Hepatocyte growth factor enhances IL-1β stimulated IL-8 secretion by Caco-2 epithelial cells

Hepatocyte growth factor (HGF) can induce proliferation and migration of intestinal epithelial cells and has also been shown to be important in wound healing of inflamed mucosal tissues. HGF is known to be expressed along with interleukin-1 (IL-1) by inflamed mucosal tissues, yet the effect of HGF on IL-1-induced proinflammatory cytokine responses by colonic epithelial cells is unknown. In this report, we have examined the effect of HGF on IL-1-induced secretion of IL-8 by the Caco-2 colonic epithelial cell line. HGF stimulation alone had no effect on the secretion of IL-8 by the Caco-2 cells. However, culture of the cells with HGF and suboptimal levels of IL-1 resulted in a significant enhancement of IL-8 secretion compared to cells cultured with IL-1 alone. A similar effect was seen with HGF and IL-1 simulation of monocyte chemoattractant protein-1 secretion by the rat IEC-6 intestinal epithelial cell line. The enhancing effect of HGF was seen regardless of whether the culture medium contained serum or not. Simultaneous stimulation with HGF and IL-1 was required for the enhancing effect as cells pretreated with HGF for 24 h and then stimulated with IL-1 alone secreted IL-8 levels similar to that of cells stimulated with IL-1 alone. These results suggest that in addition to wound healing, HGF may play a role in the IL-1-induced chemokine response of epithelial cells in inflamed mucosal tissues.     

The loss of local HGF, an endogenous gastrotrophic factor, leads to mucosal injuries in the stomach of mice

The stomach is constantly exposed to mechanical and chemical stresses. Under persistent damages, epithelial cell proliferation is required to maintain mucosal integrity. Nevertheless, which ligand system(s) is physiologically involved in gastric defense remains unclear. Herein, we provide evidence that HGF is a key "natural ligand" to reverse gastric injury. The injection of cisplatin in mice led to the loss of HGF in the gastric interstitium, associated with the decrease in proliferating epithelium and the progression of mucotitis. When c-Met tyrosine phosphorylation was abolished by anti-HGF IgG, mucosal cell proliferation became faint, leading to delayed recovery from mucotitis, and vice versa in cases of HGF supplementation. Our findings indicate that: (1) HGF/c-Met signal on mucosa is needed to restore gastric injuries; and (2) the loss of local HGF leads to manifestation of gastric lesions. This study provides a rationale that explains why HGF supplement is useful for reversing gastric diseases.     

Overexpression of gastrokine 1 in gastric cancer cells induces Fas-mediated apoptosis

Gastrokine 1 (GKN1) is involved in the replenishment of the surface lumen epithelial cell layer, in maintaining the mucosal integrity, and could play a role in cell proliferation and differentiation. In fact, after injury of the gastric mucosa, restoration may occur very rapidly in the presence of GKN1. In contrast, if the protein is downregulated, the repair process may be hampered; however, application of GKN1 to gastrointestinal cells promoted epithelial restoration. Because GKN1 possesses some mitogenic effects on intestinal epithelial cells (IEC-6) whereas this protein was also capable of inhibiting proliferation in gastric cancer cells (MKN28), we decided to study its involvement in apoptosis to understand the role of GKN1 in the modulation of inflammatory damage or tumorigenesis in gastric mucosa. We found by cytofluorimetry, Western blot and RT-PCR that the overexpression of GKN1 in gastric cancer cell lines (AGS and MKN28) stimulated the expression of Fas receptor. Moreover, compared to control cells, a significant increase of apoptosis, evaluated by TUNEL, was observed when GKN1 transfected cells were treated with a monoclonal antibody (IgM) anti-Fas. The activation of Fas expression was also observed by the overexpression of GKN1 in other cancer cell lines. Moreover, in GKN1-overexpressing gastric cancer cells exposed to FasL, the activation of caspase-3 was also observed by Western blot and fluorescence assays. Our data represent the first report for GKN1 as modulator of apoptotic signals and suggest that GKN1 might play an important role for tissue repair during the early stages of neoplastic transformation.     

Airway epithelial cell migration and wound repair by ATP-mediated activation of dual oxidase 1

The airway epithelium is continuously subjected to environmental pollutants, airborne pathogens, and allergens and relies on several intrinsic mechanisms to maintain barrier integrity and to promote epithelial repair processes following injury. Here, we report a critical role for dual oxidase 1 (Duox1), a newly identified NADPH oxidase homolog within the tracheobronchial epithelium, in airway epithelial cell migration and repair following injury. Activation of Duox1 during epithelial injury is mediated by cellular release of ATP, which signals through purinergic receptors expressed on the epithelial cell surface. Purinergic receptor stimulation by extracellular ATP is a critical determinant of epithelial cell migration and repair following injury and is associated with activation of extracellular signal-regulated kinases (ERK1/2) and matrix metalloproteinase-9 (MMP-9). Stimulation of these integral features of epithelial cell migration and repair processes was found to require the activation of Duox1. Our findings demonstrate a novel role for Duox1 in the tracheobronchial epithelium, in addition to its proposed role in antimicrobial host defense, by participating in epithelial repair processes to maintain epithelial integrity and barrier function in the face of environmental stress.     

HGF triggers activation of the COX-2 gene in rat gastric epithelial cells: action mediated through the ERK2 signaling pathway.

Although it is established that growth factors and prostaglandins function in the maintenance of gastric mucosal integrity and in the healing of gastric mucosal injury and ulceration, the regulatory relationship between growth factors and prostaglandins in the gastric mucosa is not well characterized. Therefore, we investigated whether hepatocyte growth factor (HGF) affects expression of COX-2 (the inducible form of the prostaglandin synthesizing enzyme, cyclooxygenase) in gastric epithelial cells and whether this action is mediated through the MAP (ERK) kinase signaling pathway. In RGM1 cells (an epithelial cell line derived from normal rat gastric mucosa), HGF caused an increase in COX-2 mRNA and protein by 236% and 175%, respectively (both P<0.05). This induction of COX-2 expression was abolished by pretreatment with the MAPK kinase (MEK) inhibitor PD98059. HGF also triggered a 13-fold increase in c-Met/HGF receptor phosphorylation (P<0.005) and increased ERK2 activity by 684% (P<0.01). Pretreatment with PD98059 abolished the HGF-induced increase in ERK2 activity, but not c-Met/HGF receptor phosphorylation. The specific inhibitor of p38 MAP kinase, SB203580, had no effect on HGF-induced COX-2 expression. Thus, HGF triggers activation of the COX-2 gene in gastric epithelial cells through phosphorylation of c-Met/HGF receptor and activation of the ERK2 signaling pathway.-Jones, M. K., Sasaki, E., Halter, F., Pai, R., Nakamura, T., Arakawa, T., Kuroki, T., Tarnawski, A. S. HGF triggers activation of the COX-2 gene in rat gastric epithelial cells: action mediated through the ERK2 signaling pathway.     

肠上皮快速复原过程中的细胞信号传递: 多胺和K+通道的影响

胃肠道粘膜上皮细胞具有重要的屏障作用,可以保护次上皮组织抵御一系列的有害物质,包括过敏原、病毒以及微生物病原体。粘膜损伤后的修复有赖于上皮细胞对信号网络的调节,而这一网络系统控制着基因的表达、细胞的存活、迁移及增殖。近几年的研究结果显示,在胃肠道粘膜的修复中,多胺起到关键作用;且细胞多胺的调控是众多信号传递路径的焦点。本文简要综述了多胺在肠粘膜上皮快速复原中的功能和机制,特别是对K^ 通道活性的影响。     

Modulation of stem cell fate in intestinal homeostasis,injury and repair

The mammalian intestinal epithelium constitutes the largest barrier against the external environment and makes flexible responses to various types of stimuli. Epithelial cells are fast-renewed to counteract constant damage and disrupted barrier function to maintain their integrity. The homeostatic repair and regeneration of the intestinal epithelium are governed by the Lgr5⁺ intestinal stem cells (ISCs) located at the base of crypts, which fuel rapid renewal and give rise to the different epithelial cell types. Protracted biological and physicochemical stress may challenge epithelial integrity and the function of ISCs. The field of ISCs is thus of interest for complete mucosal healing, given its relevance to diseases of intestinal injury and inflammation such as inflammatory bowel diseases. Here, we review the current understanding of the signals and mechanisms that control homeostasis and regeneration of the intestinal epithelium. We focus on recent insights into the intrinsic and extrinsic elements involved in the process of intestinal homeostasis, injury, and repair, which fine-tune the balance between self-renewal and cell fate specification in ISCs. Deciphering the regulatory machinery that modulates stem cell fate would aid in the development of novel therapeutics that facilitate mucosal healing and restore epithelial barrier function.     

Oxidative stress involvement and gene expression in indomethacin-induced gastropathy

It has been proposed that neutrophil- and oxygen radical-dependent microvascular injuries are important prime events that lead to gastric mucosal injury induced by indomethacin. Reactive oxygen species (ROS) produced by activated neutrophils after indomethacin treatment cause gastric mucosal injury via ROS-mediated oxidation of important biomolecules such as lipid, protein, and DNA. In addition, it has been revealed that indomethacin-induced gastric mucosal injury occurs via gastric epithelial cell apoptosis. However, there is little known about the mechanism of indomethacin-triggered cellular response and apoptotic signaling in gastric mucosal cells. In the present study, we summarize the evidence that supports the involvement of oxidative stress and apoptosis in indomethacin-induced gastropathy, and review the gene expression profiles of gastric epithelial cells after indomethacin treatment determined by DNA microarray analysis.     

Oxidative stress involvement and gene expression in indomethacin-induced gastropathy

Abstract

It has been proposed that neutrophil- and oxygen radical-dependent microvascular injuries are important prime events that lead to gastric mucosal injury induced by indomethacin. Reactive oxygen species (ROS) produced by activated neutrophils after indomethacin treatment cause gastric mucosal injury via ROS-mediated oxidation of important biomolecules such as lipid, protein, and DNA. In addition, it has been revealed that indomethacin-induced gastric mucosal injury occurs via gastric epithelial cell apoptosis. However, there is little known about the mechanism of indomethacin-triggered cellular response and apoptotic signaling in gastric mucosal cells. In the present study, we summarize the evidence that supports the involvement of oxidative stress and apoptosis in indomethacin-induced gastropathy, and review the gene expression profiles of gastric epithelial cells after indomethacin treatment determined by DNA microarray analysis.     

Contribution of spermidine to stimulation by hepatocyte growth factor in repair after damage of rabbit gastric mucosal cells in primary culture

Wound-healing of the gastric mucosa is suggested to be stimulated by hepatocyte growth factor (HGF). Polyamines are shown to contribute to repair after damage in the gastric mucosa. The present study was designed to elucidate whether HGF can stimulate wound-healing of the gastric mucosa via polyamine production, using rabbit gastric mucosal cells in primary culture. A wound was made as a round cell-free area in the cell sheet of confluent cultured cells. When HGF was added to the culture medium, such denuded area was significantly reduced in size compared with the control, but the reduction was inhibited by addition of D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of a rate-limiting enzyme (ornithine decarboxylase) of polyamine biosynthesis, to the culture medium. However, the inhibitory effect by DFMO was reversed by pretreatment with spermidine, but not with putrescine. Intracellular levels of polyamines in the whole confluent cells including the cells around the denuded area were not changed by addition of HGF, but putrescine and spermidine levels were decreased by further addition of DFMO. We conclude that spermidine may be involved in stimulation by HGF in the repair after damage of gastric mucosal cells.     

Protective role of adiponectin against ethanol-induced gastric injury in mice

Adiponectin is an anti-inflammatory molecule released from adipocytes, and serum adiponectin concentrations are reduced in obesity. We previously reported that gastric erosion occurs in association with obesity and low serum adiponectin levels. In the present study, we examined adiponectin-knockout (APN-KO) mice to elucidate the role of adiponectin in gastric mucosal injury. Gastric injury was induced by oral administration of ethanol in wild-type (WT) and APN-KO mice. Ethanol treatment induced severe gastric injury in APN-KO mice compared with WT mice. In APN-KO mice, increased apoptotic cells and decreased expression of prostaglandin E(2) (PGE(2)) were detected in the injured stomach. We next assessed the effect of adiponectin on the cellular response to ethanol treatment and wound repair in rat gastric mucosal cells (RGM1). Adiponectin induced the expression of PGE(2) and cyclooxygenase 2 (COX-2) in ethanol-treated RGM1 cells. RGM1 cells exhibited efficient wound repair accompanied by increased PGE(2) expression in the presence of adiponectin. Coadministration of adiponectin with celecoxib, a COX-2 inhibitor, inhibited efficient wound repair. These findings indicate that adiponectin has a protective role against ethanol-induced gastric mucosal injury in mice. This effect may be partially mediated by the efficient wound repair of epithelial cells via increased PGE(2) expression.     

Dynamics of cell movement during the wound repair of human surface respiratory epithelium.

Epithelial wound repair represents an important process by which the epithelial barrier integrity recovers after wounding. To evaluate and quantify the dynamics of surface airway cell movement during the wound repair process, we developed an in vitro wounding model of human respiratory cells in culture and we analyzed the wound repair by using videomicroscopic and image analysis techniques. We observed that wound closure occurred within 6 hours, due to the spreading and migration of the cells surrounding the wounded surface. The migration rate of the cells at the leading edge of the wound surface increased progressively up to 26 microns/h during the repair process which was characterized by a uniform centripetal direction of cell movement. The distance travelled by these cells was 2.5 fold longer than the distance travelled by ciliated cells which were located far from the wound area. These results suggest that cell migration after wounding is an important process by which the respiratory epithelial barrier integrity is maintained.     

Differential growth factor induction and modulation of human gastric epithelial regeneration

While several autocrine/paracrine growth factors (GFs) can all stimulate epithelial regeneration in experimentally wounded primary gastric cultures, clinical relevance for their non-redundant cooperative actions in human gastric ulcer healing is suggested by the sequential pattern of GF gene induction in vivo. Using new HGE cell lines able to form a coherent monolayer with tight junctions as well as using primary human gastric epithelial cultures, we show that EGF, TGFalpha, HGF and IGFs accelerate epithelial restitution upon wounding, independently of the TGFbeta pathway (as opposed to intestinal cells). However, they differently modulate cell behavior: TGFalpha exerts strong effects (even more than EGF) on cytoplasmic spreading and non-oriented protruding activity of bordering cells whereas HGF preferentially coordinates single lamella formation, cell elongation and migration into the wound. IGF-I and IGF-II rather induce the alignment of bordering cells and maintain a compact monolayer front. The number of mitotic cells maximally increases with EGF, followed by TGFalpha and IGF-I,-II. The current study demonstrates that GFs differentially regulate the regeneration of human gastric epithelial cells through specific modulation of cell shape adaptation, migration and proliferation, further stressing that a coordination of GF activities would be necessary for the normal progression of post-wounding epithelial repair.     

Helicobacter pylori vacuolating cytotoxin (VacA) alters cytoskeleton-associated proteins and interferes with re-epithelialization of wounded gastric epithelial monolayers

In previous studies, we demonstrated that Helicobacter pylori vacuolating cytotoxin (VacA) inhibits gastric epithelial cell proliferation and inhibits epidermal growth factor (EGF)-activated signal transduction. Cell proliferation and migration, both essential for mucosal healing are dependent on the cell cytoskeleton. Other investigators demonstrated that VacA induces vacuolation of eukaryotic cells. Since in some cells, control of actin cytoskeleton involves GTP-binding proteins of Rho family, in this study we examined whether VacA affects wound re-epithelialization, cell cytoskeleton-associated proteins Rho, Rac1 in a gastric epithelial (RGM1) cell monolayer wound model, and whether these changes correlate with vacuolation. VacA treatment significantly inhibited wound re-epithelialization, cell proliferation vs control. VacA-induced cell vacuolation strongly correlated with inhibition of wound re-epithelialization. Furthermore, VacA reduced Rac-1 protein expression and distribution, and C3-mediated ADP-ribosylation of Rho. These findings suggest that VacA may interfere with repair of gastric mucosal injury and ulcer re-epithelialization by altering cytoskeleton-dependent cell functions and signaling.     

Enteric glia promote intestinal mucosal healing via activation of focal adhesion kinase and release of proEGF

Wound healing of the gastrointestinal mucosa is essential for the maintenance of gut homeostasis and integrity. Enteric glial cells play a major role in regulating intestinal barrier function, but their role in mucosal barrier repair remains unknown. The impact of conditional ablation of enteric glia on dextran sodium sulfate (DSS)-induced mucosal damage and on healing of diclofenac-induced mucosal ulcerations was evaluated in vivo in GFAP-HSVtk transgenic mice. A mechanically induced model of intestinal wound healing was developed to study glial-induced epithelial restitution. Glial-epithelial signaling mechanisms were analyzed by using pharmacological inhibitors, neutralizing antibodies, and genetically engineered intestinal epithelial cells. Enteric glial cells were shown to be abundant in the gut mucosa, where they associate closely with intestinal epithelial cells as a distinct cell population from myofibroblasts. Conditional ablation of enteric glia worsened mucosal damage after DSS treatment and significantly delayed mucosal wound healing following diclofenac-induced small intestinal enteropathy in transgenic mice. Enteric glial cells enhanced epithelial restitution and cell spreading in vitro. These enhanced repair processes were reproduced by use of glial-conditioned media, and soluble proEGF was identified as a secreted glial mediator leading to consecutive activation of epidermal growth factor receptor and focal adhesion kinase signaling pathways in intestinal epithelial cells. Our study shows that enteric glia represent a functionally important cellular component of the intestinal epithelial barrier microenvironment and that the disruption of this cellular network attenuates the mucosal healing process.     

Hepatocyte Growth Factor (HGF)-Induced Cell Migration Is Negatively Modulated by Epidermal Growth Factor through Tyrosine Phosphorylation of the HGF Receptor

Hepatocyte growth factor (HGF) stimulated cell migration of human gastric carcinoma cell lines MKN1, MKN7, and MKN28. Epidermal growth factor (EGF) also stimulated the cell migration of these three cell lines. In MKN7 cells, HGF-stimulated cell migration was rather reduced in the presence of EGF, whereas such an observation was not made with MKN1 and MKN28 cells. Therefore, we compared the effect of EGF on HGF-stimulated HGF receptor phosphorylation in these cell lines. HGF induced a rapid tyrosine phosphorylation of the HGF receptor in all these cell lines. In MKN7 cells, the increased phosphorylation was further enhanced by EGF, although EGF alone did not affect tyrosine phosphorylation of the HGF receptor. In MKN1 and MKN28 cells, EGF did not influence tyrosine phosphorylation of the HGF receptor, whether HGF was present or not. The data presented here suggest that EGF negatively modulates the cellular response to HGF by increasing tyrosine phosphorylation of the HGF receptor in certain types of epithelial cells, e.g., MKN7 cells.     

Motogenic effect of recombinant HGF on airway epithelial cells during the in vitro wound repair of the respiratory epithelium

Cell migration is the earliest mechanism involved in the wound repair process of the respiratory epithelium and could be potentially enhanced by growth factors. In the present work, we investigated the localisation of the hepatocyte growth factor (HGF) receptor (c-Met) during wound repair and evaluated the effect of recombinant HGF (rHGF) on cell migration by using an in vitro model of airway epithelial wound repair. By using immunohistochemical methods, we observed that the immunoreactivity of the c-Met proto-oncogene was increased in epithelial cells engaged in the process of tissue repair. The incubation of wounded cultures with increasing concentrations of rHGF (0.2, 2, 20, and 200 ng/ml) induced a significant (P < 0.02) dose-dependent effect on the wound repair index, with a maximum effect produced at 20 ng/ml (+31.3%). The cell migration speed reached 50.2 micrometer/h at this concentration, compared to 20.4 micrometer/h in the absence of rHGF. No significant effect on cell proliferation was observed in the repairing area in the presence of rHGF. These results suggest that rHGF is able to improve the wound repair process of the airway epithelium by increasing cell migration.     

Expression of neutrophil gelatinase-associated lipocalin regulates epithelial morphogenesis in vitro

Growth factors such as hepatocyte growth factor (HGF) are highly up-regulated during development and following renal injury and are known to induce marked morphogenic actions in cultured tubular epithelial cells, including scattering, migration, single cell branching morphogenesis, and multicellular branching tubulogenesis. In the present study, we demonstrate that HGF stimulates epithelial cells to express neutrophil gelatinase-associated lipocalin (Ngal), a member of the lipocalin family of secreted proteins that has recently been shown to participate in mesenchymal-epithelial transformation via its ability to augment cellular iron uptake. At concentrations below those found to mediate iron transport, purified Ngal can induce a promigratory and probranching effect that is dependent on ERK activation. The suppression of Ngal expression using short hairpin RNA results in increased cyst formation by tubular cells. However, the simultaneous addition of Ngal and HGF leads to direct association of the two proteins, and results in a partial inhibition of HGF-mediated activation of c-Met and the downstream MAPK and phosphatidylinositol 3-kinase signaling pathways. This inhibitory effect down-regulates HGF-stimulated single cell migration, and limits branching morphogenesis at both the single cell and multicellular level. These experiments demonstrate that the local expression of Ngal can play a regulatory role in epithelial morphogenesis by promoting the organization of cells into tubular structures while simultaneously negatively modulating the branching effects of HGF.     

Trefoil factor 3 peptide regulates migration via a Twist-dependent pathway in gastric cell

Trefoil factor 3 (TFF3) is a member of the TFF-domain peptide family and essential in regulating cell migration and maintaining mucosal integrity in gastrointestinal tract. However, the role of TFF3 and its downstream regulating mechanisms in cancer cell migration remain unclear. We previously reported that TFF3 prolonged the up-regulation of Twist protein to modulate IL-8 secretion in intestinal epithelial cells. In this study, we investigated the role of Twist protein in TFF3-induced migration of SGC7901 cells. While Twist was activated by TFF3, siRNA-mediated knockdown of Twist abolished TFF3-induced cell migration. Furthermore, the migration related marker CK-8 as well as ZO-1 and MMP-9 was also regulated by TFF3 via a Twist-dependent mechanism. Our study suggests that Twist, as an important potential downstream effector, plays a key role in TFF3-modulated metastasis in gastric cancer and can be a promising therapeutic target against intestinal-type gastric cancer.