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Am J Physiol Lung Cell Mol Physiol 294: L1076-L1084, 2008. First published April 4, 2008; doi:10.1152/ajplung.00489.2007
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MMP-12 induces IL-8/CXCL8 secretion through EGFR and ERK1/2 activation in epithelial cells

Catherine Le Quément,1 Isabelle Guénon,1 Jean-Yves Gillon,2 Vincent Lagente,1 and Elisabeth Boichot1

1Institut National de la Santé et de la Recherche Médicale (INSERM) U620, Université de Rennes 1, Rennes, France, and 2Merck-Serono International S.A., Geneva, Switzerland

Submitted 28 November 2007 ; accepted in final form 3 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Macrophage metalloelastase (MMP-12) is described to be involved in pulmonary inflammatory response. To determine the mechanisms linking MMP-12 and inflammation, we examined the effect of recombinant human MMP-12 (rhMMP-12) catalytic domain on IL-8/CXCL8 production in cultured human airway epithelial (A549) cells. Stimulation with rhMMP-12 resulted in a concentration-dependent IL-8/CXCL8 synthesis 6 h later. Similar results were also observed in cultured BEAS-2B bronchial epithelial cells. In A549 cells, synthetic matrix metalloproteinase (MMP) inhibitors prevented rhMMP-12-induced IL-8/CXCL8 release. We further demonstrated that in A549 cells, rhMMP-12 induced transient, peaking at 5 min, activation of ERK1/2. Selective MEK inhibitors (U0126 and PD-98059) blocked both IL-8/CXCL8 release and ERK1/2 phosphorylation. IL-8/CXCL8 induction and ERK1/2 activation were preceded by EGF receptor (EGFR) tyrosine phosphorylation, within 2 min, and reduced by selective EGFR tyrosine kinase inhibitors (AG-1478 and PD168393) by a neutralizing EGFR antibody and by small interfering RNA oligonucleotides directed against EGFR, implicating EGFR activation. In addition, we observed an activation of c-Fos in A549 cells stimulated by rhMMP-12, dependent on ERK1/2. Using small interfering technique, we showed that c-Fos is involved in rhMMP-12-induced IL-8/CXCL8 production. From these results, we conclude that one mechanism, by which MMP-12 induces IL-8/CXCL8 release from the alveolar epithelium, is the EGFR/ERK1/2/activating protein-1 pathway.

metalloelastase; alveolar epithelium; chemokine

IN ADDITION TO ITS ROLE AS barrier function, alveolar epithelium also participates to lung defense mechanisms and to the development of pulmonary inflammation through the release of various inflammatory mediators including cytokines. Alveolar epithelial cells have been shown to secrete cytokines and chemokines that participate in innate immunity or in response to bacterial or viral infection (4, 13) or diesel exhaust (41).

Among others mediators, IL-8/CXCL8 is a member of the CXC family of chemokines and is a major chemoattractant and activator of neutrophils at site of inflammation (19, 34). IL-8/CXCL8 plays a pivotal role in several lung pathologies like asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, sarcoidosis, and acute respiratory disease syndrome (29). In vitro cultured epithelial cells have also been reported to secrete IL-8/CXCL8 in response to diesel exhaust (26), ozone (17), and cigarette smoke (15) as well as to mycoplasma, virus, or bacterial infection (4, 12, 28, 39). The IL-8/CXCL8 production by lung epithelial cells seems to be controlled by a regulation of the IL-8/CXCL8 gene expression by NF-{kappa}B and activating protein-1 (AP-1) transcription factors (18, 37), although some other mechanisms have also been described, e.g., an increased stability of the IL-8/CXCL8 mRNA (40). Neutrophil elastase, a serine protease released by neutrophils after infection (2), also interacts with alveolar epithelium to induce IL-8/CXCL8 release (22).

MMP-12, another elastase also called macrophage metalloelastase, is a member of the matrix metalloproteinases (MMPs) that are zinc-dependent endopeptidases. MMP-12 is a 54-kDa proenzyme that is secreted extracellularly and then processed into 45- and 22-kDa active forms (3). Although cultured bronchial epithelial cells have been shown to secrete MMP-12 (24), macrophages seem to be the major source of MMP-12 in the alveolar space (38). MMP-12 is overexpressed in the airways of patients affected by COPD (9, 27) and seems to also play a prominent role in the development of lung inflammation. In rodent preclinical models, mice lacking the gene encoding MMP-12 were shown to have significantly less airway inflammation following cigarette smoke exposure compared with their wild-type controls (7, 8, 14, 25). Recently, it has been reported that the recombinant human MMP-12 (rhMMP-12) catalytic domain instilled directly in the airways of mice elicited an intense inflammatory response characterized by a rapid accumulation of neutrophils, associated with an enhanced production of cytokines and chemokines, including of keratinocyte-derived chemokine (KC)/CXCL1, the murine equivalent of IL-8/CXCL8 (32).

As the mechanism by which MMP-12 triggers cell activation and recruitment associated with inflammatory process is not established, we have examined the effects of rhMMP-12 catalytic domain on human alveolar type II-like epithelial cells (A549) and on human bronchial epithelial cells (BEAS-2B). We have shown that rhMMP-12 enhanced the release of several chemokines by A549 cells, in particular, monocyte chemoattractant protein-1 (MCP-1)/CCL2, growth-related oncogene-{alpha} (GRO-{alpha})/CXCL1, and IL-8/CXCL8. In BEAS-2B cells, we have also observed a concentration-dependent increase of IL-8/CXCL8 after incubation with rhMMP-12. Focusing our study on IL-8/CXCL8, we have shown in A549 cells that MMP-12 induced its gene expression and release via EGF receptor (EGFR) transactivation and further activation of the MAPK ERK1/2 signal transduction pathway, also involving the AP-1 transcription factor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Reagents and antibodies. rhMMP-12 was provided by Pfizer Global Research and Development and is described in detail in Nénan et al. (32). Batimastat is a nonselective MMP inhibitor and was also provided by Pfizer Global Research and Development. AS111793, provided by Serono Pharmaceutical Research Institute (Geneva, Switzerland), is a novel and structurally unique derivative of succinyl hydroxamic acid (1) that selectively inhibits MMP-12 with a Ki of 5 nM.

F-12K Nutrient Mixture-Kaighn's Modification cell culture medium, antibiotics, glutamine, and trypsin-EDTA were purchased from Invitrogen. FCS was from HyClone (Logan, UT). SB-203580, SP-600125, PD-98059, and U0126 were purchased from Sigma-Aldrich (St. Louis, MO). AG-1478 and PD168393 were from Calbiochem (La Jolla, CA). TransAM AP-1 family and NF-{kappa}B p65 kits were from Active Motif (Rixensart, Belgium). The specific antibodies against phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-JNK, JNK, phospho-EGFR (Tyr845), EGFR, c-Fos, and p65 NF-{kappa}B were from Cell Signaling Technology (Beverly, MA). The monoclonal neutralizing antibody against EGFR (clone LA1) and nonimmune mouse IgG1 that was used as a negative control were purchased from Upstate Biotechnology (Lake Placid, NY). Acrylamide, SDS, Tris, HEPES, and BSA were from Eurobio (Les Ulis, France). Bradford protein assay and Precision Plus Protein Dual Color Standards were from Bio-Rad (Hercules, CA).

Cell culture. The human alveolar epithelial cell line, A549 [European Collection of Cell Cultures (ECACC), Salisbury, United Kingdom], and bronchial epithelial cell line, BEAS-2B (courteously provided by Dr. M. Si-Tahar, Unité Pasteur, Paris, France), were cultured in F-12K supplemented with 10% FCS, 1% antibiotics, 1% L-glutamine, and 10 mM HEPES in a humidified incubator at 37°C and an atmosphere of 5% CO2. Cells (1 x 105) were transferred to 12-well plates and grown to confluence for the experiments.

Treatments. A549 and BEAS-2B cells were stimulated with various concentrations of rhMMP-12 (3.10–3 to 1.10–1 U/ml). Also, A549 cells were incubated for 6 h in the absence or presence of pretreatment (1 h before the addition of 3.10–2 U/ml rhMMP-12 in that case) with either AS111793 (1–30 µM), the nonselective MMP inhibitor Batimastat (1–30 µM), the P38MAPK-specific inhibitor SB-203580 (20 µM), the JNK inhibitor SP-600125 (2 µM), or the MEK/ERK inhibitors PD-98059 (10 µM) and U0126 (1–5 µM). Alternatively, preincubation of A549 cells with the EGFR tyrosine kinase inhibitors tyrphostin AG-1478 (5–10 µM) and PD168393 (10 nM to 10 µM) or with the neutralizing antibody against EGFR (LA; 10–50 µg/ml) was performed for 30 min. All experiments were performed with serum-free medium.

After treatment, the culture supernatants were harvested and stored at –80°C until further analysis. For the analysis of IL-8/CXCL8 mRNA expression, cells were collected in TRIzol and stored at –20°C until analysis.

Measurement of chemokine proteins and mRNAs. The concentrations of IL-8/CXCL8, MCP-1/CCL2, GRO-{alpha}/CXCL1, IL-6, TNF-{alpha}, macrophage inflammatory protein-1{alpha} (MIP-1{alpha})/CCL3, and TGF-{alpha} in the culture supernatants were measured by ELISA using kits from R&D Systems (Abingdon, United Kingdom). ELISA quantifications were performed according to manufacturer's instructions.

Total RNAs were isolated from A549 cells using the TRIzol method (Invitrogen). Total RNAs (1 µg) were then subjected to reverse transcription/real-time quantitative PCR (RT-qPCR) using the fluorescent dye SYBR Green methodology and an ABI Prism 7000 detector (Applied Biosystems, Foster City, CA) as previously described (26a). Gene-specific primers were designed with the Primer3 software (Whitehead Institute for Biomedical Research, Cambridge, MA), and known intron-exon boundary informations were taken into account for each target gene to avoid detection of genomic DNA (IL-8/CXCL8 primers, forward 5'-AAG AAA CCA CCG GAA GGA AC-3', reverse 5'-AAA TTT GGG GTG GAA AGG TT-3'; GAPDH primers, forward 5'-GGC ATG GAC TGT GGT CAT GAG-3', reverse 5'-TGC ACC ACC AAC TGC TTA GC-3'). The specificity of each gene amplification was moreover checked at the end of each RT-qPCR reaction by analysis of dissociation curves of the PCR products. The curves of amplification were read with ABI Prism 7000 SDS software using the comparative cycle threshold method. Relative quantification of the steady-state target mRNA levels was calculated after normalization of the total amount of cDNA tested to a GAPDH RNA endogenous reference.

Nuclear protein extraction. Nuclear extracts were prepared with the Nuclear Extract Kit from Active Motif according to manufacturer's instructions. Briefly, 1 h after rhMMP-12 stimulation in presence or absence of inhibitors, 8 x 105 A549 cells were washed twice with PBS containing phosphatase inhibitors, centrifuged at 500 g for 5 min at 4°C, and then resuspended in hypotonic buffer (20 mM HEPES containing 5 mM NaF, 10 µM Na2MoO4, 0.1 mM EDTA) and left on ice for 15 min. Nonidet P-40 (0.5% final) was added, and, after vigorous shaking, samples were centrifuged at 14,000 g at 4°C for 30 s. Nuclei pelleted were resuspended in complete lysis buffer (lysis buffer, 10 mM DTT, protease inhibitor cocktail), rocked on ice for 30 min, vortexed, and centrifuged at 14,000 g for 10 min at 4°C. Supernatants containing the nuclear proteins were stored at –80°C until further analysis. Protein concentrations were determined using the method of Bradford with Bio-Rad protein assay.

Measurement of NF-{kappa}B (p65) and AP-1 (c-Fos) DNA binding. DNA binding of p65 NF-{kappa}B and c-Fos AP-1 was analyzed using the ELISA-based TransAM NF-{kappa}B and AP-1 family kits (Active Motif) according to manufacturer's instructions.

In brief, nuclear A549 cell extracts (5 or 10 µg) were incubated for 1 h in a 96-well plate to which oligonucleotide containing either an NF-{kappa}B or an AP-1 consensus binding site was immobilized. After washing, the plate was incubated for 1 h with an anti-NF-{kappa}B p65 antibody or an anti-c-Fos antibody (diluted 1:1,000 in each case), which specifically detected epitopes accessible only when p65 NF-{kappa}B and c-Fos activated and bound to their cognate oligonucleotides. The plate was then washed and incubated with a horseradish peroxidase-conjugated secondary antibody (1:1,000) for 1 h at room temperature. After washing, colorimetric readout was quantified by spectrophotometry at 450 nm. In each experiment, 8 x 105 A549 cells were stimulated with IL-1β (7 ng/ml) for 1 h, and DNA binding of transcriptional factors in these IL-1β-stimulated cells was used as reference, results in other experiments being expressed as percentage of DNA binding in these conditions. To assess the specificity of these assays, wild-type and mutated consensus oligonucleotides were used as competitors for NF-{kappa}B and c-Fos binding.

Analysis of protein kinase phosphorylation by Western blotting. A549 cells pretreated with or without inhibitors were incubated with rhMMP-12 for 2, 5, 15, 30, 60, 120, and 240 min. Then, cells were washed with cold PBS, lysed with RIPA lysis buffer (1 mM sodium orthovanadate, 2 mM DTT, 1 mM PMSF, 1 mM NaF, 1% protease inhibitor cocktail) for 30 min on ice and sonicated for 3 s. Equal amounts of cell lysate (100 µg) were separated by a 5% or 10% SDS-PAGE gel and then transferred onto a nitrocellulose membrane, which was further incubated for 30 min with 4% BSA in TBS containing Tween 20 and then for 2 h at room temperature with an antibody specific for phospho-ERK1/2, phospho-p38, phospho-JNK, or phospho-EGFR (diluted 1:1,000 in each case). After washing, the membranes were incubated for 1 h with a horseradish peroxidase-conjugated anti-mouse/rabbit antibody (1:2,000). Blots were then incubated with an enhanced chemiluminescence solution for 1 min and exposed.

Small interfering RNA transfection assay. Small interfering RNA (siRNA) oligonucleotides directed against c-Fos (sequences 5'-rCUrGrCUUrArCrArCrGUrCUUrCrCUUTT-3' and 5'-rArArGrGrArArGrArCrGUrGUrArArGrCrArGTT-3'), against p65 NF-{kappa}B subunit (sequences 5'-rGrArCrAUUrGrArGrGUrrGUrAUUUrCrATT-3' and 5'-UrGrArArAUrArCrArCrCUrCrArAUrGUrCTT-3'), and against EGFR (sequences 5'-rCUrAUrGUrGrCrArGrArGrGrArAUUrAUTT-3' and 5'-rAUrArAUUrCrCUrCUrGrCrArCrAUrArGTT-3') and a nontargeting siRNA (siNT1) were purchased from Sigma-Aldrich.

Transfections of siRNAs were performed in 12-well plates in the presence of DharmaFECT 1 transfection reagent (Dharmacon, Lafayette, CO). siRNAs (125 nM final) and 1.6 µl of DharmaFECT 1 per well were applied in a final volume of 320 µl of Opti-MEM (Life Technologies) before the addition of 4 x 105 A549 cells. Twenty-four hours after transfection, the medium was renewed with F-12K supplemented with 10% FCS, 1% antibiotics, 1% L-glutamine, and 10 mM HEPES. After 48 h, serum-starved A549 cells were stimulated by rhMMP-12 (3.10–2 U/ml) for 6 h.

Statistical analysis. The results are expressed as means ± SE. Analysis of treatment effects between groups was performed with a two-way ANOVA. Comparison of treatment interactions was done by t-tests. For each analysis, P values less than 5% were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Activation of A549 and BEAS-2B cells by rhMMP-12. Incubation of A549 epithelial cells with rhMMP-12 (3.10–2 U/ml) for 6 h enhanced IL-8/CXCL8, MCP-1/CCL2, GRO-{alpha}/CXCL1, and IL-6 secretion in the culture medium by 3.5-, 2.5-, 1.5-, and 28.5-fold over control levels, respectively, and all these changes were statistically significant (Fig. 1). TNF-{alpha}, MIP-1{alpha}/CCL3, and TGF-{alpha} were also quantified 6 h after incubation of the cultured cells with rhMMP-12 but were not detected (data not shown). Stimulation of A549 epithelial cells with various concentrations of rhMMP-12 (3.10–3 to 1.10–1 U/ml) for 6 h induced a concentration-dependent release of IL-8/CXCL8 (Fig. 2A). The release of IL-8/CXCL8 was associated with a transient increase of intracellular IL-8/CXCL8 mRNA that reached a maximum (4-fold) 2 h after addition of rhMMP-12 (3.10–2 U/ml) in the present experimental conditions (Fig. 2B). Stimulation of cultured BEAS-2B cells with various concentrations of rhMMP-12 (3.10–3 to 1.10–1 U/ml) also induced a concentration-dependent release of IL-8/CXCL8, although the concentrations in the culture medium, both in absence and in the presence of rhMMP-12, were much lower than these observed with A549 cells (Fig. 2C).


Figure 1
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  		Fig. 1. A–D: activation of A549 cells by recombinant human macrophage metalloelastase (rhMMP-12). Serum-starved A549 cells were stimulated with serum-free medium alone (control, white bars) or with rhMMP-12 catalytic site (3.10–2 U/ml, black bars) for 6 h. Then, the cell culture supernatants were collected, and concentrations of monocyte chemoattractant protein-1 (MCP-1)/CCL2, IL-8/CXCL8, IL-6, and growth-related oncogene-{alpha} (GRO-{alpha})/CXCL1 were measured by ELISA. Results are expressed as means ± SE, n = 3; ***P < 0.001, **P < 0.01 compared with controls.

 

Figure 2
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  		Fig. 2. Induction of mRNA expression and release of IL-8/CXCL8. Serum-starved A549 cells (A) or BEAS-2B bronchial epithelial cells (C) were stimulated with serum-free medium alone (control, white bar) or with rhMMP-12 (3.10–3 to 1.10–1 U/ml, black bars) for 6 h. Then, the cell culture supernatants were collected, and concentrations of IL-8/CXCL8 were measured by ELISA. Results are expressed as means ± SE, n = 4; **P < 0.01, ***P < 0.001 compared with controls. B: A549 cells were stimulated with rhMMP-12 (3.10–2 U/ml) for 2 and 4 h. mRNA expression was then determined by real-time quantitative PCR (RT-qPCR). Data were normalized relative to GAPDH mRNA. D and E: effect of matrix metalloproteinase (MMP) inhibitors on rhMMP-12-induced IL-8/CXCL8 production. Serum-starved A549 cells were pretreated with either 0.1% DMSO (vehicle), Batimastat (1–30 µM; D), or AS111793 (1–30 µM; E) for 1 h and then stimulated or not with rhMMP-12 (3.10–2 U/ml, black bars) for 6 h. Then, the cell culture supernatants were collected and IL-8/CXCL8 concentrations were measured by ELISA. Results are expressed as means ± SE, n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 compared with vehicle/rhMMP-12.

 
These results show that rhMMP-12 was able to concentration-dependently induce IL-8/CXCL8 production in both A549 and BEAS-2B cells and that this increase in A549 cells was related to an increase in IL-8/CXCL8 gene expression. The precise molecular mechanism that underlines the rhMMP-12 action is unknown; however, rhMMP-12-induced IL-8/CXCL8 production was prevented by 1-h preincubation of A549 cells with a broad-spectrum MMP inhibitor, Batimastat (1–30 µM; Fig. 2D), and with the selective MMP-12 inhibitor AS111793 (1–30 µM; Fig. 2E). This suggests that the secretion of IL-8/CXCL8 is linked to the proteolytic activity of rhMMP-12. Indeed, both MMP inhibitors have a similar mechanism of action that is to chelate the zinc ion into the enzyme catalytic site, therefore blocking the rhMMP-12 enzymatic activity.

rhMMP-12-induced IL-8/CXCL8 production requires EGFR activation. Preincubation of A549 cells with EGFR tyrosine kinase inhibitors, tyrphostin AG-1478 (5, 7.5, 10 µM), or PD168393 (10 nM to 10 µM) concentration-dependently and significantly decreased the rhMMP-12-induced IL-8/CXCL8 production (Fig. 3, A and B). Preincubation of the cells with LA (10–50 µg/ml) also significantly reduced IL-8/CXCL8 release induced by rhMMP-12 (Fig. 3C). Pretreatment with a control antibody (10 µg/ml IgG1) had no effect on the basal and stimulated levels of IL-8/CXCL8. Transfection with siRNAs directed against EGFR markedly reduced EGFR protein (data not shown) and significantly decreased IL-8/CXCL8 release after rhMMP-12 stimulation compared with cells transfected with siNT1 (Fig. 3D).


Figure 3
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  		Fig. 3. Involvement of EGF receptor (EGFR) in rhMMP-12-induced IL-8/CXCL8 release. Serum-starved A549 cells were pretreated with vehicle (0.1% DMSO), EGFR activation inhibitors, tyrphostin AG-1478 (5, 7.5, or 10 µM; A) or PD168393 (0.1, 1, or 10 µM; B), neutralizing EGFR antibody LA1 clone (LA; 10–50 µg/ml), or with a control antibody IgG1 (10 µg/ml; C) for 30 min and then stimulated or not with rhMMP-12 (3.10–2 U/ml, black bars) for 6 h. Then, cell culture supernatants were collected, and IL-8/CXCL8 concentrations were quantified by ELISA. Results are expressed as means ± SE, n = 3; ###P < 0.001 compared with controls; ***P < 0.001, **P < 0.01, *P < 0.05 compared with vehicle/rhMMP-12. D: A549 cells were transfected with small interfering RNA (siRNA) oligonucleotides directed against EGFR (Si EGFR) or with a nontargeting siRNA (Si NT1) as control. Seventy-two hours after transfection, serum-starved A549 cells were stimulated by rhMMP-12 (3.10–2 U/ml, black bars) for 6 h. Then, cell culture supernatants were collected, and IL-8/CXCL8 concentrations were determined by ELISA. Results are expressed as means ± SE of 3 independent experiments; ###P < 0.001 compared with controls, **P < 0.01 compared with cells transfected with Si NT1 and stimulated with rhMMP-12.

 
To confirm the EGFR tyrosine kinase phosphorylation in response to rhMMP-12, serum-starved A549 cells were incubated with rhMMP-12 (3.10–2 U/ml) for 2, 5, 15, 30, and 60 min, and then immunoblotting analysis of phosphorylated EGFR was performed. rhMMP-12 induced an increase in phosphorylated EGFR with a maximum at 5 min (Fig. 4A). The phosphorylation was prevented by tyrphostin AG-1478 (5 µM) and by PD168393 (1 µM) (Fig. 4B).


Figure 4
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  		Fig. 4. Immunoblotting for phosphorylated EGFR (P-EGFR, phospho-EGFR). A: serum-starved A549 cells were stimulated with serum-free medium alone for 1 h (control) or containing rhMMP-12 (3.10–2 U/ml) for 2, 5, 15, 30, and 60 min and then lysed. Total cell lysates were immunoblotted with antibodies against phospho-EGFR (Y845) and total EGFR. B: A549 cells were pretreated with tyrphostin AG-1478 (AG; 7.5 µM), PD168393 (PD; 1 µM), or 0.1% DMSO (Veh) for 30 min and then stimulated for 5 min with rhMMP-12 (3.10–2 U/ml). Results are representative of 3 independent experiments.

 
Involvement of MAPK in rhMMP-12-induced IL-8/CXCL8 production. The stimulation of A549 epithelial cells with rhMMP-12 (3.10–2 U/ml) for 5 min up to 4 h resulted in a time-dependent phosphorylation of ERK1/2 that occurred as early as 5 min and decreased after 30 min (Fig. 5A). In contrast, neither p38 MAPK nor JNK phosphorylation occurred when A549 cells were stimulated with rhMMP-12 (Fig. 5, B and C). Also, specific inhibitors of p38 MAPK and of JNK, i.e., SB-203580 (20 µM) and SP-600125 (2 µM), failed to inhibit IL-8/CXCL8 production (data not shown).


Figure 5
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  		Fig. 5. Involvement of MAPK ERK1/2, p38, and JNK in A549 cells stimulated with rhMMP-12. Serum-starved A549 cells were stimulated with serum-free medium alone for 1 h (control) or containing rhMMP-12 (3.10–2 U/ml) for 5, 15, 30, 60, 120, or 240 min. Total cell lysates were immunoblotted with antibodies specific for phospho-ERK1/2 and total ERK1/2 (A), phospho-p38 and total p38 (B), or phospho-JNK and total JNK (C). A549 cells stimulated with IL-1β (7 ng/ml) for 15 min were used as positive controls. Results are representative of 4 independent experiments.

 
The analysis of total A549 cell lysates by Western blot showed that pretreatment with the MEK inhibitors U0126 (1 µM) and PD-98059 (10 µM) clearly abrogated the activation of ERK1/2 observed 5 min after addition of MMP-12 to the cells (Fig. 6A). Furthermore, U0126 (1 µM) or PD-98059 (10 µM) also significantly inhibited the rhMMP-12-induced IL-8/CXCL8 secretion by A549 epithelial cells (Fig. 6B), suggesting an involvement of ERK1/2 in IL-8/CXCL8 production. These results were also confirmed by RT-qPCR, as U0126 also reduced IL-8/CXCL8 mRNAs (Fig. 6C). Inhibitors of the EGFR tyrosine kinase AG-1478 (7.5 µM) and PD168393 (10 µM) also markedly reduced the rhMMP-12-induced phosphorylation of ERK1/2, suggesting a central role of EGFR in this phenomenon (Fig. 6D).


Figure 6
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  		Fig. 6. Involvement of MAPK ERK1/2 in rhMMP-12-induced IL-8/CXCL8 release. Serum-starved A549 cells were pretreated with Vehicle (Veh; 0.1% DMSO, white bars) or MEK inhibitors PD-98059 (PD98; 10 µM, gray bars) or U0126 (U0; 1 µM, hatched bars) for 1 h or with EGFR inhibitors tyrphostin AG-1478 (7.5 µM) or PD168393 (1 µM) for 30 min and then stimulated with rhMMP-12 (3.10–2 U/ml) for 5 min or 6 h. Control cells were not stimulated with rhMMP-12. After 5 min of stimulation, part of cells were then lysed and total cell lysates were immunoblotted with antibodies specific for phospho-ERK1/2 and total ERK1/2 (A and D). The remainder of the cells was stimulated in the same conditions: for 6 h, cell culture supernatants were collected after for IL-8/CXCL8 quantification using a commercial ELISA kit (B); or, for 2 h, cells were collected to evaluate IL-8/CXCL8 mRNA levels by RT-qPCR (C). Data are expressed as means ± SE of 4 independent experiments; ###P < 0.001, cells pretreated with vehicle and stimulated with rhMMP-12 compared with control; ***P < 0.001, significantly different from cells pretreated with vehicle and stimulated with rhMMP-12.

 
rhMMP-12-induced transcriptional factors DNA binding. rhMMP-12 significantly increased DNA binding of p65 subunit to NF-{kappa}B consensus sites in A549 cells after 1 h of stimulation (Fig. 7A). Also, it increased DNA binding of c-Fos to AP-1 consensus sites (Fig. 7B). The MEK/ERK1/2 inhibitor U0126 (5 µM) did not inhibit p65 NF-{kappa}B DNA binding while it prevented c-Fos DNA binding induced by rhMMP-12 stimulation.


Figure 7
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  		Fig. 7. Involvement of NF-{kappa}B and activating protein-1 (AP-1) in rhMMP-12 stimulated A549 epithelial cells. Serum-starved A549 cells were cultured in absence or presence of U0126 (5 µM, hatched bars) for 1 h and then stimulated with rhMMP-12 (3.10–2 U/ml) for 1 h. Nuclear extracts were prepared, and measurements of DNA-binding activity of nuclear p65 NF-{kappa}B (A) or c-Fos AP-1 (B) were performed as described in MATERIALS AND METHODS. Results are expressed as percentage of DNA binding of the transcriptional factors in A549 cells stimulated with IL-1β (7 ng/ml) for 1 h (the stimulation was performed in each experiment). Results are expressed as means ± SE of 4 independent experiments. ##P < 0.01 compared with control; ***P < 0.001 compared with rhMMP-12. A549 cells were transfected with siRNA oligonucleotides directed against c-Fos (si c-Fos), p65 NF-{kappa}B (si P65), or with Si NT1 as control. Seventy-two hours after transfection, serum-starved A549 cells were stimulated by rhMMP-12 (3.10–2 U/ml, black bars) for 6 h. Then, cell culture supernatants were collected, and IL-8/CXCL8 concentrations were determined by ELISA. Results are expressed as means ± SE of 3 independent experiments; ###P < 0.001 compared with control; ***P < 0.001, compared with cells transfected with Si NT1 and stimulated with rhMMP-12.

 
Involvement of NF-{kappa}B and AP-1 in rhMMP-12-induced IL-8/CXCL8 synthesis. To confirm whether IL-8/CXCL8 release induced by rhMMP-12 is linked to p65 NF-{kappa}B and c-Fos transcriptional factors, transfections of A549 cells were performed with siRNAs directed against p65 (si p65) NF-{kappa}B subunit or with siRNA directed against c-Fos (si c-Fos). Transfection with si p65 and si c-Fos markedly reduced p65 and c-Fos protein (data not shown). Transfection with si p65 was devoid of effect (Fig. 7C), whereas cells transfected with si c-Fos show a significantly reduced IL-8/CXCL8 release after rhMMP-12 stimulation compared with cells transfected with siNT1.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
The present study reports that rhMMP-12 catalytic domain was able to activate alveolar A549 epithelial cells, leading 6 h later to the production of the cytokines IL-6, GRO-{alpha}/CXCL1, MCP-1/CCL2, and IL-8/CXCL8. Neither TNF-{alpha}, TGF-{alpha}, nor MIP-1{alpha} was detected in the culture medium, suggesting that MMP-12 can trigger the release of a specific panel of cytokines.

Our results are consistent with those observed in vivo in mice, which received an instillation of the same rhMMP-12 catalytic domain in their airways (32). Indeed, Nénan et al. (32) have reported increased concentrations of IL-6, MCP-1/CCL2, and KC/CXCL1 (the murine counterpart of IL-8/CXCL8) in the bronchoalveolar lavage fluids of mice instilled with rhMMP-12 compared with controls, associated with a marked recruitment of neutrophils in the airways. These in vivo results in mice suggest that accumulation of MMP-12 in alveolar space can trigger proinflammatory signals in airway diseases.

Furthermore, we focused our attention to IL-8/CXCL8 because of the well-established role of this chemokine in the activation and migration of neutrophils as well as in pulmonary infections and in various pulmonary diseases. We observed that rhMMP-12 concentration-dependently enhanced IL-8/CXCL8 protein release by A549 cells in the culture medium after 6 h and that this increase was preceded by the IL-8/CXCL8 gene expression 2 h after addition of the rhMMP-12 to the medium. In addition, we observed that rhMMP-12 induced a concentration-dependent release of IL-8/CXCL8 also in BEAS-2B epithelial cells, suggesting that rhMMP-12 effects are not restricted to the A549 lung carcinoma cell line. In addition, the finding that pretreatments with actinomycin D largely attenuated the response of A549 cells to rhMMP-12 (data not shown) further confirmed the involvement of transcriptional mechanisms in the MMP-12-induced IL-8/CXCL8 release by A549 cells.

Interestingly, human pulmonary epithelial cells stimulated by neutrophil elastase (NE), a serine proteinase secreted by neutrophils and to a lesser extent by monocytes, have been reported to also release IL-8/CXCL8 (31). Moreover, Kuwahara and colleagues (22) have shown that in A549 cells the IL-8/CXCL8 release depends on an enhanced IL-8/CXCL8 gene transcription via EGFR transactivation. Thus, in addition to its major role in promoting cell proliferation and tissue healing following injury, the EGFR transactivation pathway is also involved in expression of proinflammatory cytokines, including IL-8/CXCL8. We therefore investigated whether the rhMMP-12-induced IL-8/CXCL8 production was mediated via an EGFR signaling pathway, and we showed that incubation of the A549 epithelial cells with rhMMP-12 lead to EGFR tyrosine phosphorylation. EGFR tyrosine phosphorylation occurred within 2 min with a maximum at 5 min. Both the induced EGFR phosphorylation and the induced IL-8/CXCL8 production were blocked when A549 cells were preincubated with the selective EGFR tyrosine kinase inhibitors AG-1478 and PD168393 for 30 min before addition of rhMMP-12. Moreover, cells transfected with siRNA oligonucleotides directed against EGFR showed a markedly reduced IL-8/CXCL8 release after rhMMP-12 stimulation compared with cells transfected with siNT1. Taken together, these data suggest the transactivation of EGFR in response to MMP-12 induced the IL-8/CXCL8 release by A549 cells.

EGFR activation has been reported to occur mainly in response to EGFR ligand binding that further activates the receptor and induces tyrosine phosphorylation (16, 36). In the present study, rhMMP-12-induced IL-8/CXCL8 production was also partly inhibited by the preincubation of A549 cells with a neutralizing EGFR antibody that prevented the ligand binding and subsequent activation of the extracellular domain of EGFR. The fact that the inhibitors of MMP, Batimastat and AS111793, concentration-dependently blocked the IL-8/CXCL8 release by A549 cells suggests that EGFR activation was dependent on the proteolytic activity of the MMP-12 enzyme. However, the precise mechanism by which EGFR activation occurred is unknown. Some studies have established that external stimuli transactivate EGFR via a metalloproteinase-catalyzed processing of cell surface EGF-like factors (10, 35). Indeed, studies on human colonic epithelial cells and exotoxins from Clostridium difficile have shown that MMP-dependent TGF-{alpha} release can activate EGFR with subsequent IL-8/CXCL8 secretion (30, 43). Moreover, Zhao and colleagues (44) have also demonstrated that the lysophosphatidic acid-induced IL-8/CXCL8 release in human bronchial epithelial cells is partly dependent on EGFR transactivation regulated by MMPs and pro-heparin binding EGR shedding. In airway epithelial NCI-H292 cells, the transactivation of EGFR by NE has already been described to induce mucin production via activation of an EGFR cascade, involving the proteolytic cleavage of pro-TGF-{alpha} on the surface of the cells (20). Another study has also demonstrated that NE can rapidly release EGF, which recruits EGFR and MEK to downregulate tropoelastin mRNA in lung fibroblasts (11). Thus the rhMMP-12-induced EGFR activation and IL-8/CXCL8 production could putatively be a consequence of the binding of an EGFR ligand released from the cell surface by the shedding activity of MMP-12.

Similarly, TNF-{alpha} also stimulates IL-8/CXCL8 gene expression and protein production via a transcriptional mechanism. Pro-TNF-{alpha} is normally converted to active TNF-{alpha} by the membrane-bound metalloproteinase TNF-{alpha} converting enzyme (TACE, ADAM-17), but several MMPs, including MMP-12, also have TNF-{alpha} converting enzyme activity to greater or lesser degrees (6). To exclude the possibility that rhMMP-12 may release membrane-bound TNF-{alpha}, which, in turn, activate IL-8/CXCL8 production, we measured TNF-{alpha} levels in the culture supernatants using ELISA and found no TNF-{alpha} after rhMMP-12 stimulation. Furthermore, preincubation of cells with a neutralizing TNF-{alpha} antibody had no inhibitory effect on rhMMP-12-induced IL-8/CXCL8 production (data not shown). Thus we suggest that TNF-{alpha} release is unlikely to be involved in the rhMMP-12-induced IL-8/CXCL8 production by A549 cells.

The MAPK transduction pathways are well-described to be involved in the production of various cytokines. Thus we examined by Western blot analysis the activities of the kinases ERK1/2, p38, and JNK in response to rhMMP-12 stimulation of A549 epithelial cells. rhMMP-12 only induced a significant ERK1/2 activation, which occurred within 5 min of stimulation. Pretreatment with MEK inhibitors (PD-98059 and U0126) abrogated ERK1/2 phosphorylation and strongly inhibited IL-8/CXCL8 protein release as well as IL-8/CXCL8 mRNA expression. These results indicate that ERK1/2 plays a critical role in transduction regulating the IL-8/CXCL8 expression induced by rhMMP-12 in A549 cells. Here, we showed that rhMMP-12-induced ERK1/2 phosphorylation was reduced by preincubation with the EGFR tyrosine kinase inhibitors, AG-1478 and PD168393, showing that the MEK-MAPK transduction pathway is downstream to EGFR activation. These results are consistent with those showing an involvement of MAPK in the IL-8/CXCL8 gene expression following EGFR MMP-dependent transactivation (30, 43). However, the relatively low level of inhibition in our conditions suggests that other unidentified pathways also activated by MMP-12 may contribute to the ERK1/2 phosphorylation.

The promoter of the gene encoding IL-8/CXCL8 contains sequences for binding of several transcription factors, including NF-{kappa}B and AP-1 that participate to various extents in the inducible expression of the gene encoding IL-8/CXCL8 (21, 37). In the present study, rhMMP-12 induced both NF-{kappa}B (p65 subunit) and AP-1 (c-Fos subunit) nuclear localization and activation after 1 h of stimulation. However, our results also showed that the MEK/ERK1/2 inhibitor U0126 could not inhibit NF-{kappa}B p65 DNA binding, whereas it prevented AP-1 c-Fos DNA binding, after stimulation by rhMMP-12. These results indicate that, in contrast to NF-{kappa}B, the activation of AP-1 is a possible consequence of ERK1/2 activation. Using the technique of siRNA, we further demonstrated that IL-8/CXCL8 production, in response to rhMMP-12, was indeed dependent on activation of c-Fos (AP-1) but not of the p65 subunit (NF-{kappa}B). As, in addition to IL-8/CXCL8, numerous cytokines are partly NF-{kappa}B-dependent (33), the NF-{kappa}B activation observed after rhMMP-12 stimulation could possibly be linked to the expression of other genes like the ones encoding IL-6, GRO-{alpha}/CXCL1, and MCP-1/CCL2.

Here, we underlined that rhMMP-12 catalytic domain was able to activate the EGFR pathway and to induce IL-8/CXCL8 gene expression and protein release from human alveolar epithelial cells. MMP-12, a metalloproteinase secreted by activated macrophages, is known to be upregulated in lungs in inflammatory conditions, especially after cigarette smoke exposure (5, 8, 14, 23, 42). Our results suggest that MMP-12 can stimulate the alveolar epithelial cells to synthesize and release, among other cytokines, IL-8/CXCL8 that, in turn, could further attract neutrophils, participating in the airway inflammatory process.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
J.-Y. Gillon is employed by Merck-Serono International. V. Lagente received research grants from Merck-Serono International.


   ACKNOWLEDGMENTS
 
We thank M. Le Vée for expert technical assistance with siRNA.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. Lagente, Laboratoire de Pharmacodynamie et de Pharmacologie Moléculaire, INSERM U620, Université de Rennes 1, 2. Ave. du Professeur Léon Bernard, CS 34317, 35043 Rennes Cedex, France (e-mail: vincent.lagente{at}univ-rennes1.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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