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.     

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.     

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.     

Gene profile changes after Pseudomonas aeruginosa exposure in immortalized airway epithelial cells

To test the hypothesis that the excess inflammatory response in cystic fibrosis airway epithelial cells is the result of differential activation of genes in response to a laboratory strain of Pseudomonas aeruginosa (PAO1), a 48-h time course of genes expressed following PAO1 stimulation (10(9) CFU for 1 h) was studied in two pairs of airway epithelial cells: 9/HTEo- and 16HBE14o-, each with a matched normal and CF phenotype pair. cRNA was hybridized to Affymetrix HG-U95Av2 chips and pairwise comparisons against zero time (no PAO1) were calculated for each time point. PAO1 elicited profound changes in both cell pairs: for 9/HTEo-, 144 genes changed significantly in the normal pair, and 116 for the CF pair; for the 16HBE14o- pair, 57 genes changed significantly for the normal pair and 53 for the CF pair. Changes were much greater in the 9/HTEo- than in the 16HBE14o- pair, but basal levels of expression of inflammatory genes are higher in the 16HBE14o- pair, so 16HBE14o- was used mainly to corroborate the results of 9/HTEo-. Clustering analysis indicated that the pattern of gene expression is similar in the CF cells and their normal counterparts. However, there were substantial quantitative differences in gene expression. Thus, the difference between CF and normal resides in the magnitude, not the pattern, of the changes.     

An immunohistochemical study of the expression of adhesion molecules in gallbladder lesions.

We investigated the expression of 10 adhesion molecules (alpha-catenin, beta-catenin, gamma-catenin, CD44, CD44v6, ICAM-1, CD56, CEA, E-cadherin, and CD99) in 46 gallbladder carcinomas, 14 adenomas, 15 low-grade dysplasias, nine intestinal metaplasias, and 20 samples of normal gallbladder epithelium by immunohistochemistry. The expression of adhesion molecules was altered in gallbladder carcinomas and adenomas. In gallbladder carcinomas, increased expression of ICAM-1, CEA, and CD44v6 was observed, together with decreased expression of alpha/beta/gamma-catenin and CD99. In adenomas, aberrant expression of CD44v6 and CD56, as well as reduced expression of alpha/beta/gamma- and E-cadherins, was noted. Expression of alpha/beta/gamma-catenin was reduced in low-grade dysplasia, whereas there was no change in the expression of these adhesion molecules in metaplasia. Expression of ICAM-1, CD99, E-cadherin, and CD56 was correlated with clinical stage. In addition a correlation was noted between expression of ICAM-1 and E-cadherin and lymph node metastasis (p<0.05). These results suggest that altered expression of these adhesion molecules is involved in the progression and metastasis of gallbladder carcinomas.     

Influence of apical fluid volume on the development of functional intercellular junctions in the human epithelial cell line 16HBE14o-: implications for the use of this cell line as an in vitro model for bronchial drug absorption studies

Air-interfaced culture (AIC) versus liquid-covered culture (LCC) conditions are known to have different effects on the differentiated phenotype of several cell types, including lung epithelial cells. We report the influence of culture conditions such as apical medium volume on the development of intercellular junctions in the human epithelial cell line 16HBE14o-. Immunofluorescence staining of the tight-junctional protein, ZO-1, has revealed its presence in cells grown in both AIC and LCC. However, only LCC-grown cells exhibit protein ZO-1 localized as a zonula-occludens-like regular belt connecting neighboring cells. The presence of typical tight junctions has been confirmed by electron microscopy. Immunostaining for occludin, claudin-1, connexin43, and E-cadherin has demonstrated intercellular junction structures only in the cells in LCC. These morphological findings have been paralleled by higher transepithelial electrical resistance values and similar fluxes of the hydrophilic permeability marker, fluorescein-Na, under LCC compared with AIC conditions. We conclude that the formation of functional 16HBE14o- cell layers requires the presence of an apical fluid volume, in contrast to other culture conditions for airway epithelial cells.     

In vivo glycosylation of MUC1 in airway epithelial cells

The O-glycans that decorate mucin glycoproteins contribute to the biophysical and biochemical properties of these molecules and hence their function as a barrier and lubricant on epithelial surfaces. Alterations in mucin O-glycosylation in certain diseases may contribute to pathology. It is known that both the host cell type and the amino acid sequence of the mucin tandem repeat contribute to the O-glycosylation of a mucin molecule. We expressed an epitope-tagged MUC1 mucin cDNA construct in the airway cell line 16HBE14o- and the colon carcinoma cell line Caco2 and used Fast Atom Bombardment Mass Spectrometry to evaluate the contribution of the host cell to differences in O-glycosylation of a single mucin. Many of the glycans detected on the MUC1 mucin were common to both cell types, as would be predicted from biosynthetic constraints. However, MUC1 synthesized in the airway cell line showed comparatively low levels of sialylation but carried a range of oligo-N-acetyllactosamine structures that were not seen in the colon carcinoma cell line.     

AMPK induces MUC5B expression via p38 MAPK in NCI-H292 airway epithelial cells

Adenosine monophosphate-activated protein kinase (AMPK) is a well-known serine/threonine kinase that has been implicated in modulation of glucose and fatty acid metabolism. Recent reports have also implicated AMPK in modulation of mucin secretion. In this study, the effects and signaling pathways of AMPK on MUC5B expression were investigated in human NCI-H292 airway epithelial cells. Metformin, as an activator of AMPK, induced MUC5B expression in a dose-dependent manner. Compound C, as an inhibitor of AMPK, inhibited metformin-induced MUC5B expression in a dose-dependent manner. Metformin significantly activated phosphorylation of AMPK; compound C inhibited metformin-activated phosphorylation of AMPK. Without treatment with metformin, there was no difference in MUC5B mRNA expression between Ad-dnAMPK transfected and wild-type adenovirus transfected NCI-H292 cells. However, after treatment with metformin, MUC5B mRNA expression was increased in wild-type adenovirus transfected NCI-H292 cells; MUC5B mRNA expression was significantly decreased in Ad-dnAMPK transfected NCI-H292 cells. Metformin activated phosphorylation of p38 mitogen-activated protein kinase (MAPK); compound C inhibited metformin-activated phosphorylation of p38 MAPK. SB203580, as an inhibitor of p38 MAPK, significantly inhibited metformin-induced MUC5B mRNA expression, while U0126, as an inhibitor of ERK1/2 MAPK, had no effect. In addition, knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked metformin-induced MUC5B mRNA expression. In conclusion, results of this study show that AMPK induces MUC5B expression through the p38 MAPK signaling pathway in airway epithelial cells.     

Effect of cytokines on ICAM-1 and ZO-1 expression on human airway epithelial cells

The presence of adhesion molecules on airway epithelial cells may be important in recruiting leukocytes to the epithelium. The study aimed at investigating the effects of interleukin (IL)-4, IL-8, IL-13 and interferon-gamma (IFN-gamma) on cell viability and intracellular adhesion molecule (ICAM)-1 and zonula occludens protein (ZO)-1 expression on cultured human basal and columnar airway epithelial cells. Cycloheximide (CHX) induced cell death in both cell lines. The cytokines IL-4, IL-8, IL-13 and IFN-gamma had only minor effects on cell proliferation in the columnar 16HBE14o-cells, and inhibited the effects of CHX on cell death. IFN-gamma increased ICAM-1 expression in both cell lines. Western blot analysis showed that CHX inhibited both ICAM-1 and ZO-1 expression in the basal cell line. A combination of IL-4 and IFN-gamma appeared to break the tight junctions. IL-4 and IL-13 potentiated CHX-induced apoptosis in basal cells but not in columnar cells, possibly due to low levels of the IL-4 receptor. It is concluded that cytokines produced by airway epithelium may have a role in regulating sequestering of leukocytes to the airways during airway inflammation.     

Maintenance of the epithelial barrier in a bronchial epithelial cell line is dependent on functional E-cadherin local to the tight junctions

Tight junctions (TJ) are essential components of polarized epithelia, and E-cadherin is important for their formation and maintenance. The bronchial epithelial cell line, 16HBE14o- expresses E- and P-cadherin, but not N-cadherin. E- and P-cadherin levels changed during culture, the former increasing after confluence, and the latter were markedly reduced. All detectable E-cadherin was bound to β- and γ-catenins. We investigated involvement of E-cadherin with epithelial integrity using an E-cadherin specific, function-blocking antibody, SHE78-7. Surprisingly, apical SHE78-7 exposure caused a prompt fall in transepithelial resistance (TER), while TER remained unchanged for 8 hrs after basal exposure then dropped. SHE78-7 exposure increased epithelial permeability to mannitol, inulin, and 9.5 kDa and 77 kDa dextrans and caused fragmentation and loss of the tight junction protein, ZO-1, from the cell borders in some areas. Ultrastructural studies showed that all junctional intercellular contact was lost in the center of SHE78-7 induced lesions. Near the lesion periphery, epithelial structure was maintained, but TJs were dysfunctional as shown by ruthenium red penetration. Analysis of epithelial penetration by SHE78-7 revealed discrete, local defects in the apical barrier at the top of some cell hills that permitted rapid access of the antibody to E-cadherin near the apical surface. In contrast, after basal exposure, antibody initially engaged with E-cadherin nearer the basal surface and only accessed apical E-cadherin later. Taken together with the TER measurements, these data suggest compartmentalization of E-cadherin function within 16HBE14o- cells, with only the apical E-cadherin adjacent to the tight junctions contributing to the function of the latter.     

Direct and monocyte-induced innate immune response of human lung epithelial cells to Brucella abortus infection

Although Brucella frequently infects humans through inhalation, its interaction with pulmonary cells has been overlooked. We examined whether human lung epithelial cells produce proinflammatory mediators in response to Brucella infection. Infection with smooth or rough strains of Brucella abortus induced the secretion of IL-8 and GM-CSF by the bronchial epithelial cell lines Calu-6 and 16HBE14o-, but not by the alveolar epithelial cell line A549. Infected Calu-6 cells also produced low levels of MCP-1. Since monocyte-derived cytokines may induce chemokine secretion in epithelial cells, cocultures of human monocytes (THP-1 cell line) and respiratory epithelial cells were used to study such interaction. IL-8 and MCP-1 levels in B. abortus-infected THP-1:A549 and THP-1:Calu-6 cocultures, and MCP-1 levels in THP-1:16HBE14o- cocultures, were higher than those detected in infected epithelial monocultures. Conditioned medium from infected monocytes induced the secretion of IL-8 and/or MCP-1 by A549 and Calu-6 cells, and these effects were mainly mediated by IL-1 (in A549 cells) or TNF-α (in Calu-6 cells). Conversely, culture supernatants from Brucella-infected bronchial epithelial cells induced MCP-1 production by monocytes, an effect largely mediated by GM-CSF. This study shows that human lung epithelial cells mount a proinflammatory response to Brucella, either directly or after interaction with Brucella-infected monocytes.     

Naringin as a plant-derived bitter tastant promotes proliferation of cultured human airway epithelial cells via activation of TAS2R signaling

BackgroundBitter tastants can activate bitter taste receptors (TAS2Rs) and thus initiate relaxation of airway smooth muscle cells (ASMCs), which have great potential in the development of novel bronchodilator drugs for asthma therapy. However, the canonical bitter substance, denatonium is known to induce apoptosis of airway epithelial cells (AECs), indicating that other bitter tastants may also impair the epithelial integrity to prevent hazardous particulate matters such as coronaviruses. Therefore, any bitter tastants intended for treating airway disease should be carefully evaluated for potential toxicity to AECs.Hypothesis/PurposeConsidering the vast diversity of bitter tastants in nature and different types of TAS2Rs expressed in airway cells, we hypothesized that there must be some natural bitter tastants to be not only potent in inducing relaxation of ASMCs but also unharmful to AECs.Study design and methodsHere we evaluated a group of bitter flavonoids that are derived from fruits and commonly used in traditional herbal medicine, including apigenin, hesperetin, kaempferol, naringenin, quercetin, and naringin, for their effects on the proliferation of human airway epithelial-like (16HBE14o-, BEAS-2B, and A549) cells cultured in vitro. Cell proliferation and associated signaling pathways were assessed by cell counting, ATP assay, cell cycling assay, quantitative RT-PCR, Fluo-4 labeling, and fluorescence resonance energy transfer, respectively.ResultsThe results show that five of the six tested bitter tastants inhibited, but only naringin promoted the proliferation of the 16HBE14o-, BEAS-2B, and A549 cells at the dose of a few hundred micromoles. Furthermore, the naringin-promoted proliferation of the 16HBE14o- cells was associated with enhanced cell cycle progression, mRNA expression of cyclin E, and evoked calcium signaling/ERK signaling, which were all attenuated by inhibition of the TAS2R signaling pathways with specific blockers.ConclusionThese findings indicate that although the majority of the bitter flavonoids may inhibit the proliferation of AECs, naringin emerged as one to promote the proliferation of AECs via cell cycle progression and TAS2R-activated intracellular signaling. It suggests that naringin and not a few other bitter tastants can be proven with nontoxicity to the airway epithelial structure and function, which provides further confidence in the development of safe and effective TAS2R-based bronchodilators for asthma therapy.     

Molecular mechanisms controlling CFTR gene expression in the airway

The low levels of CFTR gene expression and paucity of CFTR protein in human airway epithelial cells are not easily reconciled with the pivotal role of the lung in cystic fibrosis pathology. Previous data suggested that the regulatory mechanisms controlling CFTR gene expression might be different in airway epithelium in comparison to intestinal epithelium where CFTR mRNA and protein is much more abundant. Here we examine chromatin structure and modification across the CFTR locus in primary human tracheal (HTE) and bronchial (NHBE) epithelial cells and airway cell lines including 16HBE14o- and Calu3. We identify regions of open chromatin that appear selective for primary airway epithelial cells and show that several of these are enriched for a histone modification (H3K4me1) that is characteristic of enhancers. Consistent with these observations, three of these sites encompass elements that have cooperative enhancer function in reporter gene assays in 16HBE14o- cells. Finally, we use chromosome conformation capture (3C) to examine the three-dimensional structure of nearly 800 kb of chromosome 7 encompassing CFTR and observe long-range interactions between the CFTR promoter and regions far outside the locus in cell types that express high levels of CFTR.     

A crucial role of Flagellin in the induction of airway mucus production by Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. Flagellin is a P. aeruginosa virulence factor involved in host response to this pathogen. We examined the role of flagellin in P. aeruginosa-induced mucus secretion. Using a mouse model of pulmonary infection we showed that PAK, a wild type strain of P. aeruginosa, induced airway mucus secretion and mucin muc5ac expression at higher levels than its flagellin-deficient mutant (ΔFliC). PAK induced expression of MUC5AC and MUC2 in both human airway epithelial NCI-H292 cell line and in primary epithelial cells. In contrast, ΔFliC infection had lower to no effect on MUC5AC and MUC2 expressions. A purified P. aeruginosa flagellin induced MUC5AC expression in parallel to IL-8 secretion in NCI-H292 cells. Accordingly, ΔFliC mutant stimulated IL-8 secretion at significantly lower levels compared to PAK. Incubation of NCI-H292 cells with exogenous IL-8 induced MUC5AC expression and pre-incubation of these cells with an anti-IL-8 antibody abrogated flagellin-mediated MUC5AC expression. Silencing of TLR5 and Naip, siRNA inhibited both flagellin-induced MUC5AC expression and IL-8 secretion. Finally, inhibition of ERK abolished the expression of both PAK- and flagellin-induced MUC5AC. We conclude that: (i) flagellin is crucial in P. aeruginosa-induced mucus hyper-secretion through TLR5 and Naip pathways; (ii) this process is mediated by ERK and amplified by IL-8. Our findings help understand the mechanisms involved in mucus secretion during pulmonary infectious disease induced by P. aeruginosa, such as in cystic fibrosis.     

Interferon-gamma-induced epithelial ICAM-1 expression and monocyte adhesion. Involvement of protein kinase C-dependent c-Src tyrosine kinase activation pathway.

Interferon-gamma (IFN-gamma) induced intercellular adhesion molecule-1 (ICAM-1) expression in human NCI-H292 epithelial cells, as shown by enzyme-linked immunosorbent assay and immunofluorescence staining. The enhanced ICAM-1 expression resulted in increased adhesion of U937 cells to NCI-H292 cells. Tyrosine kinase inhibitors (genistein or herbimycin), Src family inhibitor (PP2), or a phosphatidylinositol-phospholipase C inhibitor (U73122) attenuated the IFN-gamma-induced ICAM-1 expression. Protein kinase C (PKC) inhibitors (staurosporine or Ro 31-8220) also inhibited IFN-gamma-induced response. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression; this effect was inhibited by tyrosine kinase or Src inhibitor. ICAM-1 promoter activity was enhanced by IFN-gamma and TPA in cells transfected with pIC339-Luc, containing the downstream NF-kappaB and gamma-activated site (GAS) sites, but not in cells transfected with GAS-deletion mutant, pIC135 (DeltaAP2). Electrophoretic gel mobility shift assay demonstrated that GAS-binding complexes in IFN-gamma-stimulated cells contained STAT1alpha. The IFN-gamma-induced ICAM-1 promoter activity was inhibited by tyrosine kinase inhibitors, a phosphatidylinositol-phospholipase C inhibitor, or PKC inhibitors, and the TPA-induced ICAM-1 promoter activity was also inhibited by tyrosine kinase inhibitors. Cotransfection with a PLC-gamma2 mutant inhibited IFN-gamma- but not TPA-induced ICAM-1 promoter activity. However, cotransfection with dominant negative mutants of PKCalpha or c-Src inhibited both IFN-gamma- and TPA-induced ICAM-1 promoter activity. The ICAM-1 promoter activity was stimulated by cotransfection with wild type PLC-gamma2, PKCalpha, c-Src, JAK1, or STAT1. An immunocomplex kinase assay showed that both IFN-gamma and TPA activated c-Src and Lyn activities and that these effects were inhibited by staurosporine and herbimycin. Thus, in NCI-H292 epithelial cells, IFN-gamma activates PLC-gamma2 via an upstream tyrosine kinase to induce activation of PKC-alpha and c-Src or Lyn, resulting in activation of STAT1alpha, and GAS in the ICAM-1 promoter, followed by initiation of ICAM-1 expression and monocyte adhesion.