The role of the XBP-1/AGR2 signaling pathway in the regulation of airway Mucin5ac hypersecretion under hypoxia.

Oversecretion of Mucin5ac (MUC5AC), which is primarily synthesized by goblet cells and is the major gel-forming mucin, is a hallmark of various pulmonary inflammatory diseases. Hypoxia is considered a common pathophysiologic feature in various pulmonary inflammatory diseases. It has been suggested that hypoxia-inducible factor 1α (HIF-1α) acts as a key factor in hypoxia-induced MUC5AC hypersecretion; however, the exact mechanisms that maintain the stability of HIF-1α and support oversecretion by airway epithelial cells under hypoxia are still unclear. With immunohistochemistry, we found overexpression of anterior gradient 2 (AGR2) in the bronchial epithelial cells of hypoxia-treated mice. With specific shRNA transduction, AGR2 was demonstrated to be a key factor in MUC5AC hypersecretion in vitro. Additionally, co-immunoprecipitation, cell immunochemistry and confocal microscopy experiments were performed to explore the interaction between HIF-1α and AGR2 during hypoxia-induced MUC5AC hypersecretion in vitro. The results indicated increased binding and intracytoplasmic colocation of HIF-1α and AGR2. Our findings suggest that AGR2 acts as a key regulator in hypoxia-induced airway MUC5AC hypersecretion by increasing the stability of HIF-1α. Additionally, the elevated expression of AGR2 induced by hypoxia in bronchial epithelial cells likely depends on an XBP-1-associated pathway.

Ezrin/Exocyst complex regulates mucin 5AC secretion induced by neutrophil elastase in human airway epithelial cells.

BACKGROUND/AIM: Increased mucin secretion is a characteristic feature of many chronic airway diseases, particularly during periods of exacerbation; however, the exact mechanism of mucin secretion remains unclear. Ezrin, which is a specific marker of apical membranes, is predominantly concentrated in exocyst-rich cell surface structures, crosslinking the actin cytoskeleton with the plasma membrane. In the present study, we examined whether Ezrin is involved in mucin 5AC (MUC5AC) secretion after neutrophil elastase (NE) attack, and we investigated the role of the exocyst complex docking protein Sec3 in this process. METHODS: NE was used as a stimulator in a 16HBE14o- cell culture model. The expression and location of Ezrin and Sec3 were investigated, and the interaction between Ezrin and Sec3 in 16HBE14o-cells was assayed after treatment with NE, Ezrin siRNA, Sec3 siRNA, neomycin or PIP2-Ab. RESULTS: We found that Ezrin was highly expressed in the bronchi of humans with chronic airway diseases. NE induced robust MUC5AC protein secretion. The Ezrin siRNA, Sec3 siRNA, and neomycin treatments led to impaired MUC5AC secretion in cells. Both Ezrin and Sec3 were recruited primarily to the cytoplasmic membrane after NE stimulation, and the neomycin and PIP2-Ab treatments abrogated this effect. Immunoprecipitation analysis revealed that Ezrin and Sec3 combined to form complexes; however, these complexes could not be detected in Ezrin∆1-333 mutant-transfected cells, even when PIP2 was added. CONCLUSIONS: These results demonstrate that Ezrin/Sec3 complexes are essential for MUC5AC secretion in NE-stimulated airway epithelial cells and that PIP2 is of critical importance in the formation of these complexes.

Transient receptor potential melastatin 8 gene polymorphism is associated with cold-induced airway hyperresponsiveness in bronchial asthma.

BACKGROUND AND OBJECTIVE: Cold-induced airway hyperresponsiveness (CAH) is common in bronchial asthma (BA) patients and represents a problem for those living in cold climate. Transient receptor potential melastatin 8 (TRPM8) channel is the main cold temperature sensor in humans that could mediate cold response in asthmatics with CAH. No associations between TRPM8 gene polymorphisms and CAH have been reported. METHODS: The present study involved 123 BA patients. CAH was assessed by 3-min isocapnic (5% CO2 ) cold air (-20°C) hyperventilation challenge. The c.750G > C (rs11562975), c.1256G > A (rs7593557), c.3048C > T (rs11563208) and c.3174C > G (rs11563071) polymorphisms of TRPM8 gene were genotyped by allele-specific polymerase chain reaction (PCR) and PCR with subsequent restriction fragment length polymorphism analysis. RESULTS: GC genotype and C allele carriers of the c.750G > C (rs11562975) polymorphism were more frequently observed to exhibit CAH. The estimated odds ratio for the GC genotype was 3.73 95%CI (1.48; 9.37), P = 0.005. Furthermore, GC heterozygotes had a prominent decrease in forced expiratory volume in 1 s after the challenge as compared to GG homozygotes (-12% (-16; -8.1) vs -6.45% (-11; -2.1), P < 0.001). GC carriers also had a marked reduction in other spirometric parameters. CONCLUSIONS: The GC variant of the TRPM8:c.750G > C (rs11562975) polymorphism is associated with CAH in patients with BA, which suggests a potential role of TRPM8 in CAH development.

Secretoneurin induces airway mucus hypersecretion by enhancing the binding of EGF to NRP1.

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.

The chitinase-like protein YKL-40 increases mucin5AC production in human bronchial epithelial cells.

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.

Human airway trypsin-like protease induces mucin5AC hypersecretion via a protease-activated receptor 2-mediated pathway in human airway epithelial cells.

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 Ca(2+) 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.

Neutrophil elastase induces MUC5AC secretion via protease-activated receptor 2.

Mucus hypersecretion is a major manifestation in patients with chronic inflammatory airway diseases, and mucin5AC (MUC5AC) protein is a major component of airway mucus. Previous studies have demonstrated that neutrophil elastase (NE) stimulates the secretion of MUC5AC from airway epithelial cells, however, the mechanism is poorly understood. NE is a known ligand for protein active receptors (PARs), which have been confirmed to participate in releasing MUC5AC in the airways. However, the role of PARs in NE-induced MUC5AC secretion remains unclear. We demonstrated that airway goblet-like Calu-3 cells express PAR1, PAR2, and PAR3 with a predominant level of PAR2. NE can increase PAR2 expression and MUC5AC release. In our study, we showed that NE binding to PAR2 can increase the cytosolic calcium concentration and subsequently activate PKC, leading to MUC5AC secretion. In order to investigate the mechanism of increased cytosolic calcium in Calu-3 cells, thapsigargin was used to exhaust the endoplasmic reticulum (ER) calcium pools, and 2-aminoethoxydiphenyl borate was used to inhibit the function of the store-operated calcium entry (SOCE) channels in the plasma membrane. We found that the NE-induced increase in intracellular calcium concentration is derived from release of the ER calcium pool and its subsequent calcium internal flux from the extracellular space via SOCE channels, which is dependent on sufficient levels of extracellular calcium.

Oxidative stress mediates the disruption of airway epithelial tight junctions through a TRPM2-PLCγ1-PKCα signaling pathway.

Oxidative stress has been implicated as an important contributing factor in the pathogenesis of several pulmonary inflammatory diseases. Previous studies have indicated a relationship between oxidative stress and the attenuation of epithelial tight junctions (TJs). In Human Bronchial Epithelial-16 cells (16HBE), we demonstrated the degradation of zonula occludens-1 (ZO-1), and claudin-2 exhibited a great dependence on the activation of the transient receptor potential melastatin (TRPM) 2 channel, phospholipase Cγ1 (PLCγ1) and the protein kinase Cα (PKCα) signaling cascade.

[Main mediated molecules of airway shear stress-stimulated MUC5AC extracellular secretion].

OBJECTIVE: To explore the main mediated molecules of mucin (MUC) 5AC extracellular secretion stimulated by airway shear stress (SS). METHODS: The 16 human bronchial epithelial (HBE) cells were cultured and randomized divided by Stata software into 5 groups: A. control group; B. SS stimulated group; C. SS stimulated & NSC23766 (a specific inhibitor of Rac-1) incubated group; D. SS stimulated & Cytochalasin D incubated group; E. Cortactin-siRNA (a small interfering RNA of Cortactin) transfected & SS stimulated group. Each group consisted of 6 parallel wells. Triplicate experiments were performed for statistical analysis. Rhythmic rotating device was used to simulate the breathing air flow mediated shear stress. The function of Cortactin was inhibited by Cortactin-siRNA. The relative content of MUC5AC in supernatant was measured by enzyme linked immunosorbent assay (ELISA). The p-Cortactin (phosphorylation Cortactin) relative level, Cortactin relative level and the effect of transfection were measured with Western blotting. And laser confocal microscope was used to observe the polymerization of F-actin. RESULTS: The transfection of Cortactin-siRNA successfully inhibited the function of Cortactin. The relative content of MUC5AC was (0.210 ± 0.013), (0.631 ± 0.025), (0.473 ± 0.112), (0.330 ± 0.067), (0.272 ± 0.019) in groups A, B, C, D and E, the group B was significantly higher than any other group (P = 0.000, 0.043, 0.000, 0.000). The Cortactin relative level in group B (0.670 ± 0.048) was significantly higher than that in group E (0.132 ± 0.014) (P < 0.01). But as compared with groups A, C, D (0.641 ± 0.016, 0.622 ± 0.012, 0.653 ± 0.027), there was no significance (all P > 0.05). The p-Cortactin relative level in group B (0.582 ± 0.067) was significantly higher than that in groups A, C, E (0.131 ± 0.011, 0.393 ± 0.045, 0.170 ± 0.016) (P = 0.000, 0.021, 0.000). But as compared with group D (0.511 ± 0.029), there was no significance (P = 0.246). CONCLUSION: Rac-1, Cortactin and F-actin are the main mediated molecules of airway shear stress-stimulated MUC5AC extracellular secretion.

[Effects of glycyrrhizin on airway mucus hypersecretion induced by interleukin-13 in rats].

OBJECTIVE: To explore the effects of glycyrrhizin on airway mucus hypersecretion induced by interleukin-13 (IL-13) in rats. METHODS: A total of 50 SD rats were divided randomly into 5 groups with a random digit table: control group, IL-13 group, and different dosage (25, 50, 75 mg/kg) glycyrrhizin groups. The integral of expression intensity in positive cells of airway epithelium under mucus histochemical stain was calculated with modality-quantitative method. HBE-16 cells were divided into 6 groups: negative control (physiological saline), IL-13 control (10 µg/L IL-13+physiological saline), different concentration glycyrrhizin interference (10 µg/L IL-13+25, 50 and 75 µmol/L glycyrrhizin, respectively) and positive control (10 µg/L IL-13+25 µmol/L zopolrestat). The expression of mucin (MUC) 5AC mRNA, MUC5AC protein, aldose reductase (AR) activity and reactive oxygen species (ROS) content were detected by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, fluorometric method and fluorescence intensity with General Oxidative Stress Indicator (CM-H2DFDA) catheter respectively. RESULTS: In vivo, the integral of expression intensity in positive stain cells of airway epithelium were 0.12 ± 0.03, 0.87 ± 0.13, 0.56 ± 0.08, 0.46 ± 0.06 and 0.35 ± 0.04 respectively while the integral of different dosage glycyrrhizin groups was significantly lower than that of IL-13 group (all P < 0.05) with dose depentency and the IL-13 group was stronger than control group (P < 0.05). In vitro, the index of AR activity and ROS at 48 h of HBE16 cells in every group were 0.156 ± 0.021, 0.692 ± 0.039, 0.436 ± 0.019, 0.323 ± 0.042 and 0.290 ± 0.027; 5.127 ± 0.033, 24.257 ± 3.263, 11.966 ± 0.283, 8.892 ± 0.521 and 6.426 ± 0.173 respectively. The indices of IL-13 control group were higher than those of negative control group (P < 0.05) while those of different concentration glycyrrhizin interference groups were lower than those of IL-13 control group (all P < 0.05). The expressions of MUC5AC mRNA and protein of HBE16 cells in every group were 0.82 ± 0.05, 3.22 ± 0.12, 2.57 ± 0.34, 2.09 ± 0.54 and 1.58 ± 0.22; 0.18 ± 0.04, 0.65 ± 0.15, 0.48 ± 0.11, 0.33 ± 0.19 and 0.26 ± 0.06 respectively. The indices of IL-13 control group were higher than those of negative control group (P < 0.05) and those of different concentration glycyrrhizin interference groups were lower than those of IL-13 control group (P < 0.05). CONCLUSION: Glycyrrhizin may inhibit the expression of MUC5AC mRNA and MUC5AC protein induced by IL-13 and control the hypersecretion of airway mucus.

[Effect of rhythmic pressure waves on balancing airway basic mucus secretion in dogs].

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.

Triiodothyronine represses MUC5AC expression by antagonizing Sp1 binding to its promoter in human bronchial epithelial HBE16 cells.

Mucus hypersecretion is a distinguished feature of chronic inflammatory airway diseases. Interestingly, in this condition thyroid function is impaired with decreased level of triiodothyronine (T3), indicating potential link between low level of T3 and mucus hypersecretion. But the underlying mechanisms are poorly understood. In this study we aimed to elucidate the effect of T3 on MUC5AC secretion in human bronchial epithelial HBE16 cells and further investigate how T3 regulates MUC5AC gene expression at transcriptional level. By RT-PCR and ELISA we showed that T3 inhibited MUC5AC mRNA expression and protein secretion in HBE16 cells. Furthermore, luciferase assay and site-directed mutagenesis analysis demonstrated that T3 repressed MUC5AC expression at transcriptional level and the mechanism might partly lie in the specific inhibition of Sp1 binding to the promoter. Our results suggest that decreased T3 level leads to the release of repression of MUC5AC expression and thus contributes to mucus hypersecretion.

Regulation of PMA-induced MUC5AC expression by heparin in human bronchial epithelial cells.

Mucus hypersecretion is a major pathophysiologic feature in chronic inflammatory airway diseases. Oxidative stress plays a pivotal role in this process. Recent studies have found that heparin has antioxidant effects which can reduce free radical damage. Here, we hypothesized that heparin has some influence on the expression of mucin 5AC (MUC5AC) induced by phorbol myristate acetate (PMA) in a bronchial epithelial cell line (HBE16), also we have investigated the potential mechanism involved in the process. We found that ROS, the mRNA of Duox1, EGFR and MUC5AC, as well as the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC in the PMA group were significantly increased when compared with the control group (all P < 0.01). After pretreatment with heparin however, there was a significant decrease in ROS levels, the mRNA of Duox1, EGFR, and MUC5AC, and the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC, when compared with the PMA group (all P < 0.01). MUC5AC protein in the supernatant was inhibited in a dose-dependent manner by heparin. Pretreatment with DMTU resulted in a significant decrease in ROS content, the mRNA of Duox1, EGFR, and MUC5AC as well as the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC when compared with the PMA group (all P < 0.01). When cells were pretreated with both heparin and DMTU, there was a further reduction in ROS content, the mRNA of Duox1, EGFR, and MUC5AC as well as the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC, when compared with either the PMA group, heparin group, or DMTU group (all P < 0.01). Our results show that PMA can induce MUC5AC expression by activation of the Duox1-ROS-TACE-TGF-α-EGFR signaling pathway. Heparin can decrease the level of Duox1, ROS production and block the PMA-induced activation of EGFR, thus inhibiting the overexpression of mucin MUC5AC in a dose-dependent manner. In addition to reducing ROS production, heparin may also inhibit the expression of MUC5AC through other signal mechanisms.

Chronic mechanical stress induces mucin 5AC expression in human bronchial epithelial cells through ERK dependent pathways.

Mucus hypersecretion is a common pathological change in chronic inflammatory diseases of the airway. These conditions are usually accompanied by chronic mechanical stress due to airway constriction. Our objective was to study the molecular mechanisms and physical effects of chronic mechanical stress on mucin 5AC (MUC5AC) expression in airway epithelial cells. We exposed normal human bronchial epithelial (NHBE) cells cultured at an air-liquid interface to different degrees of chronic compressive mechanical stress (10, 20, 30 cmH(2)O) for 7 days(1 h per day). MUC5AC protein content was detected by enzyme-linked immunosorbent assay (ELISA). MUC5AC mRNA expression was detected by reverse transcription PCR (RT-PCR) and real-time PCR. The effects of chronic mechanical stress on phosphorylated ERK1/2 (p-ERK1/2), phosphorylated JNK (p-JNK), phosphorylated P38 (p-P38), and phosphorylation of FAK at Tyr397 (p-FAK-Y397), were assessed by Western blot. We also assessed the impact of, an EGFR kinase inhibitor (AG1478), an ERK kinase inhibitor (PD-98059), and short interfering RNA (siRNA) targeted to FAK. We found that transcriptional and protein expression levels of MUC5AC were elevated significantly in the 30 cmH(2)O compressive stress group. p-ERK1/2 increased significantly in response to compressive stress and PD-98059 could attenuated stress-induced MUC5AC expression. p-FAK-Y397 increased significantly in response to compressive stress and FAK siRNA attenuated stress-induced ERK activation strongly. AG1478 attenuated stress-induced ERK activation and MUC5AC expression significantly, but incompletely. Combination of FAK siRNA and AG1478 led to complete attenuation of ERK activation and MUC5AC expression. These results suggest that chronic mechanical stress can enhance MUC5AC expression in human bronchial epithelial cells through the ERK signal transduction pathway. Both FAK and EGFR mediate the mitogenic response induced by mechanical stress in human bronchial epithelial cells through an ERK signaling cascade.

Transient receptor potential vanilloid 1 receptors mediate acid-induced mucin secretion via Ca2+ influx in human airway epithelial cells.

Mucin hypersecretion is a key pathological feature of inflammatory respiratory diseases. Previous studies have reported that acids (gastroesophageal reflux or environmental exposure) induce many respiratory symptoms and are implicated in the pathophysiology of obstructive airway diseases. To understand these mechanisms, we measured acid-induced mucin secretion in human bronchial epithelial cells. In the present study, acid induced inward currents of transient receptor potential vanilloid (TRPV)1 and mucin 5AC (MUC5AC) secretion dose dependently, which were inhibited by TRPV1 antagonist capsazepine in a concentration-dependent manner. TRPV1 agonist capsaicin mediated a concentration-dependent increase in TRPV1 inward currents and MUC5AC secretion. Furthermore, capsaicin enhanced acid-induced TRPV1 inward currents and MUC5AC secretion. Acid-induced Ca(2+) influx was prevented by capsazepine dose dependently and enhanced by capsaicin. Pretreatment only with capsaicin also increased the Ca(2+) concentration in a concentration-dependent manner. These data suggest that pharmacological inhibition of calcium-permeable TRPV1 receptors could be used to prevent acid-induced mucin secretion, thereby providing a potential mechanism to reduce their toxicity.

Regulation of cigarette smoke-mediated mucin expression by hypoxia-inducible factor-1α via epidermal growth factor receptor-mediated signaling pathways.

Cigarette smoking is strongly implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Mucus hypersecretion is the key manifestation in patients with COPD and mucin 5AC (MUC5AC) is a major component of airway mucus. Hypoxia inducible factor-1 (HIF-1) is a transcriptional factor which can be stimulated to bind to the MUC5AC promoter and induce MUC5AC promoter activation. Previous studies have reported that activation of HIF-1α pathways by cigarette smoke contributes to the development of COPD. We hypothesize that cigarette smoke up-regulates HIF-1α production and HIF-1 activity through epidermal growth factor receptor (EGFR)-activated signal cascades pathways, leading to mucin production in human airway epithelial cells (16HBE). We show that cigarette smoke increases HIF-1α production, HIF-1 activity and MUC5AC expression. These effects are prevented by small interfering RNA (siRNA) for HIF-1α, indicating that cigarette smoke-induced mucin production is HIF-1α-dependent. Cigarette smoke activates extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K) signal pathways, both of which are inhibited by gefitinib (an inhibitor of EGFR), suggesting that cigarette smoke-activated signal pathways are mediated by EGFR in 16HBE cells. Furthermore, pretreatment with gefitinib and the pharmacological inhibitors of PI3K (LY294002) and ERK1/2 (PD98059) prevented cigarette smoke-mediated Akt and ERK1/2 phosphorylation responses, HIF-1α production, HIF-1 activity and MUC5AC expression. These observations demonstrate an important role for EGFR-mediated signaling pathways in regulating cigarette smoke-induced HIF-1 activation and MUC5AC expression. Our results suggest that cigarette smoke activates EGFR-mediated signaling pathways, leading to HIF-1α production and HIF-1 activation, resulting in mucin expression in human airway epithelial cells.

Cold temperature induces mucin hypersecretion from normal human bronchial epithelial cells in vitro through a transient receptor potential melastatin 8 (TRPM8)-mediated mechanism.

BACKGROUND: Cold air stimulus is a major environmental factor that exacerbates chronic inflammatory airway diseases, such as chronic obstructive pulmonary disease (COPD) and asthma. At the molecular level, cold is detected by transient receptor potential melastatin 8 (TRPM8). To date, TRPM8 expression has not been characterized in the airway epithelium of patients with COPD. The role of TRPM8 channels in a series of airway responses induced by cold stimuli and the molecular and biochemical pathways of TRPM8 in regulating cold-induced responses are largely unknown. OBJECTIVE: We sought to explore the role of TRPM8 in cold air-provoked mucus hypersecretion and the potential signaling pathway involved in this process. METHODS: The expression of TRPM8 in the bronchial epithelium was examined by means of immunohistochemistry, RT-PCR, and Western blotting. TRPM8 receptor function and hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) were characterized by means of Ca(2+) imaging and spatiotemporal dynamics of phospholipase C (PLC) δ1-pleckstrin homology-green fluorescent protein, respectively. The expression of MUC5AC mRNA and MUC5AC mucin protein was measured by using real-time PCR and ELISA, respectively. Four serine residues in the myristoylated alanine-rich C kinase substrate (MARCKS)-phosphorylation site domain were mutated to identify the function of MARCKS in TRPM8-mediated airway mucus hypersecretion. RESULTS: TRPM8 protein and mRNA expression were significantly increased in patients with COPD compared with expression seen in healthy subjects. Cold produced robust increases in intracellular Ca(2+) levels and promoted translocation of PLCδ1-pleckstrin homology-green fluorescent protein. Cold increased expression of MUC5AC mRNA and intracellular and secreted MUC5AC protein in a nonsustained way. Phosphorylation site domain-mutant MARCKS cDNA hindered MUC5AC secretion induced by cold. CONCLUSIONS: These results indicate that the TRPM8 receptor is involved in cold-induced mucus hypersecretion through the Ca(2+)-PLC-PIP2-MARCKS signaling pathway.

[Role of neuregulin-1ß in airway hypersecretion induced by in interleukin-1ß].

OBJECTIVE: To explore the role of neuregulin 1ß (NRG-1ß) in airway hypersecretion induced by interleukin (IL)-1ß. METHODS: After stimulating the airway epithelial cell line HBE16 with IL-1ß, the expressions of NRG-1ß mRNA and mucin (MUC) 5AC mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR), the proteins of NRG-1ß and MUC5AC measured by enzyme-linked immunosorbent assay (ELISA) and phosphorylated erythroblastic leukemia viral oncogene homolog (ErbB)1-4 detected by Western blot. The cells were pre-treated with antibodies of ErbB1-4, inhibitors of p38 mitogen-activated protein kinase (MAPK), ERK1/2, mitogen- and stress-activated protein kinase (MSK)1 and antibody of cAMP-response element-binding protein (CREB). After the addition of stimulant NRG-1ß, MUC5AC was measured by ELISA. RESULTS: IL-1ß could increase markedly the levels of NRG-1ß mRNA and MUC5AC mRNA and also the proteins of NRG-1ß and MUC5AC in a dose-dependent fashion. NRG-1ß at concentrations of 1, 10, 100, 200 nmol/L increased the expression of MUC5AC (0.328 ± 0.055, 0.364 ± 0.086, 0.650 ± 0.134, 0.586 ± 0.068) versus the control group (0.227 ± 0.019). And the results had statistical significances (P < 0.05). The expressions of phosphorylated ErbB2 and ErbB3 stimulated by NRG-1ß were positive while those of phosphorylated ErbB1 and ErbB 4 negative. After a pretreatment of antibodies of ErbB2, ErbB3, inhibitors of p38MAPK, ERK1/2, MSK1 and antibody of CREB and a stimulation of NRG-1ß, the expression of MUC5AC decreased (0.221 ± 0.033, 0.238 ± 0.044, 0.386 ± 0.021, 0.352 ± 0.022, 0.294 ± 0.017, 0.252 ± 0.019) versus the NRG-1ß group (0.650 ± 0.134). And the results had statistical significances (P < 0.05). CONCLUSION: IL-1ß may cause airway hypersecretion probably through the combination of NRG-1ß with ErbB2 and ErbB3 heterodimers and the activation of MAPK/MSK1/CREB signal conduction.

[Acrolein decreases SUMO modification of glucocorticoid receptor and reduces its sensitivity to corticosteroids in airway hypersecretion].

OBJECTIVE: To explore the variation of small ubiquitin-related modification (SUMO) level of glucocorticoid receptors (GRs) exposed to acrolein stimulation as well as the influence of the variation on mucus hypersecretion. METHODS: The recombinant plasmid was constructed by inserting small ubiquitin-like modifier 1 (SUMO1) cDNA into eukaryotic expression plasmid pcDNA3.1-EGFP with a flag sequence marker. The recombinant plasmid pcDNA3.1-EGFP-flag-SUMO1 was identified by enzyme digestion analysis. The 16HBE cells were cultured and randomized divided into 9 following groups: A. control; B. acrolein stimulated; C. dexamethasone incubated; D. acrolein stimulated and dexamethasone incubated; E. SUMO1 transfected; F. pcDNA3.1-EGFP vector transfected; G. SUMO1 transfected and acrolein stimulated; H. SUMO1 transfected, acrolein stimulated and dexamethasone incubated; I. pcDNA3.1-EGFP vector transfected, acrolein stimulated and dexamethasone incubated. Each group consisted of 5 parallel wells and experiments were repeated for 4 times for statistical analysis. The transfection efficiency was evaluated by the expression of enhanced green fluorescent protein (EGFP) via fluorescence microscope. The SUMO modification level of GRs was measured with co-immunoprecipitation and Western blot. The transcription level of mucin (MUC) 5AC was evaluated with reverse transcription-polymerase chain reaction (RT-PCR). The MUC5AC secreted in supernatant was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The SUMO modification level was much lower in group B (0.079 ± 0.023) than that in group A (0.446 ± 0.068) (t = -23.13, P = 0.000) and in group D (0.574 ± 0.018) than that in group C (0.843 ± 0.028) (t = -36.34, P = 0.000). The transcription level of MUC5AC was significantly lower in group H (0.330 ± 0.063) than group D (0.617 ± 0.190) (q = -7.80, P = 0.000). Through ELISA, there was no significance between groups G and B in the term of secretion level of MUC5AC. While the secretion level of MUC5AC was much lower in group H ((0.416 ± 0.092) µg/L) than group D ((0.663 ± 0.104) µg/L) and group G ((0.740 ± 0.343) µg/L) (q = -7.31, -9.59; P = 0.001, 0.000). CONCLUSION: Acrolein decreases the SUMO modification of GRs and reduces the inhibitory effect of dexamethasone on the transcription of MUC5AC.

[Mechanisms of mucus hypersecretion in airway of rats induced by synergies between cold air and cigarette smoke inhalation and intervention effects of drugs].

OBJECTIVE: To explore the mechanisms of mucus hypersecretion in airway of rats induced by the synergies between cold air and cigarette smoke inhalation and understand the intervention effects of saussurea and budesonide in this process. METHODS: A total of 70 SD rats were randomly divided into 7 groups. Group A: control; Group B: cold stimulation group receiving cold air inhalation for 3 h daily for 40 d; Group C: cigarette smoke inhalation group receiving cigarette smoke inhalation for 0.5 h daily for 40 d; Group D: cigarette smoke inhalation + cold stimulation group; Group E: cigarette smoke inhalation + cold stimulation + saussurea (0.8 mg/kg saussurea intraperitoneally injected once daily for 40 d); Group F: cigarette smoke inhalation + cold stimulation + inhaled budesonide (0.5 mg/kg inhaled once daily for 40 d); Group G: cigarette smoke inhalation + cold stimulation + saussurea + inhaled budesonide. The relative quantities of TRPM8 mRNA within bronchial epithelium of each group were detected by real-time polymerase chain reaction (PCR) and TRPM8 protein was detected by immunohistochemical assay and Western blot. The levels of mucin (MUC) 5AC, interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) in bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay. RESULTS: TRPM8 mRNA of groups A-G were 1.00 ± 0.00, 0.98 ± 0.07, 2.27 ± 0.29, 2.31 ± 0.30, 1.55 ± 0.38, 1.66 ± 0.40 and 1.31 ± 0.23; TRPM8 protein 0.16 ± 0.05, 0.16 ± 0.04, 0.22 ± 0.06, 0.25 ± 0.05, 0.17 ± 0.04, 0.18 ± 0.03, 0.15 ± 0.05, 0.25 ± 0.04, 0.24 ± 0.03, 0.58 ± 0.06, 0.56 ± 0.09, 0.41 ± 0.09, 0.39 ± 0.07 and 0.20 ± 0.06 respectively. TRPM8 mRNA and protein of groups C and D were significantly higher than those of group A. And groups E, F and G were significantly lower than those of group D (all P < 0.05). In BALF of groups A-G, MUC5AC were (57 ± 6), (69 ± 5), (66 ± 4), (185 ± 43), (142 ± 30), (147 ± 36) and (60 ± 11) µg/mg, IL-8 (58 ± 14), (146 ± 38), (134 ± 29), (379 ± 101), (262 ± 67), (294 ± 70) and (81 ± 27) ng/L, TNF-α(153 ± 28), (208 ± 90), (274 ± 64), (600 ± 113), (458 ± 96), (498 ± 84) and (169 ± 65) ng/L respectively. The values of groups B, C and D were significantly higher than those of group A (all P < 0.05) while groups E, F and G were significantly lower than those of group D (all P < 0.05). Cigarette smoke inhalation and cold stimulation synergistically enhanced the expression of MUC5AC, IL-8 and TNF-α. Saussurea and inhaled budesonide synergistically inhibited the expression of MUC5AC, IL-8 and TNF-α. CONCLUSIONS: Cold air inhalation evokes the release of proinflammatory cytokines and MUC5AC via activated TRPM8 channel up-regulated by cigarette smoke inhalation. Saussurea and inhaled budesonide synergistically inhibits the above mentioned process.