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Fine particulate matter (PM2.5) is a type of small particle that is <2.5 µm in diameter that may cause airway inflammation. Thus, the present study aimed to explore the effects of PM2.5 on endoplasmic reticulum (ER) stress and airway inflammation in human airway epithelial cells. For this purpose, HBE135‑E6E7 airway epithelial cells were cultured and exposed to specific concentrations of PM2.5 for various periods of time, and cell viability was determined using a Cell Counting Kit‑8 assay. The results of the present study demonstrated that exposure to PM2.5 increased the mRNA and protein expression levels of interleukin (IL)‑6, tumor necrosis factor (TNF)‑α and mucin 5AC (MUC5AC). Moreover, the expression levels of ER stress‑related proteins, such as glucose‑regulated protein 78, CCAAT‑enhancer binding protein homologous protein, activating transcription factor 6, protein kinase R‑like ER kinase (PERK), phosphorylated (p‑)PERK, inositol‑requiring enzyme 1α (IRE1α) and p‑IRE1α, and nucleotide‑binding oligomerization domain 1 (NOD1) expression levels were increased following exposure to PM2.5. Transfection with IRE1α small interfering RNA (siRNA) led to the increased production of IL‑6, TNF‑α and MUC5AC. Moreover, the expression of NOD1 and the translocation of NF‑κB p65 were inhibited following transfection with IRE1α siRNA. In addition, the results of the present study demonstrated that transfection with NOD1 siRNA decreased the production of IL‑6, TNF‑α and MUC5AC, and decreased the translocation of NF‑κB p65. The expression levels of IL‑6, TNF‑α and MUC5AC were increased in the HBE135‑E6E7 cells following treatment with C12‑iE‑DAP, a NOD1 agonist. Moreover, treatment with C12‑iE‑DAP led to the activation of NF‑κB p65. Collectively, the results of the present study suggest that PM2.5 promotes airway inflammation and mucin production by activating ER stress in HBE135‑E6E7 airway epithelial cells, and that the IRE1α/NOD1/NF‑κB pathway may be involved in this process.

Mucus overproduction is an important manifestation of chronic airway inflammatory diseases, however, the mechanisms underlying the association between cold air and mucus overproduction remain unknown. We found that the expression of the cold-inducible RNA binding protein (CIRP) was increased in patients with chronic obstructive pulmonary disease (COPD). In the present study, we tested whether CIRP was involved in inflammatory factors and mucin5AC (MUC5AC) expression after cold stimulation and investigated the potential signaling pathways involved in this process. We found that CIRP was highly expressed in the bronchi of COPD patients. The expression of CIRP, interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) were increased, and the CIRP was localized in cytoplasm after cold stimulation. MUC5AC mRNA and protein expression levels were elevated in a temperature- and time-dependent manner after cold stimulation and were associated with the phosphorylation of ERK and NF-κB, which reflected their activation. These responses were suppressed by knockdown of CIRP with a specific siRNA or the ERK and NF-κB inhibitors. These results demonstrated that CIRP was expressed in the bronchi of human COPD patients and was involved in inflammatory factors and MUC5AC expression after cold stimulation through the ERK and NF-κB pathways., (Copyright © 2016. Published by Elsevier Inc.)

Objective: To explore the mechanism of calcium-sensing receptor (CaSR) in hypoxia-induced airway mucous hypersecretion., Methods: Cultured human airway epithelial cells HBE16 by hypoxia incubator (94%N₂, 1%O₂, 5%CO₂, 37 °C). HBE16 were transfected with CaSR targeted small interfering RNA (CaSR-siRNA), pretreated with a specific activator of CaSR CaCl₂ and preincubated with various inhibitors [Gαq/11 protein inhibitor YM-254890, phospholipase C (PLC) inhibitor U73122, inositol 1, 4, 5-triphosphate receptors (IP 3R) inhibitor 2-APB and cell-permeable intracellular calcium chelator BAPTA-AM] before hypoxia. The level of MUC5AC mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). And the relative content of CaSR protein was detected by Western blot. The relative content of MUC5AC protein was measured by enzyme-linked immunosorbent assay (ELISA). The intracellular calcium concentration ([Ca²⁺]i) was measured by laser confocal microscopy., Results: The relative contents of [Ca²⁺]i and MUC5AC protein were obviously higher in hypoxic group ((154.2 ± 11.4) nmol/L, (0.624 ± 0.063) µg/L) than those in control group ((67.5 ± 2.8) nmol/L, (0.257 ± 0.051) µg/L) (all P < 0.01). The relative levels of CaSR protein and MUC5AC mRNA were significantly higher in hypoxic group (0.423 ± 0.028, 0.736 ± 0.045) than those in control group (0.185 ± 0.036, 0.321 ± 0.034) (all P < 0.01). CaCl₂ enhanced the effect of hypoxia. And the results had statistical significances (all P < 0.01). Transfection with CaSR-siRNA significantly decreased the effect of hypoxia (all P < 0.01). Pretreatments with Gαq/11 protein inhibitor, PLC inhibitor, IP 3R inhibitor or intracellular calcium chelator all significantly attenuated the hypoxia-induced MUC5AC hypersecretion and [Ca²⁺]i (all P < 0.05)., Conclusion: CaSR mediates hypoxia-induced airway mucous hypersecretion through a signaling pathway of Gαq/11/PLC/inositol 1, 4, 5-triphosphate (IP 3)/[Ca²⁺]i.

Exposure to airborne particulate matter (PM) is a worldwide health problem. Previous studies have reported that PMs induced depolarizing currents and increased intracellular Ca(2+) in human bronchial epithelial cells. Ca(2+) plays important role in the regulation of mucus exocytosis, and mucin hypersecretion is a key pathological feature of inflammatory respiratory diseases. To explore more mechanisms underlying PM toxicity, we measured PM-induced mucin secretion in human bronchial epithelial (16HBE) cells. MUC5AC secretion and cyclic adenosine monophosphate (cAMP) level were detected by ELISA. Transient receptor potential vanilloid (TRPV)1 inward currents were examined by electrophysiology. Ca(2+) concentration was assessed by laser scanning confocal microscope. Exposure of PMs to 16HBE cells was found to induce mucin secretion, as a consequence of sustained Ca(2+) influx and cAMP increase through TRPV1 receptors. Mucin secretion was completely inhibited by TRPV1 receptor antagonist capsazepine. Removal of Ca(2+) by Ca(2+) chelator BAPTA or inhibition of protein kinase A (PKA) by the PKA inhibitors H-89 each partially reduced PC(2)s-induced mucin secretion. The combination of BAPTA and H-89 completely prevented mucin secretion mediated by PMs. These results suggest that PM induces mucin secretion through Ca(2+) influx and cAMP/PKA pathway by TRPV1 receptors in human bronchial epithelial cells, thereby providing a potential mechanism to reduce PM toxicity.

Mucus hypersecretion is an important manifestation in patients with chronic obstructive pulmonary diseases (COPD). Cigarette smoke is importantly implicated in the pathogenesis of COPD. Previous studies have shown that cigarette smoke-induced MUC5AC (a major component of airway mucus) expression involving ErbB1 (EGF receptor) signalling pathway. Recently, it has been reported that cigarette smoke induces ErbB3 activation in airway epithelia to secret mucus, and the ligand of ErbB3, neuregulin (NRG) 1β, induces MU5AC expression in human bronchial epithelial cells. In the present study, we have suggested that NRG1β/ErbB3 signalling is activated by cigarette smoke, resulting in the activation of a variety of signal cascade pathways, leading to mucin production in human bronchial epithelial (16HBE) cells. We show that cigarette smoke increases NRG1β release, ErbB3 phosphorylation and MUC5AC production. These effects are prevented by an ErbB3-neutralizing antibody and by specific knockdown using small interfering RNA (siRNA) for NRG1β, implicating NRG1β-dependent ErbB3 activation in the responses. Cigarette smoke activates ERK1/2, c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3-K) signalling pathways, which are also inhibited by an ErbB3-neutralizing antibody and NRG1β siRNA, indicating the regulation of cigarette smoke-activated pathways by NRG1β/ErbB3 signalling. Furthermore, pre-treatments with metalloprotease inhibitor (TNF-α protease inhibitor-1) and specific knockdown of TNF-α-converting enzyme (TACE) with TACE siRNA prevented cigarette smoke-induced NRG1β release, ErbB3 phosphorylation and mucin production, suggesting the role of TACE in cigarette smoke-mediated NRG1β/ErbB3 signalling activation. These results suggest that NRG1β/ErbB3 signalling regulates cigarette smoke-induced mucin overproduction via the MAPK and PI3K signal pathways in 16HBE cells., (© 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.)

Rhythmic pressure waves (RPW), mimicking the mechanical forces generated during normal breathing, play a key role in airway surface liquid (ASL) homeostasis. As a major component of ASL, we speculated that the mucin5AC (MUC5AC) expression must also be regulated by RPW. However, fewer researches have focused on this question. Therefore, our aim was to test the effect and mechanism of RPW on MUC5AC expression in cultured human bronchial epithelial cells. Compared with the relevant controls, the transcriptional level of MUC5AC and the protein expressions of MUC5AC, the phospho-epidermal growth factor receptor (p-EGFR), phospho-extracellular signal-related kinase (p-ERK), and phospho-Akt (p-Akt) were all significantly increased after mechanical stimulation. However, this effect could be significantly attenuated by transfecting with EGFR-siRNA. Similarly, pretreating with the inhibitor of ERK or phosphatidylinositol 3-kinases (PI3K)/Akt separately or jointly also significantly reduced MUC5AC expression. Collectively, these results indicate that RPW modulate MUC5AC expression via the EGFR-PI3K-Akt/ERK-signaling pathway in human bronchial epithelial cells. Physiologic rhythmic pressure waves regulate gene expression in human airway epithelial cells. This genetic regulation results in increased protein expression of mucin5AC, p-EGFR, p-ERK, and p-Akt. [ABSTRACT FROM AUTHOR]

ABSTRACT 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-1 α pathways by cigarette smoke contributes to the development of COPD. We hypothesize that cigarette smoke up-regulates HIF-1 α 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-1 α production, HIF-1 activity and MUC5AC expression. These effects are prevented by small interfering RNA (siRNA) for HIF-1 α, indicating that cigarette smoke-induced mucin production is HIF-1 α-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-1 α 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-1 α production and HIF-1 activation, resulting in mucin expression in human airway epithelial cells. Copyright © 2011 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Mucus hypersecretion is commonly observed in many chronic airway inflammatory diseases. Mucin 5AC (MUC5AC) is a major airway mucin because of its high expression in goblet cells. Here, the authors identified a gene called SAM domain--containing prostate-derived Ets factor (SPDEF) that was induced by interleukin (IL)-13. Their results showed that specific knockdown of SPDEF reduced IL-13-induced MUC5AC expression in human airway epithelial cells. This finding was associated with decreased expression of anterior gradient 2 (AGR2) and Ca2++-activated Cl−− channel (CLCA1), which regulate IL-13-mediated MUC5AC overproduction. Furthermore, transfection with SPDEF siRNA enhanced expression of forkhead box a2 (Foxa2), a key transcription factor that is known to prevent mucus production. The authors also demonstrated that the repression of STAT6 inhibited expression of SPDEF and MUC5AC induced by IL-13. These results show that SPDEF plays a critical role in regulating a transcriptional network mediating IL-13-induced MUC5AC synthesis dependent on STAT6. [ABSTRACT FROM AUTHOR]

Highlights: [•] MUC5AC is the major secreted mucin in airway obstructive diseases. [•] Actin polymerization induces MUC5AC secretion in response to shear stress. [•] Actin-binding protein cortactin is expressed in human airway epithelial cells. [•] Shear stress activates cortactin and then polymerizes actin filamentous. [•] Shear stress induces MUC5AC exocytosis via cortactin-mediated actin polymerization. [Copyright &y& Elsevier]

Abstract: Mucus hypersecretion is a common pathological feature of inflammatory airway diseases. Previous studies have shown that acidic microenvironment of inflamed airways may provoke the pathophysiology of inflammatory airway diseases. However, the acidic-sensing and negative regulatory mechanisms that mediate mucus hypersecretion in inflamed airway remain elusive. Thus, we sought to explore the role of ovarian cancer G-protein-coupled receptor 1 (OGR1) in acid-induced mucin5AC (MUC5AC) hypersecretion in human airway epithelium and the inhibitory effect of regulator of G-protein signaling 2 (RGS2) in this process. We found that airway acidification increased [Ca2+]i, which was required for MUC5AC secretion. Knocking-down OGR1 and Gq with siRNAs and pretreating cells with phospholipase C inhibitor effectively attenuated acid-induced cellular responses. Moreover, the overexpression of wild-type RGS2 attenuated acid-induced cellular responses. In contrast, reducing RGS2 with siRNA enhanced the increases in acid-induced cellular responses. These data suggest that airway acidification can induce MUC5AC hypersecretion through an OGR1-mediated mechanism and RGS2 can inhibit acid-induced MUC5AC hypersecretion at OGR1 receptor level. [Copyright &y& Elsevier]

Mucus hypersecretion is the key manifestation in patients with chronic inflammatory airway diseases and mucin 5AC (MUC5AC) is a major component of airway mucus. Matrix metalloproteinases (MMP)-9, have been found to be involved in the pathogenesis of inflammatory airway diseases. Hyperglycemia has been shown to be an independent risk factor for respiratory infections. We hypothesize that high glucose (HG)-regulates MMP-9 production and MMP-9 activity through nicotinamide adenine dinucleotide phosphate (NADPH)/reactive oxygen species (ROS) cascades pathways, leading to mucin production in human airway epithelial cells (16HBE). We show that HG increases MMP-9 production, MMP-9 activity and MUC5AC expression. These effects are prevented by small interfering RNA (siRNA) for MMP-9, indicating that HG-induced mucin production is MMP-9-dependent. HG activates MMP-9 production, MMP-9 activity and MUC5AC overproduction, which is inhibited by nPG, DMSO and DPI (inhibitors of ROS and NADPH), suggesting that HG-activated mucin synthesis is mediated by NADPH/ROS in 16HBE cells. These observations demonstrate an important role for MMP-9 activated by NADPH/ROS signaling pathways in regulating HG-induced MUC5AC expression. These findings may bring new insights into the molecular pathogenesis of the infections related to diabetes mellitus and lead to novel therapeutic intervention for mucin overproduction in chronic inflammatory airway diseases. [ABSTRACT FROM AUTHOR]