Activation of epidermal growth factor receptors is responsible for mucin synthesis induced by cigarette smoke

Mucus hypersecretion from hyperplastic airway goblet cells is a hallmark of chronic obstructive pulmonary disease (COPD). Although cigarette smoking is thought to be involved in mucus hypersecretion in COPD, the mechanism by which cigarette smoke induces mucus overproduction is unknown. Here we show that activation of epidermal growth factor receptors (EGFR) is responsible for mucin production after inhalation of cigarette smoke in airways in vitro and in vivo. In the airway epithelial cell line NCI-H292, exposure to cigarette smoke upregulated the EGFR mRNA expression and induced activation of EGFR-specific tyrosine phosphorylation, resulting in upregulation of MUC5AC mRNA and protein production, effects that were inhibited completely by selective EGFR tyrosine kinase inhibitors (BIBX1522, AG-1478) and that were decreased by antioxidants. In vivo, cigarette smoke inhalation increased MUC5AC mRNA and goblet cell production in rat airways, effects that were prevented by pretreatment with BIBX1522. These effects may explain the goblet cell hyperplasia that occurs in COPD and may provide a novel strategy for therapy in airway hypersecretory diseases.     

The human cathelicidin LL-37 enhances airway mucus production in chronic obstructive pulmonary disease

Airway mucus overproduction is a distinguishing feature of chronic obstructive pulmonary disease (COPD). LL-37 is the only member of human cathelicidins family of antimicrobial peptides and plays a central role in many immune and inflammatory reactions. Increasing evidence suggests the involvement of LL-37 in the pathogenesis of COPD. Here, we investigated the effects of LL-37 on airway mucus overproduction in COPD. We observed overexpression of both LL-37 and MUC5AC mucin (a major mucin component of mucus) in airways of COPD patients and found a correlation between them. We showed in vitro that LL-37 induces MUC5AC mucin production by airway epithelial NCI-H292 cells in the absence and presence of cigarette smoke extract, with TNF-α converting enzyme (TACE)–EGFR–ERK1/2 pathway and IL-8 required for the induction. Therefore, we concluded that LL-37 enhances the mucus production in COPD airways, thus contributing to the progression of COPD.     

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.     

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.     

CCL20/CCR6 feedback exaggerates epidermal growth factor receptor-dependent MUC5AC mucin production in human airway epithelial (NCI-H292) cells

Mucous hypersecretion is an important feature of obstructive airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Multiple stimuli induce mucin production via activation of an epidermal growth factor receptor (EGFR) cascade, but the mechanisms that exaggerate mucin production in obstructive airway diseases remain unknown. In this study, we show that binding of CCL20, a G protein-coupled receptor (GPCR) ligand that is upregulated in the airways of subjects with obstructive airway diseases, to its unique GPCR CCR6 induces MUC5AC mucin production in human airway epithelial (NCI-H292) cells via metalloprotease TNF-α-converting enzyme (TACE)-dependent EGFR activation. We also show that EGFR activation by its potent ligand TGF-α induces reactivation of EGFR via binding of endogenously produced CCL20 to its receptor CCR6 in NCI-H292 cells but not in normal human bronchial epithelial (NHBE) cells, exaggerating mucin production in the NCI-H292 cells. In NCI-H292 cells, TGF-α stimulation induced two phases of EGFR phosphorylation (EGFR-P). The second EGFR-P was TACE-dependent and was responsible for most of the total mucin induced by TGF-α. Binding of endogenously produced CCL20 to CCR6 increased the second EGFR-P and subsequent mucin production induced by TGF-α. In NHBE cells, TGF-α-induced EGFR activation did not lead to significant CCL20 production or to EGFR rephosphorylation, and less mucin was produced. We conclude that NCI-H292 cells but not NHBE cells produce CCL20 in response to EGFR activation, which leads to a second phase of EGFR-P and subsequent exaggerated mucin production. These findings have potentially important therapeutic implications in obstructive airway diseases.     

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.     

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.     

Peroxisome proliferator-activated receptor-gamma inhibits cigarette smoke solution-induced mucin production in human airway epithelial (NCI-H292) cells

The main etiologic factor for chronic bronchitis is cigarette smoke. Exposure to cigarette smoke is reported to induce goblet cell hyperplasia and mucus production. Mucin synthesis in airways has been reported to be regulated by the EGFR system. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the ligand-activated nuclear receptor superfamily. PPAR-gamma is implicated in anti-inflammatory responses, but mechanisms underlying these varied roles remain ill-defined. Recently, reports have shown that upregulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) might be one of the mechanisms through which PPAR-gamma agonists exert their anti-inflammatory actions. However, no data are available on the role of PPAR-gamma in smoke-induced mucin production. In this study, we investigated the effect of PPAR-gamma agonist (rosiglitazone) on smoke-induced mucin production in NCI-H292 cells. Exposure to cigarette smoke causes a significant decrease in PTEN expression and increases dose-dependent EGFR-specific tyrosine phosphorylation, resulting in MUC5AC mucin production in NCI-H292 cells. PPAR-gamma agonists or specific inhibitors of phosphoinositide 3-kinase exert inhibition of cigarette smoke-induced mucin production, with the upregulation of PTEN signaling and downregulation of Akt expression. This study demonstrates that PPAR-gamma agonist functions as a regulator of epithelial cell inflammation that may result in reduction of mucin-producing cells in airway epithelium.     

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.     

Epidermal growth factor receptor reactivation induced by E-prostanoid-3 receptor- and tumor necrosis factor-alpha-converting enzyme-dependent feedback exaggerates interleukin-8 production in airway cancer (NCI-H292) cells

Airway epithelial cancer cells produce increased amounts of the chemokine interleukin-8 (IL-8), inducing pro-tumor responses. Multiple stimuli induce airway epithelial IL-8 production epidermal growth factor receptor (EGFR) dependently, but the mechanisms that exaggerate IL-8 production in airway cancers remain unknown. Here we show that direct activation of EGFR (EGFR-P) by its ligand transforming growth factor (TGF)-alpha induces a second EGFR-P in human airway (NCI-H292) cancer cells but not in normal human bronchial epithelial (NHBE) cells, exaggerating IL-8 production in these cancer cells. The second EGFR-P in NCI-H292 cells was caused by metalloprotease TNF-alpha-converting enzyme (TACE)-dependent cleavage of EGFR pro-ligands and was responsible for most of the total IL-8 induced by TGF-alpha. In NCI-H292 cells, TGF-alpha induced cyclooxygenase (COX)-2-dependent prostaglandin (PG)E2 production and release. PGE2 increased the second EGFR-P and IL-8 production via binding to its Gi-protein-coupled E-prostanoid (EP)3 receptor. In NHBE cells, TGF-alpha-induced EGFR-P did not lead to PGE2 production or to a second EGFR-P, and less IL-8 was produced. Thus, we conclude that a positive feedback pathway involving COX-2/PGE2/EP3 receptor-dependent EGFR reactivation exaggerates IL-8 production in NCI-H292 cancer cells but not in NHBE (normal) cells.     

Multiple TLRs activate EGFR via a signaling cascade to produce innate immune responses in airway epithelium

Toll-like receptors (TLRs) are critical for the recognition of inhaled pathogens that deposit on the airway epithelial surface. The epithelial response to pathogens includes signaling cascades that activate the EGF receptor (EGFR). We hypothesized that TLRs communicate with EGFR via epithelial signaling to produce certain innate immune responses. Airway epithelium expresses the highest levels of TLR2, TLR3, TLR5, and TLR6, and here we found that ligands for these TLRs increased IL-8 and VEGF production in normal human bronchial epithelial cells. These effects were prevented by treatment with a selective inhibitor of EGFR phosphorylation (AG-1478), a metalloprotease (MP) inhibitor, a reactive oxygen species (ROS) scavenger, and an NADPH oxidase inhibitor. In an airway epithelial cell line (NCI-H292), TNF-alpha-converting enzyme (TACE) small interfering RNA (siRNA) was used to confirm that TACE is the MP involved in TLR ligand-induced IL-8 and VEGF production. We show that transforming growth factor (TGF)-alpha is the EGFR ligand in this signaling cascade by using TGF-alpha neutralizing antibody and by showing that epithelial production of TGF-alpha occurs in response to TLR ligands. Dual oxidase 1 (Duox1) siRNA was used to confirm that Duox1 is the NADPH oxidase involved in TLR ligand-induced IL-8 and VEGF production. We conclude that multiple TLR ligands induce airway epithelial cell production of IL-8 and VEGF via a Duox1--> ROS--> TACE--> TGF-alpha--> EGFR phosphorylation pathway. These results show for the first time that multiple TLRs in airway epithelial cells produce innate immune responses by activating EGFR via an epithelial cell signaling cascade.     

Protease-activated receptor-2 (PAR-2) is a weak enhancer of mucin secretion by human bronchial epithelial cells in vitro

PAR-2, a member of a family of G-protein-coupled receptors, can be activated by serine proteases via proteolytic cleavage. PAR-2 expression is known to be upregulated in respiratory epithelium subsequent to inflammation in asthma and chronic obstructive pulmonary disease (COPD). Since these diseases also are characterized by excessive mucus production and secretion, we investigated whether PAR-2 could be linked to mucin hypersecretion by airway epithelium. Normal human bronchial epithelial (NHBE) cells in primary culture or the human bronchial epithelial cell lines, NCI-H292 and HBE-1, were used. NHBE, NCI-H292, and HBE-1 cells expressed prominent levels of PAR-2 protein. Short-term (30min) exposure of cells to the synthetic PAR-2 agonist peptide (SLIGKV-NH2) elicited a small but statistically significant increase in mucin secretion at high concentrations (100microM and 1000microM), compared to a control peptide with reversed amino acid sequence (VKGILS-NH2). Neither human lung tryptase nor bovine pancreatic trypsin, both PAR-2 agonists, affected NHBE cell mucin secretion when added over a range of concentrations. Knockdown of PAR-2 expression by siRNA blocked the stimulatory effect of the AP. The results suggest that, since PAR-2 activation only weakly increases mucin secretion by human airway epithelial cells in vitro, PAR-2 probably is not a significant contributor to mucin hypersecretion in inflamed airways.     

Normal CFTR Inhibits Epidermal Growth Factor Receptor-Dependent Pro-Inflammatory Chemokine Production in Human Airway Epithelial Cells

Mutations in cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis, a disease characterized by exaggerated airway epithelial production of the neutrophil chemokine interleukin (IL)-8, which results in exuberant neutrophilic inflammation. Because activation of an epidermal growth factor receptor (EGFR) signaling cascade induces airway epithelial IL-8 production, we hypothesized that normal CFTR suppresses EGFR-dependent IL-8 production and that loss of CFTR at the surface exaggerates IL-8 production via activation of a pro-inflammatory EGFR cascade. We examined this hypothesis in human airway epithelial (NCI-H292) cells and in normal human bronchial epithelial (NHBE) cells containing normal CFTR treated with a CFTR-selective inhibitor (CFTR-172), and in human airway epithelial (IB3) cells containing mutant CFTR versus isogenic (C38) cells containing wild-type CFTR. In NCI-H292 cells, CFTR-172 induced IL-8 production EGFR-dependently. Pretreatment with an EGFR neutralizing antibody or the metalloprotease TACE inhibitor TAPI-1, or TACE siRNA knockdown prevented CFTR-172-induced EGFR phosphorylation (EGFR-P) and IL-8 production, implicating TACE-dependent EGFR pro-ligand cleavage in these responses. Pretreatment with neutralizing antibodies to IL-1R or to IL-1alpha, but not to IL-1beta, markedly suppressed CFTR-172-induced EGFR-P and IL-8 production, suggesting that binding of IL-1alpha to IL-1R stimulates a TACE-EGFR-IL-8 cascade. Similarly, in NHBE cells, CFTR-172 increased IL-8 production EGFR-, TACE-, and IL-1alpha/IL-1R-dependently. In IB3 cells, constitutive IL-8 production was markedly increased compared to C38 cells. EGFR-P was increased in IB3 cells compared to C38 cells, and exaggerated IL-8 production in the IB3 cells was EGFR-dependent. Activation of TACE and binding of IL-1alpha to IL-1R contributed to EGFR-P and IL-8 production in IB3 cells but not in C38 cells. Thus, we conclude that normal CFTR suppresses airway epithelial IL-8 production that occurs via a stimulatory EGFR cascade, and that loss of normal CFTR activity exaggerates IL-8 production via activation of a pro-inflammatory EGFR cascade.     

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.     

Regulation of interleukin-8 via an airway epithelial signaling cascade

Airways function as an innate immune organ against airborne bacteria that are inhaled and deposited in airways. One of the mechanisms of host defense is to recruit neutrophils into airways to clear the invaders. Airway epithelial cells produce neutrophil chemoattractant interleukin (IL)-8 in response to invading bacteria. In this study we show a signaling pathway on the plasma surface of human airway epithelial NCI-H292 cells that regulate IL-8 production in response to a model inflammatory stimulus, phorbol 12-myristate 13-acetate, and a pathophysiological stimulus, gram-negative bacterial lipopolysaccharide. First, we show that EGF receptor (EGFR) and MAP kinase ERK1/2 are involved in IL-8 expression by these stimuli. Second, we show that EGFR ligand transforming growth factor (TGF)-alpha mediates IL-8 production. Third, we show that tumor necrosis factor-alpha-converting enzyme (TACE) is required for IL-8 production by cleaving EGFR proligand proTGF-alpha into soluble TGF-alpha, activating EGFR. Last, we show that dual oxidase 1 (Duox1), a homolog of NADPH oxidase in airways, mediates TACE activation and IL-8 expression via generation of reactive oxygen species. In summary, we describe a signaling pathway, Duox1-TACE-TGF-alpha-EGFR, on the surface of airway epithelial (NCI-H292) cells that mediates airway epithelial defense against bacterial infection by producing IL-8. This pathway, which also regulates mucin production in human airways, provides mechanisms for killing foreign organisms and for their clearance.     

Metalloprotease-dependent amphiregulin release mediates tumor necrosis factor-alpha-induced IL-8 secretion in the human airway epithelial cell line NCI-H292

Tumor necrosis factor-alpha (TNF-alpha) is a potent multifunctional cytokine that plays a central role in the pathogenesis of many inflammatory diseases. Interleukin-8 (IL-8) is a principle neutrophil chemoattractant and activator in humans. The alveolar macrophage-derived TNF-alpha initiates lung inflammation through its ability to stimulate IL-8 synthesis in airway epithelial cells. Since recent studies demonstrated that the stimulation of epidermal growth factor receptor (EGFR) could induce IL-8 secretion, the involvement of EGFR in TNF-alpha-induced IL-8 secretion in airway epithelium-like NCI-H292 cells was investigated in this study. TNF-alpha and epidermal growth factor (EGF) stimulated IL-8 secretion in a time- and concentration-dependent manner. Inhibition of the EGFR by either an anti-EGFR neutralizing antibody or by its specific inhibitor AG1478 (1 microM) blocked TNF-alpha-induced IL-8 secretion. In addition, TNF-alpha stimulated tyrosine phosphorylation of the EGFR within 5 min after stimulation. Further, TNF-alpha-induced IL-8 secretion was completely inhibited by the neutralizing antibody against amphiregulin (AR), an EGFR ligand, suggesting that TNF-alpha-induced IL-8 secretion was mediated by the AR-EGFR pathway. Furthermore, TNF-alpha stimulated the release of AR in a concentration-dependent manner. Finally, both AR and IL-8 release-induced by TNF-alpha were eliminated by pretreatment with either GM6001, a broad-spectrum inhibitor for metalloprotease, or TAPI-1, relatively selective inhibitor for TNF-alpha converting enzyme (TACE). These findings indicate that metalloprotease-mediated AR shedding and subsequent activation of EGFR play a critical role in TNF-alpha-induced IL-8 secretion from the human airway epithelium-like NCI-H292 cells, and that TACE is one of the most possible candidates for metalloprotease responsible for TNF-alpha-induced AR shedding.     

Inhibition of E-cadherin dependent cell-cell contact promotes MUC5AC mucin production through the activation of epidermal growth factor receptors

Mucin production by epithelial cells is modulated by many soluble factors, including epidermal growth factor (EGF). E-Cadherin promotes EGF receptor (EGFR)-mediated MUC5AC mucin production in airway epithelial cells in dense cultures, suggesting the involvement of E-cadherin in activating EGFRs and mucin production. However, the role of E-cadherin in modulating mucin production is not completely understood. We examined its role in MUC5AC production in a human lung epithelial cell line, NCI-H292. Treatment of low density NCI-H292 cells with an anti-E-cadherin monoclonal antibody (SHE78-7) inhibited cell-cell contact in the dispersed colonies, but promoted MUC5AC production. Furthermore, treatment of the NCI-H292 cells with anti-E-cadherin antibody stimulated phosphorylation of extracellular signal-regulated kinase (ERK). The enhanced production of MUC5AC was inhibited with an EGFR inhibitor and with a MEK inhibitor, but not with a Src family kinase inhibitor. These results suggest that inhibition of E-cadherin activates EGFRs independently of Src and promotes MUC5AC production through the ERK signaling pathway in sparsely cultured NCI-H292 cells.     

Inhibition of E-Cadherin Dependent Cell-Cell Contact Promotes MUC5AC Mucin Production through the Activation of Epidermal Growth Factor Receptors

Mucin production by epithelial cells is modulated by many soluble factors, including epidermal growth factor (EGF). E-Cadherin promotes EGF receptor (EGFR)-mediated MUC5AC mucin production in airway epithelial cells in dense cultures, suggesting the involvement of E-cadherin in activating EGFRs and mucin production. However, the role of E-cadherin in modulating mucin production is not completely understood. We examined its role in MUC5AC production in a human lung epithelial cell line, NCI-H292. Treatment of low density NCI-H292 cells with an anti-E-cadherin monoclonal antibody (SHE78-7) inhibited cell-cell contact in the dispersed colonies, but promoted MUC5AC production. Furthermore, treatment of the NCI-H292 cells with anti-E-cadherin antibody stimulated phosphorylation of extracellular signal-regulated kinase (ERK). The enhanced production of MUC5AC was inhibited with an EGFR inhibitor and with a MEK inhibitor, but not with a Src family kinase inhibitor. These results suggest that inhibition of E-cadherin activates EGFRs independently of Src and promotes MUC5AC production through the ERK signaling pathway in sparsely cultured NCI-H292 cells.     

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.