Double-Stranded RNA and TGF-{alpha} Promote M...

Double-Stranded RNA and TGF- Promote MUC5AC Induction in Respiratory Cells

Hiromi Tadaki^*,, Hirokazu Arakawa^1,*, Takahisa Mizuno^*, Tomoko Suzuki^*, Kiyoshi Takeyama, Hiroyuki Mochizuki^*, Kenichi Tokuyama, Shumpei Yokota and Akihiro Morikawa^*

^* Department of Pediatrics and Developmental Medicine, Gunma University Graduate School of Medicine, Gunma, Japan; Department of Pediatrics, Yokohama City University School of Medicine, Kanagawa, Japan; First Department of Medicine, Tokyo Women’s Medical University, Tokyo, Japan; and Department of Pharmacy, Takasaki University of Health and Welfare, Gunma, Japan

   Abstract Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Disclosures
References
 
Viral infection is a major trigger for exacerbation of asthma and induces overproduction of mucins. We investigated whether dsRNA could amplify the induction of mucin by TGF- in human bronchial epithelial cells, as well as the molecular mechanisms regulating MUC5AC expression. Human pulmonary mucoepidermoid carcinoma (NCI-H292) cells and normal human bronchial epithelial cells were exposed to polyinosinic-cytidyric acid (poly(I:C)) and TGF-. Then, MUC5AC protein production, mRNA expression, and promoter activity were evaluated. Cells were pretreated with a selective inhibitor of ERK, and phosphorylation of ERK was examined by Western blotting. Furthermore, the expression of MAPK phosphatase 3 (MKP3) mRNA was evaluated and the effect of MKP3 overexpression was assessed. Poly(I:C) synergistically increased MUC5AC induction by TGF- in both NCI-H292 and normal human bronchial epithelial cells. This increase was dependent on MUC5AC gene transcription. A MEK1/2 inhibitor (U0126) significantly inhibited MUC5AC production. Phosphorylation of ERK was enhanced by poly(I:C). TGF- stimulation up-regulated MKP3 mRNA expression, while costimulation with poly(I:C) inhibited this up-regulation dose-dependently. Enhanced expression of MUC5AC mRNA by poly(I:C) in wild-type cells was completely suppressed in cells transfected with the MKP3 expression vector. dsRNA can synergistically amplify the induction of MUC5AC mucin by TGF-. This synergistic effect on MUC5AC production may be due to enhanced activation of ERK through inhibition of MKP3 by poly(I:C).

   Introduction Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Disclosures
References
 
In chronic airway diseases such as asthma, goblet-cell hyperplasia is an important feature (1). Excessive secretion of mucus by hyperplastic goblet cells causes airway plugging and contributes to morbidity and mortality in asthma patients (2, 3). To date, 19 different mucin genes have been identified. Among these, MUC5AC mucin is a major component of the mucus produced by airway epithelial cells (4), and its production is regulated by epidermal growth factor receptor (EGFR) signaling pathway (5, 6). EGFR and its ligands are not only expressed in patients with malignant lung tumors, but also in those with airway inflammatory diseases such as asthma (7). TGF- is one of the ligands of EGFR, and it is known to play a critical role in phosphorylation of EGFR that leads to MUC5AC production in the airways (5).

Viral infection is a common cause of the exacerbation of asthma. Among the many viruses that infect the airways, human rhinovirus, respiratory syncytial virus, influenza virus, and parainfluenza virus are particularly common pathogens that induce the hypersecretion of mucus and exacerbation of asthma (8, 9, 10). These are RNA viruses that synthesize dsRNA during replication in infected cells. TLR3 recognizes dsRNA and was the first antiviral TLR identified (11). Because dsRNA is a universal viral molecule, TLR3 has been assumed to have a central role in the host response to infection by viruses (11). Previous studies have shown that stimulation with a synthetic analog of viral dsRNA (poly inosinic-cytidyric acid, poly(I:C))² is mediated by a pathway involving TLR3 that induces airway inflammation due to various cytokines and chemokines such as IL-8, IL-6, and RANTES (12). Despite the importance of excessive mucin production due to viral infection in triggering the exacerbation of asthma, the mechanisms causing such overproduction remain unknown.

We hypothesized that viral infection might synergistically amplify respiratory mucin gene expression and protein production induced by growth factors that are involved in the pathogenesis of asthma. Here, we demonstrate that a synthetic analog of viral dsRNA (poly(I:C)) synergistically increases the induction of respiratory mucin MUC5AC by TGF- in human airway epithelial cells, both at the level of mRNA expression and protein production. This action depends on the activation of ERK, and the ERK pathway is enhanced through inhibition of MAPK phosphatase 3 (MKP3) by poly(I:C).

   Materials and Methods Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Disclosures
References
 
Cell culture and stimulation

A human pulmonary mucoepidermoid carcinoma cell line (NCI-H292) was maintained in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C in a humidified atmosphere with 5% CO₂. NCI-H292 cells were seeded into 12-well plates for the ELISA and luciferase assay, and into 6-cm dishes for Western blotting and mRNA analysis. Cells were grown until 70% confluence was reached, and they were maintained overnight in serum-free medium before stimulation. Cells were exposed to poly(I:C) (Sigma-Aldrich) at 25 µg/ml or TGF- (R&D Systems) at 4 ng/ml, or to a combination of both agents.

Normal human bronchial epithelial (NHBE) cells were purchased from Lonza. NHBE cells were seeded at density of 1.3 x 10⁵/cm² into 12-well plates containing bronchial epithelial growth medium (Lonza) supplemented with defined growth factors and retinoic acid from the SingleQuot kit (Lonza), and were incubated at 37°C in a humidified atmosphere with 5% CO₂. Cells were exposed to poly(I:C) (25 µg/ml) or TGF- (4 ng/ml), or a combination of both agents, for 24 h.

Analysis of mucin

NCI-H292 cells were stained with Alcian blue and periodic acid-Schiff stains (AB-PAS). MUC5AC protein was measured as described previously (5). In brief, supernatants were collected at 24 h after stimulation and cell lysates were prepared with PBS, and 50 µl of each sample was incubated with bicarbonate-carbonate buffer (50 µl) at 40°C in a 96-well plate (Nunc) overnight. Plates were washed three times with PBS and blocked with 2% BSA for 1 h at 37°C. Plates were again washed three times with PBS and then incubated with 50 µl of mouse monoclonal anti-MUC5AC Ab (1/100) (Lab Vision/NeoMarkers), which was diluted with PBS containing 0.05% Tween 20 and dispensed into each well. After 1 h, the plates were washed three times with PBS, and 100 µl of HRP-sheep anti-mouse IgG conjugate (1/10,000) (Amersham Biosciences) was added to each well. After 1 h, the plates were washed three times with PBS. Color was developed with 3,3',5,5'-tetramethylbenzidine peroxidase solution (Kirkegaard & Perry Laboratories) and the reaction was stopped with 1 M H₂SO₄. The data were expressed as a fold induction on the same experimental day due to various mucin production with cell passage in NCI-H292 cells.

Real-time quantitative PCR analysis

Expression of MUC5AC, MKP3, and EGFR mRNA by NCI-H292 cells was determined by reverse transcription (RT), followed by the real-time quantitative PCR. Total RNA was extracted from lysates of differentiated NCI-H292 cells using Isogen (Nippon Gene) at 12 h after stimulation. RT was performed with 1 µg of total RNA and oligo(dT) primers in a 25-µl reaction mixture according to the manufacturer’s protocol (Applied Biosystems). The sequences of the specific primer sets that were used for PCR are listed in Table I (13, 14).

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  Table I. Primers used for quantitative real-time PCR analysis of gene expression

 
Real-time PCR was performed with an ABI Prism 7900HT sequence detection system (Applied Biosystems) using SYBR Green (Applied Biosystems) as a dsDNA-specific binding dye. For MUC5AC and β-actin, initial denaturation was done at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing and extension at 60°C for 1 min. The threshold cycle (C_T) was recorded for each sample to reflect the level of mRNA expression. A validation experiment confirmed linear dependence of the C_T value on the concentrations of MUC5AC and β-actin and the consistency of C_T (mean C_T for MUC5AC – mean C_T for β-actin) in a given sample at different RNA concentrations. C_T was therefore used as an indicator of relative mRNA expression. To determine the effects of different stimuli on MUC5AC gene expression compared with unstimulated cells, C_T was calculated (C_T = C_T for stimulated cells – C_T for unstimulated cells). MUC5AC mRNA expression was indexed to β-actin mRNA expression by using the formula 1/(2C_T) x 100%. 2C_T was calculated to demonstrate the fold change of MUC5AC gene expression in stimulated cells compared with unstimulated cells.

Expression of MKP3 and EGFR mRNA by NCI-H292 cells was determined in the same manner.

Reporter assay for the MUC5AC promoter

To investigate the regions of the MUC5AC promoter that were activated by poly(I:C) and TGF-, the full-length human MUC5AC promoter was cloned into pGL3basic (a promoterless luciferase vector). This was then serially truncated using a combination of restriction enzyme digestion and PCR amplification to successively isolate regions of the promoter containing a large variety of potential transcription factor-binding sites (–1330 to –63).

NCI-H292 (0.8 x 10⁵) cells were seeded into 12-well plates and grown overnight in complete medium. At 60% confluence, cells were rinsed with 1 ml of serum-free medium and incubated for 1 h. Then the cells were transfected using 1.3 µl of FuGENE 6 (Roche Applied Science) in 50 µl of RPMI 1640 medium per well plus 4 µl of MUC5AC promoter-luciferase plasmid DNA. At 1 h after transfection, cells were stimulated with poly(I:C) (25 µg/ml) and then incubated for 12 h before stimulation with TGF- (4 ng/ml). Cell lysates were prepared, and reporter gene activity was determined by using a luciferase assay kit (Promega). The total protein concentration of samples was measured by spectrophotometry (NanoDrop from Thermo Scientific) to adjust for variations in harvesting of cells.

Western blot analysis

Cells (3.0 x 10⁵) were washed with PBS and lysed in 300 µl of lysis buffer (0.5% Nonidet P-40, 10 mM Tris-Cl (pH 7.4), 150 mM NaCl, 3 mM p-amidinophenylmethanesulfonyl fluoride (Sigma-Aldrich), 5 mg/ml aprotinin (Sigma-Aldrich), 2 mM sodium orthovanadate (Sigma-Aldrich), 5 mM EDTA). Whole-cell extracts were subjected to electrophoresis on 7.5–12% Tris-glycine gel (XV Pantera gel; DRC) and then transferred to Sequi-Blot polyvinylidene difluoride membranes (Immobilon-P; Millipore). Membranes were blocked with 5% skim milk in Tris-buffered saline with 0.05% Tween 20 (TBS-T (pH 7.5)) for 30 min at room temperature (RT) and probed with primary anti-human phospho-p44/42 MAPK (Thr²⁰²/Tyr²⁰⁴) Ab and p44/42 MAPK Ab (Cell Signaling Technology) for 1 h at RT. The membranes were then washed with TBS-T and incubated with secondary donkey anti-rabbit Ig Ab conjugated to HRP (Amersham Biosciences) for 1 h at RT. Finally, Ab-Ag complexes were detected using an ECL chemiluminescent detection system according to the manufacturer’s instructions (ECL plus Western blot detection system; Amersham Biosciences).

Cloning of MKP3 expression vector and transfection into NCI-H292 cells

A DNA fragment of the coding sequence of MKP3 was amplified by PCR using cDNA from poly(I:C)-treated NCI-H292 cells. The purified PCR product was digested with BamHI and SalI and cloned into the pAcGFP1-C1 vector (Clontech Laboratories). The plasmid was analyzed by digestion with restriction enzymes and DNA sequencing. Plasmids for transfection were purified with HiSpeed Plasmid Maxi kit (Qiagen). H292 cells were seeded into 6-well plates and grown to 50% confluence. Cells were transfected with 4 µg of expression vector with 10 µl of Lipofectamine 2000 (Promega) and grown in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). After 24 h, the medium was changed to RPMI 1640 supplemented with 10% FBS without antibiotics. Then, the cells were exposed to poly(I:C) (25 µg/ml), TGF- (4 ng/ml), or a combination of both agents. After 12 h, the expression of MUC5AC and MKP3 mRNA was evaluated.

Other reagents

U0126 (a MEK1/2 inhibitor) was purchased from Sigma-Aldrich and monoclonal anti-human CXCL8/IL-8 Ab was purchased from R&D Systems. U0126 was dissolved in DMSO, while the monoclonal anti-human CXCL8/IL-8 Ab was dissolved in PBS. In all studies, the concentration of DMSO was 0.02–0.06%. U0126 (20 µM) (15, 16) and the anti-IL-8 Ab (2 µg/ml) were preincubated with cells for 1 h before adding poly(I:C) and TGF-.

Phosphoprotein assay

Cells (3.0 x 10⁵/ml) were seeded into 6-cm dishes and were treated with poly(I:C) for 1 h and then with TGF- for 15 min. Protein lysates were prepared by using a cell lysis kit (Bio-Rad), and phosphorylated EGFR was detected with an EGFR (Tyr) assay kit (Bio-Rad) and a phosphoprotein testing reagent kit (Bio-Rad) according to the manufacturer’s protocol. Briefly, 50 µl of cell lysate (adjusted to a protein concentration of 200–400 µg/ml) was plated into a 96-well filter plate coated with EGFR Ab-coupled beads and incubated overnight on a platform shaker at 300 rpm at RT. Total protein was measured with a Bio-Rad DC protein assay kit.

Statistical analysis

All data are expressed as the means ± SD. Results were analyzed by using the paired Student’s t test or ANOVA as appropriate. Analyses were done with SPSS II software (SPSS Japan), and p values of <0.05 were considered significant.

   Results Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Disclosures
References
 
Poly(I:C) synergistically enhances MUC5AC protein production induced by TGF-

First, we examined the ability of TGF- and poly(I:C) to induce mucous glycoconjugate production assessed by AB-PAS staining in NCI-H292 cells (Fig. 1A). Twenty-four hours of incubation with TGF- (4 ng/ml) increased PAS-positive staining, while poly(I:C) (25 µg/ml) alone did not affect staining. However, poly(I:C) enhanced the stimulatory effect of TGF- on mucous glycoconjugate production (Fig. 1A). To quantify the MUC5AC mucin production, an ELISA was performed. TGF- alone caused a 5-fold increase in MUC5AC mucin protein in cell supernatant (Fig. 1B) and a 1.5-fold increase in cell lysate (Fig. 1C) from NCI-H292 cells 24 h after stimulation. Poly(I:C) alone caused little increase in MUC5AC mucin protein; however, poly(I:C) strongly potentiated the effect of TGF-. Thereafter, we evaluated MUC5AC mucin protein only in cell supernatant, because it was more prominent than cell lysate.

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  FIGURE 1. Synergistic effect of poly(I:C) and TGF- on MUC5AC production. A, AB-PAS staining of NCI-H292 cells for identification of mucus glycoconjugates. Incubation with poly(I:C) (25 µg/ml) and TGF- (4 ng/ml) for 24 h increased positive staining. Effect of poly(I:C) (25 µg/ml) on TGF- (4 ng/ml)-induced MUC5AC mucin production in NCI-H292 cell supernatant (n = 9) (B) and cell lysate (n = 9) (C). Cells were incubated with poly(I:C) and TGF- for 24 h. Data are shown as means ± SD. ++, p < 0.01 compared with nonstimulated control cells. _*, p < 0.05 and _**, p < 0.01.

 

 
Next, we determined effects of dose responses of poly(I:C) (2.5–75 µg/ml) and TGF- (0.4–12 ng/ml) on MUC5AC mucin production (Fig. 2, A and B). Although poly(I:C) alone did not significantly induce MUC5AC mucin production in every dose, costimulation with TGF- caused an increase in MUC5AC mucin production with regard to poly(I:C) in a dose-dependent manner (Fig. 2A). TGF- alone induced a dose-dependent increase in MUC5AC mucin production, and poly(I:C) enhanced the effect of TGF- (Fig. 2B). Subsequent studies were focused on the time course of MUC5AC mucin production. Costimulation with poly(I:C) (25 µg/ml) and TGF- (4 ng/ml) caused a small increase in MUC5AC mucin production 12 h after stimulation, with maximal levels of MUC5AC at 24 h (Fig. 2C). These results may imply that poly(I:C) synergistically up-regulates MUC5AC mucin production induced by TGF-.

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  FIGURE 2. Dose responsiveness and time course of MUC5AC protein production. A, Effect of the poly(I:C) concentration on TGF- (4 ng/ml)-induced MUC5AC mucin production in NCI-H292 cells (n = 6). Cells were incubated with poly(I:C) and TGF- for 24 h. B, Effect of the TGF- concentration on poly(I:C) (25 µg/ml)-induced MUC5AC mucin production in NCI-H292 cells (n = 6). Cells were incubated with poly(I:C) and TGF- for 24 h. C, Effect of poly(I:C) (25 µg/ml) on the time course of induction of MUC5AC mucin production by TGF- (4 ng/ml) in NCI-H292 cells (n = 6). Data are shown as means ± SD. +, p < 0.05 and ++, p < 0.01 compared with nonstimulated control cells. _**, p < 0.01.

 

 
Poly(I:C) synergistically enhances MUC5AC mRNA expression induced by TGF-

To determine whether induction of MUC5AC mucin protein induced by poly(I:C) and TGF- was a result of increased MUC5AC gene transcription, we investigated levels of MUC5AC mRNA, determined by real-time quantitative RT-PCR in NCI-H292 and NHBE cells. TGF- (4 ng/ml) alone caused little increase in MUC5AC mRNA expression in NCI-H292 cells and NHBE cells upon 24 h of stimulation (Fig. 3A). Poly(I:C) (25 µg/ml) alone induced a small but significant increase in MUC5AC mRNA expression in both NCI-H292 cells and NHBE cells, and poly(I:C) strongly potentiated the effect of TGF- (Fig. 3A). A clear dose response was observed at 12 h following stimulation with both poly(I:C) (2.5–75 µg/ml) (Fig. 3B) and TGF- (0.4–12 ng/ml) (data not shown). Costimulation with poly(I:C) and TGF- caused a small increase in MUC5AC mRNA expression 6 h after stimulation, which continued to a peak at 12 h after stimulation (Fig. 3C).

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  FIGURE 3. Dose responsiveness and time course of MUC5AC mRNA expression. A, Effect of poly(I:C) (25 µg/ml) on TGF- (4 ng/ml)-induced MUC5AC gene expression in NCI-H292 cells and NHBE cells. Cells were incubated with poly(I:C) and TGF- for 24 h. Data are presented as the fold induction over the level in control NHBE cells. The right side of the y-axis is for NCI-H292 cells and the left side is for NHBE cells (n = 6). B, Effect of the poly(I:C) concentration on TGF- (4 ng/ml)-induced MUC5AC mRNA expression in NCI-H292 cells (n = 6). Cells were incubated with poly(I:C) and TGF- for 12 h. C, Effect of poly(I:C) (25 µg/ml) on the time course of induction of MUC5AC mRNA expression by TGF- (4 ng/ml) in NCI-H292 cells (n = 4). Data are shown as means ± SD. +, p < 0.05 and ++, p < 0.01 compared with nonstimulated control cells. _*, p < 0.05.

 

 
Poly(I:C) and TGF- cause synergistic trans-activation of the MUC5AC promoter

We next investigated whether the MUC5AC promoter was activated by poly(I:C) and TGF-. After 8 h, TGF- alone induced a small but significant activation of the full-length MUC5AC promoter construct (–1330) (Fig. 4). Poly(I:C) did not activate the full-length MUC5AC promoter construct (–1330), but poly(I:C) strongly enhanced the activation induced by TGF-, with 6-fold induction over that in unstimulated transfected control cells (p < 0.05) (Fig. 4). This was observed when poly(I:C) was added 12 h before TGF- stimulation (Fig. 4), but not when the two agents were added at the same time (data not shown). There was no difference in the level of activation of the short-length MUC5AC promoter construct (–63) among TGF-, poly(I:C), and both stimulations (Fig. 4). These results indicate that the –1330 to –63 region contains the elements regulating induction of the MUC5AC promoter by poly(I:C) and TGF-.

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  FIGURE 4. Effect of poly(I:C) (25 µg/ml) on TGF- (4 ng/ml)-induced trans-activation of the full-length (–1330) and the short-length (–63) MUC5AC promoter in NCI-H292 cells (n = 6). Data are shown as means ± SD. _*, p < 0.05.

 

 
Transactivation of the MUC5AC promoter by poly(I:C) and TGF- is mediated via an ERK signaling pathway

Since it was shown that induction of MUC5AC-specific mucin protein by poly(I:C) and TGF- was a result of increased MUC5AC gene transcription, we next investigated the upstream signaling leading to activation of the promoter.

First, since TGF- induces MUC5AC mucin production through the ligand-dependent trans-activation of EGFR in NCI-H292 cells (5), we examined the importance of EGFR activation for synergistic induction of MUC5AC mucin production by poly(I:C). We evaluated EGFR mRNA expression and phosphorylation of EGFR by RT-PCR and the Bio-Plex phosphoprotein assay, respectively. As a result, we found that poly(I:C) did not up-regulate EGFR mRNA expression upon 12 h of stimulation (data not shown) or increase the phosphorylation of EGFR (Fig. 5A).

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  FIGURE 5. A, Relative phosphorylation of EGFR by poly(I:C) and TGF- in NCI-H292 cells. Proteins extracted from samples collected were tested for the presence of phosphorylated EGFR by a Bio-Plex phosphoprotein assay kit using the Bio-Rad Luminex machine. The values plotted show the ratios of phosphorylated EGFR to total EGFR expressed as fold increase over control (n = 6). B, Phosphorylation of ERK1/2 by poly(I:C) (25 µg/ml) and TGF- (4 ng/ml) in NCI-H292 cells assessed by performing Western blot analysis. C, Phospho-ERK was expressed as the fold increase in relative intensity (n = 3). Data are shown as means ± SD. ++, p < 0.01 compared with nonstimulated control cells.

 

 
Second, since previous studies have demonstrated that increased production of MUC5AC mucin protein after activation of the EGFR signaling pathway was exclusively MEK/ERK-dependent (17), we investigated the requirement of ERK. Western blot analysis revealed that poly(I:C) synergistically enhanced the phosphorylation of ERK by TGF- stimulation (Fig. 5, B and C). This finding was compatible with the result of chemical inhibition by MEK1/2 inhibitor (U0126). U0126 inhibited the induction of MUC5AC protein production by poly(I:C) (75 µg/ml) and TGF- (4 ng/ml) compared with absence of the inhibitor at 24 h after stimulation (Fig. 6). These data suggest that trans-activation of the MUC5AC promoter by poly(I:C) and TGF- is mediated via an ERK signaling pathway.

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  FIGURE 6. Effect of U0126 on poly(I:C)- and TGF--induced MUC5AC mucin production in NCI-H292 cells. Cells were preincubated with U0126 (a specific inhibitor of MEK1/2) at 20 µM (15 16 ) for 1 h before adding poly(I:C) (75 µg/ml) and TGF- (4 ng/ml), and cells were analyzed 24 h after stimulation (n = 6). Data are shown as means ± SD. +, p < 0.05 and ++, p < 0.01 compared with nonstimulated control cells. _**, p < 0.01.

 

 
Poly(I:C) inhibits TGF--induced MKP3 mRNA expression

Having demonstrated that the ERK-dependent signaling was required in MUC5AC induction, still unclear is the mechanism interacting between TLR3-dependent signaling stimulated by poly(I:C) and EGFR-dependent signaling stimulated by TGF-. Since MKP3 is known to be a member of the phosphatase family that inactivates ERK1/2, we examined the effect of poly(I:C) on MKP3 mRNA expression. A real-time quantitative RT-PCR showed that expression of MKP3 mRNA was up-regulated upon 12 h of stimulation with TGF-, and MKP3 mRNA up-regulation by TGF- was inhibited by stimulation with poly(I:C) (Fig. 7A). Stimulation with TGF- led to a moderate increase in MKP3 mRNA expression at 6 h, followed by a decrease at 24 h (Fig. 7B). Costimulation with poly(I:C) dose-dependently inhibited this up-regulation, and inhibition was seen from 6 h after stimulation (Fig. 7, B and C).

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  FIGURE 7. A, Upper, Effect of poly(I:C) (25 µg/ml) on TGF- (4 ng/ml)-induced MKP3 mRNA expression in NCI-H292 cells (n = 8). Lower, To verify MKP3 expression in NCI-H292 cells, expression of MKP3 after stimulation with poly(I:C) (25 mg/ml) and TGF- (4 ng/ml) was examined by standard RT-PCR. In brief, 25 µl of reaction mixture consisted of 1 µl cDNA, 1 pmol MKP3 primer sets, and 12.5 µl AmpliTaq Gold PCR Master mix (Applied Biosystems). PCR was performed by an initial denaturation step at 95°C for 5 min followed by 30 cycles with a denaturation step at 95°C for 30 s, an annealing step at 60°C for 30 s, and an extension step at 72°C for 30 s. B, Time course of induction of MKP3 mRNA expression by poly(I:C) (25 µg/ml) and TGF- (4 ng/ml) in NCI-H292 cells (n = 4). C, Effect of the poly(I:C) concentration on TGF- (4 ng/ml)-induced expression of MKP3 mRNA in NCI-H292 cells (n = 4). Data are shown as means ± SD. +, p < 0.05 and ++, p < 0.01 compared with nonstimulated control cells. _*, p < 0.05 and _**, p < 0.01.

 

 
Effect of the MKP3 expression vector

To further demonstrate the role of MKP3 on MUC5AC mucin induction, we investigated the effect of MKP3 expression vector. MKP3 mRNA levels were significantly enhanced in cells transfected with the MKP3 expression plasmid cloned into the pAcGFP1-C1 vector when compared with wild-type cells (Fig. 8A). Enhanced expression of MUC5AC mRNA was noted in wild-type cells 12 h after coincubation with TGF- (4 ng/ml) and poly(I:C) (25 µg/ml), but was completely abolished in cells transfected with the MKP3 expression vector (Fig. 8B). These data suggest that the inhibition of MKP3 mRNA expression by poly(I:C) leads to synergistic MUC5AC mucin induction.

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  FIGURE 8. A, Effect of a MKP3 expression plasmid cloned into the pAcGFP1-C1 vector on MUC5AC mRNA expression in NCI-H292 cells. The level of MKP3 mRNA was enhanced significantly in cells transfected with the MKP3 expression plasmid. B, Enhanced expression of MUC5AC mRNA was noted in wild-type cells after 12 h of coincubation with TGF- (4 ng/ml) and poly(I:C) (25 µg/ml), but it was completely abolished in cells transfected with the MKP3 expression vector (n = 6). Data are shown as means ± SD. +, p < 0.05 and ++, p < 0.01 compared with nonstimulated control cells. _*, p < 0.05 and _**, p < 0.01.

 

 
Anti-IL-8 Ab does not inhibit poly(I:C)- and TGF--induced MUC5AC mRNA expression

Poly(I:C) is known to increase the expression of mRNA for various chemokines and cytokines (18, 19). In our preliminary study, we measured the cytokine and chemokine levels in the supernatant after stimulation with costimulation of TGF- and poly(I:C) by using a Bio-Plex cytokine assay. In that study, only the IL-8 level was synergistically high due to costimulation of TGF- and poly(I:C). Therefore, we chose IL-8, and to investigate the role of IL-8, NCI-H292 cells were preincubated with anti-IL-8 Ab 1 h before stimulation with poly(I:C) (75 µg/ml) and TGF- (4 ng/ml). Anti-IL-8 Ab did not inhibit the increase in the expression of MUC5AC mRNA induced by 12 h of stimulation with poly(I:C) and TGF- (Fig. 9).

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  FIGURE 9. Effect of the anti-IL-8 Ab on poly(I:C) (75 µg/ml) and TGF- (4 ng/ml)-induced MUC5AC mRNA expression in NCI-H292 cells (n = 4). Data are shown as means ± SD. ++, p < 0.01 compared with nonstimulated control cells.

 

 

   Discussion Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Disclosures
References
 
In this study, we found that poly(I:C) synergistically increased the production of MUC5AC induced by TGF- in both NCI-H292 and NHBE cells. This increase was dependent on activation of the MUC5AC promoter, and the upstream signaling pathway was ERK-dependent. The most interesting finding of this study was that expression of MKP3, which is one of the negative regulators of MAPK, was up-regulated by TGF- and this up-regulation was inhibited by poly(I:C), indicating that MKP3 has a central role in the synergistic induction of MUC5AC production by poly(I:C) and TGF-.

Mucin hypersecretion and goblet cell hyperplasia are characteristic features of airway inflammatory diseases such as asthma (1, 2). Since hypersecretory diseases are associated with abnormal epithelial cell growth and differentiation, and epithelial damage leads to repair and remodeling (19, 20), both inflammatory mediators and growth factors may be involved in stimulating mucin production from goblet cells. It has been postulated that activation of the EGFR pathway is a common denominator in the induction of MUC5AC mucin, a major component of mucus in the airways. Takeyama et al. have shown that stimulation of EGFR by its ligands, EGF and TGF-, causes MUC5AC production by airway epithelial cells both in vitro and in vivo (5), and this effect is potentiated by TNF- (5). In the present study, we found that using AB-PAS staining, ELISA, and RT-PCR, poly(I:C) synergistically amplified the induction of MUC5AC mucin induced by TGF- at both the mRNA and protein levels in NCI-H292 cells.

In NHBE cells, MUC5AC mRNA expression was much lower than that in NCI-H292 cells, but poly(I:C) still synergistically amplified the expression of MUC5AC mRNA induced by TGF-, indicating that synergic induction of MUC5AC by poly(I:C) and TGF- may be generalizable to normal human epithelial cells. The lower expression of MUC5AC mRNA may be explained by not using an air-liquid interface in culturing NHBE cells. Indeed, studies done in air-liquid interface or monolayers would provide us important results. However, previous studies have demonstrated that both NCI-H292 and NHBE cells share key components of the signaling pathways upstream and downstream of EGFR responsible for mucin production (21), suggesting that NCI-H292 cells are a valid model of mucin production in normal cells. Therefore, our further studies investigating the mechanisms of the signaling pathway were done in NCI-H292 cells.

In the present study, we found that synergistic induction of MUC5AC mucin production by poly(I:C) and TGF- was dependent on activation of the MUC5AC promoter within the proximal –1330/–63 region. Additionally, we investigated upstream signaling by using an inhibitor and Western blot analysis, and we found that the process was ERK-dependent. Our data are in agreement with findings reported by Hewson and coworkers, showing that increased production of MUC5AC mucin protein after activation of the EGFR signaling pathway was exclusively MEK/ERK-dependent (17). Furthermore, we found that poly(I:C) synergistically enhanced the phosphorylation of ERK induced by TGF-. Therefore, we concluded that trans-activation of the MUC5AC promoter by poly(I:C) and TGF- occurs exclusively via an ERK signaling pathway.

Receptor regulation has an important role in controlling the actions of several mediators. Yamamoto et al. demonstrated that IL-4-induced production of eotaxin-3 in airway epithelium was enhanced due to up-regulation of the IL-4 receptor by IFN- (22). In the present study, to determine whether the synergistic effect of poly(I:C) was due to up-regulation of the EGFR, we evaluated EGFR mRNA expression and EGFR phosphorylation. However, up-regulation of EGFR mRNA expression and the phosphorylation of this receptor by stimulation with poly(I:C) were not observed.

Since we had found that ERK was required for the synergistic effect of poly(I:C) on MUC5AC production induced by TGF-, we proceeded to investigate this further by evaluating the role of MKP3, which is a member of the phosphatase family that inactivates ERK1/2. MKP3 is predominantly localized in the cytoplasm and has a highly specific role in the dephosphorylation and inactivation of ERK1/2 (23, 24, 25, 26). MKP3 is an immediate early gene and is transcriptionally up-regulated after ERK2 activation (27, 28). Our present finding that MKP3 mRNA expression was 37-fold higher following stimulation with TGF- is in agreement with previous reports that MKP3 is up-regulated after activation of the ERK2 pathway (27, 28, 29). Additionally, we found that this up-regulation was inhibited by stimulation with poly(I:C), and that overexpression of MKP3 completely abolished the increase in expression of MUC5AC mRNA. These data indicate that when NCI-H292 cells are stimulated by TGF- alone, MUC5AC protein production remains under autoregulation to a certain extent by negative feedback via MKP3. However, when additional stimulation with poly(I:C) is added, MKP3 mRNA expression is partially down-regulated. This leads to synergistic activation of ERK, synergistic trans-activation of the MUC5AC promoter, and finally to synergistic production of MUC5AC protein.

Posttranscriptional events are also important in regulation of gene expression. A detailed examination of the time course of MUC5AC mRNA expression revealed that it was maximal at 12 h and decreased at 24 h after treatment with TGF- alone. In contrast, costimulation with poly(I:C) and TGF- caused a significant time-dependent increase in MUC5AC mRNA expression for up to 24 h. Furthermore, analysis of mRNA stability by real-time quantitative RT-PCR demonstrated that poly(I:C) did not alter the stability of MUC5AC mRNA (data not shown). Accordingly, the additional stimulation with poly(I:C) significantly increased and prolonged the induction of MUC5AC mRNA expression induced by TGF- without affecting the rate of MUC5AC mRNA degradation.

Poly(I:C) is known to increase the expression of mRNA for various chemokines (IP-10, RANTES, LARC, MIP1, IL-8, GRO-, and ENA-78) and cytokines (IL-1β, GM-CSF, and IL-6), as well as the cell adhesion molecule ICAM-1 (18, 19). To determine whether IL-8 has an important role in the synergistic effect of poly(I:C) and TGF- on MUC5AC production, we investigated the potential role of IL-8 by preincubation with an anti-IL-8 Ab in the cells. The anti-IL-8 Ab did not inhibit MUC5AC mRNA expression, indicating that IL-8 has no role in the process. This finding was consistent with a previous report showing that IL-8 alone had no effect on MUC5AC protein production in NCI-H292 cells (30).

Also, the role of IFN may be an important point particularly in the context of poly(I:C) and asthma. We have not done studies directly on IFN- and IFN-β. However, to further investigate whether extracellular factors (such as chemokines and cytokines) released by poly(I:C) stimulation up-regulated TGF--induced MUC5AC production, we changed the culture medium at 12 h after poly(I:C) stimulation and then stimulated the cells with TGF-. Although the extracellular factors had been removed, it did not alter the synergic expression of MUC5AC mRNA (data not shown), suggesting that extracellular factors including IFN- and IFN-β released by poly(I:C) may not contribute to the enhanced MUC5AC expression.

In conclusion, poly(I:C) synergistically increases the production of MUC5AC induced by TGF- in airway epithelial cells, due to inhibition of MKP3 expression. Studies completed with viruses, especially rhinovirus and also inactivated viruses, would provide us with important perspectives. Further studies will be needed to analyze the interaction between viruses and TGF-. Viral respiratory tract infections are the most common triggers for the exacerbation of asthma (31, 32), and mucin overproduction is one of the mechanisms involved. The present findings may help to explain the excessive production of mucus in asthmatic patients with viral infection. Mucus plugging of the airways is a feature of fatal asthma in both adults and children (33, 34). At present, there are no effective therapies to relieve the symptoms induced by hypersecretion of mucus due to viral infection in asthmatic patients. Our findings may provide a mechanism to explain mucin overproduction and a potential strategy for therapy.

   Acknowledgments  
We thank Tomoko Endo and Chinori Iijima for their excellent technical assistance.

   Disclosures Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Disclosures
References
 
The authors have no financial conflicts of interest.

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

¹ Address correspondence and reprint requests to Dr. Hirokazu Arakawa, Department of Pediatrics and Developmental Medicine, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511, Japan. E-mail address: harakawa@showa.gunma-u.ac.jp

² Abbreviations used in this paper: poly(I:C), polyinosinic-cytidyric acid; AB-PAS, Alcian blue/periodic acid-Schiff; C_T, threshold cycle; EGFR, epidermal growth factor receptor; MKP, MAPK phosphatase; NHBE, normal human bronchial epithelial; RT, room temperature.

Received for publication July 10, 2008. Accepted for publication October 23, 2008.

   References Top
Abstract
Introduction
Materials and Methods
Results
Discussion
Disclosures
References
 

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