Metalloproteinases Mediate Mucin 5AC Expression by Epidermal Growth Factor Receptor Activation
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美国呼吸和危急护理医学 2005年第2期
Departments of Environmental Health and Pulmonary and Critical Care Medicine, University of Cincinnati, Cincinnati, Ohio
Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina
Cardiovascular Research Institute and Departments of Medicine and Physiology, University of CaliforniaeCSan Francisco, San Francisco, California
ABSTRACT
Chronic obstructive pulmonary disease is marked by alveolar enlargement and excess production of airway mucus. Acrolein, a component of cigarette smoke, increases mucin 5AC (MUC5AC), a prevalent airway mucin in NCI-H292 cells by transcriptional activation, but the signal transduction pathways involved in acrolein-induced MUC5AC expression are unknown. Acrolein depleted cellular glutathione at doses of 10 e or greater, higher than those sufficient (0.03 e) to increase MUC5AC mRNA, suggesting that MUC5AC expression was independent of oxidative stress. In contrast, acrolein increased MUC5AC mRNA levels by phosphorylating epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase 3/2, or MAPK 3/2(ERK1/2). Pretreating the cells with an EGFR-neutralizing antibody, or a metalloproteinase inhibitor, decreased the acrolein-induced MUC5AC mRNA increase. Small, interfering RNA directed against ADAM17 or MMP9 inhibited the acrolein-induced MUC5AC mRNA increase. Acrolein increased the release and subsequent activation of pro-MMP9. Acrolein increased MMP9 and decreased tissue inhibitor of metalloproteinase 3 (TIMP3), an endogenous inhibitor of ADAM17, transcripts. Together, these data suggest that acrolein induces MUC5AC expression via an initial ligand-dependent activation of EGFR mediated by ADAM17 and MMP9. In addition, a prolonged effect of acrolein may be mediated by altering MMP9 and TIMP3 transcription.
Key Words: bronchitis emphysema human bronchial epithelial cells mucin
Chronic obstructive pulmonary disease affects more than 16 million people, and is the fourth leading cause of death in the United States (1). It is characterized by airflow obstruction, chronic bronchitis, and emphysema (2). Chronic obstructive pulmonary disease is marked by pathologic abnormalities in the submucosal glands and surface epithelium, which lead to excessive airway mucus production (3). Mucus is a viscoelastic gel that lines the respiratory tract epithelium and protects against infectious and environmental agents. Mucus consists of water (95%) combined with salts, lipids, and various proteins, including mucin glycoproteins (4, 5). Mucins are large, heterogeneous molecules (> 20,000 kD), consisting of a protein backbone (apomucin) to which multiple carbohydrate side chains are attached at serine and theronine residues (5). Mucins are encoded by at least 15 genes, including a cluster localized to human chromosome 11p15 (6), with at least nine genes expressed in the lungs (5). Mucin 5AC (MUC5AC) constitutes the majority of mucin glycoproteins in the airway secretions of humans (7) and is highly inducible (8eC10). Specialized epithelial (goblet) cells are the major source of MUC5AC in the airways.
Cigarette smoking is the most common cause of chronic obstructive pulmonary disease (3). In humans, chronic exposure to tobacco smoke results in an increase in the number of goblet cells because of hyperplasia and metaplasia (11). Acrolein (CH2 = CHCHO) is a potent irritant aldehyde present in tobacco smoke and is a constituent of wood smoke, diesel exhaust, and photochemical smog (12). Acrolein reacts rapidly with cellular nucleophiles (e.g., sulphydryl-containing cysteines and peptides) and depletes cellular thiols, including reduced glutathione (GSH), thereby inducing oxidative stress in primary airway epithelial cells (13). Acrolein increases matrix metalloproteinase 12eCdependent mucus metaplasia in mice (14) and MUC5AC mRNA in NCI-H292 cells (9). Increased MUC5AC expression can result from increased transcription of MUC5AC (8) and stabilization of mRNA (9). However, the mechanism by which acrolein increases MUC5AC expression remains unknown.
Multiple agents, including cigarette smoke (15), activated eosinophils (16), interleukin-13 (17), neutrophil elastase (18), Pseudomonas aeruginosa (19), and phorbol 12-myristate 13-acetate (PMA) (20) increase MUC5AC in airway epithelial cells (NCI-H292) by activating the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) cascade. Inhibition of EGFR activity decreases mucin production and reduces goblet cell metaplasia in response to various stimuli (21). Binding of the EGFR ligands to EGFR results in receptor dimerization and subsequent autophosphorylation of specific tyrosine residues in the cytoplasmic domains of the receptors (22). EGFR is also phosphorylated by treatment with ionizing radiation (23) in the absence of ligand binding to EGFR.
Endogenous EGFR ligands are synthesized as glycosylated membrane-bound precursors (24), which are cleaved by proteinases to release functional ligands (25). Pro-EGFR ligands are cleaved by matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase domain proteins (ADAMs) (26eC28). Cigarette smoke increases the expression and release of EGFR ligands, such as transforming growth factor , amphiregulin, and diphtheria toxin receptor (DTR), also known as heparin binding growth factor (HB-EGF), in airway epithelial cells (29). Cigarette smoke (30) and PMA (20) increased MUC5AC production in NCI-H292 cells via transforming growth factor-eCdependent EGFR activation mediated by ADAM17. In addition to ADAM17, other members of the ADAM and MMP protein subfamilies are involved in release of the EGFR ligands in response to treatment with various agents (31). MMP2 and MMP9 mediate EGFR activation in mouse pituitary gonadotrope cells (T3-1) (32). MMPs and ADAMs are regulated at the level of transcription (33) by activation of the precursor zymogens (34) and by action of endogenous inhibitors, tissue inhibitors of metalloproteinase proteins (TIMPs). An increase in the levels of TIMP3 and TIMP1 can inhibit the activity of ADAM17 (35) and MMP9 (36), respectively.
The present study was designed to determine the mechanism of MUC5AC expression by acrolein. First, we examined whether acrolein increases MUC5AC mRNA by depleting GSH (i.e., inducing oxidative stress). Second, we examined the role of EGFR/MAPK cascade in acrolein-induced MUC5AC mRNA increase. We determined the role of EGFR ligands in MUC5AC expression by acrolein. The role of ADAM17 or MMP9 was examined by using specific small, interfering RNA (siRNA) sequences and by measuring the MMP9 activity in conditioned medium after acrolein treatment. We also determined whether acrolein alters the expression of ADAM17 and MMP9 or their respective endogenous inhibitors, TIMP3 and TIMP1.
METHODS
Cell Culture and Acrolein Exposure
NCI-H292 cells (Cat. No. CRL-1848; American Type Culture Collection, Manassas, VA) were grown in 75-cm2 plastic tissue culture flasks (Cat. No. 3376; Corning, Corning, NY). NCI-H292 cells were maintained in RPMI 1640 medium (Cat. No. 30-2001; ATCC), supplemented with 10% fetal calf serum (Cat. No. 30-2020; ATCC), penicillin (100 U/ml), and streptomycin (100 e/ml; both from Sigma, St. Louis, MO; 37°C, pH 7.4). The cells were seeded at a density of 5,000 cells/cm2 and passaged at an approximately 90% confluence. For acrolein treatment, NCI-H292 cells were seeded (5,000 cells/cm2) into 30-mm six-well plates (Cat. No. 3506; Corning). Once confluent, the cells were incubated (37°C, pH 7.4) for 24 hours in serum-free medium (RPMI 1640). In a few studies, normal human bronchial epithelial (NHBE) cells (Cat. No. CC2540; Cambrex Biosciences, Baltimore, MD) were cultured in 75-cm2 plastic tissue culture flasks and maintained in bronchial epithelial cell growth medium (Cat. No. CC3170, Cambrex Biosciences). For acrolein treatment, NHBE cells were seeded (5,000 cells/cm2) into 30-mm six-well plates. Once confluent, the cells were incubated (37°C, pH 7.4) for 24 hours in bronchial epithelial cell basal medium.
The cells were treated with 0.03 e acrolein (Cat. No. 36520; Alfa Aesar, Ward Hill, MA), 25 ng/ml epidermal growth factor (EGF) (Cat. No. 9908; Cell Signaling Technologies, Beverly, MA) in phosphate-buffered saline (PBS; Cat. No. 14287eC080; Invitrogen, Carlsbad, CA) for 4 hours (37°C, pH 7.4). After exposure, the solution was removed, and the cells were washed with PBS and lysed by Trizol reagent (Cat. No. 15596eC026; Invitrogen). Total RNA was isolated by isopropanol/chloroform (Cat. No A416-4; Fischer, St. Louis, MO) precipitation and suspended in RNAase-free water.
Reverse Transcription and Polymerase Chain Reaction
Total RNA from each sample was reverse transcribed into cDNA using the following reaction mixture: 2.5 e total RNA from each sample in 10 e RNAse-free water, 1 e of oligo dT-15 (Cat. No. C1101; Promega, Madison, WI), 1 e of 10 mM deoxynucleotide triphosphate (Cat. No. 10297; Invitrogen). The reaction mixture was incubated (65°C, 5 minutes) and then chilled (4°C). First strand buffer (5x, 4 e), 2 e 0.1 M dithiothreitol (DTT), 1 e SuperScript II (Cat. No. 180640; Invitrogen), and 1 e RNase inhibitor (Cat. No. N2111; Promega) were then added to the reaction and further incubated (42°C, 1 hour). The reaction was terminated by heating the mixture (70°C, 5 minutes) and stored at 4°C. cDNA (2 e) was used in the subsequent polymerase chain reaction (PCR) using hot-start polymerase (Hotstar Taq, Cat. No. 203205; Qiagen, Valencia, CA) in a 50-e reaction mixture containing the following components: 5 e of PCR buffer (10x), 10 e of Q solution, 200 e of each deoxynucleotide triphosphate, 0.2 e of each primer, and 0.625 e of Hotstar Taq polymerase. Primers used for PCR were from Sigma-Genosys (Austin, TX). The sequence of primers used were as follows:
-actin: 3': GGG GTC TAC ATG GCA ACT GTG AGG AGG GGA, and 5': AAA CCT GCC AAA TAT GAT GAC ATC AAG AAG
MUC5AC: 3': TCA CAG CCG GGT ACG CGT TGG CAC AAG TGG, and 5': TGC TAT TAT GCC CTG TGT AGC CAG GAC TGC (37)
ADAM17: 3': AAT GAG AGC AAA GAA TCA AGC CCT GTC TC-3', and 5': AAG CTT GAT TCT TTG CTC TCA CCT GTC TC (38)
MMP9: 3': GGA GAC CTG AGA ACC AAT CTC, and 5': TCC AAT AGG TGA TGT TGT CGT (39)
TIMP1: 3': GGC CAT CGC CGC AGA TCC, and 5': GCT GGG TGG TAA CTC TTT ATT TCA
TIMP3: 3': CTG ACA GGT CGC GTC TAT GA, and 5': GGC GTA GTG TTT GGA CTG GT (40)
The PCR protocol used was as follows: (1) 15 minutes, 95°C (2); n cycles 30 seconds, 95°C (3); 30 seconds, 57°C (4); and 30 seconds, 72°C; after cycling, the sample was heated (10 minutes, 72°C) and cooled (4°C; total number of cycles: n = 30 for MUC5AC, n = 18 for -actin, n = 27 for TIMP1 and TIMP3, n = 30 for ADAM17, and n = 34 for MMP9).
Quantitation of PCR Products
PCR products were quantitated by densitometry. DNA (10 e) was electrophoresed on a 2% agarose gel containing 0.5 e/ml of ethidium bromide in Tris-acetateeC ethylenediaminetetraacetic acid buffer (Cat. No. BP1355; Fisher Biotech, Fair Lawn, NJ). After electrophoresis, DNA was by scanned by a Typhoon 8600 phosphor imager (Amersham Biosciences, Piscataway, NJ) and analyzed by an image quant software program (Amersham Biosciences). For each RT-PCR, a serial dilution (0.5eC0.032 e) of total mRNA from the NCI-H292 or NHBE cells was amplified and included on each gel to obtain an internally consistent reference curve. Each sample was analyzed in the linear portion of the curve. The relative amount of mRNA was determined by comparing the total intensity of each sample against the standard curve. Each sample was analyzed in duplicate, and MUC5AC, TIMP1, TIMP3, ADAM17, and MMP9 mRNA levels were expressed as fold increase or decrease according to control levels after each was normalized to -actin.
Acrolein Induces Oxidative Stress
NCI-H292 cells were exposed to increasing concentrations of acrolein (0.01eC100 e) for 4 hours (37°C, pH 7.4). After exposure, the reagents were removed, the cells were washed with PBS, and reduced GSH was measured as previously described (41). Briefly, the cells were lysed with ice-cold homogenization solution containing 154 mM KCl, 5 mM diethylenetriaminepentaacetic acid, and 0.1 M (K3[PO]4) buffer (pH 6.8) using a homogenizer (Tekmar, Cincinnati, OH) at maximum speed. An aliquot was removed for protein determination using the bicinchoninic acid method (Pierce, Rockford, IL). An equal volume of solution containing 40 mM HCl, 10 mM diethylenetriaminepentaacetic acid, 20 mM ascorbic acid, and 10% trichloroacetic acid was added to the homogenate. The suspension was centrifuged at 12,000 x g for 10 minutes. The supernatant solution was centrifuged through a 0.45-e micro-centrifuge filter (Cat. No. 78976; Millipore, Billerica, MA), and GSH was measured by fluorescent spectrophotometry using o-phthalaldehyde (Cat. No. ICN216717; Fischer).
Role of EGFR Activation in Acrolein-induced MUC5AC mRNA Increase
NCI-H292 cells were pretreated (1 hour, 37°C) with 0.25 e AG1478, an EGFR kinase inhibitor (Cat. No. 658552; Calbiochem, San Diego, CA). Cells were then treated with 0.03 e acrolein or EGF (25 ng/ml) in PBS. To measure MUC5AC mRNA level, cells were washed with ice-cold PBS after 4 hours and RNA was isolated. To determine EGFR activation, the cells were washed with ice-cold PBS after 1 hour and lysed with ice-cold radioimmunoprecipitation assay (RPA) lysis solution (Cat. No. 20eC188; Upstate, Waltham, MA) containing the following: 0.05 M Tris-HCl (pH 7.4), 0.15 M NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulphonylfluoride, 1 mM Na3VO4, 1 mM NaF, 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 1 mg/ml pepstatin. Cell lysates were centrifuged (12,000 x g, 5 minutes, 4°C) and precleared by incubating (1 hour, 4°C) with washed 60 e protein G agarose bead slurry (Cat. No.16eC266; Upstate). Protein concentration was determined by the bicinchoninic acid method, and cell lysates were diluted to 1 e/e. Cell lysate (500 e) was incubated (1 hour, 4°C) with 2 e of anti-EGFR antibody (LA-22; Cat. No. 05eC104; Upstate) and 60 e protein G agarose bead slurry. Agarose beads were collected by pulsing (12,000 x g, 5 seconds), and the supernatant was drained. The beads were washed three times with ice-cold cell lysis solution. The agarose beads were resuspended in 50 e 2x sodium dodecyl sulfate (SDS) sample buffer (Cat. No. LC2676; Invitrogen). The beads were boiled (5 minutes) and the supernatant was resolved by SDS polyacrylamide gel electrophoresis using 4 to 12% Tris-glycine gels (Cat. No. EC6028; Invitrogen). The protein was transferred electrophoretically to polyvinyldifluoride membrane (Cat. No. LC2005; Invitrogen), which was incubated with 5% fat-free skimmed milk in Tris-buffered saline (TBS) containing 0.05% Tween 20 (room temperature, 1 hour) (Cat. No. P7949, Sigma, St. Louis, MO) and incubated overnight at 4°C with 2 e/ml antiphosphotyrosine antibodies (Cat. No. 05eC321; Upstate). The membrane was washed twice with TBS containing 0.05% Tween 20 and then incubated (1 hour, room temperature) with 1:5000 rabbit anti-goat IgG horse radish peroxidaseeClinked secondary antibody (Cat. No.12eC349; Upstate). The membrane was washed twice with TBS and bound antibodies were visualized using an enhanced chemiluminescent kit (Cat. No. RPN 2108; Amersham Biosciences) according to the manufacturer's instructions. The membrane was stripped using a stripping solution containing 2% SDS, 16 mM Tris-HCl (pH 6.7) at 60°C for 1 hour. The membrane was incubated overnight at 4°C with 1:1000 anti-EGFR antibody (Cat. No. 05eC321; Upstate) using an enhanced chemiluminescent kit.
Role of MAPK Activation in Acrolein-induced MUC5AC mRNA Increase
NCI-H292 cells were pretreated (1 hour, 37°C) with 10 e PD98059, a MAP2K (MEK) inhibitor (Cat. No. 51300; Calbiochem). Cells were then treated with acrolein (0.03 e) or EGF (25 ng/ml) in PBS. To measure MUC5AC mRNA levels, the cells were washed with ice-cold PBS after 4 hours and RNA was isolated. To determine MAPK activation, the cells were washed with ice-cold PBS after 1 hour and lysed with ice-cold RPA lysis solution. Cell lysates were centrifuged, and protein concentration was determined by the bicinchoninic acid method. Cell lysates containing equal amounts of proteins were then mixed with 2x SDS sample buffer and boiled. The protein was resolved by SDS polyacrylamide gel electrophoresis and transferred electrophoretically to polyvinylidene fluoride membrane, which was incubated with 5% fat-free skimmed milk in TBS containing 0.05% Tween 20 (1 hour) (Cat No. P7949, Sigma) and incubated overnight at 4°C with 1:1,000 antieCphospho-MAPK3/2 (ERK1/2), antieCphospho-MAPK8 (JNK), or antieCphospho-MAPK14 (p38; Cat. No. 9910; Cell Signaling Technologies). The membrane was washed and then incubated at room temperature for 1hour with 1: 5,000 goat anti-rabbit IgG horse radish peroxidaseeClinked secondary antibody (Cat. No. 9211; Cell Signaling Technologies). Bound antibody was visualized using an enhanced chemiluminescent kit. The membrane was stripped and then incubated overnight at 4°C with 1:1,000 anti-MAPK3/2 (ERK1/2), anti-MAPK8 (JNK), or anti-MAPK14 (p38) antibodies (Cat. No. 9911; Cell Signaling Technologies) and visualized using an enhanced chemiluminescent kit.
Mechanism of Epithelial Growth Factor Receptor Activation by Acrolein
NCI-H292 cells were pretreated (1 hour, 37°C) with 10 e/ml of LA-1, a neutralizing antibody to the EGFR (Cat. No. 05eC101; Upstate) and treated (4 hours, 37°C) with 0.03 e acrolein or 25 ng/ml EGF in PBS. RNA was isolated and the level of MUC5AC mRNA was determined as before. To determine whether acrolein-induced MUC5AC mRNA increase involves a metalloproteinase, cells were pretreated (1 hour, 37°C) with 10 e GM 6001, a broad-spectrum metalloproteinase inhibitor (Cat. No. 364205; Calbiochem) and treated with 0.03 e acrolein or 25 ng/ml EGF in PBS. RNA was isolated and the level of MUC5AC mRNA was determined. To further determine the identity of metalloproteinase involved in acrolein-induced MUC5AC mRNA increase, cells were pretreated (1 hour, 37°C) with recombinant 2 e/ml TIMP3 (Cat. No. PF095; Calbiochem). Cells were treated (4 hours, 37°C) with 0.03 e acrolein or 25 ng/ml EGF in PBS. RNA was isolated and the level of MUC5AC mRNA was determined.
Role of ADAM17 and MMP9 in Acrolein-induced Mucin 5AC mRNA Increase
To determine the role of ADAM17 in acrolein-induced MUC5AC mRNA increase, 21-nt siRNA sequences of ADAM17; sense 5'-AAGCTTGATTCTTTGCTCTCA-3' antisense, 5'-AATGAGAGCAAAGAATCAAGC-3' (20) were used. The 21-nt siRNA was prepared in vitro by a Silencer siRNA construction kit (Cat. No. 1620; Ambion, Austin, TX) according to the manufacturer's instructions. To determine the role of MMP9 in acrolein-induced MUC5AC mRNA increase, the siRNA was synthesized chemically (Ambion) from sequences described previously (42). The sense siRNA sequence was 5'-CAUCACCUAUUGGAUCCAAdTdT-3'. The antisense siRNA was 5'- UUGGAUCCAAUAGGUGAUGdTdT-3'. The sequences were annealed according to the manufacturer's instructions. ADAM17 and MMP9 siRNA (0.03 e) were transfected into 40% confluent NCI-H292 cells using the Silencer siRNA transfection kit (Cat. No. 1630; Ambion) according to the manufacturer's instructions. As a control, cells were transfected with scrambled siRNA (Cat. No. 4800; Ambion). Gene silencing was confirmed 48 hours later by RT-PCR.
Gelatin Zymography
Cells were treated (4 hours, 37°C) with 0.03 e acrolein or 5 e H2O2 (Cat. No. 202460250; Arcos Organics) in PBS. The medium was collected, centrifuged (12,000 x g, 5 minutes, room temperature) to remove cell debris, and concentrated 20-fold using concentration devices (Cat. No. 42416; Millipore). In some experiments, after acrolein or H2O2 (4 hours, 37°C) treatment, the reagents were removed and fresh serum-free RPMI 1640 medium was added to the cells. The cells were incubated (37°C) for an additional 20 hours. The medium was collected, centrifuged (12,000 x g, 5 minutes, room temperature) to remove cell debris, and concentrated 20-fold using concentration devices. Protein concentration was determined by the bicinchoninic acid method. Samples containing 15 e of protein were mixed with 2x SDS sample buffer.
Protein was resolved by SDS polyacrylamide gel electrophoresis using 10% Tris- glycine gels containing 0.1% gelatin as a substrate (Cat. No. EC6175; Invitrogen). Gels were washed two times in zymogram renaturing solution (Cat. No. LC2670; Invitrogen; 30 minutes, room temperature). Gels were preincubated (30 minutes, 37°C) in zymogram developing solution (Cat. No. LC2671; Invitrogen) and subsequently incubated (18 hours, 37°C) in zymogram developing solution. Gels were stained in 0.5% Coomassie blue R-250 (Cat. No. 24567; Sigma) in 40% methanol, 10% acetic acid (1 hour, room temperature), and destained in 40% methanol, 10% acetic acid (1 hour, room temperature) with a rinse and two changes of destaining solution to visualize digested bands in the gelatin matrix. Gels were photographed using a digital camera.
Western Blotting for MMP9 Protein
Cells were treated (4 hours, 37°C) with 0.03 e acrolein or 5 e H2O2 in PBS. After 4 hours, the reagents were removed and fresh serum-free RPMI 1640 medium was added to the cells. The cells were incubated (37°C) for an additional 20 hours. The medium was collected, centrifuged (12,000 x g, 5 minutes, room temperature) to remove cell debris, and concentrated 20-fold using concentration devices. The protein concentration was determined by bicinchoninic acid method. Samples containing 15 e of protein were mixed with 2x SDS sample buffer containing 2.5% betamercaptoethanol (Cat. No. 516732; Sigma) and boiled (5 minutes). Protein was resolved by SDS polyacrylamide gel electrophoresis using 4 to 12% Tris-glycine gels and transferred electrophoretically to polyvinylidene fluoride membrane, which was incubated with 5% fat-free skimmed milk in TBS containing 0.05% Tween 20 (1 hour, room temperature) and incubated (overnight, 4°C) with 1:100 anti-MMP9 antibody (Cat. No. SA-106; Biomol International, Plymouth Meeting, PA). The membrane was washed twice with TBS containing 0.05% Tween 20 and then incubated (room temperature, 1hour) with 1:1,000 goat anti-rabbit IgG horse radish peroxidaseeClinked secondary antibody (Cat. No. A6154; Sigma). The membrane was washed twice with TBS. Bound antibody was visualized using an enhanced chemiluminescent kit.
Effect of Acrolein on ADAM17, MMP9, TIMP1, and TIMP3 mRNA Expression
Metalloproteinases are regulated principally at the levels of transcription and by the activity of their endogenous inhibitors, TIMPs. To determine the role of TIMPs in acrolein-induced MUC5AC mRNA increase, NCI-H292 or NHBE cells were treated (4 hours, 37°C) with 0.03 e acrolein or 25 ng/ml EGF in PBS. After exposure, the solution was removed, and the cells were washed with PBS and lysed by Trizol reagent. Total RNA was isolated and suspended in RNAase-free water. The level of ADAM17, MMP9, TIMP1, and TIMP3 transcript was measured by RT-PCR and quantitated as described before.
Statistical Analysis
For analysis of the results of mRNA measurements of MUC5AC, two-way analysis of variance for repeated measurements was used to determine statistically significant differences among group, followed by a Student-Newman-Keuls test for multiple comparisons. A probability of less than 0.05 was accepted as a statistically significant difference. All data are expressed as mean ± SEM.
RESULTS
MUC5AC mRNA Increase by Acrolein Is Independent of Oxidative Stress
Previous studies have indicated that acrolein depletes GSH at concentrations 3 e or greater and protein thiols at 10 e or greater in primary bronchial epithelial cells (13). In NCI-H292 cells, the threshold concentration of acrolein was 10 e or more, with 30 e significantly decreasing GSH levels (Figure 1). This was in excess of concentration of acrolein (0.03 e) that induced MUC5AC mRNA increase in NCI-H292 cells (Figure 2B), suggesting that acrolein-induced MUC5AC expression may be independent of oxidative stress.
MUC5AC mRNA Increase by Acrolein Involves Phosphorylation of EGFR and MAPK2/3
Acrolein at a concentration of 0.03 e increased the tyrosine phosphorylation of the EGFR in NCI-H292 cells (Figure 2A). The increase in tyrosine phosphorylation was reduced by pretreating the cells with AG1478, an EGFR tyrosine kinase inhibitor (Figure 2A). Pretreatment with AG1478 decreased the levels of MUC5AC mRNA induced by acrolein or EGF (Figures 2B and 2C). EGFR phosphorylation leads to activation of MAP2K (MEK), which in turn phosphorylates and activates MAPK. Acrolein (0.03 e) increased the phosphorylation of the MAPK3/2 (ERK1/2) and MAPK8 (JNK) but had no effect on MAPK14 (p38). Pretreating the cells with a MAP2K inhibitor, PD98059, decreased the MAPK3/2 phosphorylation induced by acrolein or EGF (Figure 3A). Pretreating the cells also decreased the levels of MUC5AC mRNA induced by acrolein or EGF (Figure 3B). Thus, acrolein-induced MUC5AC expression in NCI-H292 cells is mediated by EGFR phosphorylation and MAPK3/2 phosphorylation.
MUC5AC mRNA Increase by Acrolein Involves an Endogenous EGFR Ligand and Is Dependent on a TIMP3-sensitive Metalloproteinase
Pretreatment with a neutralizing antibody to EGFR (LA-1) decreased the acrolein or exogenous EGF-induced MUC5AC mRNA increase (Figure 4A). Pretreatment with a broad-spectrum metalloproteinase inhibitor (GM6001) decreased the acrolein-induced but not the exogenous EGF-induced MUC5AC mRNA increase (Figure 4B). Pretreatment with TIMP3, an inhibitor of ADAM17, partially inhibited the increase in acrolein-induced MUC5AC mRNA levels (Figure 4C). Thus, acrolein-induced MUC5AC expression in NCI-H292 cells involves an EGFR ligandeCdependent mechanism mediated, in part, by a TIMP3-sensitive metalloproteinase.
Acrolein-induced MUC5AC mRNA Increase Is Mediated by ADAM17 and MMP9
ADAM17 or MMP9 siRNAs inhibited the increase in acrolein-induced MUC5AC mRNA levels (Figure 5A). Cells cotransfected with ADAM17 and MMP9 siRNA showed an approximately 80% inhibition of acrolein-induced MUC5AC mRNA level (Figure 5B). The EGF-induced increase of MUC5AC mRNA levels was not inhibited (Figures 5A and 5B). Cells transfected with non-sense (scrambled) siRNA had MUC5AC mRNA levels comparable to the control and responded appropriately to acrolein (Figures 5A and 5B). Thus, acrolein-induced MUC5AC expression in NCI-H292 cells is mediated by ADAM17 and MMP9.
Acrolein Increases Release and Subsequent Activation of Pro-MMP9
Two gelatinases with a molecular weight of approximately 90 kD (pro-MMP9) and approximately 70 kD (pro-MMP2) increased in the conditioned medium after acrolein or H2O2 treatment (Figure 6A). An additional gelatinase with a molecular weight of approximately 82 kD, representing the active form of MMP9, was observed in acrolein- or H2O2-treated samples, which was absent in the control samples (Figure 6A). Extending the incubation period to 24 hours also increased MMP2 and MMP9 activity in the conditioned medium after acrolein or H2O2 treatment (Figure 6B). Western blots demonstrated protein levels of pro-MMP9 (92 kD) and MMP9 (82 kD) in the conditioned medium increased following acrolein or H2O2 treatment as compared with the PBS-treated (control) samples (Figure 6C).
Acrolein Increases MMP9 mRNA and Decreases TIMP3 mRNA in NCI-H292 and Normal Human Bronchial Epithelial Cells
When the acrolein treatment was extended to 24 hours, the MMP9 mRNA level in NCI-H292 cells increased (Figure 7). Moreover, MMP9 mRNA levels increased in NHBE cells after acrolein treatment for 24 hours. Acrolein also decreased TIMP3 mRNA in NHBE cells and NCI-H292 cells (Figure 7). The level of TIMP1 mRNA and ADAM17 mRNA remained unchanged (Figure 7).
DISCUSSION
Acrolein is a constituent of cigarette smoke, wood smoke, diesel exhaust, and cooking fumes (43). Acrolein levels are higher in secondhand as compared with mainstream cigarette smoke, because of lower combustion temperatures of smoldering cigarettes (44). Acrolein can penetrate the upper respiratory passages (45) and react with macromolecules as highly reactive zwitterions (+CH2CH = CHOeC) through electron rearrangement of an - unsaturated bond (46). Previously, Borchers and coworkers (47) reported that rats exposed to acrolein develop mucus metaplasia and that mucus hypersecretion in the airways was preceded by an increase in MUC5AC mRNA level (47). Acrolein increased MUC5AC mRNA in NCI-H292 cells at concentrations between 0.001 and 0.03 e (9).
Acrolein-induced MUC5AC expression is independent of oxidative stress. Acrolein reacts directly with protein and nonprotein sulfhydryl groups, and with primary and secondary amines found in proteins and nucleic acids (48). The conjugation of the acrolein with sulfhydryl groups is rapid and essentially irreversible (49), and leads to a decrease in cellular GSH stores. Acrolein treatment also decreased the availability of precursor amino acids used in GSH and protein synthesis in pulmonary endothelial cells (50). In NHBE cells, Grafstrom and colleagues (13) found that acrolein induced oxidative stress by depleting GSH. We found that acrolein decreased the level of GSH in a concentration-dependent manner in NCI-H292 cells. However, the threshold concentration of acrolein that decreased GSH was 10 to 30 e (Figure 1). In contrast, the threshold dose of acrolein sufficient to increase MUC5AC mRNA was 0.03 e or less, approximately a 300- to 1,000-fold lower concentration (Figure 2). Because oxidative stress occurred at concentrations of 10 e or greater, oxidative stress seems unlikely to be involved in MUC5AC expression by acrolein.
Acrolein increases MUC5AC expression by phosphorylating EGFR and by activating downstream MAPK signaling. Multiple agents, including cigarette smoke (15), interleukin-13 (16), and PMA (20), induce MUC5AC production in airway epithelial cells by phosphorylating EGFR. We found that the acrolein-induced MUC5AC mRNA increase was accompanied by and dependent on EGFR phosphorylation, because pretreating the cells with an EGFR kinase inhibitor, AG1478, inhibited this effect (Figures 2A and 2B). Previously, Takeuchi and coworkers (51) demonstrated that acrolein induced EGFR phosphorylation at a concentration of 50 e and, importantly, this concentration of acrolein led to apoptosis in keratinocytes. We found that acrolein depleted GSH and induced oxidative stress at concentrations greater than 10 e (Figure 1). Oxidative stress in the form of H2O2 phosphorylates EGFR but the pattern of tyrosine phosphorylation is different from that of ligand-induced EGFR phosphorylation (52). Importantly, this type of EGFR phosphorylation does not lead to phosphorylation (activation) of downstream MAPKs (53). We found that acrolein led to phosphorylation of MAPKs, including MAPK3/2 (ERK1/2) and MAPK8 (JNK; Figure 3A), and acrolein-induced MUC5AC mRNA increase is dependent on MAP2K phosphorylation, as indicated by PD98059 inhibition (Figure 3B). Thus, acrolein increases MUC5AC expression by phosphorylating EGFR and phosphorylating MAP2K, which in turn phosphorylates MAPK3/2 and MAPK8, independent of oxidative stress (Figure 8).
Acrolein-induced MUC5AC expression is dependent on EGFR ligand release mediated by metalloproteinase. EGFR ligands are synthesized as glycosylated membrane-bound precursors (24). Various EGFR ligands, such as transforming growth factor-, amphiregulin, and diphtheria toxin receptor, are expressed in NCI-H292 cells (29). Shao and coworkers showed that transforming growth factor- mediated MUC5AC increase by PMA (20) and cigarette smoke (30). After pretreating the NCI-H292 cells with an EGFR-neutralizing antibody (LA-1), we found that MUC5AC mRNA increase by acrolein depends on binding of an EGFR ligand to the receptor (Figure 4A). Endogenous EGFR ligands are released by proteolytic cleavage of membrane-bound proforms mediated by metalloproteinases such as MMPs and ADAMs (26, 27). By pretreating the NCI-H292 cells with a broad-spectrum metalloproteinase inhibitor (GM6001), we found that MUC5AC mRNA increase after acrolein treatment depends on a metalloproteinase (Figure 4B). The present study did not measure the release of EGFR ligands in the medium. However, Richter and coworkers (29) previously have demonstrated that EGFR ligands, including transforming growth factor-, amphiregulin, and diphtheria toxin receptor, are released from NCI-H292 cells into the cell culture medium on treatment with cigarette smoke.
Acrolein-induced MUC5AC expression involves ADAM17 and MMP9. We sought to determine the identity of metalloproteinases involved in acrolein-induced MUC5AC expression. Previously, Shao and colleagues found that ADAM17 mediated EGFR activation in NCI-H292 cells on treatment with PMA (20) or cigarette smoke (30). We found that pretreating the cells with TIMP3, an inhibitor of ADAM17, inhibited partially the acrolein-induced MUC5AC mRNA increase (Figure 4C). We used siRNA directed against ADAM17 in NCI-H292 cells and confirmed the role of ADAM17 in acrolein-induced MUC5AC expression (Figure 5). Because the inhibition of acrolein-induced MUC5AC mRNA increase by TIMP3 pretreatment or ADAM17 siRNA was partial, there was a possibility that more than one metalloproteinase may be involved in acrolein-induced MUC5AC mRNA increase. Various MMPs, including MMP2 and MMP9, are expressed in airway epithelial cells (53). In mouse pituitary gonadotrope (T3-1) cells, MMP2 and MMP9 can mediate EGFR transactivation (32). We used siRNA directed against MMP9 in NCI-H292 cells and found inhibition of the MUC5AC mRNA increase by acrolein (Figure 5A). Thus, in addition to ADAM17, MMP9 may also be involved in MUC5AC mRNA increase by acrolein (Figure 8). These results complement the previous observations by Shao and coworkers (20, 30) that ADAM17 is involved in MUC5AC mRNA increase by PMA or cigarette smoke. Moreover, cells cotransfected with siRNA against ADAM17 and MMP9 showed a greater inhibition of MUC5AC mRNA increase by acrolein compared with transfecting the cells with siRNA against either ADAM17 or MMP9 alone (Figure 5B).
Acrolein increases MMP9 protein and gelatinase activity in the cell culture medium. We observed two gelatinases, pro-MMP9 (92 kD) and pro-MMP2 (66 kD), in the cell culture medium after acrolein treatment (Figure 6A). MMP9 is synthesized as pro-MMP9 (92 kD), which is kept inactive by interaction between cysteine-sulphydryl groups in the propeptide domain and the zinc ion bound to the catalytic domain (34). Activation to MMP9 (82 kD) requires proteolytic removal of the prodomain (54) outside the cell by other proteinases. We found that control samples had only the proform (inactive) of MMP9, whereas the acrolein- or H2O2-treated samples showed the proform and the activated form of MMP9 (Figure 6A). We also found that protein level of pro-MMP9 in acrolein- or H2O2-treated samples was greater than the control samples (Figure 6C). Thus, acrolein, in addition to increasing the MMP9 protein and gelatinase activity in the cell culture medium, can also activate pro-MMP9. Extracellular activation of pro-MMP9 can be initiated by a proteinase cascade involving already activated MMPs (34), including MMP2 (55). We found that acrolein treatment increased the MMP2 activity in the cell culture medium (Figure 6A). MMP2 could activate pro-MMP9 in NCI-H292 cells after acrolein treatment; however, further studies are necessary to confirm the role of MMP2 in activating pro-MMP9 after acrolein treatment.
The mechanism by which MMP9 cleaves cell-surface pro-EGFR ligands is unknown. Surface-bound MMP9 may be responsible for processing pro-EGFR ligands. MMP9 binds with high affinity to various substrates, including hylaluronan receptor (CD-44) (56, 57), -2 chain of collagen IV (58), intracellular adhesion molecule (59), and docking proteins such as -integrin (60). In the airways, CD-44 is increased on bronchial epithelial cells in areas of damage (61). CD-44 coimmunoprecipitates with EGFR (62) and recruits active MMP7 and proeCDTR to form a complex on the cell surface (63). Further studies are necessary to determine the mechanism by which MMP9 can cleave pro-EGFR ligands.
With prolonged treatment, acrolein alters MMP9 and TIMP3 transcript levels. MMPs are tightly regulated at the transcriptional and post-transcriptional level and are also controlled at the protein level via their activators, inhibitors, and cell-surface localization (54). MMP9 is regulated at the level of transcription by several cytokines and growth factors, including EGF (34). The transcript level of MMP9 is elevated in interleukin-13eCinduced emphysema (64). We found that acrolein increased MMP9 mRNA (Figure 7) in NCI-H292 cells. Because metalloproteinases are often increased in tumor cell lines (65), we used NHBE cells to confirm increase in MMP9 mRNA after acrolein treatment (Figure 7). We also observed increased MMP9 gelatinolytic activity in NCI-H292 cells after 4 hours (Figure 6A) and 24 hours (Figure 6B). TIMP1 is an endogenous inhibitor of MMP9 (36). TIMP1 binds MMP9 in a 1:1 stoichoimetric fashion and keeps it inactive (66). We found that acrolein had no effect on TIMP1 mRNA in both NCI-H292 cells and NHBE cells (Figure 7). TIMP3, a natural inhibitor of ADAM17 (35), is also regulated at the transcriptional level (67). We found that acrolein decreased TIMP3 mRNA (Figure 7) in NCI-H292 and NHBE cells but had no effect on ADAM17 mRNA (Figure 7). Thus, in addition to rapid ADAM17- and MMP9-mediated ligand-dependent activation of EGFR, acrolein increases MMP9 activity and alters transcription of proteins critical to this pathway, which in turn may prolong the effect on MUC5AC production (Figure 8).
In summary, acrolein rapidly induces MUC5AC expression through an EGFR-MAPK pathway mediated by metalloproteinases ADAM17 and MMP9. In addition, acrolein can produce a prolonged increase in MUC5AC expression through an increase in MMP9 (transcript and activity) and a decrease in TIMP3 (transcript). Together, these interactions would be consistent with extended mucin production following exposure to this irritant, a component of cigarette smoke. Samples of lung tissues from patients with cigarette smokeeCrelated emphysema show an increase in MMP9 (68). Thus, in addition to playing an important role in alveolar enlargement and matrix degradation, MMP9 may also be involved in mucus hypersecretion.
Acknowledgments
The authors thank Jay Tichelaar for the critical review of the manuscript and Mary-Beth Genter for assistance with the MMP analyses.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina
Cardiovascular Research Institute and Departments of Medicine and Physiology, University of CaliforniaeCSan Francisco, San Francisco, California
ABSTRACT
Chronic obstructive pulmonary disease is marked by alveolar enlargement and excess production of airway mucus. Acrolein, a component of cigarette smoke, increases mucin 5AC (MUC5AC), a prevalent airway mucin in NCI-H292 cells by transcriptional activation, but the signal transduction pathways involved in acrolein-induced MUC5AC expression are unknown. Acrolein depleted cellular glutathione at doses of 10 e or greater, higher than those sufficient (0.03 e) to increase MUC5AC mRNA, suggesting that MUC5AC expression was independent of oxidative stress. In contrast, acrolein increased MUC5AC mRNA levels by phosphorylating epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase 3/2, or MAPK 3/2(ERK1/2). Pretreating the cells with an EGFR-neutralizing antibody, or a metalloproteinase inhibitor, decreased the acrolein-induced MUC5AC mRNA increase. Small, interfering RNA directed against ADAM17 or MMP9 inhibited the acrolein-induced MUC5AC mRNA increase. Acrolein increased the release and subsequent activation of pro-MMP9. Acrolein increased MMP9 and decreased tissue inhibitor of metalloproteinase 3 (TIMP3), an endogenous inhibitor of ADAM17, transcripts. Together, these data suggest that acrolein induces MUC5AC expression via an initial ligand-dependent activation of EGFR mediated by ADAM17 and MMP9. In addition, a prolonged effect of acrolein may be mediated by altering MMP9 and TIMP3 transcription.
Key Words: bronchitis emphysema human bronchial epithelial cells mucin
Chronic obstructive pulmonary disease affects more than 16 million people, and is the fourth leading cause of death in the United States (1). It is characterized by airflow obstruction, chronic bronchitis, and emphysema (2). Chronic obstructive pulmonary disease is marked by pathologic abnormalities in the submucosal glands and surface epithelium, which lead to excessive airway mucus production (3). Mucus is a viscoelastic gel that lines the respiratory tract epithelium and protects against infectious and environmental agents. Mucus consists of water (95%) combined with salts, lipids, and various proteins, including mucin glycoproteins (4, 5). Mucins are large, heterogeneous molecules (> 20,000 kD), consisting of a protein backbone (apomucin) to which multiple carbohydrate side chains are attached at serine and theronine residues (5). Mucins are encoded by at least 15 genes, including a cluster localized to human chromosome 11p15 (6), with at least nine genes expressed in the lungs (5). Mucin 5AC (MUC5AC) constitutes the majority of mucin glycoproteins in the airway secretions of humans (7) and is highly inducible (8eC10). Specialized epithelial (goblet) cells are the major source of MUC5AC in the airways.
Cigarette smoking is the most common cause of chronic obstructive pulmonary disease (3). In humans, chronic exposure to tobacco smoke results in an increase in the number of goblet cells because of hyperplasia and metaplasia (11). Acrolein (CH2 = CHCHO) is a potent irritant aldehyde present in tobacco smoke and is a constituent of wood smoke, diesel exhaust, and photochemical smog (12). Acrolein reacts rapidly with cellular nucleophiles (e.g., sulphydryl-containing cysteines and peptides) and depletes cellular thiols, including reduced glutathione (GSH), thereby inducing oxidative stress in primary airway epithelial cells (13). Acrolein increases matrix metalloproteinase 12eCdependent mucus metaplasia in mice (14) and MUC5AC mRNA in NCI-H292 cells (9). Increased MUC5AC expression can result from increased transcription of MUC5AC (8) and stabilization of mRNA (9). However, the mechanism by which acrolein increases MUC5AC expression remains unknown.
Multiple agents, including cigarette smoke (15), activated eosinophils (16), interleukin-13 (17), neutrophil elastase (18), Pseudomonas aeruginosa (19), and phorbol 12-myristate 13-acetate (PMA) (20) increase MUC5AC in airway epithelial cells (NCI-H292) by activating the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) cascade. Inhibition of EGFR activity decreases mucin production and reduces goblet cell metaplasia in response to various stimuli (21). Binding of the EGFR ligands to EGFR results in receptor dimerization and subsequent autophosphorylation of specific tyrosine residues in the cytoplasmic domains of the receptors (22). EGFR is also phosphorylated by treatment with ionizing radiation (23) in the absence of ligand binding to EGFR.
Endogenous EGFR ligands are synthesized as glycosylated membrane-bound precursors (24), which are cleaved by proteinases to release functional ligands (25). Pro-EGFR ligands are cleaved by matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase domain proteins (ADAMs) (26eC28). Cigarette smoke increases the expression and release of EGFR ligands, such as transforming growth factor , amphiregulin, and diphtheria toxin receptor (DTR), also known as heparin binding growth factor (HB-EGF), in airway epithelial cells (29). Cigarette smoke (30) and PMA (20) increased MUC5AC production in NCI-H292 cells via transforming growth factor-eCdependent EGFR activation mediated by ADAM17. In addition to ADAM17, other members of the ADAM and MMP protein subfamilies are involved in release of the EGFR ligands in response to treatment with various agents (31). MMP2 and MMP9 mediate EGFR activation in mouse pituitary gonadotrope cells (T3-1) (32). MMPs and ADAMs are regulated at the level of transcription (33) by activation of the precursor zymogens (34) and by action of endogenous inhibitors, tissue inhibitors of metalloproteinase proteins (TIMPs). An increase in the levels of TIMP3 and TIMP1 can inhibit the activity of ADAM17 (35) and MMP9 (36), respectively.
The present study was designed to determine the mechanism of MUC5AC expression by acrolein. First, we examined whether acrolein increases MUC5AC mRNA by depleting GSH (i.e., inducing oxidative stress). Second, we examined the role of EGFR/MAPK cascade in acrolein-induced MUC5AC mRNA increase. We determined the role of EGFR ligands in MUC5AC expression by acrolein. The role of ADAM17 or MMP9 was examined by using specific small, interfering RNA (siRNA) sequences and by measuring the MMP9 activity in conditioned medium after acrolein treatment. We also determined whether acrolein alters the expression of ADAM17 and MMP9 or their respective endogenous inhibitors, TIMP3 and TIMP1.
METHODS
Cell Culture and Acrolein Exposure
NCI-H292 cells (Cat. No. CRL-1848; American Type Culture Collection, Manassas, VA) were grown in 75-cm2 plastic tissue culture flasks (Cat. No. 3376; Corning, Corning, NY). NCI-H292 cells were maintained in RPMI 1640 medium (Cat. No. 30-2001; ATCC), supplemented with 10% fetal calf serum (Cat. No. 30-2020; ATCC), penicillin (100 U/ml), and streptomycin (100 e/ml; both from Sigma, St. Louis, MO; 37°C, pH 7.4). The cells were seeded at a density of 5,000 cells/cm2 and passaged at an approximately 90% confluence. For acrolein treatment, NCI-H292 cells were seeded (5,000 cells/cm2) into 30-mm six-well plates (Cat. No. 3506; Corning). Once confluent, the cells were incubated (37°C, pH 7.4) for 24 hours in serum-free medium (RPMI 1640). In a few studies, normal human bronchial epithelial (NHBE) cells (Cat. No. CC2540; Cambrex Biosciences, Baltimore, MD) were cultured in 75-cm2 plastic tissue culture flasks and maintained in bronchial epithelial cell growth medium (Cat. No. CC3170, Cambrex Biosciences). For acrolein treatment, NHBE cells were seeded (5,000 cells/cm2) into 30-mm six-well plates. Once confluent, the cells were incubated (37°C, pH 7.4) for 24 hours in bronchial epithelial cell basal medium.
The cells were treated with 0.03 e acrolein (Cat. No. 36520; Alfa Aesar, Ward Hill, MA), 25 ng/ml epidermal growth factor (EGF) (Cat. No. 9908; Cell Signaling Technologies, Beverly, MA) in phosphate-buffered saline (PBS; Cat. No. 14287eC080; Invitrogen, Carlsbad, CA) for 4 hours (37°C, pH 7.4). After exposure, the solution was removed, and the cells were washed with PBS and lysed by Trizol reagent (Cat. No. 15596eC026; Invitrogen). Total RNA was isolated by isopropanol/chloroform (Cat. No A416-4; Fischer, St. Louis, MO) precipitation and suspended in RNAase-free water.
Reverse Transcription and Polymerase Chain Reaction
Total RNA from each sample was reverse transcribed into cDNA using the following reaction mixture: 2.5 e total RNA from each sample in 10 e RNAse-free water, 1 e of oligo dT-15 (Cat. No. C1101; Promega, Madison, WI), 1 e of 10 mM deoxynucleotide triphosphate (Cat. No. 10297; Invitrogen). The reaction mixture was incubated (65°C, 5 minutes) and then chilled (4°C). First strand buffer (5x, 4 e), 2 e 0.1 M dithiothreitol (DTT), 1 e SuperScript II (Cat. No. 180640; Invitrogen), and 1 e RNase inhibitor (Cat. No. N2111; Promega) were then added to the reaction and further incubated (42°C, 1 hour). The reaction was terminated by heating the mixture (70°C, 5 minutes) and stored at 4°C. cDNA (2 e) was used in the subsequent polymerase chain reaction (PCR) using hot-start polymerase (Hotstar Taq, Cat. No. 203205; Qiagen, Valencia, CA) in a 50-e reaction mixture containing the following components: 5 e of PCR buffer (10x), 10 e of Q solution, 200 e of each deoxynucleotide triphosphate, 0.2 e of each primer, and 0.625 e of Hotstar Taq polymerase. Primers used for PCR were from Sigma-Genosys (Austin, TX). The sequence of primers used were as follows:
-actin: 3': GGG GTC TAC ATG GCA ACT GTG AGG AGG GGA, and 5': AAA CCT GCC AAA TAT GAT GAC ATC AAG AAG
MUC5AC: 3': TCA CAG CCG GGT ACG CGT TGG CAC AAG TGG, and 5': TGC TAT TAT GCC CTG TGT AGC CAG GAC TGC (37)
ADAM17: 3': AAT GAG AGC AAA GAA TCA AGC CCT GTC TC-3', and 5': AAG CTT GAT TCT TTG CTC TCA CCT GTC TC (38)
MMP9: 3': GGA GAC CTG AGA ACC AAT CTC, and 5': TCC AAT AGG TGA TGT TGT CGT (39)
TIMP1: 3': GGC CAT CGC CGC AGA TCC, and 5': GCT GGG TGG TAA CTC TTT ATT TCA
TIMP3: 3': CTG ACA GGT CGC GTC TAT GA, and 5': GGC GTA GTG TTT GGA CTG GT (40)
The PCR protocol used was as follows: (1) 15 minutes, 95°C (2); n cycles 30 seconds, 95°C (3); 30 seconds, 57°C (4); and 30 seconds, 72°C; after cycling, the sample was heated (10 minutes, 72°C) and cooled (4°C; total number of cycles: n = 30 for MUC5AC, n = 18 for -actin, n = 27 for TIMP1 and TIMP3, n = 30 for ADAM17, and n = 34 for MMP9).
Quantitation of PCR Products
PCR products were quantitated by densitometry. DNA (10 e) was electrophoresed on a 2% agarose gel containing 0.5 e/ml of ethidium bromide in Tris-acetateeC ethylenediaminetetraacetic acid buffer (Cat. No. BP1355; Fisher Biotech, Fair Lawn, NJ). After electrophoresis, DNA was by scanned by a Typhoon 8600 phosphor imager (Amersham Biosciences, Piscataway, NJ) and analyzed by an image quant software program (Amersham Biosciences). For each RT-PCR, a serial dilution (0.5eC0.032 e) of total mRNA from the NCI-H292 or NHBE cells was amplified and included on each gel to obtain an internally consistent reference curve. Each sample was analyzed in the linear portion of the curve. The relative amount of mRNA was determined by comparing the total intensity of each sample against the standard curve. Each sample was analyzed in duplicate, and MUC5AC, TIMP1, TIMP3, ADAM17, and MMP9 mRNA levels were expressed as fold increase or decrease according to control levels after each was normalized to -actin.
Acrolein Induces Oxidative Stress
NCI-H292 cells were exposed to increasing concentrations of acrolein (0.01eC100 e) for 4 hours (37°C, pH 7.4). After exposure, the reagents were removed, the cells were washed with PBS, and reduced GSH was measured as previously described (41). Briefly, the cells were lysed with ice-cold homogenization solution containing 154 mM KCl, 5 mM diethylenetriaminepentaacetic acid, and 0.1 M (K3[PO]4) buffer (pH 6.8) using a homogenizer (Tekmar, Cincinnati, OH) at maximum speed. An aliquot was removed for protein determination using the bicinchoninic acid method (Pierce, Rockford, IL). An equal volume of solution containing 40 mM HCl, 10 mM diethylenetriaminepentaacetic acid, 20 mM ascorbic acid, and 10% trichloroacetic acid was added to the homogenate. The suspension was centrifuged at 12,000 x g for 10 minutes. The supernatant solution was centrifuged through a 0.45-e micro-centrifuge filter (Cat. No. 78976; Millipore, Billerica, MA), and GSH was measured by fluorescent spectrophotometry using o-phthalaldehyde (Cat. No. ICN216717; Fischer).
Role of EGFR Activation in Acrolein-induced MUC5AC mRNA Increase
NCI-H292 cells were pretreated (1 hour, 37°C) with 0.25 e AG1478, an EGFR kinase inhibitor (Cat. No. 658552; Calbiochem, San Diego, CA). Cells were then treated with 0.03 e acrolein or EGF (25 ng/ml) in PBS. To measure MUC5AC mRNA level, cells were washed with ice-cold PBS after 4 hours and RNA was isolated. To determine EGFR activation, the cells were washed with ice-cold PBS after 1 hour and lysed with ice-cold radioimmunoprecipitation assay (RPA) lysis solution (Cat. No. 20eC188; Upstate, Waltham, MA) containing the following: 0.05 M Tris-HCl (pH 7.4), 0.15 M NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulphonylfluoride, 1 mM Na3VO4, 1 mM NaF, 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 1 mg/ml pepstatin. Cell lysates were centrifuged (12,000 x g, 5 minutes, 4°C) and precleared by incubating (1 hour, 4°C) with washed 60 e protein G agarose bead slurry (Cat. No.16eC266; Upstate). Protein concentration was determined by the bicinchoninic acid method, and cell lysates were diluted to 1 e/e. Cell lysate (500 e) was incubated (1 hour, 4°C) with 2 e of anti-EGFR antibody (LA-22; Cat. No. 05eC104; Upstate) and 60 e protein G agarose bead slurry. Agarose beads were collected by pulsing (12,000 x g, 5 seconds), and the supernatant was drained. The beads were washed three times with ice-cold cell lysis solution. The agarose beads were resuspended in 50 e 2x sodium dodecyl sulfate (SDS) sample buffer (Cat. No. LC2676; Invitrogen). The beads were boiled (5 minutes) and the supernatant was resolved by SDS polyacrylamide gel electrophoresis using 4 to 12% Tris-glycine gels (Cat. No. EC6028; Invitrogen). The protein was transferred electrophoretically to polyvinyldifluoride membrane (Cat. No. LC2005; Invitrogen), which was incubated with 5% fat-free skimmed milk in Tris-buffered saline (TBS) containing 0.05% Tween 20 (room temperature, 1 hour) (Cat. No. P7949, Sigma, St. Louis, MO) and incubated overnight at 4°C with 2 e/ml antiphosphotyrosine antibodies (Cat. No. 05eC321; Upstate). The membrane was washed twice with TBS containing 0.05% Tween 20 and then incubated (1 hour, room temperature) with 1:5000 rabbit anti-goat IgG horse radish peroxidaseeClinked secondary antibody (Cat. No.12eC349; Upstate). The membrane was washed twice with TBS and bound antibodies were visualized using an enhanced chemiluminescent kit (Cat. No. RPN 2108; Amersham Biosciences) according to the manufacturer's instructions. The membrane was stripped using a stripping solution containing 2% SDS, 16 mM Tris-HCl (pH 6.7) at 60°C for 1 hour. The membrane was incubated overnight at 4°C with 1:1000 anti-EGFR antibody (Cat. No. 05eC321; Upstate) using an enhanced chemiluminescent kit.
Role of MAPK Activation in Acrolein-induced MUC5AC mRNA Increase
NCI-H292 cells were pretreated (1 hour, 37°C) with 10 e PD98059, a MAP2K (MEK) inhibitor (Cat. No. 51300; Calbiochem). Cells were then treated with acrolein (0.03 e) or EGF (25 ng/ml) in PBS. To measure MUC5AC mRNA levels, the cells were washed with ice-cold PBS after 4 hours and RNA was isolated. To determine MAPK activation, the cells were washed with ice-cold PBS after 1 hour and lysed with ice-cold RPA lysis solution. Cell lysates were centrifuged, and protein concentration was determined by the bicinchoninic acid method. Cell lysates containing equal amounts of proteins were then mixed with 2x SDS sample buffer and boiled. The protein was resolved by SDS polyacrylamide gel electrophoresis and transferred electrophoretically to polyvinylidene fluoride membrane, which was incubated with 5% fat-free skimmed milk in TBS containing 0.05% Tween 20 (1 hour) (Cat No. P7949, Sigma) and incubated overnight at 4°C with 1:1,000 antieCphospho-MAPK3/2 (ERK1/2), antieCphospho-MAPK8 (JNK), or antieCphospho-MAPK14 (p38; Cat. No. 9910; Cell Signaling Technologies). The membrane was washed and then incubated at room temperature for 1hour with 1: 5,000 goat anti-rabbit IgG horse radish peroxidaseeClinked secondary antibody (Cat. No. 9211; Cell Signaling Technologies). Bound antibody was visualized using an enhanced chemiluminescent kit. The membrane was stripped and then incubated overnight at 4°C with 1:1,000 anti-MAPK3/2 (ERK1/2), anti-MAPK8 (JNK), or anti-MAPK14 (p38) antibodies (Cat. No. 9911; Cell Signaling Technologies) and visualized using an enhanced chemiluminescent kit.
Mechanism of Epithelial Growth Factor Receptor Activation by Acrolein
NCI-H292 cells were pretreated (1 hour, 37°C) with 10 e/ml of LA-1, a neutralizing antibody to the EGFR (Cat. No. 05eC101; Upstate) and treated (4 hours, 37°C) with 0.03 e acrolein or 25 ng/ml EGF in PBS. RNA was isolated and the level of MUC5AC mRNA was determined as before. To determine whether acrolein-induced MUC5AC mRNA increase involves a metalloproteinase, cells were pretreated (1 hour, 37°C) with 10 e GM 6001, a broad-spectrum metalloproteinase inhibitor (Cat. No. 364205; Calbiochem) and treated with 0.03 e acrolein or 25 ng/ml EGF in PBS. RNA was isolated and the level of MUC5AC mRNA was determined. To further determine the identity of metalloproteinase involved in acrolein-induced MUC5AC mRNA increase, cells were pretreated (1 hour, 37°C) with recombinant 2 e/ml TIMP3 (Cat. No. PF095; Calbiochem). Cells were treated (4 hours, 37°C) with 0.03 e acrolein or 25 ng/ml EGF in PBS. RNA was isolated and the level of MUC5AC mRNA was determined.
Role of ADAM17 and MMP9 in Acrolein-induced Mucin 5AC mRNA Increase
To determine the role of ADAM17 in acrolein-induced MUC5AC mRNA increase, 21-nt siRNA sequences of ADAM17; sense 5'-AAGCTTGATTCTTTGCTCTCA-3' antisense, 5'-AATGAGAGCAAAGAATCAAGC-3' (20) were used. The 21-nt siRNA was prepared in vitro by a Silencer siRNA construction kit (Cat. No. 1620; Ambion, Austin, TX) according to the manufacturer's instructions. To determine the role of MMP9 in acrolein-induced MUC5AC mRNA increase, the siRNA was synthesized chemically (Ambion) from sequences described previously (42). The sense siRNA sequence was 5'-CAUCACCUAUUGGAUCCAAdTdT-3'. The antisense siRNA was 5'- UUGGAUCCAAUAGGUGAUGdTdT-3'. The sequences were annealed according to the manufacturer's instructions. ADAM17 and MMP9 siRNA (0.03 e) were transfected into 40% confluent NCI-H292 cells using the Silencer siRNA transfection kit (Cat. No. 1630; Ambion) according to the manufacturer's instructions. As a control, cells were transfected with scrambled siRNA (Cat. No. 4800; Ambion). Gene silencing was confirmed 48 hours later by RT-PCR.
Gelatin Zymography
Cells were treated (4 hours, 37°C) with 0.03 e acrolein or 5 e H2O2 (Cat. No. 202460250; Arcos Organics) in PBS. The medium was collected, centrifuged (12,000 x g, 5 minutes, room temperature) to remove cell debris, and concentrated 20-fold using concentration devices (Cat. No. 42416; Millipore). In some experiments, after acrolein or H2O2 (4 hours, 37°C) treatment, the reagents were removed and fresh serum-free RPMI 1640 medium was added to the cells. The cells were incubated (37°C) for an additional 20 hours. The medium was collected, centrifuged (12,000 x g, 5 minutes, room temperature) to remove cell debris, and concentrated 20-fold using concentration devices. Protein concentration was determined by the bicinchoninic acid method. Samples containing 15 e of protein were mixed with 2x SDS sample buffer.
Protein was resolved by SDS polyacrylamide gel electrophoresis using 10% Tris- glycine gels containing 0.1% gelatin as a substrate (Cat. No. EC6175; Invitrogen). Gels were washed two times in zymogram renaturing solution (Cat. No. LC2670; Invitrogen; 30 minutes, room temperature). Gels were preincubated (30 minutes, 37°C) in zymogram developing solution (Cat. No. LC2671; Invitrogen) and subsequently incubated (18 hours, 37°C) in zymogram developing solution. Gels were stained in 0.5% Coomassie blue R-250 (Cat. No. 24567; Sigma) in 40% methanol, 10% acetic acid (1 hour, room temperature), and destained in 40% methanol, 10% acetic acid (1 hour, room temperature) with a rinse and two changes of destaining solution to visualize digested bands in the gelatin matrix. Gels were photographed using a digital camera.
Western Blotting for MMP9 Protein
Cells were treated (4 hours, 37°C) with 0.03 e acrolein or 5 e H2O2 in PBS. After 4 hours, the reagents were removed and fresh serum-free RPMI 1640 medium was added to the cells. The cells were incubated (37°C) for an additional 20 hours. The medium was collected, centrifuged (12,000 x g, 5 minutes, room temperature) to remove cell debris, and concentrated 20-fold using concentration devices. The protein concentration was determined by bicinchoninic acid method. Samples containing 15 e of protein were mixed with 2x SDS sample buffer containing 2.5% betamercaptoethanol (Cat. No. 516732; Sigma) and boiled (5 minutes). Protein was resolved by SDS polyacrylamide gel electrophoresis using 4 to 12% Tris-glycine gels and transferred electrophoretically to polyvinylidene fluoride membrane, which was incubated with 5% fat-free skimmed milk in TBS containing 0.05% Tween 20 (1 hour, room temperature) and incubated (overnight, 4°C) with 1:100 anti-MMP9 antibody (Cat. No. SA-106; Biomol International, Plymouth Meeting, PA). The membrane was washed twice with TBS containing 0.05% Tween 20 and then incubated (room temperature, 1hour) with 1:1,000 goat anti-rabbit IgG horse radish peroxidaseeClinked secondary antibody (Cat. No. A6154; Sigma). The membrane was washed twice with TBS. Bound antibody was visualized using an enhanced chemiluminescent kit.
Effect of Acrolein on ADAM17, MMP9, TIMP1, and TIMP3 mRNA Expression
Metalloproteinases are regulated principally at the levels of transcription and by the activity of their endogenous inhibitors, TIMPs. To determine the role of TIMPs in acrolein-induced MUC5AC mRNA increase, NCI-H292 or NHBE cells were treated (4 hours, 37°C) with 0.03 e acrolein or 25 ng/ml EGF in PBS. After exposure, the solution was removed, and the cells were washed with PBS and lysed by Trizol reagent. Total RNA was isolated and suspended in RNAase-free water. The level of ADAM17, MMP9, TIMP1, and TIMP3 transcript was measured by RT-PCR and quantitated as described before.
Statistical Analysis
For analysis of the results of mRNA measurements of MUC5AC, two-way analysis of variance for repeated measurements was used to determine statistically significant differences among group, followed by a Student-Newman-Keuls test for multiple comparisons. A probability of less than 0.05 was accepted as a statistically significant difference. All data are expressed as mean ± SEM.
RESULTS
MUC5AC mRNA Increase by Acrolein Is Independent of Oxidative Stress
Previous studies have indicated that acrolein depletes GSH at concentrations 3 e or greater and protein thiols at 10 e or greater in primary bronchial epithelial cells (13). In NCI-H292 cells, the threshold concentration of acrolein was 10 e or more, with 30 e significantly decreasing GSH levels (Figure 1). This was in excess of concentration of acrolein (0.03 e) that induced MUC5AC mRNA increase in NCI-H292 cells (Figure 2B), suggesting that acrolein-induced MUC5AC expression may be independent of oxidative stress.
MUC5AC mRNA Increase by Acrolein Involves Phosphorylation of EGFR and MAPK2/3
Acrolein at a concentration of 0.03 e increased the tyrosine phosphorylation of the EGFR in NCI-H292 cells (Figure 2A). The increase in tyrosine phosphorylation was reduced by pretreating the cells with AG1478, an EGFR tyrosine kinase inhibitor (Figure 2A). Pretreatment with AG1478 decreased the levels of MUC5AC mRNA induced by acrolein or EGF (Figures 2B and 2C). EGFR phosphorylation leads to activation of MAP2K (MEK), which in turn phosphorylates and activates MAPK. Acrolein (0.03 e) increased the phosphorylation of the MAPK3/2 (ERK1/2) and MAPK8 (JNK) but had no effect on MAPK14 (p38). Pretreating the cells with a MAP2K inhibitor, PD98059, decreased the MAPK3/2 phosphorylation induced by acrolein or EGF (Figure 3A). Pretreating the cells also decreased the levels of MUC5AC mRNA induced by acrolein or EGF (Figure 3B). Thus, acrolein-induced MUC5AC expression in NCI-H292 cells is mediated by EGFR phosphorylation and MAPK3/2 phosphorylation.
MUC5AC mRNA Increase by Acrolein Involves an Endogenous EGFR Ligand and Is Dependent on a TIMP3-sensitive Metalloproteinase
Pretreatment with a neutralizing antibody to EGFR (LA-1) decreased the acrolein or exogenous EGF-induced MUC5AC mRNA increase (Figure 4A). Pretreatment with a broad-spectrum metalloproteinase inhibitor (GM6001) decreased the acrolein-induced but not the exogenous EGF-induced MUC5AC mRNA increase (Figure 4B). Pretreatment with TIMP3, an inhibitor of ADAM17, partially inhibited the increase in acrolein-induced MUC5AC mRNA levels (Figure 4C). Thus, acrolein-induced MUC5AC expression in NCI-H292 cells involves an EGFR ligandeCdependent mechanism mediated, in part, by a TIMP3-sensitive metalloproteinase.
Acrolein-induced MUC5AC mRNA Increase Is Mediated by ADAM17 and MMP9
ADAM17 or MMP9 siRNAs inhibited the increase in acrolein-induced MUC5AC mRNA levels (Figure 5A). Cells cotransfected with ADAM17 and MMP9 siRNA showed an approximately 80% inhibition of acrolein-induced MUC5AC mRNA level (Figure 5B). The EGF-induced increase of MUC5AC mRNA levels was not inhibited (Figures 5A and 5B). Cells transfected with non-sense (scrambled) siRNA had MUC5AC mRNA levels comparable to the control and responded appropriately to acrolein (Figures 5A and 5B). Thus, acrolein-induced MUC5AC expression in NCI-H292 cells is mediated by ADAM17 and MMP9.
Acrolein Increases Release and Subsequent Activation of Pro-MMP9
Two gelatinases with a molecular weight of approximately 90 kD (pro-MMP9) and approximately 70 kD (pro-MMP2) increased in the conditioned medium after acrolein or H2O2 treatment (Figure 6A). An additional gelatinase with a molecular weight of approximately 82 kD, representing the active form of MMP9, was observed in acrolein- or H2O2-treated samples, which was absent in the control samples (Figure 6A). Extending the incubation period to 24 hours also increased MMP2 and MMP9 activity in the conditioned medium after acrolein or H2O2 treatment (Figure 6B). Western blots demonstrated protein levels of pro-MMP9 (92 kD) and MMP9 (82 kD) in the conditioned medium increased following acrolein or H2O2 treatment as compared with the PBS-treated (control) samples (Figure 6C).
Acrolein Increases MMP9 mRNA and Decreases TIMP3 mRNA in NCI-H292 and Normal Human Bronchial Epithelial Cells
When the acrolein treatment was extended to 24 hours, the MMP9 mRNA level in NCI-H292 cells increased (Figure 7). Moreover, MMP9 mRNA levels increased in NHBE cells after acrolein treatment for 24 hours. Acrolein also decreased TIMP3 mRNA in NHBE cells and NCI-H292 cells (Figure 7). The level of TIMP1 mRNA and ADAM17 mRNA remained unchanged (Figure 7).
DISCUSSION
Acrolein is a constituent of cigarette smoke, wood smoke, diesel exhaust, and cooking fumes (43). Acrolein levels are higher in secondhand as compared with mainstream cigarette smoke, because of lower combustion temperatures of smoldering cigarettes (44). Acrolein can penetrate the upper respiratory passages (45) and react with macromolecules as highly reactive zwitterions (+CH2CH = CHOeC) through electron rearrangement of an - unsaturated bond (46). Previously, Borchers and coworkers (47) reported that rats exposed to acrolein develop mucus metaplasia and that mucus hypersecretion in the airways was preceded by an increase in MUC5AC mRNA level (47). Acrolein increased MUC5AC mRNA in NCI-H292 cells at concentrations between 0.001 and 0.03 e (9).
Acrolein-induced MUC5AC expression is independent of oxidative stress. Acrolein reacts directly with protein and nonprotein sulfhydryl groups, and with primary and secondary amines found in proteins and nucleic acids (48). The conjugation of the acrolein with sulfhydryl groups is rapid and essentially irreversible (49), and leads to a decrease in cellular GSH stores. Acrolein treatment also decreased the availability of precursor amino acids used in GSH and protein synthesis in pulmonary endothelial cells (50). In NHBE cells, Grafstrom and colleagues (13) found that acrolein induced oxidative stress by depleting GSH. We found that acrolein decreased the level of GSH in a concentration-dependent manner in NCI-H292 cells. However, the threshold concentration of acrolein that decreased GSH was 10 to 30 e (Figure 1). In contrast, the threshold dose of acrolein sufficient to increase MUC5AC mRNA was 0.03 e or less, approximately a 300- to 1,000-fold lower concentration (Figure 2). Because oxidative stress occurred at concentrations of 10 e or greater, oxidative stress seems unlikely to be involved in MUC5AC expression by acrolein.
Acrolein increases MUC5AC expression by phosphorylating EGFR and by activating downstream MAPK signaling. Multiple agents, including cigarette smoke (15), interleukin-13 (16), and PMA (20), induce MUC5AC production in airway epithelial cells by phosphorylating EGFR. We found that the acrolein-induced MUC5AC mRNA increase was accompanied by and dependent on EGFR phosphorylation, because pretreating the cells with an EGFR kinase inhibitor, AG1478, inhibited this effect (Figures 2A and 2B). Previously, Takeuchi and coworkers (51) demonstrated that acrolein induced EGFR phosphorylation at a concentration of 50 e and, importantly, this concentration of acrolein led to apoptosis in keratinocytes. We found that acrolein depleted GSH and induced oxidative stress at concentrations greater than 10 e (Figure 1). Oxidative stress in the form of H2O2 phosphorylates EGFR but the pattern of tyrosine phosphorylation is different from that of ligand-induced EGFR phosphorylation (52). Importantly, this type of EGFR phosphorylation does not lead to phosphorylation (activation) of downstream MAPKs (53). We found that acrolein led to phosphorylation of MAPKs, including MAPK3/2 (ERK1/2) and MAPK8 (JNK; Figure 3A), and acrolein-induced MUC5AC mRNA increase is dependent on MAP2K phosphorylation, as indicated by PD98059 inhibition (Figure 3B). Thus, acrolein increases MUC5AC expression by phosphorylating EGFR and phosphorylating MAP2K, which in turn phosphorylates MAPK3/2 and MAPK8, independent of oxidative stress (Figure 8).
Acrolein-induced MUC5AC expression is dependent on EGFR ligand release mediated by metalloproteinase. EGFR ligands are synthesized as glycosylated membrane-bound precursors (24). Various EGFR ligands, such as transforming growth factor-, amphiregulin, and diphtheria toxin receptor, are expressed in NCI-H292 cells (29). Shao and coworkers showed that transforming growth factor- mediated MUC5AC increase by PMA (20) and cigarette smoke (30). After pretreating the NCI-H292 cells with an EGFR-neutralizing antibody (LA-1), we found that MUC5AC mRNA increase by acrolein depends on binding of an EGFR ligand to the receptor (Figure 4A). Endogenous EGFR ligands are released by proteolytic cleavage of membrane-bound proforms mediated by metalloproteinases such as MMPs and ADAMs (26, 27). By pretreating the NCI-H292 cells with a broad-spectrum metalloproteinase inhibitor (GM6001), we found that MUC5AC mRNA increase after acrolein treatment depends on a metalloproteinase (Figure 4B). The present study did not measure the release of EGFR ligands in the medium. However, Richter and coworkers (29) previously have demonstrated that EGFR ligands, including transforming growth factor-, amphiregulin, and diphtheria toxin receptor, are released from NCI-H292 cells into the cell culture medium on treatment with cigarette smoke.
Acrolein-induced MUC5AC expression involves ADAM17 and MMP9. We sought to determine the identity of metalloproteinases involved in acrolein-induced MUC5AC expression. Previously, Shao and colleagues found that ADAM17 mediated EGFR activation in NCI-H292 cells on treatment with PMA (20) or cigarette smoke (30). We found that pretreating the cells with TIMP3, an inhibitor of ADAM17, inhibited partially the acrolein-induced MUC5AC mRNA increase (Figure 4C). We used siRNA directed against ADAM17 in NCI-H292 cells and confirmed the role of ADAM17 in acrolein-induced MUC5AC expression (Figure 5). Because the inhibition of acrolein-induced MUC5AC mRNA increase by TIMP3 pretreatment or ADAM17 siRNA was partial, there was a possibility that more than one metalloproteinase may be involved in acrolein-induced MUC5AC mRNA increase. Various MMPs, including MMP2 and MMP9, are expressed in airway epithelial cells (53). In mouse pituitary gonadotrope (T3-1) cells, MMP2 and MMP9 can mediate EGFR transactivation (32). We used siRNA directed against MMP9 in NCI-H292 cells and found inhibition of the MUC5AC mRNA increase by acrolein (Figure 5A). Thus, in addition to ADAM17, MMP9 may also be involved in MUC5AC mRNA increase by acrolein (Figure 8). These results complement the previous observations by Shao and coworkers (20, 30) that ADAM17 is involved in MUC5AC mRNA increase by PMA or cigarette smoke. Moreover, cells cotransfected with siRNA against ADAM17 and MMP9 showed a greater inhibition of MUC5AC mRNA increase by acrolein compared with transfecting the cells with siRNA against either ADAM17 or MMP9 alone (Figure 5B).
Acrolein increases MMP9 protein and gelatinase activity in the cell culture medium. We observed two gelatinases, pro-MMP9 (92 kD) and pro-MMP2 (66 kD), in the cell culture medium after acrolein treatment (Figure 6A). MMP9 is synthesized as pro-MMP9 (92 kD), which is kept inactive by interaction between cysteine-sulphydryl groups in the propeptide domain and the zinc ion bound to the catalytic domain (34). Activation to MMP9 (82 kD) requires proteolytic removal of the prodomain (54) outside the cell by other proteinases. We found that control samples had only the proform (inactive) of MMP9, whereas the acrolein- or H2O2-treated samples showed the proform and the activated form of MMP9 (Figure 6A). We also found that protein level of pro-MMP9 in acrolein- or H2O2-treated samples was greater than the control samples (Figure 6C). Thus, acrolein, in addition to increasing the MMP9 protein and gelatinase activity in the cell culture medium, can also activate pro-MMP9. Extracellular activation of pro-MMP9 can be initiated by a proteinase cascade involving already activated MMPs (34), including MMP2 (55). We found that acrolein treatment increased the MMP2 activity in the cell culture medium (Figure 6A). MMP2 could activate pro-MMP9 in NCI-H292 cells after acrolein treatment; however, further studies are necessary to confirm the role of MMP2 in activating pro-MMP9 after acrolein treatment.
The mechanism by which MMP9 cleaves cell-surface pro-EGFR ligands is unknown. Surface-bound MMP9 may be responsible for processing pro-EGFR ligands. MMP9 binds with high affinity to various substrates, including hylaluronan receptor (CD-44) (56, 57), -2 chain of collagen IV (58), intracellular adhesion molecule (59), and docking proteins such as -integrin (60). In the airways, CD-44 is increased on bronchial epithelial cells in areas of damage (61). CD-44 coimmunoprecipitates with EGFR (62) and recruits active MMP7 and proeCDTR to form a complex on the cell surface (63). Further studies are necessary to determine the mechanism by which MMP9 can cleave pro-EGFR ligands.
With prolonged treatment, acrolein alters MMP9 and TIMP3 transcript levels. MMPs are tightly regulated at the transcriptional and post-transcriptional level and are also controlled at the protein level via their activators, inhibitors, and cell-surface localization (54). MMP9 is regulated at the level of transcription by several cytokines and growth factors, including EGF (34). The transcript level of MMP9 is elevated in interleukin-13eCinduced emphysema (64). We found that acrolein increased MMP9 mRNA (Figure 7) in NCI-H292 cells. Because metalloproteinases are often increased in tumor cell lines (65), we used NHBE cells to confirm increase in MMP9 mRNA after acrolein treatment (Figure 7). We also observed increased MMP9 gelatinolytic activity in NCI-H292 cells after 4 hours (Figure 6A) and 24 hours (Figure 6B). TIMP1 is an endogenous inhibitor of MMP9 (36). TIMP1 binds MMP9 in a 1:1 stoichoimetric fashion and keeps it inactive (66). We found that acrolein had no effect on TIMP1 mRNA in both NCI-H292 cells and NHBE cells (Figure 7). TIMP3, a natural inhibitor of ADAM17 (35), is also regulated at the transcriptional level (67). We found that acrolein decreased TIMP3 mRNA (Figure 7) in NCI-H292 and NHBE cells but had no effect on ADAM17 mRNA (Figure 7). Thus, in addition to rapid ADAM17- and MMP9-mediated ligand-dependent activation of EGFR, acrolein increases MMP9 activity and alters transcription of proteins critical to this pathway, which in turn may prolong the effect on MUC5AC production (Figure 8).
In summary, acrolein rapidly induces MUC5AC expression through an EGFR-MAPK pathway mediated by metalloproteinases ADAM17 and MMP9. In addition, acrolein can produce a prolonged increase in MUC5AC expression through an increase in MMP9 (transcript and activity) and a decrease in TIMP3 (transcript). Together, these interactions would be consistent with extended mucin production following exposure to this irritant, a component of cigarette smoke. Samples of lung tissues from patients with cigarette smokeeCrelated emphysema show an increase in MMP9 (68). Thus, in addition to playing an important role in alveolar enlargement and matrix degradation, MMP9 may also be involved in mucus hypersecretion.
Acknowledgments
The authors thank Jay Tichelaar for the critical review of the manuscript and Mary-Beth Genter for assistance with the MMP analyses.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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