Chuen-Mao Yang, PhD, Department of Pharmacology, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-San, Tao-Yuan, 33302, Taiwan. E-mail: email@example.com
BACKGROUND AND PURPOSE Interleukin (IL)-1β-induced matrix metalloproteinase (MMP-9) expression is regulated by mitogen activated protein kinases (MAPKs) and NF-κB. IL-1β also stimulates transactivation of growth factor receptors and phosphatidylinositol 3-kinase (PI3K)/Akt., leading to the expression of inflammatory proteins. Here, we investigated whether these transactivation mechanisms participated in IL-1β-induced MMP-9 expression in A549 cells.
EXPERIMENTAL APPROACH A549 cells were treated with/without pharmacological inhibitors and neutralizing antibody or transfected with dominant negative mutants and siRNA of particular protein kinases before stimulation with IL-1β. Cell migration was measured by in vitro scratch assay. Expression and enzymatic activity of MMP-9 were analysed by Western blot and gelatin zymography. Transcriptional activity of MMP-9 was analysed by RT-PCR, chromatin immunoprecipitation and promoter assays.
KEY RESULTS Inhibition of MMP-9 expression by inhibitors of Src (PP1), platelet-derived growth factor (PDGF) receptor and epithelial growth factor (EGF) receptor or transfection with siRNA for Src and Akt prevented IL-1β-induced migration of A549 cells. These tyrosine kinases were involved through phosphorylation of Src, PDGF, or EGF receptors (EGFRs) via the formation of Src/PDGFR or Src/EGFR complexes, attenuated by PP1. IL-1β-induced MMP-9 expression through EGFR transactivation was diminished by inhibitors of MMPs and heparin-binding EGF-like factor (HB-EGF), or a neutralizing HB-EGF antibody. IL-1β-stimulated activation and translocation of Akt and NF-κB (p65); the recruitment of activated NF-κB (p65) to the MMP-9 promoter region was attenuated by LY294002.
CONCLUSIONS AND IMPLICATIONS IL-1β-induced MMP-9 expression and cell migration was mediated through c-Src-dependent transactivation of EGFR/PDGFR/PI3K/Akt linking to the NF-κB pathway in A549 cells.
Lung cancer is the major cause of malignancy-related deaths worldwide, and its incidence is rising in many countries (Greenlee et al., 2001). Approximately 25–40% of lung cancers are adenocarcinoma, which belongs to the subgroup of the non-small-cell lung cancers (NSCLCs) and is the most common type in Taiwan (Chen et al., 1990; Lee et al., 2004). All of the pathologic changes of cancer progression involve extensive alterations of lung extracellular matrix (ECM). ECM is primarily degraded in tissue remodelling via two distinct pathways: the matrix metalloproteases (MMPs) pathway (Visse and Nagase, 2003) and the plasminogen activator (PAs) proteolytic axis (Pepper, 2001). MMPs are responsible for ECM degradation and implicated in several important physiological and pathogenic events such as cancer invasion and metastasis (Bieniasz et al., 2008). A subfamily of MMPs, gelatinases (i.e. MMP-2 and MMP-9), contain repeats of fibronectin motifs within their catalytic domain, allowing the ability to bind gelatin, their major substrate. These enzymes in lung are produced by structural cells of the bronchial tree and alveolae and by inflammatory cells upon stimulation (Gueders et al., 2006). Although MMP-2 and MMP-9 have similar substrate specificities (Senior et al., 1991), there is a difference in the regulation of their expression by cytokines or growth factors.
Evidence supports the premise that inflammation is a crucial component of tumour progression (Balkwill and Mantovani, 2001). Interleukin-1β (IL-1β), a pro-inflammatory cytokine, has been reported to evaluate in the lungs of asthmatic patients and plays a key role in airway inflammation (Alexander et al., 2008). Raised levels of MMP-9 have been detected in bronchoalveolar lavage fluid, blood and sputum from individuals with allergic asthma (Kelly et al., 2000). MMP-2 expressed from airway smooth muscle cells is also activated by thrombin and contributes to airway remodelling (Elshaw et al., 2004). The uncontrolled remodelling of the lung architecture is a hallmark of many types of lung cancer. In many types of neoplasm, including lung cancer, higher levels of activated MMPs have been demonstrated in more invasive and/or metastatic tumours which may give prognostic information independent of stage (Egeblad and Werb, 2002). It has been reported that the levels of IL-1β (De et al., 1998) and MMPs, such as MMP-2 and MMP-9 (Iniesta et al., 2007; Kopczynska et al., 2007), in plasma of the patients with both small-cell lung cancer and NSCLC are significantly elevated and link to the invasion and metastasis of tumour cells (Gueders et al., 2006; Kopczynska et al., 2007), which have been proposed as reliable prognostic markers (Reichenberger et al., 2001; Jumper et al., 2004; Asada et al., 2006). In addition, IL-1β has been reported to regulate the expression of a variety of proteases including MMP-9 (Lin et al., 2009) and urokinase-plasminogen activators (uPA) (Cheng et al., 2009) in lung diseases. Moreover, adenovirus-mediated inhibition of MMP-9 can abolish invasion and metastasis in NSCLCs (Rao et al., 2005). These findings imply that IL-1β-induced matrix proteolytic enzyme production, MMP-9 especially, may contribute to lung cancer progression.
Several lines of evidence have shown that IL-1β induces MMP-9 expression through MAPKs and NF-κB pathways in A549 cells (Lin et al., 2009) and fibroblasts (Furuyama et al., 2008). In addition to MAPKs and NF-κB, some alternative signalling pathways such as non-receptor tyrosine kinases (e.g. c-Src), and transactivation of PDGF receptor (PDGFR), and phosphatidylinositol 3-kinase (PI3K)/Akt are also implicated in the expression of MMP-9 in RBA cells (Wu et al., 2008). In addition, IL-1β has been shown to activate c-Src, PDGFR and PI3K/Akt associated with ICAM-1 expression in A549 cells (Lin et al., 2007). PI3K/Akt is activated through a c-Src-dependent transactivation of epithelial growth factor receptor (EGFR) and leading to MMP-9 expression induced by arsenite (Cooper et al., 2004). In fact, PI3K/Akt pathway may eventually regulate the activation and translocation of NF-κB (p65) through targeting IKKα or phosphorylation of NF-κB (p65) (Datta et al., 1999; Ozes et al., 1999; Reddy et al., 2000) and stimulate histone acetyltransferases (HAT) activity (Darieva et al., 2004). Although MMP-9 up-regulation was induced by a variety of stimuli such as IL-1β, the mechanisms of intracellular signallings leading to MMP-9 expression are not completely defined in A549 cells.
In the present study, we demonstrated that PI3K/Akt cascade was activated by c-Src-dependent transactivations of EGFR and PDGFR in response to IL-1β. Subsequently, the activated Akt led to NF-κB activation and translocation, recruited the transcriptional machinery to the MMP-9 promoter, and enhanced MMP-9 expression in A549 cells. These findings suggested that up-regulation of MMP-9 through transactivation of c-Src/EGFR/PDGFR/NF-κB pathway enhances its proteolytic functions to IL-1β-challenged A549 cells.
Materials and methods
DMEM/F-12 medium, fetal bovine serum (FBS) and Trizol were purchased from Invitrogen (Carlsbad, CA, USA). Hybond C membrane, enhanced chemiluminescence (ECL) Western blotting detection system and Hyperfilms were from GE Healthcare Biosciences (Buckinghamshire, England). Anti-MMP-9 monoclonal antibody was from NeoMarkers (Fremont, CA, USA). Neutralized heparin-binding EGF-like factor (HB-EGF) antibody was from R&D Systems (Minneapolis, MN, USA). Anti-GAPDH antibody was from Affinity BioReagentsTM (Golden, CO, USA). Polyclonal Ab c-Src, PDGFRα, EGFR, Akt, p65 and Lamin A were from Santa Cruz (Santa Cruz, CA, USA). Anti-PhosphoPlus Src, PDGFR, EGFRY845, EGFRY1068 and AktSer473 Ab kits were from Cell Signaling (Danvers, MA, USA). MMP-3 inhibitor VIII and MMP-2/MMP-9 inhibitor II were from Calbiochem (San Diego, CA, USA). AG1478, AG1296, PP1, LY294002 and GM6001 were from Biomol (Plymouth Meeting, PA, USA). Bicinchoninic acid (BCA) protein assay kit was from Pierce (Rockford, IL, USA). CRM197, enzymes and other chemicals were from Sigma (St. Louis, MO, USA). The nomenclatures of all drugs and molecular targets conform to the British Journal of Pharmacology's Guide to Receptors and Channels (Alexander et al., 2008).
Cell culture of A549
A549 cells (human alveolar epithelial carcinoma cells) were from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM/F-12 supplemented with 10% FBS and antibiotics (100 U·mL−1 penicillin G, 100 mg·mL−1 streptomycin and 250 ng·mL−1 fungizone) at 37°C in a humidified 5% CO2 atmosphere. When the cultures reached confluence (4 days), cells were treated with 0.05% (w/v) trypsin/1 mM EDTA for 3 min at 37°C. The cell suspension (2 × 105 cells·mL−1) was plated onto (1 mL/well) 12-well culture plates and (10 mL/dish) 10-cm culture dishes for the measurement of enzymatic activity, protein expression, and mRNA accumulation. Culture medium was changed after 24 h and then every 3 days. In these experiments, stimulants such as IL-1β were added to the serum-free medium for the indicated time intervals. When the inhibitors were used, they were added 1 h before IL-1β treatment. The concentrations of these inhibitors used had no toxic effect on A549 cells, excluded by LDH release test or XTT assay (data not shown).
Gelatin zymography of MMP-9 activity
A549 cells were plated onto 12-well culture plates and made quiescent by incubation in serum-free DMEM/F-12 for 24 h. The cells were incubated with IL-1β at 37°C for 48 h. When inhibitors were used, they were added 1 h prior to the application of IL-1β. The culture medium was collected and centrifuged at 10 000 ×g for 5 min at 4°C to remove cell debris. The MMP-9 expression was analysed as previously described (Lin et al., 2009). Moreover, we have performed the XTT assay or LDH release test to examine the cell survival after treatment with these inhibitors for 48 h. These data showed the A549 cell survival rate was not changed by treatment with these inhibitors for 48 h (data not shown).
Preparation of cell extracts and Western blot analysis
Growth-arrested A549 cells were incubated with IL-1β at 37°C for the indicated time intervals. The cells were washed with ice-cold PBS, scraped, collected and centrifuged at 45 000 × g for 1 h at 4°C to yield the whole cell extract, as previously described (Lin et al., 2009). Samples were denatured, subjected to SDS-PAGE using a 10% running gel and transferred to nitrocellulose membrane. Membranes were incubated overnight at 4°C using an anti-phospho-PDGFR, anti-phospho-EGFRY845, anti-phospho-EGFRY1068, anti-phospho-Src, anti-phospho-AktSer473, anti-p65, anti-GAPDH, anti-Lamin A or anti-MMP-9 antibody used at a dilution of 1:1000 in 5% (w/v) BSA in TTBS [(50 mM Tris-HCl, 150 mM NaCl, 0.05% (w/v) Tween 20, pH 7.4)]. Membranes were incubated with a 1:2000 dilution of anti-rabbit or anti-mouse horseradish peroxidase antibody for 1 h. The immunoreactive bands detected by ECL reagents were developed by Hyperfilm-ECL.
Growth-arrested cells were incubated with IL-1β at 37°C for 6 h. When inhibitors were used, they were added 1 h prior to the application of IL-1β. Total RNA was isolated with Trizol according to the protocol of the manufacturer as previously described (Lin et al., 2009). MMP-9 and β-actin sequences were amplified for 35 and 30 cycles. Amplification of β-actin, a relatively invariant internal reference RNA, was performed in parallel, and cDNA amounts were standardized to equivalent β-actin mRNA levels. Oligonucleotide primers for MMP-9 and β-actin were as follows:
MMP-9 (468 bp)
Sense: 5′- GGCGCTCATGTACCCTATGT-3′
Anti-sense: 5′- TCAAAGACCGAGTCCAGCTT-3′
β-actin (636 bp)
Cell lysates containing 1 mg of protein were incubated with 2 µg of anti-EGFR, anti-PDGFR or anti-c-Src antibody at 4°C for 24 h, and then 10 µL of 50% protein A-agarose beads was added and mixed for 24 h at 4°C. The immunoprecipitates were collected and washed three times with a lysis buffer without Triton X-100 (Sigma, St Louis, MO, USA). Following wash, 5X Laemmli buffer was added and subjected to electrophoresis on 10% SDS-PAGE, and then blotted using an anti-phospho-EGFRY845, anti-phospho-PDGFRα, anti-phospho-c-Src, anti-EGFR, anti-PDGFR or anti-c-Src antibody.
Transient transfection with siRNAs and dominant negative mutants
Stealth RNAi duplexes corresponding to human siRNAs of Akt, Src and scrambled #2 siRNA were from Invitrogen (Carlsbad, CA, USA), and MMP-9 was kindly provided by Dr C.P. Tseng (Chang Gung University, Taiwan). The plasmids encoding dominant negative mutants of Src, p85 and Akt, cloned into pcDNA3, were kindly provided by Dr R. D. Ye (Department of Pharmacology, University of Illinois at Chicago, USA). All mutants were prepared by using QIAGEN (Valencia, CA, USA) plasmid DNA preparation kits. Transient transfection of siRNAs and dominant negative mutants were carried out using Metafectene transfection reagent (Biontex Laboratories GmbH, Martinsried/Planegg, Germany). The dominant negative mutant (1 µg) or siRNA (100 nM) was formulated with Metafectene transfection reagent according to the manufacturer's instruction. Transfected cells were analysed at 48 h after IL-1β-treatment by Western blot using an anti-MMP-9, Src or Akt antibody.
Translocation of p65 and Akt
A549 cells were seeded in a 10-cm dish. After they reached confluence, the cells were starved for 24 h in serum-free DMEM/F-12 medium. When inhibitors were used, they were added 1 h prior to the application of IL-1β. After stimulation with 15 ng·mL−1 of IL-1β for the indicated time intervals, the cells were washed, scraped, and centrifuged to prepare cytosolic and nuclear fractions, as previously described (Cheng et al., 2009). The translocation of NF-κB and Akt (nucleus and cytoplasm) were determined by Western blot using an anti-p65 or Akt antibody.
A549 cells were plated on six-well culture plates with coverslips. When inhibitors were used, they were added 1 h prior to the application of 15 ng·mL−1 of IL-1β. After stimulation with IL-1β for 30 min, the cells were washed, fixed, and labelled with an anti-Akt or anti-p65 antibody and followed by FITC- and Rhodamine (TRITC)-conjugated secondary antibodies (Jackson ImmunoResearch Lab., West Grove, PA, USA) staining, respectively. The coverslips mounted with aqueous mounting medium with DAPI. The images were observed using a fluorescence microscope (Zeiss, Axiovert 200M).
Measurement of MMP-9 and NF-κB-luciferase activity
For construction of the human MMP-9 promoter-Luc (a region spanning −720 to −11 bp of MMP-9 promoter) and NF-κB-Luc reporter plasmids were cloned into pGL3-basic vector (Promega, Madison, WI, USA) and prepared by using QIAGEN plasmid DNA preparation kits. A549 cells were transfected with a MMP-9 promoter-Luc or NF-κB-Luc plasmid using Metafectene reagent. To test the effect of siRNAs transfection, A549 cells were cotransfected with the siRNA of Src, Akt, or scrambled and MMP-9-luciferase, β-galactosidase plasmids. When the inhibitors were used, they were added 1 h prior to the application of 15 ng·mL−1 of IL-1β. After incubation for 6 h, the cells were collected and then determined for luciferase activity using a luciferase assay system (Promega, Madison, WI, USA). The luciferase activities were standardized for β-galactosidase activity as previously described (Cheng et al., 2009).
Chromatin immunoprecipitation (ChIP) assay
These assays were performed as previously described (Lin et al., 2009). DNA immunoprecipitated by anti-p65 antibody was purified. The DNA pellet was re-suspended in H2O and subjected to PCR amplification with the forward primer 5′-TGTCCCTTTACTGCCCTGA-3′ and the reverse primer 5′-ACTCCAGGCTCTGTCCTCCTCTT-3′, which were specifically designed from the MMP-9 promoter region (−657 to −484). PCR products were analysed on ethidium bromide-stained agarose gels (2%). The input represents PCR products from chromatin pellets prior to immunoprecipitation.
A549 cells were cultured in six-well plates and starved with serum-free DMEM/F-12 medium for 24 h. The monolayer cells were scratched manually with a pipette tip, and the detached cells were removed with PBS. Serum-free DMEM/F-12 medium with or without IL-1β was added to each dish as indicated after pretreatment of inhibitors for 1 h (containing 10 µM hydrourea during the period of incubation). Images were observed and taken at 48 h with a digital camera and a microscope (Olympus, Japan) as previously described (Lin et al., 2009).
Concentration-effect curves were made and EC50 values were estimated using the GraphPad Prism Program (GraphPad, San Diego, CA, USA). Data were expressed as the mean ± SEM and analysed with a one-way anova at a P < 0.05 level of significance.
IL-1β induces cell migration in an MMP-9-dependent manner in A549 cells
We determined the effect of IL-1β on MMP-9 expression and activity in a concentration- and time-dependent manner in A549 cells. As shown in Figure 1A and B, exposure to IL-1β induced MMP-9 (92 kDa) expression and activity in a concentration- and time-dependent manner, analysed by gelatin zymography and Western blot. There was a maximal response within 48 h during the period of observation. In contrast, incubation with IL-1β failed to change the expression of MMP-2 (72 kDa). These results demonstrated that IL-1β-induced MMP-9 activity and expression was dependent on the concentration and incubation time of IL-1β.
To further examine the functional response of MMP-9 induced by IL-1β, A549 cell migration was evaluated. In addition, to rule out the interference with cell proliferation, cells were treated with a proliferating inhibitor hydroxyurea (10 µM) during exposure to IL-1β for 48 h. As shown in Figure 1C, IL-1β-induced cell migration was significantly attenuated by pretreatment with a selective MMP-2/9 inhibitor or transfection with MMP-9 siRNA, suggesting that MMP-9, at least in part, participated in the induction of cell migration by IL-1β in A549 cells. Analyses of the conditioned media and whole cell protein showed that MMP-9 activity and protein expression were attenuated by these treatments (Figure 1D).
Involvement of c-Src, EGFR, PDGFR and PI3K/Akt in IL-1β-induced MMP-9 expression and cell migration
To determine whether transactivation of c-Src, EGFR, PDGFR and PI3K/Akt participated in IL-1β-induced MMP-9 promoter activity and mRNA expression, the inhibitors of c-Src (PP1), EGFR (AG1478), PDGFR (AG1296) and PI3K (LY294002) were used. Pretreatment with these inhibitors blocked IL-1β-stimulated MMP-9 promoter activity (Figure 2A) and mRNA expression (Figure 2B). To ensure the involvement of c-Src and PI3K/Akt in IL-1β-induced MMP-9 expression, as shown in Figure 2C, transfection cells with dominant negative mutants of c-Src (ΔSrc), p85 (the regulatory domain of PI3K, Δp85) or Akt (ΔAkt) significantly attenuated IL-1β-induced MMP-9 protein expression. Furthermore, to clarify whether c-Src, EGFR, PDGFR, and PI3K/Akt were involved in IL-1β-induced MMP-9 expression and cell migration, as shown in Figure 2D, the images demonstrated that IL-1β significantly stimulated A549 cell migration which was blocked by pretreatment with AG1478, AG1296, PP1 or LY294002. These results concluded that IL-1β-induced MMP-9 expression and cell migration was mediated through transactivation of c-Src, EGFR, PDGFR, and PI3K/Ak in A549 cells.
IL-1β-induced MMP-9 expression and activity is mediated through c-Src-dependent EGFR/PDGFR transactivation
The alternative signal pathways such as c-Src-dependent EGFR transactivation have been shown to involve in expression of several genes, including MMP-9 (Cooper et al., 2004). To investigate whether c-Src activation was involved in the expression of MMP-9 induced by IL-1β, as shown in Figure 3A and B, pretreatment with PP1 or transfection with c-Src siRNA abolished IL-1β-induced MMP-9 expression and activity, and promoter activity, respectively, in A549 cells. In addition, we also demonstrated that transfection with c-Src siRNA significantly down-regulated c-Src protein expression (Figure 3B, inset panel).
Next, to determine whether IL-1β-induced responses were mediated through transactivation of c-Src and growth factor receptors such as EGFR and PDGFR, the cells were stimulated with IL-1β for the indicated time intervals and analysed by immunoprecipitation and Western blot. As shown in Figure 3C–E, IL-1β stimulated c-Src, PDGFR, and EGFR phosphorylation in a time-dependent manner and enhanced the association between the phosphorylation of c-Src with PDGFR or EGFR in these immunoprecipitated complexes using their respective antibody, which was abolished by pretreatment with PP1. We found that IL-1β-stimulated a maximal c-Src/PDGFR or c-Src/EGFR association occurred within 3–5 min. These results suggested that IL-1β triggered c-Src activation leading to the association with EGFR or PDGFR which resulted in MMP-9 expression in A549 cells.
IL-1β induces MMP-9 expression and activity via a Src/PDGFR/EGFR/PI3K/Akt cascade
To determine the lineage activation of Src, PDGFR, EGFR, and PI3K/Akt in IL-1β-induced MMP-9 expression, as shown in Figure 4A and B, pretreatment with AG1296 and AG1478 significantly attenuated IL-1β-induced MMP-9 expression and activity in a concentration-dependent manner, implying that IL-1β-induced MMP-9 expression and activity was mediated through PDGFR and EGFR. Moreover, to determine whether phosphorylation of PDGFR and EGFR was involved in the IL-1β-induced responses, as shown in Figure 4C, IL-1β stimulated phosphorylation of PDGFR and EGFR in a time-dependent manner with a maximal response within 1–3 min (p-PDGFR) and 5–10 min (p-EGFR), respectively. Pretreatment with either AG1296 or PP1 significantly attenuated phosphorylation of PDGFR stimulated by IL-1β (Figure 4D). In addition, pretreatment with AG1478 but not with LY294002 significantly attenuated phosphorylation of EGFR by IL-1β (Figure 4E), implying that PI3K/Akt was a downstream component of c-Src/EGFR. Next, to determine whether Akt was a downstream molecule of PDGFR and EGFR involved in these responses, the phosphorylation of Akt was detected. The data showed that IL-1β stimulated Akt phosphorylation in a time-dependent manner (Figure 4F). Pretreatment with PP1, AG1296, AG1478 or LY294002 significantly attenuated IL-1β-stimulated Akt phosphorylation (Figure 4F). These results indicated that c-Src-dependent PDGFR-EGFR/PI3K/Akt cascade was involved in IL-1β-induced MMP-9 expression and activity in A549 cells.
PI3K/Akt cascade is involved in IL-1β-induced MMP-9 expression and activity
Transactivation of EGFR and PDGFR has been shown to regulate MMP-9 expression mediated via Src and Akt phosphorylation stimulated by IL-1β. We further confirmed that Akt activation was essential for IL-1β-induced MMP-9 expression. As shown in Figure 5A, pretreatment of A549 cells with LY294002 attenuated MMP-9 expression and activity in a concentration-dependent manner. Moreover, we found that IL-1β stimulated phosphorylation and translocation of Akt into nuclear fraction (Figure 5B). To further ensure the involvement of Akt in the expression of MMP-9 induced by IL-1β, cells were transfected with Akt siRNA (si-Akt), effectively knocked down the expression of Akt protein (Figure 5C, inset part) and significantly decreased IL-1β-stimulated MMP-9 promoter activity (Figure 5C). These results suggested that PI3K/Akt is involved in IL-1β-induced MMP-9 expression and activity in A549 cells.
Involvement of MMPs in IL-1β-induced MMP-9 expression and activity
Moreover, the cleavage of pro-HB-EGF by MMPs has been shown to mediate EGFR transactivation by external stimuli in various cell types (Graves et al., 2002). We therefore investigated the effects of GM6001, a broad-spectrum inhibitor of MMPs which was used to inhibit ectodomain shedding of transmembrane EGF family precursor from the cell surface, on IL-1β-induced responses. As shown in Figure 6A, pretreatment with GM6001 caused a concentration-dependent attenuation of MMP-9 expression and activity. To ascertain the effect of MMP-dependent shedding activity on IL-1β-stimulated phosphorylation of c-Src, PDGFR, EGFR, and Akt, as shown in Figure 6B, pretreatment with CRM197 (an inhibitor of HB-EGF), GM6001, MMP-3 in. or MMP2/9 in. attenuated IL-1β-stimulated EGFR and Akt phosphorylation but had no effect on PDGFR and c-Src phosphorylation. Incubation of cells with (30 ng·mL−1) HB-EGF also stimulated EGFR phosphorylation, but not PDGFR (data not shown). These data confirmed that IL-1β-induced EGFR transactivation was mediated through MMP-3 or MMP-2/9-catalysed release of HB-EGF. To ensure these signal molecules of MMP-dependent shedding pathway involved in IL-1β-mediated responses, as shown in Figure 6C, pretreatment with GM6001, MMP-2/9 in., MMP-3 in., CRM197 or neutralized HB-EGF antibody significantly decreased IL-1β-stimulated MMP-9 promoter activity, suggesting that MMP-3 and MMP-2/9 were involved in transactivation of EGFR and Akt leading to MMP-9 induction by IL-1β in A549 cells. These results indicated that MMPs-mediated proteolytic cleavage of pro-HB-EGF led to transactivation of the EGFR resulting in MMP-9 induction by IL-1β.
NF-κB (p65) is required for IL-1β-induced MMP-9 expression
To determine whether NF-κB participated in IL-1β-induced MMP-9 expression and activity, as shown in Figure 7A and B, IL-1β stimulated NF-κB (p65) translocation from cytosol into nucleus, determined by Western blot and immunofluorescence staining, which was attenuated by pretreatment with CRM197, GM6001, MMP-2/9 in., MMP-3 in., AG1478, AG2196, PP1 or LY294002 (Figure 7A). In addition, the images of immunofluorescence staining showed that pretreatment with LY294002 significantly blocked the activation and translocation of Akt and NF-κB (p65) (Figure 7B), suggesting that NF-κB (p65) translocation was mediated through the same pathways as those of MMP-9 expression and activity, including c-Src- or MMP-dependent EGFR and PDGFR transactivation and PI3K/Akt in response to IL-1β.
To further investigate whether activation of NF-κB (p65) by IL-1β was recruited to MMP-9 promoter region, the ChIP-PCR assay was performed. As shown in Figure 7C, IL-1β stimulated recruitment of NF-κB (p65) to MMP-9 promoter region with a maximal response within 30 min and sustained up to 120 min. Pretreatment with LY294002 significantly inhibited IL-1β-induced recruitment of NF-κB (p65) to MMP-9 promoter, implying that IL-1β-stimulated NF-κB (p65) translocation and recruitment to MMP-9 promoter region was mediated through Akt.
To examine whether NF-κB transcriptional activity was regulated by IL-1β in A549 cells, we used a luciferase reporter gene containing NF-κB binding sites (pNF-κB-Luc) to determine the activity of NF-κB. As shown in Figure 7D, IL-1β-stimulated NF-κB transcriptional activity was attenuated by pretreatment with CRM197, GM6001, MMP-2/9 in., MMP-3 in., AG1478, AG2196, PP1, LY294002 or helenalin (a selective NF-κB inhibitor), suggesting that NF-κB (p65) is essential for IL-1β-induced MMP-9 gene expression in A549 cells.
Discussion and conclusions
MMPs have been shown to contribute to the formation of a complex microenvironment that promotes malignant transformation and cancer progression (Sounni and Noel, 2005). Secretion of PAs and MMPs, such as MMP-9, by structural or inflammatory cells is thought to take part in the processes of lung cancer metastasis to distant sites (Gueders et al., 2006; Offersen et al., 2007). In addition, the level of IL-1β has been shown to be elevated in plasma of the patients with lung cancer (De et al., 1998). In a previous study, we have demonstrated that IL-1β induces uPA expression and cell migration through PKCα, JNK1/2, and NF-κB in A549 cells (Cheng et al., 2009). We also demonstrate that IL-1β-induced MMP-9 expression for cell migration is mediated through both MAPKs/AP1- and NF-κB-dependent pathways (Lin et al., 2009). Therefore, we further explored whether alternative mechanism(s) to MAPKs and NF-κB downstream of IL-1β, such as RTK transactivation pathway, are involved in MMP-9 expression and cell migration in these cells. As depicted in Figure 8, we first demonstrated that IL-1β-induced MMP-9 expression and activity in a concentration-dependent fashion and c-Src was required for EGFR and PDGFR transactivation and MMP-9 expression and activity induced by IL-1β. Next, the data showed that transactivation of PDGFR and EGFR by IL-1β was linked to the PI3K/Akt cascade leading to MMP-9 expression and activity. Moreover, both PDGFR and EGFR phosphorylation stimulated by IL-1β were mediated through c-Src. EGFR phosphorylation was also triggered by MMP-dependent cleavage of HB-EGF in A549 cells. The results revealed by Western blotting, immunofluorescence staining, promoter reporter gene, and ChIP assays showed that IL-1β-stimulated NF-κB (p65) nuclear translocation and recruitment to MMP-9 promoter region was mediated through Akt. The enhanced recruitment of NF-κB (p65) to the promoter region of MMP-9 led to an increase of MMP-9 promoter activity and expression in A549 cells. These results support the notion that in A549 cells, IL-1β-induced MMP-9 expression and activity is mediated through c-Src-dependent transactivation of PDGFR, EGFR/PI3K/Akt and NF-κB signaling pathways. On the basis of reported observations from the literature and our findings, a schematic pathway depicts a model for the roles of Src/EGFR-PDGFR/PI3K/Akt and NF-κB activation associated with MMP-9 expression and activity in A549 cells exposed to IL-1β (Figure 8).
EGFR has been shown to be transactivated by various G protein-coupled receptor (GPCR) agonists, phorbol esters, cytokines, chemokines, estrogen, and cell stress signals, which was mediated through a disintegrin and metalloprotease (ADAM) or MMP activation (Graves et al., 2002; Berasain et al., 2009). Elevation of the intracellular levels of Ca2+ or reactive oxygen species (ROS), as well as phosphorylation of PKC, ERK1/2 or c-Src, are likely to be involved (Graves et al., 2002; Berasain et al., 2009). Several lines of evidence have indicated that activation of GPCRs initiates the Akt pathway through the recruitment of scaffold proteins such as Src, which in turn mediates the transactivation of the EGFR or PDGFR in various cell types (Lee et al., 2007; Lin et al., 2007). Transactivation of growth factor receptors results in regulation of gene expression involved in cell proliferation, survival, migration or amplification of inflammatory responses (Berasain et al., 2009). Moreover, c-Src has been implicated in IL-1β/IL-1 receptor-induced transactivation of PDGFR, which in turn activates downstream PI3K/Akt and ERK1/2 cascades in RBA-1 cells (Wu et al., 2008). In addition, EGFR transactivation has also been demonstrated to be initiated by other receptor tyrosine kinases, including the insulin-like growth factor-1 receptor and the platelet-derived growth factor receptor beta (PDGFβR) (Graves et al., 2002). Thus, crosstalk between receptor tyrosine kinase families provides yet another mechanism for diverse stimuli to tap into unique EGFR signalling and associated mitogenic pathways. Saito and coworkers suggest that a Src- or ROS-dependent constitutive heterodimerization of the PDGFβR-EGFR exists and facilitates PDGF-stimulated increases in EGFR tyrosine phosphorylation. The formation of the heterodimer provides a scaffold for other molecules that induces EGFR transactivation and downstream signalling, such as p21-activated kinase and ERK1/2 (Saito et al., 2001). In this aspect of ligand-independent EGFR transactivation, c-Src has been shown to stimulate tyrosine residues Y845 and Y1101 phosphorylation on EGFR and contributes to tumour progression (Biscardi et al., 1999). Although activation of IL-1R has been shown to phosphorylate tyrosine residues of c-Src (Lin et al., 2007; Wu et al., 2008), little was known about the mechanisms underlying IL-1β-induced MMP-9 expression mediated through c-Src-dependent transactivation of RTK in A549 cells. Our results demonstrated that in A549 cells, IL-1β-induced MMP-9 expression and activity was mediated through a c-Src component which was attenuated by transfection with dominant negative mutant of c-Src and c-Src siRNA or pretreatment with a c-Src inhibitor PP1 (Figures 2A–C and 3A–B). This hypothesis was further supported by the findings that pretreatment with PP1 significantly inhibited c-Src, EGFR or PDGFR activation and their association, indicating that IL-1β-induced c-Src activation precedes PDGFR and EGFR transactivation (Figures 3C–E and 4D lower panel). These results were confirmed with the studies indicating that c-Src-dependent EGFR or PDGFR transactivation is necessary and sufficient for the expression of several genes, including MMP-9 (Cooper et al., 2004; Wu et al., 2008).
In addition to c-Src-mediated EGFR transactivation, the transactivation process may be mediated through activation of a family of secreted and membrane-anchored MMPs, which induces ectodomain shedding of EGF ligands to produce bioactive soluble factors (Gschwind et al., 2001). Up-regulation of EGFR signalling has been correlated with the formation and progression of human cancers associated with MMP-mediated release of EGF-like precursors in oncogenesis and progression (Gschwind et al., 2001). To elucidate the molecular mechanisms of the MMP-mediated EGFR transactivation, we found that shedding of pro-HB-EGF by MMP-3 or MMP-2/9 involved in IL-1β-induced MMP-9 expression and activity was mediated through transactivation of EGFR and Akt phosphorylation, but not through Src phosphorylation and PDGFR transactivation. These findings further confirmed that IL-1β-induced MMP-9 expression was mediated through either MMP-dependent transactivation of EGFR or c-Src-dependent activation of the EGFR and PDGFR pathways. However, the mechanisms by which IL-1β-activated MMPs lead to the transactivation of growth factor receptors will be interesting to purse.
Several lines of evidence have shown that PI3K/Akt is involved in IL-1β-mediated signalling events. PI3K/Akt pathway is central in the transmission of growth regulatory signals originating from cell surface receptors, such as PDGFR and EGFR, and frequently aberrantly regulated in various cell types, including NSCLC (Metro et al., 2006; Bauman et al., 2007). In addition, PI3K/Akt has been reported to be essential for MMP-9 expression. For example, Akt phosphorylation is required for TNF-α or IL-1β-induced MMP-9 expression in various cell types (Lee et al., 2007; Wu et al., 2008). Our results were consistent with these studies demonstrating that Akt is a pivotal regulator for MMP-9 expression and activity in response to IL-1β in A549 cells. As elucidated in Figures 2, 3 and 5, pretreatment with pharmacological inhibitors of Src (PP1), PDGFR (AG1296), EGFR (AG1478) or PI3K (LY294002) and transfection with dominant negative mutants and siRNA attenuated IL-1β-induced MMP-9 expression and promoter activity in A549 cells. Moreover, the activation of Akt by IL-1β led to its translocation from cytosol into nucleus (Figure 5B) which was attenuated by pretreatment with PP1, AG1478, AG1296 and LY294002 in A549 cells (Figure 4F). These results suggested that IL-1β-induced MMP-9 expression and activity mediated through transactivation of growth factor receptors such as EGFR or PDGFR is linked to the PI3K/Akt pathway.
Moreover, phosphorylation of Akt may eventually regulate NF-κB activation and translocation through targeting IKKs or phosphorylation of p65 (Datta et al., 1999; Reddy et al., 2000) and stimulate HAT activity (Darieva et al., 2004). Our results also showed that IL-1β-enhanced NF-κB (p65) translocation and promoter activity was significantly attenuated by pretreatment with the inhibitors of Src, PDGFR, EGFR, PI3K and MMPs (Figure 7), indicating that transactivation of growth factor receptors plays an important role in IL-1β-stimulated NF-κB (p65) activation. In addition, NF-κB binding sites have been identified in the MMP-9 promoter region (Hatamochi et al., 1998), which appear to be essential components for the MMP-9 gene expression upon exposure to cytokines (Lin et al., 2008; 2009). Our previous studies have documented that NF-κB-dependent MMP-9 gene transcription requires the presence of HAT co-activators in cytokine-stimulated cells (Lee et al., 2007). Moreover, Akt phosphorylation induces recruitment of p300 (a HAT co-activator) to MMP-9 gene promoters (Lee et al., 2007) and leads to acetylation of histones in chromatin and association with the basal transcriptional machinery RNA polymerase II. Similarly, in A549 cells, IL-1β-stimulated activation and translocation of Akt and NF-κB (p65) (Figures 5B, 7A and B), and then the activated NF-κB (p65) was recruited to MMP-9 promoter, which resulted in MMP-9 gene expression (Figure 7C). These results suggested that NF-κB (p65) transcriptional activity is dependent on Akt phosphorylation and may induce chromatin remodelling in A549 cells. IL-1β has been shown to induce MMP-9 expression in various cell types but has no effect on glomerular mesangial cells and down-regulates MMP-9 in adult lung fibroblasts (Sasaki et al., 2000; Nee et al., 2007). Therefore, different cells in response to the same cytokine have diverse effects.
MMPs are believed to be mainly involved in degradation of ECM, thereby facilitating cell migration. However, many non-ECM proteins are substrates for MMP-9 as well. These include proteins involved in growth, immune response and angiogenesis independent of ECM degradation. The discovery of such mechanisms of MMP-9 has put an important role in cancer progression into a different perspective (Mook et al., 2004). The evidence is accumulating that MMP-9 can cleave growth factors or degrade binding proteins in the target cells, which results in an increase in bioactive growth factors. The majority of growth factors are present in an inactive pro-form, such as insulin growth factor (IGF), transforming growth factor β (TGFβ) or HB-EGF. MMP-9 has been found to activate TGFβ by association with CD44 at the cell surface and to modulate IGF bioactivity by degradation of IGF binding proteins. In addition, pro-HB-EGF has been reported to be cleaved by MMP-9 proteolytic activity, which leads to modulate EGFR signalling (Hurtado et al., 2007). However, HB-EGF is a potent inducer of tumour progression, which not only induces the expression and activities of MMP-9 and MMP-3 but also enhances cell migration (Ongusaha et al., 2004). In this study, we found that IL-1β induces cell migration by triggering a proteolytic cascade that sequentially activates MMP-2/3/9 and HB-EGF (Figure 6). Therefore, MMP-9 may participate in the processing of pro-HB-EGF leading to a positive feedback loop that elevates EGFR signals and MMP-9 expression. These events could provide an explanation for how MMP-9 might facilitate IL-1β-induced cell migration.
In conclusion, we have demonstrated that IL-1β directly induces MMP-9 expression via transactivation of c-Src-dependent EGFR, PDGFR/PI3K/Akt and NF-κB pathway, which results from MMP proteolytic activity-dependent HB-EGF leading to EGFR signalling. These findings suggested that IL-1β-induced MMP-9 expression and activity might play a critical role in the progression of NSCLC and its risk.
This work was supported by grants NSC98-2314-B-182-021 (CCL), NSC96-2320-B-182-003 and NSC96-2320-B-182-047-MY3 (CMY) from National Science Council, Taiwan; and CMRPD32043, CMRPD170332, CMRPG381521 and CMRPG350653 from Chang Gung Medical Research Foundation. The authors appreciate Mr Li-Der Hsiao for his technical assistance during the preparation of manuscript.
Conflicts of interest
The authors have no financial conflict of interest.