cag+ Helicobacter pylori induce transactivation of the epidermal growth factor receptor in AGS gastric epithelial cells

The gastric pathogen Helicobacter pylori is known to activate epithelial cell signaling pathways that regulate numerous inflammatory response genes. The aim of this study was to elucidate the pathway leading to extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in H. pylori-infected AGS gastric epithelial cells. We find that H. pylori, via activation of the epidermal growth factor (EGF) receptor activates the small GTP-binding protein Ras, which in turn, mediates ERK1/2 phosphorylation. cag+ strains of H. pylori are able to induce greater EGF receptor activation than cag- strains, and studies with isogenic mutants indicate that an intact type IV bacterial secretion system is required for this effect. Blockade of EGF receptor activation using tyrphostin AG1478 prevents H. pylori-mediated Ras activation, inhibits ERK1/2 phosphorylation, and substantially decreases interleukin-8 gene expression and protein production. Investigations into the mechanism of EGF receptor activation, using heparin, a metalloproteinase inhibitor and neutralizing antibodies reveal that H. pylori transactivates the EGF receptor via activation of the endogenous ligand heparin-binding EGF-like growth factor. Transactivation of gastric epithelial cell EGF receptors may be instrumental in regulating both proliferative and inflammatory responses induced by cag+ H. pylori infection.     

Helicobacter pylori protein HP0175 transactivates epidermal growth factor receptor through TLR4 in gastric epithelial cells

The pathophysiology of Helicobacter pylori-associated gastroduodenal diseases, ulcerogenesis, and carcinogenesis is intimately linked to activation of epidermal growth factor receptor (EGFR) and production of vascular endothelial growth factor (VEGF). Extracellular virulence factors, such as CagA and VacA, have been proposed to regulate EGFR activation and VEGF production in gastric epithelial cells. We demonstrate that the H. pylori secretory protein, HP0175, by virtue of its ability to bind TLR4, transactivates EGFR and stimulates EGFR-dependent VEGF production in the gastric cancer cell line AGS. Knock-out of the hp0175 gene attenuates the ability of the resultant H. pylori strain to activate EGFR or to induce VEGF production. HP0175-induced activation of EGFR is preceded by translocation of TLR4 into lipid rafts. In lipid rafts, the Src kinase family member Lyn interacts with TLR4, leading to tyrosine phosphorylation of TLR4. Knockdown of Lyn prevents HP0175-induced activation of EGFR and VEGF production. Tyrosine-phosphorylated TLR4 interacts with EGFR. This interaction is necessary for the activation of EGFR. Disruption of lipid rafts with methyl beta-cyclodextrin prevents HP0175-induced tyrosine phosphorylation of TLR4 and activation of EGFR. This mechanism of transactivation of EGFR is novel and distinct from that of metalloprotease-dependent shedding of EGF-like ligands, leading to autocrine activation of EGFR. It provides new insight into our understanding of the receptor cross-talk network.     

Nucleotide-binding oligomerization domain-2 (NOD2) regulates type-1 cytokine responses to Mycobacterium avium but is not required for host control of infection

Nucleotide-binding oligomerization domain-2 (NOD2) is an innate immune receptor that recognizes peptidoglycan-derived muramyl dipeptide from intracellular bacteria and triggers proinflammatory signals. In this study, we sought to evaluate the role played by this receptor during early and late stages of infection with Mycobacterium avium in mice. We demonstrated that NOD2 knockout (KO) animals were able to control M. avium infection similarly to wild-type mice at all time points studied, even though IL-12 and TNF-α production was impaired in NOD2-deficient macrophages. At 100 days following infection with this bacterium, but not at 30 days post-infection, NOD2-deficient mice showed significantly diminished production of IFN-γ, as confirmed by reduced accumulation of IFN-γ and IL-12 mRNA in the spleens of KO mice. Additionally, a reduction in the size and in the number of lymphocytes/granulocytes of hepatic granulomas from NOD2 KO animals was observed only during late time points of M. avium infection. Taken together, these data demonstrate that NOD2 regulates type-1 cytokine responses to M. avium but is not required for the control of infection with this bacterium in vivo.     

Activation of the epidermal growth factor receptor tyrosine protein kinase in the absence of receptor oligomerization

The epidermal growth factor (EGF) receptor exists in a monomeric (170 kDa) form and in several aggregated states (360 kDa, greater than 500 kDa). The hypothesis that the oligomerization of the receptor is required for the stimulation of the kinase was tested by correlating the oligomeric state of the receptor with the protein kinase activity. EGF and sphingosine stimulate the phosphorylation of an exogenous peptide substrate by the receptor to an equal extent. Chemical cross-linking using disuccinimidyl suberate and the analysis of EGF receptor complexes by Western blotting demonstrated that EGF caused the aggregation of receptors. Similar results were obtained when [32P]phosphate-labeled receptors were cross-linked using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. These results were confirmed by sucrose density gradient sedimentation analysis. In contrast to the effects of EGF, incubation of EGF receptors with sphingosine did not cause the oligomerization of the receptors. These data demonstrate that the EGF receptor kinase can be stimulated independently of the aggregation of the receptors.     

Plant growth regulators and virus infection: A critical review

Virus infection can severely inhibit plant growth and distort development. This article reviews changes in plant growth regulator metabolism caused by infection. In general, virus infection decreases auxin and gibberellin concentrations and increases abscisic acid concentration. Ethylene production is stimulated in necrotic or chlorotic reactions to infection, but not where the virus spreads systemically without necrosis. While these broad trends are true for most host-virus combinations studied, several situations are recorded where the virus had other effects on growth substance concentration. Cytokinin changes do not show any common pattern: both increases and decreases after infection have been reported.The extent to which virus-induced changes in growth substance concentration could be responsible for observed alterations in host growth and development is discussed. While changes in abscisic acid, gibberellin and ethylene production seem potentially important, the experimental evidence does not provide conclusive proof for control of growth by these changes.The numerous investigations of effects of exogenous regulators on virus multiplication and pathogenesis are reviewed. Different regulators, or the same regulator applied at different times or concentrations, had very diverse effects, and in some cases did significantly alter virus multiplication and pathogenesis. However, such studies seem to have yielded disappointingly little understanding of the biochemistry of the host-virus interaction, and the possible involvement of growth substances in this.Possible uses of plant growth regulators in chemotherapy of virus disease, and their possible involvement in natural or induced resistance mechanisms are discussed.     

MICAL-like1 mediates epidermal growth factor receptor endocytosis

Small GTPase Rabs are required for membrane protein sorting/delivery to precise membrane domains. Rab13 regulates epithelial tight junction assembly and polarized membrane transport. Here we report that Molecule Interacting with CasL (MICAL)-like1 (MICAL-L1) interacts with GTP-Rab13 and shares a similar domain organization with MICAL. MICAL-L1 has a calponin homology (CH), LIM, proline rich and coiled-coil domains. It is associated with late endosomes. Time-lapse video microscopy shows that green fluorescent protein-Rab7 and mcherry-MICAL-L1 are present within vesicles that move rapidly in the cytoplasm. Depletion of MICAL-L1 by short hairpin RNA does not alter the distribution of a late endosome/lysosome-associated protein but affects the trafficking of epidermal growth factor receptor (EGFR). Overexpression of MICAL-L1 leads to the accumulation of EGFR in the late endosomal compartment. In contrast, knocking down MICAL-L1 results in the distribution of internalized EGFR in vesicles spread throughout the cytoplasm and promotes its degradation. Our data suggest that the N-terminal CH domain associates with the C-terminal Rab13 binding domain (RBD) of MICAL-L1. The binding of Rab13 to RBD disrupts the CH/RBD interaction, and may induce a conformational change in MICAL-L1, promoting its activation. Our results provide novel insights into the MICAL-L1/Rab protein complex that can regulate EGFR trafficking at late endocytic pathways.     

Dependence of epidermal growth factor activation on its affinity and oligomerization]

Epidermal growth factor receptor (EGF-R) oligomerization has been followed on A-431 cells using covalent labeling by 125I-EGF and EGF-dependent autophosphorylation of receptor-kinase. High molecular weight complexes corresponding to monomeric, dimeric, and trimeric forms of EGF-R are detected. The process of oligomerization occurs effectively at 37 degrees C while at 4 degrees C no oligomer formation is detected. PMA or ATP treatment reduces the number of high-affinity EGF-binding sites but has no influence on dimer formation. Dimerisation of the EGF-R in the absence of the ligand has been established on formalin-fixed A-431 cells.     

Ligand-mediated autophosphorylation activity of the epidermal growth factor receptor during internalization

The association of EGF with its receptor in endosomes isolated from rat liver homogenates was assessed biochemically by polyethylene glycol precipitation and morphologically by electron microscope radioautography. The proportion of receptor-bound ligand in endosomes at 15 min after the injection of doses of 0.1 and 1 microgram EGF/100 g body weight was 57%. This value increased to 77% for the dose of 10 micrograms EGF injected. Quantitative electron microscope radioautography carried out on endosomes isolated at 15 min after the injection of 10 micrograms 125I-EGF demonstrated that most radiolabel was over the endosomal periphery thereby indicating that ligand- receptor complexes were in the bounding membrane but not in intraluminal vesicles of the content. EGF receptor autophosphorylation activity during internalization was evaluated in plasmalemma and endosome fractions. This activity was markedly but transiently reduced on the cell surface shortly after the administration of saturating doses of EGF. The same activity, however, was augmented and prolonged in endosomes for up to 30 min after EGF injection. The transient desensitization of cell surface activity was not due to prior in vivo phosphorylation since receptor dephosphorylation in vitro failed to restore autophosphorylation activity. Transient desensitization of cell surface autophosphorylation activity coincided with a diminished capacity for endocytosis of 125I-EGF with endocytosis returning to normal after the restoration of cell surface autophosphorylation activity. The inhibition of cell surface autophosphorylation activity and the activation of endosomal autophosphorylation activity coincident with downregulation suggest that EGF receptor traffic is governed by ligand-regulated phosphorylation activity.     

Echovirus 1 internalization negatively regulates epidermal growth factor receptor downregulation

We have demonstrated previously that the human picornavirus Echovirus 1 (EV1) triggers an infectious internalization pathway that follows closely, but seems to stay separate, from the epidermal growth factor receptor (EGFR) pathway triggered by epidermal growth factor (EGF). Here, we confirmed by using live and confocal microscopy that EGFR and EV1 vesicles are following intimately each other but are distinct entities with different degradation kinetics. We show here that despite being sorted to different pathways and located in distinct endosomes, EV1 inhibits EGFR downregulation. Simultaneous treatment with EV1 and EGF led to an accumulation of EGFR in cytoplasmic endosomes, which was evident already 15 min p.i. and more pronounced after 2 hr p.i. EV1 treatment led to reduced downregulation, which was proven by increased total cellular amount of EGFR. Confocal microscopy studies revealed that EGFR accumulated in large endosomes, presumably macropinosomes, which were not positive for markers of the early, recycling, or late endosomes/lysosomes. Interestingly, EV1 did not have a similar blocking effect on bulk endosomal trafficking or transferrin recycling along the clathrin pathway suggesting that EV1 did not have a general effect on cellular trafficking pathways. Importantly, EGF treatment increased EV1 infection and increased cell viability during infection. Simultaneous EV1 and EGF treatment seemed to moderately enhance phosphorylation of protein kinase C α. Furthermore, similar phenotype of EGFR trafficking could be produced by phorbol 12‐myristate 13‐acetate treatment, further suggesting that activated protein kinase C α could be contributing to EGFR phenotype. These results altogether demonstrate that EV1 specifically affects EGFR trafficking, leading to EGFR downregulation, which is beneficial to EV1 infection.     

Transactivation of epidermal growth factor receptor by insulin-like growth factor 1 requires basal hydrogen peroxide

Insulin-like growth factor (IGF-1) plays an important role in prostate cancer development. Recent studies suggest that IGF-1 has mitogenic action through epidermal growth factor receptor (EGFR). However, the mechanism remains largely unknown. Here, we demonstrated in prostate cancer DU145 cells that IGF-1 induced EGFR transactivation, leading to ERK activation. Matrix metalloproteinase-mediated shedding of heparin-binding EGF is involved in this process. Antioxidants and catalase inhibited IGF-1-stimulated EGFR phosphorylation, indicating that H(2)O(2) is required for EGFR activation. However, exogenous H(2)O(2) did not activate EGFR or IGF-1R in DU145 cells. IGF-1 did not induced production of H(2)O(2) in DU145 cells. Our results suggest that transactivation of EGFR by IGF-1 requires basal intracellular H(2)O(2) in DU145 cells.     

Signaling by the Drosophila epidermal growth factor receptor pathway during development

In 1997 we wrote a review entitled "A thousand and one roles for the Drosophila epidermal growth factor (EGF) receptor (DER/EGFR)." We are not there yet in terms of the number of developmental roles assigned to this receptor in Drosophila. Nevertheless, DER has certainly emerged as one of the key players in development, since it is used repeatedly to direct cell fate choices, cell division, cell survival, and migration. A battery of activating ligands and an inhibitory ligand achieves this versatility. For the ligands that are produced as membrane-bound precursors, trafficking and processing are the key regulatory steps, determining the eventual temporal and spatial pattern of receptor activation. In most cases DER is activated at a short range, in the cells adjacent to the ones producing the active ligand. This activation dictates a binary choice. In some instances DER is also activated over a longer range, and multiple cell fate choices may be induced, according to its level of activation. A battery of negative feedback loops assures the limited range of DER induction. The distinct responses to DER activation in the different tissues depend upon combinatorial interactions with other signaling pathways and tissue-specific factors, at the level of target-gene regulation.     

Role of somatostatin receptor 1 and 5 on epidermal growth factor receptor mediated signaling

Epidermal growth factor (EGF) regulates normal and tumor cell proliferation via epidermal growth factor receptor (EGFR) phosphorylation, homo- or heterodimerization and activation of mitogen-activated protein kinases (MAPKs) and PI3K/AKT cell survival pathways. In contrast, SST via activation of five different receptor subtypes inhibits cell proliferation and has been potential target in tumor treatment. To gain further insight for the effect of SSTRs on EGFR activated signaling, we determine the role of SSTR1 and SSTR1/5 in human embryonic kidney (HEK) 293 cells. We here demonstrate that cells transfected with SSTR1 or SSTR1/5 negatively regulates EGF mediated effects attributed to the inhibition of EGFR phosphorylation, MAPKs as well as the cell survival signaling. Furthermore, SSTR effects were significantly enhanced in cells when EGFR was knock down using siRNA or treated with selective antagonist (AG1478). Most importantly, the presence of SSTR in addition to modulating signaling pathways leads to the dissociation of the constitutive and EGF induced heteromeric complex of EGFR/ErbB2. Furthermore, cells cotransfected with SSTR1/5 display pronounced effect of SST on the signaling and dissociation of the EGFR/ErbB2 heteromeric complex than the cells expressing SSTR1 alone. Taken together this study provides the first evidence that the presence of SSTR controls EGF mediated cell survival pathway via dissociation of ErbB heteromeric complex. We propose that the activation of SSTR and blockade of EGFR might serve novel therapeutic approach in inhibition of tumor proliferation.     

Molecular targeting in radiotherapy: epidermal growth factor receptor

Radiation therapy is utilized as a treatment to cure or manage cancer; however, because of risk to local healthy tissue-and a modest success rate of some radiotherapy-strategies have been sought that would increase the therapeutic index of the treatment while reducing damage to surrounding tissue. Cell and tissue irradiation stimulates a series of biochemical and molecular signals; various components of this ionizing radiation (IR)-inducible signal transduction cascade can promote the survival of tumor cells. Identification of interactions between IR and a signaling pathway creates an opportunity to target those signaling intermediates to improve the outcome of radiotherapy. The epidermal growth factor receptor (EGFR, also termed ErbB1) is involved in normal development and differentiation of epithelial cells as well as in tumorigenesis. The EGFR is activated by IR, thus making this receptor and other members of the ErbB family important targets for radiosensitizing molecular interventions. Recent approaches have utilized monoclonal antibodies, small molecules, and transgenic technologies to undermine the kinase activity of EGFR.     

STAT3: a critical component in the response to Helicobacter pylori infection

STAT3 imparts a profound influence on both the epithelial and immune components of the gastric mucosa, and through regulation of key intracellular signal transduction events, is well placed to control inflammatory and oncogenic outcomes in the context of Helicobacter (H.) pylori infection. Here we review the roles of STAT3 in the host immune response to H. pylori infection, from both gastric mucosal and systemic perspectives, as well as alluding more specifically to STAT3‐dependent mechanisms that might be exploited as drug targets.     

Extracellular HspBP1 and Hsp72 synergistically activate epidermal growth factor receptor

Background information. Heat‐inducible Hsp72 is the founding member of the Hsp70 (heat shock proteins of 70 kDa) family of molecular chaperones. It is localized primarily in cytoplasm and nucleus but is also found extracellularly. The source of e‐Hsp72 (extracellular Hsp72) is not precisely identified and may not be the same in every situation. A number of studies demonstrated that e‐Hsp72 plays an important role in cell survival, tumour rejection and immune response. However, currently little is known about regulation of e‐Hsp72 function. In cells, Hsp72 is controlled by co‐chaperones. An abundant co‐chaperone, HspBP1 (Hsp72‐binding protein 1) was found extracellularly in the serum. In the present study we analysed the secretion and function of e‐HspBP1 (extracellular HspBP1). Results. A431 human squamous carcinoma cells accumulated Hsp72 and HspBP1 in chromogranin A‐positive granules following heat stress or in the presence of U73122, an inhibitor of phospholipase C. Following these treatments, A431 cells also increased the secretion of both proteins into the culture medium. The secreted e‐Hsp72 and e‐HspBP1 were co‐immunoprecipitated from the conditioned medium. Purified recombinant HspBP1 augmented e‐Hsp72‐mediated phosphorylation of EGFR (epidermal growth factor receptor) and its down‐stream targets, ERK1 (extracellular signal‐regulated kinase 1) and ERK2 in a concentration‐dependent manner. Finally, a HspBP1 N‐terminal domain deletion mutant and boiled recombinant HspBP1 did not affect the e‐Hsp72‐mediated activity. Conclusions. Heat stress and PLC (phospholipase C) inhibition result in the enhanced secretion of both Hsp72 and HspBP1. In an extracellular environment, the two chaperones interact both physically and functionally, leading to the activation of th EGFR—ERK1/2 signalling pathway. However, the magnitude of EGFR activation depends on the e‐HspBP1/e‐Hsp72 ratio in the medium. Extracellular chaperone‐mediated activation of EGFR can provide a survival advantage to cells under heat shock and other stresses.     

Interdependent epidermal growth factor receptor signalling and trafficking

Epidermal growth factor (EGF) receptor (EGFR) signalling regulates diverse cellular functions, promoting cell proliferation, differentiation, migration, cell growth and survival. EGFR signalling is critical during embryogenesis, in particular in epithelial development, and disruption of the EGFR gene results in epithelial immaturity and perinatal death. EGFR signalling also functions during wound healing responses through accelerating wound re-epithelialisation, inducing cell migration, proliferation and angiogenesis. Upregulation of EGFR signalling is often observed in carcinomas and has been shown to promote uncontrolled cell proliferation and metastasis. Therefore aberrant EGFR signalling is a common target for anticancer therapies. Various reports indicate that EGFR signalling primarily occurs at the plasma membrane and EGFR degradation following endocytosis greatly attenuates signalling. Other studies argue that EGFR internalisation is essential for complete activation of downstream signalling cascades and that endosomes can serve as signalling platforms. The aim of this review is to discuss current understanding of intersection between EGFR signalling and trafficking.