Distinct activation of epidermal growth factor receptor by UTP contributes to epithelial cell wound repair

The release of nucleotides after injury activates purinergic receptors, leading to phosphorylation of site-specific residues on epidermal growth factor receptor (EGFR). To elucidate the differences between the injury-induced response and that induced by exogenous EGF, we examined recruitment of docking proteins, internalization of EGFR, and migration after injury. Injury induced by scratch wounds or stimulation by addition of UTP caused a brief internalization of EGFR, which paralleled the lesser association with growth factor receptor-bound protein 2 (Grb2) and phosphorylation of EGFR. The internalization caused by EGF was sustained and detected for longer than 60 minutes and correlated with phosphorylation of the receptor. The EGF caused recruitment of Grb2, phospholipase C-γ-1 (PLCγ1), Shc, and Src to EGFR. Glutathione S-transferase pull downs were performed, and glutathione S-transferase-PLCγ1 showed binding of Grb2 when stimulated with EGF but not with UTP or injury. Furthermore, UTP did not induce PLCγ1 phosphorylation, and the phosphorylation induced by EGF was attenuated by costimulation with UTP. The response to heparin-binding EGF was equivalent to that of EGF. Site-directed mutagenesis showed that phosphorylation of Y1068 and Y1086 of EGFR is required for repair. Together, our results show that injury and activation of purinergic receptors and direct activation of EGFR via EGF induce distinct downstream pathways.     

Enhanced wound healing by recombinant Escherichia coli Nissle 1917 via human epidermal growth factor receptor in human intestinal epithelial cells: therapeutic implication using recombinant probiotics

The gastrointestinal mucosa has a remarkable ability to repair damage with the support of epidermal growth factor (EGF), which stimulates epithelial migration and proliferative reepithelialization. For the treatment of mucosal injuries, it is important to develop efficient methods for the localized delivery of mucoactive biotherapeutics. The basic idea in the present study came from the assumption that an intestinal probiotic vehicle can carry and deliver key recombinant medicinal proteins to the injured epithelial target in patients with intestinal ulcerative diseases, including inflammatory bowel disease. The study was focused on the use of the safe probiotic E. coli Nissle 1917, which was constructed to secrete human EGF in conjunction with the lipase ABC transporter recognition domain (LARD). Using the in vitro physically wounded monolayer model, ABC transporter-mediated EGF secretion by probiotic E. coli Nissle 1917 was demonstrated to enhance the wound-healing migration of human enterocytes. Moreover, the epithelial wound closure was dependent on EGF receptor-linked activation, which exclusively involved the subsequent signaling pathway of the mitogen-activated protein kinase kinase (MEK) extracellular-related kinases 1 and 2 (ERK1/2). In particular, the migrating frontier of the wounded edge displayed the strongest EGF receptor-linked signaling activation in the presence of the recombinant probiotic. The present study provides a basis for the clinical application of human recombinant biotherapeutics via an efficient, safe probiotic vehicle.     

Phosphatidylinositol-4,5-bisphosphate regulates epidermal growth factor receptor activation

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2) or PIP(2)] is a direct modulator of a diverse array of proteins in eukaryotic cells. The functional integrity of transmembrane proteins, such as ion channels and transporters, is critically dependent on specific interactions with PIP(2) and other phosphoinositides. Here, we report a novel requirement for PIP(2) in the activation of the epidermal growth factor receptor (EGFR). Down-regulation of PIP(2) levels either via pharmacological inhibition of PI kinase activity, or via manipulation of the levels of the lipid kinase PIP5K1α and the lipid phosphatase synaptojanin, reduced EGFR tyrosine phosphorylation, whereas up-regulation of PIP(2) levels via overexpression of PIP5K1α had the opposite effect. A cluster of positively charged residues in the juxtamembrane domain (basic JD) of EGFR is likely to mediate binding of EGFR to PIP(2) and PIP(2)-dependent regulation of EGFR activation. A peptide mimicking the EGFR juxtamembrane domain that was assayed by surface plasmon resonance displayed strong binding to PIP(2). Neutralization of positively charged amino acids abolished EGFR/PIP(2) interaction in the context of this peptide and down-regulated epidermal growth factor (EGF)-induced EGFR auto-phosphorylation and EGF-induced EGFR signaling to ion channels in the context of the full-length receptor. These results suggest that EGFR activation and downstream signaling depend on interactions of EGFR with PIP(2) and point to the basic JD's critical involvement in these interactions. The addition of this very different class of membrane proteins to ion channels and transporters suggests that PIP(2) may serve as a general modulator of the activity of many diverse eukaryotic transmembrane proteins through their basic JDs.     

Decoding epithelial signals: critical role for the epidermal growth factor receptor in controlling intestinal transport function

The intestinal epithelium engages in bidirectional transport of fluid and electrolytes to subserve the physiological processes of nutrient digestion and absorption, as well as the elimination of wastes, without excessive losses of bodily fluids that would lead to dehydration. The overall processes of intestinal ion transport, which in turn drive the secretion or absorption of water, are accordingly carefully regulated. We and others have identified the epidermal growth factor receptor (EGFr) as a critical regulator of mammalian intestinal ion transport. In this article, we focus on our studies that have uncovered the intricate signalling mechanisms downstream of EGFr that regulate both chloride secretion and sodium absorption by colonocytes. Emphasis will be placed on the EGFr-associated regulatory pathways that dictate the precise outcome to receptor activation in response to signals that may seem, on their face, to be quite similar if not identical. The concepts to be discussed underlie the ability of the intestinal epithelium to utilize a limited set of signalling effectors to produce a variety of outcomes suitable for varying physiological and pathophysiological demands. Our findings therefore are relevant not only to basic biological principles, but also may ultimately point to new therapeutic targets in intestinal diseases where ion transport is abnormal.     

Connective tissue growth factor induces renal fibrosis via epidermal growth factor receptor activation

Connective tissue growth factor (CCN2/CTGF) is a matricellular protein that is overexpressed in progressive human renal diseases, mainly in fibrotic areas. In vitro studies have demonstrated that CCN2 regulates the production of extracellular matrix (ECM) proteins and epithelial–mesenchymal transition (EMT), and could therefore contribute to renal fibrosis. CCN2 blockade ameliorates experimental renal damage, including diminution of ECM accumulation. We have reported that CCN2 and its C‐terminal degradation product CCN2(IV) bind to epidermal growth factor receptor (EGFR) to modulate renal inflammation. However, the receptor involved in CCN2 profibrotic actions has not been described so far. Using a murine model of systemic administration of CCN2(IV), we have unveiled a fibrotic response in the kidney that was diminished by EGFR blockade. Additionally, in conditional CCN2 knockout mice, renal fibrosis elicited by folic acid‐induced renal damage was prevented, and this was linked to inhibition of EGFR pathway activation. Our in vitro studies demonstrated a direct effect of CCN2 via the EGFR pathway on ECM production by fibroblasts and the induction of EMT in tubular epithelial cells. Our studies clearly show that the EGFR regulates CCN2 fibrotic signalling in the kidney, and suggest that EGFR pathway blockade could be a potential therapeutic option to block CCN2‐mediated profibrotic effects in renal diseases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Protease-activated receptor-2 activation in gastric cancer cells promotes epidermal growth factor receptor trans-activation and proliferation

Dysregulated epidermal growth factor receptor (EGFR) signaling is involved in gastric cancer (GC) cell growth. However, the mechanism that sustains EGFR signaling in GC remains unknown. Since protease-activated receptor-2 (PAR-2), a G protein-coupled receptor, has been shown to trans-activate EGFR in several cell types, we examined the role of PAR-2 in GC. We show here that in vitro activation of PAR-2 enhances the growth of two GC cell lines, AGS and MKN28. In both these cell lines, PAR-2 trans-activated EGFR and inhibition of EGFR tyrosine kinase activity by AG1478 or specific EGFR siRNA completely prevented PAR-2-driven proliferation. Antibody blockade of EGF-like ligands to EGFR did not modify EGFR signaling or cell growth induced by PAR-2 activation. In contrast, PAR-2 promoted Src activation and interaction of this kinase with EGFR. In support of this, inhibition of Src kinase activity by PP1 or siRNA blocked PAR-2-induced EGFR signaling cascade and cell growth. Finally, PAR-2 was detectable in both normal and GC specimens, but its expression was more pronounced in GC than controls and correlated with activated EGFR. These data show that PAR-2 is overexpressed in GC and suggest a role of PAR-2 in EGFR trans-activation and cell growth.     

Heparin-binding epidermal growth factor cleavage mediates zinc-induced epidermal growth factor receptor phosphorylation

We have previously shown that exposure to zinc ions can activate epidermal growth factor (EGF) receptor (EGFR) signaling in murine fibroblasts and A431 cells through a mechanism involving Src kinase. While studying the effects of zinc ions in normal human bronchial epithelial cell, we uncovered evidence for an additional mechanism of Zn(2+)-induced EGFR activation. Exposure to Zn(2+) induced phosphorylation of EGFR at tyrosine 1068, a major autophosphorylation site, in a dose- and time-dependent fashion. This effect of Zn(2+) on EGFR was significantly blocked with an antibody against the ligand-binding domain of the receptor. Neutralizing antibodies against EGFR ligands revealed the involvement of heparin-binding EGF (HB-EGF) in Zn(2+)-induced EGFR phosphorylation. This observation was further supported by immunoblots showing elevated levels of HB-EGF released by Zn(2+)-exposed cells. Zymography showed the existence of matrix metalloproteinase-3 in Zn(2+)-challenged cells. Incubation with a specific matrix metalloproteinase-3 inhibitor suppressed Zn(2+)-induced EGFR phosphorylation as well as HB-EGF release. Therefore, these data support an autocrine or paracrine mechanism whereby Zn(2+) induces EGFR phosphorylation through the extracellular release of EGFR ligands, which may be mediated by metalloproteinases.     

Enteropathogenic Escherichia coli type III effectors EspG and EspG2 disrupt the microtubule network of intestinal epithelial cells

Enteropathogenic Escherichia coli infection of intestinal epithelial cells leads to localized depletion of the microtubule cytoskeleton, an effect that is dependent on delivery of type III translocated effector proteins EspG and Orf3 (designated EspG2) to the site of depletion. Microtubule depletion involved disruption rather than displacement of microtubules.     

Macrophage migration inhibitory factor and interleukin-8 produced by gastric epithelial cells during Helicobacter pylori exposure induce expression and activation of the epidermal growth factor receptor

While a link between Helicobacter pylori exposure and gastric cancer has been established, the underlying mechanisms remain unclear. H. pylori induces a chronic inflammatory response in infected individuals. A link between chronic inflammation and carcinogenesis has long been suggested but never elucidated. Epidermal growth factor receptor (EGFR) signaling plays an important role in both proinflammatory and procarcinogenic mechanisms and is upregulated on gastric epithelial cells (GECs) during H. pylori exposure. The aim of this study was to examine the effects of two important proinflammatory cytokines released during H. pylori infection, macrophage migration inhibitory factor (MIF) and interleukin-8 (IL-8), on the expression and transactivation of EGFR and on the proliferation of GECs during H. pylori exposure. The expression of EGFR by GECs was increased by exposure to either H. pylori, recombinant MIF, or recombinant IL-8. However, cag pathogenicity island knockout strains of H. pylori had very little effect on expression. MIF and IL-8 also induced phosphorylation of EGFR, signaling events, and proliferation during H. pylori exposure, all of which were decreased when they were neutralized by these cytokines or were blocked from their receptors. The overall role of EGFR in these responses to H. pylori exposure was assessed by knocking down EGFR expression by small interfering RNA.     

Autocrine ligands for the epidermal growth factor receptor mediate interleukin-8 release from bronchial epithelial cells in response to cigarette smoke

Surfactant replacement preparations containing either surfactant protein (SP)-B or SP-C significantly improve lung function in surfactant-deficient infants, suggesting that these peptides may be functionally redundant. SP-B is absent and SP-C is greatly diminished in the airspaces of SP-B (-/-) mice, which die of respiratory distress syndrome (RDS) shortly after birth. The goal of this study was to determine if elevated expression of SP-C mature peptide could reverse the neonatal lethality in SP-B (-/-) mice. SP-C peptide (residues 24-57 of mouse SP-C proprotein) with a hemagglutinin epitope (SP-C(24-57)HA) was expressed in type II cells of transgenic mice, with the goal of crossing these animals into the SP-B (-/-) background. Unexpectedly, expression of the SP-C(24-57)HA transgene resulted in delayed/arrested lung development and lethal, neonatal RDS of all transgenic progeny in two independent transgenic lines. In transgenic mice, SP-C(24-57)HA was localized predominantly to the endoplasmic reticulum and Golgi; in contrast, SP-B and SP-C were very difficult to detect in the endoplasmic reticulum of wild-type mice. These results suggest that elevated expression of SP-C(24-57)HA in type II cells resulted in aggregation of SP-C in the early secretory pathway, leading to cytotoxicity and, ultimately, altered lung development.     

Epidermal growth factor receptor activation by epidermal growth factor mediates oxidant-induced goblet cell metaplasia in human airway epithelium

Mucus overproduction in inflammatory and obstructive airway diseases is associated with goblet cell (GC) metaplasia in airways. Although the mechanisms involved in GC metaplasia and mucus hypersecretion are not completely understood, association with oxidative stress and epidermal growth factor receptor (EGFR) signaling has been reported. To explore the mechanisms involved in oxidative stress-induced GC metaplasia, cultures of differentiated normal human bronchial epithelial cells grown at the air-liquid interface were exposed to reactive oxygen species (ROS) generated by xanthine/xanthine oxidase. EGFR activation and signaling was assessed by measuring EGF and transforming growth factor-alpha release and EGFR and (44/42)MAPK phosphorylation. The GC population was evaluated by confocal microscopy. ROS-induced EGFR activation resulted in GC proliferation and increased MUC5AC gene and protein expression. Signaling was due to pro-EGF processing by tissue kallikrein (TK), which was activated by ROS-induced hyaluronan breakdown. It was inhibited by catalase, a TK inhibitor, and EGF-blocking antibodies. Exposure to recombinant TK mimicked the ROS effects, increasing the expression of MUC5AC and lactoperoxidase. In addition, ROS induced the antiapoptotic factor Bcl-2 in a TK-dependent fashion. In conclusion, ROS-induced GC metaplasia in normal human bronchial epithelial cells is associated with HA depolymerization and EGF processing by TK followed by EGFR signaling, suggesting that increases in TK activity could contribute to GC metaplasia and mucus hypersecretion in diseases such as asthma and chronic bronchitis. The data also suggest that increases in GC population could be sustained by the associated upregulation of Bcl-2 in airway epithelial cells.     

Heparin alters epidermal growth factor metabolism in cultured rat glomerular epithelial cells.

Extracellular matrix plays an important role in regulating cell growth. The authors have previously shown that heparin and heparan sulfate inhibit glomerular visceral epithelial cell (GEC) growth and that epidermal growth factor (EGF) can partially reverse the effect of heparin. The authors studied EGF processing by GEC in an attempt to clarify the mechanism by which heparin inhibits GEC growth. Control and heparin-treated GEC rapidly internalized 125I-EGF (within 15 minutes). In heparin-treated cells, 125I-EGF reappeared on the cell surface during the course of a 1-hour incubation and the percent internalized dropped significantly to 59.0% +/- 8.6%, suggesting recycling of 125I-EGF-occupied receptors. After incubation with 125I-EGF, heparin-treated cells also released significantly more cpm of 125I into EGF-free medium (2526 +/- 68 cpm-H; 903 +/- 32-C). Analysis of the released 125I by gel filtration chromatography showed more totally degraded 125I-EGF in media from heparin-treated cells (30.8% +/- 1.6% in heparin-treated versus 17.8% +/- 3.2 in control; P less than 0.05). Analysis of EGF-induced dimerization of receptors showed no effect of heparin on this ratio. These studies suggest that heparin decreases GEC response to EGF by accelerating its uptake and degradation. Matrix alterations in disease states may thus play a role in altering cell responsiveness to growth factors.     

Bacterial virulence factor inhibits caspase-4/11 activation in intestinal epithelial cells

The human pathogen enteropathogenic Escherichia coli (EPEC), as well as the mouse pathogen Citrobacter rodentium, colonize the gut mucosa via attaching and effacing lesion formation and cause diarrheal diseases. EPEC and C. rodentium type III secretion system (T3SS) effectors repress innate immune responses and infiltration of immune cells. Inflammatory caspases such as caspase-1 and caspase-4/11 are crucial mediators of host defense and inflammation in the gut via their ability to process cytokines such as interleukin (IL)-1β and IL-18. Here we report that the effector NleF binds the catalytic domain of caspase-4 and inhibits its proteolytic activity. Following infection of intestinal epithelial cells (IECs) EPEC inhibited caspase-4 and IL-18 processing in an NleF-dependent manner. Depletion of caspase-4 in IECs prevented the secretion of mature IL-18 in response to infection with EPECΔnleF. NleF-dependent inhibition of caspase-11 in colons of mice prevented IL-18 secretion and neutrophil influx at early stages of C. rodentium infection. Neither wild-type C. rodentium nor C. rodentiumΔnleF triggered neutrophil infiltration or IL-18 secretion in Cas11 or Casp1/11-deficient mice. Thus, IECs have a key role in modulating early innate immune responses in the gut via a caspase-4/11—IL-18 axis, which is targeted by virulence factors encoded by enteric pathogens.     

反义表皮生长因子受体cDNA转染抑制胶质母细胞瘤生长的机制研究

目的 探讨反义表皮生长因子受体(EGFR)cDNA转染抑制胶质母细胞瘤细胞株U87MG生长的机制。方法 反义EGFR cDNA转染胶质母细胞瘤细胞株U87MG后,筛选出低表达EGFR的细胞株;通过观察肿瘤细胞的形态和Western蛋白印迹免疫化学方法检测胶质纤维酸性蛋白(GFAP)的表达,来研究反义,EGFR cDNA转染对胶质母细胞瘤分化的影响;通过流式细胞仪进行细胞周期分析及免疫组织化学方法检测细胞周期调控蛋白p53、Rb、p16和CDK4等,以研究反义EGFR cDNA转染对细胞周期的影响及机制;用telomeric repeat amplification protocol(TRAP分析)检测肿瘤细胞的端粒酶活性。结果 反义EGFR cDNA转染胶质母细胞瘤U87MG后,肿瘤细胞的突起延长,细胞的GFAP表达增高;肿瘤细胞的G0／G1期细胞百分率明显增高,而S期细胞百分率明显减少;肿瘤细胞的野生型,p53蛋白表达明显增高,而Rb、p16和CDK4等的蛋白表达水平未发生明显改变;肿瘤细胞的端粒酶活性明显降低。结论 反义EGFR cDNA转染可以通过诱导胶质母细胞瘤细胞分化、诱导野生型p53的表达、G1细胞周期阻滞及抑制端粒酶活性等机制而抑制肿瘤细胞生长,而且这些作用机制之间是相互作用、相互协调而共同发挥抑制肿瘤细胞生长作用的。     

Sequential changes in epidermal growth factor receptor/ligand function in cultured rat liver epithelial cells transformed chemically in vitro

Diploid WB rat liver epithelial cells contain abundant, rapidly internalized epidermal growth factor receptors, and respond pleiotropically to ligand binding. Signal transduction pathways downstream from the EGF receptor involve activation of elements that are both dependent on and independent of protein kinase C activation. Neoplastic transformation of wild-type WB rat liver epithelial cells by exposure to N-methyl-N'-nitro-N-nitrosoguanidine is associated with progressive alterations in the responses of affected cells to binding of EGF to EGF receptors, including heightened cell proliferation and the expression of several other phenotypic properties. Tumorigenic rat liver epithelial cells acquire the ability to express transforming growth factor-alpha (TGF-alpha), and to secrete this growth factor in a regulated and then unregulated manner. TGF-alpha expression, together with the presence of abundant EGF receptors, provides affected cells with an autocrine growth cycle. The ability of transformed WB rat liver epithelial cells to produce tumors cosegregates clonally with TGF-alpha expression and with heightened expression of c-myc, c-Ha-ras and c-Ki-ras proto-oncogenes.