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Objective: 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.

Aims: Secretoneurin(SN), a neuropeptide, has been considered a reliable marker of allergenic stimulation. However, the relationship between SN and the secretion of airway mucin remains unclear. In this study, we aimed to examine the in vitro relationship between SN and airway mucin over synthesis, as well as the signaling pathways involved., Methods and Results: Exogenous SN was added to two human airway epithelial cell lines (16HBE and NCI-H292). Measured by real-time quantitative polymerase chain reaction (qPCR) and enzyme-linked immuno sorbent assay (ELISA) respectively, the intracellular mucin(MUC)5AC mRNA and MUC5AC protein of culture supernates exhibited a time- and dose-dependent increase after stimulation of SN. Based on the evidence of an increased phosphorylation of ERK1/2 induced by exogenous SN, we performed the radioactive binding assay. We failed to find direct binding of SN to either epidermal growth factor receptor (EGFR) or Neuropilin-1(NRP1), the co-receptor of EGFR. But we detected an enhanced binding of EGF to NRP1 in the two airway epithelial cell lines induced by exogenous SN. Either EGF neutralizing antibody or MEK specific inhibitor (PD-98059) could attenuate the over synthesis of MUC5AC induced by exogenous SN, indicating an endogenous EGF dependent mechanism in MUC5AC over synthesis induced by SN., Conclusions: We conclude that SN induces MUC5AC hypersecretion in a dose- and time-dependent manner; moreover, the MUC5AC over synthesis induced by SN is strongly associated with the enhanced binding of EGF to NRP1 and the activation of EGFR and ERK1/2 subsequently., (© 2014 S. Karger AG, Basel.)

Objective: To investigate the effect of different pressure on mucin (MUC) secretion in dog airway mucus layer and explore the participation mechanisms., Methods: Totally 24 healthy dogs were randomly divided into 4 groups (n=6) after double-lumen endobronchial tube intubation. In group A the dogs were ventilated bilaterally with normal breathing frequency and pressure; In group B: one side of the dog lung did not ventilate, while the other side was excessively ventilated. In group C: the tension-sensitive cation channel (TRPV) 4 blocker Ruthenium Red (RR) was injected in advance, and ventilated as in group A. In group D: TRPV4 blocker RR was injected firstly, and then ventilated as in group B. After 12 h ventilation, we collected bronchoalveolar lavage fluid (BALF), tested MUC (2, 5AC, 5B) protein content by ELISA, and detected the transcription of MUC (2, 5AC, 5B) mRNA of bronchial lung tissue by RTPCR., Results: Compared with the normal ventilation (A2) group, the protein level of MUC of excessive ventilation (B2) group was significantly higher, mainly MUC5AC (P<0.05); compared with the normal ventilation (A1) group, the protein level of MUC of no ventilation (B1) group was significantly decreased (P<0.05). After pretreatment with RR, the MUC protein level of group C1, C2 were significantly lower than that of group A1, A2 (P<0.05); the MUC protein level of group B2were significantly lower than that of group D2 (P<0.05). The MUC (2, 5AC and 5B) mRNA level was higher in excessive ventilation group (B2) than in normal ventilation group (A2). After pretreatment with RR, in comparison with group A1, A2, the MUC (2, 5AC and 5B) mRNA level of group C1, C2 declined; in comparison with group B2, the MUC (2, 5AC and 5B) mRNA level of group D2 declined., Conclusion: Rhythmic pressure waves may regulate and balance basic airway mucin secretion through activating the TRPV4 channel in dogs.

Mucus hypersecretion is an important manifestation in patients with chronic inflammatory airway diseases. Excessive production of mucin leads to airway mucus obstruction and contributes to morbidity and mortality in these diseases. The molecular mechanisms underlying mucin overproduction, however, still remain largely unknown. Here, we report that the bacterium Pseudomonas aeruginosa (P. aeruginosa), an important human respiratory pathogen, induced MUC5AC mucin expression via an epithelial cell signaling cascade in human airway epithelial cells. The flagellin purified from P. aeruginosa up-regulated MUC5AC expression by activating its receptor Toll-like receptor 5 (TLR5) in 16HBE cells. This effect was inhibited by NADPH oxidase inhibitor (DPI), small interfering RNA of dual oxidase 2 (Duox2) and reactive oxygen species (ROS) scavengers (nPG and DMSO). Flagellin induced TGF-α release, and stimulated phosphorylated epidermal growth factor receptor (EGFR) and MUC5AC overproduction. These effects were prevented by EGFR and TGF-α neutralizing antibodies, metalloprotease inhibitors (GM6001 and TNF-α protease inhibitor-1) and specific knockdown of TNF-α-converting enzyme (TACE) with TACE siRNA. These findings may bring new insights into the molecular pathogenesis of P. aeruginosa infections and lead to novel therapeutic intervention for mucin overproduction in patients with P. aeruginosa infections., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This study investigated whether aldose reductase (AR) inhibition affects interleukin (IL)-13-induced mucus production in the human bronchial epithelial cell line-16 (HBE16) cells. The HBE16 cells were cultured with AR inhibitors (zopolrestat) or were transfected with an AR small interfering (si)RNA. Subsequently, the cells were stimulated with 10 ng/ml IL-13 for 2h. The levels of mucin (MUC)5AC mRNA and protein were measured by using RT-PCR or ELISA. Intracellular reactive oxygen species (ROS) were measured fluorimetrically with the CM-H2DCFDA probe. Western blotting was performed to determine the levels of AR, phosphorylated signal transducer and activator of transcription 6 (p-STAT6) and phosphorylated Janus kinase 2 (p-JAK2). The results show that treatment with zopolrestat or transfection with AR siRNA significantly suppressed IL-13-stimulated MUC5AC mRNA and protein in the HBE16 cells (P<0.05). AR inhibition could suppress IL-13-induced ROS generation, the phosphorylation of JAK2/STAT6 pathway and the activation of nuclear factor (NF)-kappa B, thereby decreasing mucus production in vitro (all P<0.05). Therefore, the inhibition of AR could be a therapeutic target for mucus hypersecretion in chronic inflammation lung disease., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Theaflavins isolated from black tea have been used in studies on the prevention of tumor growth. The aim of this study was to investigate whether treatment with theaflavins influences the mucus hypersecretion induced by cigarette smoke in the lungs of experimental rats. Firstly, cigarette smoke was aerosolized using a machine designed for inhalation by rats. The rats were divided into the negative control group, the cigarette smoke inhalation group, the theaflavins (TFs) treatment group, and the TFs +cigarette smoke inhalation group. The animals were sacrificed on day 60 of the experiment. Secondly, the rats were treated with theaflavins at different doses via a gastric tube and sacrificed on day 30. The changes in the levels of mucin 5AC (MUC5AC) and epidermal growth factor receptor (EGFR) in the airway were evaluated. Cigarette smoke induced a significant increase in the levels of MUC5AC and EGFR in all groups. These increases could be reversed by intragastric administration of theaflavins. The effect was more pronounced with the duration of treatment and coincided with a decrease in the expression of both targets. The rats showed various degrees of reduction in the expression of these parameters, which correlated with the theaflavin dose. TFs could inhibit the activation of EGFR, decrease the level of MUC5AC, and relieve airway mucous hypersecretion via the EGFR signaling pathway. These effects correlated directly with the duration of action and the dosage. In the future, oral theaflavins might be valuable in the treatment of chronic airway inflammation.

Mucus hypersecretion is an important manifestation in patients with chronic inflammatory airway diseases. Mucin 5AC (MUC5AC) is a major component of airway mucus. MUC5AC expression is regulated by epidermal growth factor receptor (EGFR) which can be activated by reactive oxygen species (ROS). Hyaluronan (HA), a linear glycosaminoglycan with molecular weights ranging from 2 × 10(5) to 1 × 10(7), is expressed in airway epithelium and can be depolymerized by ROS into hyaluronan fragments. The mechanisms through which fragmented HA exerts its biologic functions have been elucidated by interactions with its receptor CD44. The aim of our study was to examine the role of HA and CD44 in ROS-induced EGFR activation and MUC5AC expression. We exposed NCI-H292 cells to ROS generated by xanthine/xanthine oxidase (X/XO). ROS-induced EGFR phosphorylation, which was activated by tissue kallekrein (TK) activation and EGF release. We found ROS promoted CD44 co-immunoprecipitation with EGFR and MUC5AC up-regulation. These effects were mimicked by hyaluronan fragments. All the effects were inhibited by blocking CD44 or EGFR, suggesting that CD44 plays a critical role in ROS-induced MUC5AC up-regulation. These results show that ROS depolymerizes hyaluronan into fragments, and these fragments bind their receptor CD44 to induce TK activation, which cleaves EGF precursors into mature EGF to activate its receptor EGFR. Furthermore, we provide evidence that hyaluronan fragments are sufficient to induce CD44/EGFR interaction and EGFR signaling which lead to MUC5AC expression. The results indicate that the regulation of ROS-induced MUC5AC expression by hyaluronan and CD44 may provide important insights in the mechanism of mucus hypersecretion.

Abstract: Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases. However, the regulatory mechanisms that mediate excessive mucin production remain elusive. Recently, the level of YKL-40, a chitinase-like protein, has been found to be significantly increased in chronic inflammatory airway diseases and has been shown to be associated with the severity of these diseases. In this study, we sought to explore the effect of YKL-40 on mucin5AC (MUC5AC) production in chronic inflammatory airway diseases and the potential signaling pathways involved in this process. We found that elevated YKL-40 levels increased the mRNA and protein expression of MUC5AC in a dose- and time-dependent manner, in association with the phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB), reflecting their activation. These responses were significantly suppressed by the knockdown of protease-activating receptor 2 (PAR2) with specific small interfering RNA or the inhibitors of ERK and NF-κB. YKL-40-induced MUC5AC overproduction was also effectively attenuated by the inhibitor of focal adhesion kinase (FAK). Taken together, these results imply that YKL-40 can stimulate excessive MUC5AC production through PAR2- and FAK-mediated mechanisms. [Copyright &y& Elsevier]

Abstract: Mucus hypersecretion is a common feature in chronic airway diseases, and serine proteases play a critical role in this process. However, the mechanisms by which serine proteases induce mucin5AC hypersecretion have not been fully explored. In this study, we characterized human airway trypsin-like protease (HAT), a serine protease that is found in the mucoid sputum of patients with chronic airway diseases and is an agonist of protease-activated receptor 2 (PAR2)-induced cellular responses in human bronchial epithelial cells (16HBE). We also investigated the potential involvement of PAR2 in this process. We found that both HAT and PAR2-AP enhance the exocytosis of mucin5AC protein, whereas HAT, but not PAR2-AP, enhances the expression of mucin5AC mRNA. PAR2 is expressed at a much higher level in the cells than the other three PARs. Transfection with an siRNA against the PAR2 receptor or Gαq/11 protein or pretreatment with the Gαq/11 protein inhibitor YM-254890, the PLC inhibitor U73122 or the intracellular Ca2+ chelator BAPTA-AM all effectively attenuated the HAT-induced cellular responses. Taken together, these results indicate that HAT can stimulate mucin5AC hypersecretion through a PAR2-mediated signaling pathway in 16HBE cells. Thus, PAR2 could represent a novel therapeutic target for chronic airway diseases with mucus hypersecretion. [Copyright &y& Elsevier]