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Am J Physiol Lung Cell Mol Physiol 293: L170-L181, 2007. First published April 20, 2007; doi:10.1152/ajplung.00445.2006
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Diesel exhaust particles induce matrix metalloprotease-1 in human lung epithelial cells via a NADP(H) oxidase/NOX4 redox-dependent mechanism

Nadia Amara,1 Rafik Bachoual,1,3 Mathieu Desmard,1 Slawomir Golda,1,4 Cecile Guichard,2,5 Sophie Lanone,1 Michel Aubier,1,6 Eric Ogier-Denis,2 and Jorge Boczkowski1,6

1Institut National de la Santé et de la Recherche Médicale (INSERM) U700 and 2INSERM U773 and Centre de Recherche Bichat Beaujon CRB3, Paris, France; Université Paris 7 Denis Diderot, Faculté de Médecine, site Bichat, Paris, France; 3Faculté des Sciences de Gabès, Tunisia; 4Department of Medical Biotechnology, Jagiellonian University, Krakow, Poland; 5Department of Pathology, Immunology and Clinical Pathology, Centre Medical Universitaire, Geneva, Switzerland; and 6Assistance Publique Hopitaux de Paris, CIC 007, Hôpital Bichat, Paris, France

Submitted 10 November 2006 ; accepted in final form 17 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chronic exposure to particulate air pollution is associated with lung function impairment. To determine the molecular mechanism(s) of this phenomenon, we investigated, in an alveolar human epithelial cell line (A549), whether diesel exhaust particles (DEPs), a main component of particulate air pollution, modulates the expression and activity of the matrix metalloprotease (MMP)-1, a collagenase involved in alveolar wall degradation. Interaction of DEPs with cigarette smoke, which also produces structural and functional lung alterations, was also investigated. A noncytotoxic concentration of DEPs induced an increase in MMP-1 mRNA and protein expression and activity in A549 cells without modifying the expression of the MMP inhibitors TIMP-1 and -2. This effect was not potentiated when cells were coexposed to noncytotoxic concentrations of cigarette smoke condensate. DEP-induced MMP-1 was associated with increased ERK 1/2 phosphorylation and upregulation of expression and activity of the NADPH oxidase analog NOX4. Cell transfection with a NOX4 small interfering RNA prevented these phenomena, showing the critical role of a NOX4 ERK 1/2 pathway in DEP-induced MMP-1 expression and activity. Similar results to those observed in A549 cells were obtained in another human lung epithelial cell line, NCI-H292. Furthermore, experiments in mice intratracheally instilled with DEPs confirmed the in vitro findings, showing the induction of NOX4 and MMP-1 protein expression in alveolar epithelial cells. We conclude that alveolar alterations secondary to MMP-1 induction could explain lung function impairment associated with exposure to particulate pollution.

chronic obstructive pulmonary disease; environment; pollution; nanoparticles

ATMOSPHERIC POLLUTION PARTICLES with an aerodynamic diameter <2.5 µm (PM2.5) have been recently found to be associated with increased respiratory- and cardiovascular-related death and disease (26). In urban areas, diesel exhaust particles (DEPs) are probably a major component of PM2.5, especially in Europe, where many people drive diesel-powered vehicles (23). DEPs are composed of a carbonaceous core, on which molecules such as organic compounds or bacterial LPS are adsorbed. The two main families of organic compounds are polycyclic aromatic hydrocarbons and quinones, both of which are involved in reactive oxygen species (ROS) production (5, 37). At least two mechanisms could be potentially involved in ROS production, 1) an immediate oxidation-reduction cycling of quinones or quinone-like compounds and/or 2) a subsequent metabolization of polycyclic aromatic hydrocarbons by the cytochrome P450 (2). However, production of ROS under pathophysiological conditions is a complex process that involves different mechanisms, and little is known about sources of ROS production other than that after cell exposure to DEPs as we mentioned.

A main respiratory consequence of exposure to air pollution particulate matter is progressive impairment in lung function. Epidemiological studies have shown the association between the level of exposure to PM2.5 and progressive airflow limitation (1). One possible mechanism for this phenomenon could be a reduction in the lumen of distal airways related to wall inflammation and fibrosis secondary to an effect of PM2.5 on airway epithelial cells (8, 11). However, other mechanisms could be involved, since maintenance of distal airway patency depends on not only the thickness of the wall, but also airway radial traction by attachment to pulmonary alveolar walls. Since PM2.5 particles can reach and be deposited on the alveolar wall (7), an effect of DEPs on alveolar epithelial cells can be postulated. Indeed, although the contribution of abnormalities in bronchial and alveolar epithelial cells to in vivo respiratory response to DEP is difficult to assess, inflammatory and genotoxic alterations in alveolar epithelial cells have been reported after in vivo exposure to DEPs (4, 19).

Matrix metalloproteases (MMPs) and their inhibitors (TIMPs) are important players in the protease/antiprotease balance in the lung. Notably, overexpression of interstitial collagenase MMP-1 in alveolar epithelial cells (type II pneumocytes) has been shown to be involved in the loss of alveolar wall integrity in chronic obstructive pulmonary disease (COPD) (12, 32), a disease secondary to chronic exposure to cigarette smoke and characterized by airflow limitation that is not fully reversible.

Increased MMP-1 expression and activity in alveolar epithelial cells after exposure to DEPs could be a mechanism explaining, at least in part, the deleterious effects of PM2.5 on lung function. The rationale for this hypothesis is that 1) DEPs can reach and be deposited on the alveolar wall (7), 2) DEPs have oxidative properties that mediate several of their biological effects (2), and 3) MMP-1 expression is tightly regulated by ROS (6, 44) through an ERK 1/2 or p38 MAPK pathway (32, 36). DEP-induced MMP-1 expression could degrade collagen in alveolar walls, reducing the number or the force of alveolar attachment to distal airways, resulting in a loss of strength in airway radial traction. However, the effect of DEPs on MMP-1 expression in alveolar epithelial cells and the mechanism(s) involved are unknown.

We aimed first to investigate the in vitro effects of DEPs on the expression and activity of MMP-1 and its inhibitors (TIMP-1 and -2) in alveolar epithelial cells. Increasing evidence suggests a role for endogenous pro-oxidant systems, such as homologs of NADPH oxidase (NOX), in ROS production under pathophysiological conditions (28). Therefore, we investigated whether NOX enzymes are endogenous sources of oxidants involved in DEP-induced MMP-1 expression in alveolar epithelial cells. Indeed, DEPs can activate NOX in microglial cells (3), and alveolar epithelial cells express different NOX proteins, such as NOX1 and NOX4 (16, 17).

In addition to its effects on respiratory function, exposure to PM2.5 can aggravate preexisting pulmonary diseases such as COPD (24). The molecular mechanisms of this effect are not well known. Since distal airway alterations are similar in smokers with COPD and in people living in highly polluted areas (8), our second aim was to investigate whether DEPs potentiated or not an effect of cigarette smoke on MMP-1 expression and activity in alveolar epithelial cells.

We used the human cell line A549 as representative of human type II cells because the morphology and cell functionality of this cell line (i.e., surfactant synthesis, oxidative metabolism, transport proteins) are consistent with that of type II pneumocytes in vivo (15). Critical results observed in A549 cells were verified in another human lung epithelial cell line, NCI-H292 (31). Furthermore, relevant in vitro findings were validated in an in vivo model in mice exposed to DEPs by intratracheal instillation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and particles. The diesel particulate matter SRM 2975 (45) was purchased from the National Institute of Standards and Technology (Gaithersburg, MD), and carbon black particles (CBPs), 95 nm in diameter (FR103), were purchased from Degussa (Frankfurt, Germany). Unless specified, all chemicals were from Sigma (St. Louis, MO).

The A549 and NCI-H292 cell lines were purchased from the American Type Culture Collection (CCL 185 line; Rockville, MD). Cells were kept at 37°C in a humidified incubator under 5% CO2 in air and grown in Ham's F-12 culture medium containing 4 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin supplemented with 10% FCS until they reached 80–90% confluence.

Cells were placed in medium without FCS and stimulated with DEPs [5–10 µg/cm2, dispersed in 0.04% dipalmitoyl lecithin (DPL)] and/or cigarette smoke condensate (CSC, 10 µg/ml) for 6 or 24 h. To examine the effect of the carbonaceous core of DEPs, cells were also exposed to CBPs (at 10 µg/cm2, also dispersed in 0.04% DPL). DEP and CBP concentrations were expressed in micrograms per centimeter squared because particles rapidly sediment onto the culture. In all experiments, 10 µg/cm2 was equivalent to 50 µg/ml with 10 µg/cm2 being relevant to concentrations of PM2.5 found in urban air (34). CSC concentrations were expressed in micrograms per milliliter for comparison with literature data (see, for example, Ref. 14).

CSC was prepared by use of a smoking machine (Anitech, Paris, France). The particulate matter from Kentucky standard cigarettes (2R4F; University of Kentucky) was collected on Cambridge glass fiber filters, and the amount obtained was determined by weight increase of the filter. The collected smoke particulates were dissolved in dimethyl sulfoxide (DMSO) to yield a 20 mg/ml solution, and aliquots were kept at –80°C.

The control condition consisted of cells incubated with 0.04% DPL plus DMSO (10 µg/ml).

Cellular toxicity. Cellular toxicity and viability were assessed by trypan blue exclusion, lactate dehydrogenase (LDH) release in the medium, and incorporation of the fluorescent dye propidium iodide (PI). Fluorescence intensity was quantified by use of a Coulter EPICS XL flow cytometer with Expo32 software (both from Beckman Coulter, Villepinte, France).

Real-time RT-PCR. NOX1 and 4, MMPs, TIMPs, and transforming growth factor-beta (TGF-beta) mRNA expression was quantified by quantitative real-time RT-PCR by use of the PCR ABI 7700 apparatus (Applied Biosystems). The following sets of primers were used: NOX1, 5'-GTACAAATTCCAGTGTGCAGACCAC-3' and 5'-CAGACTGGAATATCGGTGACAGCA-3'; NOX4, 5'-CTCAGCGGAATCAATCAGCTGTG-3' and 5'-AGAGGAACA CGACAATCAGCCTTAG-3'; MMP-1, 5'-CATGCGCACAAATCCCTTCTA-3' and 5'-TGTCCCTGAACAGCCCAGTACT-3'; MMP-2, 5'-CTGCTGGCTGCCTTAGAACCT-3' and 5'-AACCATCACTATGTGGGCTGAGA-3'; TIMP-1, 5'-CAACCGCAGCGAGGAGTTT-3' and 5'-TGCATTCCTCACAGCAACA-3'; TIMP-2, 5'-CCCTATCAAGAGGATCCAGTATGAGA-3' and 5'-TTCCTGCAATGAGATATTCCTTCTTT-3'; and TGF-beta, 5'-ACTCATTCAGTCACCATAGCAACACT-3' and 5'-CGCCTGGCCTGAACTACTATCTT-3'.

Expression of mRNA was normalized to that of ubiquitin. In previous experiments, we verified that ubiquitin mRNA expression did not change during the different experimental conditions.

Western blot analysis of MMP-1 protein expression. Western blot analysis was performed as described previously (43) with use of a monoclonal anti-human MMP-1 antibody (1:400; R&D Systems, Lille, France). The expression of the housekeeping protein beta-actin was evaluated with use of a monoclonal anti-beta-actin antibody. The results are expressed as the ratio of the expression of MMP-1 to that of beta-actin.

Zymography assay. Samples of cell culture supernatants were separated under nonreducing conditions on 12% polyacrylamide gels containing 1 mg/ml casein as described previously (9, 13, 21). Gels were loaded with 20-µl sample and 10-µl loading buffer and processed under Laemmli conditions. MMP-1 activity was assessed by incubation of the gels in 45% acetic acid and 10% methanol.

Intracellular ROS production. Intracellular ROS production was quantified by measuring 2',7'-dichlorodihydrofluorescein diacetate (H2DCFH-DA) oxidation as described previously (42). Briefly, cells were cultured in 96-well plates. H2DCFH-DA (final concentration in DMSO, 10 µM) was added 1 h before addition of the different particles. Fluorescence was measured at 480–555 nm with use of a multiwell fluorescence plate reader (Fluorostar BMG) 1 h after the addition of stimuli. Results are expressed in fluorescent arbitrary units.

Immunohistochemical analysis. Immunohistochemistry was performed as described previously (35, 41). We used a rabbit polyclonal anti-NOX4 antibody for which specificity was verified previously (35) at 1:200 dilution. Positive cells were revealed by use of the Vectastain ABC-alkaline phosphatase kit system (Vector Laboratories, Burlingame, CA) and fast red substrate.

To test the specificity of immunostaining, NOX4 antibody was replaced by normal rabbit serum with no positive cells identified (data not shown). Furthermore, we ensured the specificity of the antibody by performing immunohistochemistry in cells transfected with a NOX4 small interfering RNA (siRNA; see below).

Cell transfection with siRNA. To suppress endogenous NOX4 expression, experiments involved a specific siRNA (Xeragon-Qiagen) against NOX4 designed on the target region from the NOX4 gene (5'-AAACCGGCAGGAGUUUACCCAG-3'). Scrambled siRNA (nonhomologous to the human genome, 5'-AACCAGCAAGGUGUAUCGCCAC-3') was used as a control. The specificity of the NOX4 siRNA was verified previously (35). A549 cells were placed onto six-well plates (5 x 105 cells per well) and transfected with 0.8 µg of each siRNA duplex by use of Lipofectamine transfection reagent for 4 h in medium devoid of serum and antibiotics. This procedure did not affect cell viability (data not shown). Cells were then washed once with PBS and grown in complete medium. Gene silencing was monitored after incubation for 24 h.

Immunohistochemical detection of MMP-1 and NOX4 in mice alveolar epithelial cells. Male mice from the C57BL6 strain were instilled intratracheally with 50 µg of DEPs suspended in 100 µl of sterile saline solution or saline solution alone. Then, 24 h after instillation, animals were anesthetized with a lethal dose of sodium pentobarbital (50 mg/kg). The lungs were removed and immediately frozen in liquid nitrogen for immunohistochemical studies.

Four 6-µm-thick cryostat sections of lung were fixed in acetone and incubated with appropriate dilutions of antibody. Positive cells were revealed by use of the Vectastain ABC-alkaline phosphatase kit system and fast red substrate.

Antibodies used were a monoclonal anti-MMP-1 (GenWay Biotech, San Diego, CA) at 1:400 dilution and a rabbit polyclonal anti-NOX4 (35) at 1:300 dilution.

To test the specificity of the immunostaining, MMP-1 and NOX4 antibodies were replaced by a control isotype antibody and normal rabbit serum, respectively, with no positive cells identified (data not shown).

Quantification of immunostaining involved counting the number of cells positive for both MMP-1 and NOX4. Results are expressed as the number of positive cells per optical field. At least 10 fields in each slide were counted. The quantitative image analysis of all slides was performed by three investigators (N. Amara, M. Desmard, and J. Boczkowski1) blinded to the experimental groups. When the differences between the individual counts were >10%, the sections were analyzed together, and a consensual result was obtained.

These experiments were approved by the local Institutional Animal Care and Use Committee, and the experimental protocol was in agreement with French legal recommendations related to animal studies.

Statistical analysis. Values are given as means ± SE. The data were analyzed by one-way ANOVA or nonparametric tests as appropriate. Statistical significance was defined as P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of particles on cytotoxicity and cell viability. Stimulation with DEPs or other stimuli did not induce cytotoxicity after 6- or 24-h incubation (without removing the particles from the media) as evaluated by trypan blue dye staining. Indeed, the percentage of cells taking up trypan blue (nonviable cells) was 2.26 ± 1.02% in untreated control cells and ranged from 2.98 ± 0.93% to 4.75 ± 1.07% in cells treated with the different stimuli (differences between treated and untreated cells were not significant; data not shown).

Incubation of A549 cells with DEPs or CBPs at 10 µg/cm2 did not increase LDH activity in the media at 6 or 24 h (Fig. 1, A and B). By contrast, incubation with CSC at concentrations higher than 10 µg/ml significantly increased LDH activity after both 6 and 24 h (Fig. 1; P < 0.05 vs. control). However, incubation with 10 µg/cm2 DEPs plus 10 µg/ml CSC did not increase LDH activity (Fig. 1, A and B). Similar results were observed on PI staining (Fig. 1, C and D).


Figure 1
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  		Fig. 1. Effect of particles on cytotoxicity and cell viability in A549 cells. A and B: lactate dehydrogenase (LDH) activity. Control consisted of cells incubated with particle vehicles [0.04% dipalmitoyl lecithin (DPL) plus 10 µg/ml DMSO], cigarette smoke condensate 10 (CSC10), CSC20, and CSC100 at 10, 20, and 100 µg/ml, respectively, carbon black particles 10 (CBP10) at 10 µg/cm2, and diesel exhaust particles 10 (DEP10) at 10 µg/cm2. Values are means ± SE, n = 5; *P < 0.05 vs. control. C and D: propidium iodide (PI) staining. Results are expressed as proportion of positive cells. Values are means ± SE, n = 5; *P < 0.05 vs. control.

 
Collectively, these results show an absence of cytotoxicity of DEPs and CBPs at the concentrations used and a cytotoxic effect of CSC at concentrations higher than 10 µg/ml. To avoid the influence of cytotoxicity in the evaluation of the effects of DEPs and CSC on MMP-1 expression, we used the noncytotoxic concentration of 10 µg/ml CSC.

Effect of particles on MMP-1 and TIMP expression and activity. Real-time RT-PCR revealed that 6- and 24-h exposure of A549 cells to DEPs induced a significant and dose-dependent increase in MMP-1 mRNA expression compared with the control condition (P < 0.05), whereas CBPs and CSC elicited no effect (Fig. 2, A and B). Treatment with DEPs combined with CSC increased MMP-1 expression in a similar response to treatment with DEPs alone (Fig. 2B). MMP-1 protein expression and activity was induced after 24-h incubation with DEPs (Fig. 2, C and D). mRNA expression of MMP-2, the MMP inhibitors TIMP-1 and -2, and the profibrogenic cytokine TGF-beta was not changed under the different experimental conditions (data not shown).


Figure 2
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  		Fig. 2. Effect of particles on matrix metalloprotease-1 (MMP-1) expression and activity in A549 cells. A: effect of different concentrations of DEP on MMP-1 mRNA expression. MMP-1 mRNA levels are expressed as the ratio to ubiquitin mRNA levels. Values are means ± SE, n = 5; *P < 0.05 vs. control at 6 h. B: effect of different particles on MMP-1 mRNA expression after 6-h incubation with the different particles. MMP-1 mRNA content is expressed as in A. Values are means ± SE, n = 5; *P < 0.05 vs. control. C: representative Western blot of MMP-1 expression. Histograms represent means ± SE of 4 different experiments; *P < 0.05 vs. control condition. D: representative zymographic analysis of MMP-1 activity. Data represent results of 4 experiments.

 
Role of ROS in DEP-induced MMP-1 expression. We first examined ROS production by the different types of particles by quantifying H2DCFH-DA oxidation. Compared with the control condition, incubation for 1 h with DEPs alone or in combination with CSC gave a significant and similar increase in fluorescence (Fig. 3A; P < 0.05). By contrast, incubation of cells with CBPs or CSC alone did not increase fluorescence intensity (Fig. 3A).


Figure 3
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  		Fig. 3. Role of reactive oxygen species (ROS) on DEP-induced MMP-1 in A549 cells. A: effect of the antioxidant N-acetylcysteine (NAC) on ROS production in A549 cells assessed by measurement of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFH-DA) oxidation. Cells were incubated with NAC 1 h before the addition of particles to the culture media, and ROS production was assessed 1 h later. The values expressed as arbitrary fluorescence units are means ± SE, n = 5; *P < 0.05 vs. control. B: effect of NAC on MMP-1 induction by the different particles. Cells were incubated with NAC 1 h before the addition of particles to the culture media, and MMP-1 expression was examined 6 h later. MMP-1 mRNA levels are expressed as ratio to ubiquitin mRNA levels. Values are means ± SE, n = 5; *P < 0.05 vs. control.

 
Next, we examined the effect of the antioxidant N-acetylcysteine on DEP-induced MMP-1 expression. Pretreatment of A549 cells for 1 h with N-acetylcysteine (1 mM) reversed the increased ROS production and MMP-1 mRNA expression induced by DEPs (Fig. 3, A and B). These data suggest that the DEP-induced increase in MMP-1 expression occurs through an oxidative pathway.

Role of MAPKs in DEP-induced MMP-1 expression. Treatment of A549 cells with CBPs did not increase ERK 1/2 phosphorylation, whereas treatment with CSC resulted in phosphorylation, which peaked at 10 min (Fig. 4A). With DEP treatment, phosphorylation of ERK 1/2 began at 10 min but was delayed, peaking at 30 min, and was still elevated at 40 min (Fig. 4A). The degree of increase at 30 min was higher on incubation with DEPs than with CSC compared with the control condition (5.51 ± 0.34 vs. 0.81 ± 0.12 increase, respectively; P < 0.05).


Figure 4
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  		Fig. 4. Effect of particles on MAP kinase activation in A549 cells. A–C: representative Western blots showing the time course of phosphorylation of ERK 1/2, p38, and JNK, respectively, induced by the different particles. Histograms represent means ± SE of 4 different experiments; *P < 0.05 vs. control condition (0 min); #P < 0.05 vs. CSC. pERK1/2, phospho ERK1/2; p-p38, phospho p38; t-p38, total p38.

 
JNK and p38 phosphorylation was increased 10 min after treatment with the different particles (Fig. 4, B and C). The increase was similar for all particle types except for the increase in JNK, which was higher with DEP treatment than with CBPs or CSC (Fig. 4C; P < 0.05).

To define the role of the different MAPKs on DEP-induced MMP-1 expression, we used specific inhibitors and showed that preincubation for 1 h with PD-98059 (50 µM, a selective inhibitor of MAPK 1/2, the kinase upstream of ERK 1/2) reduced DEP-mediated increase in MMP-1 (Fig. 5A). By contrast, preincubation with SB-203580 (10 µM), which selectively blocks the p38 pathway (Fig. 5B), and with SP-600125 (40 nM), the JNK inhibitor, did not prevent DEP-induced MMP-1 expression (Fig. 5C). These data support a specific role for ERK 1/2 in DEP-induced MMP-1 expression in A549 cells.


Figure 5
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  		Fig. 5. Effect of MAP kinase inhibitors on MMP-1 expression in A549 cells. A–C: effect of inhibitors of ERK 1/2 (PD-98059), p38 (SP-600125), and JNK (SB-203580) MAP kinases (A, B, and C, respectively) on MMP-1 induction by the different particles. Cells were incubated with inhibitors 1 h before the addition of particles to the culture media, and MMP-1 expression was examined 6 h later. MMP-1 mRNA levels are expressed as ratio to ubiquitin mRNA levels. Values are means ± SE, n = 5; *P < 0.05 vs. respective control.

 
Role of NOX4 in DEP-induced MMP-1 expression. Preincubation of cells for 1 h with diphenylene iodonium (10 µM), an inhibitor of flavoprotein-dependent oxidases including NOX (18), prevented increased MMP-1 mRNA expression induced by DEPs (Fig. 6A), which suggests an involvement of NADPH oxidase/NOX in this process.


Figure 6
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  		Fig. 6. Role of NOX on DEP-induced MMP-1 expression in A549 cells. A: effect of diphenylene iodonium (DPI) on MMP-1 induction by the different particles. Cells were incubated with DPI 1 h before the addition of particles to the culture media, and MMP-1 expression was examined 6 h later. MMP-1 mRNA levels are expressed as ratio to ubiquitin mRNA levels. Values are means ± SE, n = 5; *P < 0.05 vs. respective control. B and C: NOX4 and NOX1 mRNA levels expressed as ratio to ubiquitin mRNA levels. Control values were similar at the different time points; the figure represents control values at 6 h. Values are means ± SE, n = 4; *P < 0.05 vs. control. D: NOX4 protein expression revealed by immunochemistry in control cells and cells incubated for 24 h with 10 µg/cm2 DEPs. No staining was observed when the anti-NOX4 antibody was replaced by normal rabbit serum (data not shown).

 
As demonstrated previously, A549 cells express both NOX1 and NOX4 at baseline (Fig. 6, B and C; Refs. 16, 17). DEPs increased NOX4 expression at the mRNA and protein level (Fig. 6, B and D) but did not modify NOX1 mRNA expression (Fig. 6C). CBPs and CSC elicited no effect on NOX1 and NOX4 mRNA expression (data not shown).

Accordingly, cell transfection with a NOX4 siRNA reduced NOX4 mRNA level (by 76.8 ± 2.45%) and NOX4 protein expression at 24 h, but no such effect was observed with Lipofectamine or scrambled siRNA (Fig. 7, A and B). Furthermore, the NOX4 siRNA did not modify mRNA expression of NOX1 (Fig. 7C).


Figure 7
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  		Fig. 7. Effect of silencing NOX4 expression by small interfering RNA (siRNA) in NOX4 and NOX1 expression in A549 cells. A: NOX4 mRNA levels expressed as ratio to ubiquitin mRNA levels after cell incubation with specific NOX4 siRNA or controls. Values are means ± SE, n = 4; *P < 0.05 vs. control. B: representative NOX4 protein expression detected by immunohistochemistry in cells incubated with specific NOX4 siRNA or controls. Positive red staining disappeared in cells incubated with NOX4 siRNA but not with Lipofectamine or scrambled siRNA. C: NOX1 mRNA levels expressed as ratio to ubiquitin mRNA levels after cell incubation with specific NOX4 siRNA or controls. Values are means ± SE, n = 4.

 
Transfection with NOX4 siRNA abolished the effect of DEPs on ROS production, MMP-1 mRNA expression, and ERK 1/2 phosphorylation, whereas treatment with scrambled siRNA had no effect (Fig. 8, A–C). These findings suggest a critical role of NOX4 in DEP-induced, oxidant-mediated MMP-1 expression.


Figure 8
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  		Fig. 8. Effect of silencing NOX4 expression by siRNA in DEP-induced MMP-1 expression in A549 cells. A: effect of DPI, scrambled siRNA, or NOX4 siRNA on ROS production 1 h after incubation with DEP or vehicle as assessed by measurement of H2DCFH-DA oxidation. The values expressed as arbitrary fluorescence units are means ± SE, n = 5; *P < 0.05 vs. control. B: effect of scrambled siRNA or NOX4 siRNA on MMP-1 induction after incubation of cells with DEP for 6 h. MMP-1 mRNA levels are expressed as ratio to ubiquitin mRNA levels. Values are means ± SE, n = 5; *P < 0.05 vs. control. C: representative Western blot showing the effect of scrambled siRNA or NOX4 siRNA on DEP-induced ERK 1/2 phosphorylation. Histograms represent means ± SE of 4 different experiments; *P < 0.05 vs. control condition. D: representative Western blot showing the effects of scrambled siRNA or NOX4 siRNA on DEP-induced JNK phosphorylation. Histograms represent means ± SE of 4 different experiments; *P < 0.05 vs. control condition.

 
Since DEP exposure increased the phosphorylation of JNK (Fig. 4C), we examined the effect of NOX4 on DEP-induced JNK phosphorylation. NOX4 siRNA prevented DEP-mediated activation of JNK (Fig. 8D), which suggests a widespread role of NOX4 in cell signaling processes.

Effect of DEPs on MMP-1 and NOX4 expression in NCI-H292 cells. Incubation of NCI-H292 cells with DEPs or CBP at 10 µg/cm2 did not increase LDH activity in the media at 6 or 24 h (data not shown). This concentration of DEP induced a significant increase in MMP-1 and NOX4 mRNA expression at 6 h (Fig. 9). No change in the expression of NOX1 was observed (data not shown).


Figure 9
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  		Fig. 9. Effect of DEPs on MMP-1 and NOX4 expression in NCI-H292 cells. A: MMP-1 mRNA levels are expressed as ratio to ubiquitin mRNA levels. Values are means ± SE, n = 4; *P < 0.05 vs. control. B: NOX4 mRNA levels are expressed as ratio to ubiquitin mRNA levels. Values are means ± SE, n = 4; *P < 0.05 vs. control.

 
Effect of DEPs on MMP-1 and NOX4 expression in mice. Finally, we examined MMP-1 and NOX4 protein expression in mice lungs after intratracheal instillation of DEPs. DEPs induced MMP-1 and NOX4 expression in alveolar epithelial cells of mice compared with controls (Fig. 10). The percentage of NOX4- and MMP-1-positive cells per field in control animals were 1.20 ± 0.50% and 2.34 ± 0.89% and, in animals instilled with DEP, 30.81 ± 1.89% and 23.21 ± 1.45%, respectively (P < 0.05 for NOX4 and MMP-1, respectively). These data confirm in vivo the effect of DEPs on MMP-1 and NOX4 expression in the alveolar epithelium.


Figure 10
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  		Fig. 10. MMP-1 and NOX4 expression in mice intratracheally instilled with DEPs or saline solution. A and B: pulmonary MMP-1 and NOX4 protein expression assessed by immunohistochemistry 24 h after intratracheal instillation of sterile saline solution (Control) or DEPs in mice. Positive cells are indicated by an arrow. No staining was observed when the anti-MMP-1 and anti-NOX4 antibodies were replaced by a control isotype antibody or a normal rabbit serum, respectively (data not shown).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Exposure of A549 cells to noncytotoxic concentrations of DEPs induced MMP-1 mRNA and protein expression and activity via a pathway involving oxidants produced by the NADPH oxidase analog NOX4 and subsequent ERK 1/2 phosphorylation. Furthermore, NOX4 also mediated DEP-induced JNK MAPK phosphorylation. By contrast, CSC treatment at nontoxic concentrations alone or with DEPs did not induce MMP-1 expression or activity. DEPs also induced MMP-1 and NOX4 expression in the human lung epithelial cell line NCI-H292. To the best of our knowledge, these data provide the first evidence that 1) noncytotoxic concentrations of DEPs induce MMP-1 expression and activity in epithelial cells, and 2) NOX4 plays a critical role in this phenomenon, showing a new role for this NADPH oxidase analog. Furthermore, these results could be extrapolated to the in vivo situation, since we found induced MMP-1 and NOX4 protein expression in alveolar epithelial cells of mice receiving DEPs intratracheally.

DEP exposure is well known to induce ROS production in cells such as bronchial epithelial cells and macrophages (2, 20), a key event underlying the DEP-elicited inflammatory reaction (2). DEP-induced ROS production can be explained by direct extracellular production by quinoid redox cycling (38) or intracellular production as a consequence of metabolism of polycyclic aromatic hydrocarbons by CYP enzymes such as CYP1A1 (2). However, the exact mechanism of this production is complex and probably involves different pathways. The present data are the first evidence of increased ROS production induced by DEPs in A549 cells. Quantifying increased ROS production in A549 cells is particularly relevant, given their high content of antioxidant enzymes such as heme oxygenase-1, glutamate cysteine ligase, and thioredoxin (27), which could degrade ROS immediately after production. Transfection of cells with an siRNA against NOX4 suppressed ROS production elicited by DEPs early (1 h) after incubation, which demonstrates that activation of this NADPH oxidase was responsible for immediate ROS generation after DEP incubation and defines a new pathway of ROS production after cell exposure to DEPs. However, we cannot exclude that DEP metabolism by CYP1A1 could be involved in a late production of ROS, since evidence of induced CYP1A1 expression and activity has been shown after 24-h incubation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in A549 cells (22).

Our evidence of DEP activation of NOX4 agrees with in vitro data of rat microglial cells showing NOX2 activation by these particles (3). Although LPS is one of the common chemicals adsorbed on the surface of DEPs, this mechanism is unlikely to explain NOX4 activation, because the expression of TGF-beta, a cytokine induced by LPS, was not modified in our study (data not shown). Alternatively, quinones can activate NOX4 by ROS produced by redox cycling (38) as demonstrated in adult cardiac fibroblasts incubated with H2O2 (10). Finally, although CBPs did not induce an immediate increase in ROS production, we cannot completely rule out a direct effect of a particle core in NOX4 activation as was proposed recently by Zhao and coworkers (45) concerning activation of NOX2 in rat lung. In addition to inducing activation, DEPs induced NOX4 mRNA and protein expression, a phenomenon mediated by TGF-beta in human vascular smooth muscle cells (40). However, we did not find an increase in TGF-beta mRNA expression after DEP exposure, so this mechanism was unlikely in our study. Alternatively, DEP-mediated NOX4 induction could occur through JNK activation as demonstrated by Pedruzzi and colleagues in smooth muscle cells exposed to 7-ketocholesterol (35). Our results showing DEP-induced JNK activation supports this hypothesis. Interestingly, we found that DEP-induced JNK activation was suppressed by transfection with NOX4 siRNA, which suggests the existence of a positive autocrine loop: DEPs induce NOX4 activation, which mediates JNK activation, which in turn induces NOX4 expression. Clearly, the mechanism(s) of NOX4 activation and induction by DEPs in A549 cells deserve further studies.

A potential limitation of this study is that the results obtained in A549 cells could reflect a particular response of this cell line to DEPs and not a general response of alveolar epithelial cells to this compound. However, we think this situation unlikely because MMP-1 and NOX4 were also induced in response to DEPs in another lung epithelial cell line, NCI-H292. Furthermore, we are confident of the relevance of these findings in an integrated system, since, similar to our findings of DEP-induced MMP-1, NOX4 protein was induced by DEP treatment in situ in alveolar epithelial cells of mice lung.

NOX4 is widely expressed in various tissues such as kidney, lung, placenta, pancreas, bone, and blood vessels, where it is involved in different pathophysiological processes (28). Our data are the first demonstration of a role of NOX4 in the modulation of the protease/antiprotease balance. Furthermore, induction of MMP-1 and NOX4 by DEPs was demonstrated in two different lung epithelial cell lines, A549 and NCI-H292. NOX4 modulated MMP-1 expression through ERK 1/2 activation, which agrees with previous data showing ERK 1/2 activation by oxidative stimuli in bronchial epithelial cells (32). Moreover, the evidence of ERK 1/2 and JNK activation by NOX4 in A549 cells expands on recent data from Li and coworkers (29) showing that NOX4 activated p38 MAPK in cardiac myocytes.

The increase in MMP-1 expression and activity in cells incubated with DEP was 4–5x greater than that under the control conditions, whereas induction by inflammatory stimuli such as interleukin-1beta could be between 60–100x greater (25). This moderate induction of MMP-1 could be consistent with a low but continuous degradation of interstitial collagen, which could lead to a progressive and chronic degradation of lung function as observed when people are chronically exposed to high levels of particulate air pollution. Moreover, our results fit with data published by Nagai and coworkers (33) showing that chronic exposure of guinea pigs to diesel exhaust causes alveolar destruction, the particulate fraction of the exhaust being particularly involved in this effect.

The absence of the effect of cigarette smoke on MMP-1 expression contrasts with data from Mercer and colleagues (32) showing that whole cigarette smoke induced MMP-1 mRNA and protein expression and activity in human small-airway epithelial cells and potentiated induction by proinflammatory cytokines. The difference in MMP-1 expression was paralleled by differences in ERK 1/2 phosphorylation: transient in our case (10 min) and prolonged in the above authors' study (lasting at least 60 min). Differences in the type of cigarette smoke material could explain this discrepancy, since we used CSC and not whole cigarette smoke. However, use of CSC overestimates instead of underestimates the oxidative and inflammatory effects of cigarette smoke (39). Furthermore, Mercer and colleagues (32) did not report any data on cytotoxicity, whereas we used a noncytotoxic concentration of condensate to avoid interference with death-signaling pathways, since MMP-1 has been recently reported to be involved in apoptosis (30). Further investigations are needed to evaluate the mechanisms of MMP-1 induction by cigarette smoke and the potential involvement of death-signaling pathways.

In conclusion, this study shows that DEP-induced MMP-1 expression is mediated by NOX4 via ERK 1/2 activation in human lung epithelial cells. Furthermore, DEP activation of JNK was dependent on NOX4. These results provide a new molecular basis for understanding respiratory cell responses to environmental aggression and the effect of chronic exposure to particulate airway pollution on lung function. Furthermore, they provide the basis for designing strategies to protect lung function by modulating NOX4 expression or activity.


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N. Amara was supported by Association pour la recherche sur les nicotianées and Chancellerie des Universités de Paris (legs Poix), M. Desmard by Fondation pour la Recherche Médicale Française and Institut National de la Santé et de la Recherche Médicale (INSERM; Poste d'Accueil pour Internes), S. Golda by Région Ile de France, and J. Boczkowski by INSERM and Assistance Publique-Hôpitaux de Paris (Contrat d'Interface). Part of this study was supported by PRIMEQUAL2 Grant CV02000158.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Boczkowski, INSERM U700, Faculté de Médecine Paris 7, site X. Bichat, BP416, 75870 Paris Cedex 18, France (e-mail: jbb2{at}bichat.inserm.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.


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