Induction of MMP-9 in normal human bronchial epithelial cells by TNF-alpha via NF-kappa B-mediated pathway

Atsuko Hozumi, Yoshihiro Nishimura, Teruaki Nishiuma, Yoshikazu Kotani, and Mitsuhiro Yokoyama

First Department of Internal Medicine, Kobe University School of Medicine, Kobe 650-0017, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we determined whether the proinflammatory cytokines tumor necrosis factor (TNF)-alpha and interleukin-1beta contribute to the regulation of matrix metalloproteinase (MMP)-9 in human bronchial epithelial cells and whether the induction of MMP-9 is regulated by the transcription factor nuclear factor (NF)-kappa B. We demonstrated that TNF-alpha induced MMP-9 at both the protein and mRNA levels in human bronchial epithelial cells and that interleukin-1beta did not. In contrast, induction of the tissue inhibitor of metalloproteinase-1 by TNF-alpha was less than that of interleukin-1beta . Increased expression of MMP-9 and NF-kappa B activation induced by TNF-alpha were inhibited by pyrrolidine dithiocarbamate and N-acetyl-L-cysteine but were not inhibited by curcumin. These results suggest that TNF-alpha induces the expression of MMP-9 in human bronchial epithelial cells and that this induction is mediated via the NF-kappa B-mediated pathway.

matrix metalloproteinase-2; matrix metalloproteinase-9; tumor necrosis factor-alpha ; nuclear factor-kappa B; proinflammatory cytokine; airway inflammation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A RECENT STUDY (39) has reported that human bronchial epithelial cells are capable of expressing matrix metalloproteinase-2 and matrix metalloproteinase-9 under basal conditions. Matrix metalloproteinases are a family of extracellular matrix-degrading enzymes and are induced by different stimuli including growth factors, cytokines, and tumor promoters. Matrix metalloproteinases play important roles in inflammation, tissue remodeling, angiogenesis, wound healing, tumor invasion, and metastatic progression (22, 24). Recent evidence (23) suggests that matrix metalloproteinase-9 is induced by airway inflammation. Matrix metalloproteinases are thought to be secreted in physical association with their specific inhibitor tissue inhibitor of metalloproteinase (TIMP-1, TIMP-2, TIMP-3, or TIMP-4). Matrix metalloproteinase-9 activity is inhibited by forming a 1:1 complex with TIMP-1 (3, 7, 12, 18).

Tumor necrosis factor (TNF)-alpha and interleukin-1beta are increased in the lungs of patients with asthma pathophysiologically (4, 15) and may be key cytokines in airway inflammation. Their effects on the expression of matrix metalloproteinases and TIMPs have been studied in various cell lines (6, 8, 36, 38), and these cytokines induce matrix metalloproteinases. It has been demonstrated that TNF-alpha exerts its effects via nuclear factor-kappa B, a transcription factor of diametric complex, in various cell types. TNF-alpha -induced nuclear factor-kappa B activation has been suggested to be mediated by reactive oxygen intermediates such as hydrogen peroxide because antioxidants such as pyrrolidine dithiocarbamate and N-acetyl-L-cysteine inhibit nuclear factor-kappa B activation (30, 31, 34). Several studies have shown that a conserved proximal activator protein (AP)-1 binding site is required in the induction of matrix metalloproteinase-9 (1, 11, 29, 41), and analysis of the matrix metalloproteinase-9 promoter has identified an essential proximal AP-1 element and an upstream nuclear factor-kappa B site (29).

We hypothesized that the inflammatory cytokines TNF-alpha and interleukin-1beta induce matrix metalloproteinase-9 in human bronchial epithelial cells and that these are induced via nuclear factor-kappa B activation. To investigate the expression and activity of matrix metalloproteinase-9 in human bronchial epithelial cells, we performed Northern blotting and gelatin zymography and examined the expression of TIMP-1 by enzyme-linked immunosorbent assay (ELISA). The effects of nuclear factor-kappa B activation on the expression of metalloproteinase-9 induced by TNF-alpha were investigated by electrophoretic mobility shift assay.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Normal human bronchial epithelial cells (passage 1) were purchased from Clonetics (San Diego, CA). Human bronchial epithelial cells were grown as monolayers in tissue culture flasks or dishes at 100% humidity and 5% CO2 at 37°C in serum-free modified LHC-9 medium (Clonetics) supplemented with 7.5 mg/ml of bovine pituitary extract, 0.5 mg/ml of hydrocortisone, 0.5 µg/ml of human recombinant epidermal growth factor, 0.5 mg/ml of epinephrine, 10 mg/ml of transferrin, 5 mg/ml of insulin, 0.1 µg/ml of retinoic acid, 6.5 µg/ml of triiodothyronine, 50 mg/ml of bovine serum albumin, fatty acid free, and 50 mg/ml of gentamicin sulfate-amphotericin B (Clonetics). For subculturing, the cells from the monolayers were harvested with trypsin (0.025%) and EDTA (0.01%) in HEPES-buffered saline solution, centrifuged at low speed (800 rpm for 5 min), and resuspended in fresh medium before they were grown on 60-mm plastic culture dishes. The cells were grown to confluence over 7-10 days. Human bronchial epithelial cells grown to 80% confluence were incubated in growth factor-free medium for 24 h. These confluent cells were incubated with interleukin-1beta or TNF-alpha for the indicated times. To investigate the relationship between matrix metalloproteinase-9 and nuclear factor-kappa B, pyrrolidine dithiocarbamate and N-acetyl-L-cysteine were added 1.5 h before stimulation with interleukin-1beta and TNF-alpha .

Northern blot analysis. Total cellular RNA was extracted from human bronchial epithelial cells with ISOGEN (Nippon Gene, Tokyo, Japan). RNA samples were applied to a 1% denaturing agarose gel, electrophoresed, and blotted onto a Hybond-N filter. The blotted filters were hybridized with 32P-labeled human matrix metalloproteinase-9 cDNA probe generated by random priming with a multiprime DNA labeling system (Amersham International).

After hybridization, the filters were washed twice with 1× saline-sodium citrate (SSC)-1% sodium dodecyl sulfate (SDS) at 65°C for 20 min and once with 1× SSC-0.1% SDS at 65°C for 20 min. The filters were exposed to an imaging plate (BAS 2, Fuji Xerox, Tokyo, Japan) for 2 h at room temperature. The relative intensities of signals were determined by an autoimage analyzer (BAS 2000, Fuji Xerox). Equal RNA loading was confirmed by ethidium bromide staining of 28S and 18S rRNA.

Zymography. The human bronchial epithelial cell culture medium was harvested and stored at -20°C until used. Aliquots of each sample were subjected to SDS-PAGE in 10% polyacrylamide gels containing 1 mg/ml of gelatin. The method of Laemmli (17) was followed, excluding any reducing agents or boiling products. After electrophoresis, the gels were washed twice in 2.5% Triton X-100 at room temperature for 20 min to remove SDS. The gel was then incubated in reaction buffer [100 mM Tris · HCl (pH 7.5), 10 mM CaCl2, and 1 mM Brij 35] overnight at 37°C and stained with Coomassie brilliant blue R-250. The positions of matrix metalloproteinases that contained gelatinolytic activity appeared as clear bands. Molecular masses of gelatinolytic bands were estimated with prestained molecular mass markers (Bio-Rad Laboratories). For detection of zymogen, samples were incubated for 4 h at 37°C with 1 mM aminophenylmercuric acetate (Sigma) before undergoing SDS-PAGE. To determine the inhibition profile of the enzymatic activities, we also used incubation in reaction buffer containing 10 mM EDTA, a metalloproteinase inhibitor, and 2 mM phenylmethylsulfonyl fluoride (Sigma), a serine proteinase inhibitor. Gelatinolytic activity was measured on zymography-digitalized images with NIH Image shareware V.1.55 as described in a recent work by Shan et al. (33).

Quantification of TIMP-1 protein by ELISA. TIMP-1 protein concentrations in human bronchial epithelial cell culture medium were measured with a human TIMP-1 sandwich ELISA kit (Amersham). The assays were performed in triplicate following instructions of the manufacturer.

Cell fractionation and preparation of nuclear extracts. Nuclear extracts were prepared with a modified version of the method of Schreiber et al. (32). Confluent human bronchial epithelial cells in a 60-mm dish were washed with ice-cold phosphate-buffered saline (PBS), harvested by scraping into 1 ml of PBS, and centrifuged in a 1.5-ml microtube at 5,000 rpm for 1 min. The pellet was resuspended in 400 µl of cold hypotonic buffer [10 mM HEPES buffer (pH 7.8) containing 10 mM KCl, 0.1 mM EDTA, 0.1% Nonidet P-40, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml of aprotinin, 2 µg/ml of pepstatin, and 2 µg/ml of leupeptin] and pelleted at 5,000 rpm for 1 min. Then the pellet was resuspended in 100 µl of hypertonic buffer [50 mM HEPES-saline buffer (pH 7.8) containing 420 mM KCl, 0.1 mM EDTA, 5 mM MgCl2, 20% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml of aprotinin, 2 µg/ml of pepstatin, and 2 µg/ml of leupeptin] and incubated for 30 min at 4°C. The nuclear pellet was isolated by centrifugation at 15,000 rpm for 15 min. The supernatant was the nuclear extract. Protein concentrations were determined with the method of Bradford (5). The extracts were then stored at -80°C.

Electrophoretic mobility shift assay. Nuclear extracts (2 µg of protein) were incubated with 60,000 counts/min of a 25-bp oligonucleotide containing the nuclear factor-kappa B consensus sequence 5'-T CGA CAG AGG GAC TTT CCG A-3' that was prelabeled with 32P by random priming with a multiprime DNA labeling system (Amersham). Incubation was performed for 15 min at room temperature in the presence of 2 µg of poly(dI-dC) as a nonspecific competitor and 4 mM Tris · HCl (pH 7.9) containing 12 mM HEPES (pH 7.9), 60 mM KCl, 1 mM EDTA, 12% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 4.5 mg/ml of nuclease-free bovine serum albumin. For competition studies, unlabeled wild-type oligonucleotides were added to the binding reaction before addition of the radiolabeled probe. To examine specific binding, anti-p50 nuclear factor-kappa B antibody (Transduction Laboratories) was preincubated with nuclear extracts for 2 h. All incubation mixtures were subjected to electrophoresis on gels containing 25 mM Tris, 52.6 mM glycine, 1.3 mM EDTA, 4% acrylamide, 0.05% bis-acrylamide, and 2.5% glycerol, which were subsequently dried and exposed to an imaging plate (BAS 2, Fuji Xerox) for 1 h at room temperature. The relative intensities of signals were determined with an autoimage analyzer (BAS 2000, Fuji Xerox).

Reagents. TNF-alpha , pyrrolidine dithiocarbamate, N-acetyl-L-cysteine, Brij 35, aminophenylmercuric acetate, and phenylmethylsulfonyl fluoride were purchased from Sigma (St. Louis, MO). Interleukin-1beta was purchased from Immugenex. The matrix metalloproteinase-9 probe was kindly provided by Dr. K. Tryggvason (Biocenter and Department of Biochemistry, University of Oulu, Oulu, Finland).

Data and statistical analysis. Data are expressed as means ± SE. Statistical evaluation of the data was performed with Student's t-test for unpaired observations with StatView 4.5 (Abacus Concepts, Berkeley, CA) for Macintosh.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TNF-alpha induced production of matrix metalloproteinase-9 by human bronchial epithelial cells. After stimulation with TNF-alpha (10 ng/ml) or interleukin-1beta (10 ng/ml), total RNAs of human bronchial epithelial cells were collected at each time point (0, 8, 24, 36, and 48 h) and the expression of matrix metalloproteinase-9 was analyzed by Northern blot analysis. The level of matrix metalloproteinase-9 mRNA was increased in a time-dependent manner and the expression peaked at 24 h, indicating a 11.9 ± 0.15-fold increase compared with the control value (n = 3 experiments; Fig. 1, A and E). However, interleukin-1beta did not consistently increase the expression of matrix metalloproteinase-9 mRNA compared with TNF-alpha (1.93 ± 0.67-fold increase compared with the control value; n = 3 experiments; Fig. 1, B and E).


View larger version (49K):
[in this window]
[in a new window]
 
Fig. 1.   Tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta stimulation increases matrix metalloproteinase (MMP)-9 mRNA in human bronchial epithelial cells. Human bronchial epithelial cells were stimulated with (+) and with out (-) TNF-alpha (10 ng/ml; A) and interleukin-1beta (10 ng/ml; B) for indicated times. Total RNA was isolated as in MATERIALS AND METHODS and analyzed by Northern blotting. Human bronchial epithelial cells were stimulated with indicated concentrations of TNF-alpha (C) and interleukin-1beta (D) for 24 h. A representative Northern blot from 1 of 3 experiments is shown. E and F: time course and dose response, respectively, of MMP-9 mRNA expression. Data are means ± SE of 3 separate experiments. Significantly different from control: * P < 0.01; ** P < 0.001.

To determine dose responsiveness, human bronchial epithelial cells were incubated for 24 h with TNF-alpha and interleukin-1beta at concentrations from 0.1 to 20 ng/ml. TNF-alpha induced matrix metalloproteinase-9 mRNA in a dose-dependent manner (Fig. 1, C and F). Peak induction of matrix metalloproteinase-9 mRNA was observed with 10 ng/ml of TNF-alpha , indicating a 4.05 ± 0.46-fold increase compared with the control value. Interleukin-1beta increased matrix metalloproteinase-9 mRNA at a low level compared with TNF-alpha , but this induction did not show dose dependency (Fig. 1, B and D).

With zymography, gelatinolytic activities were detected as a major band of 92 kDa produced by the pro form of matrix metalloproteinase-9 and a minor band of 72 kDa corresponding to the pro form of matrix metalloproteinase-2 under basal conditions (Fig. 2, A and B). The active forms of matrix metalloproteinase-9 and matrix metalloproteinase-2 were not observed. EDTA completely inhibited all gelatinase activities, whereas phenylmethylsulfonyl fluoride had slightly inhibitory effect (Fig. 3A). Moreover, after incubation of aliquots of human bronchial epithelial cell culture medium in the presence of aminophenylmercuric acetate, the major band of 92-kDa gelatinase was activated into a smaller band of 88 kDa (Fig. 3B) and the minor band of 72-kDa gelatinase migrated to that of 68 kDa (data not shown). These results are consistent with previous findings showing that the 92- and 72-kDa gelatinases belong to the matrix metalloproteinase family. After TNF-alpha stimulation, the pro form of matrix metalloproteinase-9 was increased in a time-dependent manner (Fig. 2, A and C), but interleukin-1beta did not increase expression of the pro form of matrix metalloproteinase-9 compared with that in untreated cells (Fig. 2B). To determine whether this induction was in a dose-dependent manner, human bronchial epithelial cells were incubated for 24 h with TNF-alpha and interleukin-1beta at concentrations from 0.1 to 20 ng/ml. TNF-alpha induced the pro form of matrix metalloproteinase-9 in a dose-dependent manner, but interleukin-1beta did not (Fig. 4). Peak induction of the pro form of matrix metalloproteinase-9 was observed with 10 ng/ml of TNF-alpha .


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 2.   Gelatinase production by cultured human bronchial epithelial cells after stimulation with and without TNF-alpha (A) or interleukin-1beta (B) for indicated times. Then, each culture medium (20 µl) of human bronchial epithelial cells was subjected to gelatin zymography. Nos. at left, molecular mass. C: gelatinase activity. Values are means ± SE of 3 separate experiments. * P < 0.05 vs. control.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 3.   Identification of metalloproteinases. A: after PAGE or gelatin electrophoresis of conditioned medium from human bronchial epithelial cells, gelatin substrate gels were incubated in the incubation buffer with and without EDTA or phenylmethylsulfonyl fluoride (PMSF). B: activation of progelatinase by p-aminophenylmercuric acetate (APMA). Conditioned medium samples were incubated with APMA for 4 h at 37°C. The conditioned medium was analyzed by zymography. Nos. at left, molecular mass.



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 4.   Gelatinase production by cultured human bronchial epithelial cells after stimulation with TNF-alpha (A) or IL-1beta (B) for 24 h at indicated concentrations. Then, each culture medium (20 µl) of human bronchial epithelial cells was subjected to gelatin zymography. Nos. at left, molecular mass. C and D: gelatinase activity after treatment with TNF-alpha and IL-1beta , respectively. Values are means ± SE of 3 separate experiments.

TIMP-1 production by human bronchial epithelial cells. TNF-alpha or interleukin-1beta stimulation increased TIMP-1 production in a time-dependent manner (Fig. 5A). However, TNF-alpha did not increase induction compared with interleukin-1beta . To determine whether this induction was in a dose-dependent manner, human bronchial epithelial cells were incubated for 24 h with TNF-alpha and interleukin-1beta at concentrations from 0.1 to 20 ng/ml. These inductions were increased statistically but not in a dose-responsive manner (Fig. 5, B and C).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5.   Quantitative analysis of tissue inhibitor of metalloproteinase-1 (TIMP-1) secretion by human bronchial epithelial cells cultured in the presence of TNF-alpha or IL-1beta . TIMP-1 secretion from culture medium was quantified by ELISA. A: time course of TIMP-1 protein release. Human bronchial epithelial cells were stimulated with TNF-alpha (10 ng/ml) or IL-1beta (10 ng/ml) for indicated times. B and C: dose response of TIMP-1 protein release. Human bronchial epithelial cells were stimulated with indicated concentrations of TNF-alpha (B) and IL-1beta (C). Values are means ± SE in percentage of unstimulated sample (control) from 3 separate experiments. Significantly different from control: * P < 0.05; **P < 0.005.

Matrix metalloproteinase-9 production by TNF-alpha -stimulated human bronchial epithelial cells was inhibited by pyrrolidine dithiocarbamate or N-acetyl-L-cysteine. The intracellular redox state of the lung cells may have a key role in the regulation of the inflammatory immune responses in many inflammatory lung diseases (28). To examine the contribution of nuclear factor-kappa B, human bronchial epithelial cells were treated with antioxidants such as N-acetyl-L-cysteine or pyrrolidine dithiocarbamate before TNF-alpha stimulation. In Northern blotting, N-acetyl-L-cysteine or pyrrolidine dithiocarbamate showed a dose-dependent inhibition of TNF-alpha -induced matrix metalloproteinase-9 mRNA expression (Fig. 6, A and B). As a control, 28S rRNA levels were not altered by pyrrolidine dithiocarbamate and N-acetyl-L-cysteine.


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of pyrrolidine dithiocarbamate (PDTC) or N-acetyl-L-cysteine (NAC) on TNF-alpha -induced MMP-9 in human bronchial epithelial cells pretreated with indicated concentrations of PDTC or NAC for 1.5 h before stimulation with TNF-alpha . mRNA was isolated as in MATERIALS AND METHODS and analyzed by Northern blotting. A and B: induction of MMP-9 mRNA in human bronchial epithelial cells pretreated with PDTC and NAC, respectively, and stimulated with TNF-alpha for 24 h. Human bronchial epithelial cells were pretreated with PDTC (C) and NAC (D). Each culture medium (20 µl) of human bronchial epithelial cells was substrated for SDS-PAGE or gelatin electrophoresis. E and F: MMP-9 production after treatment with PDTC and NAC, respectively. All results are representative of 3 experiments.

In zymography, the band of 92-kDa gelatinolytic activity in response to TNF-alpha was also inhibited by N-acetyl-L-cysteine and pyrrolidine dithiocarbamate (Fig. 6, C and D). The AP-1 inhibitor curcumin did not inhibit 92-kDa gelatinolytic activity (data not shown). The band of 72-kDa gelatinase induced by TNF-alpha was also inhibited by pyrrolidine dithiocarbamate and N-acetyl-L-cysteine. These results suggest that the expression of matrix metalloproteinase-9 mRNA and 92-kDa gelatinase was induced by TNF-alpha via nuclear factor-kappa B activation.

TNF-alpha induced nuclear factor-kappa B activation in human bronchial epithelial cells. To examine whether TNF-alpha induced nuclear factor-kappa B activation in human bronchial epithelial cells, we performed an electrophoretic mobility shift assay with a nuclear factor-kappa B oligonucleotide (25 bp). The nuclear factor-kappa B oligonucleotide prelabeled with 32P was incubated with the nuclear extracts prepared from human bronchial epithelial cells (Fig. 7A). Stimulation of human bronchial epithelial cells with TNF-alpha induced the expression of nuclear factor-kappa B (3.5 ± 1.9-fold; Fig. 7A, lane 2). Pyrrolidine dithiocarbamate treatment before stimulation with TNF-alpha showed inhibition of nuclear factor-kappa B activation in nuclear extracts (P < 0.05; Fig. 7A, lane 3), and N-acetyl-L-cysteine treatment before stimulation with TNF-alpha partially inhibited nuclear factor-kappa B activation (Fig. 7A, lane 5). Pyrrolidine dithiocarbamate alone inhibited nuclear factor-kappa B activation compared with that in control cells (P < 0.001; Fig. 7A, lane 4), but N-acetyl-L-cysteine alone did not change nuclear factor-kappa B activation (Fig. 7A, lane 6). To elucidate that this activation is specific for the nuclear factor-kappa B pathway, anti-p50 nuclear factor-kappa B antibody was preincubated with nuclear extracts before binding with nuclear factor-kappa B oligonucleotides (Fig. 7B). Supershift assay showed that the binding of anti-p50 antibody with a nuclear factor-kappa B element induced by TNF-alpha caused an upward shift (Fig. 7B, lane 3) from the baseline (Fig. 7B, lane 2) and was inhibited by pyrrolidine dithiocarbamate (Fig. 7B, lane 4). N-acetyl-L-cysteine showed partial inhibition of the upper shift caused by TNF-alpha treatment (Fig. 7B, lane 5). However, the AP-1 inhibitor curcumin did not inhibit this reaction (Fig, 7B, lane 8). To explore the relationship with protein kinase C activity, the nonselective inhibitors calphostin C, H-7, and staurosporine were added. Calphostin C (data not shown) and H-7 (Fig. 7, lane 6) partially inhibited nuclear factor-kappa B expression induced by TNF-alpha , whereas staurosporine totally inhibited it (Fig. 7, lane 7).


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of antioxidant or protein kinase C inhibitor on the TNF-alpha -induced DNA binding activities of nuclear factor (NF)-kappa B. A: nuclear extracts from human bronchial epithelial cells were treated with TNF-alpha (10 ng/ml), PDTC (20 µM), NAC (30 mM), curcumin (10 µM), H-7 (100 µM), or staurosporine (10 µM). Lane 1, control; lane 2, TNF-alpha ; lane 3, PDTC plus TNF-alpha ; lane 4, PDTC; lane 5, NAC plus TNF-alpha ; lane 6, NAC; lane 7, excess cold NF-kappa B oligonucleotide. B: for specific binding, nuclear extracts were preincubated with (lanes 1 and 3-8) and without (lane 2) anti-p50 NF-kappa B antibody (Ab) before the binding reaction. Lane 1, control; lanes 2 and 3, TNF-alpha , lane 4, PDTC plus TNF-alpha ; lane 5, NAC plus TNF-alpha ; lane 6, H-7 plus TNF-alpha ; lane 7, staurosporine plus TNF-alpha ; lane 8, curcumin plus TNF-alpha .


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrated that TNF-alpha induced an increase in matrix metalloproteinase-9 expression and nuclear factor-kappa B activation in human bronchial epithelial cells and that these inductions were inhibited by pyrrolidine dithiocarbamate and N-acetyl-L-cysteine. On the other hand, interleukin-1beta did not consistently induce matrix metalloproteinase-9 expression.

Matrix metalloproteinases and their inhibitors are implicated in the degradation of extracellular matrix components and hence in the mechanism of cellular migration (20, 25, 37). Inflammatory cytokines, particularly interleukin-1beta and TNF-alpha , induce the production of matrix metalloproteinase-9 in normal human fibroblasts (38) and osteosarcoma and fibrosarcoma cell lines (27). It is well known that interleukin-1beta and TNF-alpha play a central role in acute inflammation in the lung. Our data indicated that TNF-alpha increased matrix metalloproteinase-9 expression at both the protein and mRNA levels in human bronchial epithelial cells. Yao et al. (39) demonstrated matrix metalloproteinase-9 production and activation induced by lipopolysaccharide and supported the fact that matrix metalloproteinase-9 may be involved in inflammatory pulmonary processes. These data suggest that acute inflammation increases production of matrix metalloproteinase-9 in human bronchial epithelial cells and that matrix metalloproteinase-9 production may contribute to the pathogenesis of several inflammatory diseases in the human airway.

Recent studies have indicated that matrix metalloproteinase-9 is increased in bronchoalveolar lavage fluid (23) and bronchial tissues (26) in patients with bronchial asthma, suggesting that matrix metalloproteinase-9 may play an important role in bronchial asthma. In addition, a recent study by Shan et al. (33) demonstrated that the expression and release of matrix metalloproteinase-9 in human bronchial epithelial cells were stimulated by platelet-activating factor, which plays a role as a mediator in pathological states of asthma. In contrast, matrix metalloproteinase-2 was not significantly increased after stimulation by TNF-alpha , interleukin-1beta , and platelet-activating factor in the present or in the recent study by Shan et al. These data indicate that matrix metalloproteinase-2 expression induced by these inflammatory cytokines is constitutive at a low level compared with matrix metalloproteinase-9 expression. Previous studies have demonstrated that the source of matrix metalloproteinase-9 in airway inflammation is eosinophils (14, 26), neutrophils (19), and bronchial epithelial cells (39, 40). This study also shows that the source of matrix metalloproteinase-9 is bronchial epithelial cells in an inflammatory model in vitro.

TIMP-1 expression in human bronchial epithelial cells did not increase after TNF-alpha treatment compared with interleukin-1beta treatment. These data suggest that human bronchial epithelial cells produce matrix metalloproteinase-9 and TIMP-1 in the basal condition. However, the expression of matrix metalloproteinase-9 induced by TNF-alpha was higher than that of TIMP-1. These results correspond with a previous report (40) that TNF-alpha and interleukin-1beta induced an increase in the latent form of 92-kDa gelatinase production and mRNA level, whereas TIMP-1 production and mRNA level were unchanged in the presence of interleukin-1beta and that these were decreased in the presence of TNF-alpha . An imbalance between matrix metalloproteinase and TIMP activities has been reported under various conditions. In patients with asthma, matrix metalloproteinase-9 and TIMP-1 immunoreactivities were significantly increased in both the epithelium and submucosa and the expression of matrix metalloproteinase-9 was stronger than that of TIMP-1 in the submucosa (14), suggesting that the increased expression of matrix metalloproteinase-9 may be produced by eosinophils in chronic asthmatic patients. However, our investigation indicated that matrix metalloproteinase-9 and TIMP-1 were produced by bronchial epithelial cells in an inflammation model in vitro. Because matrix metalloproteinase-9 activity is inhibited by forming a 1:1 complex with TIMP-1, our data suggest that inflammatory changes induce both matrix metalloproteinase-9 activation and TIMP-1 secretion and that the overflow of matrix metalloproteinase-9 may cause cell invasion, matrix degradation, and tissue remodeling.

A previous study (29) indicated that analysis of the matrix metalloproteinase-9 promoter has identified an essential proximal AP-1 element and an upstream nuclear factor-kappa B site. We hypothesized that nuclear factor-kappa B may mediate the induction of matrix metalloproteinase-9 in human bronchial epithelial cells stimulated by TNF-alpha . Because a recent study (13) demonstrated the presence of activated nuclear factor-kappa B in asthmatic airways and inflammatory cells and that nuclear factor-kappa B is responsible for the regulation of a number of cytokines (interleukin-1, interleukin-2, interleukin-6, interleukin-8, granulocyte-macrophage colony-stimulating factor, regulated on activation normal T cell expressed and secreted, and TNF-alpha ), nuclear factor-kappa B may play a role in airway inflammation. It has been demonstrated that TNF-alpha exerts its effects via nuclear factor-kappa B. Nuclear factor-kappa B activation may be regulated at several potential points, including signal transduction, Ikappa B degradation, and nuclear translocation of nuclear factor-kappa B. Although antioxidants inhibit nuclear factor-kappa B-mediated cytokine production in some cell lines, the role of nuclear factor-kappa B in airway epithelial cells has not yet been determined (9, 21). Our data showed that TNF-alpha increased nuclear factor-kappa B activation in human bronchial epithelial cells and that this expression is inhibited by the antioxidants pyrrolidine dithiocarbamate and N-acetyl-L-cysteine. In addition, the expression of matrix metalloproteinase-9 induced by TNF-alpha was inhibited by pyrrolidine dithiocarbamate and N-acetyl-L-cysteine. However, an AP-1 inhibitor, curcumin, did not show the inhibitory effect of nuclear factor-kappa B activation. These data suggest that activation of nuclear factor-kappa B induces an increase in the expression of matrix metalloproteinase-9 after stimulation of TNF-alpha in human bronchial epithelial cells, although we could not show where the point of nuclear factor-kappa B activation is regulated. In airway epithelial cells from the human lung carcinoma cell line A549, TNF-alpha induces Ikappa B degradation, nuclear factor-kappa B activation, and interleukin-8 gene transcription (10). Moreover, in primary rabbit and human dermal fibroblasts, matrix metalloproteinase-9 mRNA expression induced by interleukin-1beta and platelet-derived growth factor is inhibited by Ikappa B-alpha overexpression (2). Recently, it has been reported (16) that protein kinase C activation stimulated by TNF-alpha induces Ikappa B kinase activation and Ikappa B degradation. Our data showed that treatment with a nonselective protein kinase C inhibitor inhibited the nuclear factor-kappa B activation induced by TNF-alpha . Therefore, matrix metalloproteinase-9 may be expressed via activation of nuclear factor-kappa B through protein kinase C activation.

Although these data indicated that activation of nuclear factor-kappa B induces expression of matrix metalloproteinase-9 after treatment with TNF-alpha , we should examine the possibility that the antioxidants pyrrolidine dithiocarbamate and N-acetyl-L-cysteine affected other transcription factors and signal transduction systems. In bacteria and eukaryotic cells, oxidative stress-responsive transcription factors unrelated to nuclear factor-kappa B are likely to exist (35). Their activation should also be suppressed by pyrrolidine dithiocarbamate and N-acetyl-L-cysteine. Although our data showed that the AP-1 inhibitor did not decrease matrix metalloproteinase-9 secretion by TNF-alpha , further studies will be required to explain the pathway of expression of matrix metalloproteinase-9 in detail after stimulation with TNF-alpha .

In conclusion, TNF-alpha increased the expression of matrix metalloproteinase-9 and induced an imbalance between matrix metalloproteinase-9 and TIMP-1 in human bronchial epithelial cells. The nuclear factor-kappa B-mediated pathway plays a role in matrix metalloproteinase-9 expression induced by TNF-alpha in human bronchial epithelial cells.


    ACKNOWLEDGEMENTS

We thank Dr. Karl Tryggvason (Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden) for providing the matrix metalloproteinase-9 cDNA probe. We also thank Dr. T. Ishida and Dr. T. Takahashi (First Department of Internal Medicine, Kobe University School of Medicine, Kobe, Japan) for teaching us the methods of investigation.


    FOOTNOTES

Address for reprint requests and other correspondence: Y. Nishimura, First Dept. of Internal Medicine, Kobe Univ. School of Medicine, 7-5-1 Kusunoki-cho, Chuo-Ku, Kobe 650-0017, Japan (E-mail: nishiy{at}med.kobe-u.ac.jp).

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.

Received 28 July 2000; accepted in final form 14 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Benbow, U, and Brinckerhoff CE. The AP-1 site and MMP gene regulation: what is all the fuss about? Matrix Biol 15: 519-526, 1997[ISI][Medline].

2.   Bond, M, Fabunmi RP, Baker AH, and Newby AC. Synergistic upregulation of metalloproteinase-9 by growth factors and inflammatory cytokines: an absolute requirement for transcription factor NF-kappa B. FEBS Lett 435: 29-34, 1998[ISI][Medline].

3.   Boone, TC, Johnson MJ, De Clerck YA, and Langley KE. cDNA cloning and expression of a metalloproteinase inhibitor related to tissue inhibitor of metalloproteinases. Proc Natl Acad Sci USA 87: 2800-2804, 1990[Abstract].

4.   Borish, L, Mascali JJ, Dishuck J, Beam WR, Martin RJ, and Rosenwasser LJ. Detection of alveolar macrophage-derived IL-1 beta in asthma. Inhibition with corticosteroids. J Immunol 149: 3078-3082, 1992[Abstract/Free Full Text].

5.   Bradford, MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976[ISI][Medline].

6.   Campbell, EJ, Cury JD, Lazarus CJ, and Welgus HG. Monocyte procollagenase and tissue inhibitor of metalloproteinases. Identification, characterization, and regulation of secretion. J Biol Chem 262: 15862-15868, 1987[Abstract/Free Full Text].

7.   Carmichael, DF, Sommer A, Thompson RC, Anderson DC, Smith CG, Welgus HG, and Stricklin GP. Primary structure and cDNA cloning of human fibroblast collagenase inhibitor. Proc Natl Acad Sci USA 83: 2407-2411, 1986[Abstract].

8.   Docherty, AJ, Lyons A, Smith BJ, Wright EM, Stephens PE, Harris TJ, Murphy G, and Reynolds JJ. Sequence of human tissue inhibitor of metalloproteinases and its identity to erythroid-potentiating activity. Nature 318: 66-69, 1985[ISI][Medline].

9.   Fiedler, MA, Wernke-Dollries K, and Stark JM. Effects of N-acetyl cysteine on RSV induced IL-8 gene expression and NF-kappa B activation in A549 cells (Abstract). Am J Respir Crit Care Med 153: A18, 1996.

10.   Fiedler, MA, Wernke-Dollries K, and Stark JM. Inhibition of TNF-alpha-induced NF-kappa B activation and IL-8 release in A549 cells with the proteasome inhibitor MG-132. Am J Respir Cell Mol Biol 19: 259-268, 1998[Abstract/Free Full Text].

11.   Fini, ME, Bartlett JD, Matsubara M, Rinehart WB, Mody MK, Girard MT, and Rainville M. The rabbit gene for 92-kDa matrix metalloproteinase. Role of AP1 and AP2 in cell type-specific transcription. J Biol Chem 269: 28620-28628, 1994[Abstract/Free Full Text].

12.   Greene, J, Wang M, Liu YE, Raymond LA, Rosen C, and Shi YE. Molecular cloning and characterization of human tissue inhibitor of metalloproteinase 4. J Biol Chem 271: 30375-30380, 1996[Abstract/Free Full Text].

13.   Hart, LA, Krishnan VL, Adcock IM, Barnes PJ, and Chung KF. Activation and localization of transcription factor, nuclear factor-kappa B, in asthma. Am J Respir Crit Care Med 158: 1585-1592, 1998[Abstract/Free Full Text].

14.   Hoshino, M, Nakamura Y, Sim JJ, Shimojo J, and Isogai S. Bronchial subepithelial fibrosis and expression of matrix metalloproteinase-9 in asthmatic airway inflammation. J Allergy Clin Immunol 102: 783-788, 1998[ISI][Medline].

15.   Kips, JC, Tavernier J, and Pauwels RA. Tumor necrosis factor causes bronchial hyperresponsiveness in rats. Am Rev Respir Dis 145: 332-336, 1992[ISI][Medline].

16.   Lallena, M, Diaz-Meco MT, Bren G, Paya CV, and Moscat J. Activation of Ikappa B kinase b by protein kinase C isoform. Mol Cell Biol 19: 2180-2188, 1999[Abstract/Free Full Text].

17.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[ISI][Medline].

18.   Leco, KJ, Khokha R, Pavloff N, Hawkes SP, and Edwards DR. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an extracellular matrix-associated protein with a distinctive pattern of expression in mouse cells and tissues. J Biol Chem 269: 9352-9360, 1994[Abstract/Free Full Text].

19.   Lemjabbar, H, Gosset P, Lamblin C, Tillie I, Hartmann D, Wallaert B, Tonnel AB, and Lafuma C. Contribution of 92 kDa gelatinase type IV collagenase in bronchial inflammation during status asthmaticus. Am J Respir Crit Care Med 159: 1298-1307, 1999[Abstract/Free Full Text].

20.   Maeda, A, and Sobel RA. Matrix metalloproteinases in the normal human central nervous system, microglial nodules, and multiple sclerosis lesions. J Neuropathol Exp Neurol 55: 300-309, 1996[ISI][Medline].

21.   Mastronarde, JG, Monick MM, Mukaida N, Matsushima K, and Hunninghake GW. AP-1 is the primary transcription factor which cooperatively interacts with NF-kB in RSV-induced IL-8 gene expression in airway epithelium (Abstract). Am J Respir Crit Care Med 155: A648, 1997.

22.   Matrisian, LM. Metalloproteinases and their inhibitors in matrix remodeling. Trends Genet 6: 121-125, 1990[ISI][Medline].

23.   Mautino, G, Oliver N, Chanez P, Bousquet J, and Capony F. Increased release of matrix metalloproteinase-9 in bronchoalveolar lavage fluid and by alveolar macrophages of asthmatics. Am J Respir Cell Mol Biol 17: 583-591, 1997[Abstract/Free Full Text].

24.   Mignatti, P, and Rifkin DB. Biology and biochemistry of proteinases in tumor invasion. Physiol Rev 73: 161-195, 1993[Free Full Text].

25.   Mignatti, P, and Rifkin DB. Plasminogen activators and matrix metalloproteinases in angiogenesis. Enzyme Protein 49: 117-137, 1996[ISI][Medline].

26.   Ohno, I, Ohtani H, Nitta Y, Suzuki J, Hoshi H, Honma M, Isoyama S, Tanno Y, Tamura G, Yamauchi K, Nagura H, and Shirato K. Eosinophils as a source of matrix metalloproteinase-9 in asthmatic airway inflammation. Am J Respir Cell Mol Biol 16: 212-219, 1997[Abstract].

27.   Okada, Y, Tsuchiya H, Shimizu H, Tomita K, Nakanishi I, Sato H, Seiki M, Yamashita K, and Hayakawa T. Induction and stimulation of 92-kDa gelatinase/type IV collagenase production in osteosarcoma and fibrosarcoma cell lines by tumor necrosis factor alpha. Biochem Biophys Res Commun 171: 610-617, 1990[ISI][Medline].

28.   Rahman, I, and MacNee W. Role of transcription factors in inflammatory lung diseases. Thorax 53: 601-612, 1998[Free Full Text].

29.   Sato, H, and Seiki M. Regulatory mechanism of 92 kDa type IV collagenase gene expression which is associated with invasiveness of tumor cells. Oncogene 8: 395-405, 1993[ISI][Medline].

30.   Sato, M, Miyazaki T, Nagaya T, Murata Y, Ida N, Maeda K, and Seo H. Antioxidants inhibit tumor necrosis factor-alpha mediated stimulation of interleukin-8, monocyte chemoattractant protein-1, and collagenase expression in cultured human synovial cells. J Rheumatol 23: 432-438, 1996[ISI][Medline].

31.   Schreck, R, Rieber P, and Baeuerle PA. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. EMBO J 10: 2247-2258, 1991[Abstract].

32.   Schreiber, E, Matthias P, Muller MM, and Schaffner W. Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells. Nucleic Acids Res 17: 6419, 1989[ISI][Medline].

33.   Shan, L, Nishimura Y, Kotani Y, and Yokoyama M. Platelet activating factor increases the expression of metalloproteinase-9 in human bronchial epithelial cells. Eur J Pharmacol 374: 147-156, 1999[ISI][Medline].

34.   Shibanuma, M, Kuroki T, and Nose K. Inhibition by N-acetyl-L-cysteine of interleukin-6 mRNA induction and activation of NF kappa B by tumor necrosis factor alpha in a mouse fibroblastic cell line, Balb/3T3. FEBS Lett 353: 62-66, 1994[ISI][Medline].

35.   Storz, G, Tartaglia LA, and Ames BN. Transcriptional regulator of oxidative stress-inducible genes: direct activation by oxidation. Science 248: 189-194, 1990[ISI][Medline].

36.   Stricklin, GP, and Welgus HG. Human skin fibroblast collagenase inhibitor. Purification and biochemical characterization. J Biol Chem 258: 12252-12258, 1983[Abstract/Free Full Text].

37.   Thorgeirsson, UP, Lindsay CK, Cottam DW, and Gomez DE. Tumor invasion, proteolysis, and angiogenesis. J Neurooncol 18: 89-103, 1993[ISI].

38.   Unemori, EN, Hibbs MS, and Amento EP. Constitutive expression of a 92-kD gelatinase (type V collagenase) by rheumatoid synovial fibroblasts and its induction in normal human fibroblasts by inflammatory cytokines. J Clin Invest 88: 1656-1662, 1991[ISI][Medline].

39.   Yao, PM, Buhler JM, d'Ortho MP, Lebargy F, Delclaux C, Harf A, and Lafuma C. Expression of matrix metalloproteinase gelatinases A and B by cultured epithelial cells from human bronchial explants. J Biol Chem 271: 15580-15589, 1996[Abstract/Free Full Text].

40.   Yao, PM, Maitre B, Delacourt C, Buhler JM, Harf A, and Lafuma C. Divergent regulation of 92-kDa gelatinase and TIMP-1 by HBECs in response to IL-1beta and TNF-alpha . Am J Physiol Lung Cell Mol Physiol 273: L866-L874, 1997[Abstract/Free Full Text].

41.   Yokoo, T, and Kitamura M. Dual regulation of IL-1beta -mediated matrix metalloproteinase-9 expression in mesangial cells by NF-kappa B and AP-1. Am J Physiol Renal Fluid Electrolyte Physiol 270: F123-F130, 1996[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 281(6):L1444-L1452
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society