Protective Effect Of Epidermal Growth Factor Research Articles

Epidermal Growth Factor Protects Against High Glucose-Induced Podocyte Injury Possibly via Modulation of Autophagy and PI3K/AKT/mTOR Signaling Pathway Through DNA Methylation.

AimDiabetic nephropathy (DN) is a serious health problem worldwide. Epidermal growth factor (EGF) has suggested as a potential biomarker for the progression of chronic kidney disease. In this study, we examined the effects of EGF on the high glucose (HG)-induced podocyte injury and explored the underlying molecular mechanisms.MethodsThe cell proliferation, toxicity, and cell apoptosis of podocytes were determined by CCK-8 assay, lactate dehydrogenase release assay, and flow cytometry, respectively, and protein levels in the podocytes were determined by Western blot assay. Mechanistically, DNA methylation analysis, bioinformatic analysis, methylation‑specific PCR and quantitative real-time PCR were used to analyze functional pathways in differentially methylated genes and the expression of the key methylated genes in the podocytes after different interventions.ResultsEGF treatment significantly increased the protein expression level of LC3 and decreased the protein level of P62 in HG-stimulated podocytes, which was attenuated by autophagy inhibitor, 3-methyladenine. EGF increased the cell proliferation and the protein expression levels of nephrin and synaptopodin, but reduced cell toxicity and cell apoptosis and protein expression level of cleaved caspase-3, which was partially antagonized by 3-methyladenine. DNA methylation expression profiles revealed the differential hypermethylation sites and hypomethylation sites among podocytes treated with normal glucose, HG and HG+EGF. GO enrichment analysis showed that DNA methylation was significantly enriched in negative regulation of phosphorylation, cell-cell junction and GTPase binding. KEGG pathway analysis showed that these genes were mainly enriched in PI3K-Akt, Hippo and autophagy pathways. Further validation studies revealed that six hub genes (ITGB1, GRB2, FN1, ITGB3, FZD10 and FGFR1) may be associated with the protective effects of EGF on the HG-induced podocyte injury.ConclusionIn summary, our results demonstrated that EGF exerted protective effects on HG-induced podocytes injury via enhancing cell proliferation and inhibiting cell apoptosis. Further mechanistic studies implied that EGF-mediated protective effects in HG-stimulated podocytes may be associated with modulation of autophagy and PI3K/AKT/mTOR signaling pathway.

Epidermal growth factor attenuates blood-spinal cord barrier disruption via PI3K/Akt/Rac1 pathway after acute spinal cord injury.

After spinal cord injury (SCI), disruption of blood–spinal cord barrier (BSCB) elicits blood cell infiltration such as neutrophils and macrophages, contributing to permanent neurological disability. Previous studies show that epidermal growth factor (EGF) produces potent neuroprotective effects in SCI models. However, little is known that whether EGF contributes to the integrity of BSCB. The present study is performed to explore the mechanism of BSCB permeability changes which are induced by EGF treatment after SCI in rats. In this study, we demonstrate that EGF administration inhibits the disruption of BSCB permeability and improves the locomotor activity in SCI model rats. Inhibition of the PI3K/Akt pathways by a specific inhibitor, LY294002, suppresses EGF‐induced Rac1 activation as well as tight junction (TJ) and adherens junction (AJ) expression. Furthermore, the protective effect of EGF on BSCB is related to the activation of Rac1 both in vivo and in vitro. Blockade of Rac1 activation with Rac1 siRNA downregulates EGF‐induced TJ and AJ proteins expression in endothelial cells. Taken together, our results indicate that EGF treatment preserves BSCB integrity and improves functional recovery after SCI via PI3K‐Akt‐Rac1 signalling pathway.

Role of mitochondrial Bax, caspases, and MAPKs for ceramide-induced apoptosis in renal proximal tubular cells

It remains elusive whether crosstalk exists among mitochondrial Bax, caspases, and mitogen-activated protein kinases (MAPKs), and whether epidermal growth factor (EGF), which may activate MAPKs, affects ceramide-induced apoptosis through the crosstalk in renal proximal tubular cells (RPTCs). Effect of ceramide on expression of mitochondrial Bax and phosphorylated (p)-ERK, p38MAPK and JNK, that of MAPKs inhibition, and of EGF in the presence or absence of MAPKs inhibition on ceramide-induced apoptosis were examined in HK-2 cells. Apoptosis and expression of mitochondrial Bax and p-MAPKs were measured by Hoechst 33258 staining and Western blotting. C2-ceramide, but not dihydroC2-ceramide, inactive C2-ceramide, induced apoptosis at 24 h. C2-ceramide enhanced the mitochondrial Bax expression at 1 h, which was peaked at 3-6 h and decreased at 24 h, but remained increased, compared to control. An inhibitor of caspases, zVAD-fmk, ameliorated ceramide-induced apoptosis, suggesting a role of caspases for ceramide-induced apoptosis. C2-ceramide enhanced the expression of p-ERK and p-p38MAPK, but not p-JNK, at 1 h, which was increased till 24 h. An inhibitor of ERK, PD98059, or of p38MAPK, SB202190, failed to affect C2-ceramide-induced apoptosis. EGF, which enhanced the expression of p-ERK and p-p38MAPK but not p-JNK, ameliorated C2-ceramide-induced apoptosis without affecting mitochondrial Bax. Inhibition of ERK or p38MAPK failed to abolish the protective effect of EGF on C2-ceramide-induced apoptosis. Mitochondrial Bax and caspases, but not MAPKs, play a role for ceramide-induced apoptosis in RPTCs. EGF ameliorates ceramide-induced apoptosis in Bax- and MAPKs-independent pathways. The mechanism of ceramide-induced apoptosis and anti-apoptotic effect of EGF deserves further investigations.

Epidermal growth factor protects the apical junctional complexes from hydrogen peroxide in bile duct epithelium

The tight junctions of bile duct epithelium form a barrier between the toxic bile and liver parenchyma. Disruption of tight junctions appears to have a crucial role in the pathogenesis of various liver diseases. In this study, we investigated the disruptive effect of hydrogen peroxide and the protective effect of epidermal growth factor (EGF) on the tight junctions and adherens junctions in the bile duct epithelium. Oxidative stress in NRC-1 and Mz-ChA-1 cell monolayers was induced by administration of hydrogen peroxide. Barrier function was evaluated by measuring electrical resistance and inulin permeability. Integrity of tight junctions, adherens junctions and the actin cytoskeleton was determined by imunofluorescence microscopy. Role of signaling molecules was determined by evaluating the effect of specific inhibitors. Hydrogen peroxide caused a rapid disruption of tight junctions and adherens junctions leading to barrier dysfunction without altering the cell viability. Hydrogen peroxide rapidly increased the levels of p-MLC (myosin light chain) and c-Src(pY418). ML-7 and PP2 (MLCK and Src kinase inhibitors) attenuated hydrogen peroxide-induced barrier dysfunction, tight junction disruption and reorganization of actin cytoskeleton. Pretreatment of cell monolayers with EGF ameliorated hydrogen peroxide-induced tight junction disruption and barrier dysfunction. The protective effect of EGF was abrogated by ET-18-OCH3 and the Ro-32-0432 (PLCγ and PKC inhibitors). Hydrogen peroxide increased tyrosine phosphorylation of ZO-1, claudin-3, E-cadherin and β-catenin, and pretreatment of cells with EGF attenuated tyrosine phosphorylation of these proteins. These results demonstrate that hydrogen peroxide disrupts tight junctions, adherens junctions and the actin cytoskeleton by an MLCK and Src kinase-dependent mechanism in the bile duct epithelium. EGF prevents hydrogen peroxide-induced tight junction disruption by a PLCγ and PKC-dependent mechanism.

Ileal cytokine dysregulation in experimental necrotizing enterocolitis is reduced by epidermal growth factor.

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants. We have shown in previous studies that proinflammatory interleukin-18 and interleukin-12 are up-regulated in the ileum of rats with experimental NEC and that epidermal growth factor (EGF) reduces the development of disease. Here we investigated whether the protective effects of EGF are a result of changes in ileal interleukin-18, interleukin-12 and/or antiinflammatory interleukin-10. Newborn rats were artificially fed with either growth-factor-free rat milk substitute (RMS) or RMS supplemented with 500 ng/mL EGF (RMS + EGF) and NEC was induced via exposure to asphyxia and cold stress. Cytokine expression and localization were assessed using reverse-transcription real-time polymerase chain reaction and immunohistology/confocal microscopy. Enteral administration of EGF (RMS + EGF) decreased overproduction of interleukin-18 and increased interleukin-10 production in the ileum. Furthermore, increased interleukin-10 production was associated with up-regulation of the transcription factor Sp1 in RMS + EGF rats. These data suggest that EGF may reduce NEC via increased interleukin-10 and decreased interleukin-18 and that EGF-mediated up-regulation of Sp1 may account for the increased interleukin-10.

Ileal Cytokine Dysregulation in Experimental Necrotizing Enterocolitis Is Reduced by Epidermal Growth Factor

ABSTRACTBackgroundNecrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants. We have shown in previous studies that proinflammatory interleukin‐18 and interleukin‐12 are up‐regulated in the ileum of rats with experimental NEC and that epidermal growth factor (EGF) reduces the development of disease. Here we investigated whether the protective effects of EGF are a result of changes in ileal interleukin‐18, interleukin‐12 and/or antiinflammatory interleukin‐10.MethodsNewborn rats were artificially fed with either growth‐factor–free rat milk substitute (RMS) or RMS supplemented with 500 ng/mL EGF (RMS + EGF) and NEC was induced via exposure to asphyxia and cold stress. Cytokine expression and localization were assessed using reverse‐transcription real‐time polymerase chain reaction and immunohistology/confocal microscopy.ResultsEnteral administration of EGF (RMS + EGF) decreased overproduction of interleukin‐18 and increased interleukin‐10 production in the ileum. Furthermore, increased interleukin‐10 production was associated with up‐regulation of the transcription factor Sp1 in RMS + EGF rats.ConclusionsThese data suggest that EGF may reduce NEC via increased interleukin‐10 and decreased interleukin‐18 and that EGF‐mediated up‐regulation of Sp1 may account for the increased interleukin‐10.

The Effect of Portal Hypertension on the Glycoprotein Biosynthesis of Rat Gastric Mucosa

The aim of this study was to assess the influence of epidermal growth factor (EGF) on glycoprotein biosynthesis in portal hypertensive (PHT) gastric mucosa. Portal-vein ligation (PVL) for a period of 4 weeks was applied to 40 male Wistar rats to produce experimental portal hypertension. The rats were subdivided into four groups. Human EGF was administrated to these four groups of animals at a does of 0, 10, 25, and 50 w g/kg/day for 7 days. An additional group of 10 rats without PVL and EGF pretreatment was employed as a control. The severity of gross gastric mucosal lesions was evaluated macroscopically by a gross ulcer index. Glycoprotein biosynthesis of the gastric mucosa was determined by the incorporation rate of [3 H]glucosamine. Quantitative changes of gastric mucosal hexosamines were also used for mucosal glycoproteins analyses. The gross mucosal damage was considerably greater in the PVL group without EGF pretreatment than in the EGF-pretreated groups (p < .05). The incorporation rate of [3 H]glucosamine was significantly higher in the control group and the EGF-pretreated groups than in the PVL group without EGF pretreatment (p < .05). Moreover, the incorporation rate of [3 H]glucosamine and the gastric mucosal hexosamine content were closely relevant to administration does of human EGF (p < .001). In addition, the reduction of glycoprotein biosynthesis was closely related to the increase in portal pressure (p = .001) and the severity of portal hypertensive gastropathy (p < .001). Our current study shows that the rate of incorporation of glucosamine is decreased in the PHT gastric mucosa and that EGF significantly stimulated glycoprotein synthesis in the PHT gastric mucosa. Accordingly, these findings may be helpful to explain the protective effect of EGF on the PHT gastric mucosa via increased glycoprotein biosynthesis in the stomach.

Gastroprotective and vasodilatory effects of epidermal growth factor: the role of sensory afferent neurons.

Epidermal growth factor (EGF) has been shown to exert gastric hyperemic and gastroprotective effects via capsaicin-sensitive afferent neurons, including the release of calcitonin gene-related peptide (CGRP). We examined the protective and vasodilatory effects of EGF on the gastric mucosa and its interaction with sensory nerves, CGRP, and nitric oxide (NO) in anesthetized rats. Intragastric EGF (10 or 30 microg) significantly reduced gastric mucosal lesions induced by intragastric 60% ethanol (50.6% by 10 microg EGF and 70.0% by 30 microg EGF). The protective effect of EGF was significantly inhibited by pretreatment with capsaicin desensitization, human CGRP1 antagonist hCGRP-(8-37), or N(omega)-nitro-L-arginine methyl ester (L-NAME). Intravital microscopy showed that topically applied EGF (10-1,000 microg/ml) dilated the gastric mucosal arterioles dose dependently and that this vasodilatory effect was significantly inhibited by equivalent pretreatments. These findings suggest that EGF plays a protective role against ethanol-induced gastric mucosal injury, possibly by dilating the gastric mucosal arterioles via capsaicin-sensitive afferent neurons involving CGRP and NO mechanisms.

Cytotoxic effects of tumour necrosis factor (TNF)-alpha and interferon-gamma on cultured human trophoblast are modulated by fibronectin.

Tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma, produced by maternal inflammatory cells, may compromise trophoblast survival at the trophoblast-maternal interface and notably in the placental bed which is invaded by trophoblast. Extracellular matrix components, e.g. fibronectin, may enhance trophoblast survival. A possible protective effect of fibronectin against toxic effects of TNF-alpha and IFN-gamma was investigated in cultured trophoblasts isolated from six human term placentas, grown on uncoated and fibronectin-coated plastics. IFN-gamma and increasing doses of TNF-alpha resulted in decreasing viability of trophoblast on uncoated as well as fibronectin-coated dishes, as shown by 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assays, but for each TNF/IFN treatment condition viability on fibronectin was higher (P < 0.001). Epidermal growth factor (EGF), a growth factor reported to protect against TNF-alpha/IFN-gamma induced toxicity, resulted in further increased viability, but not if IFN-gamma was included in the treatment. EGF caused increased fibronectin secretion into the medium (P < 0.001), and double cytokeratin/fibronectin immunostaining confirmed the trophoblastic nature of fibronectin secreting cells. We conclude that fibronectin increases viability, but does not completely abolish the cytotoxic action of TNF-alpha and IFN-gamma on trophoblast. The protective effect of EGF may be related to stimulation of fibronectin secretion by trophoblast.

Inhibitory effect of epidermal growth factor and hepatocyte growth factor on endothelin-1 release by rabbit proximal tubule cells.

Several studies have demonstrated an upregulation of endothelin-1 (ET-1) synthesis in acute and chronic renal failure. Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) have been shown to stimulate renal tubular cell proliferation and to accelerate renal regeneration after drug-induced and ischemia-induced renal injury. This study aimed to investigate the effect of EGF and HGF on ET-1 release, and whether the effect of EGF and HGF is antagonized by the tyrosine kinase inhibitor lavendustin A. Rabbit proximal tubule cells were incubated for 48 h with EGF or HGF (0.1-10.0 nM), lavendustin A (0.1-10.0 microM) or co-incubated with EGF or HGF (1 nM) and lavendustin A. ET-1 concentrations in the culture medium were measured with a specific enzyme-linked immunosorbent assay (ELISA). EGF and HGF exerted a significant (p < 0.001) dose-dependent inhibitory effect on ET-1 release. Lavendustin A induced a dose-dependent stimulation of ET-1 release and antagonized the inhibitory effect of EGF and HGF on ET-1 release. The inhibition of EGF and HGF receptor tyrosine kinase activity by lavendustin A was confirmed by Western blotting. These data suggest that EGF and HGF reduce ET-1 release via EGF and HGF receptor tyrosine kinase activity. The inhibitory action of EGF and HGF on ET-1 release might be involved in mediating the protective effects of EGF and HGF in renal injury.

NGF protects PC12 cells against ischemia by a mechanism that requires the N-kinase.

Nerve growth factor (NGF), which has been shown to act as a morphological and neurochemical differentiating factor in PC12 cells, also protects PC12 cells from the toxicity of serum withdrawal and ischemia. By using a previously established in vitro model of ischemia, which incorporates the combination of anoxia with glucose deprivation (Boniece and Wagner: J Neurosci 13:4220-4228, 1993), we have been able to study the signal transduction pathways upon which NGF-induced survival is dependent. Here we demonstrate that inhibitors of the N-kinase and NGF-induced neuritogenesis, 6-thioguanine and 2-aminopurine, prevent the protective effects of NGF, while they have little, if any, effect on the protection conferred by epidermal growth factor (EGF) or dbcAMP. This suggests that only NGF acts by a mechanism that depends strongly on the N-kinase. Furthermore, the methyltransferase inhibitor 5'-deoxy-5'-methylthioadenosine (MTA), which also inhibits NGF-induced neuritogenesis, inhibits the protective effect of NGF but not the protective effects of EGF or dbcAMP. Thus, the neuroprotective effect of NGF requires some of the same signal transduction steps used by NGF to promote differentiation and neurite formation. Furthermore, we found that exposure of PC12 cells to retinoic acid, which promotes the differentiation and inhibits the growth of PC12 cells, also improves cell survival during ischemia. In addition, a combination of NGF and retinoic acid was more effective than either agent alone. It is likely that these two agents confer protection by independent pathways.

Protection from 1-methyl-4-phenylpyridinium (MPP +) toxicity and stimulation of regrowth of MPP +-damaged dopaminergic fibers by treatment of mesencephalic cultures with EGF and basic FGF

Several peptide growth factors can maintain survival or promote recovery of injured central neurons. In the present study, the effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the toxicity produced by the dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium (MPP +), were investigated in rat mesencephalic dopaminergic neurons in culture. High affinity [ 3H]DA uptake and morphometric analyses of tyrosine hydroxylase immunostained neurons were used to assess the extent of MPP + toxicity, dopaminergic neuronal survival and growth of neurites. Consistent with previous reports, EGF and bFGF treatments stimulated neuritic outgrowth in dopaminergic neurons, increased DA uptake and enhanced their long-term survival in vitro. These growth factors also stimulated proliferation of astrocytes. The time course of EGF and bFGF effects on dopaminergic neurons coincided with the increase in glial cell density, suggesting that proliferation of glia mediates their trophic effects. Several findings from our study support this possibility. When MPP + was applied to cultures at 4 days in vitro, before glial cells had proliferated, the damage to dopaminergic neurons was not affected by EGF or bFGF pretreatments. However, when cultures maintained in the presence of the growth factors for 10 days were exposed to MPP +, after they had become confluent with dividing glial cells, the MPP +-induced decreases in DA uptake and cell survival were significantly attenuated. Furthermore, when glial cell proliferation was inhibited by 5-fluoro-2′-deoxyuridine, the protective effects of EGF and bFGF against MPP + toxicity were abolished. Continuous treatment of MPP +-exposed cultures with EGF or bFGF resulted in the stimulation of process regrowth of damaged dopaminergic neurons with concomitant recovery of DA uptake, suggesting that the injured neurons are able to respond to the trophic effects of EGF and bFGF. In summary, our study shows that the trophic effects of EGF and bFGF on mesencephalic dopaminergic neurons include protection from the toxicity produced by MPP + and promotion of recovery of MPP +-damaged neurons. Stimulation of glial cell proliferation is necessary for these effects.

Protective effect of epidermal growth factor on glutamate neurotoxicity in cultured cerebellar neurons

The effects of epidermal growth factor (EGF) on glutamate-induced neuronal death were investigated in primary cultures of dissociated cerebellar neurons from fetal rats. Addition of an excess concentration of l-glutamate (5 mM) to the culture medium greatly decreased the number of surviving neurons 24 h later. When EGF was added to the culture medium 20 h prior to exposure to glutamate, glutamate-induced neuronal death was significantly reduced. The protective effects of EGF on glutamate neurotoxicity were concentration-dependent in the range of 0.01–10 ng/ml. When EGF was added 1 h prior to exposure to glutamate, it did not prevent glutamate-induced neuronal death, indicating that a longer exposure period is required for EGF to exert its protective effects. Furthermore, the protective effects of EGF on glutamate neurotoxicity disappeared in the presence of cycloheximide (0.1 μM), a protein synthesis inhibitor. These results suggest that EGF can protect brain neurons against glutamate toxicity through some protein synthesis.

Role of Polyamines and Prostaglandins in Gastroprotective Action of Epidermal Growth Factor Against Ethanol Injury

Epidermal growth factor (EGF) exhibits its gastroprotective action against a variety of irritants and ulcerogens and plays an important role in healing of acute and chronic gastroduodenal ulcerations, but the mechanisms of these effects are not known. The present study was undertaken to determine whether polyamines (such as spermine or putrescine) and prostaglandins (PGs), which also show protective properties, contribute to the gastroprotective effect of EGF against ethanol injury in rats. It was found that both EGF and polyamines significantly and dose-dependently prevented the formation of gastric lesions induced by absolute ethanol, the effect being similar to that obtained with 16,16-dimethylprostaglandin E2. Pretreatment with indomethacin failed to affect the gastroprotective action of EGF and polyamines, suggesting that endogenous PGs may not play any major role in this protection. Our finding that the protective effect of EGF can be abolished by pretreatment with DFMO, an inhibitor of the polyamine biosynthetic pathway, suggests that gastroprotection by EGF is due, at least in part, to stimulation of biosynthesis of protective polyamines.

Role of epidermal growth factor in gastroduodenal mucosal protection.

Epidermal growth factor (EGF) is a polypeptide produced in the submandibular glands, Brunner's glands, and the kidneys. The peptide is secreted in an exocrine fashion into saliva, duodenal juice, and urine. EGF stimulates cellular growth and differentiation and inhibits gastric acid secretion. Removal of the submandibular glands decreases the amount of EGF in saliva and gastric juice and subsequently the synthesis of DNA in the gastric mucosa is reduced as well as its resistance to bile-salt-induced gastric lesions. Intragastric instillation of EGF can prevent gastric ulcerations induced by aspirin as well as cysteamine in rats. EGF also accelerates the healing of chronic gastric ulcers induced by acetic acid. Cysteamine is a duodenal ulcerogen in rats. After cysteamine administration, the secretion of EGF from Brunner's glands decreases and the glands become depleted of mucus. Intraduodenal instillation of EGF can partly prevent formation of cysteamine-induced duodenal ulcers. Oral administration of EGF can accelerate healing of chronic duodenal ulcers in rats. The beneficial effect of EGF on healing of chronic gastroduodenal ulcers is probably due to the delayed effects of EGF such as stimulation of RNA and DNA synthesis. The protective effects of EGF are probably related to the early actions of the peptide such as activation of cell surface proteins and increased glycosaminoglycan synthesis.

Antiproliferative effects of heparin on vascular smooth muscle cells are reversed by epidermal growth factor.

Heparin and related glycosaminoglycans are potent inhibitors of both in vivo and in vitro smooth muscle cell (SMC) proliferation. We have found that epidermal growth factor (EGF) reverses the antiproliferative effects of heparin. Other known SMC mitogens, including platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), and thrombin, were unable to prevent heparin action. The EGF specificity was further demonstrated by developing a biological growth assay in which EGF or PDGF, at concentrations as low as 1 ng/ml, stimulated SMC growth in the absence of other serum components. Under these conditions, EGF, but not PDGF, suppressed heparin inhibition as well. The ability of EGF to reverse heparin inhibition was only observed when mitogen and glycosaminoglycan were added to SMC at similar times. If SMC were pretreated with heparin for 48 hours prior to EGF addition, the protective effects of EGF were lost. Heparin did not directly prevent 125I-EGF or platelet-derived EGF-like peptides from binding to the EGF receptor on SMC. However, cultures that were pretreated with heparin for 48 hours bound 49% less 125I-EGF than cultures that had been pretreated with the mucopolysaccharide for only 2 hours or that had not been preexposed to heparin. In previous studies, we have established that heparin exerts its maximal inhibitory activity after a 48-hour treatment of SMC (Reilly et al. 1986). Taken together, these data suggest that heparin may exert its antiproliferative potential by slowly and specifically altering SMC response to EGF-like mitogens of platelet origin.