Search

Salidroside is an effective component of the traditional Chinese herb, Rhodiola rosea, that is known to have the ability to protect individuals from cold attacks. In the present study, we investigated the effects of salidroside on respiratory epithelial cells exposed to cold temperatures. We wished to determine whether salidroside exerts any effect on cold-induced mucin (MUC) production and the possible mechanisms involved in this process. We incubated HBE16 cells with salidroside, exposed them to a cold stimulus (18˚C), and assayed the following endpoints: MUC production (the expression of MUC5AC), concentration intracellular of free calcium ([Ca2+]i), the activation of the transient receptor potential melastatin 8 (TRPM8) channel and the cAMP response element-binding protein (CREB). Our results revealed a significant increase in the [Ca2+]i concentration, as well as in TRPM8 and CREB expression in the cold-stimulated cells. MUC5AC expression was also increased. Treatment of the cells with salidroside at concentrations of 50 and 100 µM decreased the [Ca2+]i concentration, with a maximal effect detected in the cells treated with 100 µM salidroside. The expression of TRPM8 and TRPM8 channel conductivity were also repressed by salidroside; salidroside decreased the high levels of CREB activity and phosphorylation observed in the cold-stimulated cells. Furthermore, we transfected the cold-stimulated cells with CREB small interfering RNA (siRNA) to analyze TRPM8 gene expression in the absence of CREB activity. The results revealed that the cells treated with either CREB siRNA or salidroside expressed low levels of TRPM8 mRNA and protein. These results indicate that salidroside reduces MUC overproduction induced by cold stimuli and that salidroside exerts its protective effects by inhibiting TRPM8 activation, mainly by decreasing CREB activity.

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.)

The overexpression and hypersecretion of mucus is a hallmark of chronic pulmonary inflammatory disease. Mucin5AC (MUC5AC) is a major component of airway gel-forming mucin. Members of the Unc13 (Munc13) protein family act as important activators of granule exocytosis from various types of mammalian cells. The present study aimed to determine the role of Munc13 family proteins in MUC5AC secretion via an in vitro study with BEAS-2B and Calu-3 cell lines. Reverse transcription-quantitative polymerase chain reaction and western blotting indicated that stimulation of the cells with 100 nM human neutrophil elastase (hNE) for 1 h did not affect the expression of either unc13 homolog B (Munc13-2) or unc13 homolog D (Munc13-4), but immunofluorescence analysis demonstrated that hNE treatment was associated with the recruitment of Munc13-4 to the plasma membrane. Co-immunoprecipitation analysis indicated increased binding between Munc13-4 and syntaxin2 followingh NE stimulation; however, Munc13-2 formed a stable interaction with syntaxin2 with or without hNE stimulation. Subsequently, Munc13-2 and Munc13-4 expression levels were downregulated in BEAS-2B and Calu-3 cells using small interfering RNA (siRNA). ELISAs and immunofluorescence analysis were performed to assess MUC5AC secretion and intracellular retention, respectively. Munc13-2 siRNA transfection did not alter the expression levels of intracellular or secreted MUC5AC following hNE stimulation in either cell line; however, it increased the baseline intracellular levels of MUC5AC and decreased the amount of secreted MUC5AC. Conversely, Munc13-4 siRNA transfection increased the intracellular levels of MUC5AC and decreased the amount of secreted MUC5AC following hNE stimulation, but did not affect their baseline quantities. The results of the present study indicate that Munc13-2 may be an essential regulator of basal MUC5AC exocytosis, while Munc13-4 appears to be a Munc13 protein subtype that may to be sensitive to hNE stimulation during airway MUC5AC hypersecretion. [ABSTRACT FROM AUTHOR]

The cyclic mechanical effect of airflow during breathing creates the optimal airway hydration state. MUC (mucin) 5AC is an important component of the airway mucus. The formation of MUC5AC is related to ATP and intracellular calcium in the epithelial cells. In this study, we evaluated the effect of ATP release from intracellular calcium in epithelial cells on cyclic pressure-induced mucus secretion in the airway. 16HBE (human bronchial epithelial cells) were cultured in vitro on cyclically tilted cultured plates and divided into five groups: control, tilt, tilt and BAPTA-AM (1,2-bis-(oaminophenoxy) ethane-N,N,N',N'-tetra-acetic acid-acetoxymethyl ester), tilt and EGTA and tilt and RB-2 (reactive blue- 2). The shear stress and compressive stress were induced by the surface tension of the liquid, atmospheric pressure and liquid gravity. Cell activity, MUC5AC mRNA expression level, MUC5AC protein expression level and ATP release and intracellular calcium changes were measured with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, RT-PCR (reverse transcription-PCR), HPLC and inverted fluorescence microscope, respectively. We detected that cyclic pressure significantly increased MUC5AC secretion and ATP release. The enhanced ATP release could be inhibited by both BAPTA-AM and RB-2, while EGTA did not have a suppressive effect. BAPTA-AM, EGTA and RB-2 did not obviously inhibit MUC5AC mRNA expression. Cyclic pressure did not induce MUC5AC secretion in the airway mucus epithelium via Ca2+ -dependent ATP release, and nearly all Ca2+ was provided by stored intracellular Ca2+. [ABSTRACT FROM AUTHOR]

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]

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. [ABSTRACT FROM AUTHOR]

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 Ca2+ 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 Ca2+ 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 Ca2+-PLC-PIP2-MARCKS signaling pathway. [Copyright &y& Elsevier]

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]