Src-JNK-dependent pathway in cigarette smoke-induced mucous hypersecretion in airway epithelial cells

The aim of this work was to find out whether Src kinase family and c-Jun N-terminal kinase (JNK) are involved in the ROS signaling pathway that could induce mucin MUC5AC expression in cultured cells of airway epithelia (BEAS-2B). For this purpose, the impact of cigarette smoke extract (CSE) on ROS production and MUC5AC expression in BEAS-2B cells was studied. Effects of ROS scavenger dimethylthiourea (DMTU), JNK specific inhibitor SP600125, and Src specific inhibitor PP2 in the CSE-induced ROS generation and MUC5AC expression were also assessed. A dose-dependent increase of ROS production in cells exposed to different concentrations of CSE was detected. DMTU inhibited cigarette smoke-induced Src phosphorylation, suggesting the ROS involvement in activation of Src kinase. Furthermore, SP600125 reduced the expression of MUC5AC. The activation of JNK was suppressed by PP2 but not by TACE inhibitor TAPI-1 or EGFR inhibitor PD153035. These results suggest that Src kinase participate in JNK activation and MUC5AC synthesis, which is independent of the TACE/EGFR activation. We conclude that ROS-Src-JNK signal cascade plays a particular role in cigarette smoke-induced mucin MUC5AC expression in BEAS-2B cells.     

人中性粒细胞弹性蛋白酶诱导气道黏液高分 泌细胞模型的建立及其机制的初步研究

目的：以人中性粒细胞弹性蛋白酶(HNE)为诱导因素,研究建立黏蛋白(MUC)5AC和5B高表达的细胞模型,同时对黏蛋白高表达机制进行初步研究。方法：培养人肺腺癌细胞A549,以HNE为刺激因素,EGFR中和抗体、表皮细胞生长因子受体（EGFR）磷酸化阻断剂AG1478为干预因素,分组培养。采用四甲基偶氮唑盐光吸收法（MTT法）检测HNE对细胞活性的影响;逆转录-聚合酶链反应（RT-PCR）检测MUC5AC mRNA、MUC5B mRNA的变化;酶联免疫吸附测定法(ELISA)定量分析MUC5AC和MUC5B蛋白含量的差异;细胞免疫化学以及激光共聚焦技术进一步直观观察MUC5AC、MUC5B、p-EGFR蛋白表达的变化。结果：HNE对A549细胞活力的影响呈剂量依赖性;HNE刺激组的MUC5AC、MUC5B基因转录和蛋白表达水平均明显高于对照组,差异有统计学意义（均P<0.01）;HNE刺激组p-EGFR蛋白表达显著增多,EGFR中和抗体、AG1478能显著降低HNE诱导的MUC5AC高表达,但对MUC5B高表达无干预作用。结论：人肺腺癌细胞A549同时表达MUC5AC和MUC5B,HNE能有效刺激A549细胞高表达MUC5AC和MUC5B,黏蛋白高表达细胞模型的建立为研究气道粘液高分泌疾病提供了实验基础。HNE通过激活EGFR信号转导通路诱导MUC5AC的高表达,但MUC5B高表达机制与之不同,有待进一步研究。     

Flagellin/TLR5 responses induce mucus hypersecretion by activating EGFR via an epithelial cell signaling cascades

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.     

Neutrophil elastase induces MUC5AC mucin production in human airway epithelial cells via a cascade involving protein kinase C, reactive oxygen species, and TNF-alpha-converting enzyme

Mucus hypersecretion is a prominent manifestation in patients with chronic inflammatory airway diseases and contributes to their morbidity and mortality by plugging airways and causing recurrent infections. Human neutrophil elastase (HNE) exists in high concentrations (1-20 microM) in airway secretions of these patients and induces overproduction of MUC5AC mucin, a major component of airway mucus. Previous studies showed that HNE induces MUC5AC mucin production involving reactive oxygen species (ROS) generation and TGF-alpha-dependent epidermal growth factor receptor (EGFR) activation in human airway epithelial cells. However, the molecular mechanisms involved in these responses are not defined. TNF-alpha-converting enzyme (TACE) cleaves pro-TGF-alpha into soluble TGF-alpha and can be activated by ROS. We hypothesize that HNE activates TACE via ROS generation, resulting in cleavage of pro-TGF-alpha, EGFR activation, and MUC5AC mucin expression in airway epithelial cells. Here we show that in human airway epithelial cells HNE increases TGF-alpha release, EGFR phosphorylation, and MUC5AC mucin expression, effects that were attenuated by TACE inhibitor TAPI-1 and by specific knockdown of TACE expression with small interfering RNA, implicating TACE in HNE-induced responses. These responses to HNE were also reduced by pretreatment with ROS scavengers, implicating ROS. Furthermore, we show that HNE causes protein kinase C (PKC) activation and translocation from cytosol to plasma membrane; blockade of this effect by PKC inhibitors reduced HNE-induced ROS generation and other responses, implicating PKC. We conclude that HNE induces MUC5AC mucin expression via a cascade involving PKC-ROS-TACE in human airway epithelial cells.     

Human eosinophils induce mucin production in airway epithelial cells via epidermal growth factor receptor activation.

Eosinophil recruitment and mucus hypersecretion are characteristic of asthmatic airway inflammation, but eosinophils have not been shown to induce mucin production. Because an epidermal growth factor receptor (EGFR) cascade induces MUC5AC mucin in airways, and because EGFR is up-regulated in asthmatic airways, we examined the effect of eosinophils on MUC5AC mucin production in NCI-H292 cells (a human airway epithelial cell line that produces mucins). Eosinophils were isolated from the peripheral blood of allergic patients, and their effects on MUC5AC mucin gene and protein synthesis were assessed using in situ hybridization and ELISAs. When IL-3 plus GM-CSF or IL-3 plus IL-5 were added to eosinophils cultured with NCI-H292 cells, MUC5AC mucin production increased; eosinophils or cytokines alone had no effect. Eosinophil supernatant obtained by culturing eosinophils with IL-3 plus GM-CSF or IL-3 plus IL-5 also increased MUC5AC synthesis in NCI-H292 cells, an effect that was prevented by selective EGFR inhibitors (AG1478, BIBX1522). Supernatant of activated eosinophils induced EGFR phosphorylation in NCI-H292 cells. Supernatant of activated eosinophils contained increased concentrations of TGF-alpha protein (an EGFR ligand) and induced up-regulation of TGF-alpha expression and release in NCI-H292 cells. A blocking Ab to TGF-alpha reduced activated eosinophil-induced MUC5AC synthesis in NCI-H292 cells. These results show that activated eosinophils induce mucin synthesis in human airway epithelial cells via EGFR activation, and they implicate TGF-alpha produced by eosinophils and epithelial cells in the EGFR activation that results in mucin production in human airway epithelium.     

Oxidative stress causes mucin synthesis via transactivation of epidermal growth factor receptor: role of neutrophils

Oxidative stress has been implicated in the pathogenesis of inflammatory diseases of airways. Here we show that oxidative stress causes ligand-independent activation of epidermal growth factor receptors (EGFR) and subsequent activation of mitogen-activated protein kinase kinase (MEK)-p44/42 mitogen-activated protein kinase (p44/42mapk), resulting in mucin synthesis in NCI-H292 cells. Exogenous hydrogen peroxide and neutrophils activated by IL-8, FMLP, or TNF-alpha increased EGFR tyrosine phosphorylation and subsequent activation of p44/42mapk and up-regulated the expression of MUC5AC at both mRNA and protein levels in NCI-H292 cells. These effects were blocked by selective EGFR tyrosine kinase inhibitors (AG1478, BIBX1522) and by a selective MEK inhibitor (PD98059), whereas a selective platelet-derived growth factor receptor tyrosine kinase inhibitor (AG1295), a selective p38 MAPK inhibitor (SB203580), and a negative compound of tyrosine kinase inhibitors (A1) were without effect. Neutrophil supernatant-induced EGFR tyrosine phosphorylation, activation of p44/42mapk, and MUC5AC synthesis were inhibited by antioxidants (N-acetyl-cysteine, DMSO, dimethyl thiourea, or superoxide dismutase); neutralizing Abs to EGFR ligands (EGF and TGF-alpha) were without effect, and no TGF-alpha protein was found in the neutrophil supernatant. In contrast, the EGFR ligand, TGF-alpha, increased EGFR tyrosine phosphorylation, activation of p44/42mapk, and subsequent MUC5AC synthesis, but these effects were not inhibited by antioxidants. These results implicate oxidative stress in stimulating mucin synthesis in airways and provide new therapeutic approaches in airway hypersecretory diseases.     

Distinct roles for the NK cell-activating receptors in mediating interactions with dendritic cells and tumor cells

Hyperproduction of goblet cells and mucin in the airway epithelium is an important feature of airway inflammatory diseases. We investigated the involvement of Notch signaling in MUC5AC expression in NCI-H292 cells, a human lung carcinoma cell line. Epidermal growth factor (EGF) stimulated generation of the Notch intracellular domain (NICD) in a RBP-Jκ-dependent manner. Treatment with γ-secretase inhibitors L-685,458 or DAPT or introduction of small interfering RNA directed against Notch1 reduced EGF-induced MUC5AC expression. The inhibitory effect of L-685,458 on EGF-induced MUC5AC mRNA and protein expression was also observed in primary human bronchial epithelial cells. Blockage of Notch signaling with L-685,458 or Notch siRNA resulted in a decrease in EGF-induced phosphorylation of ERK. These results suggested that ERK activation is necessary for the regulation of EGF receptor (EGFR)-mediated MUC5AC expression by Notch signaling. Conversely, forced expression of NICD induced both EGFR and ERK phosphorylation with MUC5AC expression even in the absence of EGF. Treatment of the NICD-expressing cells with EGF further augmented ERK phosphorylation in an additive manner. The ERK phosphorylation induced by exogenous NICD was inhibited by treatment with an Ab that antagonizes EGFR activity as well as by inhibitors of EGFR and ERK, implying that Notch signaling induces MUC5AC expression by activating the EGFR pathway. Collectively, these results suggest that MUC5AC expression is regulated by a bidirectional circuit between Notch and EGFR signaling pathways.     

Variation of human mucin gene expression in gastric cancer cell lines and gastric mucous cell primary cultures

Human gastric mucous cells - gastric cancer cell lines mucin gene expression - TNFalpha - RT-PCR immunocytochemistry Little is known on the expression pattern of mucin genes in human gastric cancer cell lines in relation to mucin expression in normal gastric epithelial cells. Thus, the aim of this study was to compare gastric cancer cell lines and non-transformed epithelial cells in their expression of the different mucin genes, in order to use these cells as models for physiological MUC expression in human stomach. Human gastric mucous cell primary cultures which were obtained from surgical specimen by collagenase/pronase treatment and a panel of six human gastric cancer cells were screened for mRNA expression of the mucin genes MUC1, MUC2, MUC5AC, MUC5B, and MUC6. Mucin gene expression was analyzed by semi-quantitative RT-PCR, and by Western blotting and immunocytochemistry. Primary cultured human gastric mucous cells retained the stomach-specific pattern of mRNA expression found in gastric mucosal biopsies (MUC1, MUC5AC, MUC6), whereas any gastric cancer cell line exhibited an aberrant mucin gene expression. Mucin gene expression showed large variations in levels and patterns from cell line to cell line, but MUC2 was aberrantly expressed in all cancer cells. Immunocytochemistry confirmed aberrant MUC2 protein expression in cancer cells. The expression of the secretory mucin genes MUC2 and MUC5AC varied in relation to the length of cultivation of the cancer cell lines. Treatment of the gastric cancer cells with TNFalpha resulted in an enhanced mRNA expression of MUC1, MUC2, and MUC5AC (2-fold increase within 3 hours; p <0.05). In contrast, immunocytochemistry disclosed a decrease in MUC2 and MUC5AC staining intensity. Our results indicate that primary cultured human gastric mucous cells provide a physiological in vitro system for investigations of gastric mucin gene regulation. In gastric cancer cells marked changes in the mucin gene expression pattern are found with coexpression of non-gastric type mucins. Gastric mucin gene expression may be regulated by proinflammatory cytokines which could have implications in gastritis.     

Neutrophil elastase induces mucin production by ligand-dependent epidermal growth factor receptor activation

Neutrophil products are implicated in hypersecretory airway diseases. To determine the mechanisms linking a proteolytic effect of human neutrophil elastase (HNE) and mucin overproduction, we examined the effects of HNE on MUC5AC mucin production in human airway epithelial (NCI-H292) cells. Stimulation with HNE for 5-30 min induced MUC5AC production 24 h later, which was prevented by HNE serine active site inhibitors, implicating a proteolytic effect of HNE. MUC5AC induction was preceded by epidermal growth factor receptor (EGFR) tyrosine phosphorylation and was prevented by selective EGFR tyrosine kinase inhibitors, implicating EGFR activation. HNE-induced MUC5AC production was inhibited by a neutralizing transforming growth factor-alpha (TGF-alpha, an EGFR ligand) antibody and by a neutralizing EGFR antibody but not by oxygen free radical scavengers, further implicating TGF-alpha and ligand-dependent EGFR activation in the response. HNE decreased pro-TGF-alpha in NCI-H292 cells and increased TGF-alpha in cell culture supernatant. From these results, we conclude that HNE-induced MUC5AC mucin production occurs via its proteolytic activation of an EGFR signaling cascade involving TGF-alpha.     

The tyrosine phosphatase, SHP-1, is involved in bronchial mucin production during oxidative stress

Mucus hypersecretion is a clinically important manifestation of chronic inflammatory airway diseases, such as asthma and Chronic obstructive pulmonary disease (COPD). Mucin production in airway epithelia is increased under conditions of oxidative stress. Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 suppression is related to the development of airway inflammation and increased ROS levels. In this study, we investigated the role of SHP-1 in mucin secretion triggered by oxidative stress. Human lung mucoepidermoid H292 carcinoma cells were transfected with specific siRNA to eliminate SHP-1 gene expression. Cultured cells were treated with hydrogen peroxide (H₂O₂), and Mucin 5AC(MUC5AC) gene expression and mucin production were determined. Activation of p38 mitogen activated protein kinase (MAPK) in association with MUC5AC production was evaluated. N-acetylcysteine (NAC) was employed to determine whether antioxidants could block MUC5AC production. To establish the precise role of p38, mucin expression was observed after pre-treatment of SHP-1-depleted H292 cells with the p38 chemical blocker. We investigated the in vivo effects of oxidative stress on airway mucus production in SHP-1-deficient heterozygous (mev/+) mice. MUC5AC expression was enhanced in SHP-1 knockdown H292 cells exposed to H₂O₂, compared to that in control cells. The ratio between phosphorylated and total p38 was significantly increased in SHP-1-deficient cells under oxidative stress. Pre-treatment with NAC suppressed both MUC5AC production and p38 activation. Blockage of p38 MAPK led to suppression of MUC5AC mRNA expression. Notably, mucin production was enhanced in the airway epithelia of mev/+ mice exposed to oxidative stress. Our results clearly indicate that SHP-1 plays an important role in airway mucin production through regulating oxidative stress.     

E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC mucin expression in cultured human airway epithelial cells

In previous work, we showed that epidermal growth factor receptor (EGFR) activation causes mucin expression in airway epithelium in vivo and in human NCI-H292 airway epithelial cells and normal human bronchial epithelial (NHBE) cells in vitro. Here we show that the cell surface adhesion molecule, E-cadherin, promotes EGFR-mediated mucin production in NCI-H292 cells in a cell density- and cell cycle-dependent fashion. The addition of the EGFR ligand, transforming growth factor (TGF)-alpha, increased MUC5AC protein expression markedly in dense, but not in sparse, cultures. MUC5AC-positive cells in dense cultures contained 2 N DNA content and did not incorporate bromodeoxyuridine, suggesting that they develop via cell differentiation and that a surface molecule involved in cell-cell contact is important for EGFR-mediated mucin production. In support of this hypothesis, in dense cultures of NCI-H292 cells and in NHBE cells at air-liquid interface, blockade of E-cadherin-mediated cell-cell contacts decreased EGFR-dependent mucin production. E-cadherin blockade also increased EGFR-dependent cell proliferation and TGF-alpha-induced EGFR tyrosine phosphorylation in dense cultures of NCI-H292 cells, suggesting that E-cadherin promotes EGFR-dependent mucin production and inhibits EGFR-dependent cell proliferation via modulation of EGFR phosphotyrosine levels. Furthermore, in dense cultures, E-cadherin blockade decreased the rate of EGFR tyrosine dephosphorylation, implicating an E-cadherin-dependent protein tyrosine phosphatase in EGFR dephosphorylation. Thus E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC production, and our results suggest that this occurs via a pathway involving protein tyrosine phosphatase-dependent EGFR dephosphorylation.     

LPS-stimulated MUC5AC production involves Rac1-dependent MMP-9 secretion and activation in NCI-H292 cells

Chronic obstructive pulmonary disease (COPD) is an inflammatory process characterized by airway mucus hypersecretion. Previous studies have reported that lipopolysaccharides (LPS) stimulate mucin 5AC (MUC5AC) production via epidermal growth factor receptor (EGFR) in human airway cells. Moreover, this production was shown to depend on the expression and activity of matrix metalloproteinase 9 (MMP-9), which is increased in COPD patients’ serum. In the present study we investigated the signaling pathway mediating LPS-stimulated secretion and activation of MMP-9, and the regulatory effects of this pathway on the production of MUC5AC in the human airway cells NCI-H292. Using specific inhibitors, we found that LPS-stimulated cells secreted and activated MMP-9 via EGFR. Our results also indicate that signaling events downstream of EGFR involved PI3K-dependent activation of Rac1, which mediated the NADPH-generated reactive oxygen species responsible for MMP-9 secretion and activation. Finally, we observed that EGFR/PI3K/Rac1/NADPH/ROS/MMP-9 regulate MUC5AC production in LPS-challenged NCI-H292 cells.     

Regulation of MUC5AC expression by NAD(P)H:quinone oxidoreductase 1

Neutrophil elastase (NE), a potent neutrophil inflammatory mediator, increases MUC5AC mucin gene expression through undefined pathways involving reactive oxygen species. To determine the source of NE-generated reactive oxygen species, we used pharmacologic inhibitors of oxidoreductases to test whether they blocked NE-regulated MUC5AC mRNA expression. We found that dicumarol, an inhibitor of the NADP(H):quinone oxidoreductase 1 (NQO1), inhibited MUC5AC mRNA expression in A549 lung adenocarcinoma cells and primary normal human bronchial epithelial cells. We further tested the role of NQO1 in mediating NE-induced MUC5AC expression by inhibiting NQO1 expression using short interfering RNA (siRNA). Transfection with siRNA specific for NQO1 suppressed NQO1 expression and significantly abrogated MUC5AC mRNA expression. NE treatment caused lipid peroxidation in A549 cells; this effect was inhibited by pretreatment with dicumarol, suggesting that NQO1 also regulates oxidant stress in A549 cells after NE exposure. NE exposure increased NQO1 protein and activity levels; NQO1 expression and activity were limited to the cytosol and did not translocate to the plasma membrane. Our results indicate that NQO1 has an important role as a key mediator of NE-regulated oxidant stress and MUC5AC mucin gene expression.     

Purification and characterization of a human pancreatic adenocarcinoma mucin.

Pancreatic mucins consist of core proteins that are decorated with carbohydrate structures. Previous studies have identified at least two physically distinct populations of mucins produced by a pancreatic adenocarcinoma cell line (HPAF); one is the MUC1 core protein, which includes an oligosaccharide structure identified by a monoclonal antibody (MAb) recognizing the DU-PAN-2 epitope. In this study, we purified and characterized a second mucin fraction, which also shows reactivity with the DU-PAN-2 antibody, but which has an amino acid composition that is not consistent with the MUC1 core protein. This new mucin was purified by ammonium sulfate precipitation, molecular sieve chromatography, and density gradient centrifugation. It eluted in the void volume of a Sepharose 4B column together with an associated low molecular weight protein, which could be further resolved. The mucin is highly polyanionic due to numerous sulfated and sialylated saccharide chains. Carbohydrate analyses of the purified mucin showed the presence of galactose, glucosamine, galactosamine, and sialic acid, but no mannose, glucose, or uronic acid. The purified and deglycosylated mucin shows no reactivity with anti-MUC1 apomucin antibody, but reacts with antiserum against deglycosylated tracheal mucins and antiserum against the MUC4 tandem repeat peptide. Analysis of mucin expression in HPAF cells revealed high levels of MUC1 and MUC4 mRNA, and moderate levels of MUC5AC and MUC5B mRNA. The amino acid composition of the purified mucin shows a high degree of similarity to the MUC4 core protein.