Ibudilast Suppresses MUC5AC Mucus Production through Inhibition of ERK1/2 Phosphorylation

Abstract

Mucus hypersecretion is a hallmark of respiratory diseases, and excess airway mucus can worsen these conditions. Therefore, it is important to control the production of airway mucus in the treatment of respiratory diseases. The phosphodiesterase inhibitor ibudilast has been reported to be effective in treating sputum and postnasal drip in patients with chronic airway inflammation. On the basis of the hypothesis that ibudilast could inhibit mucus production in the airway, in the present study, we examined the effects of ibudilast on the production of MUC5AC, a major protein component of mucus. In in vitro studies using NCI-H292 cells, ibudilast suppressed MUC5AC production induced by various stimuli. In addition, ibudilast inhibited extracellular signal-regulated kinase (ERK)1/2 phosphorylation and MUC5AC gene transcription. Furthermore, it attenuated MUC5AC production and Muc5ac mRNA expression in lipopolysaccharide-treated mice in vivo. Collectively, these findings demonstrate that ibudilast has an inhibitory effect on mucus production, which could at least partly be attributed to the inhibition of ERK1/2 phosphorylation and the repression of MUC5AC gene transcription.

INTRODUCTION

Mucus overproduction is a common feature of inflammatory respiratory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis.^1–3) While airway mucus physiologically serves as a defense mechanism against pathogens, excessive or viscous mucus seen in pathological conditions can cause airway obstruction and inhibition of mucociliary clearance, resulting in exacerbation of respiratory disease. Normal mucus is composed of 97% water and 3% solid components. Secreted mucin is a major solid component that contributes to the physical properties of mucus.²⁾ Secreted mucin is a large glycoprotein encoded by MUC5AC and MUC5B, which are predominantly expressed in the lungs.⁴⁾ MUC5B is mainly expressed in healthy individuals and contributes to clearance of pathogens.⁵⁾ In contrast, MUC5AC is expressed less in the healthy airway and more in the inflamed airways along with goblet cell hyperplasia. It has been shown that deficiency of Muc5ac attenuates neutrophil trafficking into the lungs during acute lung injury in mice and airway hyper-responsiveness in ovalbumin-induced asthmatic mice.^6,7) This finding suggests that MUC5AC not only forms mucus plaques in small airways but also contributes to inflammation and airway hyper-responsiveness. Therefore, it is important to reduce MUC5AC production in the treatment of respiratory diseases.

Ibudilast (also known as AV411, MN-166, and 3-isobutyryl-2-isopropyl-pyrazolo-[1,5-a]pyridine) is a phosphodiesterase-4 (PDE4) inhibitor which is clinically used to treat bronchial asthma and cerebrovascular disorders. A previous clinical study has shown that ibudilast is effective in treating sputum and postnasal drip in patients with chronic airway inflammation.⁸⁾ However, few pharmacological studies have addressed the inhibitory effect of ibudilast on MUC5AC production. In the present study, we investigated whether ibudilast inhibits MUC5AC production both in vitro and in vivo.

MATERIALS AND METHODS

Reagents and Animals

Ibudilast was obtained from Tokyo Chemical Industry (>98% purity, TCI, Tokyo, Japan). Lipopolysaccharide (LPS) from Escherichia coli and dexamethasone were procured from Sigma-Aldrich (MO, U.S.A.). Transforming growth factor (TGF)-α was purchased from R&D Systems (MN, U.S.A.). Male ICR (Institute of Cancer Research) mice (8 weeks old) were obtained from the Sankyo Labo Service Corporation (SLC, Tokyo, Japan).

Animal Experiments

The mice were anaesthetized by isoflurane inhalation, and LPS (100 µg) in sterilized saline (50 µL) was intratracheally administered. Ibudilast (20 mg/kg) or dexamethasone (1 mg/kg) was intraperitoneally injected 1 h before LPS administration and then daily until Day 6. Subsequently, 24 h after the last administration of LPS, bronchoalveolar lavage fluid (BALF) and lung tissue were collected. Animal experiments were performed according to the animal care guidelines of Tokyo University of Science (Y19024).

Cell Culture and Stimulation

Human pulmonary mucoepidermoid carcinoma (NCI-H292) cells were obtained from the American Type Culture Collection (Rockville, MD, U.S.A.). The cells were maintained in RPMI1640 medium (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum, 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 MUC5AC protein and mRNA analysis and into 35-mm dishes for Western blot analysis. The cells were grown until 100% confluence and maintained in serum-free medium for 12 h prior to stimulation. The cells were co-treated with TGF-α (10 ng/mL) and ibudilast (1–100 µM).

Enzyme Linked Immunosorbent Assay (ELISA) for MUC5AC Protein Determination

The secreted MUC5AC protein in the culture media or BALF was measured by ELISA. In brief, 100 µL of the sample was added to a Nunc-immuno™ Plate and incubated at 42 °C until the sample dried. The plates were washed thrice with 0.05% Tween-20/phosphate-buffered saline (PBS) and blocked with 2% bovine serum albumin in PBS for 1 h at room temperature. The plates were washed again and incubated with 100 µL of mouse monoclonal anti-MUC5AC antibody (45M1, 1 : 100) or rabbit polyclonal anti-MUC5AC antibody (H160, 1 : 1000) diluted in 0.05% Tween-20/PBS overnight at 4 °C. The plates were washed and 100 µL of horseradish peroxidase (HRP)-conjugated anti-mouse or -rabbit immunoglobulin G (IgG) antibody was added to each well. Color was developed using 3,3′,5,5′-tetramethylbenzidine peroxidase solution for 30 min at room temperature, and the reaction was stopped with 2 M H₂SO₄. The absorbance at 450 nm was measured using ARVO™ X4 (PerkinElmer, Inc., Waltham, MA, U.S.A.).

Real-Time Quantitative PCR (RT-qPCR) Analysis

The expression of MUC5AC mRNA was determined by RT-qPCR. Total RNA was extracted from cell lysates or lung tissue using RNAisoplus (TaKaRa Bio, Shiga, Japan). Reverse transcription was performed with 0.5 µg of total RNA in 10 µL-reaction mixtures using PrimeScript™ RT Master Mix (TaKaRa Bio) according to the manufacturer’s protocol. Real-time PCR was performed with a CFX Connect™ Real-Time PCR Detection System (Bio-Rad, CA, U.S.A.). For the amplification of target genes, the following primers were used: human-MUC5AC, forward-5′-TCC ACC ATA TAC CGC CAC AGA-3′, reverse-5′-TGG ACC GAC AGT CAC TGT CAA C-3′; human-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward-5′-GTC TCC TCT GAC TTC AAC AGC G-3′, reverse-5′-ACC ACC CTG TTC TGT AGC CAA-3′; murine-Muc5ac, forward-5′-CAG CCG AGA GGA GGG TTT GAT CT-3′ reverse-5′-AGT CTC TCT CCG CTC CTC TCA AT-3′; murine-Gapdh, forward-5′-ACC ATC TTC CAG GAG CGA GA-3′, reverse-5′-CAG TCT TCT GGG TGG CAG TG-3′. The obtained value of cycle threshold was normalized to that of GAPDH amplification according to the ΔCt method.

Luciferase Assay

A reporter plasmid containing the 5′-flanking region of the human MUC5AC gene (ranging from −3752 to +68 bp) was kindly gifted by Dr. Carol B. Basbaum (University of California, San Francisco, CA, U.S.A.). Transient transfection was performed using FuGENE HD (Promega, CA, U.S.A.) in accordance with the manufacturer’s recommendations. Briefly, NCI-H292 cells cultured for 24 h in 24-well plates were transfected with DNA-transfection reagent mixture, after which they were harvested. Luciferase activity was measured with a luminometer (Lumat LB9507; EG&G Berthold, Bad Wildbad, Germany) using the Dual Luciferase Assay kit (Promega). Co-transfection with the pGL4.74 [hRluc/TK] vector, which expressed renilla luciferase, was performed to enable normalization of data for transfection efficiency.

Western Blotting

Phosphorylated-extracellular signal-regulated kinase (ERK)1/2 and total-ERK1/2 protein levels were determined by Western blot analysis. The total protein (15 µg) in the cell lysate was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride membrane. After blocking with 5% skim milk/0.05% Tween-20 in PBS for 1 h at room temperature, the membrane was incubated with primary antibodies (1 : 1000) against phosphorylated-ERK1/2 or total-ERK1/2 (CST, MA, U.S.A.) overnight at 4 °C. After washing three times with 0.05% Tween-20/PBS, the membrane was treated with HRP-conjugated anti-rabbit IgG monoclonal secondary antibodies (1 : 20000) for 1 h at room temperature. After three washes, the immunocomplexes on the membrane were detected by SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, MA, U.S.A.) on a ChemiDoc™ MP (Bio-Rad).

Statistical Analysis

All data were expressed as mean ± standard error of the mean (S.E.M.). Significant differences were assessed using one-way ANOVA followed by a Student–Newman–Keuls test. For all experiments, p-values of <0.05 were regarded as significant.

RESULTS

Ibudilast Suppressed MUC5AC Production in NCI-H292 Cells

Epidermal growth factor (EGF) receptor (EGFR) ligands, such as TGF-α, EGF and amphiregulin have been known to increase MUC5AC production.^9,10) Among these EGFR ligands, TGF-α is contributed to the pathophysiological increase in airway mucus production in asthmatic airway. In the airway epithelial cells, TGF-α is synthesized as membrane-bound pro-TGF-α and is released following cleavage by proteases, such as matrix metalloproteinases and tumor necrosis factor-α-converting enzyme, which contributed to MUC5AC expression.^11–15) In addition, expression of EGF receptor is increased in the lung of asthmatic patients.^16,17) Therefore, in the present study, we used TGF-α to stimulate MUC5AC production. To determine whether ibudilast directly inhibits mucus secretion by the airway epithelial cells, we first measured the amount of MUC5AC in NCI-H292 cells treated with TGF-α (10 ng/mL) for 24 h. The treatment increased MUC5AC protein levels compared with those in control cells. Dexamethasone (DEX, 0.1 µM) inhibited this TGF-α-induced increase in MUC5AC, as reported previously.¹⁸⁾ Similar to the effect of DEX, ibudilast (1–100 µM) suppressed MUC5AC levels in a dose-dependent manner (Fig. 1A, Supplementary Fig. 1). The most pronounced inhibition occurred with 100 µM ibudilast. The amount of MUC5AC in the culture media was found to increase independent of TGF-α concentration, and the maximum increase was observed at >50 ng/mL. Ibudilast suppressed the increase in MUC5AC released from the cells treated with any concentration of TGF-α; this effect was observed even at the highest concentration (100 ng/mL) (Fig. 1B).

Fig. 1. Ibudilast Suppressed TGF-α-Induced MUC5AC Production in Dose-Dependent Manner

(A) NCI-H292 cells were co-treated with TGF-α (10 ng/mL) and indicated dose of ibudilast or dexamethasone (DEX, 0.1 µM) for 24 h. (B) NCI-H292 cells were co-treated with TGF-α (5, 10, 25, 50, or 100 ng/mL) and ibudilast (100 µM) for 24 h. Secreted MUC5AC protein in the culture media were measured by ELISA. Results represent the mean ± standard error of the mean (S.E.M.) (n = 3; * p < 0.05, ** p < 0.01, vs. group treated with indicated concentration of TGF-α alone, ^† p < 0.05, ^†† p < 0.01, ^††† p < 0.001 vs. non-treated group).

Next, we examined the effect of ibudilast on the increase in MUC5AC release induced by other stimuli. H₂O₂ (1 mM) and cigarette smoke extract + LPS (10 µg/mL) as well as TGF-α increased the amount of MUC5AC in the spent culture media of the treated cells. Ibudilast inhibited these hikes in MUC5AC secretion (Figs. 2A, B), suggesting that the effect of ibudilast was not dependent on the method of stimulation.

Fig. 2. Ibudilast Suppressed MUC5AC Production Induced by Various Stimuli

NCI-H292 cells were co-treated with H₂O₂ (1 mM) or Cigarette Smoke Extract (CSE) + LPS (10 µg/mL) and ibudilast (100 µM) or dexamethasone (0.1 µM) for 24 h. Secreted MUC5AC protein in the culture media were measured by ELISA. Results represent the mean ± S.E.M. (n = 3; *** p < 0.001, vs. group treated with each stimulus alone, ^††† p < 0.001 vs. non-treated group).

Ibudilast Repressed MUC5AC Gene Transcription

To further evaluate the inhibitory effect of ibudilast, we investigated its effect on TGF-α-induced MUC5AC mRNA expression. NCI-H292 cells were treated with TGF-α in combination with ibudilast. TGF-α caused a 6-fold increase in MUC5AC mRNA levels when compared with the control cells (Fig. 3A). Co-treatment of ibudilast significantly (p < 0.05) inhibited the increase in MUC5AC mRNA (Fig. 3A). We also examined whether ibudilast inhibited MUC5AC transcription. The cells were transiently transfected with a luciferase reporter plasmid containing the human MUC5AC promoter, and the effect of ibudilast was examined. In agreement with the changes in the levels of MUC5AC protein and mRNA, TGF-α activated the MUC5AC reporter gene (Fig. 3B). Ibudilast or DEX co-treatment suppressed the TGF-α-induced increase in luciferase activity (Fig. 3B), suggesting that both ibudilast and DEX inhibit MUC5AC gene transcription.

Fig. 3. Ibudilast Repressed TGF-α-Induced MUC5AC Gene Transcription

(A) NCI-H292 cells were co-treated with TGF-α (10 ng/mL) and ibudilast (100 µM) or dexamethasone (0.1 µM) for 12 h. MUC5AC mRNA were measured by qRT-PCR and normalized to GAPDH. (B) NCI-H292 cells were transfected with luciferase reporter plasmid containing human MUC5AC promoter (−3752/ + 68). Transfected cells were co-treated with TGF-α (10 ng/mL) and ibudilast (100 µM) or dexamethasone (0.1 µM) for 12 h. Cell lysates were collected and luciferase activity was measured. Results represent the mean ± S.E.M. (n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001, vs. group treated with TGF-α alone, ^††† p < 0.001 vs. non-treated group).

Ibudilast Inhibited ERK1/2 Phosphorylation Induced by TGF-α

ERK1/2 phosphorylation is a major signaling event downstream of epidermal growth factor receptor activation and it is well known to be essential for MUC5AC production. Thus, the effect of ibudilast on ERK1/2 phosphorylation was examined. TGF-α increased the levels of phosphorylated ERK1/2, which peaked at 1 h after treatment, although the increase persisted for up to 12 h. Ibudilast considerably decreased the TGF-α-induced phosphorylation of ERK1/2 at 6 and 12 h (Fig. 4A). In addition, decrease in ERK1/2 phosphorylation at 12 h was dependent on the concentration of ibudilast (Fig. 4B). To confirm that inhibition of ERK1/2 caused the decrease in MUC5AC production, the effect of U0126, a mitogen-activated protein extracellular kinase (MEK)-ERK inhibitor, was also examined. While ibudilast and DEX inhibited the increased production of MUC5AC by TGF-α, U0126 (20 µM) also showed a significant inhibitory effect (Fig. 4C). These data suggest that phosphorylation of ERK1/2 is involved in MUC5AC production by TGF-α and that ibudilast suppressed MUC5AC production through inhibition of ERK1/2 phosphorylation.

Fig. 4. Ibudilast Inhibited ERK1/2 Phosphorylation in Time- and Dose-Dependent Manner

(A) NCI-H292 cells were co-treated with TGF-α (10 ng/mL) and ibudilast (100 µM) for indicated time. (B) NCI-H292 cells were co-treated with TGF-α (10 ng/mL) and ibudilast (1–100 µM) for 12 h. Phosphorylated-signal-regulated kinase (ERK)1/2 and total-ERK1/2 in cell lysate were analyzed by Western-blotting. (C) NCI-H292 cells were co-treated with TGF-α (10 ng/mL) and ibudilast (100 µM) or MEK-ERK inhibitor U0126 (20 µM) for 24 h. Secreted MUC5AC protein in the culture media were measured by ELISA. Results represent the mean ± S.E.M. (n = 3; *** p < 0.001, vs. group treated with TGF-α alone, ^††† p < 0.001 vs. non-treated group).

Ibudilast Suppressed MUC5AC mRNA and Protein Expression in LPS-Treated Mice

To determine whether ibudilast suppresses MUC5AC production in vivo, we evaluated its effect in a mouse model of goblet cell hyperplasia induced by intratracheal instillation of LPS. The amount of MUC5AC protein in BALF was evaluated by ELISA, and the amount of Muc5ac mRNA in lung tissue was evaluated by qRT-PCR. As reported previously, MUC5AC protein and mRNA expression were markedly increased 7 d after LPS instillation, both of which were suppressed by dexamethasone.¹⁹⁾ Besides, ibudilast significantly suppressed MUC5AC protein and mRNA expression (Figs. 5A, B). These results indicated that ibudilast inhibited MUC5AC production not only in vitro but also in vivo.

Fig. 5. Ibudilast Suppressed MUC5AC mRNA and Protein Expression in LPS-Treated Mice

(A) Schematic diagram of experimental protocol. (B) MUC5AC protein in BALF was measured by ELISA. (C) The expression level of Muc5ac mRNA in lung were measured by qRT-PCR and normalized to Gapdh. Results represent the mean ± S.E.M. (n = 8–13; * p < 0.05, ** p < 0.01, vs. vehicle administered group, ^†† p < 0.01, ^††† p < 0.001 vs. healthy group).

DISCUSSION

Mucus hypersecretion is a hallmark of inflammatory airway diseases and a prominent pathophysiological feature of inflammatory respiratory diseases. Therefore, therapeutic agents that can control mucus production are important for the treatment of respiratory diseases in combination with anti-inflammatory drugs. The major purpose of this study was to determine whether ibudilast exerted any effect on MUC5AC production in NCI-H292 airway goblet-like cells. Our findings showed that ibudilast can suppress MUC5AC production and mRNA expression, both in vitro and in vivo. The effect of ibudilast is likely to be mediated, at least in part, through the inhibition of ERK1/2 phosphorylation, which is a major signaling event downstream of EGFR activation. It has been known that EGFR ligands and EGFR plays an important role in MUC5AC production, and its expression has been shown to be increased in those with inflammatory airways, such as asthmatic patients.¹⁷⁾ Therefore, inhibitors of EGFR signaling may display efficacy in treating mucus hyperproduction. In this study, ibudilast inhibited MUC5AC production induced not only by TGF-α but also by H₂O₂ and a combination of LPS and cigarette smoke extract. These data also suggest that the ERK1/2 phosphorylation, a common pathway leading to MUC5AC gene expression, is inhibited by ibudilast treatment.

In this study, we examined the effect of ibudilast on TGF-α-induced MUC5AC expression and found that this drug significantly inhibits the maximal expression of MUC5AC even at the highest dose of TGF-α used (100 ng/mL) (Fig. 1B), while the effective dose range of TGF-α seemed unaffected. Therefore, the possibility that ibudilast acted as a simple antagonist for EGFR was excluded. We also examined the effect of ibudilast on ERK1/2 phosphorylation at various incubation times. This drug decreased ERK1/2 phosphorylation at 6 and 12 h but did not inhibit phosphorylation at shorter incubation times (Fig. 4A); therefore, the possibility of ibudilast acting as a simple ERK1/2 inhibitor was also excluded.

Similar effects of PDE inhibitors have been reported previously. Mata et al. have shown that roflumilast, cilomilast, and rolipram, all of which are PDE4 inhibitors, dose-dependently inhibit MUC5AC mRNA expression in A549 lung epithelial cells.²⁰⁾ Lee et al. have also shown that rolipram inhibited MUC5AC mRNA expression in human middle ear epithelial cells that were infected with Streptococcus pneumoniae.²¹⁾ In these reports, the effects of PDE inhibitors were observed to be dependent on cAMP and protein kinase A. However, in our study, the effect of ibudilast was not abolished even in the presence of an inhibitor of protein kinase A (H-89) and dibutyryl cAMP, a cAMP analog, did not inhibited the production of MUC5AC (Supplementary Fig. 3). The reason for this discrepancy between the current and previous reports is unclear, but it may be due to difference of the cell types and the different stimuli used in these experiments. The effect of cAMP-dependent signaling on MUC5AC expression is still under debate; for example, the cAMP-response element has been identified in the 5′-promoter region of the MUC5AC gene, and IL-1ß has been shown to induce an increase in MUC5AC gene transcription through this element in NCI-H292 cells.²²⁾ In addition, blockage of the ß2-adrenergic receptor has been reported to inhibit cigarette smoke extract-induced MUC5AC mRNA expression in NCI-H292 cells by inhibiting ERK1/2 phosphorylation.²³⁾ Furthermore, the inhibition of isolated guinea pig tracheal smooth muscle contraction and thromboxane generation in eosinophils by ibudilast has been reported at much lower concentrations (about 1 µM); however, this dose of ibudilast did not affect MUC5AC production in this study.²⁴⁾ Therefore, although ibudilast is a PDE inhibitor, we hypothesize that the inhibitory effect of ibudilast on MUC5AC production and ERK1/2 phosphorylation may occur via mechanisms other than cAMP-dependent pathway.

In conclusion, we have demonstrated that ibudilast has an inhibitory effect on MUC5AC production both in vitro and in vivo and that the effect might be mediated via the inhibition of ERK1/2 phosphorylation and the subsequent inhibition of MUC5AC gene transcription. Further studies to confirm the underlying mechanism are required. Nonetheless, ibudilast has the potential to serve as a novel therapeutic option to treat mucus overproduction in airway diseases.

Acknowledgments

We are grateful to Dr. Jun Tamaoki and Dr. Mitsuko Kondo at Tokyo Women’s Medical University who greatly contributed to this study. This work was in part supported by Grant-in-Aid for Scientific Research (20K07795) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

REFERENCES

• 1) Kreda SM, Davis CW, Rose MC. CFTR, mucins, and mucus obstruction in cystic fibrosis. Cold Spring Harb. Perspect. Med., 2, a009589 (2012).
• 2) Fahy JV, Dickey BF. Airway mucus function and dysfunction. N. Engl. J. Med., 363, 2233–2247 (2010).
• 3) Dunican EM, Elicker BM, Gierada DS, et al. Mucus plugs in patients with asthma linked to eosinophilia and airflow obstruction. J. Clin. Invest., 128, 997–1009 (2018).
• 4) Rose MC, Voynow JA. Respiratory tract mucin genes and mucin glycoproteins in health and disease. Physiol. Rev., 86, 245–278 (2006).
• 5) Roy MG, Livraghi-Butrico A, Fletcher AA, et al. Muc5b is required for airway defence. Nature, 505, 412–416 (2014).
• 6) Koeppen M, McNamee EN, Brodsky KS, Aherne CM, Faigle M, Downey GP, Colgan SP, Evans CM, Schwartz DA, Eltzschig HK. Detrimental role of the airway mucin Muc5ac during ventilator-induced lung injury. Mucosal Immunol., 6, 762–775 (2013).
• 7) Evans CM, Raclawska DS, Ttofali F, Liptzin DR, Fletcher AA, Harper DN, McGing MA, McElwee MM, Williams OW, Sanchez E, Roy MG, Kindrachuk KN, Wynn TA, Eltzschig HK, Blackburn MR, Tuvim MJ, Janssen WJ, Schwartz DA, Dickey BF. The polymeric mucin Muc5ac is required for allergic airway hyperreactivity. Nat. Commun., 6, 6281 (2015).
• 8) Tagaya E, Tamaoki J, Arai N, Kondo M, Nagaoka M, Isono K, Nagaoka M, Nagai A. Effect of the PDE4 inhibitor ibudilast on sputum and postnasal drip symptoms in patients with chronic airway inflammation. The Journal of the Japanese Respiratory Society., 48, 412 (2010).
• 9) Takeyama K, Dabbagh K, Lee HM, Agustí C, Lausier JA, Ueki IF, Grattan KM, Nadel JA. Epidermal growth factor system regulates mucin production in airways. Proc. Natl. Acad. Sci. U.S.A., 96, 3081–3086 (1999).
• 10) Perrais M, Pigny P, Copin MC, Aubert JP, Van Seuningen I. Induction of MUC2 and MUC5AC mucins by factors of the epidermal growth factor (EGF) family is mediated by EGF receptor/Ras/Raf/extracellular signal-regulated kinase cascade and Sp1. J. Biol. Chem., 277, 32258–32267 (2002).
• 11) Yoshisue H, Hasegawa K. Effect of MMP/ADAM inhibitors on goblet cell hyperplasia in cultured human bronchial epithelial cells. Biosci. Biotechnol. Biochem., 68, 2024–2031 (2004).
• 12) Shao MX, Nakanaga T, Nadel JA. Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor-alpha-converting enzyme in human airway epithelial (NCI-H292) cells. Am. J. Physiol. Lung Cell. Mol. Physiol., 287, L420–L427 (2004).
• 13) Shao MX, Nadel JA. Dual oxidase 1-dependent MUC5AC mucin expression in cultured human airway epithelial cells. Proc. Natl. Acad. Sci. U.S.A., 102, 767–772 (2005).
• 14) Shao MX, Nadel JA. Neutrophil elastase induces MUC5AC mucin production in human airway epithelial cells via a cascade involving protein kinase C, reactive oxygen species, and TNF-alpha-converting enzyme. J. Immunol., 175, 4009–4016 (2005).
• 15) Shao MX, Ueki IF, Nadel JA. Tumor necrosis factor alpha-converting enzyme mediates MUC5AC mucin expression in cultured human airway epithelial cells. Proc. Natl. Acad. Sci. U.S.A., 100, 11618–11623 (2003).
• 16) Puddicombe SM, Polosa R, Richter A, Krishna MT, Howarth PH, Holgate ST, Davies DE. Involvement of the epidermal growth factor receptor in epithelial repair in asthma. FASEB J., 14, 1362–1374 (2000).
• 17) Takeyama K, Fahy JV, Nadel JA. Relationship of epidermal growth factor receptors to goblet cell production in human bronchi. Am. J. Respir. Crit. Care Med., 163, 511–516 (2001).
• 18) Takami S, Mizuno T, Oyanagi T, Tadaki H, Suzuki T, Muramatsu K, Takizawa T, Arakawa H. Glucocorticoids inhibit MUC5AC production induced by transforming growth factor-α in human respiratory cells. Allergol. Int., 61, 451–459 (2012).
• 19) Nishimoto Y, Hisatsune A, Katsuki H, Miyata T, Yokomizo K, Isohama Y. Glycyrrhizin attenuates mucus production by inhibition of MUC5AC mRNA expression in vivo and in vitro. J. Pharmacol. Sci., 113, 76–83 (2010).
• 20) Mata M, Sarriá B, Buenestado A, Cortijo J, Cerdá M, Morcillo EJ. Phosphodiesterase 4 inhibition decreases MUC5AC expression induced by epidermal growth factor in human airway epithelial cells. Thorax, 60, 144–152 (2005).
• 21) Lee J, Komatsu K, Lee BC, Lim JH, Jono H, Xu H, Kai H, Zhang ZJ, Yan C, Li JD. Phosphodiesterase 4B mediates extracellular signal-regulated kinase-dependent up-regulation of mucin MUC5AC protein by Streptococcus pneumoniae by inhibiting cAMP-protein kinase A-dependent MKP-1 phosphatase pathway. J. Biol. Chem., 287, 22799–22811 (2012).
• 22) Chen Y, Garvin LM, Nickola TJ, Watson AM, Colberg-Poley AM, Rose MC. IL-1β induction of MUC5AC gene expression is mediated by CREB and NF-κB and repressed by dexamethasone. Am. J. Physiol. Lung Cell. Mol. Physiol., 306, L797–L807 (2014).
• 23) Zhou Y, Zhang Y, Guo Y, Zhang Y, Xu M, He B. β2-Adrenoceptor involved in smoking-induced airway mucus hypersecretion through β-arrestin-dependent signaling. PLOS ONE, 9, e97788 (2014).
• 24) Souness JE, Villamil ME, Scott LC, Tomkinson A, Giembycz MA, Raeburn D. Possible role of cyclic AMP phosphodiesterases in the actions of ibudilast on eosinophil thromboxane generation and airways smooth muscle tone. Br. J. Pharmacol., 111, 1081–1088 (1994).