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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 463-473, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.2002-0039OC

S-Nitrosothiols Inhibit Cytokine-Mediated Induction of Matrix Metalloproteinase-9 in Airway Epithelial Cells

Tatsuya Okamoto, Giuseppe Valacchi, Kishorchandra Gohil, Takaaki Akaike and Albert van der Vliet

Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of California Davis School of Medicine, Davis, California; and Department of Microbiology, Kumamoto University School of Medicine, Kumamoto, Japan.

Address correspondence to: Albert van der Vliet, Department of Pathology, University of Vermont College of Medicine, Burlington, VT 05405. E-mail: Albert.van-der-Vliet{at}uvm.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inflammatory lung diseases are associated with increased production of matrix metalloproteinase-9 (MMP-9) from infiltrating granulocytes or from the respiratory epithelium, and inappropriate expression and activation of MMP-9 may be associated with tissue injury and airway remodeling. Inflammatory conditions also result in increased expression of inducible nitric oxide synthase (iNOS), and nitric oxide (NO·) has been reported to have variable effects on MMP-9 gene expression and activation in various cell types. We investigated the involvement of NO· or its metabolites on MMP-9 expression in human bronchial and alveolar epithelial cells by studying effects of NOS inhibition or exogenous NO· donors on cytokine-induced MMP-9 expression. Although inhibition of NOS, transfection with iNOS, or addition of NO· donors did not affect MMP-9 induction by inflammatory cytokines, addition of S-nitrosothiols dramatically inhibited MMP-9 expression, which was potentiated by depletion of cellular GSH. Cytokine-induced MMP-9 expression involves the activation of the transcription factor NF-{kappa}B, and S-nitrosothiols, in contrast to NO·, were found to inhibit cytokine-induced nuclear translocation and DNA binding of NF-{kappa}B. The inhibitory effects of S-nitrosothiols on cytokine-induced lung epithelial MMP-9 expression illustrate an additional mechanism by which nitrosative stress may affect epithelial injury and repair processes during conditions of airway inflammation.

Abbreviations: activator protein-1, AP-1 • bronchoalveolar lavage fluids, BALF • bovine serum albumin, BSA • L-buthionine-[S,R]-sulfoximine, BSO • colony-forming units, CFU • chronic obstructive pulmonary disease, COPD • diethylenetriamine NONOate, DETA NONOate • enhanced chemiluminescence, ECL • electrophoretic mobility shift assay, EMSA • fetal bovine serum, FBS • glyceraldehyde-3-phosphate dehydrogenase, GAPDH • S-nitrosoglutathione, GSNO • interferon {gamma}, IFN{gamma} • immunoglobulin G, IgG • inhibitor of NF-{kappa}B, I{kappa}B • interleukin-1ß, IL-1ß • inducible nitric oxide synthase, iNOS • NG-monomethyl-L-arginine, L-NMMA • lipopolysaccharide, LPS • matrix metalloproteinase, MMP • nuclear factor-kappa B, NF-{kappa}B • primary normal human bronchial epithelial cells, NHBE • nitric oxide, NO· • phosphate-buffered saline, PBS • proform of MMP, proMMP • reactive nitrogen species, RNS • reverse transcription-polymerase chain reaction, RT-PCR • 3-morpholinosydnonimine, SIN-1 • S-nitroso-N-acetyl-[D,L]-penicillamine, SNAP • tumor necrosis factor {alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Matrix metalloproteinases (MMPs) are a family of zinc neutral endopeptidases that are critical for disintegrating and remodeling of extracellular matrix during inflammation, wound healing, angiogenesis, and tumor invasion and metastasis (1). Among 23 different vertebrate MMPs cloned to date, MMP-2 (72-kD gelatinase) and MMP-9 (92-kD gelatinase) can degrade type IV collagen, a major component of the epithelial basement membrane, and they are considered to be important in injury and subsequent repair processes in the lung. Elevated levels of MMP-9 have been observed in idiopathic pulmonary fibrosis, acute respiratory distress syndrome, pulmonary emphysema, asthma, and cystic fibrosis (1–3). Several lines of evidence support a role for MMP-9 in neutrophil emigration in the lung (4, 5), although neutrophil emigration was unaffected in MMP-9–deficient mice (6). Studies with MMP-9 inhibitors or MMP-9 knockout mice, and genetic polymorphisms have implicated MMP-9 in respiratory tract injury in immune complex alveolitis (5), lipopolysaccharide (LPS)-induced lung injury (7), or pulmonary emphysema (8). However, MMP-9 has also been shown to be essential for upper or lower respiratory epithelial cell migration (9) and serves a critical role in repair and remodeling processes of the injured respiratory tract (10).

MMPs are produced by a variety of cell types, including neutrophils, macrophages, and fibroblasts (1), and the elevated MMP-9 levels in inflammatory lung diseases are thought to originate from infiltrating granulocytes or alveolar macrophages. However, human bronchial epithelial cells are an important source of MMPs (primarily MMP-2 and MMP-9) in the lung. Although epithelial cells constitutively express MMP-2, MMP-9 expression can be induced by various proinflammatory cytokines, growth factors, and LPS (11) through activation of the transcription factors nuclear factor kappa B (NF-{kappa}B) and activator protein-1 (AP-1) (12, 13). All MMPs are released as inactive proforms (proMMPs) and require activation in the extracellular space by limited proteolysis or chemical modification of the conserved cysteine residue in the prodomain (cysteine switch) (14, 15). Thus, the biologic activity of MMPs is controlled by gene expression and by post-translational mechanisms.

Inflammatory conditions of the respiratory tract are commonly characterized by elevated production of nitric oxide (NO·) through increased expression of inducible NO· synthase (iNOS) within the respiratory epithelium and in inflammatory cells, such as monocytes/macrophages and neutrophils (16–18). Although NO· possesses a wide array of regulatory and protective functions in inflammatory processes and has been shown to downregulate inflammatory cytokine production through the inhibition of NF-{kappa}B activation (17), active inflammatory conditions promote the oxidative metabolism of NO· to more reactive nitrogen species (RNS), which can affect biochemical pathways by unique covalent modifications in biologic targets, such as S-nitrosylation or tyrosine nitration (19, 20).

Several recent studies have identified various regulatory properties of NO· and/or RNS on gene expression and activation of MMPs. For instance, several RNS are capable of activating MMPs, including human neutrophil collagenase (MMP-8) and MMP-9 (21, 22), via oxidative modification of the cysteine switch. In addition, NO· has been reported to induce MMP-9 expression in chondrocytes (23) but was found to downregulate MMP-9 expression in mesangial cells (24) and aortic smooth muscle cells (25, 26). One problem with these studies is that it was often unclear whether NO· itself or RNS were responsible for these effects, and variable metabolism of NO· in these different experimental designs may have yielded disparate effects on MMP expression and activation. Moreover, the potential effects of NO· on MMP expression and activation in airway epithelial cells have not been studied.

The present study was performed to explore potential regulatory roles of NO· and RNS on MMP expression in human pulmonary epithelial cells. Our results demonstrate that, in contrast to NO·, S-nitrosothiols are capable of downregulating cytokine-mediated induction of MMP-9, indicating that regulatory effects of NO· on MMP-9 are related to enhanced oxidative metabolism under inflammatory conditions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture and Transfection
Experiments were performed with primary normal human bronchial epithelial cells (NHBE) (Clonetics, Cambrex, Walkersville, MD), which were used at passage 2–4 and grown in designed media (Bronchial/Tracheal Epithelial Cell Growth Medium Bullet Kit; BEGM; Cambrex) and with papilloma virus-immortalized human bronchial epithelial cells from a normal subject (HBE1) and from a patient with cystic fibrosis (CFT1) (27) (provided by Dr. R. Wu, University of California Davis, Davis, CA and J. Yankaskas, University of North Carolina, Chapel Hill, NC). HBE1 or CFT1 cells (passage 25–30) were cultured in serum-free Ham's F12 medium (GibcoBRL Life Technologies, Grand Island, NY) containing 0.6% penicillin-streptomycin (GibcoBRL) and 60 µg/ml gentamycin (GibcoBRL), 5 µg/ml transferrin (Sigma, St. Louis, MO), 0.1 µM dexamethasone (Sigma), 0.2 µg/ml cholera toxin (List Biologic Labs, Campbell, CA), 5 µg/ml insulin (Sigma), 20 ng/ml epidermal growth factor (Upstate Biotechnology, Lake Placid, NY), and 40 µg/ml bovine hypothalamus extract (GibcoBRL). Human type II alveolar epithelial carcinoma cells (A549; ATCC, Manassas, VA) were used and grown in Ham's F12 medium containing 0.6% penicillin-streptomycin, 60 µg/ml gentamycin, and 10% fetal bovine serum (FBS).

To investigate a role for endogenous NOS activity, HBE1 cells were stably transfected with a retrovirus vector (pLXSN/iNOS), encoding rat iNOS gene constructed in plasmid pLXSN under the control of a long terminal repeat promoter. The iNOS retrovirus vector-producing cell line (PA317/iNOS; kindly provided by Drs. T. Ogura and F. Tamura, National Cancer Center Research Institute, East, Chiba, Japan) was grown in Dulbecco's modified Eagle's medium (GibcoBRL) supplemented with 10% FBS until they were subconfluent monolayers. The culture medium was harvested, filtered, and stored at -80°C as a pLXSN/iNOS stock solution. The stock solution had titers of 1 x 105 colony-forming units (CFU) per ml. Transfection of the retrovirus vector to HBE1 cells was performed as previously described (28). Briefly, HBE1 cells were plated at 2 x 106 cells in a T75 flask the previous day and infected with 4 x 105 CFU of pLXSN/iNOS in the presence of 8 µg/ml of hexadimethrine bromide (Polybrene; Sigma) for 3 h. Infected HBE1 cells were selected in the presence of 500 µg/ml of G418 sulfate (Calbiochem, La Jolla, CA) for 2 wk. Expression of iNOS in the transfected cells (HBE1iNOS) was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, and functional iNOS protein expression was confirmed by ozone chemiluminescence analysis of nitrite (NO2-) and nitrate (NO3-) accumulation in the culture media.

Cell Treatments
Cells were placed in 24-well cell culture plates at a density of 5 x 104 cells/well and were grown until subconfluence (~ 90%). The medium was changed, and cells were pretreated with various concentrations (0–1,000 µM) of NO·-donor compounds for 30 min, followed by stimulation with human recombinant tumor necrosis factor {alpha} (TNF-{alpha}) (PeproTech Inc., Rocky Hill, NJ), interferon {gamma} (IFN-{gamma}) (PeproTech), or interleukin-1ß (IL-1ß) (Alexis, San Diego, CA) in the absence or presence of 1 mM NG-monomethyl-L-arginine (L-NMMA) (Sigma) or various NO·-releasing agents and incubated for up to 24 h at 37°C in humidified 5% CO2 atmosphere. NO· compounds used in this study were spermine NONOate and diethylenetriamine (DETA) NONOate (Cayman Chemical, Ann Arbor, MI) as NO·-releasing agents, 3-morpholinosydnonimine chloride (SIN-1) (Cayman) as a ONOO- donor, and S-nitroso-N-acetyl-[D,L]-penicillamine (SNAP) (Alexis) and S-nitrosoglutathione (GSNO) (Sigma) as S-nitrosothiol compounds. These NO· compounds did not significantly affect cell morphology or viability at the concentrations used. At the end of culture periods, the conditioned media were harvested and centrifuged at 6,000 rpm for 10 min to remove floating cells, and the supernatants were stored at -80°C until assay. For Western blot analysis and determination of cellular GSH, cells were lysed with lysing buffer (50 mM Hepes, 250 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1 mM ethyleneglycol-bis-(ß-aminoethyl ether)-N,N'-tetraacetic acid, 2 mM Na3CO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1% Triton X-100, pH 7.2) and centrifuged at 12,000 rpm for 10 min, and the supernatants were stored at -80°C until assay. Alternatively, cells were treated with TRIzol reagent for extraction of total RNA for RT-PCR analysis of MMP-9 or iNOS expression.

Gelatin Zymography
Secretion of MMP-2 and MMP-9 into the culture media was analyzed by gelatin zymography and quantified using NIH image shareware as described previously (2). Briefly, unconcentrated conditioned media (30 µl) were separated on NOVEX Gelatin Zymogram Pre-Cast Gels (Invitrogen, Carlsbad, CA) at a constant current of 25 mA at 4°C under nonreducing conditions. After electrophoresis, gels were washed three times with 40 mM Tris-HCl (pH 7.6), 10 mM CaCl2, 2 µM ZnCl2, 0.1% polyoxyethylene 23 lauryl ether (Brij 35; Sigma), and 3% Triton X-100 for 15 min to remove sodium dodecyl sulfate and then incubated for 24 h at 37°C in the same buffer without Triton X-100. After incubation, the gels were fixed in 10% acetic acid/50% methanol and stained with Coomassie brilliant blue R-250 for 60 min. MMP-induced gelatin digestion was identified as a clear lytic zone against a blue background. The dried gels were scanned and converted to digitized images for quantitation using NIH image v.1.61 shareware.

RT-PCR Analysis of MMP-2, MMP-9, and iNOS
The mRNA expression of MMP-2, MMP-9, iNOS, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were evaluated by semiquantitative RT-PCR. Total RNA was extracted and purified with TRIzol reagent (GibcoBRL) according to the manufucturer's instructions. The first strand cDNA was prepared from 5 µg of total RNA in first-strand buffer containing 0.5 µg oligo (dT) 15 primer (Promega, Madison, WI), 10 mM dithiothreitol, 0.5 mM dNTP mix, and 200 U Moloney murine leukemia virus reverse transcriptase (GibcoBRL) (20 µl reaction volume) and incubated at 37°C for 50 min. The PCR reactions were performed in a 20-µl reaction volume (5 µl of first-strand cDNA, 2 µl of 1 µM of both sense and antisense primers, and 13 µl of PCR buffer containing 1.5 mM of MgCl2, 1 U of Platinum Taq-DNA polymerase, and 200 µM dNTP mix) (GibcoBRL). Each cycle consisted of 1 min at 94°C for denaturation, 2 min at 60°C for annealing, and 2 min at 72°C for extension. As an internal control, GAPDH was amplified to normalize the starting amount of cDNA for each sample. The following primers were designed based on the cDNA sequences: MMP-2, 5'-ATA CCA TCG AGA CCA TGC-3' (sense) and 5'-CAG CTG TCA TAG GAT GTG-3' (antisense); MMP-9, 5'-ACG ATG ACG AGT TGT GGT-3' (sense) and 5'-CAT TCA CGT CGT CCT TAT-3' (antisense); iNOS, 5'-GTG AGG ATC AAA AAC TGG GG-3' (sense) and 5'-ACC TGC AGG TTG GAC CAC-3' (antisense); and GAPDH, 5'-TTC ATT GAC CTC AAC TAC AT-3' (sense) and 5'-GAG GGG CCA TCC ACA GTC TT-3' (antisense) (Operon Technologies, Alameda, CA). The expected sizes of the PCR products were 640 bp for MMP-2, 699 bp for MMP-9, 380 bp for iNOS, and 467 bp for GAPDH. Amplification was stopped in the linear phase of the PCR to quantitate the amount of mRNA in each sample. cDNA was diluted 10-fold for MMP-2, MMP-9, and iNOS and 100-fold for GAPDH, and 30 cycles were used. PCR products were resolved by 3% agarose gel electrophoresis and visualized by ethidium bromide staining, and band densities were quantified by NIH image shareware.

Western Blot Analysis for iNOS
For analysis of iNOS protein, aliquots of cell lysate containing 40 µg of protein were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis using NOVEX 4–12% Tris-Glycine Pre-Cast Gel (Invitrogen). Proteins were electroblotted onto polyvinylidene difluoride (Immobilon-P) transfer membrane (Millipore, Bedford, MA), which was incubated with TTBS/milk buffer (20 mM Tris-HCl, 137 mM NaCl, 0.05% Tween 20, 3% milk, pH 7.6) for 1 h followed by incubation (4°C overnight) with rabbit anti-iNOS polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted (1:2,000) with TTBS/milk buffer. This antibody recognizes human and rat iNOS protein. Membranes were finally incubated (1 h) with anti-rabbit immunoglobulin G (IgG)-peroxidase conjugate (Sigma) in TTBS/milk buffer (1:2,000), and immunoreactive bands were visualized using ECL Plus (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Nitrite and Nitrate Assay
To quantitate the cellular production of NO·, its metabolites NO2- and NO3- were measured in the conditioned cell culture media by chemical reduction in 122 mM vanadium (III) in 2M HCl at 95°C and analysis of the resulting NO· with an ozone chemiluminescence NO· analyzer (model 7020; ANTEK Instruments, Houston, TX) (29).

Nuclear Translocation of NF-{kappa}B
To investigate effects on TNF-{alpha}-induced NF-{kappa}B activation, we followed nuclear translocation of NF-{kappa}B by immunofluorescence staining using a rabbit anti-human NF-{kappa}B (p50) antibody (Santa Cruz Biotechnology) (30). HBE1 cells (1 x 105 cells/well) were plated in 1 ml of medium in a 24-well cell culture plate on 12-mm circle cover slips coated with human fibronectin (Sigma) 24 h before stimulation. Cells were pre-incubated with 500 µM of SNAP, GSNO, or spermine NONOate for 30 min and stimulated with TNF-{alpha} (100 ng/ml) for an additional 30 min. The medium was removed, and cells were washed with phosphate-buffered saline (PBS) and fixed with 500 µl/well of 4% paraformaldehyde for 20 min at room temperature on a plate shaker. After three washes with PBS, cells were permeabilized with 500 µl/well of cold (-20°C) methanol for 6 min and washed three more times with PBS. To block nonspecific antigenic sites, cells were incubated for 30 min with 500 µl/well of 1% bovine serum albumin (BSA) and 10% goat serum in PBS at 37°C. Cells were incubated overnight at 5°C with 300 µl rabbit anti-NF-{kappa}B (p50) antibody (1:3,000 dilution in 1% BSA/PBS), rinsed with 1% BSA/PBS, and blocked with 500 µl/well of 1% BSA and 10% goat serum in PBS at 37°C for 30 min. Cells were incubated for 1 h at room temperature with 300 µl of goat biotinylated anti-rabbit IgG (Vector Laboratories, Burlingame, CA) (1:2,500 dilution in 1% BSA/PBS) on a plate shaker. The cells were washed three times for 5 min in 1% BSA/PBS and incubated 1 h at room temperature with 200 µl/well of avidin-fluorescein isothiocyanate (Sigma) (1:1,000 dilution in 1% BSA/PBS) in the dark. The cells were washed three times for 5 min with 1% BSA/PBS and rinsed with PBS. Coverslips were transferred face down on microscope slides and examined under a fluorescence microscope (model BH2; Olympus America, Melville, NY).

Electrophoretic Mobility Shift Assay
As an alternative measure of NF-{kappa}B activation, we measured the NF-{kappa}B binding activity of cell nuclear extracts by electrophoretic mobility shift assay (EMSA). HBE1 cells were plated on 100-mm dishes (7.5 x 105 cells/dish) and treated with 500 µM of SNAP or GSNO for 30 min followed by stimulation with TNF-{alpha} (100 ng/ml) for 30 min. To prepare nuclear extracts, cells were washed three times with ice-cold PBS, placed on dry ice/ethanol, collected in 500 µl/dish of buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.2 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1 mM leupeptin), and transferred to microcentrifuge tubes. After incubation for 10 min on ice, Nonidet P-40 (final concentration 0.02%) was added; suspensions were passed six times through a 26.5-G needle, and nuclei were pelleted by centrifugation for 2 min at 10,000 rpm. The supernatant (cytoplasmic extract) was collected, and the pellet was resuspended in 50 µl of buffer B (20 mM Hepes, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 mM leupeptin). After incubation for 30 min on ice, samples were cleared by centrifugation for 5 min at 10,000 rpm at 4°C, and the supernatant (nuclear extract) was collected. An NF-{kappa}B consensus oligonucleotide (5'-AGT TGA GGG GAC TTT CCC AGG C-3') and an NF-{kappa}B mutant oligonucleotide (5'-AGT TGA GGC GAC TTT CCC AGG C-3') (Santa Cruz Biotechnology) were labeled with digoxygenin using DIG Genius gel shift kit (Boehringer Mannheim, Indianapolis, IN) and incubated overnight at 5°C with nuclear extract (20 µg protein). The complexes were electrophoresed (0.5x TBE buffer, 150 V for 2 h at room temperature) on NOVEX 6% nondenaturing polyacrylamide gels (Invitrogen) and electroblotted (400 mA for 1 h) onto nitrocellulose (protein) and nylon (oligonucleotide) membranes (31). The digoxygenin-labeled oligonucleotides were detected by anti-digoxygenin alkaline phosphatase-conjugated Fab fragments and enhanced chemiluminescence (ECL). The p50 subunit of NF-{kappa}B on the nitrocellulose membrane was probed by rabbit anti-NF-{kappa}B (p50) antibody and detected by ECL Plus.

Determination of Cellular GSH
Cellular GSH levels were determined by reversed phase-high performance liquid chromatography as described (32). Cell lysates were mixed with an equal volume of Thiolyte monobromobimane reagent (Calbiochem) (4 mM in 50 mM sodium N-ethylmorpholine, pH 8.0) and incubated for 5 min at room temperature in the dark. Proteins were precipitated with 5% trichloroacetic acid and removed by centrifugation at 6,000 x g for 15 min, and the supernatants were injected onto a 5-µm Spherisorb RP-18 column and eluted with 8% acetonitrile in 0.25% acetic acid at a flow rate of 1 ml/min. GSH was detected using fluorescence detection (excitation, 394 nm; emission, 480 nm) (model 2690; Waters, Milford, MA) and quantified using external standards.

Statistical Analysis
Data are expressed as means ± standard error of the mean. Statistical evaluation of the data was performed with Student's t test for unpaired observations, and differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cytokine Stimulation Induces MMP-9 Expression in Lung Epithelial Cells
All lung epithelial cells tested were found to express MMP-9 and MMP-2, which are secreted in their proform (proMMP) (Figure 1) . Proteolytically active forms of these MMPs (MMP-2, 64 kD and MMP-9, 84 kD) were not detected (Figure 1A). Basal MMP-9 and MMP-2 expression was higher in CFT1 cells compared with corresponding HBE1 cells. In accordance with previous studies (12, 33), cell stimulation with TNF-{alpha} dose-dependently increased MMP-9 production by all cell types, but MMP-2 expression was not affected by TNF-{alpha} stimulation (Figure 1B). Stimulation of cells with IL-1ß (up to 100 ng/ml) did not increase MMP-9 activity, consistent with earlier studies (12). TNF-{alpha}-induced MMP-9 expression and secretion was detectable after a 6-h lag period, and maximal levels were detected after 18–24 h (Figure 2) . The time course of mRNA expression and the secreted protein was completely parallel, indicating that the induction of MMP-9 by TNF-{alpha} occurs at the transcriptional level. Based on these findings, analysis of MMP-9 activity and mRNA in subsequent experiments were analyzed 24 h after TNF-{alpha} stimulation.



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  		Figure 1. Induction of MMP-9 in various pulmonary epithelial cells by TNF-{alpha}. HBE1, CFT1, NHBE, and A549 cells were grown until 90% confluence and stimulated with 0, 10, or 100 ng/ml TNF-{alpha} for 24 h. (A) MMP-9 and MMP-2 activities in the culture medium were analyzed by gelatin zymography, and a typical zymogram is shown. (B) Densitometric analysis of the gelatinolytic activity in the zymography gel. Data represent the mean ± SEM of five separate determinations. Significantly different from control (without TNF-{alpha}): *P < 0.05; **P < 0.005.

 


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  		Figure 2. Time-dependent induction of MMP-9 by TNF-{alpha} stimulation of HBE1 and NHBE cells. (A) HBE1 and NHBE cells were stimulated with TNF-{alpha} (100 ng/ml) for various time periods, and MMP-9 mRNA expression and gelatinolytic activity was measured as described. (B) Densitometry analysis of MMP-9 gelatinolytic activity and mRNA expression by RT-PCR. Data are represented as percent of control (time 0; immediately after the addition of TNF-{alpha}) and represent the mean ± SEM of three separate determinations. Significantly different from control (time 0): *P < 0.05; **P < 0.005.

 
Effect of iNOS Activity on Epithelial MMP-9 Expression
Stimulation of cells with a mixture of cytokines (CM: 100 ng/ml TNF-{alpha}, 50 ng/ml IL-1ß, and 50 ng/ml IFN{gamma}) resulted in less MMP-9 expression compared with stimulation with TNF-{alpha} alone (Figure 3) . While unstimulated or TNF-{alpha}-stimulated cells did not express iNOS, CM stimulation induced iNOS protein and mRNA in all cell types except CFT1 cells (Figure 3A). We used a mixture of cytokines to optimize induction of iNOS (16, 34). We postulated that the reduced MMP-9 expression by CM compared with TNF-{alpha} might be caused by iNOS-derived NO·, and this should then be corrected by pharmacologic inhibition of iNOS, except in CFT1 cells, which do not express iNOS. Stimulation with CM significantly increased NO2-/NO3- accumulation in the culture media of NHBE and A549 cells, illustrating elevated NO· production, which was prevented by pre-incubation with L-NMMA (1 mM) (Figure 3B, upper panel). However, NOS inhibition by L-NMMA did not affect MMP-9 expression by CM stimulation in any of the cell types tested (Figure 3). The lower MMP-9 expression by CM, compared with that by TNF-{alpha} alone, is most likely caused by inhibitory effects of IFN{gamma} because TNF-{alpha}-mediated MMP-9 induction in HBE1 cells was found to be reduced by co-stimulation with IFN{gamma} (not shown).



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  		Figure 3. Effects of NOS inhibition on MMP-9 expression in pulmonary epithelial cells. (A) HBE1, CFT1, NHBE, and A549 cells were stimulated with TNF-{alpha} alone (100 ng/ml) or with a mixture of cytokines (CM: 100 ng/ml TNF-{alpha}, 50 ng/ml IL-1{alpha}, and 50 ng/ml IFN{alpha}) in the presence or absence of L-NMMA (1 mM) for 24 h. MMP-9 mRNA and activity were analyzed as described earlier, and iNOS mRNA and protein were determined by RT-PCR and Western blot analysis, respectively. (B) NO2-/NO3- measurement in the culture medium (upper panel) and densitometry analysis of the MMP-9 gelatinolytic activity (lower panel). Data represent the mean ± SEM of five separate determinations. Significant differences between groups: *P < 0.05; **P < 0.005; NS, not significant.

 
Whereas iNOS is constitutively expressed in upper and lower airway epithelium in vivo, this expression is lost upon epithelial cell culture (16, 18). Hence, cultured airway epithelial cells may not provide a suitable model to investigate a role for endogenous NOS activity on epithelial gene regulation by inflammatory stimuli. In an attempt to correct this deficiency, we stably transfected iNOS into HBE1 cells by infection with a retrovirus vector encoding rat iNOS gene. Thus, transfected HBE1 cells (HBE1iNOS) expressed iNOS protein (Western blot) and mRNA (RT-PCR) and produced NO2-/NO3- into the culture media, even without cytokine stimulation (Figure 4) . TNF-{alpha} stimulation did not significantly affect iNOS expression or NO2-/NO3- production (Figure 4A and 4B upper panel) but markedly increased MMP-9 activity (zymogram) and mRNA (RT-PCR). Stimulation with CM caused somewhat reduced induction of MMP-9 and suppressed cellular production of NO2-/NO3-, the latter perhaps caused by upregulation of arginase. In all cases, co-incubation with L-NMMA (1 mM) dramatically inhibited cellular NO2-/NO3- production but did not significantly affect MMP-9 induction in response to TNF-{alpha} or CM (Figure 4A and 4B lower panel), although there was a slight tendency to reduction. These data suggest that cytokine-induced MMP-9 expression in airway epithelial cells is not regulated by endogenously produced NO·, which at most contributes marginally to cytokine-induced MMP-9 expression.



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  		Figure 4. Effect of NOS inhibition on MMP-9 expression and induction in iNOS-transfected HBE1 (HBE1iNOS) cells. (A) HBE1iNOS cells were grown until they were subconfluent and were stimulated with TNF-{alpha} (100 ng/ml) or CM (100 ng/ml TNF-{alpha}, 50 ng/ml IL-1ß, and 50 ng/ml IFN{alpha}) in the presence or absence of L-NMMA (1 mM) for 24 h. MMP-9 activity (in the culture medium) and MMP-9 and iNOS mRNA were analyzed by gelatin zymography and RT-PCR, respectively. The presence of iNOS protein was confirmed by Western blot analysis. (B) NO2-/NO3- measurement in the culture medium (upper panel) and densitometry analysis of MMP-9 activity, which was expressed relative to TNF-{alpha}-stimulated activity (lower panel). All data represent the mean ± SEM of five separate determinations. Significant differences between groups: *P < 0.05; NS, not significant.

 
Effects of Exogenous NO· and RNS on Cytokine-Induced MMP-9 Expression
To explore the possibility that cytokine-induced MMP-9 expression by airway epithelial cells is affected by exogenously generated NO· or RNS, HBE1 and NHBE cells were exposed to NO·-releasing agents (spermine NONOate or DETA NONOate), to an ONOO--generating agent (SIN-1), or to S-nitrosothiols (GSNO or SNAP) before and during stimulation with TNF-{alpha} (100 ng/ml). Spermine NONOate, DETA NONOate, and SIN-1 (up to 500 µM) had little or no effect on TNF-{alpha}-mediated MMP-9 expression (Figure 5) . In contrast, SNAP and GSNO dose-dependently suppressed TNF-{alpha}-mediated induction of MMP-9 mRNA levels and protein production in both cell types and were capable of completely inhibiting TNF-{alpha}-mediated induction of MMP-9. These findings indicate that the inhibitory effects of S-nitrosothiols on TNF-{alpha}-mediated MMP-9 expression are not caused by release of NO· but perhaps involve other more direct mechanisms.



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  		Figure 5. Effects of exogenous NO· or RNS on cytokine-induced MMP-9 expression. (A) NHBE cells were grown until they were subconfluent and were preincubated with the indicated concentrations of S-nitrosothiols (GSNO/SNAP), NO·-releasing agents (spermine NONOate, DETA NONOate), or the peroxynitrite-generating compound SIN-1 for 30 min. Cells were then stimulated with TNF-{alpha} (100 ng/ml) for 24 h, and MMP-9 activity in the culture media and MMP-9 mRNA expression were analyzed by zymography and RT-PCR, respectively. (B) MMP-9 gelatinolytic activity and MMP-9 mRNA expression were quantified densitometrically and expressed relative to TNF-{alpha}-stimulated values (%). (C) Similar results obtained from HBE1 cells. Data represent the mean ± SEM of three separate determinations. Significantly different from TNF-{alpha}-stimulated value (without NO· compounds): *P < 0.05.

 
Effects of NO· and RNS on NF-{kappa}B Activation
Because the activation of NF-{kappa}B is a critical event in TNF-{alpha}-induced expression of MMP-9 (12, 35), we explored the effects of NO· and S-nitrosothiols on NF-{kappa}B activation by TNF-{alpha}. Immunofluorescence staining of NF-{kappa}B (p50) in unstimulated HBE1 cells shows primarily cytoplasmic localization with relatively little nuclear staining (Figure 6) . Following a 30-min stimulation with TNF-{alpha} (100 ng/ml), large amounts of p50 are translocated to the nucleus, consistent with NF-{kappa}B activation. Pretreatment of cells with SNAP or GSNO (500 µM) significantly suppressed TNF-{alpha}-induced p50 nuclear translocation, whereas pretreatment with spermine NONOate (500 µM) had no effect (Figure 6A). To confirm the immunofluorescence results, we examined nuclear extracts for NF-{kappa}B binding activity by EMSA using consensus NF-{kappa}B binding sequences and observed increased NF-{kappa}B binding activity after TNF-{alpha} stimulation, which was again inhibited by cell pretreatment with SNAP or GSNO (Figure 6B). These findings are consistent with the inhibitory effects on MMP-9 expression and suggest that GSNO and SNAP suppress MMP-9 induction by preventing NF-{kappa}B activation through the inhibition of nuclear translocation and/or DNA binding.



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  		Figure 6. Effects of NO· and RNS on TNF-{alpha}-induced NF-{kappa}B activation in HBE1 cells. (A) Subconfluent HBE1 cells were pretreated with 500 µM of S-nitrosothiols (GSNO/SNAP) or spermine NONOate for 30 min and stimulated with TNF-{alpha} (100 ng/ml) for 30 min. After cell fixing, the cellular localization of NF-{kappa}B was analyzed by immunofluorescence staining using anti–NF-{kappa}B (p50) antibody. (B) Nuclear extracts (20 µg of protein) from TNF-{alpha} and/or S-nitrosothiol-treated HBE1 cells were analyzed by EMSA, and the NF-{kappa}B–oligonucleotide complex was detected by antidigoxygenin (upper panel) or anti-p50 antibodies (lower panel).

 
Effects of S-Nitrosothiols on Cellular GSH Status
Based on the redox properties of S-nitrosothiols, we anticipated that they might affect cellular redox status by altering cellular GSH levels. Cell exposure to SNAP (1 mM) rapidly depleted cellular GSH levels during the first hour, after which GSH levels gradually recovered and reached initial levels after 24 h (Figure 7A) . This transient GSH depletion by SNAP was dose dependent (Figure 7B). In contrast, exposure to GSNO (1 mM) did not reduce cellular GSH levels (Figure 7A), but GSH levels somewhat increased after 24 h. Hence, the effects of these S-nitrosothiols on MMP-9 expression do not seem to be a direct consequence of effects on cellular GSH levels.



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  		Figure 7. Modulation of cellular GSH levels by S-nitrosothiols. Subconfluent HBE1 cells were treated with various concentrations (0–1,000 µM) of SNAP or GSNO for various time periods (1, 6, and 24 h), and cellular GSH was determined after derivatization with monobromobimane and HPLC analysis and expressed as nmol GSH/mg protein. (A) Time-dependent changes in cellular GSH levels after incubation with 1 mM SNAP or GSNO. (B) Dose-dependent effects of SNAP or GSNO on the cellular GSH levels after 1 h. Data represent the mean ± SEM of three separate determinations. Significantly different from control (without SNAP or GSNO): *P < 0.05.

 
We next investigated whether depletion of cellular GSH affects the ability of S-nitrosothiols to suppress MMP-9 expression by preincubating HBE1 cells with 500 µM of L-buthionine-[S,R]-sulfoximine (BSO) (Sigma) for 18 h before treatment with SNAP or GSNO and stimulation with TNF-{alpha}. Cell pretreatment with BSO dramatically reduced cellular GSH levels to 15% of initial levels (31.2 ± 6.9 versus 4.8 ± 1.2 nmol/mg protein) (Figure 8A) . Although prior GSH depletion did not affect the ability of TNF-{alpha} to induce MMP-9, the inhibitory effects of SNAP and GSNO on TNF-{alpha}-mediated MMP-9 expression were markedly potentiated (Figure 8B). These findings suggest that GSH is indirectly involved in the modulation of MMP-9 expression by S-nitrosothiols, perhaps by its ability to reverse their biologic actions.



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  		Figure 8. Effect of depletion of cellular GSH on S-nitrosothiol-mediated inhibition of MMP-9 expression. (A) Subconfluent HBE1 cells were treated with or without BSO (500 µM) for 18 h, and cellular GSH levels were measured. (B) BSO-treated or untreated HBE1 cells were incubated with the indicated concentrations of SNAP or GSNO for 30 min and stimulated with TNF-{alpha} (100 ng/ml) for 24 h. MMP-9 activity in the culture media was analyzed by zymography and is represented as a percentage of TNF-{alpha}-stimulated activity of untreated cells. All data represent the mean ± SEM of three separate determinations. Significantly different from TNF-{alpha}-stimulated activity (without SNAP or GSNO): *P < 0.05; **P < 0.005.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
MMP family enzymes are important in tissue development and regeneration, and inappropriate MMP regulation and activation during, for example, chronic inflammation, may lead to disturbances in the turnover and remodeling of pulmonary extracellular matrix, which could contribute to the structural "remodeling" that occurs in chronic respiratory tract diseases such as asthma or chronic obstructive pulmonary disease (COPD) (36). Particularly, MMP-2 and MMP-9 possess type IV collagenolytic activity, which is important in the destruction/repair of the epithelial basement membrane in the lung. Whereas MMP-2 is constitutively expressed in various cell types, MMP-9 is strongly induced in epithelial cells by inflammatory cytokines, particularly TNF-{alpha} (11, 12). Inflammatory conditions are also associated with increased activation of oxidant-producing enzymes and NO·, which could directly affect MMP expression or activation (21–23, 37). In the present study, we have demonstrated that TNF-{alpha}-induced MMP-9 expression in human bronchial epithelial cells can be completely suppressed by S-nitrosothiols, although it seems to be insensitive to NO·. These findings and our earlier studies (21, 22) suggest that the regulatory properties of NO· on MMPs are related to its oxidative metabolism to RNS (e.g., under inflammatory conditions).

Analysis of the promoter region of the MMP-9 gene has identified an essential proximal AP-1 element and an upstream NF-{kappa}B binding site (13), and NF-{kappa}B activation is essential for TNF-{alpha}-mediated transcription of MMP-9 (12, 35). We demonstrated that the inhibitory effects of S-nitrosothiols on MMP-9 expression were associated with diminished nuclear translocation and activation of NF-{kappa}B. NF-{kappa}B activation is involved in the induction of various pro-inflammatory genes and is critical in inflammatory-immune processes in the lung. Various studies have indicated that NO· (or S-nitrosothiols) may regulate inflammatory processes by suppressing NF-{kappa}B activation (e.g., in neutrophils or alveolar macrophages) (17, 38). Consequently, the inhibitory effects of S-nitrosothiols on MMP-9 expression may be caused by inhibition of NF-{kappa}B activation, and the effects on MMP-9 are most likely not specific but may be common to other NF-{kappa}B–regulated genes.

One of the most significant findings of our results is that they distinguish effects of S-nitrosothiols from those of NO·. This is important because the biologic or cellular effects of S-nitrosothiols are often wrongly attributed to NO·, although they are distinctly different with regard to the redox state of the NO moiety (NO+ versus NO·), which endows S-nitrosothiols with biologic activities (e.g., thiolation, transnitrosation) not shared by NO· (39). The inhibitory effects on MMP-9 expression by S-nitrosothiols, in contrast to NO· donors, may be related to their ability to induce changes in cellular redox status, as demonstrated indirectly by a transient reduction in cellular GSH levels after exposure to SNAP. However, such a reduction was not observed with GSNO because its reactions with cellular GSH would regenerate GSH and not result in a net depletion of GSH. The disparity in cellular GSH depletion by SNAP and GSNO implies that GSH depletion alone is not responsible for their inhibitory effects on NF-{kappa}B activation and MMP-9 expression. Cell exposure to these S-nitrosothiols possibly results in modification of redox-sensitive protein cysteine residues through transnitrosation or mixed disulfide formation, and such modifications may be reversed by cellular GSH. Consistent with this, depletion of cellular GSH by BSO potentiated the inhibitory effects of S-nitrosothiols, suggesting that GSH is involved in the elimination of S-nitrosothiols or in the reversal of protein S-nitrosylation or oxidation. GSH is involved in various enzyme systems that protect cells against nitrosative stress, such as a cellular GSNO reductase activity recently identified in bacteria (GSH-dependent formaldehyde dehydrogenase), which uses NADH and GSH as co-factors (40). Other cellular mechanisms that detoxify S-nitrosothiols may include thioredoxin reductase (41), various heme proteins, or other cellular thiols (39).

Although SNAP is able to freely enter target cells, transfer of transnitrosating activity of GSNO into the intracellular compartment seems to involve the periplasmic {gamma}-glutamyl transpeptidase ({gamma}GTP), which converts GSNO to glutamate and S-nitrosocysteinylglycine, because GSNO cannot passively diffuse into cells (42, 43). Inhibition of {gamma}GTP by 50–100 µm acivicin was found to partially prevent GSNO-mediated inhibition of TNF-{alpha}-induced MMP-9 induction (not shown), suggesting that the effects of GSNO are, at least in part, mediated by actions on intracellular targets, such as proteins involved in NF-{kappa}B activation.

The activation of NF-{kappa}B by inflammatory cytokines involves various distinct steps, including the activation of I{kappa}B kinase, which catalyzes the phosphorylation of two N-terminal serine residues of inhibitor of NF-{kappa}B (I{kappa}B); polyubiquitination and proteosomal degradation of I{kappa}B; and translocation of activated NF-{kappa}B into the nucleus, where it can bind to promotor regions of target genes (44). Although detailed mechanisms of the nuclear translocation of NF-{kappa}B are unknown, cytoskeletal proteins such as tubulin, which are critical for intracellular kinetics and cellular morphology and movement, seem to have important roles in this process (44). Recently, tubulin S-nitrosylation has been observed in response to endogenous NO· activation (45), which may alter structure and function of microtubules. Other pathways by which NO· or RNS can affect NF-{kappa}B activation may involve induction of I{kappa}B expression (38) or direct S-nitrosylation of a cysteine residue within the NF-{kappa}B p50 subunit, which prevents DNA binding activity (46, 47). In addition, it was recently demonstrated that S-nitrosothiols are capable of inhibiting I{kappa}B kinase activity (48), presumably through nitrosation of a redox-sensitive cysteine residue within this enzyme.

Nitrosylation of cysteine residues is increasingly being recognized as a ubiquitous regulatory mechanism, comparable to phosphorylation, and represents a post-translational modification that can influence many cellular pathways involved in signal transduction, DNA repair, and host defense in vivo (20). For example, a family of thiol proteases that play central roles in apoptosis (collectively known as caspases) are inhibited by S-nitrosylation of its active site cysteine residue, and this has been proposed as a regulatory mechanism in vivo (49). Similarly, S-nitrosylation may also regulate NF-{kappa}B activity in intact cells. However, the precise critical cellular targets involved in such regulation remain to be identified.

Because of their chemical instability, the presence of S-nitrosothiols in vivo has been difficult to demonstrate, and there are relatively few reports documenting the presence of S-nitrosothiols in intact biologic systems. Nevertheless, S-nitrosothiols have been detected in nM to µM levels in tracheal aspirates or bronchoalveolar lavage fluids (BALF) from healthy subjects (50–52). Interestingly, levels of S-nitrosothiols in tracheal aspirates or BALF from patients with asthma or cystic fibrosis were found to be lower compared with those in healthy subjects, even though overall NO· production was normal or even enhanced (50–52). In addition, protein tyrosine nitration has been found to be enhanced in these conditions (52, 53), indicating altered or enhanced metabolism to RNS that promotes protein nitration but reduces other biologic activities associated with NO·, including S-nitrosylation (19, 52). Such conditions may also reduce the anti-inflammatory properties of NO·, perhaps by minimizing its regulatory effects on NF-{kappa}B activation. In addition, altered NO· metabolism in inflammatory conditions, which suppresses S-nitrosylation and favors formation of RNS, could enhance MMP-9 expression (present study) and activation (21, 22) and thereby contribute to progressive tissue injury.

In conclusion, our results show that, in contrast to NO·, S-nitrosothiols are capable of suppressing cytokine-mediated induction of MMP-9 in airway epithelial cells, possibly through a thiol-to-thiol NO+ transfer and consequent inhibition of the NF-{kappa}B signaling pathway. Inhibition of the NF-{kappa}B pathway by S-nitrosothiols could represent a protective system to terminate the excessive inflammation and may depend on NO· metabolic pathways that operate under inflammatory conditions. Overall, disturbances in lung epithelial expression or activation of MMPs in association with altered NO· metabolism during airway inflammation may affect epithelial injury and repair process in the lung and could be involved in the airway remodeling that is observed in asthma or COPD.


   Acknowledgments
 
The authors thank Drs. Tsutomu Ogura and Fumio Tamura (National Cancer Center Research Institute, East, Chiba, Japan) for providing the iNOS retrovirus vector producing cell line (PA317/iNOS) and Dr. Yvonne Janssen-Heininger for her contributions to the discussion. This work was supported by research grants from NIH (HL60812), the Cystic Fibrosis Foundation, the University of California Tobacco Related Disease Research Program (7RT-0167), and by a grant from the Uehara Memorial Foundation, Japan.

Received in original form April 2, 2002

Received in final form May 14, 2002


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 Materials and Methods
 Results
 Discussion
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 Am. J. Respir. Cell Mol. Bio.Home page
P. F. Bove, U. V. Wesley, A.-K. Greul, M. Hristova, W. R. Dostmann, and A. van der Vliet
Nitric Oxide Promotes Airway Epithelial Wound Repair through Enhanced Activation of MMP-9
Am. J. Respir. Cell Mol. Biol., February 1, 2007; 36(2): 138 - 146.
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 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
G. Candiano, M. Bruschi, N. Pedemonte, L. Musante, R. Ravazzolo, S. Liberatori, L. Bini, L. J. V. Galietta, and O. Zegarra-Moran
Proteomic analysis of the airway surface liquid: modulation by proinflammatory cytokines
Am J Physiol Lung Cell Mol Physiol, January 1, 2007; 292(1): L185 - L198.
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F. L. M. Ricciardolo, P. J. Sterk, B. Gaston, and G. Folkerts
Nitric Oxide in Health and Disease of the Respiratory System
Physiol Rev, July 1, 2004; 84(3): 731 - 765.
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V. Lagente, E. Naline, I. Guenon, M. Corbel, E. Boichot, J.-L. Burgaud, P. Del Soldato, and C. Advenier
A Nitric Oxide-Releasing Salbutamol Elicits Potent Relaxant and Anti-Inflammatory Activities
J. Pharmacol. Exp. Ther., July 1, 2004; 310(1): 367 - 375.
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