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MUC4 is a predominant membrane-tethered mucin lubricating and protecting the epithelial surface and playing various biological roles in the renewal and differentiation of epithelial cells, cell signaling, cell adhesion, and carcinogenesis. Interestingly, recent studies have demonstrated that MUC4 expression regulates the epithelial-mesenchymal transition (EMT) of cancer cells in ovarian, pancreatic, and lung cancer. However, the effects of MUC4 expression on EMT in human airway epithelial cells are not yet well known. Here, we describe the effects of transforming growth factor beta 1 (TGF-β1)-induced MUC4 expression on EMT and evaluate its downstream signaling pathway in human airway epithelial cells. In human airway epithelial NCI-H292 cells, exposure to TGF-β1 induced expression of MUC4, CDH2, VIM and SNAI1 genes and encoded by them proteins, MUC4, N-cadherin, vimentin and Snail, and reduced the level of CDH1 and its product, E-cadherin. In MUC4-knockdown cells, TGF-β1-induced expression levels of MUC4, CDH2, VIM and SNAI1 and corresponding proteins were suppressed, but CDH1 and E-cadherin levels were not. In addition, TGF-β1-induced phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2) was suppressed, but that of Smad2/3, Akt, and p38 was not. The results of this study suggest that MUC4 silencing inhibits TGF-β1 -induced EMT via the ERK1/2 pathway, and a possible role of MUC4 in the induction of EMT in human airway epithelial cells.

Mucus plays an important role in protecting the respiratory tract from irritants. However, mucus hypersecretion is a major indicator of airway diseases. 1,2-Benzisothiazolin-3-one (BIT), as a microbicide, induces asthmatic inflammation. Therefore, we focused on the effects of BIT-related mucin secretion in airway epithelial cells. Our in vivo study showed increased mucus and MUC5AC expressions in the bronchioles of mice that inhaled BIT. For investigating the signaling pathways, we performed experiments in human airway epithelial cells. BIT induced the MUC5AC expression and significantly increased the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The specific inhibitors of ERK1/2, p38, and NF-κB blocked the BIT-induced MUC5AC expression. Therefore, these results suggest that BIT induces the MUC5AC expression via the ERK1/2, p38, and NF-κB pathways in human airway epithelial cells, which may be involved in mucus hypersecretion associated with airway inflammatory diseases.

Background Glyoxal (GO), and methylglyoxal (MGO) are among the most toxic compounds emitted by electronic cigarette (E-cig) and regular tobacco cigarette smoke. Airway diseases presented mucus over production as their major pathophysiologic feature. However, the effects of GO and MGO on pro-inflammatory cytokines and mucin expression in human nasal epithelial cells, as well as the underlying signaling pathway, have not yet been studied. Objective This study is to determine whether GO and MGO induce pro-inflammatory cytokines, and MUC5AC/5B expression via mitogen-activated protein kinase (MAPK)s and nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways. Methods The effect of GO, and MGO on pro-inflammatory cytokines, mucins expression and the signalling pathway of GO and MGO were investigated using water-soluble tetrazolium salt-1, enzyme immunoassays, and immunoblot analysis with specific inhibitors and small interfering RNA. Results GO and MGO did not affect cell viability up to 2 mM in human nasal epithelial cells. GO and MGO increased production of pro-inflammatory such as interleukin (IL)-1β and IL-6) and MUC5AC/5B. Additionally, GO and MGO significantly activated extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, and NF-κB. Whether ERK1/2, p38 MAPK, and NF-κB signaling pathway were involved in GO and MGO-induced production of pro-inflammatory cytokines (IL-1β and IL-6) and MUC5AC/5B, we used specific inhibitors and siRNA transfection. These significantly repressed GO- and MGO-induced expression of pro-inflammatory cytokines (IL-1β and IL-6) and MUC5AC/5B. Conclusions GO and MGO induced pro-inflammatory cytokines and MUC5AC/5B expression via ERK1/2, p38 MAPK, and NF-κB in human nasal epithelial cells. These results suggested that GO and MGO may be involved in mucus hypersecretion-related airway diseases.

Background: Mucin 5AC, oligomeric mucus/gel-forming (MUC5AC) expression is significantly increased in allergic and inflammatory airway diseases. Interleukin (IL) 36 gamma is predominantly expressed in airway epithelial cells and plays an important role in innate and adaptive immune responses. IL-36 gamma is induced by many inflammatory mediators, including cytokines and bacterial and viral infections. However, the association between IL-36 gamma and mucin secretion in human airway epithelial cells has not yet been fully investigated. Objective: The objective of this study was to determine whether IL-36 gamma might play a role in the regulation of mucin secretion in airway epithelial cells. We investigated the effect and brief signaling pathway of IL-36 gamma on MUC5AC expression in human airway epithelial cells. Methods: Enzyme immunoassay, immunoblot analysis, immunofluorescence staining, reverse transcriptase–polymerase chain reaction (PCR), and real-time PCR were performed in mucin-producing human airway epithelial NCI-H292 cells and in human nasal epithelial cells after pretreatment with IL-36 gamma, several specific inhibitors, or small interfering RNAs (siRNA). Results: IL-36 gamma induced MUC5AC expression and activated the phosphorylation of extracellular signal regulated kinase (ERK) 1 and 2, p38, and nuclear factor-kappa-light-chain-enhancer of activated B cells (NF–kappa B). IL-36 receptor antagonist significantly attenuated these effects. The specific inhibitor and siRNA of ERK1, ERK2, p38, and NF–kappa B significantly attenuated IL-36 gamma induced MUC5AC expression. Conclusion: These results indicated that IL-36 gamma induced MUC5AC expression via the IL-36 receptor–mediated ERK1/2 and p38/NF–kappa B pathway in human airway epithelial cells.

Mucus plays an important role in protecting the respiratory tract from irritants. However, mucus hypersecretion is a major indicator of airway diseases. 1,2-Benzisothiazolin-3-one (BIT), as a microbicide, induces asthmatic inflammation. Therefore, we focused on the effects of BIT-related mucin secretion in airway epithelial cells. Our in vivo study showed increased mucus and MUC5AC expressions in the bronchioles of mice that inhaled BIT. For investigating the signaling pathways, we performed experiments in human airway epithelial cells. BIT induced the MUC5AC expression and significantly increased the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The specific inhibitors of ERK1/2, p38, and NF-κB blocked the BIT-induced MUC5AC expression. Therefore, these results suggest that BIT induces the MUC5AC expression via the ERK1/2, p38, and NF-κB pathways in human airway epithelial cells, which may be involved in mucus hypersecretion associated with airway inflammatory diseases.

Exposure to diesel exhaust particles (DEPs) is known to cause serious health problems, owing to a steady increase in the number of diesel vehicles worldwide. DEPs comprise approximately 90% particle mass existing in the fine size range (≤2.5 μm) and are mainly absorbed in the respiratory tract. However, limited information is available on the effects of DEP exposure on the respiratory tract in humans. Here, we investigated the effect and signaling pathways of DEPs on the expression of mucin, especially MUC5AC and MUC5B, in human airway epithelial cells by reverse-transcriptase polymerase chain reaction (PCR), real-time PCR, enzyme-linked immunosorbent assay, western blotting, and immunofluorescence staining. The signaling pathways activated following DEP-induced expression of MUC5AC and MUC5B in airway epithelial cells were analyzed by evaluating Toll-like receptor 4 (TLR4), mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase 1/2 [ERK1/2] and p38), and nuclear factor kappa B (NF-κB) phosphorylation with western blot and small-interfering RNA (siRNA) analyses. DEPs significantly increased MUC5AC and MUC5B expression in human airway epithelial cells that was closely related to TLR4, MAPK (ERK 1/2 and p38), and NF-κB pathway activation. This is the first report to demonstrate the DEP-mediated increase in MUC5AC and MUC5B expression via the TLR4-mediated activation of ERK1/2, p38 MAPK, and NF-κB signaling pathways in human airway epithelial cells.

Objectives Endoplasmic reticulum (ER) stress is known to be associated with inflammatory airway diseases, and three major transmembrane receptors: double-stranded RNA-activated protein kinase-like ER kinase, inositol requiring enzyme 1, and activating transcription factor 6 (ATF6) play important roles in ER stress-related proinflammatory signaling. However, the effects of ER stress and these three major signaling pathways on the regulation of the production of airway mucins in human nasal airway epithelial cells have not been elucidated. Methods In primary human nasal epithelial cells, the effect of tunicamycin (an ER stress inducer) and 4-phenylbutyric acid (4-PBA, ER stress inhibitor) on the expression of MUC5AC and MUC5B was investigated by reverse transcriptase-polymerase chain reaction, real-time polymerase chain reaction, enzyme immunoassay, and immunoblot analysis. Small interfering RNA (siRNA) transfection was used to identify the mechanisms involved. Results Tunicamycin increased the expressions of MUC5AC and MUC5B and the mRNA expressions of ER stress-related signaling molecules, including spliced X-box binding protein 1 (XBP-1), transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP), and ATF6. In addition, 4-PBA attenuated the tunicamycin-induced expressions of MUC5AC and MUC5B and the mRNA expressions of ER stress-related signaling molecules. Furthermore, siRNA knockdowns of XBP-1, CHOP, and ATF6 blocked the tunicamycin-induced mRNA expressions and glycoprotein productions of MUC5AC and MUC5B. Conclusion These results demonstrate that ER stress plays an important role in the regulation of MUC5AC and MUC5B via the activations of XBP-1, CHOP, and ATF6 in human nasal airway epithelial cells.

Background β-Glucan is found in the cell walls of fungi, bacteria, and some plant tissues, and is detected by the innate immune system. Furthermore, this recognition is known to worsen respiratory symptoms in patients with allergic and inflammatory airway diseases. However, the means by which β-glucan affects the secretion of major mucins by human airway epithelial cells has not been elucidated. Therefore, in this study, the effect and signaling pathway of β-glucan on mucins MUC4 and MUC5B were investigated in human airway epithelial cells. Methods In NCI-H292 cells and human normal nasal epithelial cells, the effect and signaling pathway of β-glucan on MUC4 and MUC5B expression were investigated using reverse transcriptase–polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with specific inhibitors and small interfering RNA (siRNA). Results β-Glucan increased MUC4 and MUC5B expression and activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). SB203580 (a p38 MAPK inhibitor) and pyrrolidine dithiocarbamate (PDTC; a NF-κB inhibitor) inhibited β-glucan–induced MUC4 and MUC5B expression. In addition, siRNA knockdown of p38 MAPK blocked β-glucan–induced MUC4 and MUC5B mRNA expression and β-glucan–activated phosphorylation of NF-κB. Furthermore, Toll-like receptor 4 (TLR4) mRNA expression was increased by β-glucan, and siRNA knockdown of TLR4 blocked β-glucan–induced MUC4 and MUC5B mRNA expression and β-glucan–activated phosphorylation of p38 MAPK and NF-κB. Conclusion These results demonstrate that in human airway epithelial cells β-glucan induces MUC4 and MUC5B expression via the TLR4–p38 MAPK–NF-κB signaling pathway.

Adipokines, a group of proteins including leptin, visfatin, resistin, and adiponectin, are produced by adipocytes. Among adipokines, resistin is implicated in insulin resistance and inflammatory response modulation. Mucus hypersecretion has been greatly linked to airway diseases, such as asthma, chronic obstructive pulmonary disease, and rhinosinusitis. Increasing evidence has indicated that adipokines, such as leptin and visfatin, play important regulatory roles in various biological processes involved in mucus secretion. However, the effects of resistin on mucin expression in human airway epithelial cells, as well as the underlying mechanisms, have not been investigated yet. We showed that resistin affected mucin expression in human airway epithelial cells via the mitogen-activated protein kinase/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Resistin increased MUC5AC and MUC5B expression in NCI-H292 and primary human nasal epithelial cells. Additionally, it significantly increased the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and NF-κB. ERK1/2 and p38 specific inhibitors significantly attenuated resistin-induced MUC5AC/5B expression; however, NF-κB inhibitor reduced resistin-induced MUC5AC, but not MUC5B, expression. Knockdown of ERK1, ERK2, and p38 by ERK1, ERK2, and p38 small interfering RNA (siRNA), respectively, significantly blocked resistin-induced MUC5AC and MUC5B mRNA expression. In addition, NF-κB siRNA attenuated resistin-induced MUC5AC, but not MUC5B, expression. These results suggested that resistin induced MUC5AC and MUC5B expression via activation of different signaling pathways in human airway epithelial cells.

Objectives Doxycycline is commonly used in medicine for its bacteriostatic antimicrobial properties. Recent studies have reported that doxycycline also has anti-inflammatory effects. Matrix metalloproteinase (MMP)-9 has been found to be involved in the physiological and pathological process of inflammatory airway disease. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, is known to stimulate the expression of MMP and mucin genes in the airway and intestinal epithelial cells. Therefore, the effects and signal pathways of doxycycline on PMA-induced MUC5B expression dependent MMP-9 in human airway epithelial cells were investigated. Methods In human NCI-H292 airway epithelial cells, MUC5B and MMP-9 mRNA expression, MUC5B protein expression, and MMP-9 protein activity after the treatment with PMA, MMP-9 or doxycycline were determined by reverse transcriptase-polymerase chain reaction, enzyme immunoassay, gelatin zymography, and Western blot analysis. Results PMA increased MMP-9 and MUC5B expression. MMP-9 increased MUC5B expression. Doxycycline inhibited PMA-induced MUC5B expression, and PMA-induced MMP-9 mRNA expression and protein activity. Doxycycline inhibited phosphorylation of p38 induced by PMA and MMP-9. Conclusion The results of this study suggest that doxycycline inhibited PMA-induced MUC5B mRNA expression and protein production through the MMP-9 and p38 pathways in human NCI-H292 airway epithelial cells.

Garlic has been shown to have antimicrobial, hypolipidemic, antithrombotic, antitumor and immunostimulatory properties. The medicinal effects of garlic are derived from the flavonoid and organosulfur components. Diallyl disulfide (DADS), an organosulfur, is the main component responsible for the diverse biological effects of garlic. However, the effects of DADS on mucin gene expression in airway epithelial cells have not been reported to date. Therefore, this study was performed to investigate the effects and brief signaling pathway of DADS associated with MUC5B expression in NCI-H292 epithelial cells using RT-PCR, ELISA, western blot, immunocytochemistry and cell transfection with siRNA. DADS induced MUC5B expression and activated the phosphorylation of ERK1/2 MAPK. In addition, U0126 inhibited DADS-induced MUC5B expression and DADS-activated phosphorylation of ERK1/2 MAPK. Moreover, the immunopositive cells for MUC5B protein did not appear after treatment of DADS with U0126, and the knockdown of ERK2 MAPK by ERK2 MAPK siRNA significantly blocked DADS-induced MUC5B mRNA expression. However, DADS did not activate the phosphorylation of p38 MAPK, and SB203580 did not inhibit DADS-induced MUC5B expression. This is the first study to show that DADS-induced MUC5B expression appears to be regulated by activation of the ERK2 MAPK signaling pathway in human NCI-H292 airway epithelial cells. Copyright © 2011 John Wiley & Sons, Ltd.

Leptin, an adipocyte-secreted hormone that regulates food intake and metabolic response, has been recently reported to increase in the serum during inflammatory airway disease associated with mucus-hypersecretion. We investigated the effects of leptin on mucin expression in human airway epithelial cells and the signaling pathways. The expression of the leptin receptor was evaluated in human nasal mucosa and NCI-H292 cells. Leptin-induced expression of major respiratory mucins in NCI-H292 cells was analyzed. Mutant leptin, which acts as a receptor antagonist, and specific inhibitors of extracellular signal-regulated kinase (ERK1/2), p38 and Janus kinase-2 (JAK2)/signal transducer and activator of transcription-3 (STAT3) were used. Leptin receptors were expressed in the nasal mucosa and NCI-H292 cells. Treatment with leptin significantly increased the expression of MUC5AC and MUC5B in NCI-H292 cells; these effects were blocked by mutant leptin. The cells activated by leptin showed increased ERK1/2, p38, and STAT3 phosphorylation. Leptin-induced MUC5B expression was blocked by the ERK1/2 and p38 pathway inhibitors, but not by the JAK2/STAT3 pathway inhibitor. Leptin might significantly contribute to the production of major gel-forming mucins by direct stimulation of airway epithelial cells and the activation of leptin receptors coupled with the activation of ERK1/2 or p38, but not the JAK2/STAT3 pathway.

Background Glutaredoxins (GRX)-1 is glutathione-dependent oxidoreductase. However, the role of these enzymes remains unknown in airway inflammatory diseases. Therefore, we aimed to establish the expression pattern of GRX-1 in the nasal polyps (NPs) and to assess the regulatory mechanisms associated with GRX-1 expression in interleukin (IL)-1beta-treated airway epithelial cells. Methods The expression of GRX-1 in NPs and normal nasal mucosa were analyzed by reverse-transcription polymerase chain reaction and immunohistochemical staining. IL-1beta–induced reactive oxygen species (ROS) formation and GRX-1 expression in the airway epithelial cells was determined by flow cytometry and immunoassay. Results The expression level of GRX-1 in NPs was significantly higher than in the normal nasal mucosa (p < 0.05). GRX-1 was highly expressed in the surface epithelial cells and the submucosal glandular cells in the NPs. IL-1beta increased the intracellular ROS formation and GRX-1 expression in airway epithelial cells. The inhibition of IL-1beta–induced ROS production by N-acetyl-cystein, an ROS scavenger, reduced GRX-1 expression. Diphenyleneiodonium and apocynin, NADPH oxidase inhibitors, did not abolish IL-1beta–induced ROS ormation and GRX-1 expression, whereas budesonide attenuated it. Conclusion High GRX-1 expression in NPs might be a primary de ense against chronic in lammatory oxidative stress in nasal mucosa. IL-1beta–induced up-regulation o GRX-1 in airway epithelial cells is probably mediated by ROS. Glucocorticoids can regulate IL-1beta–induced ROS ormation and GRX-1 expression.

Objectives/Hypothesis: Among the airway mucin genes, the MUC4 gene is an important gene in its response to inflammatory diseases of the upper airway. However, the expression and regulation of the MUC4 gene in the nasal polyps remains unclear. Study Design: The purpose of this study was to evaluate the expression of MUC4 mRNA and synthesis of mucin glycoprotein in the nasal polyps before and after treatment with a topical steroid in vivo and in vitro. Methods: Nasal polyps were obtained from 20 patients with chronic rhinosinusitis and were subsequently cultured. The level of MUC4 mRNA was measured by reverse-transcription polymerase chain reaction, and the amount of the MUC4 mucin glycoprotein was estimated by the enzyme-linked immunosorbent assay method. Results: The expression of MUC4 mRNA was found to be significantly higher in the nasal polyps than in the inferior turbinate (P < .05). The addition of interleukin (IL)-1β and lipopolysaccharide (LPS) increased the expression of MUC4 mRNA and mucin glycoprotein synthesis in cultured nasal polyp epithelial cells. Treatment with glucocorticoid inhibited the expression of MUC4 mRNA in the nasal polyps; it also inhibited the expression of IL-1β and LPS-induced MUC4 mRNA and mucin glycoprotein synthesis in cultured nasal polyp epithelial cells. The inhibitory effects of glucocorticoid were restored by treatment with a glucocorticoid receptor antagonist (RU-486). Conclusions: These results suggest that the MUC4 gene is expressed in the nasal polyps and that glucocorticoid can control the expression of the MUC4 gene and mucin glycoprotein synthesis.

Background Among a variety of inflammatory mediators, visfatin is a proinflammatory adipocytokine associated with inflammatory reactions in obesity, metabolic syndrome, chronic inflammatory disease, and autoimmune disease. However, the biological role of visfatin in secretion of major mucins in human airway epithelial cells has not been reported. Therefore, this study was conducted in order to investigate the effect and the brief signaling pathway of visfatin on MUC8 and MUC5B expression in human airway epithelial cells. Results Visfatin significantly induced MUC8 and MUC5B expression. Visfatin significantly activated phosphorylation of p38 MAPK. Treatment with SB203580 (p38 MAPK inhibitor) and knockdown of p38 MAPK by siRNA significantly blocked visfatin-induced MUC8 and MUC5B expression.Visfatin significantly increased ROS formation. Treatment with SB203580 significantly attenuated visfatin-induced ROS formation. Treatment with NAC (ROS scavenger) and DPI (NADPH oxidase inhibitor) significantly attenuated visfatin-induced MUC8 and MUC5B expression. However, treatment with NAC and DPI did not attenuate visfatin-activated phosphorylation of p38 MAPK. Visfatin significantly activated the phosphorylation of NF-κB. Treatment with PDTC (NF-κB inhibitor) significantly attenuated visfatin-induced MUC8 and MUC5B expression. Conclusions These results suggest that visfatin induces MUC8 and MUC5B expression through p38 MAPK/ROS/NF-κB signaling pathway in human airway epithelial cells.

Thymic stromal lymphopoietin (TSLP) is an interleukin 7-like cytokine and a potent factor for B- and T-cell growth and differentiation. Recent studies have demonstrated an association of TSLP with allergic and inflammatory airway diseases. However, no study on the effect of TSLP on expression of mucin genes in airway epithelial cells has been reported. Therefore, the effects and brief signaling pathways of TSLP on expression of mucin genes in human airway epithelial cells were investigated in this study. In mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the effect and signaling pathway of TSLP on expression of mucin genes were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). In human NCI-H292 airway epithelial cells, TSLP increased MUC5B expression. TSLP significantly activated phosphorylation of ERK1/2 and p38 mitogen-activated protein kinase (MAPK). U0126 (ERK1/2 MAPK inhibitor) and SB203580 (p38 MAPK inhibitor) significantly attenuated TSLP-induced MUC5B mRNA expression. Knockdown of ERK1, ERK2, and p38 MAPK by ERK1, ERK2, and p38 MAPK siRNA significantly blocked TSLP-induced MUC5B mRNA expression. In the primary cultures of normal nasal epithelial cells, TSLP significantly increased MUC5B mRNA expression, which was significantly attenuated after pretreatment with U0126 and SB203580. These results suggest that TSLP induces MUC5B expression via the ERK1/2 and p38 MAPK signaling pathway in human airway epithelial cells.

Objective: Mucus plays an important role in protecting human airways from external environments by entrapping inhaled foreign particles and bacteria, and clearing them from the airways by ciliary movement. Epigallocatechin-3-gallate (EGCG) is a major component of green tea extract and known to provide numerous functions, such as anti-oxidant effect, anti-tumor effect, anti- diabetic effect, and anti-inflammatory effect. However, the precise mechanism of action about anti-inflammatory effect of EGCG is not fully defined.Method: The effect of EGCG on phorbol 12-myrisate 13- acetate (PMA)–induced MUC5B expression and signaling pathways were investigated in human NCI-H292 airway epithelial cells, human nasal epithelial cells, and in asthma model of mice.Results: PMA-induced MUC5B mRNA and protein levels were significantly decreased after treatment with EGCG. PMA increased phosphorylation of p38 MAPK and this was significantly decreased after pretreatment with EGCG. PMA induced MMP-9 overexpression and this wa...

Adenosine monophosphate-activated protein kinase (AMPK) is a well-known serine/threonine kinase that has been implicated in modulation of glucose and fatty acid metabolism. Recent reports have also implicated AMPK in modulation of mucin secretion. In this study, the effects and signaling pathways of AMPK on MUC5B expression were investigated in human NCI-H292 airway epithelial cells. Metformin, as an activator of AMPK, induced MUC5B expression in a dose-dependent manner. Compound C, as an inhibitor of AMPK, inhibited metformin-induced MUC5B expression in a dose-dependent manner. Metformin significantly activated phosphorylation of AMPK; compound C inhibited metformin-activated phosphorylation of AMPK. Without treatment with metformin, there was no difference in MUC5B mRNA expression between Ad-dnAMPK transfected and wild-type adenovirus transfected NCI-H292 cells. However, after treatment with metformin, MUC5B mRNA expression was increased in wild-type adenovirus transfected NCI-H292 cells; MUC5B mRNA expression was significantly decreased in Ad-dnAMPK transfected NCI-H292 cells. Metformin activated phosphorylation of p38 mitogen-activated protein kinase (MAPK); compound C inhibited metformin-activated phosphorylation of p38 MAPK. SB203580, as an inhibitor of p38 MAPK, significantly inhibited metformin-induced MUC5B mRNA expression, while U0126, as an inhibitor of ERK1/2 MAPK, had no effect. In addition, knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked metformin-induced MUC5B mRNA expression. In conclusion, results of this study show that AMPK induces MUC5B expression through the p38 MAPK signaling pathway in airway epithelial cells.

Objective To acquire basic information concerning the function of the membrane-bound mucin MUC16 in nasal mucosa compared with the best-characterized membrane-bound mucin, MUC4. Design In vitro study using semiquantatitive reverse transcription–polymerase chain reaction analysis and immunoassay. Setting Yeungnam University College of Medicine. Subjects We examined the nasal polyps obtained during endoscopic sinus surgery in 10 patients, the normal ethmoid sinus mucosa obtained from 10 patients, and human nasal polyp epithelial (HNPE) cells. Main Outcome Measures Gene expression of MUC4 and MUC16 in nasal polyps and normal nasal mucosa. In addition, we evaluated the effect of 4 physiologically relevant agents, including retinoic acid, interleukin 1β, phorbol 12-myristate 13-acetate (PMA), and dexamethasone, on the expression of MUC4 and MUC16 in HNPE cells at the gene and protein levels. Results In nasal polyps, MUC4 was upregulated compared with normal nasal mucosa ( P = .009), whereas MUC16 expression did not differ between nasal polyps and normal nasal mucosa. Retinoic acid and interleukin 1β increased MUC4 expression at the gene and protein level in HNPE cells, whereas MUC16 expression was not affected. Unlike retinoic acid and interleukin 1β, PMA and dexamethasone increased MUC16 expression, whereas they had no significant effect on MUC4 expression. Conclusions Expression of MUC4 and MUC16 are regulated differently in nasal mucosa. Dexamethasone and PMA are potent mediators for the expression of MUC16 in nasal polyps.

Objectives Mucin gene expression and mucin production are markedly increased in inflammatory airway disorders such as asthma, chronic bronchitis and rhinosinusitis. Cytokines, lipopolysaccharides and other inflammatory mediators such as prostaglandin and leukotriene are related to the secretion and production of mucin. However, the relationship of leukotrienes with mucin genes expression is not clear. The aim of this study is to evaluate MUC2/5AC gene expression and mucin secretion by the leukotriene receptor in human airway epithelial cells. Methods The effect of leukotriene D4 and the leukotriene receptor antagonist, pranlukast hydrate (ONO-1078) on the regulation of MUC2/5AC gene expression and mucin secretion were observed in human airway NCI-H292 epithelial cells. The mRNA levels of MUC2/5AC and the amount of mucin were determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunoassay. Results Leukotriene D4 upregulated MUC2/5AC gene expression and mucin secretion in a dose dependent pattern. Pranlukast hydrate (ONO-1078, 100 μM) downregulated the leukotriene D4-induced MUC2/5AC gene expression and mucin secretion. Conclusion These results suggest that the leukotriene receptor system is one of the mechanisms related to MUC2/5AC gene expression and mucin secretion in the human airway epithelium.

Received April 15, 2015 Revised June 3, 2015 Accepted June 3, 2015 Address for correspondence Yong-Dae Kim, MD, PhD Department of OtorhinolaryngologyHead and Neck Surgery, College of Medicine, Yeungnam University, 170 Hyeonchung-ro, Nam-gu, Daegu 42415, Korea Tel +82-53-620-3781 Fax +82-53-628-7884 E-mail ydkim@med.yu.ac.kr Background and ObjectivesZZPolyinosinic-polycytidylic acid (Poly I:C) is structurally similar to double-stranded RNA, and is known to induce various inflammatory mediators and to cause inflammatory reactions in airway epithelial cells. However, the effect of Poly I:C on secretion of mucins in human airway epithelial cells has been very rarely reported. In this study, the effect and brief signaling pathway of Poly I:C on the expression of mucin genes were investigated in human airway epithelial cells. Materials and MethodZZIn mucin-producing human NCI-H292 airway epithelial cells and the primary cultures of normal human nasal epithelial cells, the effect and signaling pathway of Poly I:C on expression of mucin genes were investigated using reverse transcriptase-polymerase chain reaction, real-time PCR, enzyme immunoassay, and immunoblot analysis with specific inhibitors and small interfering RNA (siRNA) for mitogen-activated protein kinase (MAPK). ResultsZZPoly I:C induced the MUC5B expression, and activated the phosphorylation of ERK1/2 and p38 MAPK. U0126 (ERK1/2 MAPK inhibitor) and SB203580 (p38 MAPK inhibitor) inhibited the Poly I:C-induced MUC5B expression. In addition, the knockdown of ERK2 and p38 MAPK by siRNA significantly blocked the Poly I:C-induced MUC5B mRNA expression. ConclusionZZPoly I:C induces the MUC5B expression via ERK2 and p38 MAPK signaling pathways in human airway epithelial cells. Therefore, Poly I:C may play a role in the regulation of mucus hypersecretion through MAPK signaling pathways in the human airway epithelial cells. Korean J Otorhinolaryngol-Head Neck Surg 2015;58(9):615-21

This study demonstrates for the first time the effects of IL-1beta on the regulation of protein production as well as MUC2 gene transcription in cultured human airway epithelial cells. The effect of IL-1beta on the regulation of MUC2 protein was determined by flow cytometric analysis. The expression level of MUC2 induced by IL-1beta increased in a dose-dependent manner. MUC2 transcripts were detected after 2 h of exposure to IL-1beta and reached maximal level after 8 h. Actinomycin D experiments indicated that the IL-1beta-mediated MUC2 expression was controlled by transcriptional regulation. Both RT-PCR and FACS analysis showed that budesonide concomitantly attenuated IL-1beta mediated MUC2 gene as well as protein production levels. Use of the glucocorticoid receptor antagonist, RU-486, restored the inhibitory effect of budesonide on the IL-1beta-mediated MUC2 protein as well as gene. The data suggest that IL-1beta up-regulates MUC2 gene by transcriptional regulation and that budesonide suppresses the IL-1beta-medicated MUC2 expression via decreased transcriptional activation.

Objectives Delphinidin is one of the anthocyanidins. It is believed to have anti-inflammatory property including antioxidant, antiangiogenic, and anti-cancer properties. However, the anti-inflammatory effect of delphinidin in mucin-producing human airway epithelial cells has not been determined. Therefore, this study was conducted in order to investigate the effect and the brief signaling pathway of delphinidin in lipopolysaccharide (LPS)-induced MUC8 and MUC5B expression in human airway epithelial cells. Methods In mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay were used for investigating the expressions of MUC8, MUC5, and Toll-like receptor 4 (TLR4), after LPS treatment and delphinidin treatment. And the signaling pathway of delphinidin on LPS-induced MUC8 and MUC5B expression was investigated using the RT-PCR, and immunoblot analysis. To confirm the involvement of TLR4 in LPS-induced MUC8 and MU5B expression, the cells were transfected with TLR4 siRNA. Results In NCI-H292 airway epithelial cells, LPS (100 ng/mL) significantly induced TLR4, MUC8, and MUC5B expression. TLR4 siRNA significantly blocked LPS-induced MUC8 and MUC5B mRNA expression. LPS (100 ng/mL) significantly activated the phosphorylation of extracellular signal related kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK). Delphinidin (50 and 100 µM) inhibited LPS-induced TLR4, MUC8, and MUC5B expression and LPS-induced phosphorylation of ERK1/2 and p38 MAPK. In the primary cultures of normal nasal epithelial cells, delphinidin (50 and 100 µM) significantly inhibited LPS-induced TLR4, MUC8, and MUC5B gene expression. Conclusion These results suggest that delphinidin attenuates LPS-induced MUC8 and MUC5B expression through the TLR4-mediated ERK1/2 and p38 MAPK signaling pathway in human airway epithelial cells. These findings indicated that delphinidin may be a therapeutic agent for control of inflammatory airway diseases.

Received June 23, 2014 Revised July 23, 2014 Accepted July 24, 2014 Address for correspondence Yong-Dae Kim, MD, PhD Department of OtorhinolaryngologyHead and Neck Surgery, College of Medicine, Yeungnam University, 170 Hyeonchung-ro, Nam-gu, Daegu 705-802, Korea Tel +82-53-620-3781 Fax +82-53-628-7884 E-mail ydkim@med.yu.ac.kr Background and ObjectivesZZRoflumilast, a selective inhibitor of phosphodiesterase type 4, has an anti-inflammatory property. It has been used in the treatment of chronic inflammatory airway diseases such as chronic obstructive pulmonary disease and asthma. However, the effect of roflumilast on mucus secretion in inflammatory airway epithelial cells has not been reported. Therefore, this study was aimed at investigating the effects of roflumilast on the inflammatory mediator-induced MUC5AC and MUC5B expression in human airway epithelial cells. Materials and MethodZZIn human mucin-producing NCI-H292 airway epithelial cells and primary cultures of nasal epithelial cells, the effects of roflumilast on lipopolysaccharide (LPS)and phorbl-12-myrsitate-13-acetate (PMA)-induced MUC5AC and MUC5B expression were analyzed by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. ResultsZZRoflumilast attenuated LPS-induced MUC5AC and MUC5B mRNA and glycoprotein expression in NCI-H292 cells. And roflumilast attenuated PMA-induced MUC5AC and MUC5B mRNA and glycoprotein expression in NCI-H292 cells. In addition, roflumilast attenuated LPS and PMA-induced MUC5AC and MUC5B mRNA expression in the primary cultures of nasal epithelial cells. ConclusionZZThese results suggest that roflumilast attenuates MUC5AC and MUC5B expressions in airway epithelial cells. Roflumilast may be a potentially ideal therapeutic agent for the control of mucus-hypersecretion in treating chronic inflammatory airway diseases. Korean J Otorhinolaryngol-Head Neck Surg 2014;57(12):830-5

Background and Objectives MUC5AC and MUC5B are representative secretory mucin genes in the human airway. MUC5AC and MUC5B expression are increased by a variety of inflammatory mediators. Betulinic acid, a naturally occurring pentacyclic triterpenoid, is known to have an anti-inflammatory property. However, the effects of betulinic acid on mucin secretion of airway epithelial cells still have not been reported. Therefore, in this study, the effect of betulinic acid on inflammatory mediators-induced MUC5AC and MUC5B expression was investigated in human airway epithelial cells. Materials and Method In the mucin-producing human NCI-H292 airway epithelial cells, the effects of betulinic acid on interleukin-1β (IL-1β)-, lipopolysaccharide (LPS)-, and phorbol myristate acetate (PMA)-induced MUC5AC and MUC5B expression were analyzed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Results Betulinic acid attenuated IL-1β-, LPS- and PMA-induced MUC5B mRNA and glycoprotein expression in NCI-H292 cells. Betulinic acid did not attenuate IL-1β-, LPS- and PMA-induced MUC5AC mRNA and glycoprotein expression in NCI-H292 cells. Conclusion These results suggest that betulinic acid attenuates IL-1β-, LPS- and PMA-induced MUC5B expression in airway epithelial cells. Therefore, betulinic acid may modulate a control of mucus-hypersecretion in airway inflammatory disease. Korean J Otorhinolaryngol-Head Neck Surg 2014;57(8):526-32

Objectives Among the inflammatory mediators, phorbol 12-myristate 13-acetate (PMA) is associated with the regulation of MUC5B expression in the airway epithelial cells. Epigallocatechin-3-gallate (EGCG) is the major component of green tea extract. The biological activity of EGCG includes reduction of cholesterol and antioxidant activity, as well as anti-inflammatory effect. However, the precise action mechanism of anti-inflammatory effect of EGCG in the airway epithelial cells has not been fully defined. This study investigates the effect and the brief signaling pathway of EGCG on PMA-induced MUC5B expression in the airway epithelial cells. Methods In NCI-H292 airway epithelial cells, the effect and signaling pathway of EGCG on MUC5B expression were investigated using real-time polymerase chain reaction analysis, enzyme immunoassay, immunohistochemical analysis, gelatin zymography assay, and immunoblot analysis. Results In NCI-H292 airway epithelial cells, PMA induced MUC5B expression, phosphorylation of p38 mitogen-activated protein kinase (MAPK), and matrix metalloproteinase-9 (MMP-9) expression and protein activity. EGCG significantly decreased PMA-induced MUC5B expression, phosphorylation of p38 MAPK, and MMP-9 expression and protein activity. SB203580 (p38 MAPK inhibitor) significantly decreased PMA-induced MMP-9 expression. In addition, SB203580 and MMP-9 I (MMP-9 inhibitor) significantly decreased PMA-induced MUC5B expression. Conclusion These results suggest that EGCG down-regulates PMA-induced MUC5B expression through the p38 MAPK dependent MMP-9 signaling pathway in human airway epithelial cells.

Received March 11, 2013 Revised April 18, 2013 Accepted April 21, 2013 Address for correspondence Yong-Dae Kim, MD, PhD Department of OtorhinolaryngologyHead and Neck Surgery, College of Medicine, Yeungnam University, 170 Hyeonchung-ro, Nam-gu, Daegu 705-703, Korea Tel +82-53-620-3781 Fax +82-53-628-7884 E-mail ydkim@med.yu.ac.kr Background and ObjectivesZZNaringenin and delphinidin are types of anthocyanidin, which are flavonoids and thus have anti-inflammatory property. Moderate consumption of natural dietary naringenin and delphinidin is believed to do anti-inflammatory action, but the action mechanism is unclear. Therefore, this study aimed to investigate the effects of naringenin and delphinidin on interleukin-1β (IL-1β)and lipopolysaccharide (LPS)-induced MUC5AC and MUC5B expressions in airway epithelial cells. Materials and MethodZZIn NCI-H292 cells and cultured nasal polyp epithelial cells, the effects of naringenin and delphinidin on IL-1βand LPS-induced MUC5AC and MUC5B expressions were analyzed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. ResultsZZDelphinidin attenuated IL-1βand LPS-induced MUC5AC and MUC5B mRNA and glycoprotein expression in a dose-dependent pattern in NCI-H292 cells and in cultured nasal polyp epithelial cells. Naringenin partially attenuated IL-1βand LPS-induced MUC5AC and MUC5B mRNA and glycoprotein expression at a high dose. ConclusionZZThese results suggest that delphinidin attenuates MUC5AC and MUC5B expressions in the airway epithelial cells. Between anthocyanidin and delphinidin, delphinidin exhibits greater potential as an ideal therapeutic agent for the control of mucus-hypersecretion in the treatment of airway inflammatory diseases. Korean J Otorhinolaryngol-Head Neck Surg 2013;56:291-6

OBJECTIVES: Phorbol 12-myristate 13-acetate (PMA) is widely used as a protein kinase C (PKC) activator, PKC is involved in the secretion of mucins. MUC16, one of the membrane-bound mucins, is produced in human airway epithelial cells. However, the effect and signaling pathway of PMA on MUC16 expression in human airway epithelial cells has not been reported. Therefore, the effect and brief signaling pathway of PMA on MUC16 expression were investigated in human airway epithelial cells in this study. METHODS: In the mucin-producing human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells, the effect and signaling pathway of PMA on MUC16 expression were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA) for p38 mitogen-activated protein kinase (MAPK). RESULTS: PMA increased MUC16 expression, and activated phosphorylation of p38 MAPK. However, it did not activate phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). SB203580 (p38 MAPK inhibitor) inhibited PMA-induced MUC16 expression, while U0126 (ERK1/2 inhibitor) did not. In addition, the knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked PMA-induced MUC16 mRNA expression. Rottlerin (PKCδ inhibitor) inhibited PMA-induced MUC16 expression, and also inhibited the phosphorylation of activated p38 MAPK by PMA. CONCLUSION: These results show for the first time that PMA-induced MUC16 expression is regulated by activation of the PKCδ and p38 MAPK signaling pathway in human airway epithelial cells.

Interleukin 1beta(IL-1beta), a proinflammatory cytokine, is related with inflammatory diseases and it up-regulates MUC2 gene expression and mucin secretion. This study was designed to investigate the signal transduction pathway of the IL-1beta-mediated MUC2 gene expression and mucin secretion in human airway epithelial cells. In cultured human airway NCI-H292 epithelial cells, the steady state of the mRNA level of MUC2 gene expression and mucin secretion induced by IL-1beta were determined by reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme immunoassay, and immunoblot analysis. To observe the signal pathway of the IL-1beta-mediated MUC2 gene expression and mucin secretion, we used several specific inhibitors. PD98059 (MEK/ERK inhibitor) suppressed IL-1beta-mediated MUC2 gene expression and mucin secretion, while SB203580 (p38 inhibitor) did not. Ro31-8220 (PKC inhibitor) inhibited IL-1beta-mediated MUC2 gene expression and mucin secretion. It inhibited ERK phosphorylation, but did not inhibit p38 phosphorylation. LY294002 (PI3K inhibitor) also suppressed MUC2 expression, but did not inhibit any MAPKs phosphorylation. These results suggest that the IL-1beta-mediated MUC2 gene expression and mucin secretion in NCI-H292 cells are regulated through activation of the PKC-MEK/ERK pathway, and that PI3K is also involved in the IL-1beta-mediated MUC2 gene expression and mucin secretion.