Epidermal growth factor receptor-dependent PI3K-activation promotes restitution of wounded human gastric epithelial monolayers

Restitution is a crucial event during the healing of superficial injury of the gastric mucosa involving epithelial cell sheet movement into the damaged area. We demonstrated that growth factors promote the restitution of human gastric epithelial cells. However, the intracellular signaling pathways that transmit extracellular cues as well as regulate basal and growth factor-stimulated gastric epithelial cell migration are still unclear. Herein, confluent human gastric epithelial cell monolayers (HGE-17) or primary cultures of gastric epithelial cells were wounded with a razor blade and the migration response was analyzed in presence or absence of TGFalpha or of pharmacological inhibitors of signaling proteins. Kinase activation profile analysis and phase-contrast microscopy were also performed in parallel. We report that ERK1/2 and Akt activities are rapidly stimulated following wounding of HGE-17 cells. Treatment of confluent HGE-17 cells or primary cultures of gastric epithelial cells with the phosphatidylinositol 3-kinase inhibitor LY294002, but not the MEK1 inhibitor, PD98059, significantly inhibits basal and TGFalpha-induced migration following wounding. Conversely, treatment of wounded HGE-17 cells with phosphatidylinositol(3,4,5)-triphosphate is sufficient to stimulate basal cell migration by 235%. In addition, pp60c-src kinase activity and tyrosine phosphorylation of epidermal growth factor receptors (EGFR) are also rapidly enhanced after wounding and pharmacological inhibition of both these activities strongly attenuates basal and TGFalpha-induced migration as well as Akt phosphorylation levels. In conclusion, the present results indicate that EGFR-dependent PI3K activation promotes restitution of wounded human gastric epithelial monolayers.     

Metalloprotease disintegrin-mediated ectodomain shedding of EGFR ligands promotes intestinal epithelial restitution

EGF receptor (EGFR) promotes intestinal epithelial restitution, an important early process in the reepithelialization of ulcers. During epithelial restitution, the mechanism of EGFR activation is not known. We evaluated the role of TNF-converting enzyme (TACE), a metalloprotease disintegrin that proteolytically processes plasma membrane-anchored EGFR ligand precursors into their mature active forms, in wound-induced EGFR activation and epithelial restitution. With the use of scrape-wounded rat intestinal epithelial-1 (RIE-1) cell monolayers to model epithelial ulceration and restitution, we observed the rapid wound-dependent release of EGFR ligands into culture medium. RIE-1 cells express TACE, and treatment with phorbol ester, an established TACE stimulus, triggered the extracellular release of an EGFR ligand, transforming growth factor-alpha. Blockade of TACE using TNF processing inhibitor (TAPI-1), a specific hydroxamate inhibitor of metalloprotease disintegrins, prevented release of EGFR ligands from wounded RIE-1 cell monolayers. The restitution of wounded RIE-1 cell monolayers was also dose-dependently inhibited by TAPI-1, establishing the role of metalloprotease disintegrins in this process. These results have established a mechanism of EGFR activation in wounded intestinal epithelium and show an important functional role for metalloprotease disintegrin-mediated ectodomain shedding during intestinal epithelial restitution. Therefore, activation of the TACE-EGFR system might promote the healing of intestinal tract ulcers in patients.     

Trefoil peptides promote restitution of wounded corneal epithelial cells

The ocular surface shares many characteristics with mucosal surfaces. In both, healing is regulated by peptide growth factors, cytokines, and extracellular matrix proteins. However, these factors are not sufficient to ensure most rapid healing. Trefoil peptides are abundantly expressed epithelial cell products which exert protective effects and are key regulators of gastrointestinal epithelial restitution, the critical early phase of cell migration after mucosal injury. To assess the role of trefoil peptides in corneal epithelial wound healing, the effects of intestinal trefoil factor (ITF/TFF3) and spasmolytic polypeptide (SP/TFF2) on migration and proliferation of corneal epithelial cells were analyzed. Both ITF and SP enhanced restitution of primary rabbit corneal epithelial cells in vitro. While the restitution-enhancing effects of TGF-alpha and TGF-beta were both inhibited by neutralizing anti-TGF-beta-antibodies, trefoil peptide stimulation of restitution was not. Neither trefoil peptide significantly affected proliferation of primary corneal epithelial cells. ITF but not SP or pS2 mRNA was present in rabbit corneal and conjunctival tissues. In summary, the data indicate an unanticipated role of trefoil peptides in healing of ocular surface and demand rating their functional actions beyond the gastrointestinal tract.     

Endotoxin inhibits intestinal epithelial restitution through activation of Rho-GTPase and increased focal adhesions

Diseases of gut inflammation such as neonatal necrotizing enterocolitis (NEC) result after an injury to the mucosal lining of the intestine, leading to translocation of bacteria and endotoxin (lipopolysaccharide). Intestinal mucosal defects are repaired by the process of intestinal restitution, during which enterocytes migrate from healthy areas to sites of injury. In an animal model of NEC, we determined that intestinal restitution was significantly impaired compared with control animals. We therefore sought to determine the mechanisms governing enterocyte migration under basal conditions and after an endotoxin challenge. Here we show that the cytoskeletal reorganization and stress fiber formation required for migration in IEC-6 enterocytes requires RhoA. Enterocytes were found to express the endotoxin receptor Toll-like receptor 4, which served to bind and internalize lipopolysaccharide. Strikingly, endotoxin treatment significantly inhibited intestinal restitution, as measured by impaired IEC-6 cell migration across a scraped wound. Lipopolysaccharide was found to increase RhoA activity in a phosphatidylinositol 3-kinase-dependent manner, leading to an increase in phosphorylation of focal adhesion kinase and an enhanced number of focal adhesions. Importantly, endotoxin caused a progressive, RhoA-dependent increase in cell matrix tension/contractility, which correlated with the observed impairment in enterocyte migration. We therefore conclude that endotoxin inhibits enterocyte migration through a RhoA-dependent increase in focal adhesions and enhanced cell adhesiveness, which may participate in the impaired restitution observed in experimental NEC.     

Nuclear factor-kappa b activation in silica-induced interleukin 8 production by human bronchial epithelial cells

Recent studies indicate that interleukin 8 (IL-8) plays an important role in interstitial lung diseases including silica-induced lung inflammation. To investigate the regulation of IL-8 expression and production in human bronchial epithelial cells, we examined the effects of silica on NF-kappaB activation. Human bronchial epithelial cell line BET-1A was cultured with hormonally defined Ham's F12 medium. The administration of silica induced IL-8 production in BET-1A dose-dependently and time-dependently. Northern blot analysis demonstrated that silica upregulated IL-8 expression in BET-1A. Moreover, electrophoretic mobility shift assays revealed that NF-kappaB activation occurred in the presence of silica, which was inhibited by antioxidants such as N-acetylcysteine (NAC). These data suggest that reactive oxygen species may be involved in the activation of NF-kappaB induced by silica.     

Polyamines regulate Rho-kinase and myosin phosphorylation during intestinal epithelial restitution

Polyamines are required for the early phase of mucosal restitution that occurs as a consequence of epithelial cell migration. Our previous studies have shown that polyamines increase RhoA activity by elevating cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) through controlling voltage-gated K(+) channel expression and membrane potential (E(m)) during intestinal epithelial restitution. The current study went further to determine whether increased RhoA following elevated [Ca(2+)](cyt) activates Rho-kinase (ROK/ROCK) resulting in myosin light chain (MLC) phosphorylation. Studies were conducted in stable Cdx2-transfected intestinal epithelial cells (IEC-Cdx2L1), which were associated with a highly differentiated phenotype. Reduced [Ca(2+)](cyt), by either polyamine depletion or exposure to the Ca(2+)-free medium, decreased RhoA protein expression, which was paralleled by significant decreases in GTP-bound RhoA, ROCK-1, and ROKalpha proteins, Rho-kinase activity, and MLC phosphorylation. The reduction of [Ca(2+)](cyt) also inhibited cell migration after wounding. Elevation of [Ca(2+)](cyt) induced by the Ca(2+) ionophore ionomycin increased GTP-bound RhoA, ROCK-1, and ROKalpha proteins, Rho-kinase activity, and MLC phosphorylation. Inhibition of RhoA function by a dominant negative mutant RhoA decreased the Rho-kinase activity and resulted in cytoskeletal reorganization. Inhibition of ROK/ROCK activity by the specific inhibitor Y-27632 not only decreased MLC phosphorylation but also suppressed cell migration. These results indicate that increase in GTP-bound RhoA by polyamines via [Ca(2+)](cyt) can interact with and activate Rho-kinase during intestinal epithelial restitution. Activation of Rho-kinase results in increased MLC phosphorylation, leading to the stimulation of myosin stress fiber formation and cell migration.     

Formyl peptide receptor-1 activation enhances intestinal epithelial cell restitution through phosphatidylinositol 3-kinase-dependent activation of Rac1 and Cdc42

Inflammatory disorders of the gastrointestinal tract result in the breakdown of the intestinal epithelial barrier in the form of erosion and ulceration. To reestablish the epithelial barrier, the epithelium must efficiently migrate to reseal wounds. Numerous signaling cascades are involved in the induction and regulation of this complex process. N-formyl peptide receptors comprise a group of Gi-coupled receptors that regulate innate immune responses. Previously, we identified the expression of functional N-formyl peptide receptors in model SK-CO15 intestinal epithelial cells and observed a role for activation of these receptors in regulating cellular invasive behavior. In these studies, we performed formyl peptide receptor-1 (FPR) localization and evaluated its role in regulating intestinal epithelial cell wound closure. Immunolocalization studies using a recently developed specific monoclonal anti-FPR Ab demonstrated its localization along the lateral membrane of crypt epithelial cells in normal human colonic epithelium. In vitro studies using the classical FPR agonist fMLF showed that FPR activation significantly enhances model intestinal epithelial cell restitution and that FPR localized along actin filaments in lamellipodial and filopodial extrusions. The increase in cell migration was associated with activation of PI3K, Rac1, and Cdc42. Pharmacologic inhibition of PI3K activity abrogated the fMLF-induced increase in wound closure and activation of both Rac1 and Cdc42. Inhibition of Rac1 and Cdc42 using pharmacologic inhibitors and dominant negative mutants also inhibited the fMLF-induced increase in cell migration. Taken together, theses results support a novel role for FPR stimulation in enhancing intestinal epithelial cell restitution through PI3K-dependent activation of Rac1 and Cdc42.     

Adrenomedullin promotes epithelial restitution of rat and human gastric mucosa in vitro

We have investigated the effect of adrenomedullin (AM) on restitution of mucosal integrity following damage in rat and human gastric mucosa, measuring the potential difference (PD) on a mucosal strip mounted on an Ussing chamber. Mucosal damage was induced by 0.5, 1.0, and 2.0 M NaCl solution, and it caused an immediate and significant decrease in PD. In the rat AM group, PD recovered significantly more than in control group at 120 min after exposure to 0.5 M (p < 0.01) and 1.0 M (p < 0.05) NaCl solution. In the human AM group, PD completely recovered at 120 min after exposure to 0.5 M (p < 0.05) NaCl solution. In rat mucosa damaged by 0.5 M NaCl solution, the effect was inhibited by human (h)-CGRP(8-37) and there was no significant difference between the h-CGRP(8-37) group and control group. On immunohistochemical examination of rat gastric mucosa, AM was detected within the chief cell. AM probably promotes epithelial restitution primarily through the CGRP receptor, but it does not ameliorate more severe damage of gastric mucosa in vitro.     

mTORC1 signaling activation increases intestinal stem cell activity and promotes epithelial cell proliferation

The crypt-villus axis of the intestine undergoes a continuous renewal process that is driven by intestinal stem cells (ISCs). However, the homeostasis is disturbed under constant exposure to high ambient temperatures, and the precise mechanism is unclear. We found that both EdU⁺ and Ki67⁺ cell ratios were significantly reduced after exposure to 41°C, as well as the protein synthesis rate of IPEC-J2 cells, and the expression of ubiquitin and heat shock protein 60, 70, and 90 were significantly increased. Additionally, heat exposure decreased enteroid expansion and budding efficiency, as well as induced apoptosis after 48 hr; however, no significant difference was observed in the apoptosis ratio after 24 hr. In the process of heat exposure, the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway was significantly inhibited in both IPEC-J2 cells and enteroids. Correspondingly, treatment of IPEC-J2 and enteroids with the mTORC1 agonist MHY1485 at 41°C significantly attenuated the inhibition of proliferation and protein synthesis, increased the ISC activity, and promoted expansion and budding of enteroid. In summary, we conclude that the mTORC1 signaling pathway regulates intestinal epithelial cell and stem cell activity during heat exposure-induced injury.     

Hydrogen inhalation reduced epithelial apoptosis in ventilator-induced lung injury via a mechanism involving nuclear factor-kappa B activation

Entry of enveloped viruses into cells is initiated by binding of their envelope glycoproteins (Envs) to cell surface-associated receptors. The Crimean-Congo hemorrhagic fever virus (CCHFV) has two Envs, Gn and Gc, with poorly understood role in binding to susceptible cells. We expressed codon optimized Gn and Gc, and identified independently folded soluble Env fragments, one of which (Gc residues 180–300) bound CCHFV susceptible cells supposedly by interacting with a putative receptor. This receptor binding domain (RBD) was used to identify its interacting partner by coimmunoprecipitation and mass spectrometry. Thus we identified the human cell surface nucleolin as a putative CCHFV entry factor. Nucleolin was expressed on all susceptible cells tested but not on the surface of cells resistant to CCHFV infection. Further studies are needed to explore the nucleolin function as a plausible CCHFV receptor and the molecular mechanisms of the Gc-nucleolin interactions. The identification of the CCHFV RBD and its binding partner could provide novel targets for therapy and tools for prevention as well as more complete understanding of the mechanisms of CCHFV entry and pathogenesis.     

TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation

Crohn's disease (CD) patients have an abnormal increase in intestinal epithelial permeability. The defect in intestinal tight junction (TJ) barrier has been proposed as an important etiologic factor of CD. TNF-alpha increases intestinal TJ permeability. Because TNF-alpha levels are markedly increased in CD, TNF-alpha increase in intestinal TJ permeability could be a contributing factor of intestinal permeability defect in CD. Our purpose was to determine some of the intracellular mechanisms involved in TNF-alpha modulation of intestinal epithelial TJ permeability by using an in vitro intestinal epithelial system consisting of filter-grown Caco-2 monolayers. TNF-alpha produced a concentration- and time-dependent increase in Caco-2 TJ permeability. TNF-alpha-induced increase in Caco-2 TJ permeability correlated with Caco-2 NF-kappa B activation. Inhibition of TNF-alpha-induced NF-kappa B activation by selected NF-kappa B inhibitors, curcumin and triptolide, prevented the increase in Caco-2 TJ permeability, indicating that NF-kappa B activation was required for the TNF-alpha-induced increase in Caco-2 TJ permeability. This increase in Caco-2 TJ permeability was accompanied by down-regulation of zonula occludens (ZO)-1 proteins and alteration in junctional localization of ZO-1 proteins. TNF-alpha modulation of ZO-1 protein expression and junctional localization were also prevented by NF-kappa B inhibitors. TNF-alpha did not induce apoptosis in Caco-2 cells, suggesting that apoptosis was not the mechanism involved in TNF-alpha-induced increase in Caco-2 TJ permeability. These results demonstrate for the first time that TNF-alpha-induced increase in Caco-2 TJ permeability was mediated by NF-kappa B activation. The increase in permeability was associated with NF-kappa B-dependent downregulation of ZO-1 protein expression and alteration in junctional localization.     

Hydrogen inhalation reduced epithelial apoptosis in ventilator-induced lung injury via a mechanism involving nuclear factor-kappa B activation

We recently demonstrated the inhalation of hydrogen gas, a novel medical therapeutic gas, ameliorates ventilator-induced lung injury (VILI); however, the molecular mechanisms by which hydrogen ameliorates VILI remain unclear. Therefore, we investigated whether inhaled hydrogen gas modulates the nuclear factor-kappa B (NFκB) signaling pathway. VILI was generated in male C57BL6 mice by performing a tracheostomy and placing the mice on a mechanical ventilator (tidal volume of 30 ml/kg or 10 ml/kg without positive end-expiratory pressure). The ventilator delivered either 2% nitrogen or 2% hydrogen in balanced air. NFκB activation, as indicated by NFκB DNA binding, was detected by electrophoretic mobility shift assays and enzyme-linked immunosorbent assay. Hydrogen gas inhalation increased NFκB DNA binding after 1 h of ventilation and decreased NFκB DNA binding after 2 h of ventilation, as compared with controls. The early activation of NFκB during hydrogen treatment was correlated with elevated levels of the antiapoptotic protein Bcl-2 and decreased levels of Bax. Hydrogen inhalation increased oxygen tension, decreased lung edema, and decreased the expression of proinflammatory mediators. Chemical inhibition of early NFκB activation using SN50 reversed these protective effects. NFκB activation and an associated increase in the expression of Bcl-2 may contribute, in part, to the cytoprotective effects of hydrogen against apoptotic and inflammatory signaling pathway activation during VILI.     

EGF promotes gastric mucosal restitution by activating Na(+)/H(+) exchange of epithelial cells

This study was conducted to determine whether the contributions of epidermal growth factor (EGF) to gastric mucosal restitution after injury are mediated by stimulation of Na(+)/H(+) exchangers in surface mucous cells (SMC). Intact sheets of guinea pig gastric mucosae were incubated in vitro. Intracellular pH (pH(i)) in SMC was measured fluorometrically, using 2',7'- bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Restitution after Triton X-100-induced injury was evaluated by recovery of electrical resistance. At neutral luminal pH, exogenous EGF (ex-EGF) increased pH(i) and enhanced restitution in the absence but not in the presence of serosal HCO. During exposure to luminal acid, ex-EGF not only prevented intracellular acidosis but also promoted restitution. These effects of ex-EGF were blocked by serosal amiloride or anti-EGF-receptor antibody. In the absence of ex-EGF, restitution was inhibited by replacement of luminal and serosal solutions with fresh solutions and was blocked more completely by serosal anti-EGF-receptor antibody. These results suggest that both endogenous and ex-EGF contribute to restitution via basolateral EGF receptors, with effects mediated, at least in part, by stimulation of basolateral Na(+)/H(+) exchangers.     

Rac1 promotes intestinal epithelial restitution by increasing Ca2+ influx through interaction with phospholipase C-(gamma)1 after wounding

Intestinal mucosal restitution occurs as a consequence of epithelial cell migration and reseals superficial wounds after injury. This rapid reepithelialization is mediated in part by a phospholipase C-gamma1 (PLC-gamma1)-induced Ca(2+) signaling, but the exact mechanism underlying such signaling and its regulation remains elusive. The small GTP-binding protein Rac1 functions as a pivotal regulator of several signaling networks and plays an important role in regulating cell motility. The current study tests the hypothesis that Rac1 modulates intestinal epithelial cell migration after wounding by altering PLC-gamma1-induced Ca(2+) signaling. Inhibition of Rac1 activity by treatment with its inhibitor NSC-23766 or Rac1 silencing with small interfering RNA decreased store depletion-induced Ca(2+) influx and suppressed cell migration during restitution, whereas ectopic overexpression of Rac1 increased Ca(2+) influx and promoted cell migration. Rac1 physically interacted with PLC-gamma1 and formed Rac1/PLC-gamma1 complex in intestinal epithelial cells. PLC-gamma1 silencing in cells overexpressing Rac1 prevented stimulation of store depletion-induced Ca(2+) influx and cell migration after wounding. Polyamine depletion inhibited expression of both Rac1 and PLC-gamma1, decreased Rac1/PLC-gamma1 complex levels, reduced Ca(2+) influx, and repressed cell migration. Overexpression of Rac1 alone failed to rescue Ca(2+) influx after store depletion and cell migration in polyamine-deficient cells, because it did not alter PLC-gamma1 levels. These results indicate that Rac1 promotes intestinal epithelial cell migration after wounding by increasing Ca(2+) influx as a result of its interaction with PLC-gamma1.     

ADAM-15 inhibits wound healing in human intestinal epithelial cell monolayers

The disintegrin metalloproteases (or ADAMs) are membrane-anchored glycoproteins that have been implicated in cell-cell or cell-matrix interactions and in proteolysis of molecules on the cell surface. The expression and/or the pathophysiological implications of ADAMs are not known in intestinal epithelial cells. Therefore, our aim was to investigate the expression and the role of ADAMs in intestinal epithelial cells. Expression of ADAMs was assessed by RT-PCR, Western blot analysis, and immunufluorescence experiments. Wound-healing experiments were performed by using the electric cell substrate impedence sensing technology. Our results showed that ADAMs-10, -12, and -15 mRNA are expressed in the colonic human cell lines Caco2-BBE and HT29-Cl.19A. An ADAM-15 complementary DNA cloned from Caco2-BBE poly(A)+ RNA, and encompassing the entire coding region, was found to be shorter and to present a different region encoding the cytoplasmic tail compared with ADAM-15 sequence deposited in the database. In Caco2-BBE cells and colonic epithelial cells, ADAM-15 protein was found in the apical, basolateral, and intracellular compartments. We also showed that the overexpression of ADAM-15 reduced cell migration in a wound-healing assay in Caco2-BBE monolayers. Our data show that 1) ADAM-15 is expressed in human intestinal epithelia, 2) a new variant of ADAM-15 is expressed in a human intestinal epithelial cell line, and 3) ADAM-15 is involved in intestinal epithelial cells wound-healing processes. Together, these results suggest that ADAM-15 may have important pathophysiological roles in intestinal cells.