Delphinidin Inhibits LPS-Induced MUC8 and MUC5B Expression Through Toll-like Receptor 4-Mediated ERK1/2 and p38 MAPK in Human Airway Epithelial Cells

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 microM) 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 microM) 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.

Roxithromycin Suppresses MUC5B/8 Mucin Genes and Mucin Production in Airway Epithelial Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Macrolide antibiotics are known to inhibit mucus hypersecretion in patients with chronic airway diseases, but its action mechanism is unclear. Several reports demonstrated that macrolides significantly inhibited gene expression of MUC2, MUC4 and MUC5AC in the airway epithelial cells, but little is known about its inhibitory effect for the other important airway mucins. In upper airway tracts, MUC5B and MUC8 are other important secreted mucin genes. Therefore, this study was aimed to investigate the effects of roxithromycin on the IL-1beta-induced gene expression and mucin production of MUC5B and MUC8 in NCI-H292 cells and cultured human nasal polyp epithelial cells. SUBJECTS AND METHOD: The effects of roxithromycin on the IL-1beta-induced MUC5B and MUC8 expression were analyzed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Roxithromycin attenuated the IL-1beta-induced MUC5B and MUC8 gene expression and mucin production with a dose-dependent pattern in NCI-H292 epithelial cells and cultured human nasal polyp epithelial cells. CONCLUSION: Roxithromycin exerts direct inhibitory effects on the gene expression of MUC5B and MUC8 in airway epithelial cells. These novel findings may explain the clinical efficacy of 14-membered macrolides in the treatment of chronic airway inflammations.

Inhibitory Effect of 15-Deoxy-Delta(12,14)-Prostaglandin J(2) on the MUC8 Expression in the Human Airway Epithelial Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Among the numerous mucin genes, MUC8 is regarded as one of the most important mucin genes which are related with upper airway disease such as a nasal polyp. 15-Deoxy-delta(12,14)-prostaglandin J(2)(15d-PGJ(2)), the most recently discovered prostaglandin, has been known to have multiple cellular functions, including anti-inflammatory and cytoprotective effects. However, the effect of 15d-PGJ(2) on mucin gene expression or mucin production has not yet been investigated. The purpose of this study was to determine the effect of 15d-PGJ(2) on interleukin-1beta (IL-1beta)-induced MUC8 gene expression and mucin secretion in the cultured NCI-H292 cells and human nasal polyp epithelial cells. SUBJECTS AND METHOD: The MUC8 mRNA levels were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and MUC8 mucin levels were measured using an enzyme-linked immunosorbent assay (ELISA) in the cultured epithelial cells stimulated by IL-1beta. 15d-PGJ(2) was added 1 hour before stimulation. RESULTS: 15d-PGJ(2) attenuated the IL-1beta-induced MUC8 mRNA expression and mucin secretion with a dose-dependent pattern in both cultured NCI-H292 cells and human nasal polyp epithelial cells. CONCLUSION: These results suggest that 15d-PGJ(2) may be considered as an effective agent for the control of airway mucus hypersecretion through the down-regulation of MUC8 gene.

MUC8 as a Ciliated Cell Marker in Human Nasal Epithelium / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: To examine the MUC8 mRNA expression patterns according to the mucociliary differentiation of the normal human nasal epithelial (NHNE) cells, and to investigate the localization of the MUC8 proteins in the nasal polyps. MATERIALS AND METHODS: The passage-2 NHNE cells were cultured using an air-liquid interface technique and nasal polyp specimens. On the 2, 7, 14, and 28 days after confluence, the ciliated cells were counted using cytospin slide immunostaining using H6C5 and beta-tubulin, and the MUC8 mRNA levels were determined using real-time quantitative PCR. After synthesizing the polyclonal anti-MUC8 peptide antibodies, MUC8 immunostaining was preformed using the nasal polyps. The MUC8 mRNA and protein levels were determined with the NHNE cells treated with IL-1beta (10 ng/ml for 24 hours) using RT-PCR and Western blot analysis. RESULTS: The increasing pattern of the number of ciliated cells as well as the MUC8 gene expression level with increasing culture time in the NHNE cells was quite similar. MUC8 was expressed in the ciliated cells of the human nasal polyps. The MUC8 protein level as well as the mRNA level was up-regulated as a result of the IL-1beta treatment. CONCLUSIONS: This study indicates that the MUC8 protein is expressed in ciliated cells from the human nasal epithelial cells and is up-regulated by the IL-1beta treatment. These results suggest that the MUC8 gene and protein expression levels might be used as a ciliated cell marker in the human nasal epithelium.

Expression of MUC5AC and MUC8 mRNA in human nasal mucosa / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: MUC5AC is known to be a major secretory mucin in goblet cells of the mucosa of human lower respiratory tract. But in our preliminary study, we found that the levels of MUC8 mRNAs were significantly increased in the biopsy specimens of the nasal polyps whereas other mucin genes were not. This suggests the possibility that MUC8 might be one of the major overexpressed mucins in the nasal polyps. The purpose of this study is to investigate the cellular location of MUC5AC and MUC8 mRNA. Material and methods : Normal posterior ethmoid mucosa and the polyp tissue were fixed in 4% paraformaldehyde and were hybridized with the RNA riboprobe for MUC8 and the oligonucleotide probe for MUC5AC in the presence of digoxigenin (DIG). RESULT: In the normal posterior ethmoid mucosa, MUC 5AC mRNA and MUC8 mRNA were barely expressed in the epithelium and the submucosal glands. In the polyp epithelium, the expression of MUC 5AC mRNA was localized in the cytoplasm of goblet cells and the expression of MUC8 mRNA was strongly localized in the nucelus of the goblet cells, and weakly localized in the cytoplasm of the goblet cells. MUC8 mRNA was also expressed in low levels in the nucleus of the submucosal glands. CONCLUSION: MUC8 mRNA is localized mainly in the nucleus of goblet cells and is one of the major mucin genes overexpressed in goblet cells of thnasal polyp.

Expression and regulation of MUC8 & MUC5AC by various cytokines in normal human nasal epithelial cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Sinusitis is one of the most commonly reported diseases in the world. A network of inflammatory mediators is known to be involved in the pathogenesis of chronic sinusitis and nasal mucus secretion may also be under the control of an inflammatory mediator network. To date, 12 human mucin genes have been identified; however, the regulation of MUC8 has not yet been found out. In this study, we described the regulation of the MUC8 mRNA expression by inflammatory mediators and investigated its cellular location. MATERIALS AND METHOD: MUC8 mRNA and MUC5AC mRNA were detected in culture using passage-2 normal human nasal epithelial (NHNE) cells after the treatment with a mixture of following inflammatory mediators; TNF-alpha, IL-1beta, LPS, IL-4, PAF. The translocation of MUC8 mRNA from the nucleus to the cytoplasm was investigated by treating the inflammatory mediators with in situ hybridization. RESULTS: We found that a mixture of inflammatory mediators increased the MUC8 mRNA expression but decreased the MUC5AC mRNA expression in cultured normal human nasal epithelial cells. Among the inflammatory mediators, Interleukin-4 was responsible for the decrease in the MUC5AC mRNA expression and the MUC5AC mucin secretion. We also found that MUC8 mRNA resides in the nucleus of goblet cells and is transported into the cytoplasm following the treatment with inflammatory mediators. CONCLUSION: These results indicate that MUC8 may play an important role in the pathogenesis of mucus hypersecretion in chronic sinusitis.

The Role of p38 MAP Kinase Signal Transduction in the Induction of MUC 8 Gene Expression in Normal Human Nasal Epithelial Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: In cystic fibrosis, chronic bronchitis, and asthma, the mucociliary mechanism is impaired when mucin is produced excessively. The mRNA encoding MUC8 has been shown to be the major up-regulated mucin under inflammatory condition and is likely to contribute to the airway mucus plugging characteristic of these diseases. The aim of this study is to determine the intracellular signaling pathway directly involved in the MUC8 regulation following inflammatory mediator treatments. MATERIALS AND METHOD: Passage-2 normal human airway epithelial cells were used in all experiments. Inflammatory signal-induced MAP kinase activity was measured by Western blot analysis using phosphospecific anti-active MAP kinase antibodies. Inflammatory signal-induced MUC8 expression was measured in the absence or presence of SB203580 by the semi-quantative RT-PCR. RESULTS: Inflammatory stimuli such as LPS, TNF-alpha, and IL-lbeta activated the p38 MAP kinase and subsequently up-regulated the MUC8 expression. Interestingly, the TNF-alpha or IL-lbeta-inducibility of the MUC8 gene expression was greatly enhanced by specific inhibition of the p38 MAP kinase by using SB 203580 CONCLUSION: These results suggest that the intracellular p38 MAP kinase activity is a negative regulator for the MUC8 up-regulation in human nasal epithelial cells following infiammatory stimuli.