated levels of circulating ET-1, and increased ET-1 production, exist in scleroderma patients that correlated with the severity of the fibrotic phenotype, suggesting that ET-1 may play a key role not only in normal wound repair but also in the pathogenesis of fibrosis [1]. ET-1 binds with two G protein-coupled receptors (GPCR), named ET_A and ET_B [3], and produces a broad range of biological properties including the mitogenic activity of fibroblasts [3], [4]. In the context of wound healing, ET-1 acts with other profibrotic mediators to recruit fibroblasts and allows for their differentiation to contractile myofibroblasts [5]. The α-smooth muscle action (α-SMA) is a hallmark of myofibroblasts and an indicator of fibroblast differentiation, which was significantly increased in lung fibrosis [6], [7]. As already mentioned, ET-1 is the principal effector of the transforming growth factor (TGF)-β, which induces the differentiation of fibroblasts into myofibroblasts (α-SMA expression) and the production of extracellular matrix (ECM) components [8], [9]. Recent evidence showed that levels of ET-1 and CTGF increased in animal models of pulmonary hypertension, and in the gastric wall of systemic sclerosis patients [10]. Furthermore, several studies verified that ET-1 could induce CTGF expression in cardiomyocytes and vascular smooth muscle cells [11], [12].

CTGF, formerly known as CCN2, is an immediate early gene belongs to CCN family, which regulates diverse biological effects including cell adhesion, matrix production, tissue modeling, proliferation, and differentiation [13], [14]. CTGF was recently identified as an agent for tissue fibrosis [14], and has been reported that highly expressed in a wide range of fibrotic conditions [4], [15], [16]. CTGF was reported to promote ECM accumulation and fibroblastic differentiation, and in vivo evidence recently suggested that CTGF overexpression can exacerbate fibrosis [17], [18]. The promoter region of the human Ctgf gene contains many transcription factor binding sites, including AP-1, STAT, SMAD, basal control element (BCE)-1, E twenty-six-specific (Ets)-1, NF-κB, and specificity protein 1 (Sp1) [19], [20]. Our previous study demonstrated that thrombin-induced CTGF expression is predominately mediated through AP-1 in human lung fibroblasts [21]. At present, however, whether the AP-1 is involved in ET-1-induced CTGF expression in human lung fibroblasts is still unknown.

Jun N-terminal kinases (JNK) is one major subfamily of mitogen-activated protein kinase (MAPK) pathways, which activated by a broad variety of extracellular stimuli such as growth factors and inflammatory cytokines [22]. JNK convert these stimuli into intracellular responses by activating downstream target proteins such as AP-1 [23]. JNK and AP-1 have been implicated in the pathogenesis of fibrosis in systemic sclerosis (SSc) [23], [24]. There is evidence that TGF-β1-induced CTGF expression is mediated through JNK in human lung fibroblasts [25]. Our previous study also demonstrated that thrombin-induced CTGF expression through ASK1-dependent JNK/AP-1 pathway in human lung fibroblasts [21]. However, the role of JNK pathway in regulating ET-1-induced CTGF expression is remained unclear. Therefore, we investigated the role of JNK/AP-1 pathway in ET-1-induced CTGF expression in human lung fibroblasts. In the present study, we demonstrated that ET-1 acts on ET_AR to activate JNK signaling pathway, which in turns induce AP-1 activation and recruitment to CTGF promoter, and promotes CTGF expression in human lung fibroblasts.