Matrix metalloprotease-9 induces transforming growth factor-β1 production in airway epithelium via activation of epidermal growth factor receptors
Introduction
Matrix metalloproteinase (MMP)-9 has been shown to be involved in diverse pathologic conditions, particularly asthma, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease (COPD) (Atkinson and Senior, 2003). MMP-9, and increased MMP-9 activity has been detected in the airways of COPD and asthma patients (Araujo et al., 2008, Tang et al., 2006, Vernooy et al., 2004). Airflow limitation is a characteristic of COPD and is caused by a combination of small airway disorders and parenchymal destruction (Hogg, 2004). An imbalance between MMP-9 and a tissue inhibitor of metalloproteinase-1 is considered to be associated with the progression of asthmatic airway remodeling (Atkinson and Senior, 2003, Kelly and Jarjour, 2003). The mechanism of MMP-9 induction of airway fibrosis remains to be elucidated.
Transforming growth factor (TGF)-β1, a critical mediator of lung fibrosis, is a strong extracellular matrix inducer and a chemoattractant for fibroblasts and neutrophils (Hannigan et al., 1998, Postlethwaite et al., 1987). Targeted overexpression of TGF-β1 leads to progressive fibrosis (Sime et al., 1997). Increased expression of TGF-β1 has been observed in the small airway epithelium of smokers and patients with COPD, and the expression of TGF-β1 mRNA has been shown to be positively associated with the degree of small airway obstruction (Takizawa et al., 2001). Moreover, it is believed that TGF-β1 may play an essential role in the airway remodeling that occurs in asthmatic patients (Bosse and Rola-Pleszczynski, 2007). In response to stimuli, the airway epithelium can produce TGF-β1 (Perng et al., 2006, Perng et al., 2007, Perng et al., 2008) and contribute greatly to the pathogenesis of airway obstruction and remodeling.
The epidermal growth factor receptor (EGFR) is expressed in most tissue and mediates a variety of biological functions, such as growth, differentiation and survival (Zwick et al., 1999). These signals are initiated by receptor binding of EGF and EGF-like ligands, which are expressed as transmembrane precursor proteins and need to be cleaved by proteases to release the mature form (Chan et al., 2006). It has been reported that angiotensin II can mediate cardiomyocyte hypertrophic growth pathways via MMP-dependent heparin-binding EGF liberation and EGFR activation (Smith et al., 2004). In addition, inhibition of EGFR tyrosine kinase (TK) can diminish TGF-α-induced pulmonary fibrosis in the animal model (Hardie et al., 2008). Whether or not EGFR is involved in lung tissue remodeling is worthy of further investigation.
The relationship between MMP-9, EGFR and TGF-β1 in obstructive airway disorders is not yet understood. The airway epithelium is likely to be exposed to high levels of MMP-9, but the response of the epithelium following this exposure is not yet clear. We hypothesized that MMP-9 may stimulate the airway epithelium to produce fibrogenic mediators through activation of membrane-bound receptors.
Section snippets
Modified air–liquid interface culture for human airway epithelial cells (HAECs)
Preparation of the modified air–liquid interface culture for HAECs has been described in detail previously (Perng et al., 2003). Briefly, human bronchus, obtained from surgical lobectomy for lung cancer, was rinsed several times with Leibovitz's L-15 medium containing penicillin, streptomycin, and amphotericin B. The tissue was cut into pieces, which were planted onto six-well culture inserts and coated with type IV collagen. Two milliliters of culture medium were added to the basal chamber,
Effect of MMP-9 on TGF-β1 release
The effect of MMP-9 on the generation of TGF-β1 from HAECs is shown in Fig. 1. No significant increase in TGF-β1 production was observed at the 3 h, 6 h and 12 h incubation time points. At 18 h of incubation, an increase of TGF-β1 production was found in the presence of MMP-9 (1 ng/ml) (95.7 ± 10.9 vs. 36.2 ± 9.1 pg/ml, stimulated vs. control, p < 0.05). The maximal TGF-β1 level (total and active form) was significantly increased after 24 h of incubation (162.7 ± 11.8 vs. 60.2 ± 11.4 pg/ml, stimulated vs.
Discussion
This is the first report to demonstrate that in a cell culture model, the key protease MMP-9 can stimulate human airway epithelial cells to produce TGF-β1 through the cleavage of membrane-bound EGF and the EGF-like ligand TGF-α, activation of EGFR, and phosphorylation of p44/42 MAP kinases. A significant increase in EGF and TGF-α release was observed in the presence of MMP-9 at 45 min and 1 h of incubation for EGF and TGF-α respectively. Protease inhibitors EGFR-mAb and EGFR-TKI suppressed
Conclusion
MMP-9 cleaves and releases EGF and EGF-like ligand TGF-α, activates EGFR, and induces TGF-β1 production in airway epithelial cells, which may contribute significantly to the pathogenesis of airway remodeling. The importance of MMP-9 and EGFR and their potential as pharmacological targets for lung remodeling deserves further investigation in animal studies and, looking further forward, clinical research. Early intervention to stop these processes may be useful in preventing lung fibrosis in
Conflict of interest statement
All authors of this paper declare that they have no financial or other potential conflicts of interest concerning the subject of this manuscript.
Declaration
This article has not been published previously and has not been under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere including electronically in the same form, in English or in any other language, without the written consent of the copyright-holder.
Acknowledgments
We thank Mo-Tzu Wu and Tun-Yun Hsueh for collection of lung tissue and Dr. Yung-Yang Liu for manuscript preparation. This work was supported by research grants from the Taipei Veterans General Hospital (V96C1-052).
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