Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by ETAR-dependent JNK/AP-1 pathway
Graphical abstract
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 ETA and ETB [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 ETAR 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.
Section snippets
Materials
ET-1 was purchased from Bachem Americas (Torrance, CA). SP600125 was purchased from Calbiochem (Ellisville, MO). JNK1DN, JNK2DN, and pcDNA were kindly provided by Dr. M.-C. Chen (Taipei Medical University, Taipei, Taiwan). pBK-CMV-Lac Z (LacZ) was kindly provided by Dr. W.-W. Lin (National Taiwan University, Taipei, Taiwan). The human CTGF promoter (−747/+214) luciferase construct (pGL3-CTGF-Luc) and a series of human CTGF promoter deletion constructs (−408/+214, −184/+214, −119/+214, and
ET-1 induced CTGF expression in human lung fibroblasts
To explore whether ET-1 induces CTGF expression in human lung fibroblasts, a human embryonic lung fibroblast cell line (WI-38) were used. Incubation of WI-38 cells for 2 h with ET-1 (1–1000 nM) produced concentration-dependent increases in CTGF expression by 581.3 ± 76.3% at 10 nM of ET-1 (n = 4), and the response abated at the highest concentration (1000 nM) (Fig. 1A). Furthermore, we found that ET-1 (10 nM) induced an increase in CTGF expression in WI-38 cells in time-dependent manners (n = 3); the
Discussion
Our findings demonstrate that ET-1 might act on ETAR to activate the JNK signaling cascade, which in turn initiates AP-1 activation and recruitment to the CTGF promoter, and finally induces CTGF expression in human lung fibroblasts. It is well known that ET-1 plays a pathological role in pulmonary diseases [3], and several lines of evdience suggest that ET-1 is also a profibrotic mediator of fibrotic diseases [1], [30]. ET-1 regulates expression of matrix-associated genes, such as collagen and
Conflict of interest
The authors have no conflicting financial interest.
Acknowledgment
This study was supported by National Science Council, Taiwan (NSC100-2320-B-038-027-MY2 and NSC100-2320-B-038-022-MY2).
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