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EGF Receptor (egf + receptor)
Selected AbstractsEGF receptor in relation to tumor development: molecular basis of responsiveness of cancer cells to EGFR-targeting tyrosine kinase inhibitorsFEBS JOURNAL, Issue 2 2010Kenji Takeuchi The function of the epidermal growth factor receptor (EGFR) is dysregulated in various types of malignancy as a result of gene amplification, mutations, or abnormally increased ligand production. Therefore, the tyrosine kinase activity of the EGFR is a promising therapeutic target. EGFR tyrosine kinase inhibitors, such as gefitinib (Iressa), show evident anticancer effects in patients with non-small cell lung cancer. The induction of apoptosis has been considered to be the major mechanism for these gefitinib-mediated anticancer effects. Lung cancer cells harboring mutant EGFRs become dependent on them for their survival and, consequently, undergo apoptosis following the inhibition of EGFR tyrosine kinase by gefitinib. Gefitinib has been shown to inhibit cell survival and growth signaling pathways such as the extracellular signal-regulated kinase 1/2 pathway and the Akt pathway, as a consequence of the inactivation of EGFR. However, the precise downstream signaling molecules of extracellular signal-regulated kinase 1/2 and Akt have not yet been elucidated. In this minireview we have highlighted the effect of tyrosine kinase inhibitors on members of the Bcl-2 family of proteins, which are downstream signaling molecules and serve as the determinants that control apoptosis. We also discuss tyrosine kinase inhibitor-induced apoptosis via c-Jun NH2 -terminal kinase and p38 mitogen-activated protein kinase. [source] Epidermal growth factor receptor expression regulates proliferation in the postnatal rat retinaGLIA, Issue 2 2006Jennie L. Close Abstract Epidermal growth factor (EGF) is known to promote proliferation of both retinal progenitors and Muller glia in vitro, but several questions remain concerning an in vivo role for this factor. In this study, we investigated whether the EGF receptor (EGFR) is necessary for the maintenance of normal levels of progenitor and Muller glial proliferation in vivo. Here, we show that (1) mice with homozygous deletion of the Egfr gene have reduced proliferation in late stages of retinal histogenesis, (2) EGF is mitogenic for Müller glia in vivo during the first two postnatal weeks in the rodent retina, (3) the effectiveness of EGF as a Müller glial mitogen declines in parallel with the decline in EGFR expression as the retina matures, and (4) following damage to the retina from continuous light exposure, EGFR expression is up-regulated in Müller glia to levels close to those in the neonatal retina, resulting in a renewed mitotic response to EGF. Together with previous results from other studies, these data indicate that the downregulation of a growth factor receptor is one mechanism by which glial cells maintain mitotic quiescence in the mature nervous system. © 2006 Wiley-Liss, Inc. [source] Significance of the Grb2 and Son of Sevenless (Sos) Proteins in Human Bladder Cancer Cell LinesIUBMB LIFE, Issue 4 2000Takafumi Watanabe Abstract The epidermal growth factor (EGF) receptor has been suggested to have an important role in tumor initiation and progression of human bladder cancers. Grb2 protein, which is the downstream effector of the EGF receptor, acts as an adaptor protein between the EGF receptor and the Ras guanine-nucleotide exchange factor, son of sevenless (Sos) protein. Sos protein regulates the action of Ras protein by promoting the exchange of GDP for GTP . However, the significance of Grb2 and Sos proteins, which is related to EGF-triggered Ras activation, has not been elucidated in human bladder cancer. The aim of the present study is to clarify the significance of these proteins in human bladder cancer cell lines. In the present study, we used four human bladder cancer cell lines (T24, KU-7, UMUC-2, UMUC-6) and two kinds of cultured normal urothelial cells (HMKU-1, HMKU-2) isolated from patients with no malignancy. We examined the expression of EGF receptor, Grb2, and Sos proteins in these cells by Western blot analysis. Furthermore, the bladder cancer cell lines were subjected to sequence analysis to identify a point mutation in the c-H-ras gene at codon 12. There was no marked difference in the expression of the EGF receptor between human bladder cancer cell lines and cultured normal urothelial cells. On the other hand, expression of Grb2 and Sos proteins was substantially increased in all human bladder cancer cell lines examined in comparison with cultured normal urothelial cells, whether codon 12 of H-ras was mutated or not. These results suggest that the amplification of both Grb2 and SOS proteins plays an important role in the carcinogenesis of human bladder cancer. [source] Two modes of ERK activation by TNF in keratinocytes: Different cellular outcomes and bi-directional modulation by vitamin D,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2008Ester Ziv Abstract Inflammation, elicited in the skin following tissue damage or pathogen invasion, may become chronic with deleterious consequences. Tumor necrosis factor (TNF) is a key mediator of cutaneous inflammation and the keratinocyte an important protagonist of skin immunity. Calcitriol, the hormonally active vitamin D metabolite, and its analogs attenuate epidermal inflammation and inhibit the hyperproliferation of keratinocytes associated with the inflammatory disorder, psoriasis. Since activation of extracellular signal-regulated kinase (ERK) promotes keratinocyte proliferation and mediates epidermal inflammation, we studied the effect of calcitriol on ERK activation in HaCaT keratinocytes exposed to the ubiquitous inflammatory cytokine TNF. By using the EGF receptor (EGFR) tyrosine kinase inhibitor, AG1487 and the Src family inhibitor, PP-1, we established that TNF activated ERK in an EGFR and Src dependent and an EGFR and Src independent modes. EGFR dependent activation resulted in the upregulation of the transcription factor, c-Fos, while the EGFR independent activation mode was of a shorter duration, did not affect c-Fos expression but induced IL-8 mRNA expression. Pretreatment with calcitriol, enhanced TNF-induced EGFR-Src dependent ERK activation and tyrosine phosphorylation of the EGFR, but abolished the EGFR-Src independent ERK activation. These effects were mirrored by enhancement of c-Fos and inhibition of IL-8 induction by TNF. Treatment with calcitriol increased the rate of the de-phosphorylation of activated ERK, accounting for the inhibition of EGFR-Src independent ERK activation by TNF. It is possible that effects on the ERK cascade contribute to the effects of calcitriol and its synthetic analogs on cutaneous inflammation and keratinocyte proliferation. J. Cell. Biochem. 104: 606,619, 2008. © 2007 Wiley-Liss, Inc. [source] Bradykinin-induced p42/p44 MAPK phosphorylation and cell proliferation via Src, EGF receptors, and PI3-K/Akt in vascular smooth muscle cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2005Chuen-Mao Yang In our previous study, bradykinin (BK) exerts its mitogenic effect through Ras/Raf/MEK/MAPK pathway in vascular smooth muscle cells (VSMCs). In addition to this pathway, the non-receptor tyrosine kinases (Src), EGF receptor (EGFR), and phosphatidylinositol 3-kinase (PI3-K) have been implicated in linking a variety of G-protein coupled receptors to MAPK cascades. Here, we investigated whether these different mechanisms participating in BK-induced activation of p42/p44 MAPK and cell proliferation in VSMCs. We initially observed that BK- and EGF-dependent activation of Src, EGFR, Akt, and p42/p44 MAPK and [3H]thymidine incorporation were mediated by Src and EGFR, because the Src inhibitor PP1 and EGFR kinase inhibitor AG1478 abrogated BK- and EGF-dependent effects. Inhibition of PI3-K by LY294002 attenuated BK-induced Akt and p42/p44 MAPK phosphorylation and [3H]thymidine incorporation, but had no effect on EGFR phosphorylation, suggesting that EGFR may be an upstream component of PI3-K/Akt and MAPK in these responses. This hypothesis was supported by the tranfection with dominant negative plasmids of p85 and Akt which significantly attenuated BK-induced Akt and p42/p44 MAPK phosphorylation. Pretreatment with U0126 (a MEK1/2 inhibitor) attenuated the p42/p44 MAPK phosphorylation and [3H]thymidine incorporation stimulated by BK, but had no effect on Akt activation. Moreover, BK-induced transactivation of EGFR and cell proliferation was blocked by matrix metalloproteinase inhibitor GM6001. These results suggest that, in VSMCs, the mechanism of BK-stimulated activation of p42/p44 MAPK and cell proliferation was mediated, at least in part, through activation of Src family kinases, EGFR transactivation, and PI3-K/Akt. Copyright © 2004 Wiley-Liss, Inc. [source] Novel EGF pathway regulators modulate C. elegans healthspan and lifespan via EGF receptor, PLC-,, and IP3R activationAGING CELL, Issue 4 2010Hiroaki Iwasa Summary Improving health of the rapidly growing aging population is a critical medical, social, and economic goal. Identification of genes that modulate healthspan, the period of mid-life vigor that precedes significant functional decline, will be an essential part of the effort to design anti-aging therapies. Because locomotory decline in humans is a major contributor to frailty and loss of independence and because slowing of movement is a conserved feature of aging across phyla, we screened for genetic interventions that extend locomotory healthspan of Caenorhabditis elegans. From a group of 54 genes previously noted to encode secreted proteins similar in sequence to extracellular domains of insulin receptor, we identified two genes for which RNAi knockdown delayed age-associated locomotory decline, conferring a high performance in advanced age phenotype (Hpa). Unexpectedly, we found that hpa-1 and hpa-2 act through the EGF pathway, rather than the insulin signaling pathway, to control systemic healthspan benefits without detectable developmental consequences. Further analysis revealed a potent role of EGF signaling, acting via downstream phospholipase C-,plc-3 and inositol-3-phosphate receptor itr-1, to promote healthy aging associated with low lipofuscin levels, enhanced physical performance, and extended lifespan. This study identifies HPA-1 and HPA-2 as novel negative regulators of EGF signaling and constitutes the first report of EGF signaling as a major pathway for healthy aging. Our data raise the possibility that EGF family members should be investigated for similar activities in higher organisms. [source] Cimetidine inhibits epidermal growth factor-induced cell signalingJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 3 2007Tatsuya Fujikawa Abstract Background:, Cimetidine, a histamine-2 (H2) receptor antagonist, has been demonstrated to have anticancer effects on colorectal cancer, melanoma and renal cell carcinoma. In the current study, we clarified that cimetidine inhibits both epidermal growth factor (EGF)-induced cell proliferation and migration in hepatocellular carcinoma (HCC) cell lines. Method:, HCC cell lines (Hep3B, HLF, SK-Hep-1, JHH-2, PLC/PRF/5 and HLE) were used and cell proliferation was assessed by [3H]-thymidine incorporation assay. Cell migration was measured by in vitro cell migration assay. Biological effects of cimetidine were assessed with human EGF receptor (EGFR)-expressing mouse fibroblast cells (NR6-WT). The autophosphorylation of EGFR and the activation of other downstream effectors were analyzed by immunoprecipitation and immunoblotting. The concentration of intracellular cyclic AMP (cAMP) was measured by competitive enzyme immunoassay. Results:, Cimetidine inhibited both EGF-induced cell proliferation and migration in Hep3B, HLF, SK-Hep-1 and JHH-2, while cimetidine did not affect EGF-induced cell proliferation and migration in PLC/PRF/5 and HLE. Cimetidine was revealed to disrupt the EGF-induced autophosphorylation of EGFR and its downstream effectors, mitogen activated protein kinases and phospholipase C-,. To define the molecular basis of this negative regulation, we identified that cimetidine significantly decreased intracellular cAMP levels and that decrement of cAMP inhibited autophosphorylation of EGFR. The cell permeable cAMP analog, CPT-cAMPS reversed the cimetidine-induced inhibition of EGF-induced cell proliferation and cell migration by restoring autophosphorylation of EGFR. Conclusion:, Cimetidine inhibited EGF-induced cell proliferation and migration in HCC cell lines by decreasing the concentration of intracellular cAMP levels. Cimetidine may be a candidate chemopreventive agent for HCC. [source] Lysophosphatidic Acid Inhibits Ca2+ Signaling in Response to Epidermal Growth Factor Receptor Stimulation in Human Astrocytoma Cells by a Mechanism Involving Phospholipase C, and a G,i ProteinJOURNAL OF NEUROCHEMISTRY, Issue 4 2000Marita Hernández Abstract: The effect of the lysophospholipid mediators lysophosphatidic acid (LPA) and sphingosine 1-phosphate and the polypeptide growth factor epidermal growth factor (EGF) on the human astrocytoma cell line 1321N1 was assessed. These agonists produced a rapid and transient increase of the intracellular Ca2+ concentration. When LPA was perfused before addition of EGF, the EGF-dependent Ca2+ transient was abrogated, whereas this was not observed when EGF preceded LPA addition. This inhibitory effect was not found for other EGF-mediated responses, e.g., activation of the mitogen-activated protein kinase cascade and cell proliferation, thus pointing to the existence of cross-talk between LPA and EGF for only a branch of EGF-induced responses. As 1321N1 cells expressed mRNA encoding the LPA receptors endothelial differentiation gene (Edg)-2, Edg-4, and Edg-7 and as sphingosine 1-phosphate did not interfere with LPA signaling, Edg-2, Edg-4, and/or Edg-7 could be considered as the LPA receptors mediating the aforementioned cross-talk. Attempts to address the biochemical mechanism involved in the cross-talk between the receptors were conducted by the immunoprecipitation approach using antibodies reacting with the EGF receptor (EGFR), phosphotyrosine, phospholipase C, (PLC,)-1, and G,i protein. LPA was found to induce coupling of PLC,-1 to the EGFR by a mechanism involving a G,i protein, in the absence of tyrosine phosphorylation of both PLC, and the EGFR. These data show a cross-talk between LPA and EGF limited to a branch of EGFR-mediated signaling, which may be explained by a LPA-induced, G,i -protein-mediated translocation of PLC,-1 to EGFR in the absence of detectable tyrosine phosphorylation of both proteins. [source] Epidermal Growth Factor Induces Oxidative Neuronal Injury in Cortical CultureJOURNAL OF NEUROCHEMISTRY, Issue 1 2000Yoo Kyung Cha Abstract : Recently, we have demonstrated that certain neurotrophic factors can induce oxidative neuronal necrosis by acting at the cognate tyrosine kinase-linked receptors. Epidermal growth factor (EGF) has neurotrophic effects via the tyrosine kinase-linked EGF receptor (EGFR), but its neurotoxic potential has not been studied. Here, we examined this possibility in mouse cortical culture. Exposure of cortical cultures to 1-100 ng/ml EGF induced gradually developing neuronal death, which was complete in 48-72 h ; no injury to astrocytes was noted. Electron microscopic findings of EGF-induced neuronal death were consistent with necrosis ; severe mitochondrial swelling and disruption of cytoplasmic membrane occurred, whereas nuclei appeared relatively intact. The EGF-induced neuronal death was accompanied by increased free radical generation and blocked by the anti-oxidant Trolox. Suggesting mediation by the EGFR, an EGFR tyrosine kinase-specific inhibitor, C56, attenuated EGF-induced neuronal death. In addition, inhibitors of extracellular signal-regulated protein kinase 1/2 (Erk-1/2) (PD98056), protein kinase A (H89), and protein kinase C (GF109203X) blocked EGF-induced neuronal death. A p38 mitogen-activated protein kinase inhibitor (SB203580) or glutamate antagonists (MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione) showed no protective effect. The present results suggest that prolonged activation of the EGFR may trigger oxidative neuronal injury in central neurons. [source] Quantitative mass spectrometry to investigate epidermal growth factor receptor phosphorylation dynamicsMASS SPECTROMETRY REVIEWS, Issue 1 2008Sven Schuchardt Abstract Identifying proteins of signaling networks has received much attention, because an array of biological processes are entirely dependent on protein cross-talk and protein,protein interactions. Protein posttranslational modifications (PTM) add an additional layer of complexity, resulting in complex signaling networks. Of particular interest to our working group are the signaling networks of epidermal growth factor (EGF) receptor, a transmembrane receptor tyrosine kinase involved in various cellular processes, including cell proliferation, differentiation, and survival. Ligand binding to the N -terminal residue of the extracellular domain of EGF receptor induces conformational changes, dimerization, and (auto)-phosphorylation of intracellular tyrosine residues. In addition, activated EGF receptor may positively affect survival pathways, and thus determines the pathways for tumor growth and progression. Notably, in many human malignancies exaggerated EGF receptor activities are commonly observed. An understanding of the mechanism that results in aberrant phosphorylation of EGF receptor tyrosine residues and derived signaling cascades is crucial for an understanding of molecular mechanisms in cancer development. Here, we summarize recent labeling methods and discuss the difficulties in quantitative MS-based phosphorylation assays to probe for receptor tyrosine kinase (RTK) activity. We also review recent advances in sample preparation to investigate membrane-bound RTKs, MS-based detection of phosphopeptides, and the diligent use of different quantitative methods for protein labeling. © 2007 Wiley Periodicals, Inc., Mass Spec Rev 27:51,65, 2008 [source] Inositol hexaphosphate downregulates both constitutive and ligand-induced mitogenic and cell survival signaling, and causes caspase-mediated apoptotic death of human prostate carcinoma PC-3 cells,MOLECULAR CARCINOGENESIS, Issue 1 2010Mallikarjuna Gu Abstract Constitutively active mitogenic and prosurvival signaling cascades due to aberrant expression and interaction of growth factors and their receptors are well documented in human prostate cancer (PCa). Epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) are potent mitogens that regulate proliferation and survival of PCa cells via autocrine and paracrine loops involving both mitogen-activated protein kinase (MAPK)- and Akt-mediated signaling. Accordingly, here we assessed the effect of inositol hexaphosphate (IP6) on constitutive and ligand (EGF and IGF-1)-induced biological responses and associated signaling cascades in advanced and androgen-independent human PCa PC-3 cells. Treatment of PC-3 cells with 2,mM IP6 strongly inhibited both growth and proliferation and decreased cell viability; similar effects were also observed in other human PCa DU145 and LNCaP cells. IP6 also caused a strong apoptotic death of PC-3 cells together with caspase 3 and PARP cleavage. Mechanistic studies showed that biological effects of IP6 were associated with inhibition of both constitutive and ligand-induced Akt phosphorylation together with a decrease in total Akt levels, but a differential inhibitory effect on MAPKs extra cellular signal-regulated kinase 1/2 (ERK1/2), c- Jun N-terminal protein kinase (JNK1/2), and p38 under constitutive and ligand-activated conditions. Under similar condition, IP6 also inhibited AP-1 DNA-binding activity and decreased nuclear levels of both phospho and total c-Fos and c- Jun. Together, these findings for the first time establish IP6 efficacy in inhibiting aberrant EGF receptor (EGFR) or IGF-1 receptor (IGF-1R) pathway-mediated sustained growth promoting and survival signaling cascades in advanced and androgen-independent human PCa PC-3 cells, which might have translational implications in advanced human PCa control and management. © 2009 Wiley-Liss, Inc. [source] Epidermal growth factor receptor and claudin-2 participate in A549 permeability and remodeling: Implications for non-small cell lung cancer tumor colonizationMOLECULAR CARCINOGENESIS, Issue 6 2009Yakov Peter Abstract Tumor colonization involves changes in cell permeability and remodeling. Paracellular permeability is regulated by claudins, integrated tight junction (TJ) proteins, located on the apicolateral portion of epithelial cells. Epidermal growth factor (EGF) was reported to modify cellular claudin levels and induce remodeling. To investigate a role for EGF receptor (EGFR) activation in tumor colonization we studied the effect of EGF and claudin-2 overexpression on permeability and cell reorganization in the human A549 non-small cell lung cancer (NSCLC) cell line. Our data demonstrated that A549 cells possess functional TJs and that EGF treatment increased levels of claudin-2 expression by 46%. Furthermore, EGFR signaling reduced monolayer permeability to choline and triggered cellular remodeling. The mitogen-activated protein kinase inhibitor PD98059 blocked the effect on A549 permeability and remodeling. EGF stimulation also exacerbated a fourfold increase in cell colonization elicited by claudin-2 upregulation. Our findings are consistent with the hypothesis that EGFR signaling plays an important role in A549 cell physiology and acts synergistically with claudin-2 to accelerate tumor colonization. Understanding the influence of EGF on A549 cell permeability and reorganization will help shed light on NSCLC tumor colonization and contribute to the development of novel anti-cancer treatments. © 2008 Wiley-Liss, Inc. [source] Low p38 MAPK and JNK activation in cultured hepatocytes of DRH rats; a strain highly resistant to hepatocarcinogenesisMOLECULAR CARCINOGENESIS, Issue 9 2007Satoshi Honmo Abstract DRH rats are a hepatocarcinogenesis-resistant strain isolated from hepatocarcinogenesis-sensitive Donryu rats, and the liver of DRH shows less histological damage and fewer/smaller neoplastic hepatic lesions by the treatment with hepatocarcinogens. To investigate the mechanism of the resistance, the properties of hepatocytes of DRH and Donryu were compared. In primary culture, DRH hepatocytes exhibited higher proliferation and less apoptosis than Donryu hepatocytes in the presence of EGF and insulin. However, such difference was not correlated to the degree of DNA damage associated with cell culture or cell cycle checkpoint function. Although the mitogen-activated protein kinases [EGF receptor (EGFR) and extracellular signal regulating kinases (ERK1/2)] were activated to the same degree, the stress-activated protein kinases [p38 mitogen-activated protein kinase (p38) and c- jun N-terminal kinase (JNK)] were activated to a lesser degree in the DRH hepatocytes. Treatment with 2-acetylaminofluorene (2-AAF) in vivo also resulted in less JNK and p38 activation in the DRH livers. Furthermore, apoptosis signal-regulating kinase 1 (ASK1) was inhibited by the lysate from the DRH but not by the Donryu hepatocytes. The low activation of the stress-activated protein kinases may be linked to the resistance to cellular stress, which may underlie the hepatocarcinogenesis-resistance in DRH rats. © 2007 Wiley-Liss, Inc. [source] Cooperative inhibitory effect of ZD1839 (Iressa) in combination with 17-AAG on glioma cell growth,MOLECULAR CARCINOGENESIS, Issue 5 2006Daniel R. Premkumar Abstract ZD1839 ("Iressa") is an orally active, selective epidermal growth factor (EGF) receptor-tyrosine kinase inhibitor. We evaluated the antitumor activity of ZD1839 in combination with HSP90 antagonist, 17-AAG in malignant human glioma cell lines. ZD1839 independently produced a dose-dependent inhibition of cellular proliferation in glioma cells grown in culture with time- and dose-dependent accumulation of cells in G1 phase of the cell cycle on flow cytometric analysis, although the concentrations required for optimal efficacy were at or above the limits of clinically achievable levels. Because the heat shock protein (HSP) is involved in the conformational maturation of a number of signaling proteins critical to the proliferation of malignant glioma cells, we hypothesized that the HSP90 inhibitor 17-AAG would potentiate ZD 1839-mediated glioma cytotoxicity by decreasing the activation status of EGF receptor, as well as downregulating the levels of other relevant signaling effectors. We, therefore, examined the effects of ZD1839 and 17-AAG, alone and in combination, on signal transduction and apoptosis in a series of malignant glioma cell lines. Simultaneous exposure to these inhibitors significantly induced cell death and quantitative analysis revealed that interaction between ZD1839 and 17-AAG-induced cytotoxicity was synergistic, leading to a pronounced increase in active caspase-3 and PARP cleavage. No significant growth inhibition or caspase activation was seen in control cells. The enhanced cytotoxicity of this combination was associated with diminished Akt activation and a significant downregulation of EGFR receptor, Raf-1 and mitogen activated protein kinase (MAPK). Cells exposed to 17-AAG and ZD1839 displayed a significant reduction in cell cycle regulatory proteins, such as CDK4 and CDK6. Taken together, these findings suggest that ZD1839, an EGF receptor tyrosine kinase inhibitor, plays a critical role in regulating the apoptotic response to 17-AAG and that multi-site targeting of growth signaling and cell survival pathways could provide a potent strategy to treat patients with malignant gliomas. © 2006 Wiley-Liss, Inc. [source] Co-regulation of B-Myb expression by E2F1 and EGF receptor,MOLECULAR CARCINOGENESIS, Issue 1 2006Norihisa Hanada Abstract Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is frequently over-expressed in human cancers and is associated with tumorigenesis, and increased tumor proliferation and progression. Also found in breast tumors with high levels is B-Myb, a transcription factor whose expression is activated by E2F1/3 at the late G1 phase and the level is sustained through the S phase. Recent reports suggest a casual correlation between EGFR and B-Myb expression in primary breast carcinomas. However, the mechanism for such co-expression remains un-investigated. Here, we report that EGFR is important for B-Myb expression and the underlying mechanism involves cooperated effects from EGFR and E2F1. EGF stimulation and forced expression of EGFR significantly increase B-Myb gene activity and such increase occurs in the G1 phase. EGF-induced B-Myb expression was not significantly suppressed following inhibition of PI-3K and ERK, two major EGFR downstream pathways. In contrast, we observed EGF-induced in vivo association of nuclear EGFR to the B-Myb promoter and the association is only detected at the G1/S phase and is abolished by EGFR kinase inhibitor. As EGFR lacks DNA-binding domain but contains transactivational activity and E2F1 activates B-Myb expression in the G1/S phase, we further reasoned that nuclear EGFR might cooperate with E2F1 leading to activation of B-Myb. Indeed, we found that EGFR co-immunoprecipitated with E2F1 in an EGF-dependent manner and that EGF activated in vivo binding of E2F1 to the B-Myb promoter. Consistently, forced expression of both EGFR and E2F1 in EGFR-null CHO cells greatly enhanced B-Myb promoter activity, compared to the vector control and expression of EGFR or E2F1 alone. Promoter mutagenesis studies showed that EGF-induced activation of B-Myb promoter required both E2F and EGFR target sites. In summary, our data suggest that deregulated EGFR signaling pathway facilitate tumor cell proliferation partly via EGFR interaction with E2F1 and subsequent activation of B-Myb gene expression. © 2005 Wiley-Liss, Inc. [source] EGF-induced EGF-receptor and MAP kinase phosphorylation in goat cumulus cells during in vitro maturationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2005Laurence Gall Abstract EGF has been shown to influence meiotic maturation and development competence of oocyte in various mammalian species. We previously reported, in goat, that the EGF receptor (EGF-R) was present both on cumulus cells and oocytes. Here, EGF-induced signaling was investigated during the in vitro maturation process in goat cumulus,oocyte complexes (COCs). Cumulus cells and oocytes were subjected to Western immunoblotting analysis using anti-MAP kinase, anti-phosphotyrosine, anti-phospho MAP kinase, and anti-phospho EGF-R antibodies. We demonstrated that treatment with EGF during the in vitro maturation process induced rapid tyrosine phosphorylation of EGF-R in a time and concentration dependent manner in cumulus cells. A similar pattern of activation by phosphorylation was observed for MAP kinase upon EGF stimulation. AG 1478, an inhibitor of the EGF kinase, suppressed EGF-stimulated phosphorylation of EGF-R and also affected the MAP kinase activation. Treatment with the MEK inhibitor PD 98059 abolished EGF-induced MAP kinase activation. We did not observe oocyte EGF-R phosphorylation in our experiments during the in vitro maturation process. Our data indicate, in goat cumulus cells, that activation of EGF-R by EGF triggers signaling through the MAP kinase pathway during in vitro maturation. This supports the hypothesis that the major site of action for EGF, that regulates oocyte maturation, is the cumulus cell. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] Several signaling pathways are involved in the control of cattle oocyte maturationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Céline Vigneron Abstract The main limit of in vitro production of domestic mammal embryos comes from the low capacity of in vitro matured oocytes to develop after fertilization. As soon as they are separated from follicular environment, oocytes spontaneously resume meiosis without completion of their terminal differentiation. Roscovitine (ROS), an inhibitor of M-phase promoting factor (MPF) kinase activity reversibly blocks the meiotic resumption in vitro. However, in cattle maturing oocytes several cellular events such as protein synthesis and phosphorylation, chromatin condensation and nuclear envelope folding escape ROS inhibition suggesting the alternative pathways in oocyte maturation. We compared the level of synthesis and phosphorylation of several protein kinases during bovine cumulus oocyte complex (COC) maturation in vitro in the presence or not of epidermal growth factor (EGF) and ROS. We showed that during the EGF-stimulated maturation, ROS neither affected the decrease of EGF receptor (EGFR) nor did inhibit totally its phosphorylation in cumulus cells and also did not totally eliminate tyrosine phosphorylation in oocytes. However, ROS did inhibit the Phosphoinositide 3-kinase (PI3) activity when oocytes mature without EGF. Accumulation of Akt/PKB (protein kinase B), JNK1/2 (jun N-terminal kinases) and Aurora-A in oocytes during maturation was not affected by ROS. However, the phosphorylation of Akt but not JNKs was diminished in ROS-treated oocytes. Thus, PI3 kinase/Akt, JNK1/2 and Aurora-A are likely to be involved in the regulation of bovine oocyte maturation and some of these pathways seem to be independent to MPF activity and meiotic resumption. This complex regulation may explain the partial meiotic arrest of ROS-treated oocytes and the accelerated maturation observed after such treatment. Mol. Reprod. Dev. 69: 466,474, 2004. © 2004 Wiley-Liss, Inc. [source] Characterization of a comparative model of the extracellular domain of the epidermal growth factor receptorPROTEIN SCIENCE, Issue 2 2000Robert N. Jorissen Abstract The Epidermal Growth Factor (EGF) receptor is a tyrosine kinase that mediates the biological effects of ligands such as EGF and transforming growth factor alpha. An understanding of the molecular basis of its action has been hindered by a lack of structural and mutational data on the receptor. We have constructed comparative models of the four extracellular domains of the EGF receptor that are based on the structure of the first three domains of the insulin-like growth factor-1 (IGF-1) receptor. The first and third domains of the EGF receptor, L1 and L2, are right-handed beta helices. The second and fourth domains of the EGF receptor, S1 and S2, consist of the modules held together by disulfide bonds, which, except for the first module of the S1 domain, form rod-like structures. The arrangement of the L1 and S1 domains of the model are similar to that of the first two domains of the IGF-1 receptor, whereas that of the L2 and S2 domains appear to be significantly different. Using the EGF receptor model and limited information from the literature, we have proposed a number of regions that may be involved in the functioning of the receptor. In particular, the faces containing the large beta sheets in the L1 and L2 domains have been suggested to be involved with ligand binding of EGF to its receptor. [source] Dihydrotestosterone activates the MAPK pathway and modulates maximum isometric force through the EGF receptor in isolated intact mouse skeletal muscle fibresTHE JOURNAL OF PHYSIOLOGY, Issue 3 2010M. M. Hamdi It is generally believed that steroid hormones have both genomic and non-genomic (rapid) actions. Although the latter form an important component of the physiological response of these hormones, little is known about the cellular signalling pathway(s) mediating these effects and their physiological functions in adult mammalian skeletal muscle fibres. Therefore, the primary aim of this study was to investigate the non-genomic actions of dihydrotestosterone (DHT) and their physiological role in isolated intact mammalian skeletal muscle fibre bundles. Our results show that treating the fibre bundles with physiological concentrations of DHT increases both twitch and tetanic contractions in fast twitch fibres. However, it decreases them in slow twitch fibres. These changes in force are accompanied by an increase in the phosphorylation of MAPK/ERK1/2 in both fibre types and that of regulatory myosin light chains in fast twitch fibres. Both effects were insensitive to inhibitors of Src kinase, androgen receptor, insulin-like growth factor 1 receptor and platelet-derived growth factor receptor. However, they were abolished by the MAPK/ERK1/2 kinase inhibitor PD98059 and the epidermal growth factor (EGF) receptor inhibitor tyrphostin AG 1478. In contrast, testosterone had no effect on force and increased the phosphorylation of ERK1/2 in slow twitch fibres only. From these results we conclude that sex steroids have non-genomic actions in isolated intact mammalian skeletal muscle fibres. These are mediated through the EGF receptor and one of their main physiological functions is the enhancement of force production in fast twitch skeletal muscle fibres. [source] Matrix metalloproteinases (MMPs) in bladder cancer: the induction of MMP9 by epidermal growth factor and its detection in urineBJU INTERNATIONAL, Issue 1 2003J.E. Nutt OBJECTIVES To investigate the matrix metalloproteinases (MMPs) 2 and 9 in bladder cancer cell lines stimulated with epidermal growth factor (EGF), and to investigate the presence of gelatinases in the urine of patients with bladder tumours, in relation to the stage and grade of tumour and the EGF receptor (EGFR) status. PATIENTS, SUBJECTS AND METHODS Conditioned media from cultured tumour cells were analysed by zymography. Urine samples from 28 patients with transitional cell carcinoma and 12 normal volunteers were also analysed. Western blotting was used to verify the bands of gelatinolytic activity. The EGFR status of the tumours was assessed by immunohistochemistry. RESULTS MMP9 was induced by EGF in the RT112 but not the RT4 bladder tumour cell line, whereas MMP2 production was unaffected by EGF. Gelatin zymography of urine samples from patients with bladder tumours showed high levels of MMP activity, with 78% positive for MMP9 and 28% positive for MMP2. The total gelatinolytic and MMP9 activity were significantly higher in patients with high-stage invasive tumours than in those with superficial tumours (P < 0.05), and were higher than in normal controls. Gelatinolytic activity at 130 and 200 kDa in urine was identified as MMP9 and MMP2. There was no significant relationship of urinary MMP9 activity to EGFR status of the tumour. CONCLUSION EGF induces MMP9 but not MMP2 in bladder cells. Analysis of urinary gelatinases is a useful noninvasive technique and both total gelatinase and MMP9 activity are associated with high stages of bladder tumours. [source] Role of the aging vasculature and Erb B-2 signaling in epidermal growth factor-dependent intravasion of breast carcinoma cells,CANCER, Issue 1 2004Daniel J. Price Ph.D. Abstract BACKGROUND The risks for developing breast carcinoma and dying from the disease increase with age. Mortality from breast carcinoma usually is due to metastatic disease. Metastatic cells are able to invade into the vascular tissue in a growth factor-dependent manner. Because breast carcinoma mortality increases with age, examination of breast carcinoma interactions with young and aged endothelial cells is essential. METHODS We studied a series of breast epithelial cells (HMT-3522 cells) that exhibited either noninvasive characteristics (S-1 cells) or epidermal growth factor (EGF)-dependent invasive characteristics (T4-2 cells). RESULTS Increased invasion of HMT-3522 cells was observed across an aged rat brain microvascular endothelial cell (BMEC) monolayer that was isolated from aged rats (24 months) compared with young rats (age 1 month). This increased invasion was inhibited by the specific EGF receptor inhibitor, AG1478, and by the Erb B-2-specific inhibitor, AG825. To analyze further the contribution of Erb B-2 to the EGF-dependent invasion of HMT-3522 cells, T4-2 cells were treated with the Erb B-2-specific therapeutic antibody trastuzumab and with the specific inhibitor AG825 and were then assayed for invasion. Both inhibitors led to a significant decrease in EGF-dependent invasion. Erb B-2 expression was found to be elevated in T4-2 cells (, 5-fold higher) compared with S-1 cells. However, treatment of T4-2 cells with the specific Erb B-2 inhibitor, AG825, failed to inhibit EGF-mediated signaling to phosphatidylinositol 3-kinase or extracellular-regulated kinases 1 and 2. CONCLUSIONS The current study findings indicate that aging of endothelium may contribute to the invasive phenotype of breast carcinoma cells and that "cross-talk" between Erb B-2 and EGF receptor is required for the intravasion of these cells into the surrounding vasculature. Cancer 2004. © 2004 American Cancer Society. [source] Transglutaminase differentially regulates growth signalling in rat perivenous and periportal hepatocytesCELL PROLIFERATION, Issue 3 2006A. Maruko PPH and PVH subpopulations have been isolated using the digitonin/collagenase perfusion technique. DNA synthesis was assessed by [3H] thymidine incorporation into hepatocytes. The assay for binding of [125I] EGF to cultured hepatocytes was analysed by Scatchard plot analysis. Pretreatment with the TG2 inhibitor monodansylcadaverine (MDC) greatly increased EGF-induced DNA synthesis in both PPH and PVH. Furthermore, [125I] EGF binding studies in PVH treated with MDC indicated that high-affinity EGF receptor expression was markedly up-regulated, whereas in PPH, there was no significant effect. Treatment with retinoic acid (RA), an inducer of TG2 expression, significantly decreased EGF-induced DNA synthesis in both PPH and PVH. Binding studies in the presence of RA revealed that the high-affinity EGF receptor was down-regulated and completely absent in both PPH and PVH. These results suggest that TG2 was involved in the differential growth capacities of PPH and PVH through down-regulation of high-affinity EGF receptors. [source] Importance of EGF receptor, HER2/Neu and Erk1/2 kinase signalling for host cell elongation and scattering induced by the Helicobacter pylori CagA protein: antagonistic effects of the vacuolating cytotoxin VacACELLULAR MICROBIOLOGY, Issue 3 2009Nicole Tegtmeyer Summary Helicobacter pylori is the causative agent of gastric pathologies ranging from chronic gastritis to peptic ulcers and even cancer. Virulent strains carrying both the cag pathogenicity island (cagPAI) and the vacuolating cytotoxin VacA are key players in disease development. The cagPAI encodes a type IV secretion system (T4SS) which forms a pilus for injection of the CagA protein into gastric epithelial cells. Injected CagA undergoes tyrosine phosphorylation and induces actin-cytoskeletal rearrangements involved in host cell scattering and elongation. We show here that the CagA-induced responses can be inhibited in strains expressing highly active VacA. Further investigations revealed that VacA does not interfere with known activities of phosphorylated CagA such as inactivation of Src kinase and cortactin dephosphorylation. Instead, we demonstrate that VacA exhibits inactivating activities on the epidermal growth factor receptor EGFR and HER2/Neu, and subsequently Erk1/2 MAP kinase which are important for cell scattering and elongation. Inactivation of vacA gene, downregulation of the VacA receptor RPTP-,, addition of EGF or expression of constitutive-active MEK1 kinase restored the capability of H. pylori to induce the latter phenotypes. These data demonstrate that VacA can downregulate CagA's effects on epithelial cells, a novel molecular mechanism showing how H. pylori can avoid excessive cellular damage. [source] Bradykinin-induced p42/p44 MAPK phosphorylation and cell proliferation via Src, EGF receptors, and PI3-K/Akt in vascular smooth muscle cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2005Chuen-Mao Yang In our previous study, bradykinin (BK) exerts its mitogenic effect through Ras/Raf/MEK/MAPK pathway in vascular smooth muscle cells (VSMCs). In addition to this pathway, the non-receptor tyrosine kinases (Src), EGF receptor (EGFR), and phosphatidylinositol 3-kinase (PI3-K) have been implicated in linking a variety of G-protein coupled receptors to MAPK cascades. Here, we investigated whether these different mechanisms participating in BK-induced activation of p42/p44 MAPK and cell proliferation in VSMCs. We initially observed that BK- and EGF-dependent activation of Src, EGFR, Akt, and p42/p44 MAPK and [3H]thymidine incorporation were mediated by Src and EGFR, because the Src inhibitor PP1 and EGFR kinase inhibitor AG1478 abrogated BK- and EGF-dependent effects. Inhibition of PI3-K by LY294002 attenuated BK-induced Akt and p42/p44 MAPK phosphorylation and [3H]thymidine incorporation, but had no effect on EGFR phosphorylation, suggesting that EGFR may be an upstream component of PI3-K/Akt and MAPK in these responses. This hypothesis was supported by the tranfection with dominant negative plasmids of p85 and Akt which significantly attenuated BK-induced Akt and p42/p44 MAPK phosphorylation. Pretreatment with U0126 (a MEK1/2 inhibitor) attenuated the p42/p44 MAPK phosphorylation and [3H]thymidine incorporation stimulated by BK, but had no effect on Akt activation. Moreover, BK-induced transactivation of EGFR and cell proliferation was blocked by matrix metalloproteinase inhibitor GM6001. These results suggest that, in VSMCs, the mechanism of BK-stimulated activation of p42/p44 MAPK and cell proliferation was mediated, at least in part, through activation of Src family kinases, EGFR transactivation, and PI3-K/Akt. Copyright © 2004 Wiley-Liss, Inc. [source] Novel biphasic traffic of endocytosed EGF to recycling and degradative compartments in lacrimal gland acinar cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2004Jiansong Xie The purpose of this study was to delineate the traffic patterns of EGF and EGF receptors (EGFR) in primary cultured acinar epithelial cells from rabbit lacrimal glands. Uptake of [125I]-EGF exhibited saturable and non-saturable, temperature-dependent components, suggesting both receptor-mediated and fluid phase endocytosis. Accumulation of [125I] was time-dependent over a 120-min period, but the content of intact [125I]-EGF decreased after reaching a maximum at 20 min. Analytical fractionation by sorbitol density gradient centrifugation and phase partitioning indicated that within 20 min at 37°C [125I] reached an early endosome, basal,lateral recycling endosome, pre-lysosome, and lysosome. Small components of the label also appeared to reach the Golgi complex and trans -Golgi network. Intact [125I]-EGF initially accumulated in the recycling endosome; the content in the recycling endosome subsequently decreased, and by 120 min increased amounts of [125I]-labeled degradation products appeared in the pre-lysosomes and lysosomes. Confocal microscopy imaging of FITC-EGF and LysoTrackerRed revealed FITC enriched in a dispersed system of non-acidic compartments at 20 min and in acidic compartments at 120 min. Both confocal immunofluorescence microscopy and analytical fractionation indicated that the intracellular EGFR pool was much larger than the plasma membrane-expressed pool at all times. Cells loaded with [125I]-EGF released a mixture of intact EGF and [125I]-labeled degradation products. The observations indicate that in lacrimal acinar cells, EGFR and EGF,EGFR complexes continually traffic between the plasma membranes and a system of endomembrane compartments; EGF-stimulation generates time-dependent signals that initially decrease, then increase, EGF,EGFR traffic to degradative compartments. J. Cell. Physiol. 199: 108,125, 2004© 2003 Wiley-Liss, Inc. [source] Influences of dopaminergic lesion on epidermal growth factor-ErbB signals in Parkinson's disease and its model: neurotrophic implication in nigrostriatal neuronsJOURNAL OF NEUROCHEMISTRY, Issue 4 2005Yuriko Iwakura Abstract Epidermal growth factor (EGF) is a member of a structurally related family containing heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor alpha (TGF,) that exerts neurotrophic activity on midbrain dopaminergic neurons. To examine neurotrophic abnormality in Parkinson's disease (PD), we measured the protein content of EGF, TGF,, and HB-EGF in post-mortem brains of patients with Parkinson's disease and age-matched control subjects. Protein levels of EGF and tyrosine hydroxylase were decreased in the prefrontal cortex and the striatum of patients. In contrast, HB-EGF and TGF, levels were not significantly altered in either region. The expression of EGF receptors (ErbB1 and ErbB2, but not ErbB3 or ErbB4) was down-regulated significantly in the same forebrain regions. The same phenomenon was mimicked in rats by dopaminergic lesions induced by nigral 6-hydroxydopamine infusion. EGF and ErbB1 levels in the striatum of the PD model were markedly reduced on the lesioned side, compared with the control hemisphere. Subchronic supplement of EGF in the striatum of the PD model locally prevented the dopaminergic neurodegeration as measured by tyrosine hydroxylase immunoreactivity. These findings suggest that the neurotrophic activity of EGF is maintained by afferent signals of midbrain dopaminergic neurons and is impaired in patients with Parkinson's disease. [source] Transglutaminase differentially regulates growth signalling in rat perivenous and periportal hepatocytesCELL PROLIFERATION, Issue 3 2006A. Maruko PPH and PVH subpopulations have been isolated using the digitonin/collagenase perfusion technique. DNA synthesis was assessed by [3H] thymidine incorporation into hepatocytes. The assay for binding of [125I] EGF to cultured hepatocytes was analysed by Scatchard plot analysis. Pretreatment with the TG2 inhibitor monodansylcadaverine (MDC) greatly increased EGF-induced DNA synthesis in both PPH and PVH. Furthermore, [125I] EGF binding studies in PVH treated with MDC indicated that high-affinity EGF receptor expression was markedly up-regulated, whereas in PPH, there was no significant effect. Treatment with retinoic acid (RA), an inducer of TG2 expression, significantly decreased EGF-induced DNA synthesis in both PPH and PVH. Binding studies in the presence of RA revealed that the high-affinity EGF receptor was down-regulated and completely absent in both PPH and PVH. These results suggest that TG2 was involved in the differential growth capacities of PPH and PVH through down-regulation of high-affinity EGF receptors. [source] Bile Acids Initiate Lineage-Addicted Gastroesophageal Tumorigenesis by Suppressing the EGF Receptor-AKT AxisCLINICAL AND TRANSLATIONAL SCIENCE, Issue 4 2009Li Gong M.D., Ph.D. Abstract While bile acids are a risk factor for tumorigenesis induced by reflux disease, the mechanisms by which they contribute to neoplasia remain undefined. Here, we reveal that in gastroesophageal junction (GEJ) cells bile acids activate a tissue-specific developmental program defining the intestinal epithelial cell phenotype characterizing GEJ metaplasia. Deoxycholic acid (DCA) inhibited phosphorylation of EGF receptors (EGFRs) suppressing the proto-oncogene AKT. Suppression of EGFRs and AKT by DCA actuated an intestine-specific cascade in which NF-,B transactivated the tissue-specifi c transcription factor CDX2. In turn, CDX2 orchestrated a lineage-specific differentiation program encompassing genes characterizing intestinal epithelial cells. Conversely, progression from metaplasia to invasive carcinoma in patients, universally associated with autonomous activation of EGFRs and/or AKT, was coupled with loss of this intestinal program. Thus, bile acids induce intestinal metaplasia at the GEJ by activating the lineage-specifi c differentiation program involving suppression of EGFR and AKT, activating the NF-,B-CDX2 axis. Induction of this axis provides the context for lineage-addicted tumorigenesis, in which autonomous activation of AKT corrupts adaptive intestinal NF-,B signaling, amplifying tumorigenic programs. 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