MUC5AC Protein Production Research Articles

Endoplasmic reticulum stress/XBP1 promotes airway mucin secretion under the influence of neutrophil elastase.

Endoplasmic reticulum (ER) stress is an important reaction of airway epithelial cells in response to various stimuli, and may also be involved in the mucin secretion process. In the present study, the effect of ER stress on neutrophil elastase (NE)-induced mucin (MUC)5AC production in human airway epithelial cells was explored. 16HBE14o-airway epithelial cells were cultured and pre-treated with the reactive oxygen species (ROS) inhibitor, N-acetylcysteine (NAC), or the ER stress chemical inhibitor, 4-phenylbutyric acid (4-PBA), or the cells were transfected with inositol-requiring kinase 1α (IRE1α) small interfering RNA (siRNA) or X-box-binding protein 1 (XBP1) siRNA, respectively, and subsequently incubated with NE. The results obtained revealed that NE increased ROS production in the 16HBE14o-cells, with marked increases in the levels of ER stress-associated proteins, such as glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), phosphorylated protein kinase R-like endoplasmic reticulum kinase (pPERK) and phosphorylated (p)IRE1α. The protein and mRNA levels of spliced XBP1 were also increased, and the level of MUC5AC protein was notably increased. The ROS scavenger NAC and ER stress inhibitor 4-PBA were found to reduce ER stress-associated protein expression and MUC5AC production and secretion. Further analyses revealed that MUC5AC secretion was also attenuated by IRE1α and XBP1 siRNAs, accompanied by a decreased mRNA expression of spliced XBP1. Taken together, these results demonstrate that NE induces ER stress by promoting ROS production in 16HBE14o-airway epithelial cells, leading to increases in MUC5AC protein production and secretion via the IRE1α and XBP1 signaling pathways.

Topical administration of ambroxol eye drops augments tear secretion in rabbits.

To investigate if topical administration of ambroxol promotes tear secretion and to compare with Diquas ophthalmic eye drop. Two consecutive studies were conducted using sixteen (32 eyes) New Zealand White rabbits. The first study compared the efficacy of ambroxol hydrochloride (0.05%, 0.2%, and 1.0%) on tear and mucin secretion when administered twice daily. Tear secretion was assessed by Schirmer test I and mucin production by conjunctival impression cytology and PAS stain. The second study compared 0.2% ambroxol hydrochloride with Diquas. A human goblet cell line and human conjunctival tissue were used to test the effect of ambroxol hydrochloride on the expression of aquaporin 5 (AQP5) and MUC5AC, using reverse transcriptase-quantitative polymerase chain reaction and immunoblotting. All three concentrations of ambroxol hydrochloride demonstrated significant efficacy on tear stimulation within 2 weeks of treatment and total mucin component appeared increased. When administered topically twice daily, 0.2% ambroxol hydrochloride was more effective in augmenting tear secretion than Diquas. With 24 h of treatment, 5 μM of ambroxol hydrochloride upregulated AQP5 and MU5AC mRNA and MUC5AC protein in a goblet cell line. When tested on preserved human conjunctiva tissue, a trend of increased production of MUC5AC protein was seen (P = 0.26). Ambroxol is effective in augmenting tear secretion at the ocular surface in rabbits. With actions desirable of a candidate dry eye drug, further investigation of ambroxol and related compounds is warranted to explore their value toward clinical application.

Ablation of IL-33 Suppresses Th2 Responses but Is Accompanied by Sustained Mucus Obstruction in the Scnn1b Transgenic Mouse Model.

Cystic fibrosis is characterized by dehydration of the airway surface liquid layer with persistent mucus obstruction. Th2 immune responses are often manifested as increased mucous cell density (mucous cell metaplasia) associated with mucus obstruction. IL-33 is a known inducer of Th2 immune responses, but its roles in mucus obstruction and related phenotypes in a cystic fibrosis-like lung disease model (i.e., Scnn1b-Tg-positive [Tg+]) mouse, remain unclear. Accordingly, IL-33 knockout (IL-33KO) Tg+ mice were examined and compared with IL-33 heterozygous (IL-33HET) Tg+ mice. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had complete absence of bronchoalveolar lavage fluid eosinophilia, accompanied with significant reduction in bronchoalveolar lavage fluid concentration of IL-5, a cytokine associated with eosinophil differentiation and recruitment, and IL-4, a major Th2 cytokine. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had significantly reduced levels of Th2-associated gene signatures (Slc26a4, Clca1, Retnla, and Chi3l4), along with complete loss of intracellular mucopolysaccharide staining in the airway epithelium. As compared with IL-33HET/Tg+ mice, although the IL-33KO/Tg+ mice had significantly reduced levels of MUC5AC protein expression, they showed no reduction in the degree of mucus obstruction, MUC5B protein expression, bacterial burden, and neonatal mortality. Interestingly, the histological features, including subepithelial airway inflammation and alveolar space enlargement, were somewhat exaggerated in IL-33KO/Tg+ mice compared with IL-33HET/Tg+ mice. Taken together, our data indicate that although IL-33 modulates Th2 inflammatory responses and MUC5AC protein production, mucus obstruction is not dependent on IL-33.

Effect of phosphorylation of cortactin at different sites on secretion by airway mucus 5AC

To explore the role of cortical actin-binding protein (cortactin) in shear stress-induced mucin (MUC) 5AC secretion in human airway epithelial cells and the effect of phosphorylation of cortactin at different sites. Methods: HBE16 airway epithelial cells were cultured, and then transfected with mutation carrier, such as pEGFP-N1-cortactin (Cort), pEGFP-N1-Cort-Y421A, pEGFP-N1-Cort-Y470A and pEGFP-N1-Cort-Y486A. The cells were divided into a normal control group, a shear stress group, a shear stress + pEGFP-N1 group, a shear stress + PEGFP-N1-Cort group, a shear stress + pEGFP-N1-Cort-Y421A group, a shear stress + pEGFP-N1-Cort-Y470A group, and a shear stress + pEGFP-N1-Cort-Y486A group. The shear stress were set at 4 dynes/cm2. The levels of MUC5AC protein and mRNA in cells and culture supernatant were assayed with enzyme-linked immunosorbent assay (ELISA) and real-time PCR. The cortactin and phosphorylated cortactin were detected by Western blot. F-actin was stained by fluorescein isothiocyanate (FITC)-phalloidin. Results: There was an obvious increase of phosphorylated cortactin in cells exposed to 4 dynes/cm2 of shear stress for 30 min, which reached climax at 2 hours concomitant with elevation of MUC5AC protein production and mRNA expression in the different experiment groups (all P<0.05). Compared with single shear stress-stimulated group, MUC5AC in supernatant was increased obviously, and the distribution of F-actin in cytomembrane was also increased in the pEGFP-N1-Cort group (both P<0.05), while there were no changes in the MUC5AC protein and mRNA levels in cytoplasm. Compared with the shear stress+pEGFP-N1-Cort group, the MUC5AC protein in the culture supernatant was decreased, and the polymerization of F-actin at cell membranes were also attenuated in the shear stress+pEGFP-N1-Cort-Y421A group and the shear stress + pEGFP-N1-Cort-Y470A group (both P<0.05), while there was no significant effect in the shear stress + pEGFP-N1-Cort-Y486A group (P>0.05). Conclusion: Cortactin is involved in shear stress-mediated MUC5AC secretion in human airway epithelial cells, and the phosphorylated site of Tyr421 and Tyr470 may play an important role in it.

Tussilagone suppressed the production and gene expression of MUC5AC mucin via regulating nuclear factor-kappa B signaling pathway in airway epithelial cells

In the present study, we investigated whether tussilagone, a natural product derived from Tussilago farfara, significantly affects the production and gene expression of airway MUC5AC mucin. Confluent NCI-H292 cells were pretreated with tussilagone for 30 min and then stimulated with EGF (epidermal growth factor) or PMA (phorbol 12-myristate 13-acetate) for 24 h or the indicated periods. The MUC5AC mucin gene expression was measured by RT-PCR. Production of MUC5AC mucin protein was measured by ELISA. To elucidate the action mechanism of tussilagone, effect of tussilagone on PMA-induced NF-κB signaling pathway was investigated by western blot analysis. Tussilagone significantly inhibited the production of MUC5AC mucin protein and down-regulated the expression of MUC5AC mucin gene, induced by EGF or PMA. Tussilagone inhibited PMA-induced activation (phosphorylation) of inhibitory kappa B kinase (IKK), and thus phosphorylation and degradation of inhibitory kappa Ba (IκBα). Tussilagone inhibited PMA-induced phosphorylation and nuclear translocation of nuclear factor kappa B (NF-κB) p65. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. These results suggest that tussilagone can regulate the production and gene expression of mucin by acting on airway epithelial cells through regulation of NF-κB signaling pathway.

Tedizolid inhibits MUC5AC production induced by methicillin-resistant Staphylococcus aureus in human airway epithelial cells.

The innate immune system plays an important role in early immunity against respiratory tract infection. Although airway epithelial cells produce mucus to eliminate pathogens and irritants, hypersecretion of mucus is harmful for the host as it may cause airway obstruction and inhibit influx of antimicrobial agents. It has been reported that several antimicrobial agents have an immunomodulatory effect invitro and invivo, but little is known about whether tedizolid, a novel oxazolidinone, can modulate immune responses. In this study, we evaluated whether tedizolid can suppress MUC5AC production in human airway epithelial cells stimulated by methicillin-resistant Staphylococcus aureus (MRSA). Compared with the control, tedizolid significantly inhibited MUC5AC protein production and mRNA overexpression at concentrations of both 2 and 10μg/mL (representative of trough and peak concentrations in human epithelial lining fluid). Among the mitogen-activated protein kinase inhibitors tested, only extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation was inhibited by tedizolid as indicated by western blot analysis. These results indicate that tedizolid inhibits the overproduction of MUC5AC protein by inhibiting phosphorylation of ERK1/2. This study revealed that tedizolid suppresses excessive mucin production in human airway epithelial cells. The immunomodulatory effect of tedizolid may improve outcomes in patients with severe respiratory infectious diseases caused by MRSA.

Inhibition of suppressor of cytokine signaling 1 on lipopolysaccharide-induced mucin5AC hypersecretion and the mechanism in human bronchial epithelial cells

To explore the inhibitory role of suppressor of cytokine signaling 1 (SOCS1) in lipopolysaccharide (LPS)-induced mucin5AC (MUC5AC) hypersecretion and the potential mechanism involved in this process. The human bronchial epithelial cells 16HBE were divided into 0, 0.5, 1, 6, 12 and 24 h groups according to the time of LPS challenge. In gain- and loss- of functions experiments, wild-type SOCS1 and SOCS1-targeted siRNA (SOCS1-siRNA) were synthesized to identify the function of SOCS1 in LPS-mediated MUC5AC hypersecretion, and named wild-type SOCS1 group and SOCS1-siRNA group, respectively, and the non-transfected group and non-targeted siRNA group were used as controls. In Filgotinib group, the specific inhibitor of Janus kinase 1 (JAK1), Filgotinib, was used to detect the role of JAK1/signal transducer and activator of transcription 1 (STAT1) signaling pathway in LPS challenge, and the aqueous physiological buffer group was used as the control. The production of MUC5AC protein was measured by enzyme linked immunosorbent assay (ELISA), and the amount of MUC5AC protein was normalized to the total protein in cell lysates and was expressed as μg/mg cell lysates. The proteins expressions of SOCS1, phosphorylation of JAK1 and STAT1 were measured by Western blot, and the total expression of its protein (for JAK1 and STAT1) or β-actin (for SOCS1) was used as the loading control. Compared to 0 h group, LPS induced a robust induction in MUC5AC expression, the expression levels of MUC5AC in 0, 0.5, 1, 6, 12 and 24 h groups were (2.86±0.20), (3.42±0.29), (3.43±0.12), (10.22±0.96), (14.56±1.12), (14.15±1.34) μg/mg, in association with a decrease of SOCS1 expression. And in 6 h group, the expressions of MUC5AC and SOCS1 were both medium up-regulated (all P<0.05). Consequently, the application of LPS for 6 h was selected as the optimal responses period in the ensuing experiments. Compared to the expression of MUC5AC protein in non-transfected group, high level of SOCS1 in wild-type SOCS1 group led to a reduced phosphorylation of JAK1/STAT1, as well as Filgotinib did, thereby suppressing excessive MUC5AC production in wild-type SOCS1 group and Filgotinib group [(4.04±0.65), (7.02±0.83) vs (10.37±1.00) μg/mg] (all P<0.05). Conversely, compared to the expression of MUC5AC in non-targeted siRNA group, down-regulation of SOCS1 in SOCS1-siRNA group promoted the phosphorylation of JAK1/STAT1, and then further increased MUC5AC production [(13.69±1.32) vs (11.01±1.41) μg/mg] (all P<0.05). These results show that SOCS1 suppresses LPS-promoted MUC5AC hypersecretion through the inhibition of JAK1/STAT1 signaling pathway.

Leukotriene E4 induces MUC5AC release from human airway epithelial NCI-H292 cells

BackgroundHypersecretion of mucin in the airway epithelium is an important feature of allergic airway diseases. Of the 3 cysteinyl leukotrienes (CysLTs; LTC4 LTD4 and LTE4), only LTE4 is sufficiently stable to be detectable in extracellular fluids. However, LTE4 has received little attention because it binds poorly to the CysLT1 and CysLT2 receptors; therefore, little is known about the effects of LTE4 on mucous secretion. Recently, studies have focused on the P2Y12 receptor as a potential receptor for LTE4, because this receptor is required for LTE4-mediated pulmonary inflammation. In our previous study, we confirmed the expression of P2Y12 receptor in human airway epithelial cells. To clarify the roles of LTE4 in airway epithelial cells, we investigated mucus secretion by LTE4 in vitro. MethodsConfluent NCI-H292 cells were stimulated with LTE4 (0.01–1 μM) for 24 h. The release and production of MUC5AC protein, a gel-forming mucin, were evaluated with an enzyme-linked immunosorbent assay. ResultsWestern blot analysis revealed that NCI-H292 cells expressed P2Y12 receptor protein. LTE4 significantly induced the release of MUC5AC mucin in a dose-dependent manner. Th2 cytokines such as IL-4 (10 ng/mL) and IL-13 (10 ng/mL) accelerated the LTE4-induced release of MUC5AC protein. MRS2935, a P2Y12 receptor antagonist, partially inhibited the LTE4-induced release of MUC5AC protein in the airway. In contrast, MK571, a CysLT1 receptor antagonist, did not affect the release of MUC5AC protein elicited by LTE4. ConclusionsThese results suggest that LTE4 may play some important roles in allergic mucus secretion partially via activation of P2Y12 receptor.

Apigenin Inhibits Tumor Necrosis Factor-α-Induced Production and Gene Expression of Mucin through Regulating Nuclear Factor-Kappa B Signaling Pathway in Airway Epithelial Cells.

In the present study, we investigated whether apigenin significantly affects tumor necrosis factor-α (TNF-α)-induced production and gene expression of MUC5AC mucin in airway epithelial cells. Confluent NCI-H292 cells were pretreated with apigenin for 30 min and then stimulated with TNF-α for 24 h or the indicated periods. The MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription - polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Apigenin significantly inhibited MUC5AC mucin production and down-regulated MUC5AC gene expression induced by TNF-α in NCI-H292 cells. To elucidate the action mechanism of apigenin, effect of apigenin on TNF-α-induced nuclear factor kappa B (NF-κB) signaling pathway was also investigated by western blot analysis. Apigenin inhibited NF-κB activation induced by TNF-α. Inhibition of inhibitory kappa B kinase (IKK) by apigenin led to the suppression of inhibitory kappa B alpha (IκBα) phosphorylation and degradation, p65 nuclear translocation. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. Apigenin also has an influence on upstream signaling of IKK because it inhibited the expression of adaptor protein, receptor interacting protein 1 (RIP1). These results suggest that apigenin can regulate the production and gene expression of mucin through regulating NF-κB signaling pathway in airway epithelial cells.

Immunomodulatory effect of linezolid on methicillin-resistant Staphylococcus aureus supernatant-induced MUC5AC overexpression in human airway epithelial cells.

Linezolid is the first member of the oxazolidinones and is active against drug-resistant Gram-positive pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA). Additionally, linezolid shows an immunomodulatory effect, such as inhibition of inflammatory cytokine production. In this study, we examined the effect of linezolid on MRSA-induced MUC5AC overexpression in airway epithelial cells. In this study, an MRSA supernatant was used to avoid the direct effect of linezolid on MRSA. MUC5AC protein production was significantly increased with a 40-fold dilution of MRSA supernatant. At the mRNA level, MUC5AC gene expression was significantly increased 6 and 9 h after stimulation. In an inhibition study, linezolid significantly reduced MRSA-induced MUC5AC protein and mRNA overexpression at concentrations of 5 and 20 μg/ml, which were the same as the trough and peak concentrations in human epithelial lining fluid. In an analysis of cell signaling, among the mitogen-activated protein kinase inhibitors, only the extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor reduced the MUC5AC protein production to the same level as that of the control; on Western blot analysis, only ERK1/2 was phosphorylated by the MRSA supernatant. In addition, the ERK1/2 phosphorylation was inhibited by linezolid. MUC5AC and MUC5B are the major barrier that traps inhaled microbial organisms, particulates, and foreign irritants. However, in patients with chronic respiratory diseases, pathogen-induced MUC5AC overexpression causes many problems, and control of the overexpression is important. Thus, this study revealed that linezolid showed a direct immunomodulatory effect in airway epithelial cells.

Inhibition of TNF‐α‐Induced MUC5AC Mucin Gene Expression and Production by Wogonin Through the Inactivation of NF‐κB Signaling in Airway Epithelial Cells

In this study, we investigated whether wogonin significantly affects MUC5AC mucin gene expression and production in human airway epithelial cells. Confluent NCI-H292 cells were pretreated with wogonin for 30 min and then stimulated with tumor necrosis factor-α (TNF-α) for 24 h or the indicated periods. The MUC5AC mucin gene expression and mucin protein production were measured by RT-PCR and ELISA, respectively. We found that incubation of NCI-H292 cells with wogonin significantly inhibited mucin production and down-regulated MUC5AC gene expression induced by TNF-α in a dose-dependent fashion. To elucidate the action mechanism of wogonin, effect of wogonin on TNF-α-induced NF-κB signaling pathway was investigated by western blot analysis. Wogonin inhibited NF-κB activation induced by TNF-α. Inhibition of IKK by wogonin led to the suppression of IκB phosphorylation and degradation, p65 nuclear translocation and NF-κB-regulated gene expression. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. Wogonin also inhibited the gene products involved in cell survival (Bcl-2) and proliferation (cyclooxygenase-2). These results suggest that wogonin inhibits the NF-κB signaling pathway, which may explain its role in the inhibition of MUC5AC mucin gene expression and production.

Glucocorticoids Inhibit MUC5AC Production Induced by Transforming Growth Factor-α in Human Respiratory Cells

Mucus hypersecretion from airway epithelium is a characteristic feature of severe asthma. Glucocorticoids (GCs) may suppress mucus production and diminish the harmful airway obstruction. We investigated the ability of GCs to suppress mRNA expression and protein synthesis of a gene encoding mucin, MUC5AC, induced by transforming growth factor (TGF)-α in human mucoepidermoid carcinoma (NCI-H292) cells and the molecular mechanisms underlying the suppression. We determined if GCs such as dexamethasone (DEX), budesonide (BUD), and fluticasone (FP) could suppress MUC5AC production induced by a combination of TGF-α and double-strand RNA, polyinosinic-polycytidylic acid (polyI:C). MUC5AC mRNA expression and MUC5AC protein production were evaluated. The signaling pathways activated by TGF-α and their inhibition by GCs were tested using a phosphoprotein assay and MUC5AC promoter assay. DEX significantly suppressed the expression of MUC5AC mRNA and MUC5AC protein induced by TGF-α. The activation of the MUC5AC promoter by TGF-α was significantly inhibited by DEX. DEX did not affect activation of downstream pathways of the EGF receptor or mRNA stability of MUC5AC transcripts. DEX, BUD, and FP suppressed MUC5AC protein expression induced by a combination of TGF-α and polyI:C in a dose-dependent manner. GCs inhibited MUC5AC production induced by TGF-α alone or a combination of TGF-α and polyI:C; the repression may be mediated at the transcriptional but not post-transcriptional level.

High-dose tobramycin inhibits lipopolysaccharide-induced MUC5AC production in human lung epithelial cells

Tobramycin inhalation therapy is an effective therapy for cystic fibrosis as well as severe bronchiectasis that is colonized with Pseudomonas aeruginosa. The mechanism responsible for the efficacy of tobramycin in the treatment of severe chronic infectious diseases has not been elucidated. We demonstrate that high-dose tobramycin decreases MUC5AC gene expression and protein production in NCI-H292 cell stimulated with lipopolysaccharide of P. aeruginosa. MUC5AC protein of NCI-H292 cell stimulated by lipopolysaccharide was analyzed by an enzyme-linked immunosorbent assay and MUC5AC expression at the mRNA level was analyzed by RT-PCR. Western blot was performed to examine a potential role for the signaling molecules upstream of NFκB. High-dose tobramycin (500 μg/ml) decreased the level of MUC5AC protein released into the supernatant of the NCI-H292 cell line at 24 h after lipopolysaccharide stimulation ( P < 0.001). The tobramycin treatment also inhibited MUC5AC mRNA expression at 12 h after lipopolysaccharide stimulation ( P < 0.05) and suppressed NFκB activation 60 min after lipopolysaccharide stimulation ( P < 0.001). Tobramycin suppressed the phosphorylation of extracellular signal-regulated protein kinase, p38 MAP kinase. These results suggest that high-dose tobramycin, such as inhalation therapy, can inhibit MUC5AC gene expression and MUC5AC protein production in NCI-H292 cells, in part through the mitogen-activated protein kinase pathway. Thus, the activation of TLR4 and the subsequent immune/inflammatory responses induced by chronic infections such as P. aeruginosa might be modulated by tobramycin. Our data may reveal one of the mechanisms responsible for the clinical effect of tobramycin inhalation therapy against severe chronic respiratory diseases due to P. aeruginosa.

Regulatory mechanism of activator protein-1 on the expression of MUC5AC induced by cigarette smoke extract

To investigate the mechanism of activator protein-1 (AP-1) on cigarette smoke-induced airway mucous hypersecretion and to explore the possible signal transduction pathway that activates AP-1. The airway epithelial cell line (BEAS-2B) was cultured in vivo and treated with cigarette smoke extract (CSE). The DNA binding activity of AP-1 was blocked by the transfection of c-Jun dominant negative mutant TAM67 into the cells. SP600125 and PD98059 were used to block the activation of c-Jun terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) respectively. MUC5AC protein was detected by enzyme-linked immunosorbent assay, MUC5AC mRNA level was analyzed by RT-PCR, while the protein contents of p-JNK, p-ERK and p-P38 were detected by Western blot, and the DNA binding activity of AP-1 was determined by electrophoretic mobility shift assay. The MUC5AC protein production and mRNA expression in the CSE group were significantly higher than those in the control group, and the DNA binding activity of AP-1 was also higher than that in the control group (P<0.01). The protein contents of p-ERK and p-JNK in the CSE group were higher than those in the control group (P<0.01), but the p-P38 level was not significantly different from that in the control group (P>0.05). After the transfection of TAM67 into the cells, the expression levels of MUC5AC protein and mRNA and the binding activity of AP-1 decreased significantly (P<0.01). The DNA binding activity of AP-1 and the expression levels of MUC5AC protein and mRNA were lower in the SP600125 group and in the PD98059 group than those in the CSE group (P<0.05). After being activated by JNK and ERK which are phosphorylated by cigarette smoke, AP-1 binds to its DNA binding elements on the promoter of MUC5AC gene and up-regulates the MUC5AC expression at the transcriptional level.

Effects of c-src/Nox1/ROS on mucin5AC expression induced by neutrophil elastase in airway epithelial cells

to explore the upper signaling pathway in neutrophil elastase (NE)-induced mucin5AC (MUC5AC) production in human BEAS-2B airway epithelial cells. the BEAS-2B airway epithelial cells were cultured and divided into 7 groups: negative control group, NE stimulation group, negative siRNA control group treated with or without NE, NADPH oxidase 1(Nox1) siRNA group treated with NE, c-src siRNA group treated with NE, and c-src specific inhibitor PP2 group treated NE. The relative content of reactive oxygen species (ROS) was assayed with a special kit. The levels of MUC5AC protein in culture medium, Nox1 protein, phosphoralyted c-src (p-c-src) kinase and MUC5AC mRNA in culture cells were detected with enzyme-linked immunosorbent assay, Western blot, and RT-PCR, respectively. there was an obvious increase of ROS production (0.76 +/- 0.09) in cells exposed to NE, with elevation of Nox1 protein (0.88 +/- 0.12) and MUC5AC protein production (0.82 +/- 0.09) and mRNA expression (0.77 +/- 0.05), all had significant differences when compared with normal control group, t = 6.13 - 9.44, P < 0.01. Nox1 siRNA inhibited ROS activity (0.19 +/- 0.06), reduced MUC5AC protein (0.31 +/- 0.05) and mRNA level (0.32 +/- 0.06), compared with single NE-stimulated group, P < 0.01. c-src siRNA and PP2 also showed the same effects, both of them decreased the NE-induced high activity of ROS, was 0.29 +/- 0.05, and 0.41 +/- 0.11, respectively, the MUC5AC protein in the c-src siRNA group and PP2 group were 0.38 +/- 0.09 and 0.48 +/- 0.08, MUC5AC mRNA level were 0.41 +/- 0.04 and 0.46 +/- 0.07, the Nox1 protein were 0.39 +/- 0.08 and 0.44 +/- 0.05, all P < 0.05, compared with single NE-treated group. C-src/Nox1/ROS transduction cascade may be upper signal regulators in NE-mediated MUC5AC expression in BEAS-2B cells.

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

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

EDITORIAL

We start this issue with the first of a set of two ‘Year-in-Review’ papers, highlighting important findings in respiratory medicine from articles published in Respirology in 2007. The first paper by Professor Bardin and Professor Ruffin reviews the clinical science publications in Respirology in 2007. The second ‘Year-in-Review’ paper, which will appear in the next issue, will review the respiratory science publications reported in the Journal in 2007. This issue features the last review in the Presidents' Series by the immediate past-President of the American College of Chest Physicians, Professor Mark Rosen. In his review ‘Pulmonary Complications of HIV Infection’, Professor Rosen discusses the incidence of different pulmonary disorders associated with HIV infection, and their diagnosis and treatment. Professor Rosen is Chief of the Divisions of Pulmonary, Critical Care and Sleep Medicine at North Shore University Hospital and Long Island Jewish Medical Center. His research interests include pulmonary disease and critical care in patients with HIV infection, respiratory failure and medical ethics. He has published extensively on the subject of pulmonary disease in patients with HIV infection and is co-editor of the textbook ‘HIV and the Lung’. I highly recommend this paper to you as it is a comprehensive review of the topic and should be of great interest. We regret to announce the cancellation of the Presidents' Review by Dr Nan Zhong from the APSR owing to unforeseen circumstances. Our next Presidents' Series with reviews by the new Presidents of the major respiratory societies will commence in a later issue this year. Three highlights in this issue are articles studying the role of mitogen-activated protein kinase pathways in airway epithelial cells. The first of these articles ‘Involvement of the p38 MAPK pathway in IL-13-induced mucous cell metaplasia in mouse tracheal epithelial cells’ by Dr Fujisawa et al. (pp. 191–202), demonstrates that the p38 MAPK pathway is involved in IL-13-induced mucous cell metaplasia and MUC5AC mRNA regulation in mouse tracheal epithelial cells and that phosphorylation of p38 MAPK may require STAT6-dependent de novo protein synthesis induced by IL-13. The same study also shows that the ERK1/2 pathway is not directly activated by IL-13. The article ‘Analysis of gene expression in human bronchial epithelial cells upon influenza virus infection and regulation by p38 MAP kinase and JNK’ by Dr Hayashi et al. (pp. 203–214) reports on the role of p38 MAPK in gene expression in human bronchial epithelial cells following influenza virus infection. In ‘Erythromycin attenuates MUC5AC synthesis and secretion in cultured human tracheal cells infected with RV14’ (pp. 215–220), Dr Inoue et al. examine the inhibitory effect of erythromycin on RV14-induced p44/42 MAPK activation and MUC5AC protein production and hypersecretion in human tracheal cells, suggesting a link between the two. Another highlight in this issue is the article by Dr Long et al. entitled ‘Rapid non-genomic effects of glucocorticoids on oxidative stress in a guinea pig model of asthma’ (pp. 227–232). In this study, the authors identify a rapid non-genomic mechanism whereby glucocorticoids reduce oxidative stress in ‘asthmatic’ guinea pigs. These findings may contribute to future developments in the clinical treatment of asthma. The article by Dr Bu et al.‘Effects of independent lung ventilation and lateral position on cytokine markers of inflammation after unilateral lung acid injury in dogs’ (pp. 233–239) compared the effects of different mechanical ventilation settings and lateral body position on inflammatory markers in an animal model of unilateral lung acid injury. Their results suggest that the release of pro-inflammatory cytokines in the injured lung is attenuated by independent lung ventilation and lateral positioning. Early initiation of this type of mechanical ventilation may be of benefit in patients with severe unilateral lung injury. Finally, the Short Communication by Dr Koh et al.‘Vascular endothelial growth factor in epithelial lining fluid of patients with acute respiratory distress syndrome’ (pp. 281–284) demonstrates that elevated levels of vascular endothelial growth factor in lung epithelial lining fluid may predict a better outcome in patients with acute lung injury and acute respiratory distress syndrome. Professor Chong-Kin Liam from the University of Malaya, Malaysia has decided to retire early from his role as Associate Editor of Respirology. Professor Liam joined the Journal in April 2006. His contribution over the past year and a half has been greatly appreciated and we are very grateful for his assistance. Professor Liam will continue to be involved with Respirology as an Editorial Board member. I hope you enjoy this issue of Respirology.

Pseudomonas aeruginosa induces MUC5AC production via epidermal growth factor receptor.

Hypersecretory disease associated with Pseudomonas aeruginosa (PA) infections is characterised by increased goblet cells and increased mucin production. Recently, an epidermal growth factor receptor (EGFR) signalling cascade was shown to be a common pathway through which many stimuli induce mucin MUC5AC expression in airways by differentiation to a goblet cell phenotype. This study looked at whether PA products induce EGFR expression and activation and thus result in mucin MUC5AC production. Human airway epithelial (NCI-H292) cells were stimulated with PA culture supernatant (Sup). MUC5AC protein production, MUC5AC and EGFR messenger ribonucleic acid (mRNA) expression, and phosphorylated EGFR and phosphorylated p44/42 mitogen-activated protein kinase (MAPK) were all examined using enzyme-linked immunosorbent assay, by in situ hybridisation and by immunoblotting. PA Sup induced MUC5AC mRNA and subsequent protein expression, EGFR and p44/42 MAPK phosphorylation and EGFR mRNA expression. Induction of MUC5AC mRNA and protein expression and EGFR and p44/42 MAPK phosphorylation were inhibited completely by pretreatment with a selective EGFR tyrosine kinase inhibitor. Pretreatment with a selective inhibitor of MAPK kinase prevented MUC5AC production and p44/42 MAPK phosphorylation but not EGFR phosphorylation. The authors conclude that PA products induce mucin MUC5AC production in human airway epithelial cells via the expression and activation of epidermal growth factor receptor.