Transcriptional regulation of increased CCL2 expression in pulmonary fibrosis involves nuclear factor-κB and activator protein-1

https://doi.org/10.1016/j.biocel.2013.04.003Get rights and content

Abstract

Chemokine (CC motif) ligand-2 (CCL2) is a member of C-C chemokine superfamily that contributes to inflammatory and fibrotic process. Studies in patients and experimental animals provide compelling evidence that increased CCL2 expression plays an important role in the development of fibroproliferative lung disease. The up-regulated CCL2 expression in pulmonary fibrosis is also involved in the potent profibrotic effects that thrombin exerts during lung injury. Here, we investigated the transcriptional mechanism involved in CCL2 production by thrombin in human primary lung fibroblasts and explored the transcriptional mechanism of increased CCL2 expression in pulmonary fibrosis. Thrombin increased CCL2 mRNA levels but not mRNA stability, suggesting it was acting transcriptionally. The increased binding of transcription factors to nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) elements in the CCL2 promoter contributed to active transcription following thrombin stimulation. Primary human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF) produced significantly higher levels of CCL2 than nonfibrotic lung fibroblasts. Furthermore, chromatin immunoprecipitation assays detected increased binding of NF-κB p65 and AP-1 subunit c-Jun to the CCL2 promoter of IPF cells both in the presence and absence of thrombin stimulation. The significantly increased binding of p65 and c-Jun to the CCL2 promoter was also observed in the lung tissue of bleomycin-induced pulmonary fibrosis murine model. Collectively, these findings strongly suggest that the increased binding of transcription factors to NF-κB and AP-1 elements in the CCL2 promoter is responsible for the active transcription expression of CCL2 in pulmonary fibrosis.

Introduction

Pulmonary fibrosis is classically characterized by progressive and irreversible destruction of lung architecture, which is caused by excessive and disorganized extracellular matrix (ECM) proteins. It ultimately leads to collapse of normal alveolar structure, disruption of gas exchange, and death from respiratory failure (Wynn, 2011). Idiopathic pulmonary fibrosis (IPF) is a particularly severe form of pulmonary fibrosis with unknown etiology. Patients with IPF only have a life expectancy of 2–6 years after diagnosis (Selman et al., 2001). Currently, lung transplantation is the only effective treatment available for progressive lung fibrosis. It is therefore necessary to further investigate pathogenesis of this disease to allow the development of novel, targeted, and effective therapeutic options.

Mononuclear cell recruitment and activation are the most characteristic histological features in early pulmonary fibrosis and are driven mainly by chemokines such as chemokine (CC motif) ligand-2 (CCL2)/monocyte chemoattractant protein-1 (MCP-1) (Gharaee-Kermani and Phan, 2005, Moore et al., 2001). CCL2 belongs to a C-C chemokine superfamily of small proteins. Although primarily considered as a potent chemoattractant for monocytes, T-cells, and natural killer cells, CCL2 is also involved in the direct activation of fibroblasts, leading to ECM generation via the induction of potent profibrotic mediator, transforming growth factor β1 (Gharaee-Kermani et al., 1996). It may also contribute to excessive collagen deposition via the recruitment of fibrocytes (Moore et al., 2005), which are believed to represent a source of fibroblasts and myofibroblasts during the fibroproliferative response to tissue damage (Phillips et al., 2004). CCL2 is produced by numerous cell types, including monocytes/macrophages, fibroblasts, and epithelial cells (Suga et al., 1999), and acts via the CC chemokine receptor 2. CCL2 mRNA and protein are strongly expressed in the lungs of patients with IPF (Moore et al., 2001). CCL2 levels in serum and bronchoalveolar lavage fluid (BALF) were significantly elevated in IPF subjects compared with healthy volunteers (Suga et al., 1999). In addition, mice deficient for CCR2, are protected from bleomycin- and fluorescein isothiocyanate-induced lung fibrosis (Smith et al., 1995), and anti-CCL2 gene therapy attenuates bleomycin-induced fibrosis (Inoshima et al., 2004). Collectively, these findings support the notion that CCL2 is upregulated in lung fibrosis and plays an important role in the development of fibroproliferative lung disease. However, the molecular mechanisms for increased CCL2 expression in pulmonary fibrosis have not been explored.

There is increasing evidence that the coagulation proteinase such as thrombin play an important role in pulmonary fibrosis. Thrombin levels are increased in BALF from patients with fibrotic lung disease and promote lung fibroblast proliferation (Hernandez-Rodriguez et al., 1995, Idell et al., 1987, Ohba et al., 1994). Furthermore, procoagulant activity and thrombin levels are also increased in mouse and rat BALF after bleomycin-induced lung injury (Howell et al., 2001, Tani et al., 1991). Anticoagulants can reduce fibrosis in experimental animal models when given either prophylactically (Howell et al., 2001) or therapeutically (Gunther et al., 2003). In addition, absence of proteinase-activated receptor-1 (PAR-1), the major thrombin receptor, reduces bleomycin-induced pulmonary fibrosis, accompanied by a significant reduction in pulmonary level of CCL2 (Howell et al., 2005). These findings strongly support the notion that up-regulated CCL2 expression in pulmonary fibrosis is involved in the potent profibrotic effects exerted by thrombin during lung injury. Thrombin can induce CCL2 expression in several cell types, including monocytes (Colotta et al., 1994), endothelial cells (Colotta et al., 1994, Marin et al., 2001), smooth muscle cells (Brandes et al., 2001) and dermal fibroblasts (Bachli et al., 2003). We have previously shown that thrombin can induce CCL2 expression in murine (Deng et al., 2008) and primary human lung fibroblasts (Ortiz-Stern et al., 2012). However, the transcriptional and post-transcriptional regulation of CCL2 expression by thrombin in human lung fibroblasts has not been studied.

CCL2 expression is primarily regulated at the level of transcription initiation. Two 5′-flanking regions of the human MCP-1 gene: a distal enhancer and a proximal promoter, separated by 2.2 kb of DNA are involved in transcriptional regulation (Cho et al., 2002, Ueda et al., 1994). The proximal promoter region is just upstream of the transcription start site and contains a GC box that binds Sp1, NF-κB, and nuclear factor (NF)-1, in addition to two AP-1 sites. The distal enhancer contains two NF-κB binding sites. Previous studies of the human CCL2 promoter have demonstrated that CCL2 expression is critically governed by different transcription factors, including NF-κB and AP-1 (Martin et al., 1997, Patel et al., 2012, Sutcliffe et al., 2009), in a highly cell type- and stimulus-specific manner.

Here we have explored the transcriptional mechanism regulating basal and thrombin-induced CCL2 expression in primary cultures of fibroblasts from nonfibrotic lungs (F-NL) and compared these with fibroblasts from lungs of IPF patients (F-IPF). Furthermore, we evaluated murine lung tissues from the bleomycin-induced pulmonary fibrosis model for evidence of transcriptional molecules found in vitro to be responsible for CCL2 overexpression. We found that thrombin regulates CCL2 expression transcriptionally via NF-κB and AP-1, and the increased binding of NF-κB subunit p65 and AP-1 subunit c-Jun to the CCL2 promoter are responsible for CCL2 overexpression in pulmonary fibrosis.

Section snippets

Materials

Human thrombin, 2-[(aminocarbonyl)amino]-5-[4-fluorophenyl]-3-thiophenecarboxamide (TPCA), actinomycin D were purchased from Sigma–Aldrich (Shanghai, China). c-Jun NH2-terminal kinase inhibitor SP600125 was from Merck (Genewindows Biotech, Guangzhou, China). Anti-NF-κB p65 antibody, anti-c-Jun antibody, anti-phospho-c-Jun antibody, anti-GAPDH antibody, anti-beta-actin antibody and polyclonal goat anti-rabbit antibody conjugated to horseradish peroxidase were from Santa Cruz (Univ-bio, Shanghai,

Thrombin induces CCL2 production in a time and dose-dependent manner in F-NL

To determine the optimal concentration and stimulation time of thrombin for use in our experiments, thrombin concentration response and time course experiments were performed. In concentration-response experiment in which fibroblasts were stimulated for 6 h with increasing concentrations of thrombin, CCL2 concentrations ranged from 9.22 ± 2.94 pg/ml in unstimulated F-NL cells to a maximum of 164.78 ± 30.93 pg/ml in response to 10 nM thrombin (Fig. 1A). Time-course experiments were performed with

Discussion

The major finding of our present study demonstrates that NF-κB and AP-1 are involved in thrombin-induced CCL2 transcriptional expression in human lung fibroblast and that increased binding of NF-κB and AP-1 to the CCL2 promoter contributes to overexpression of CCL2 in pulmonary fibrosis. This is the first description of transcriptional mechanism of thrombin-induced CCL2 expression in primary human lung fibroblasts. Furthermore, our study is the first description of transcriptional mechanisms of

Acknowledgments

This work was supported by National Natural Science Foundation of China (30900661 and 81072208); the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry; Science Foundation of Shantou University Medical College and Basic Science and Clinical Research Foundation of Shantou University Medical College.

References (48)

  • J. Zhang et al.

    Targeting chemokine (C-C motif) ligand 2 (CCL2) as an example of translation of cancer molecular biology to the clinic

    Progress in Molecular Biology and Translational Science

    (2010)
  • H.N. Antoniades et al.

    Expression of monocyte chemoattractant protein 1 mRNA in human idiopathic pulmonary fibrosis

    Proceedings of the National Academy of Sciences of the United States of America

    (1992)
  • R.P. Brandes et al.

    Thrombin-induced MCP-1 expression involves activation of the p22phox-containing NADPH oxidase in human vascular smooth muscle cells

    Thrombosis and Haemostasis

    (2001)
  • N.H. Cho et al.

    Induction of the gene encoding macrophage chemoattractant protein 1 by Orientia tsutsugamushi in human endothelial cells involves activation of transcription factor activator protein 1

    Infection and Immunity

    (2002)
  • F. Colotta et al.

    Expression of monocyte chemotactic protein-1 by monocytes and endothelial cells exposed to thrombin

    American Journal of Pathology

    (1994)
  • W.R. Coward et al.

    Repression of IP-10 by interactions between histone deacetylation and hypermethylation in idiopathic pulmonary fibrosis

    Molecular and Cellular Biology

    (2010)
  • W.R. Coward et al.

    Defective histone acetylation is responsible for the diminished expression of cyclooxygenase 2 in idiopathic pulmonary fibrosis

    Molecular and Cellular Biology

    (2009)
  • X. Deng et al.

    Thrombin induces fibroblast CCL2/JE production and release via coupling of PAR1 to Galphaq and cooperation between ERK1/2 and Rho kinase signaling pathways

    Molecular Biology of the Cell

    (2008)
  • M. Gharaee-Kermani et al.

    Molecular mechanisms of and possible treatment strategies for idiopathic pulmonary fibrosis

    Current Pharmaceutical Design

    (2005)
  • A. Gunther et al.

    Prevention of bleomycin-induced lung fibrosis by aerosolization of heparin or urokinase in rabbits

    American Journal of Respiratory and Critical Care Medicine

    (2003)
  • M. Hetzel et al.

    Different effects of growth factors on proliferation and matrix production of normal and fibrotic human lung fibroblasts

    Lung

    (2005)
  • S. Idell et al.

    Procoagulant activity in bronchoalveolar lavage in the adult respiratory distress syndrome. Contribution of tissue factor associated with factor VII

    American Review of Respiratory Disease

    (1987)
  • I. Inoshima et al.

    Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary fibrosis in mice

    American Journal of Physiology. Lung Cellular and Molecular Physiology

    (2004)
  • M. Jordana et al.

    Heterogeneous proliferative characteristics of human adult lung fibroblast lines and clonally derived fibroblasts from control and fibrotic tissue

    American Review of Respiratory Disease

    (1988)
  • Cited by (50)

    • Macrophage-secreted TSLP and MMP9 promote bleomycin-induced pulmonary fibrosis

      2019, Toxicology and Applied Pharmacology
      Citation Excerpt :

      Currently, lung transplantation is the only effective treatment available for progressive lung fibrosis. It is therefore necessary to further investigate the pathogenesis of this disease to allow the development of novel, targeted, and effective therapeutic options (Deng et al., 2013). IPF is characterized by thickened alveolar walls with mesenchymal cell proliferation and extracellular matrix deposition, remodeling, and alveolar accumulation of inflammatory cells, particularly macrophages (Zhang et al., 1993).

    • A computational approach to unraveling TLR signaling in murine mammary carcinoma

      2018, Computers in Biology and Medicine
      Citation Excerpt :

      The data showed that the size of the modeled world did not affect CCL2 expression (data not shown), yet data from the model (Fig. 3d) did not recapitulate data from the laboratory experiment (Fig. 1). Because elevated levels of NFκB have been associated with many tumors [17–19], and because NFκB is a transcriptional factor for CCL2 [20], we wanted to determine the extent to which NFκB levels were associated with TLR4-induced CCL2 expression by the tumor cells and whether modulating NFκB levels in the model would allow the modeling data to recapitulate the experimental data. To evaluate the importance of NFκB for CCL2 production in the model we ran the simulation after altering NFκB levels in 4T1 and DC.

    View all citing articles on Scopus
    View full text