EGF and K+ channel activity control normal and cystic fibrosis bronchial epithelia repair

Severe lesions of airway epithelia are observed in cystic fibrosis (CF) patients. The regulatory mechanisms of cell migration and proliferation processes, involved in the repair of injured epithelia, then need to be better understood. A model of mechanical wounding of non-CF (NuLi) and CF (CuFi) bronchial monolayers was employed to study the repair mechanisms. We first observed that wound repair, under paracrine and autocrine EGF control, was slower (up to 33%) in CuFi than in NuLi. Furthermore, EGF receptor (EGFR) activation, following wounding, was lower in CuFi than in NuLi monolayers. Cell proliferation and migration assays indicated a similar rate of proliferation in both cell lines but with reduced (by 25%) CuFi cell migration. In addition, cell migration experiments performed in the presence of conditioned medium, collected from NuLi and CuFi wounded bronchial monolayers, suggested a defect in EGF/EGFR signaling in CF cells. We (49) recently demonstrated coupling between the EGF response and K(+) channel function, which is crucial for EGF-stimulated alveolar repair. In CuFi cells, lower EGF/EGFR signaling was accompanied by a 40-70% reduction in K(+) currents and KvLQT1, ATP-sensitive potassium (K(ATP)), and Ca(2+)-activated K(+) (KCa3.1) channel expression. In addition, EGF-stimulated bronchial wound healing, cell migration, and proliferation were severely decreased by K(+) channel inhibitors. Finally, acute CFTR inhibition failed to reduce wound healing, EGF secretion, and K(+) channel expression in NuLi. In summary, the delay in CuFi wound healing could be due to diminished EGFR signaling coupled with lower K(+) channel function, which play a crucial role in bronchial repair.     

Regulation of normal and cystic fibrosis airway epithelial repair processes by TNF-α after injury

Chronic infection and inflammation have been associated with progressive airway epithelial damage in patients with cystic fibrosis (CF). However, the effect of inflammatory products on the repair capacity of respiratory epithelia is unclear. Our objective was to study the regulation of repair mechanisms by tumor necrosis factor-α (TNF-α), a major component of inflammation in CF, in a model of mechanical wounding, in two bronchial cell lines, non-CF NuLi and CF CuFi. We observed that TNF-α enhanced the NuLi and CuFi repair rates. Chronic exposure (24-48 h) to TNF-α augmented this stimulation as well as the migration rate during repair. The cellular mechanisms involved in this stimulation were then evaluated. First, we discerned that TNF-α induced metalloproteinase-9 release, epidermal growth factor (EGF) shedding, and subsequent EGF receptor transactivation. Second, TNF-α-induced stimulation of the NuLi and CuFi wound-closure rates was prevented by GM6001 (metalloproteinase inhibitor), EGF antibody (to titrate secreted EGF), and EGF receptor tyrosine kinase inhibitors. Furthermore, we recently reported a relationship between the EGF response and K(+) channel function, both controlling bronchial repair. We now show that TNF-α enhances KvLQT1 and K(ATP) currents, while their inhibition abolishes TNF-α-induced repair stimulation. These results indicate that the effect of TNF-α is mediated, at least in part, through EGF receptor transactivation and K(+) channel stimulation. In contrast, cell proliferation during repair was slowed by TNF-α, suggesting that TNF-α could exert contrasting actions on repair mechanisms of CF airway epithelia. Finally, the stimulatory effect of TNF-α on airway wound repair was confirmed on primary airway epithelial cells, from non-CF and CF patients.     

K(+) channels regulate ENaC expression via changes in promoter activity and control fluid clearance in alveolar epithelial cells

Active Na(+) absorption by alveolar ENaC is the main driving force of liquid clearance at birth and lung edema resorption in adulthood. We have demonstrated previously that long-term modulation of KvLQT1 and K(ATP) K(+) channel activities exerts sustained control in Na(+) transport through the regulation of ENaC expression in primary alveolar type II (ATII) cells. The goal of the present study was: 1) to investigate the role of the α-ENaC promoter, transfected in the A549 alveolar cell line, in the regulation of ENaC expression by K(+) channels, and 2) to determine the physiological impact of K(+) channels and ENaC modulation on fluid clearance in ATII cells. KvLQT1 and K(ATP) channels were first identified in A549 cells by PCR and Western blotting. We showed, for the first time, that KvLQT1 activation by R-L3 (applied for 24h) increased α-ENaC expression, similarly to K(ATP) activation by pinacidil. Conversely, pharmacological KvLQT1 and K(ATP) inhibition or silencing with siRNAs down-regulated α-ENaC expression. Furthermore, K(+) channel blockers significantly decreased α-ENaC promoter activity. Our results indicated that this decrease in promoter activity could be mediated, at least in part, by the repressor activity of ERK1/2. Conversely, KvLQT1 and K(ATP) activation dose-dependently enhanced α-ENaC promoter activity. Finally, we noted a physiological impact of changes in K(+) channel functions on ERK activity, α-, β-, γ-ENaC subunit expression and fluid absorption through polarized ATII cells. In summary, our results disclose that K(+) channels regulate α-ENaC expression by controlling its promoter activity and thus affect the alveolar function of fluid clearance.     

Regulation of ENaC and CFTR expression with K+ channel modulators and effect on fluid absorption across alveolar epithelial cells

In a recent study (Leroy C, Dagenais A, Berthiaume Y, and Brochiero E. Am J Physiol Lung Cell Mol Physiol 286: L1027-L1037, 2004), we identified an ATP-sensitive K(+) (K(ATP)) channel in alveolar epithelial cells, formed by inwardly rectifying K(+) channel Kir6.1/sulfonylurea receptor (SUR)2B subunits. We found that short applications of K(ATP), voltage-dependent K(+) channel KvLQT1, and calcium-activated K(+) (K(Ca)) channel modulators modified Na(+) and Cl(-) currents in alveolar monolayers. In addition, it was shown previously that a K(ATP) opener increased alveolar liquid clearance in human lungs by a mechanism possibly related to epithelial sodium channels (ENaC). We therefore hypothesized that prolonged treatment with K(+) channel modulators could induce a sustained regulation of ENaC activity and/or expression. Alveolar monolayers were treated for 24 h with inhibitors of K(ATP), KvLQT1, and K(Ca) channels identified by PCR. Glibenclamide and clofilium (K(ATP) and KvLQT1 inhibitors) strongly reduced basal transepithelial current, amiloride-sensitive Na(+) current, and forskolin-activated Cl(-) currents, whereas pinacidil, a K(ATP) activator, increased them. Interestingly, K(+) inhibitors or membrane depolarization (induced by valinomycin in high-K(+) medium) decreased alpha-, beta-, and gamma-ENaC and CFTR mRNA. alpha-ENaC and CFTR proteins also declined after glibenclamide or clofilium treatment. Conversely, pinacidil augmented ENaC and CFTR mRNAs and proteins. Since alveolar fluid transport was found to be driven, at least in part, by Na(+) transport through ENaC, we tested the impact of K(+) channel modulators on fluid absorption across alveolar monolayers. We found that glibenclamide and clofilium reduced fluid absorption to a level similar to that seen in the presence of amiloride, whereas pinacidil slightly enhanced it. Long-term regulation of ENaC and CFTR expression by K(+) channel activity could benefit patients with pulmonary diseases affecting ion transport and fluid clearance.     

Effect of tyrosine kinase inhibition on basal and epidermal growth factor-stimulated human Caco-2 enterocyte sheet migration and proliferation

Mucosal healing requires enterocyte migration (restitution) supplemented by proliferation. Proliferation and migration may be studied independently by thymidine uptake and proliferation-blocked cell migration using human Caco-2 enterocyte monolayers in culture. Since epidermal growth factor (EGF) promotes mucosal healing and the EGF receptor is a tyrosine kinase, we hypothesized that tyrosine kinases might therefore modulate enterocyte migration and proliferation. The tyrosine kinase inhibitors genistein and 2, 5-dihydroxymethylcinnamate, which block kinase ATP-binding and substrate-binding sites, respectively, were studied alone and with EGF. Proliferation was blocked with mitomycin. Although each inhibitor decreased basal and EGF-stimulated monolayer expansion when cell proliferation occurred, neither genistein nor 2, 5-dihydroxymethylcinnamate decreased migration when proliferation was blocked. However, each inhibitor prevented EGF stimulation of proliferation-blocked migration and thymidine uptake. More substantial inhibition of basal proliferation by genistein correlated with increased protein-linked DNA breaks, which may reflect nonspecific inhibition of DNA topoisomerase activity by genistein. The more specific 2,5-dihydroxymeth-ylcinnamate blocked changes in the α2 integrin subunit organization which may modulate EGF-stimulated migration. Antiproliferative effects of tyrosine kinase inhibitors decrease basal monolayer expansion but true basal enterocyte migration appears independent of tyrosine kinase regulation. However, a specific tyrosine kinase-dependent modulation of cell-matrix interaction inhibits EGF-stimulated migration. © 1994 Wiley-Liss, Inc.     

Nonsteroidal anti-inflammatory drugs inhibit re-epithelialization of wounded gastric monolayers by interfering with actin, Src, FAK, and tensin signaling.

Re-epithelialization is essential for gastrointestinal ulcer and cutaneous wound healing. It requires epithelial cell migration and proliferation, processes that are stimulated by epidermal growth factor (EGF), and dependent on the cell cytoskeleton. Activation of Src and focal adhesion kinase (FAK) has been implicated in EGF-stimulated cell migration. Nonsteroidal anti-inflammatory drugs (NSAIDs) (both nonselective and Cox2-selective) interfere with ulcer healing and re-epithelialization in vitro and in vivo, but the cellular targets and mechanisms remain unexplored forming the basis of this study. Using a wounded gastric epithelial cell monolayer model, we demonstrated that NSAIDs reduce both basal and epidermal growth factor (EGF)-induced re-epithelialization, and that this action involves disruption of actin stress fiber formation, reduced c-Src activity, decreased phosphorylation of focal adhesion kinase (FAK), tensin and their cellular re-distribution. There was a strong correlation between NSAIDs-mediated inhibitory effect on re-epithelialization and loss of stress fibers and reduced tensin signal. Furthermore, NSAIDs significantly reduced EGF-stimulated c-Src association with FAK. These findings suggest that NSAIDs can directly affect the cell cytoskeleton and signaling pathways essential for re-epithelialization.     

Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration

Regulated intestinal epithelial cell migration plays a key role in wound healing and maintenance of a healthy gastrointestinal tract. Epidermal growth factor (EGF) stimulates cell migration and wound closure in intestinal epithelial cells through incompletely understood mechanisms. In this study we investigated the role of the small GTPase Rac in EGF-induced cell migration using an in vitro wound-healing assay. In mouse colonic epithelial (MCE) cell lines, EGF-stimulated wound closure was accompanied by a doubling of the number of cells containing lamellipodial extensions at the wound margin, increased Rac membrane translocation in cells at the wound margin, and rapid Rac activation. Either Rac1 small interfering (si)RNA or a Rac1 inhibitor completely blocked EGF-stimulated wound closure. Whereas EGF failed to activate Rac in colon cells from EGF receptor (EGFR) knockout mice, stable expression of wild-type EGFR restored EGF-stimulated Rac activation and migration. Pharmacological inhibition of either phosphatidylinositol 3-kinase (PI3K) or Src family kinases reduced EGF-stimulated Rac activation. Cotreatment of cells with both inhibitors completely blocked EGF-stimulated Rac activation and localization to the leading edge of cells and lamellipodial extension. Our results present a novel mechanism by which the PI3K and Src signaling cascades cooperate to activate Rac and promote intestinal epithelial cell migration downstream of EGFR.     

Interleukin-1beta augments in vitro alveolar epithelial repair

Biologically active interleukin (IL)-1beta is present in the pulmonary edema fluid obtained from patients with acute lung injury and has been implicated as an important early mediator of nonpulmonary epithelial wound repair. Therefore, we tested the hypothesis that IL-1beta would enhance wound repair in cultured monolayers from rat alveolar epithelial type II cells. IL-1beta (20 ng/ml) increased the rate of in vitro alveolar epithelial repair by 118 +/- 11% compared with that in serum-free medium control cells (P < 0.01). IL-1beta induced cell spreading and migration at the edge of the wound but not proliferation. Neutralizing antibodies to epidermal growth factor (EGF) and transforming growth factor-alpha or inhibition of the EGF receptor by tyrphostin AG-1478 or genistein inhibited IL-1beta-induced alveolar epithelial repair, indicating that IL-1beta enhances in vitro alveolar epithelial repair by an EGF- or transforming growth factor-alpha-dependent mechanism. Moreover, the mitogen-activated protein kinase pathway is involved in IL-1beta-induced alveolar epithelial repair because inhibition of extracellular signal-regulated kinase activation by PD-98059 inhibited IL-1beta-induced alveolar epithelial repair. In conclusion, IL-1beta augments in vitro alveolar epithelial repair, indicating a possible novel role for IL-1beta in the early repair process of the alveolar epithelium in acute lung injury.     

Epidermal growth factor stimulates serine/threonine phosphorylation of the focal adhesion protein paxillin in a MEK-dependent manner in normal rat kidney cells

Epidermal growth factor (EGF)-stimulated proliferation of renal epithelial cells plays an important role in the recovery of kidney tubule epithelia following exposure to insult. Numerous studies have demonstrated that tyrosine phosphorylation of the focal adhesion protein paxillin mediates in part the effects of growth factors on cell growth, migration, and organization of the actin-based cytoskeleton. The experiments in this report were designed to determine the effect of EGF on paxillin phosphorylation in normal rat kidney (NRK) epithelial cells. Interestingly, treatment of NRK cells with EGF stimulated paxillin serine/threonine phosphorylation, which caused a reduction in the mobility of paxillin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The EGF-stimulated mobility shift of paxillin was independent of an intact cytoskeleton, phosphatidylinositol 3-kinase (PI 3-kinase) activation, protein kinase C (PKC) activation, and cellular adhesion. However, inhibitors of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase abrogated the EGF-stimulated change in paxillin mobility. In addition, the EGF-stimulated change in paxillin serine/threonine phosphorylation was not accompanied by a profound reorganization of the actin cytoskeleton. These results identify paxillin as a component EGF signaling in renal epithelial cells and implicate members of the MAP kinase pathway as critical regulators of paxillin serine/threonine phosphorylation.     

Epidermal growth factor-stimulated intestinal epithelial cell migration requires Src family kinase-dependent p38 MAPK signaling

Members of the epidermal growth factor (EGF) family of ligands and their receptors regulate migration and growth of intestinal epithelial cells. However, our understanding of the signal transduction pathways determining these responses is incomplete. In this study we tested the hypothesis that p38 is required for EGF-stimulated intestinal epithelial monolayer restitution. EGF-stimulated migration in a wound closure model required continuous presence of ligand for several hours for maximal response, suggesting a requirement for sustained signal transduction pathway activation. In this regard, prolonged exposure of cells to EGF activated p38 for up to 5 h. Furthermore genetic or pharmacological blockade of p38 signaling inhibited the ability of EGF to accelerate wound closure. Interestingly p38 inhibition was associated with increased EGF-stimulated ERK1/ERK2 phosphorylation and cell proliferation, suggesting that p38 regulates the balance of proliferation/migration signaling in response to EGF receptor activity. Activation of p38 in intestinal epithelial cells through EGF receptor was abolished by blockade of Src family tyrosine kinase signaling but not inhibition of phosphatidylinositol 3-kinase or protein kinase C. Taken together, these data suggest that Src family kinase-dependent p38 activation is a key component of a signaling switch routing EGF-stimulated responses to epithelial cell migration/restitution rather than proliferation during wound closure.     

Differential modes of action of fibronectin and epidermal growth factor on rabbit corneal epithelial migration

In order to clarify the roles of fibronectin (FN) and epidermal growth factor (EGF) in corneal wound healing, we cultured blocks of excised rabbit cornea for 24 hours in media containing one of these agents, then measured the length of the path of the epithelial layer that had migrated down the side of the block. Both FN and EGF stimulated epithelial migration significantly in a dose-dependent fashion. Responses to EGF involved a time lag of at least 12 hours before stimulation could be observed, but there was no lag-time for FN-stimulated migration. FN was maximally effective only if it was continuously present. In contrast, exposure to EGF for 6 hours did not stimulate epithelial migration, but exposure for 9 hours resulted in the same stimulatory effects as were observed after 24 hours' continuous exposure. Anti-FN antibody inhibited the FN- and EGF-stimulated migration of corneal epithelium. But anti-EGF antibody inhibited only EGF-stimulated migration and had no effect on FN-stimulated migration. These results indicate that, unlike FN, EGF need not be present, once the epithelial cells have recognized its signal. Furthermore, the stimulatory effect of EGF depended on FN, while that of FN was independent of EGF. The effects of EGF on migration of corneal epithelium may, therefore, be mediated by FN.     

Keratinocyte growth factor can enhance alveolar epithelial repair by nonmitogenic mechanisms

Pretreatment with keratinocyte growth factor (KGF) ameliorates experimentally induced acute lung injury in rats. Although alveolar epithelial type II cell hyperplasia probably contributes, the mechanisms underlying KGF's protective effect remain incompletely described. Therefore, we tested the hypothesis that KGF given to rats in vivo would enhance alveolar epithelial repair in vitro by nonproliferative mechanisms. After intratracheal instillation (48 h) of KGF (5 mg/kg), alveolar epithelial type II cells were isolated for in vitro alveolar epithelial repair studies. KGF-treated cells had markedly increased epithelial repair (96 +/- 22%) compared with control cells (P < 0.001). KGF-treated cells had increased cell spreading and migration at the wound edge but no increase in in vitro proliferation compared with control cells. KGF-treated cells were more adherent to extracellular matrix proteins and polystyrene. Inhibition of the epidermal growth factor (EGF) receptor with tyrosine kinase inhibitors abolished the KGF effect on epithelial repair. In conclusion, in vivo administration of KGF augments the epithelial repair rate of alveolar epithelial cells by altering cell adherence, spreading, and migration and through stimulation of the EGF receptor.     

Role of epidermal growth factor-stimulated protein kinase in control of proliferation of A431 cells

Epidermal growth factor (EGF), which stimulates tyrosine-specific protein kinase activity both in vivo and in vitro, inhibits proliferation of A431 human epidermoid carcinoma cells. After mutagenesis clonal cell lines that were resistant to the growth inhibitory effects of EGF were selected. All six variants examined contained decreased EGF-stimulated protein kinase. The number of EGF receptors in variant cells decreased in parallel with EGF-stimulated protein kinase activity so that the specific activity of EGF-stimulated protein kinase per EGF receptor remained constant in variant cell lines with up to tenfold reductions in both activities. This result suggests that both EGF binding and kinase activities reside in the same or closely coupled molecules. The effect of EGF on growth of two resistant variants was examined in detail. Clone 29 contains approximately 50% and clone 4 contains approximately 20% of the EGF-stimulated protein kinase activity of the parental A431 cell line. In serum-supplemented medium, EGF stimulated proliferation of clone 29 but did not affect growth of clone 4. In a 1:1 mixture of DME and F-12 medium without serum, EGF caused both clone 29 and clone 4 to grow as well as in 10% serum. These variants, which were selected for resistance to the growth inhibitory effects of EGF, thus exhibit a strong mitogenic response to EGF. This result suggests that resistance to the growth inhibitory effect of EGF may involve both a decrease in EGF-stimulated protein kinase and an alteration in the response pathway.     

ErbB2 and EGFR are downmodulated during the differentiation of 3T3-L1 preadipocytes

The expression of receptors belonging to the epidermal growth factor receptor subfamily has been largely studied these last years in epithelial cells mainly as involved in cell proliferation and malignant progression. Although much work has focused on the role of these growth factor receptors in the differentiation of a variety of tissues, there is little information in regards to normal stromal cells. We investigated erbB2 expression in the murine fibroblast cell line Swiss 3T3L1, which naturally or hormonally induced undergoes adipocyte differentiation. We found that the Swiss 3T3-L1 fibroblasts express erbB2, in addition to EGFR, and in a quantity comparable to or even greater than the breast cancer cell line T47D. Proliferating cells increased erbB2 and EGFR levels when reaching confluence up to 4- and 10-fold, respectively. This expression showed a significant decrease when growth-arrested cells were stimulated to differentiate with dexamethasone and isobutyl-methylxanthine. Differentiated cells presented a decreased expression of both erbB2 and EGFR regardless of whether the cells were hormonally or spontaneously differentiated. EGF stimulation of serum-starved cells increased erbB2 tyrosine phosphorylation and retarded erbB2 migration in SDS-PAGE, suggesting receptor association and activation. Heregulin-alpha1 and -beta1, two EGF related factors, had no effect on erbB2 or EGFR phosphorylation. Although 3T3-L1 cells expressed heregulin, its specific receptors, erbB3 and erbB4, were not found. This is the first time in which erbB2 is reported to be expressed in an adipocytic cell line which does not depend on non EGF family growth factors (thyroid hormone, growth hormone, etc.) to accomplish adipose differentiation. Since erbB2 and EGFR expression were downmodulated as differentiation progressed it is conceivable that a mechanism of switching from a mitogenic to a differentiating signaling pathway may be involved, through regulation of the expression of these growth factor receptors.     

Effect of protein and steroidal osteotropic agents on differentiation and epidermal growth factor-mediated growth of the CFK1 osseous cell line.

The effects of factors known to influence bone metabolism were examined using the osseous cell line CFK1. Parathyroid hormone (PTH) and dexamethasone (DEX) appeared to enhance the formation of cell foci of CFK1 cells in culture whereas retinoic acid (RA) caused a marked alteration in individual cell morphology. Bone morphogenetic protein (BMP-2) and PTH increased alkaline phosphatase activity, however, this index of differentiation was suppressed by epidermal growth factor (EGF), DEX, and RA. BMP-2 and EGF each stimulated DNA synthesis in a dose-dependent manner and enhanced cell numbers, but, no synergistic response of EGF and BMP-2 was observed. PTH and DEX failed to significantly alter cell number or EGF-stimulated DNA synthesis or cell proliferation. Although RA treatment of CFK1 cells resulted in a reduction in cell number compared to control, pretreatment with RA enhanced EGF-stimulated DNA synthesis and proliferative effects. At least part of this effect was by increasing the EGF receptor binding capacity of the cells. Furthermore, using cell cycle analysis, addition of EGF stimulated the progression of RA-treated cells into the DNA synthesis (S) phase with a reduced lag time. EGF and BMP-2, therefore, appear to exert a role in the expansion dynamics of the CFK1 population although BMP-2 may also enhance differentiation. PTH and DEX may act primarily to modulate the differentiated function of the CFK1 cells. RA inhibited cell proliferation and may mediate differentiation towards a less established cell population with upregulation of EGF receptors. The CFK1 cell model may, therefore, provide insight into microenvironmental control of growth and differentiation of precursor osseous cells.     

Contribution of the IsK (MinK) potassium channel subunit to regulatory volume decrease in murine tracheal epithelial cells

The cell volume regulatory response following hypotonic shocks is often achieved by the coordinated activation of K(+) and Cl(-) channels. In this study, we investigate the identity of the K(+) and Cl(-) channels that mediate the regulatory volume decrease (RVD) in ciliated epithelial cells from murine trachea. RVD was inhibited by tamoxifen and 1,9-dideoxyforskolin, two agents that block swelling-activated Cl(-) channels. These data suggest that swelling-activated Cl(-) channels play an important role in cell volume regulation in murine tracheal epithelial cells. Ba(2+) and apamin, inhibitors of K(+) channels, were without effect on RVD, while tetraethylammoniun had little effect on RVD. In contrast, clofilium, an inhibitor of the KvLQT/IsK potassium channel complex potently inhibited RVD, suggesting a role for the KvLQT/IsK channel complex in cell volume regulation by tracheal epithelial cells. To investigate further the role of KvLQT/IsK channels in RVD, we used IsK knock-out mice. When exposed to hypotonic solutions, tracheal cells from IsK(+/+) mice underwent RVD, whereas cells from IsK(-/-) failed to recover their normal size. These data suggest that the IsK potassium subunit plays an important role in RVD in murine tracheal epithelial cells.