Trefoil factor family-peptides promote migration of human bronchial epithelial cells: synergistic effect with epidermal growth factor.

A process termed "restitution" enables rapid repair of the respiratory epithelium by migration of neighbouring cells. Mucin-associated TFF-peptides (formerly P-domain peptides or trefoil factors) are typical motogens enhancing migration of cells in various in vitro models mimicking restitution of the intestine. The human bronchial epithelial cell line BEAS-2B was used as a model system of airway restitution. The motogenic activities of recombinant human TFF2 as well as porcine TFF2 were demonstrated by in vitro wound healing assays of BEAS-2B cells. TFF2 did not induce phosphorylation of the epidermal growth factor (EGF) receptor. EGF was capable of enhancing the motogenic effect of human TFF2 at a concentration of 3 x 10(-10) M whereas EGF itself (i.e., in the absence of TFF2) did not stimulate migration at this low concentration. Furthermore, TFF2 as well as monomeric and dimeric forms of TFF3 enhanced migration of BEAS-2B cells in Boyden chambers. Motogenic activity of TFF2 was also shown for normal human bronchial epithelial (NHBE) cells in Boyden chambers. These results suggest that TFF-peptides act as motogens in the human respiratory epithelium triggering rapid repair of damaged mucosa in the course of airway diseases such as asthma.     

Interleukin-31 induces cytokine and chemokine production from human bronchial epithelial cells through activation of mitogen-activated protein kinase signalling pathways: implications for the allergic response

Interleukin-31 (IL-31) is a novel T-helper-lymphocyte-derived cytokine that plays an important role in allergic skin inflammation and atopic dermatitis. It has recently been implicated in bronchial inflammation. We investigated the functions and mechanisms of IL-31-induced activation of human bronchial epithelial cells. The gene and protein expressions of candidate cytokines/chemokines from IL-31-stimulated human bronchial epithelial BEAS-2B cells were first quantified by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The activity of different mitogen-activated protein kinases (MAPKs) in IL-31-stimulated BEAS-2B cells was assessed by Western blot. The IL-31 could significantly elevate the gene and protein expressions of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1/CCL2) of BEAS-2B cells in both time-dependently and dose-dependently. Combination of IL-31 with either IL-4 or IL-13 further enhanced VEGF and CCL2 production while IL-31 could synergistically augment the release of EGF, VEGF, CCL2, IL-6 and IL-8 in cocultures of BEAS-2B cells and eosinophils. In addition, IL-31 could activate p38 MAPK, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) of BEAS-2B cells. Selective inhibitors of p38 MAPK (SB203580), ERK (PD98059), and JNK (SP600125) could differentially inhibit the production of EGF, VEGF and CCL2, thereby suggesting a role for MAPKs in IL-31 functions. In conclusion, the activation of MAPKs can be crucial for IL-31-mediated activation of bronchial epithelial cells, thereby providing an immunological role for IL-31 in bronchial inflammation, at least partly, via epithelial EGF, VEGF and CCL2 production.     

Trefoil factor family 3 peptide promotes human airway epithelial ciliated cell differentiation

Human airway surface epithelium is frequently damaged by inhaled factors (viruses, bacteria, xenobiotic substances) as well as by inflammatory mediators that contribute to the shedding of surface epithelial cells. To regain its protective function, the epithelium must rapidly repair and redifferentiate. The Trefoil Factor Family (TFF) peptides are secretory products of many mucous cells. TFF3, the major TFF in the airways, is able to enhance airway epithelial cell migration, but the role of this protein in differentiation has not been defined. To identify the specific role of TFF3 in the differentiation of the human airway surface epithelium, we analyzed the temporal expression pattern of TFF3, MUC5AC, and MUC5B mucins (goblet cells) and ciliated cell markers beta-tubulin (cilia) and FOXJ1 (ciliogenesis) during human airway epithelial regeneration using in vivo humanized airway xenograft and in vitro air-liquid interface (ALI) culture models. We observed that TFF3, MUC5AC, MUC5B, and ciliated cell markers were expressed in well-differentiated airway epithelium. The addition of exogenous recombinant human TFF3 to epithelial cell cultures before the initiation of differentiation resulted in no change in MUC5AC or cytokeratin 13 (CK13, basal cell marker)-positive cells, but induced an increase in the number of FOXJ1-positive cells and in the number of beta-tubulin-positive ciliated cells (P < 0.05). Furthermore, this effect on ciliated cell differentiation could be reversed by specific epidermal growth factor (EGF) receptor (EGF-R) inhibition. These results indicate that TFF3 is able to induce ciliogenesis and to promote airway epithelial ciliated cell differentiation, in part through an EGF-R-dependent pathway.     

Interferon-gamma inhibits hepatocyte growth factor-stimulated cell proliferation of human bronchial epithelial cells: upregulation of p27(kip1) cyclin-dependent kinase inhibitor.

Proliferation of bronchial epithelial cells is an important biologic process in a variety of physiologic and pathologic conditions. In this study, we demonstrate that hepatocyte growth factor (HGF) stimulates proliferation of human bronchial epithelial cells obtained from healthy volunteers. The mitogenic effect of HGF is dependent on costimulation with serum and is completely abrogated by interferon-gamma (IFN-gamma). In the absence of serum, HGF is capable of inducing activation of extracellular signal-regulated kinases (ERK)1 and ERK2, but fails to stimulate proliferation by itself. These effects of HGF and IFN-gamma were reproduced faithfully in BEAS-2B cells, which are an immortalized cell line derived from human bronchial epithelial cells. Further, we investigated the molecular mechanisms underlying the effects of HGF and IFN-gamma in BEAS-2B cells and found that the MEK1 inhibitor PD98059, but not the p38 M-associated protein kinase inhibitor SB203580, abrogates HGF-induced ERK activation and proliferation in response to HGF and serum. In addition, LY294002, which is the specific inhibitor of phosphatidyl inositol 3-kinase, partially inhibited HGF- and serum-stimulated proliferation. We also found that HGF by itself is capable of inducing a G1 cyclin, cyclin D1, but fails to downregulate p27(kip1) cyclin-dependent kinase (CDK) inhibitor, which is a requisite for G1 to S phase cell cycle progression. IFN-gamma does not interfere with the effects of HGF on either ERK activation or cyclin D1 induction; however, it prevents the downregulation of p27(kip1) CDK inhibitor that takes place in response to a combination of HGF and serum. These results indicate that the MEK-ERK signaling pathway is necessary but not sufficient for human bronchial epithelial cell proliferation, and implicate the significance of HGF and IFN-gamma in the repair processes of injured human bronchial epithelial cells.     

Targeted intestinal epithelial deletion of the chemokine receptor CXCR4 reveals important roles for extracellular-regulated kinase-1/2 in restitution

Barrier defects and/or alterations in the ability of the gut epithelium to repair itself are critical etiological mechanisms of gastrointestinal disease. Our ongoing studies indicate that the chemokine receptor CXCR4 and its cognate ligand CXCL12 regulate intestinal-epithelial barrier maturation and restitution in cell culture models. Gene-deficient mice lacking CXCR4 expression specifically by the cells of the intestinal epithelium were used to test the hypothesis that CXCR4 regulates mucosal barrier integrity in vivo. Epithelial expression of CXCR4 was assessed by RT-PCR, Southern blot, immunoblot and immunohistochemistry. In vivo wounding assays were performed by addition of 3% dextran sodium sulfate (DSS) in drinking water for 5 days. Intestinal damage and DAI scores were assessed by histological examination. Extracellular-regulated kinase (ERK) phosphorylation was assessed in vivo by immunoblot and immunofluorescence. CXCR4 knockdown cells were established using a lentiviral approach and ERK phosphorylation was assessed. Consistent with targeted roles in restitution, epithelium from patients with inflammatory bowel disease indicated that CXCR4 and CXCL12 expression was stable throughout the human colonic epithelium. Conditional CXCR4-deficient mice developed normally, with little phenotypic differences in epithelial morphology, proliferation or migration. Re-epithelialization was absent in CXCR4 conditional knockout mice following acute DSS-induced inflammation. In contrast, heterozygous CXCR4-depleted mice displayed significant improvement in epithelial ulcer healing in acute and chronic inflammation. Mucosal injury repair was correlated with ERK1/2 activity and localization along the crypt-villus axis, with heterozygous mice characterized by increased ERK1/2 activation. Lentiviral depletion of CXCR4 in IEC-6 cells similarly altered ERK1/2 activity and prevented chemokine-stimulated migration. Taken together, these data indicate that chemokine receptors participate in epithelial barrier responses through coordination of the ERK1/2 signaling pathway.     

Trefoil factor family domain peptides in the human respiratory tract

Trefoil factor family domain peptides (TFF) are thought to be involved in mucosal epithelial restitution and wound healing of the gastrointestinal tract and are up-regulated in ulceration and in a variety of solid tumours. It was hypothesized that TFFs are also expressed on mucosal surfaces of the human respiratory tract. Lung tissue, nasal polyps, and sputum samples from seven patients with cystic fibrosis (CF), two with chronic and acute bronchitis, and non-dysplastic material from two cases of bronchial adenocarcinoma were analysed for TFF expression by immunohistochemistry, immunofluorescence, western blot and RT-PCR. Expression of TFF1 and TFF3 was observed in material from all patients. TFFs were localized in goblet and ciliated cells, as well as in some submucosal cells of tracheobronchial tissues and nasal polyps from normal and CF individuals. In sputa of patients with CF and with chronic or acute bronchitis, TFF1 and TFF3 were detected by western blotting. Freshly cultivated nasal epithelial cells transcribed and secreted TFFs and mucins, whereas nasal cells cultivated for 6 weeks still expressed mucins, but not TFFs. Secreted TFFs and mucins also bound to the surface of Staphylococcus aureus in infected CF airways. In conclusion, TFF1 and TFF3 are expressed and secreted in normal and inflamed airways. The association of TFFs with bacteria may contribute to the anti-microbial mucociliary defence system.     

Biliary epithelial trefoil peptide expression is increased in biliary diseases

AIMS: Maintenance of the cellular integrity of the biliary epithelium may involve the production of mucins and mucin-associated peptides. In the luminal gastrointestinal tract, mucins and the mucin-associated trefoil peptides (TFF) are integral to cytoprotection and cellular repair of the mucosa. METHODS AND RESULTS: Samples of normal and diseased human liver tissue were examined using histological and immunohistochemical techniques, for the expression of TFF and mucins. Bile ducts were classified as small, medium or large depending upon the number of biliary epithelial cells. TFF expression was demonstrated in biliary epithelial cells of both normal and diseased liver tissue. TFF expression was greatest in the large bile ducts. In normal liver tissue, expression of at least one TFF was demonstrated in 2-7% of small bile ducts, 5-31% of medium bile ducts and 31-85% of large bile ducts. Seventy-seven percent of large bile ducts secreted mucins and all three TFF concurrently, compared with 3% of medium bile ducts and no small bile ducts. Biliary disease resulted in an increased expression of TFF1 and TFF3 in the medium bile ducts. CONCLUSIONS: The biliary epithelial cells in normal and diseased human liver tissue express TFF, particularly in the larger bile ducts. TFF expression may be up-regulated or induced in biliary diseases as a response to injury, as is seen in epithelial damage elsewhere in the gastrointestinal tract.     

p38 mitogen-activated protein kinase regulates growth factor-induced mitogenesis of rat pulmonary myofibroblasts

Myofibroblast proliferation is a central feature of pulmonary fibrogenesis. Several growth factors, including platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), stimulate myofibroblast growth by activating extracellular signal regulated kinases 1 and 2 (ERK1/2). In this report, we demonstrate that PDGF-BB and EGF also activate the p38 mitogen-activated protein (MAP) kinase. Inhibition of p38 activity with the pyridinylimidazole compound SB203580 enhanced both PDGF-BB and EGF-stimulated DNA synthesis in rat lung myofibroblasts. ERK1/2 phosphorylation in response to either PDGF-BB or EGF treatment was significantly increased by pretreatment of cells with SB203580. We also demonstrated that ERK1/2-induced phosphorylation of PHAS-1 substrate was enhanced by inhibition of p38 MAP kinase with SB203580. However, SB203580 did not significantly increase growth factor-induced activation of MEK, the upstream kinase that phosphorylates ERK1/2. p38 MAP kinase was co-immunoprecipitated with ERK-1/2 following growth factor stimulation. Collectively, these data demonstrate that p38 MAP kinase activation negatively regulates PDGF- and EGF-mediated growth responses by directly interacting with ERK1/2 and suppressing its phosphorylation.     

Analysis of trefoil factor family protein 1 (TFF1, pS2) expression in chronic cholecystitis and gallbladder carcinoma

Trefoil factor family protein 1 (TFF1, pS2) interacts with mucins to protect gastrointestinal epithelium against injury and contributes to mucosal repair by promoting epithelial cell migration and restitution. Moreover, TFF1 has antiproliferative and anti-apoptotic effects and promotes cell scattering and invasion. We investigated TFF1 expression in healthy and inflamed non-neoplastic gallbladder mucosa as well as in gallbladder carcinomas (n=57) and corresponding metastases (n=18), using a tissue microarray technique. TFF1 immunoreactivity was absent in healthy mucosa, focally observed in epithelium with inflammatory changes and present in 35% of primary and 24% of metastatic cancer tissues. Immunoreactivity significantly decreased with increasing tumour stage (P=0.009) and increasing tumour grade (P=0.001). Patients with TFF1 positive tumours showed a more favourable outcome compared to patients with TFF1 negative tumours in univariate analysis (P=0.006). However, multivariate analysis proved resection status and tumour grade as the only independent prognostic factors. In conclusion, TFF1 is expressed in inflamed non-neoplastic gallbladder epithelium and in low stage and low grade gallbladder carcinomas. Thus, TFF1 may be the missing link between gallstones, chronic cholecystitis and gallbladder cancer. Further studies are needed to evaluate whether TFF1 immunostaining can be used as a diagnostic tool to identify patients with a more favourable outcome.     

TGF-beta suppresses EGF-induced MAPK signaling and proliferation in asthmatic epithelial cells

Epithelial damage is an important pathophysiologic feature of asthma. Bronchial epithelium damage results in release of growth factors such as transforming growth factor (TGF)-beta(1) that may affect epithelial cell proliferation. The objective of our study is to evaluate the importance of TGF-beta(1) in regulating epithelial cell repair in asthma. We evaluated the effect of TGF-beta(1) on epidermal growth factor (EGF)-induced proliferation and downstream signaling in epithelial cells obtained from subjects with asthma compared with cells from healthy subjects. Cell proliferation was evaluated by bromodeoxyuridine incorporation. EGF receptor (EGFR), mitogen-activated protein kinase, TGF-beta receptors, Smads, Smad anchor for receptor activation (SARA), and cyclin-dependant kinase inhibitors were evaluated by Western blot. TGF-beta(1) and receptor expression were measured by RT-PCR and by enzyme-linked immunosorbent assay. Proliferation of epithelial cells at baseline and after EGF stimulation was significantly reduced in cells derived from subjects with asthma compared with cells obtained from healthy control subjects. EGF-induced ERK1/2 phosphorylation was reduced in epithelial cells from subjects with asthma compared with cells from healthy control subjects. This was paralleled with a reduced EGFR phosphorylation. Addition of TGF-beta(1) significantly decreased EGF-induced cell proliferation. TGF-beta(1) production was higher in asthmatic epithelial cells compared with normal cells. This was supported by a high expression of pSmad 3 and SARA in cells derived from individuals with asthma compared with normal subjects. Cycline-dependent kinase inhibitors were highly expressed in asthmatic compared with normal cells. Inhibition of TGF-beta(1) signaling in asthmatic epithelial cells restored EGFR, ERK1/2 phosphorylation, and cell proliferation induced by EGF. Our results suggest that TGF-beta restrains EGFR phosphorylation and downstream signaling in bronchial epithelial cells.     

The effect of epidermal growth factor on production of vascular endothelial growth factor by amnion-derived (WISH) cells

Our objective was to clarify the physiological role of vascular endothelial growth factor (VEGF) by amnion-derived (WISH) cells. WISH cells were cultured, and the effect of epidermal growth factor (EGF), mitogen-activated protein (MAP) kinase kinase or extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors (U0126) or phosphatidylinositol (PI) 3-kinase on the production of VEGF was examined. VEGF was assayed by ELISA. The activation of MAP kinase and akt, which is phosphorylated by PI 3-kinase, were detected by Western blot analysis using anti-phosphorylated MAP kinase antibody and anti-phosphorylated akt antibody. In the time course of VEGF production following EGF treatment, VEGF production showed a significant increase only after 16 (p < 0.01)-32h (p < 0.01). EGF increased the production of VEGF by WISH cells in a dose-dependent manner. The MAP kinase and akt activity were determined by treatment with EGF. VEGF production was significantly decreased following pretreatment with U0126 or wortmannin for two hours before treatment with EGF (p < 0.01, p < 0.01). WISH cells appeared to produce VEGF via a mechanism involving tyrosine kinase activation of EGF receptor and MAP kinase or PI 3-kinase. It is suggested that VEGF may contribute to the neovascularization and proliferation of the placenta and gestational tissue, and EGF may play an important role in regulation of VEGF production in the placenta.     

Epidermal growth factor as a candidate for ex vivo expansion of bone marrow-derived mesenchymal stem cells

Bone marrow mesenchymal stem cells (BMMSCs) are pluripotent cells capable of differentiating into several cell types and are thus an attractive cell source for connective tissue engineering. A challenge in such a use is expansion and directed seeding in vitro, requiring proliferation and survival, and directed migration, respectively, prior to functional differentiation. The epidermal growth factor (EGF) receptor (EGFR) is the prototypal growth factor receptor and elicits these responses from a wide variety of stromal, epithelial, and endothelial cells. Ligands for this receptor are appealing for use in tissue engineering because they are relatively resistant to biological extremes and amenable to high-volume production. Therefore, we determined whether an EGFR ligand, EGF, could be used for ex vivo expansion of BMMSCs. EGF stimulated motility in rat and immortalized human BMMSCs. EGF-induced proliferation was observed in immortalized human BMMSCs but was not apparent in rat BMMSCs under our experimental conditions. EGF did not, however, rescue either type of BMMSC from apoptosis due to lack of serum. During our examination of key signaling intermediaries, EGF caused robust phosphorylation of extracellular signal-regulated protein kinase (ERK) and protein kinase B/akt (AKT) but only minimal phosphorylation of EGFR and phospholipase C-gamma in rat BMMSCs, whereas in the human BMMSCs these intermediaries were all strongly activated. EGF also induced robust ERK activation in primary porcine mesenchymal stem cells. EGF pretreatment or cotreatment did not interfere with secondarily induced differentiation of either type of BMMSC into adipogenic or osteogenic lineages. Platelet-derived growth factor (PDGF) effects were similar to but not additive with those elicited by EGF, with some quantitative differences; however, PDGF did interfere with the differentiation of these BMMSCs. These findings suggest that EGFR ligands could be used for ex vivo expansion and direction of BMMSCs.     

Airway epithelial epidermal growth factor receptor mediates hogbarn dust-induced cytokine release but not Ca2+ response

A subset of workers in swine confinement facilities develops chronic respiratory disease. An aqueous extract of dust from these facilities (hogbarn dust extract [HDE]) induces IL-6 and IL-8 release and several other responses in isolated airway epithelial cells. The cell membrane receptors by which HDE initiates these responses have not been identified. Because several other inhaled agents induce airway epithelial cell responses through epidermal growth factor receptor (EGFR) activation, we hypothesized that HDE would activate EGFRs and that EGFRs would be required for some of the responses to HDE. Exposure of Beas-2B cells to HDE caused EGFR phosphorylation and downstream ERK activation, and both responses were blocked by the EGFR-selective kinase inhibitor AG1478. AG1478 and EGFR-neutralizing antibody reduced HDE-stimulated IL-6 and IL-8 release by about half. Similar EGFR phosphorylation and requirement of EGFRs for maximal IL-6 and IL-8 release were found with primary isolates of human bronchial epithelial cells. Because HDE-stimulated IL-6 and IL-8 release involve the Ca(2+)-dependent protein kinase Cα, we hypothesized that HDE would induce intracellular Ca(2+) mobilization. HDE exposure induced intracellular Ca(2+) mobilization in Beas-2B cells and in primary cell isolates, but this response was neither mimicked by EGF nor inhibited by AG1478. Thus, HDE activates EGFRs and their downstream signaling, and EGFR activation is required for some but not all airway epithelial cell responses to HDE.     

肺炎克雷伯杆菌荚膜多糖经EGFR途径诱导支气管上皮细胞分泌细胞因子

目的:探讨表皮生长因子受体(EGFR)在肺炎克雷伯杆菌(KP)荚膜多糖(CPS)诱导人正常支气管上皮BEAS-2B细胞分泌炎性细胞因子中的作用。方法:体外培养KP并提取CPS,用不同浓度的CPS刺激BEAS-2B细胞,通过ELISA检测细胞上清中肿瘤坏死因子α(TNF-α)和白细胞介素8(IL-8)的水平,并于刺激后不同时点通过Western blot检测EGFR的磷酸化水平;EGFR抑制剂AG1478预处理BEAS-2B细胞后,Western blot检测ERK磷酸化水平,间接免疫荧光染色检测p65的核转位,并观察细胞上清中TNF-α和IL-8的变化情况;最后经ERK抑制剂PD98059和NF-κB抑制剂PDTC分别预处理后用CPS刺激细胞,ELISA检测细胞上清中TNF-α和IL-8的水平。结果:10 mg/L CPS刺激BEAS-2B细胞12 h能够显著诱导其分泌TNF-α和IL-8;Western blot和间接免疫荧光染色检测结果显示,CPS刺激可显著诱导BEAS-2B细胞中EGFR和ERK的磷酸化及p65的核转位。经EGFR抑制剂AG1478预处理细胞后,ERK的磷酸化水平显著降低,NF-κB的核转位减少;而在EGFR、ERK及NF-κB抑制剂预处理的细胞的上清中,TNF-α和IL-8分泌水平均明显降低(P<0.05)。结论:肺炎克雷伯杆菌荚膜多糖能够通过EGFR激活ERK和NF-κB信号通路,进而诱导人正常支气管上皮细胞分泌炎性细胞因子TNF-α和IL-8,提示EGFR可能是KP感染引起炎症反应的关键因子。