Human NCI-H292 Airway Epithelial Cells Research Articles

C-C Motif Chemokine Receptor 3-Mediated Extracellular Signal-Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase Signaling: Promising Targets for Human Airway Epithelial Mucin 5AC Induction by Eotaxin-2 and Eotaxin-3

Introduction: Eotaxin-2 and -3 of the C-C chemokine subfamily function as potent chemoattractant factors for eosinophil recruitment and various immune responses in allergic and inflammatory airway diseases. Mucin 5AC (MUC5AC), a major gel-forming secretory mucin, is overexpressed in airway inflammation. However, the association between mucin secretion and eotaxin-2/3 expression in the upper and lower airway epithelial cells has not been fully elucidated. Therefore, in this study, we investigated the effects of eotaxin-2/3 on MUC5AC expression and its potential signaling mediators. Methods: We analyzed the effects of eotaxin-2 and -3 on NCI-H292 human airway epithelial cells and primary human nasal epithelial cells (HNEpCs) via reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting. Along with immunoblot analyses with specific inhibitors and small interfering RNA (siRNA), we explored the signaling pathway involved in MUC5AC expression following eotaxin-2/3 treatment. Results: In HCI-H292 cells, eotaxin-2/3 activated the mRNA expression and protein production of MUC5AC. A specific inhibitor of C-C motif chemokine receptor 3 (CCR3), SB328437, suppressed eotaxin-2/3-induced MUC5AC expression at both the mRNA and protein levels. Eotaxin-2/3 induced the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 and p38, whereas pretreatment with a CCR3 inhibitor significantly attenuated this effect. Induction of MUC5AC expression with eotaxin-2/3 was decreased by U0126 and SB203580, specific inhibitors of ERK1/2 and p38 mitogen-activated protein kinase (MAPK), respectively. In addition, cell transfection with ERK1/2 and p38 siRNAs inhibited eotaxin-2/3-induced MUC5AC expression. Moreover, specific inhibitors (SB328437, U0126, and SB203580) attenuated eotaxin-2/3-induced MUC5AC expression in HNEpCs. Conclusion: Our results imply that CCR3-mediated ERK1/2 and p38 MAPK are involved in the signal transduction of eotaxin-2/3-induced MUC5AC overexpression.

Cathelicidin induces epithelial-mesenchymal transition to promote airway remodeling in smoking-related chronic obstructive pulmonary disease

BackgroundEpithelial-mesenchymal transition (EMT) is an important characteristic in the remodeling of airways that occurs in chronic obstructive pulmonary disease (COPD). Cigarette smoke is a potential driving factor of this EMT in COPD. However, the mechanisms by which cigarette smoke induce EMT remain uncertain. Cathelicidin has been implicated as a causal factor of airway inflammation and mucus hypersecretion in smoking-related COPD. This study aimed to investigate whether cathelicidin induces EMT to promote airway remodeling in this disease.MethodsHuman lung tissue was collected from smokers with COPD and smokers without COPD. The EMT markers E-cadherin and vimentin were examined by immunohistochemistry. Mouse models of COPD were established by taking mice with airway cathelin-related antimicrobial peptide (CRAMP), the murine homologue of cathelicidin, either upregulated or downregulated by intranasal introduction of lentiviral vectors and then exposing them to cigarette smoke. E-cadherin and vimentin expression in the airways of the model mice was examined using immunofluorescence. Tumor necrosis factor alpha (TNF-α) converting enzyme (TACE), transforming growth factor alpha (TGF-α), and epidermal growth factor receptor (EGFR) expression was analyzed by Western blot. Additionally, NCI-H292 human airway epithelial cells, both with and without cathelicidin downregulation, were stimulated with cigarette smoke extract (CSE) and LL-37 synthetic peptide, a bioactive fragment of cathelicidin. This was done to confirm that the TACE/TGF-α/EGFR signaling pathway is activated in humans exposed to cigarette smoke.ResultsSignificant EMT was found in the small airways of smokers both with and without COPD, as well as in the airways of COPD model mice. Downregulation of CRAMP in COPD mice, however, ameliorated airway EMT induced by cigarette smoke. Conversely, upregulation of CRAMP enhanced airway EMT in vivo; TACE, TGF-α, and EGFR were found to be involved in this process. In vitro, EMT induced by CSE and LL-37 was inhibited by blocking TACE, TGF-α, and EGFR expression.ConclusionsCathelicidin promotes airway EMT by activating the TACE/TGF-α/EGFR signaling pathway. This mediates smoking-induced airway remodeling in the pathogenesis of COPD.

Clusterin Induces MUC5AC Expression via Activation of NF-κB in Human Airway Epithelial Cells.

Objectives Clusterin (CLU) is known as apolipoprotein J, and has three isoforms with different biological functions. CLU is associated with various diseases such as Alzheimer disease, atherosclerosis, and some malignancies. Recent studies report an association of CLU with inflammation and immune response in inflammatory airway diseases. However, the effect of CLU on mucin secretion of airway epithelial cells has not yet been understood. Therefore, the effect and brief signaling pathway of CLU on MUC5AC (as a major secreted mucin) expression were investigated in human airway epithelial cells.Methods In the tissues of nasal polyp and normal inferior turbinate, the presence of MUC5AC and CLU was investigated using immunohistochemical stain and Western blot analysis. In mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the effect and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway of CLU on MUC5AC expression were investigated using immunohistochemical stain, reverse transcription-polymerase chain reaction, real-time polymerase chain reaction, enzyme immunoassay, and Western blot analysis.Results In the nasal polyps, MUC5AC and CLU were abundantly present in the epithelium on immunohistochemical stain, and nuclear CLU (nCLU) was strongly detected on Western blot analysis. In human NCI-H292 airway epithelial cells or the primary cultures of normal nasal epithelial cells, recombinant nCLU increased MUC5AC expression, and significantly activated phosphorylation of NF-κB. And BAY 11-7085 (a specific NF-κB inhibitor) and knockdown of NF-κB by NF-κB siRNA (small interfering RNA) significantly attenuated recombinant nCLU-induced MUC5AC expression.Conclusion These results suggest that nCLU induces MUC5AC expression via the activation of NF-κB signaling pathway in human airway epithelial cells.

Hypothiocyanous Acid Suppresses PolyI:C-Induced Antiviral Responses by Modulating IRF3 Phosphorylation in Human Airway Epithelial Cells.

Pattern recognition receptors recognize RNA viruses and trigger type I and III interferon (IFN) production and apoptosis to limit viral replication and spread. Some innate immune cells produce oxidants in response to viral infection to protect against invasion. Recent studies have demonstrated the virucidal activity of hypothiocyanous acid (HOSCN), an oxidant generated by the peroxidase-catalyzed reaction of thiocyanate with hydrogen peroxide. However, the effects of HOSCN on host antiviral responses are still unknown. In this study, we aimed to clarify the role of HOSCN in host antiviral responses against RNA viruses in airway epithelial cells using polyinosinic-polycytidylic acid (polyI:C), a mimic of viral RNA. Our results show that HOSCN repressed antiviral responses in NCI-H292 human airway epithelial cells. HOSCN decreased polyI:C-induced apoptosis and the expression levels of IFNB1, IFNL1, IFNL2 and IFNL3 mRNAs. In addition, the induction of other interferon regulatory factor 3 (IRF3)-dependent genes was also suppressed by HOSCN. Further analyses focused on IRF3 revealed that HOSCN inhibited the phosphorylation of IRF3 at Ser386 and Ser396 as well as its dimerization and nuclear translocation by inhibiting the phosphorylation of TANK-binding kinase 1 (TBK1). Furthermore, HOSCN led to the phosphorylation of IRF3 at residues other than Ser386 and Ser396, implying that HOSCN may cause a conformational change in IRF3 to impair its function. Collectively, these results suggest that HOSCN plays a novel signaling role in the antiviral response, acting as a negative regulator of apoptotic and TBK1-IRF3 signaling pathways and limiting IRF3-dependent gene expression.

Tilianin Inhibits MUC5AC Expression Mediated Via Down-Regulation of EGFR-MEK-ERK-Sp1 Signaling Pathway in NCI-H292 Human Airway Cells.

In the human airway, mucus exists to protect the respiratory system as a primary barrier of the innate immune system. However, hyperexpressed mucus limits airflow, resulting in a decrease of lung function. Among more than 20 mucin family members, MUC5AC and MUC5B are major glycoproteins in human airway mucus. The epidermal growth factor receptor (EGFR) signaling pathway is one of the mechanisms of these mucins expression and specificity protein-1 (Sp1) transcription factor is the downstream signal of this pathway, playing pivotal roles in mucin expression. Even though there are some drugs for treating mucus hypersecretion, no drug has proven effects on humans. We found that the flavonoid tilianin regulated MUC5AC expression and also inhibited Sp1 phosphorylation. In this study, we investigated how tilianin would modulate EGFR signaling and regulate mucin production. In conclusion, tilianin inhibited MUC5AC expression mediated via modulating the EGFRMEK-ERK-Sp1 signaling pathway in NCI-H292 human airway epithelial cells. This study may provide the basis for the novel treatment of mucus hypersecretion.

<i>Citrus jabara</i> Extracts Suppress MUC5AC Mucin Production in Human Lung Epithelial Cells

In the human airway, the overproduction of MUC5AC mucin is a key feature of allergic asthma, and it induces airway narrowing and obstruction. The production of MUC5AC is regulated by several signals, but the mechanism is not completely understood. We investigated the effect of jabara, a citrus containing abundant flavonoids, on the regulation of MUC5AC production. When NCI-H292 human airway epithelial cells were cultured with jabara extracts, we found that the expression of Periodic acid-schiff stained mucin was suppressed with downregulated MUC5AC production. In human primary airway cells derived from asthmatic patients, MUC5AC production was also suppressed by jabara extracts. The treatment of cells with jabara extracts decreased ERK activation in NCI-H292 and in primary cells. These results show that jabara extracts contain some factors that suppress MUC5AC production and ERK activity and suggest that it will be useful for relieving asthma.

Escherichia coli-derived and Staphylococcus aureus-derived extracellular vesicles induce MUC5AC expression via extracellular signal related kinase 1/2 and p38 mitogen-activated protein kinase in human airway epithelial cells.

Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) release extracellular vesicles (EVs). E. coli-derived and S. aureus-derived EVs are associated with neutrophilic respiratory inflammation. In neutrophilic respiratory inflammation of human, expression of mucin is increased in airway epithelial cells and is associated with increased morbidity and mortality of the affected patients. However, no study on the effects of EVs on expression of mucin genes has been reported in airway epithelial cells. Therefore, this study was conducted in order to examine the effects and the brief signaling pathways of E. coli-derived and S. aureus-derived EVs on MUC5AC expression in human airway epithelial cells. In mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the effects and signaling pathways of E. coli-derived and S. aureus-derived EVs on MUC5AC expression were examined using reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). E. coli-derived and S. aureus-derived EVs induced MUC5AC expression. E. coli-derived and S. aureus-derived EVs significantly activated phosphorylation of extracellular signal related kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) and p38 MAPK. ERK1/2 MAPK inhibitor, p38 MAPK inhibitor, ERK1/2 MAPK siRNA, and p38 MAPK siRNA significantly blocked E. coli-derived and S. aureus-derived EVs induced MUC5AC messenger RNA (mRNA) expression. The results of this study suggest that E. coli-derived and S. aureus-derived EVs induced MUC5AC expression via ERK1/2 and p38 MAPK signaling pathways in human airway epithelial cells.

Cadmium induces mucin 8 expression via Toll-like receptor 4-mediated extracellular signal related kinase 1/2 and p38 mitogen-activated protein kinase in human airway epithelial cells.

Inhalation of cadmium can lead to development of inflammatory airway diseases such as acute pulmonary edema and chronic obstructive pulmonary disease. In inflammatory airway diseases, expression of mucins is increased, which leads to increased morbidity and mortality of the affected patients. However, no study on the effect of cadmium on expression of mucin genes in airway epithelial cells has been reported. Therefore, this study was conducted in order to investigate the effect and the brief signaling pathway of cadmium on expression of mucin genes in human airway epithelial cells. In mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the effect and signaling pathway of cadmium on expression of mucin genes were investigated using reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). Cadmium increased mucin 8 (MUC8) expression and Toll-like receptor (TLR) 4 messenger RNA (mRNA) expression. Cadmium significantly activated phosphorylation of extracellular signal related kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) and p38 MAPK. ERK1/2 MAPK inhibitor, p38 MAPK inhibitor, TLR4 siRNA, ERK1/2 MAPK siRNA, and p38 MAPK siRNA significantly blocked cadmium-induced MUC8 mRNA expression. TLR4 siRNA significantly blocked cadmium-activated phosphorylation of ERK1/2 MAPK and p38 MAPK. The results of this study suggest for the first time that cadmium induces MUC8 expression via TLR4-mediated ERK1/2 and p38 MAPK signaling pathway in human airway epithelial cells.

Effect of Multi-Walled Carbon Nanotubes on MUC5AC and MUC5B Expression in Airway Epithelial Cells

Received March 25, 2015 Revised May 8, 2015 Accepted May 17, 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 705-703, Korea Tel +82-53-620-3781 Fax +82-53-628-7884 E-mail ydkim@med.yu.ac.kr Background and ObjectivesZZMulti-walled carbon nanotubes (MWCNT) are one of the most commonly used nanomaterials to date. Recent studies have demonstrated that MWCNT increase immune response and allergic inflammation in airway epithelial cells. However, the effects of MWCNT on mucin in human airway epithelial cells have not been reported. Therefore, in the present study, the effect of MWCNT on MUC16, MUC5AC, and MUC5B expressions were investigated in human airway epithelial cells. Subjects and MethodZZIn mucin-producing human NCI-H292 airway epithelial cells and primary cultures of normal nasal epithelial cells, the effects of MWCNT on MUC16, MUC5AC, and MUC5B expression were analyzed by reverse transcription polymerase chain reaction, real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. ResultsZZIn human NCI-H292 airway epithelial cells, MWCNT significantly induced the expression MUC5AC and MUC5B mRNA and the production of MUC5AC and MUC5B protein. However, MWCNT did not induce the expression of MUC16 mRNA. In the primary cultures of normal nasal epithelial cells, MWCNT also induced the expression of MUC5AC and MUC5B mRNA and the production of MUC5AC and MUC5B proteins. ConclusionZZThe results of this study demonstrate that MWCNT induces MUC5AC and MUC5B expression in human airway epithelial cells. These findings provide important information about the biological role of MWCNT on mucus-secretion in human airway epithelial cells. Korean J Otorhinolaryngol-Head Neck Surg 2015;58(8):552-7

Effect of Polyinosinic-Polycytidylic Acid on MUC5B Expression in Human Airway Epithelial Cells

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

Staphylococcus Enterotoxin A Induces MUC5B Expression via TLR2‐dependent MAPK in Airway Epithelial Cells

Objectives: S taphylococcus aureus enterotoxins are known to induce an inflammatory response of the airways, increase sensitization to inhaled allergens, and decrease T-cell sensitivity to steroids. However, the effects of Staphylococcus enterotoxin A (SEA) on mucin secretion of airway epithelial cells have not yet been reported. Therefore, in this study, the effect and brief signaling pathway of SEA on MUC5B expression were investigated in human airway epithelial cells. 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 SEA on MUC5B 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). Results: SEA increased MUC5B mRNA and protein expression. Toll-like receptor 2 (TLR2) mRNA expression was significantly increased after treatment with SEA. Knockdown of TLR2 by siRNA significantly blocked SEA-induced MUC5B mRNA expression. SEA significantly activated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). U0126 (ERK1/2 MAPK inhibitor) and SB203580 (p38 MAPK inhibitor) significantly inhibited SEA-induced MUC5B mRNA expression. In addition, knockdown of ERK1/2 and p38 MAPK by siRNA significantly blocked SEA-induced MUC5B mRNA expression. Furthermore, the phosphorylation of ERK1/2 and p38 MAPK was significantly blocked by knockdown of TLR2 by siRNA. Conclusions: These results show for the first time that SEA induces MUC5B expression via TLR2, ERK1/2, and p38 MAPK signaling pathway in human airway epithelial cells.

Effect of thymic stromal lymphopoietin on MUC5B expression 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.

Akt induces down regulation of MUC5AC production in NCI-H292 human airway epithelial cells cultured on extracellular matrix.

MUC5AC mucin overproduction is a key feature of asthma as contributes to airway obstruction. The production of MUC5AC is regulated in part by signals from extracellular matrix via integrin pathways, but it remains largely unclear. We investigated the role of Akt, a typical signal transducer in the integrin pathway, in the regulation of MUC5AC production. When NCI-H292 human airway epithelial cells were cultured on laminin or Matrigel, we found that the activity of Akt was suppressed, as compared to control cells with upregulated MUC5AC production. In contrast, Akt was activated in cells cultured on type IV collagen with downregulated MUC5AC production. The Akt inhibitor induced upregulation of MUC5AC. In contrast, overexpression of active Akt induced downregulation of MUC5AC production. These results suggest that a signal from laminin or Matrigel induces upregulation of MUC5AC by suppressing Akt activity, whereas a signal from type IV collagen induces downregulation of MUC5AC, mediated by Akt activation.

Staphylococcus Enterotoxin a Induces Muc5B Expression Via Toll-Like Receptor 2, Extracellular Signal–Regulated Kinase 1/2, and P38 Mitogen-Activated Protein Kinase in Human Airway Epithelial Cells

Staphylococcus aureus enterotoxins are known to induce an inflammatory response of the airways, increase sensitization to inhaled allergens, and decrease T-cell sensitivity to steroids. However, the effects of Staphylococcus enterotoxin A (SEA) on mucin secretion of airway epithelial cells still have not been reported. Therefore, in this study, the effect and brief signaling pathway of SEA on MUC5B expression were investigated in human airway epithelial cells. 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 SEA on MUC5B expression were investigated using reverse transcriptase-polymerase chain reaction (PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). SEA increased MUC5B mRNA and protein expression. Toll-like receptor 2 (TLR2) mRNA expression was significantly increased after treatment with SEA. Knockdown of TLR2 by siRNA significantly blocked SEA-induced MUC5B mRNA expression. SEA significantly activated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). U0126 (ERK1/2 MAPK inhibitor) and SB203580 (p38 MAPK inhibitor) significantly inhibited SEA-induced MUC5B mRNA expression. In addition, knockdown of ERK1/2 and p38 MAPK by siRNA significantly blocked SEA-induced MUC5B mRNA expression. Furthermore, the phosphorylation of ERK1/2 and p38 MAPK was significantly blocked by knockdown of TLR2 by siRNA. These results show for the first time that SEA induces MUC5B expression via TLR2, ERK1/2, and p38 MAPK signaling pathway in human airway epithelial cells.

Effect of Betulinic Acid on MUC5AC and MUC5B Expression in Airway Epithelial Cells

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

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

ObjectivesDelphinidin 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.MethodsIn 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.ResultsIn 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.ConclusionThese 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.

Roflumilast Attenuates MUC5AC and MUC5B Expression in Airway Epithelial Cells

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

Effect of Onchocerca Volvulus Chitinase on MUC5B Expression in Human Airway Epithelial Cells

Objectives: Chitin is an essential structural component of the wall of fungal cells and is present in the exoskeleton of arthropods. It has been generally assumed that mammals lack the ability to produce chitinase proteins, the enzymes responsible for chitin degradation. However, recent studies have demonstrated that mammals produce chitinases and chitinase-like proteins, and chitinase plays a potential role in human asthma and allergic inflammation. In this study, the effect and brief signaling pathway of chitinase on MUC5B expression were investigated in human airway epithelial cells. 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 chitinase on MUC5B expression were investigated using the reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA). Results: In the mucin-producing human NCI-H292 airway epithelial cells, chitinase increased MUC5B expression. Chitinase significantly activated the phosphorylation of p38 MAPK but not the phosphorylation of ERK1/2. SB203580 (p38 MAPK inhibitor) significantly attenuated chitinase-induced MUC5B mRNA expression, but U0126 (ERK1/2 inhibitor) did not. Knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked chitinase-induced MUC5B expression. In the primary cultures of normal nasal epithelial cells, chitinase significantly increased MUC5B gene expression, and this was significantly attenuated after pretreatment with SB203580. Conclusions: These results suggest that chitinase induces MUC5B expression by activation of the p38 MAPK signaling pathway in human airway epithelial cells.

Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells

HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL37 in human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells(PNEC) in response to HDAC inhibitors with or without poly (I:C) stimulation was assessed using real-time PCR and western blot. In parallel, IL-6 expression was evaluated by ELISA. Our results showed that HDAC inhibitors up-regulated LL-37 gene expression independent of poly (I:C) stimulation in PNEC as well as in NCI-H292 cells. HDAC inhibitors increased LL37 protein expression in NCI-H292 cells but not in PNEC. In addition, HDAC inhibitors significantly inhibited poly (I:C)-induced IL-6 production in both of the epithelial cells. In conclusion, HDAC inhibitors directly up-regulated LL-37 gene expression in human airway epithelial cells.

Effect ofOnchocerca VolvulusChitinase on MUC5B Expression in Human Airway Epithelial Cells

Chitin is an essential structural component of the wall of fungal cells and is present in the exoskeleton of arthropods. It has been generally assumed that mammals lack the ability to produce chitinase proteins, the enzymes responsible for chitin degradation. However, recent studies have indicated that mammals produce chitinases and chitinase-like proteins, and chitinase plays a potential role in human asthma and allergic inflammation. In this study, the effect and brief signaling pathway of chitinase on MUC5B expression were investigated in human airway epithelial cells. 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 chitinase on MUC5B expression were investigated using the 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 the mucin-producing human NCI-H292 airway epithelial cells, chitinase increased MUC5B expression. Chitinase significantly activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) but not the phosphorylation of extracellular signa-l-related kinase (ERK) 1/2. The SB203580 (p38 MAPK inhibitor) significantly attenuated chitinase-induced MUC5B mRNA expression, but U0126 (ERK1/2 inhibitor) did not. Knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked chitinase-induced MUC5B expression. In the primary cultures of normal nasal epithelial cells, chitinase significantly increased MUC5B gene expression and this was significantly attenuated after pretreatment with SB203580. These results suggest that chitinase induces MUC5B expression by activation of the p38 MAPK signaling pathway in human airway epithelial cells.