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Sample records for activated egf receptor

  1. EGF stimulates the activation of EGF receptors and the selective activation of major signaling pathways during mitosis.

    PubMed

    Wee, Ping; Shi, Huaiping; Jiang, Jennifer; Wang, Yuluan; Wang, Zhixiang

    2015-03-01

    Mitosis and epidermal growth factor (EGF) receptor (EGFR) are both targets for cancer therapy. The role of EGFR signaling in mitosis has been rarely studied and poorly understood. The limited studies indicate that the activation of EGFR and downstream signaling pathways is mostly inhibited during mitosis. However, we recently showed that EGFR is phosphorylated in response to EGF stimulation in mitosis. Here we studied EGF-induced EGFR activation and the activation of major signaling pathways downstream of EGFR during mitosis. We showed that EGFR was strongly activated by EGF during mitosis as all the five major tyrosine residues including Y992, Y1045, Y1068, Y1086, and Y1173 were phosphorylated to a level similar to that in the interphase. We further showed that the activated EGFR is able to selectively activate some downstream signaling pathways while avoiding others. Activated EGFR is able to activate PI3K and AKT2, but not AKT1, which may be responsible for the observed effects of EGF against nocodazole-induced cell death. Activated EGFR is also able to activate c-Src, c-Cbl and PLC-γ1 during mitosis. However, activated EGFR is unable to activate ERK1/2 and their downstream substrates RSK and Elk-1. While it activated Ras, EGFR failed to fully activate Raf-1 in mitosis due to the lack of phosphorylation at Y341 and the lack of dephosphorylation at pS259. We conclude that contrary to the dogma, EGFR is activated by EGF during mitosis. Moreover, EGFR-mediated cell signaling is regulated differently from the interphase to specifically serve the needs of the cell in mitosis.

  2. EGF receptor ligands: recent advances

    PubMed Central

    Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J.

    2016-01-01

    Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR.

  3. EGF receptor ligands: recent advances.

    PubMed

    Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J

    2016-01-01

    Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR. PMID:27635238

  4. EGF receptor ligands: recent advances

    PubMed Central

    Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J.

    2016-01-01

    Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR. PMID:27635238

  5. Autocrine EGF receptor activation mediates endothelial cell migration and vascular morphogenesis induced by VEGF under interstitial flow

    SciTech Connect

    Semino, Carlos E. . E-mail: semino@mit.edu; Kamm, Roger D.; Lauffenburger, Douglas A.

    2006-02-01

    We show here that autocrine ligand activation of epidermal growth factor (EGF) receptor in combination with interstitial flow is critically involved in the morphogenetic response of endothelial cells to VEGF stimulation. Human umbilical vein endothelial cell (HUVEC) monolayers cultured on a collagen gel and exposed to low interstitial flow in the absence of EGF and VEGF remained viable and mitotic but exhibited little evidence of vascular morphogenesis. Addition of VEGF produced a flow-dependent morphogenetic response within 48 to 72 h, characterized by branched capillary-like structures. The response was substantially abolished by inhibitors related to the autocrine EGF receptor pathway including Galardin, AG1478, PD98059, and an EGF receptor-blocking antibody, indicating that regulation of the morphogenetic process operates via autocrine EGF receptor activation. Moreover, we observed that in our system the EGF receptor was always activated independently of the interstitial flow, and, in addition, the EGF receptor inhibitors used above reduced the phosphorylation state of the receptor, correlating with inhibition of capillary morphogenesis. Finally, 5'bromo-2'-deoxyuridine (BrdU) labeling identified dividing cells at the monolayer but not in the extending capillary-like structures. EGF pathway inhibitors Galardin and AG1478 did not reduce BrdU incorporation in the monolayer, indicating that the EGF-receptor-mediated morphogenetic behavior is mainly due to cell migration rather than proliferation. Based on these results, we propose a two-step model for in vitro capillary morphogenesis in response to VEGF stimulation with interstitial fluid flow: monolayer maintenance by mitotic activity independent of EGF receptors and a migratory response mediated by autocrine EGF receptor activation wherein cells establish capillary-like structures.

  6. Is receptor oligomerization causally linked to activation of the EGF receptor kinase?

    NASA Technical Reports Server (NTRS)

    Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Transduction of a signal from an extracellular peptide hormone to produce an intracellular response is often mediated by a cell surface receptor, which is usually a glycoprotein. The secondary intracellular signal(s) generated after hormone binding to the receptor have been intensively studied. The nature of the primary signal generated by ligand binding to the receptor is understood less well in most cases. The particular case of the epidermal growth factor (EGF) receptor is analyzed, and evidence for or against two dissimilar models of primary signal transduction is reviewed. Evidence for the most widely accepted current model is found to be unconvincing. Evidence for the other model is substantial but indirect; a direct test of this model remains to be done.

  7. EGF or PDGF receptors activate atypical PKClambda through phosphatidylinositol 3-kinase.

    PubMed Central

    Akimoto, K; Takahashi, R; Moriya, S; Nishioka, N; Takayanagi, J; Kimura, K; Fukui, Y; Osada, S i; Mizuno, K; Hirai, S i; Kazlauskas, A; Ohno, S

    1996-01-01

    Overexpression of a TPA-insensitive PKC member, an atypical protein kinase C (aPKClambda), results in an enhancement of the transcriptional activation of TPA response element (TRE) in cells stimulated with epidermal growth factor (EGF) or platelet-derived growth factor (PDGF). EGF or PDGF also caused a transient increase in the in vivo phosphorylation level and a change in the intracellular localization of aPKClambda from the nucleus to the cytosol, indicating the activation of aPKClambda in response to this growth factor stimulation. These immediate signal-dependent changes in aKPClambda were observed for a PDGF receptor add-back mutant (Y40/51) that possesses only two of the five major autophosphorylation sites and binds PI3-kinase, and were inhibited by wortmannin, an inhibitor of PI3-kinase. Furthermore, an N-terminal fragment of the catalytic subunit of PI3-kinase, p110alpha, inhibited aPKClambda-dependent activation of TRE in Y40/51 cells stimulated with PDGF. Overexpression of p110alpha resulted in an enhancement of TRE expression in response to PDGF and the regulatory domain of aPKClambda inhibited this TRE activation in Y40/51 cells. These results provide the first in vivo evidence supporting the presence of a novel signalling pathway from receptor tyrosine kinases to aPKClambda through PI3-kinase. Images PMID:8631300

  8. Targeting the EGF Receptor for Ovarian Cancer Therapy

    PubMed Central

    Zeineldin, Reema; Muller, Carolyn Y.; Stack, M. Sharon; Hudson, Laurie G.

    2010-01-01

    Ovarian carcinoma is the leading cause of death from gynecologic malignancy in the US. Factors such as the molecular heterogeneity of ovarian tumors and frequent diagnosis at advanced stages hamper effective disease treatment. There is growing emphasis on the identification and development of targeted therapies to disrupt molecular pathways in cancer. The epidermal growth factor (EGF) receptor is one such protein target with potential utility in the management of ovarian cancer. This paper will discuss contributions of EGF receptor activation to ovarian cancer pathogenesis and the status of EGF receptor inhibitors and EGF receptor targeted therapies in ovarian cancer treatment. PMID:20066160

  9. Roles of mitogen activated protein kinases and EGF receptor in arsenite-stimulated matrix metalloproteinase-9 production

    SciTech Connect

    Cooper, Karen L.; Myers, Terrance Alix; Rosenberg, Martina; Chavez, Miquella; Hudson, Laurie G. . E-mail: lghudson@unm.edu

    2004-11-01

    The dermatotoxicity of arsenic is well established and epidemiological studies identify an increased incidence of keratinocytic tumors (basal cell and squamous cell carcinoma) associated with arsenic exposure. Little is known about the underlying mechanisms of arsenic-mediated skin carcinogenesis, but activation of mitogen-activated protein (MAP) kinases and subsequent regulation of downstream target genes may contribute to tumor promotion and progression. In this study, we investigated activation of the extracellular signal regulated kinase (ERK) and the stress-associated kinase p38 by arsenite in HaCat cells, a spontaneously immortalized human keratinocyte cell line. Arsenite concentrations {>=}100 {mu}M stimulate rapid activation of p38 and ERK MAP kinases. However, upon extended exposure (24 h), persistent stimulation of p38 and ERK MAP kinases was detected at low micromolar concentrations of arsenite. Although ERK and p38 were activated with similar time and concentration dependence, the mechanism of activation differed for these two MAP kinases. ERK activation by arsenite was fully dependent on the catalytic activity of the epidermal growth factor (EGF) receptor and partially dependent on Src-family kinase activity. In contrast, p38 activation was independent of EGF receptor or Src-family kinase activity. Arsenite-stimulated MAP kinase signal transduction resulted in increased production of matrix metalloproteinase (MMP)-9, an AP-1 regulated gene product. MMP-9 induction by arsenite was prevented when EGF receptor or MAP kinase signaling was inhibited. These studies indicate that EGF receptor activation is a component of arsenite-mediated signal transduction and gene expression in keratinocytes and that low micromolar concentrations of arsenite stimulate key signaling pathways upon extended exposure. Stimulation of MAP kinase cascades by arsenic and subsequent regulation of genes including c-fos, c-jun, and the matrix degrading proteases may play an important

  10. Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor

    SciTech Connect

    Whiteley, B.; Glaser, L.

    1986-10-01

    Treatment of cells with tumor-promoting phorbol diesters, which causes activation of protein kinase C, leads to phosphorylation of the epidermal growth factor (EGF) receptor at threonine-654. Addition of phorbol diesters to intact cells causes inhibition of the EGF-induced tyrosine-protein kinase activity of the EGF receptor and it has been suggested that this effect of phorbol diesters is mediated by the phosphorylation of the receptor by protein kinase C. The authors measured the activity of protein kinase C in A431 cells by determining the incorporation of (/sup 32/P)phosphate into peptides containing threonine-654 obtained by trypsin digestion of EGF receptors. After 3 h of exposure to serum-free medium, A431 cells had no detectable protein kinase C activity. Addition of EGF to these cells resulted in (/sup 32/P) incorporation into threonine-654 as well as into tyrosine residues. This indicates that EGF promotes the activation of protein kinase C in A431 cells. The phophorylation of threonine-654 induced by EGF was maximal after only 5 min of EGF addition and the (/sup 32/P) incorporation into threonine-654 reached 50% of the (/sup 32/P) in a tyrosine-containing peptide. This indicates that a significant percentage of the total EGF receptors are phosphorylated by protein kinase C. A variety of external stimuli activate Na/sup +//H/sup +/ exchange, including EGF, phorbol diesters, and hypertonicity. To ascertain whether activation of protein kinase C is an intracellular common effector of all of these systems, the authors measured the activity of protein kinase C after exposure of A431 cells to hyperosmotic conditions and observed no effect on phosphorylation of threonine-654, therefore, activation of Na/sup +//H/sup +/ exchange by hypertonic medium is independent of protein kinase C activity.

  11. Epidermal growth factor (EGF)-receptor is phosphorylated at threonine-654 in A431 cells following EGF addition

    SciTech Connect

    Whiteley, B.; Glaser, L.

    1986-05-01

    It has been shown that activation of protein kinase C by tumor-promoting phorbol diesters causes phorphorylation of the EGF-receptor at threonine-654 and is believed to thereby regulate the EGF receptor tyrosine kinase and EGF binding activity. In their present studies, /sup 32/P-labeled A431 cells were treated with and without 10 nM phorbol 12-myristate 13-acetate (PMA), or with 200 ng/ml EGF. Analysis of /sup 32/P-labeled EGF receptor tryptic phosphopeptides by reverse-phase HPLC confirmed the known effects of PMA and revealed that EGF caused phosphorylation at threonine-654 as well as various tyrosine residues. This effect occurred as early as 1 minute after EGF addition and was maximal after 5 minutes. The magnitude of the response appears to be 50% of a 15 minute treatment with 10 nM PMA. Direct measurement of diacylglycerol using an E. coli diacylglycerol kinase confirmed that EGF-stimulated phosphoinositide turnover could cause very rapid activation of protein kinase C. These results imply that protein kinase C is playing a role in negative modulation of EGF-receptor activity following EGF addition to A431 cells.

  12. Identification of heparin-binding EGF-like growth factor (HB-EGF) as a biomarker for lysophosphatidic acid receptor type 1 (LPA1) activation in human breast and prostate cancers.

    PubMed

    David, Marion; Sahay, Debashish; Mege, Florence; Descotes, Françoise; Leblanc, Raphaël; Ribeiro, Johnny; Clézardin, Philippe; Peyruchaud, Olivier

    2014-01-01

    Lysophosphatidic acid (LPA) is a natural bioactive lipid with growth factor-like functions due to activation of a series of six G protein-coupled receptors (LPA₁₋₆). LPA receptor type 1 (LPA₁) signaling influences the pathophysiology of many diseases including cancer, obesity, rheumatoid arthritis, as well as lung, liver and kidney fibrosis. Therefore, LPA₁ is an attractive therapeutic target. However, most mammalian cells co-express multiple LPA receptors whose co-activation impairs the validation of target inhibition in patients because of missing LPA receptor-specific biomarkers. LPA₁ is known to induce IL-6 and IL-8 secretion, as also do LPA₂ and LPA₃. In this work, we first determined the LPA induced early-gene expression profile in three unrelated human cancer cell lines expressing different patterns of LPA receptors (PC3: LPA₁,₂,₆; MDA-MB-231: LPA1,2; MCF-7: LPA₂,₆). Among the set of genes upregulated by LPA only in LPA₁-expressing cells, we validated by QPCR and ELISA that upregulation of heparin-binding EGF-like growth factor (HB-EGF) was inhibited by LPA₁-₃ antagonists (Ki16425, Debio0719). Upregulation and downregulation of HB-EGF mRNA was confirmed in vitro in human MDA-B02 breast cancer cells stably overexpressing LPA₁ (MDA-B02/LPA₁) and downregulated for LPA₁ (MDA-B02/shLPA1), respectively. At a clinical level, we quantified the expression of LPA₁ and HB-EGF by QPCR in primary tumors of a cohort of 234 breast cancer patients and found a significantly higher expression of HB-EGF in breast tumors expressing high levels of LPA₁. We also generated human xenograph prostate tumors in mice injected with PC3 cells and found that a five-day treatment with Ki16425 significantly decreased both HB-EGF mRNA expression at the primary tumor site and circulating human HB-EGF concentrations in serum. All together our results demonstrate that HB-EGF is a new and relevant biomarker with potentially high value in quantifying LPA

  13. Interaction of a monoclonal antibody against hEGF with a receptor site for EGF.

    PubMed

    Valente, S; Souto, B; Balter, H; Welling, M M; Román, E; Robles, A; Pauwels, E K

    1999-11-01

    Epidermal growth factor (EGF) has been detected by radioimmunoassay (RIA) in different body fluids such as serum, amniotic fluid, and urine. Human tumor tissues with EGF receptors (EGF-Rc) may be saturated with EGF, which may be of prognostic value. An RIA was envisaged to measure human epidermal growth factor (hEGF) levels using EGF-Rc as capture agent and a monoclonal antibody anti-hEGF (MAb anti-hEGF) labeled with 125Iodine as a marker for this binding. The purpose of this work was to study the feasibility of MAb anti-hEGF to detect the receptor binding sites and to investigate the interaction between MAb anti-hEGF and the EGF-Rc. Various binding experiments were performed to study possible interference and interactions in the complex MAb anti-hEGF and the receptor. Affinity constants were determined by means of Scatchard plot analysis to interpret the complex stability challenged with other compounds for a better understanding of the interaction process. Binding constants were of the same order for all the ligands tested separately involving the EGF-Rc, but were significantly higher (t = 15.7, p < 0.05) for hEGF in its binding to MAb anti-hEGF. It was possible with equilibrium studies and competition experiments to evaluate the interaction of EGF and MAb anti-hEGF with the EGF receptor. This observation makes the MAb anti-hEGF a potential tracer for the quantitation of receptors in vitro, and possibly for the detection of membrane receptors on tumor cells in vivo.

  14. HEPARIN-BINDING EGF CLEAVAGE MEDIATES ZINC-INDUCED EGF RECEPTOR PHOSPHORYLATION

    EPA Science Inventory

    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 cel...

  15. PET imaging of EGF receptors using [18F]FBEM-EGF in a Head and Neck Squamous Cell Carcinoma model

    PubMed Central

    Li, Weihua; Niu, Gang; Lang, Lixin; Guo, Ning; Ma, Ying; Kiesewetter, Dale O.; Backer, Joseph M.; Shen, Baozhong; Chen, Xiaoyuan

    2011-01-01

    Purpose To prepare and evaluate a new radiotracer for molecular imaging of cell surface receptors for epidermal growth factor (EGF). Methods Cys tagged EGF (cEGF) was labeled with 18F by coupling the free thiol group of the Cys tag with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM) to form [18F]FBEM-cEGF. Cell uptake, internalization and efflux of [18F]FBEM-cEGF were tested in human head and neck squamous carcinoma UM-SCC1 cells. In vivo tumor targeting and pharmacokinetics of the radiotracers were evaluated in UM-SCC1 tumor-bearing athymic nude mice by static and dynamic microPET imaging. Ex vivo biodistribution assays were performed to confirm the noninvasive imaging results. Results The radiolabeling yield for [18F]FBEM-cEGF was over 60%, based on starting [18F]FBEM. [18F]FBEM-cEGF exhibited rapid blood clearance through both hepatobiliary and renal excretion. UM-SCC1 tumors were clearly visualized and showed modest tracer uptake of 2.60 ± 0.59 %ID/g at 30 min post injection. Significantly higher tumor uptake of [18F]FBEM-cEGF (5.99 ± 1.61 %ID/g at 30 min p.i., p < 0.01) and tumor/non-tumor ratio were achieved by co-injection of 50 μg of unlabeled EGF. Decreased liver uptake of [18F]FBEM-cEGF was observed when unlabeled EGF was co-administered. Conclusion With optimized liver blocking, [18F]FBEM-cEGF has the potential to be used in a non-invasive and quantitative manner for detection of malignant lesions and evaluation of EGFR activity. PMID:22109665

  16. Carboxy-terminal truncations of epidermal growth factor (EGF) receptor affect diverse EGF-induced cellular responses.

    PubMed

    Li, W; Hack, N; Margolis, B; Ullrich, A; Skorecki, K; Schlessinger, J

    1991-08-01

    The binding of epidermal growth factor (EGF) to its receptor induces tyrosine phosphorylation of phospholipase C gamma (PLC gamma), which appears to be necessary for its activation leading to phosphatidyl inositol (PI) hydrolysis. Moreover, EGF-receptor (EGF-R) activation and autophosphorylation results in binding of PLC gamma to the tyrosine phosphorylated carboxy-terminus of the receptor. To gain further insights into the mechanisms and interactions regulating these processes, we have analyzed transfected NIH-3T3 cells expressing two EGF-R carboxy-terminal deletion mutants (CD63 and CD126) with reduced capacity to stimulate PI hydrolysis, Ca2+ rises, and DNA synthesis. In fact, the CD126 mutant lacking 126 carboxy-terminal amino acids, including four tyrosine autophosphorylation sites, was unable to stimulate PI hydrolysis or Ca2+ rise in response to EGF. Surprisingly, EGF binding to the cell lines expressing CD63 or CD126 mutants was followed by similar stimulation of tyrosine phosphorylation of PLC gamma. Our results suggest that although necessary, tyrosine phosphorylation of PLC gamma may not be sufficient for stimulation and PI hydrolysis. It is clear, however, that the carboxy-terminal region of EGF-R is involved in regulation of interactions with cellular targets and therefore plays a crucial role in postreceptor signaling pathways.

  17. Src-Mediated EGF Receptor Activation Regulates Ozone-Induced Interleukin 8 Expression in Human Bronchial Epithelial Cells

    PubMed Central

    Wages, Phillip A.; Devlin, Robert B.; Diaz-Sanchez, David; Peden, David B.; Samet, James M.

    2014-01-01

    Background: Human exposure to ozone (O3) results in pulmonary function decrements and airway inflammation. The mechanisms underlying these adverse effects remain unclear. Epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of lung inflammation. Objective We examined the role of EGFR activation in O3-induced expression of the chemokine interleukin 8 (IL-8) in human bronchial epithelial cells (HBEC). Methods We detected phosphorylated EGFR using immunoblotting. EGFR dimerization was examined through cross-linking reaction and immunoblotting, and levels of IL-8 protein were measured using ELISA. Results Exposure to O3 (0.25–1.0 ppm) induced rapid and marked increase in EGFR phosphorylation at the autophosphorylation site Y1068 and the transphosphorylation site Y845, implicating the involvement of Src kinase. Further investigation showed that O3 stimulation induced phosphorylation of Src at Y416, indicative of Src activation. Pharmacological inhibition of Src kinase activity abrogated O3-induced EGFR phosphorylation at tyrosines 1068 and 845. Moreover, pretreatment of BEAS-2B cells with inhibitor of either EGFR or Src kinase activities significantly blocked O3-induced IL-8 expression. Conclusion Conclusion: O3 exposure increased IL-8 expression through Src-mediated EGFR transactivation in HBEC. Citation> Wu W, Wages PA, Devlin RB, Diaz-Sanchez D, Peden DB, Samet JM. 2015. Src-mediated EGF receptor activation regulates ozone-induced interleukin 8 expression in human bronchial epithelial cells. Environ Health Perspect 123:231–236; http://dx.doi.org/10.1289/ehp.1307379 PMID:25303742

  18. Affinity Regulates Spatial Range of EGF Receptor Autocrine Ligand Binding

    SciTech Connect

    Dewitt, Ann; Iida, Tomoko; Lam, Ho-Yan; Hill, Virginia; Wiley, H S.; Lauffenburger, Douglas A.

    2002-08-08

    Proper spatial localization of EGFR signaling activated by autocrine ligands represents a critical factor in embryonic development as well as tissue organization and function, and ligand/receptor binding affinity is among the molecular and cellular properties suggested to play a role in governing this localization. The authors employ a computational model to predict how receptor-binding affinity affects local capture of autocrine ligand vis-a-vis escape to distal regions, and provide experimental test by constructing cell lines expressing EGFR along with either wild-type EGF or a low-affinity mutant, EGF{sup L47M}. The model predicts local capture of a lower affinity autocrine ligand to be less efficient when the ligand production rate is small relative to receptor appearance rate. The experimental data confirm this prediction, demonstrating that cells can use ligand/receptor binding affinity to regulate ligand spatial distribution when autocrine ligand production is limiting for receptor signaling.

  19. IL-1beta signals through the EGF receptor and activates Egr-1 through MMP-ADAM.

    PubMed

    Sanchez-Guerrero, Estella; Chen, Elya; Kockx, Maaike; An, Si-Wei; Chong, Beng H; Khachigian, Levon M

    2012-01-01

    The immediate-early gene Egr-1 controls the inducible expression of many genes implicated in the pathogenesis of a range of vascular disorders, yet our understanding of the mechanisms controlling the rapid expression of this prototypic zinc finger transcription factor is poor. Here we show that Egr-1 expression induced by IL-1beta is dependent on metalloproteinases (MMP) and a disintegrin and a metalloproteinase (ADAM). Pharmacologic MMP/ADAM inhibitors and siRNA knockdown prevent IL-1beta induction of Egr-1. Further, IL-1beta activates Egr-1 via the epidermal growth factor receptor (EGFR). This is blocked by EGFR tyrosine kinase inhibition and EGFR knockdown. IL-1beta induction of Egr-1 expression is reduced in murine embryonic fibroblasts (mEFs) deficient in ADAM17 despite unbiased expression of EGFR and IL-1RI in ADAM17-deficient and wild-type mEFs. Finally, we show that IL-1beta-inducible wound repair after mechanical injury requires both EGFR and MMP/ADAM. This study reports for the first time that Egr-1 induction by IL-1beta involves EGFR and MMP/ADAM-dependent EGFR phosphorylation. PMID:22792188

  20. EGF receptor-targeted nanocarriers for enhanced cancer treatment

    PubMed Central

    Master, Alyssa M; Gupta, Anirban Sen

    2013-01-01

    The ‘nanomedicine’ approach has revolutionized cancer therapy by enabling the packaging of therapeutic agents within engineered nanovehicles that can specifically accumulate within the tumor stroma and then be internalized within cancer cells, to render site-selective action while minimizing nonspecific uptake and harmful side effects. While the specific accumulation within the tumor stroma is rendered by the ability of the nanovehicles to passively permeate through the tumor’s leaky vasculature, the cellular internalization is often achieved by exploiting receptor-mediated active endocytotic mechanisms using receptor-specific ligand decoration on the vehicle surface. To this end, a highly important receptor found in several cancers is the EGF receptor, which has been implicated in tumor aggression and proliferation. In this context, we provide a comprehensive review of the various approaches of ligand decorations on nanovehicles for active targeting to EGF receptors, and discuss their pros and cons towards optimizing the design of EGF receptor-targeted nanomedicine systems. PMID:23249333

  1. ACTIVATION OF THE EGF RECEPTOR SIGNALING PATHWAY IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO METALS

    EPA Science Inventory

    We have previously shown that exposure to combustion-derived metals rapidly (within 20 min) activated mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), in the human bronchial epithelial cell line BEAS. To study the mechanisms respons...

  2. The linear C-terminal regions of epidermal growth factor (EGF) and transforming growth factor-alpha bind to different epitopes on the human EGF receptor.

    PubMed Central

    Lenferink, A E; De Roos, A D; Van Vugt, M J; Van de Poll, M L; Van Zoelen, E J

    1998-01-01

    Epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) bind with similar affinities in a competitive fashion to the human EGF receptor, and basically induce similar mitogenic responses. In spite of the fact that EGF and TGFalpha are structurally alike, it is still not clear if the two growth factors bind the receptor in an identical manner. The observation that the 13A9 antibody blocks binding of TGFalpha, but not that of EGF, to the human EGF receptor [Winkler, O'Connor, Winget and Fendly (1989) Biochemistry 28, 6373-6378] suggests that their binding characteristics are not identical. In the present study we have made use of a set of EGF/TGFalpha chimaeric molecules to show that the 13A9 antibody blocks receptor binding of ligands with TGFalpha sequences, but not of ligands with EGF sequences, in their C-terminal linear regions. Using HaCaT human keratinocyte cells in culture, it was determined that ligands that are able to bind the EGF receptor in the presence of 13A9 are also able to induce calcium release from intracellular stores in these cells, indicating that these ligands have the ability to activate the EGF receptor in the presence of the antibody. From these data it is concluded that the flexible C-terminal linear domains of EGF and TGFalpha bind to separate sequences on the EGF receptor, such that the binding domain of TGFalpha, but not that of EGF, overlaps with the binding epitope of the 13A9 antibody. PMID:9806896

  3. Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting.

    PubMed

    Higginbotham, James N; Zhang, Qin; Jeppesen, Dennis K; Scott, Andrew M; Manning, H Charles; Ochieng, Josiah; Franklin, Jeffrey L; Coffey, Robert J

    2016-01-01

    Exosomes are small, 40-130 nm secreted extracellular vesicles that recently have become the subject of intense focus as agents of intercellular communication, disease biomarkers and potential vehicles for drug delivery. It is currently unknown whether a cell produces different populations of exosomes with distinct cargo and separable functions. To address this question, high-resolution methods are needed. Using a commercial flow cytometer and directly labelled fluorescent antibodies, we show the feasibility of using fluorescence-activated vesicle sorting (FAVS) to analyse and sort individual exosomes isolated by sequential ultracentrifugation from the conditioned medium of DiFi cells, a human colorectal cancer cell line. EGFR and the exosomal marker, CD9, were detected on individual DiFi exosomes by FAVS; moreover, both markers were identified by high-resolution stochastic optical reconstruction microscopy on individual, approximately 100 nm vesicles from flow-sorted EGFR/CD9 double-positive exosomes. We present evidence that the activation state of EGFR can be assessed in DiFi-derived exosomes using a monoclonal antibody (mAb) that recognizes "conformationally active" EGFR (mAb 806). Using human antigen-specific antibodies, FAVS was able to detect human EGFR and CD9 on exosomes isolated from the plasma of athymic nude mice bearing DiFi tumour xenografts. Multicolour FAVS was used to simultaneously identify CD9, EGFR and an EGFR ligand, amphiregulin (AREG), on human plasma-derived exosomes from 3 normal individuals. These studies demonstrate the feasibility of FAVS to both analyse and sort individual exosomes based on specific cell-surface markers. We propose that FAVS may be a useful tool to monitor EGFR and AREG in circulating exosomes from individuals with colorectal cancer and possibly other solid tumours. PMID:27345057

  4. Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting

    PubMed Central

    Higginbotham, James N.; Zhang, Qin; Jeppesen, Dennis K.; Scott, Andrew M.; Manning, H. Charles; Ochieng, Josiah; Franklin, Jeffrey L.; Coffey, Robert J.

    2016-01-01

    Exosomes are small, 40–130 nm secreted extracellular vesicles that recently have become the subject of intense focus as agents of intercellular communication, disease biomarkers and potential vehicles for drug delivery. It is currently unknown whether a cell produces different populations of exosomes with distinct cargo and separable functions. To address this question, high-resolution methods are needed. Using a commercial flow cytometer and directly labelled fluorescent antibodies, we show the feasibility of using fluorescence-activated vesicle sorting (FAVS) to analyse and sort individual exosomes isolated by sequential ultracentrifugation from the conditioned medium of DiFi cells, a human colorectal cancer cell line. EGFR and the exosomal marker, CD9, were detected on individual DiFi exosomes by FAVS; moreover, both markers were identified by high-resolution stochastic optical reconstruction microscopy on individual, approximately 100 nm vesicles from flow-sorted EGFR/CD9 double-positive exosomes. We present evidence that the activation state of EGFR can be assessed in DiFi-derived exosomes using a monoclonal antibody (mAb) that recognizes “conformationally active” EGFR (mAb 806). Using human antigen-specific antibodies, FAVS was able to detect human EGFR and CD9 on exosomes isolated from the plasma of athymic nude mice bearing DiFi tumour xenografts. Multicolour FAVS was used to simultaneously identify CD9, EGFR and an EGFR ligand, amphiregulin (AREG), on human plasma-derived exosomes from 3 normal individuals. These studies demonstrate the feasibility of FAVS to both analyse and sort individual exosomes based on specific cell-surface markers. We propose that FAVS may be a useful tool to monitor EGFR and AREG in circulating exosomes from individuals with colorectal cancer and possibly other solid tumours. PMID:27345057

  5. ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES SRC-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)

    EPA Science Inventory

    ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES Src-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)
    Weidong Wu1, Lee M. Graves2, Gordon N. Gill3 and James M. Samet4 1Center for Environmental Medicine and Lung Biology; 2Department of Pharmacology, University o...

  6. Lysophosphatidic Acid-induced ERK Activation and Chemotaxis in MC3T3-E1 Preosteoblasts are Independent of EGF Receptor Transactivation

    SciTech Connect

    Karagiosis, Sue A.; Chrisler, William B.; Bollinger, Nikki; Karin, Norman J.

    2009-06-01

    Growing evidence indicates that bone-forming osteoblasts and their progenitors are target cells for the lipid growth factor lysophosphatidic acid (LPA) which is produced by degranulating platelets at sites of injury. LPA is a potent inducer of bone cell migration, proliferation and survival in vitro and an attractive candidate to facilitate preosteoblast chemotaxis during skeletal regeneration in vivo, but the intracellular signaling pathways mediating the effects of this lipid on bone cells are not defined. In this study we measured the ability of LPA to stimulate extracellular signal-related kinase (ERK1/2) in MC3T3-E1 preosteoblastic cells and determined the contribution of this pathway to LPA-stimulated chemotaxis. LPA-treated cells exhibited a bimodal activation of ERK1/2 with maximal phosphorylation at 5 and 60 minutes. The kinetics of ERK1/2 phosphorylation were not coupled to Ras activation or LPA-induced elevations in cytosolic Ca2+. While LPA is coupled to the transactivation of the EGF receptor in many cell types, LPA-stimulated ERK1/2 activation in MC3T3-E1 cells was unaffected by inhibition of EGF receptor function. ERK isoforms rapidly accumulated at nuclear sites in LPA-treated cells, a process that was blocked if ERK1/2 phosphorylation was prevented with the MEK1 inhibitor U0126. Blocking ERK1/2 phosphorylation with U0126 also diminished MC3T3-E1 cell migration and altered the normal disassembly of LPA-induced stress fibers, while the inhibition of EGF receptor function had no effect on LPA-coupled preosteoblast motility. Our results identify ERK1/2 activation as a mediatora mediator of LPA-stimulated MC3T3-E1 cell migration that may be relevant to preosteoblast motility during bone repair in vivo.

  7. EGF receptor inhibitors in the treatment of glioblastoma multiform: old clinical allies and newly emerging therapeutic concepts.

    PubMed

    Gadji, Macoura; Crous, Ana-Maria Tsanaclis; Fortin, David; Krcek, Jerry; Torchia, Mark; Mai, Sabine; Drouin, Regen; Klonisch, Thomas

    2009-12-25

    Glioblastoma multiform (GBM) is the most common malignant brain tumour in adults. Despite decades of experimentation to improve the outcome of patients with GBM this highly aggressive tumour remains fatal. Primary GBM are often characterized by the over-expression of epidermal growth factor (EGF) receptor/HER1 and/or its mutational variants, with ligand-independent, constitutively active EGF receptor vIII variant most frequently observed in GBM. EGF receptor signalling can promote tumorigenesis by increasing cell proliferation, tissue invasion, neoangiogenesis, tumour cell chemoresistance, and by inhibiting apoptosis of cancer cells. EGF receptor was the first receptor to serve as target for cancer therapy of many solid tumours. After 2 decades of intensive targeting of EGF receptor for molecular therapy, several anti-EGF receptor inhibitors are now available in the clinic. Therapeutic strategies to target EGF receptor and EGF receptor mutant forms in GBM include humanized monoclonal antibodies, tyrosine kinase inhibitors, and RNAi compounds. However, despite the fact that most EGF receptor-directed glioma therapies to date have focused on single therapeutic agents, a multi-directional approach involving targeted inhibition of multiple signalling pathways has emerged as a more robust therapeutical approach. Furthermore, the emergence of the hypothesis of "brain cancer stem cells" in the bulb of GBM identifies this population of cells with self-renewal capacity as novel obligatory targets for efficient cure of GBM. Here we summarize current findings on the clinical role of these EGF receptor inhibitory therapeutic agents in the treatment of GBM.

  8. ERK phosphorylation in intact, adult brain by alpha(2)-adrenergic transactivation of EGF receptors.

    PubMed

    Du, Ting; Li, Baoman; Liu, Shufang; Zang, Peizhuo; Prevot, Vincent; Hertz, Leif; Peng, Liang

    2009-12-01

    Our previous work demonstrated dexmedetomidine-activated phosphorylation of extracellular regulated kinases 1 and 2 (ERK(1/2)) in primary cultures of mouse astrocytes and showed that it is evoked by alpha(2)-adrenoceptor-mediated transactivation of epidermal growth factor (EGF) receptors, a known response to activation of G(i/o)- or G(q)-coupled receptors [Li, B., Du, T., Li, H., Gu, L., Zhang, H., Huang, J., Hertz, L., Peng, L., 2008a. Signaling pathways for transactivation by dexmedetomidine of epidermal growth factor receptors in astrocytes and its paracrine effect on neurons. Br. J. Pharmacol. 154, 191-203]. Like most studies of transactivation, that study used cultured cells, raising the question whether a similar effect can be demonstrated in intact brain tissue and the brain in vivo. In the present study we have shown that (i) dexmedetomidine-mediated ERK(1/2) phosphorylation occurs in mouse brain slices with a similar concentration dependence as in cultured astrocytes (near-maximum effect at 50nM); (ii) intraperitoneal injection of dexmedetomidine (3microg/kg) in adult mice causes rapid phosphorylation of the EGF receptor (at Y845 and Y992) and of ERK(1/2) in the brain; (iii) both EGF receptor and ERK(1/2) phosphorylation are inhibited by intraventricular administration of (a) AG 1478, a specific inhibitor of the receptor-tyrosine kinase of the EGF receptor; (b) GM 6001, an inhibitor of metalloproteinase(s) required for release of EGF receptor agonists from membrane-bound precursors; or (c) heparin, neutralizing heparin-binding EGF (HB-EGF). Thus, in intact brain HB-EGF, known to be expressed in brain, may be the major EGF agonist released in response to stimulation of alpha(2)-adrenoceptors, the released agonist(s) activate(s) EGF receptors, and ERK(1/2) is phosphorylated as a conventional response to EGF receptor activation. Our previous paper (see above) showed that dexmedetomidine evokes no ERK(1/2) phosphorylation in cultured neurons, but neurons

  9. Manipulating the Lateral Diffusion of Surface-Anchored EGF Demonstrates that Receptor Clustering Modulates Phosphorylation Levels

    PubMed Central

    Stabley, D.; Retterer, S.; Marshall, S.; Salaita, K.

    2013-01-01

    Upon activation, the epidermal growth factor (EGF) receptor becomes phosphorylated and triggers a vast signaling network that has profound effects on cell growth. The EGF receptor is observed to assemble into clusters after ligand binding and tyrosine kinase autophosphorylation, but the role of these assemblies in the receptor signaling pathway remains unclear. To address this question, we measured the phosphorylation of EGFR when the EGF ligand was anchored onto laterally mobile and immobile surfaces. We found that cells generated clusters of ligand-receptor complex on mobile EGF surfaces, and displayed a lower ratio of phosphorylated EGFR to EGF when compared to immobilized EGF that is unable to cluster. This result was verified by tuning the lateral assembly of ligand-receptor complexes on the surface of living cells using patterned supported lipid bilayers. Nanoscale metal lines fabricated into the supported membrane constrained lipid diffusion and EGF receptor assembly into micron and sub-micron scale corrals. Single cell analysis indicated that clustering impacts EGF receptor activation, and larger clusters (> 1 µm2) of ligand-receptor complex generated lower EGF receptor phosphorylation per ligand than smaller assemblies (< 1 µm2) in HCC1143 cells that were engaged to ligand-functionalized surfaces. We investigated the mechanism of EGFR clustering by treating cells with compounds that disrupt the cytoskeleton (Latrunculin-B), clathrin-mediated endocytosis (Pitstop2), and inhibit EGFR activation (Gefitinib). These results help elucidate the nature of large-scale EGFR clustering, thus underscoring the general significance of receptor spatial organization in tuning function. PMID:23416883

  10. Manipulating the Lateral Diffusion of Surface-Anchored EGF Demonstrates that Receptor Clustering Modulates its Phosphorylation Levels

    SciTech Connect

    Stabley, Daniel; Retterer, Scott T; Marshal, Stephen; Salaita, Khalid

    2013-01-01

    Upon activation, the epidermal growth factor (EGF) receptor becomes phosphorylated and triggers a vast signaling network that has profound effects on cell growth. The EGF receptor is observed to assemble into clusters after ligand binding and tyrosine kinase autophosphorylation, but the role of these assemblies in the receptor signaling pathway remains unclear. To address this question, we measured the phosphorylation of EGFR when the EGF ligand was anchored onto laterally mobile and immobile surfaces. We found that cells generated clusters of ligand-receptor complex on mobile EGF surfaces, and generated a lower ratio of phosphorylated EGFR to EGF than when compared to immobilized EGF that is unable to cluster. This result was verified by tuning the lateral assembly of ligand-receptor complexes on the surface of living cells using patterned supported lipid bilayers. Nanoscale metal lines fabricated into the supported membrane constrained lipid diffusion and EGF receptor assembly into micron and sub-micron scale corrals. Single cell analysis indicated that clustering impacts EGF receptor activation, and larger clusters (> 1 m2) of ligand-receptor complex generated lower EGF receptor phosphorylation per ligand than smaller assemblies (< 1 m2) in HCC1143 cells that were engaged to ligand-functionalized surfaces. We investigated EGFR clustering by treating cells with compounds that disrupt the cytoskeleton (Latrunculin-B), clathrin-mediated endocytosis (Pitstop2), and inhibit EGFR activation (Gefitinib). These results help elucidate the nature of large-scale EGFR clustering, thus underscoring the general significance of receptor spatial organization in tuning function.

  11. G-protein-coupled receptor signaling and the EGF network in endocrine systems.

    PubMed

    Hsieh, Minnie; Conti, Marco

    2005-09-01

    The epidermal growth factor (EGF) network is composed of a complex array of growth factors synthesized as precursors and expressed on the cell surface. These latent growth factors are activated by cleavage and shedding from the cell surface and act by binding to various homo- and hetero-dimers of the EGF receptors (ErbBs). Although the exact molecular steps are poorly understood, ligand binding to G-protein-coupled receptors as diverse as the beta-adrenoceptors or the lysophosphatidic acid receptors leads to shedding of EGF growth factors and activation of EGF receptors. Recent observations from the pituitary and in the ovary are providing new insight into the role of this network in endocrine systems.

  12. EGF-receptor and extracellular matrix changes in mouse pulmonary carcinogenesis

    SciTech Connect

    Smith, G.J.; Morris, C.; Leigh, D.; Rhodes, G.C.; Wong, A. )

    1991-03-01

    Malignant Balb/c mouse lung cell clones related to alveologenic carcinoma exhibited low levels of epidermal growth factor (EGF) receptor activity compared to nonmalignant cell clones. Immunoprecipitation of cell homogenates and immunohistochemistry on urethane-induced lung tumors suggest that the absence of activity reflects decreased amounts of EGF receptor protein. Low levels of EGF receptor alone cannot cause neoplastic transformation, since a nonneoplastic cell cone, B5D3, exhibited low levels of EGF receptor despite its nontransformed phenotype. The reduced levels of EGF receptor in malignant clones have been mimicked by long-term (12 h) treatment of a nontransformed cell clone with 200 nM phorbol dibutyrate. The detection of mutated ras oncogene in the transformed cell lines, taken together with the EGF receptor findings, suggests that more than one alteration in the signal transduction pathway may be necessary for transformation in alveologenic adenoma and carcinoma cell systems. A further phenotypic feature of transformation, reduced expression of the extracellular matrix proteins fibronectin and laminin, may be mediated at the transcriptional level.

  13. ROLE OF RAS IN METAL-INDUCED EGF RECEPTOR AND NFKB SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    We have shown previously that EGF receptor signaling is triggered by some metals associated with ambient air particles. Western blot using phospho-specific antibodies showed that As, Zn and V activated EGF receptor tyrosine kinase and the downstream kinases, MEK1/2 and ERK1/2. Us...

  14. The novel SAM domain protein Aveugle is required for Raf activation in the Drosophila EGF receptor signaling pathway

    PubMed Central

    Roignant, Jean-Yves; Hamel, Sophie; Janody, Florence; Treisman, Jessica E.

    2006-01-01

    Activation of the Raf kinase by GTP-bound Ras is a poorly understood step in receptor tyrosine kinase signaling pathways. One such pathway, the epidermal growth factor receptor (EGFR) pathway, is critical for cell differentiation, survival, and cell cycle regulation in many systems, including the Drosophila eye. We have identified a mutation in a novel gene, aveugle, based on its requirement for normal photoreceptor differentiation. The phenotypes of aveugle mutant cells in the eye and wing imaginal discs resemble those caused by reduction of EGFR pathway function. We show that aveugle is required between ras and raf for EGFR signaling in the eye and for mitogen-activated protein kinase phosphorylation in cell culture. aveugle encodes a small protein with a sterile α motif (SAM) domain that can physically interact with the scaffold protein connector enhancer of Ksr (Cnk). We propose that Aveugle acts together with Cnk to promote Raf activation, perhaps by recruiting an activating kinase. PMID:16600911

  15. Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha.

    PubMed Central

    Lax, I; Johnson, A; Howk, R; Sap, J; Bellot, F; Winkler, M; Ullrich, A; Vennstrom, B; Schlessinger, J; Givol, D

    1988-01-01

    The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors. Images PMID:3260329

  16. An ultrasensitive sorting mechanism for EGF Receptor Endocytosis

    PubMed Central

    Schmidt-Glenewinkel, Hannah; Vacheva, Ivayla; Hoeller, Daniela; Dikic, Ivan; Eils, Roland

    2008-01-01

    Background The Epidermal Growth Factor (EGF) receptor has been shown to internalize via clathrin-independent endocytosis (CIE) in a ligand concentration dependent manner. From a modeling point of view, this resembles an ultrasensitive response, which is the ability of signaling networks to suppress a response for low input values and to increase to a pre-defined level for inputs exceeding a certain threshold. Several mechanisms to generate this behaviour have been described theoretically, the underlying assumptions of which, however, have not been experimentally demonstrated for the EGF receptor internalization network. Results Here, we present a mathematical model of receptor sorting into alternative pathways that explains the EGF-concentration dependent response of CIE. The described mechanism involves a saturation effect of the dominant clathrin-dependent endocytosis pathway and implies distinct steady-states into which the system is forced for low vs high EGF stimulations. The model is minimal since no experimentally unjustified reactions or parameter assumptions are imposed. We demonstrate the robustness of the sorting effect for large parameter variations and give an analytic derivation for alternative steady-states that are reached. Further, we describe extensibility of the model to more than two pathways which might play a role in contexts other than receptor internalization. Conclusion Our main result is that a scenario where different endocytosis routes consume the same form of receptor corroborates the observation of a clear-cut, stimulus dependent sorting. This is especially important since a receptor modification discriminating between the pathways has not been found experimentally. The model is not restricted to EGF receptor internalization and might account for ultrasensitivity in other cellular contexts. PMID:18394191

  17. H2O2 is required for UVB-induced EGF receptor and downstream signaling pathway activation.

    PubMed

    Peus, D; Meves, A; Vasa, R A; Beyerle, A; O'Brien, T; Pittelkow, M R

    1999-12-01

    Ultraviolet radiation (UVR)-induced receptor phosphorylation is increasingly recognized as a widely occurring phenomenon. However, the mechanisms, mediators, and sequence of events involved in this process remain ill-defined. We have recently shown that exposure of human keratinocytes to physiologic doses of ultraviolet B radiation (UVB) activates epidermal growth factor receptor (EGFR)/extracellular-regulated kinase 1 and 2 (ERK1/2), and p38 signaling pathways via reactive oxygen species. Here we demonstrate that UVB exposure increased intra- and extracellular H2O2 production rapidly in a time-dependent manner. An EGFR-specific monoclonal antibody abrogated EGFR autophosphorylation and markedly decreased the phosphorylation of ERK1/2 whereas p38 activation was unaffected. Overexpression of catalase strongly inhibited UVB-induced EGFR/ERK1/2 pathway activation. These findings establish the sequence of events after UVB irradiation: (i) H2O2 generation, (ii) EGFR phosphorylation, and (iii) ERK activation. Our results identify UVB-induced H2O2 as a second messenger that is required for EGFR and dependent downstream signaling pathways activation.

  18. Odin (ANKS1A) modulates EGF receptor recycling and stability.

    PubMed

    Tong, Jiefei; Sydorskyy, Yaroslav; St-Germain, Jonathan R; Taylor, Paul; Tsao, Ming S; Moran, Michael F

    2013-01-01

    The ANKS1A gene product, also known as Odin, was first identified as a tyrosine-phosphorylated component of the epidermal growth factor receptor network. Here we show that Odin functions as an effector of EGFR recycling. In EGF-stimulated HEK293 cells tyrosine phosphorylation of Odin was induced prior to EGFR internalization and independent of EGFR-to-ERK signaling. Over-expression of Odin increased EGF-induced EGFR trafficking to recycling endosomes and recycling back to the cell surface, and decreased trafficking to lysosomes and degradation. Conversely, Odin knockdown in both HEK293 and the non-small cell lung carcinoma line RVH6849, which expresses roughly 10-fold more EGF receptors than HEK293, caused decreased EGFR recycling and accelerated trafficking to the lysosome and degradation. By governing the endocytic fate of internalized receptors, Odin may provide a layer of regulation that enables cells to contend with receptor cell densities and ligand concentration gradients that are physiologically and pathologically highly variable. PMID:23825523

  19. SRC-mediated EGF Receptor Activation Regulates Ozone-induced Interleukin 8 Expression in Human Bronchial Epithelial Cells

    EPA Science Inventory

    BACKGROUND: Human exposure to ozone (03) results in pulmonary function decrements and airway inflammation. The mechanisms underlying these adverse effects remain unclear. Epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of lung inflammation. ...

  20. PTX3 gene activation in EGF-induced head and neck cancer cell metastasis.

    PubMed

    Chang, Wei-Chiao; Wu, Shuo-Lun; Huang, Wan-Chen; Hsu, Jinn-Yuan; Chan, Shih-Hung; Wang, Ju-Ming; Tsai, Jhih-Peng; Chen, Ben-Kuen

    2015-04-10

    Overexpression of the epidermal growth factor (EGF) receptor (EGFR) is associated with enhanced invasion and metastasis in head and neck squamous cell carcinoma (HNSCC). Long Pentraxin PTX3 is involved in immune escape in cancer cells. Here, we identified PTX3 as a promoting factor that mediates EGF-induced HNSCC metastasis. EGF-induced PTX3 transcriptional activation is via the binding of c-Jun to the activator protein (AP)-1 binding site of the PTX3 promoter. PI3K/Akt and NF-κB were essential for the PTX3 activation. EGF-induced PTX3 expression was blocked in c-Jun- and NF-κB-knockdown cells. EGF-mediated PTX3 secretion resulted in the enhancement of cell migration and invasion, and interactions between cancer and endothelial cells. The tail-vein injection animal model revealed that depletion of PTX3 decreased EGF-primed tumor cell metastatic seeding of the lungs. In addition, fibronectin, matrix metalloproteinase-9 (MMP9) and E-cadherin were essential components in EGFR/PTX3-mediated cancer metastasis. In conclusion, PI3K/Akt and NF-κB-dependent regulation of AP-1 mediates PTX3 transcriptional responses to EGF. Autocrine production of EGF-induced PTX3 in turn induces metastatic molecules, activating inflammatory cascades and metastasis.

  1. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    PubMed Central

    Kourouniotis, George; Wang, Yi; Pennock, Steven; Chen, Xinmei; Wang, Zhixiang

    2016-01-01

    The binding of epidermal growth factor (EGF) to EGF receptor (EGFR) stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ) and tagged a green fluorescent protein (GFP) at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc), extracellular signal-regulated kinase (ERK) and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis. PMID:27463710

  2. Id-1 expression induces androgen-independent prostate cancer cell growth through activation of epidermal growth factor receptor (EGF-R).

    PubMed

    Ling, Ming-Tat; Wang, Xianghong; Lee, Davy T; Tam, P C; Tsao, Sai-Wah; Wong, Yong-Chuan

    2004-04-01

    The failure of prostate cancer treatment is largely due to the development of androgen independence, since the androgen depletion therapy remains the front-line option for this cancer. Previously, we reported that over-expression of the helix-loop-helix protein Id-1 was associated with progression of prostate cancer and ectopic expression of Id-1 induced serum-independent proliferation in prostate cancer cells. In the present study, we investigated if exogenous Id-1 expression in the androgen sensitive LNCaP cells had any effect on androgen-dependent cell growth and studied the molecular mechanisms involved. Using stable Id-1 transfectants, we found that expression of Id-1 was able to reduce androgen-stimulated growth and S phase fraction of the cell cycle in LNCaP cells, indicating that Id-1 may be involved in the development of androgen independence in these cells. The Id-1-induced androgen-independent prostate cancer cell growth was correlated with up-regulation of EGF-R (epidermal growth factor-receptor) and PSA (prostate specific antigen) expression, as confirmed by western blotting analysis and luciferase assays. In contrast, down-regulation of Id-1 in androgen-independent DU145 cells by its antisense oligonucleotides resulted in suppression of EGF-R expression at both transcriptional and protein levels. In addition, the results from immunohistochemistry study showed that Id-1 expression was significantly elevated in hormone refractory prostate cancer tissues when compared with the hormone-dependent tumours. Our results suggest that up-regulation of Id-1 in prostate cancer cells may be one of the mechanisms responsible for developing androgen independence and this process may be regulated through induction of EGF-R expression. Inactivation of Id-1 may provide a potential therapeutic strategy leading to inhibition of androgen-independent prostate cancer cell growth.

  3. Secreted phospholipase A2-IIA-induced a phenotype of activated microglia in BV-2 cells requires epidermal growth factor receptor transactivation and proHB-EGF shedding

    PubMed Central

    2012-01-01

    Background Activation of microglia, the primary component of the innate immune response in the brain, is a hallmark of neuroinflammation in neurodegenerative disorders, including Alzheimer’s disease (AD) and other pathological conditions such as stroke or CNS infection. In response to a variety of insults, microglial cells produce high levels of inflammatory cytokines that are often involved in neuronal injury, and play an important role in the recognition, engulfment, and clearance of apoptotic cells and/or invading microbes. Secreted phospholipase A2-IIA (sPLA2-IIA), an enzyme that interacts with cells involved in the systemic immune/inflammatory response, has been found up-regulated in the cerebrospinal fluid and brain of AD patients. However, despite several approaches, its functions in mediating CNS inflammation remain unknown. In the present study, the role of sPLA2-IIA was examined by investigating its direct effects on microglial cells. Methods Primary and immortalized microglial cells were stimulated by sPLA2-IIA in order to characterize the cytokine-like actions of the phospholipase. The hallmarks of activated microglia analyzed include: mitogenic response, phagocytic capabilities and induction of inflammatory mediators. In addition, we studied several of the potential molecular mechanisms involved in those events. Results The direct exposure of microglial cells to sPLA2-IIA stimulated, in a time- and dose-dependent manner, their phagocytic and proliferative capabilities. sPLA2-IIA also triggered the synthesis of the inflammatory proteins COX-2 and TNFα. In addition, EGFR phosphorylation and shedding of the membrane-anchored heparin-binding EGF-like growth factor (pro-HB-EGF) ectodomain, as well as a rapid activation/phosphorylation of the classical survival proteins ERK, P70S6K and rS6 were induced upon sPLA2-IIA treatment. We further demonstrated that the presence of an EGFR inhibitor (AG1478), a matrix metalloproteinase inhibitor (GM6001), an ADAM

  4. In brown adipocytes, adrenergically induced β{sub 1}-/β{sub 3}-(G{sub s})-, α{sub 2}-(G{sub i})- and α{sub 1}-(G{sub q})-signalling to Erk1/2 activation is not mediated via EGF receptor transactivation

    SciTech Connect

    Wang, Yanling; Fälting, Johanna M.; Mattsson, Charlotte L.; Holmström, Therése E.; Nedergaard, Jan

    2013-10-15

    Brown adipose tissue is unusual in that the neurotransmitter norepinephrine influences cell destiny in ways generally associated with effects of classical growth factors: regulation of cell proliferation, of apoptosis, and progression of differentiation. The norepinephrine effects are mediated through G-protein-coupled receptors; further mediation of such stimulation to e.g. Erk1/2 activation is in cell biology in general accepted to occur through transactivation of the EGF receptor (by external or internal pathways). We have examined here the significance of such transactivation in brown adipocytes. Stimulation of mature brown adipocytes with cirazoline (α{sub 1}-adrenoceptor coupled via G{sub q}), clonidine (α{sub 2} via G{sub i}) or CL316243 (β{sub 3} via G{sub s}) or via β{sub 1}-receptors significantly activated Erk1/2. Pretreatment with the EGF receptor kinase inhibitor AG1478 had, remarkably, no significant effect on Erk1/2 activation induced by any of these adrenergic agonists (although it fully abolished EGF-induced Erk1/2 activation), demonstrating absence of EGF receptor-mediated transactivation. Results with brown preadipocytes (cells in more proliferative states) were not qualitatively different. Joint stimulation of all adrenoceptors with norepinephrine did not result in synergism on Erk1/2 activation. AG1478 action on EGF-stimulated Erk1/2 phosphorylation showed a sharp concentration–response relationship (IC{sub 50} 0.3 µM); a minor apparent effect of AG1478 on norepinephrine-stimulated Erk1/2 phosphorylation showed nonspecific kinetics, implying caution in interpretation of partial effects of AG1478 as reported in other systems. Transactivation of the EGF receptor is clearly not a universal prerequisite for coupling of G-protein coupled receptors to Erk1/2 signalling cascades. - Highlights: • In brown adipocytes, norepinephrine regulates proliferation, apoptosis, differentiation. • EGF receptor transactivation is supposed to mediate GPCR

  5. Nuclear envelope-localized EGF family protein amphiregulin activates breast cancer cell migration in an EGF-like domain independent manner

    SciTech Connect

    Tanaka, Hisae; Nishioka, Yu; Yokoyama, Yuhki; Higashiyama, Shigeki; Matsuura, Nariaki; Matsuura, Shuji; Hieda, Miki

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer Nuclear envelope-localized proAREG activates cancer cell migration via its cytoplasmic domain. Black-Right-Pointing-Pointer The induction of cell migration does not require the EGF-like domain or EGR function. Black-Right-Pointing-Pointer Nuclear envelope-localized proAREG suppresses breast cancer cell growth without EGFR function. Black-Right-Pointing-Pointer This study revealed a novel function mediated by the intracellular domain of proAREG. -- Abstract: Amphiregulin (AREG), an EGF family protein, is synthesized as a type I transmembrane precursor (proAREG) and expressed on the cell surface with an extracellular EGF-like domain and an intracellular short cytoplasmic tail. The ectodomain shedding yields a soluble EGF receptor ligand (soluble AREG) which binds to EGF receptor (EGFR) and concomitantly induces migration of unshed proAREG from the plasma membrane to the nuclear envelope (NE). AREG is known to play a potential role in breast cancer and has been intensively investigated as an EGF receptor ligand, while the function of the NE-localized proAREG remains unknown. In this study we used a truncated mutant that mimics NE-localized proAREG without shedding stimuli to discriminate between the functions of NE-localized and plasma membrane-localized proAREG and demonstrate that NE-localized proAREG activates breast cancer cell migration, but suppresses cell growth. Moreover, the present study shows that induction of cell migration by NE-localized proAREG does not require the extracellular growth factor domain or EGF receptor function. Collectively these data demonstrate a novel function mediated by the intracellular domain of proAREG and suggest a significant role for NE-localized proAREG in driving human breast cancer progression.

  6. Selective abrogation of the proinvasive activity of the trefoil peptides pS2 and spasmolytic polypeptide by disruption of the EGF receptor signaling pathways in kidney and colonic cancer cells.

    PubMed

    Rodrigues, Sylvie; Attoub, Samir; Nguyen, Quang-Dé; Bruyneel, Erik; Rodrigue, Christelle M; Westley, Bruce R; May, Felicity E B; Thim, Lars; Mareel, Marc; Emami, Shahin; Gespach, Christian

    2003-07-17

    Trefoil peptides (TFFs) are now considered as scatter factors, proinvasive and angiogenic agents acting through cyclooxygenase-2 (COX-2)- and thromboxane A2 receptor (TXA2-R)-dependent signaling pathways. As expression and activation levels of the epidermal growth factor receptor (EGFR) predict the metastatic potential of human colorectal cancers, the purpose of this study was to establish whether the EGF receptor tyrosine kinase (EGFR-TK) contributes to cellular invasion induced by TFFs in kidney and colonic cancer cells. Both the dominant negative form of the EGFR (HER-CD533) and the EGFR-TK inhibitor ZD1839 (Iressa) abrogated cellular invasion induced by pS2, spasmolytic polypeptide (SP) and the src oncogene, but not by ITF and the TXA2-R. Similarly, EGFR-TK inhibition by ZD1839 reversed the invasive phenotype promoted by the constitutively activated form of the EGFR (EGFRvIII) and the EGFR agonists transforming growth factor alpha (TGFalpha), amphiregulin and EGF. We also provide evidence that TFFs, EGFRvIII, and TGFalpha trigger common proinvasive pathways using the PI3'-kinase and Rho/Rho- kinase cascades. These findings identify the EGFR-TK as a key signaling element for pS2- and SP-mediated cellular invasion. It is concluded that although pS2, SP and ITF belong to the same family of inflammation- and cancer-associated regulatory peptides, they do not control identical signaling networks.

  7. Effect of the anti-receptor ligand-blocking 225 monoclonal antibody on EGF receptor endocytosis and sorting

    SciTech Connect

    Jaramillo, Maria L. . E-mail: maria.jaramillo@nrc.ca; Leon, Zully; Grothe, Suzanne; Paul-Roc, Beatrice; Abulrob, Abedelnasser; O'Connor McCourt, Maureen

    2006-09-10

    The anti-receptor antibody, 225 mAb, is known to block binding of ligand to the epidermal growth factor receptor (EGFR). However, the effect of this neutralizing antibody on EGFR endocytosis, trafficking and degradation remains unclear. Here, we demonstrate that endocytosis of {sup 125}I-225 mAb occurs, albeit with a slower rate than that of EGF. Using pulse chase assays, we show that internalized {sup 125}I-225 mAb is recycled to the surface much more efficiently than internalized {sup 125}I-EGF. Also, we found that internalization of {sup 125}I-225 mAb, in contrast to that of EGF, is independent of receptor tyrosine kinase activity, as evidenced by its insensitivity to AG1478, a specific EGFR tyrosine kinase inhibitor. Analysis of the levels of cell surface and total EGFR showed that treatment with 225 mAb results in a 30-40% decrease in surface EGFR and a relatively slow downregulation of total EGFR. Taken together, these data indicate that 225 mAb induces internalization and downregulation of EGFR via a mechanism distinct from that underlying EGF-induced EGFR internalization and downregulation.

  8. EGF-receptor phosphorylation and downstream signaling are activated by benzo[a]pyrene 3,6-quinone and benzo[a]pyrene 1,6-quinone in human mammary epithelial cells

    SciTech Connect

    Rodriguez-Fragoso, Lourdes; Melendez, Karla; Hudson, Laurie G.; Lauer, Fredine T.; Burchiel, Scott W.

    2009-03-15

    Benzo[a]pyrene (BaP) is activated by xenobiotic-metabolizing enzymes to highly mutagenic and carcinogenic metabolites. Previous studies in this laboratory have shown that benzo[a]pyrene quinones (BPQs), 1,6-BPQ and 3,6-BPQ, are able to induce epidermal growth factor receptor (EGFR) cell signaling through the production of reactive oxygen species. Recently, we have reported that BPQs have the potential to induce the expression of genes involved in numerous pathways associated with cell proliferation and survival in human mammary epithelial cells. In the present study we demonstrated that BPQs not only induced EGFR tyrosine autophosphorylation, but also induced EGFR-dependent tyrosine phosphorylation of phospholipase C-{gamma}1 and several signal transducers and activators of transcription (STATs). The effects of BPQs were evaluated in a model of EGF withdrawal in MCF10-A cells. We found that BPQs (1 {mu}M), induced EGFR tyrosine phosphorylation at positions Y845, Y992, Y1068, and Y1086. PLC-{gamma}1 phosphorylation correlated with the phosphorylation of tyrosine-Y992, a proposed docking site for PLC-{gamma}1 on the EGFR. Additionally, we found that BPQs induced the activation of STAT-1, STAT-3, STAT-5a and STAT-5b. STAT5 was shown to translocate to the nucleus following 3,6-BPQ and 1,6-BPQ exposures. Although the patterns of phosphorylation at EGFR, PLC-{gamma}1 and STATs were quite similar to those induced by EGF, an important difference between BPQ-mediated signaling of the EGFR was observed. Signaling produced by EGF ligand produced a rapid disappearance of EGFR from the cell surface, whereas BPQ signaling maintained EGFR receptors on the cell membrane. Thus, the results of these studies show that 1,6-BPQ and 3,6-BPQ can produce early events as evidenced by EGFR expression, and a prolonged transactivation of EGFR leading to downstream cell signaling pathways.

  9. Phenethyl isothiocyanate suppresses EGF-stimulated SAS human oral squamous carcinoma cell invasion by targeting EGF receptor signaling.

    PubMed

    Chen, Hui-Jye; Lin, Chung-Ming; Lee, Chao-Ying; Shih, Nai-Chen; Amagaya, Sakae; Lin, Yung-Chang; Yang, Jai-Sing

    2013-08-01

    Phenethyl isothiocyanate (PEITC) is a natural compound that is involved in chemoprevention as well as inhibition of cell growth and induction of apoptosis in several types of cancer cells. Previous studies have revealed that PEITC suppresses the invasion of AGS gastric and HT-29 colorectal cancer cells. However, the effects of PEITC on the metastasis of SAS oral cancer cells remain to be determined. Our results showed that PEITC treatment inhibited the invasion of EGF-stimulated SAS cells in a concentration-dependent manner, but appeared not to affect the cell viability. The expression and enzymatic activities of matrix metalloprotease-2 (MMP-2) and matrix metalloprotease-9 (MMP-9) were suppressed by PEITC. Concomitantly, we observed an increase in the protein expression of both tissue inhibitor of metalloproteinase-1 (TIMP-1) and -2 (TIMP-2) in treated cells. Furthermore, PEITC treatments decreased the protein phosphorylation of epidermal growth factor receptor (EGFR) and downstream signaling proteins including PDK1, PI3K (p85), AKT, phosphorylated IKK and IκB to inactivate NF-κB for the suppression of MMP-2 and MMP-9 expression. In addition, PEITC can trigger the MAPK signaling pathway through the increase in phosphorylated p38, JNK and ERK in treated cells. Our data indicate that PEITC is able to inhibit the invasion of EGF-stimulated SAS oral cancer cells by targeting EGFR and its downstream signaling molecules and finally lead to the reduced expression and enzymatic activities of both MMP-2 and MMP-9. These results suggest that PEITC is promising for the therapy of oral cancer metastasis.

  10. Active G protein-coupled receptors (GPCR), matrix metalloproteinases 2/9 (MMP2/9), heparin-binding epidermal growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) are necessary for trenbolone acetate-induced alterations in protein turnover rate of fused bovine satellite cell cultures.

    PubMed

    Thornton, K J; Kamanga-Sollo, E; White, M E; Dayton, W R

    2016-06-01

    Trenbolone acetate (TBA), a testosterone analog, increases protein synthesis and decreases protein degradation in fused bovine satellite cell (BSC) cultures. However, the mechanism through which TBA alters these processes remains unknown. Recent studies indicate that androgens improve rate and extent of muscle growth through a nongenomic mechanism involving G protein-coupled receptors (GPCR), matrix metalloproteinases (MMP), heparin-binding epidermal growth factor (hbEGF), the epidermal growth factor receptor (EGFR), erbB2, and the insulin-like growth factor-1 receptor (IGF-1R). We hypothesized that TBA activates GPCR, resulting in activation of MMP2/9 that releases hbEGF, which activates the EGFR and/or erbB2. To determine whether the proposed nongenomic pathway is involved in TBA-mediated alterations in protein turnover, fused BSC cultures were treated with TBA in the presence or absence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R, and resultant protein synthesis and degradation rates were analyzed. Assays were replicated at least 9 times for each inhibitor experiment utilizing BSC cultures obtained from at least 3 different steers that had no previous exposure to steroid compounds. As expected, fused BSC cultures treated with 10 n TBA exhibited increased ( < 0.05) protein synthesis rates and decreased ( < 0.05) protein degradation rates when compared to control cultures. Treatment of fused BSC cultures with 10 n TBA in the presence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R suppressed ( < 0.05) TBA-mediated increases in protein synthesis rate. Alternatively, inhibition of GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R in the presence of 10 n TBA each had no ( > 0.05) effect on TBA-mediated decreases in protein degradation. However, inhibition of both EGFR and erbB2 in the presence of 10 n TBA resulted in decreased ( < 0.05) ability of TBA to decrease protein degradation rate. Additionally, fused BSC cultures treated with 10 n

  11. Active G protein-coupled receptors (GPCR), matrix metalloproteinases 2/9 (MMP2/9), heparin-binding epidermal growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) are necessary for trenbolone acetate-induced alterations in protein turnover rate of fused bovine satellite cell cultures.

    PubMed

    Thornton, K J; Kamanga-Sollo, E; White, M E; Dayton, W R

    2016-06-01

    Trenbolone acetate (TBA), a testosterone analog, increases protein synthesis and decreases protein degradation in fused bovine satellite cell (BSC) cultures. However, the mechanism through which TBA alters these processes remains unknown. Recent studies indicate that androgens improve rate and extent of muscle growth through a nongenomic mechanism involving G protein-coupled receptors (GPCR), matrix metalloproteinases (MMP), heparin-binding epidermal growth factor (hbEGF), the epidermal growth factor receptor (EGFR), erbB2, and the insulin-like growth factor-1 receptor (IGF-1R). We hypothesized that TBA activates GPCR, resulting in activation of MMP2/9 that releases hbEGF, which activates the EGFR and/or erbB2. To determine whether the proposed nongenomic pathway is involved in TBA-mediated alterations in protein turnover, fused BSC cultures were treated with TBA in the presence or absence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R, and resultant protein synthesis and degradation rates were analyzed. Assays were replicated at least 9 times for each inhibitor experiment utilizing BSC cultures obtained from at least 3 different steers that had no previous exposure to steroid compounds. As expected, fused BSC cultures treated with 10 n TBA exhibited increased ( < 0.05) protein synthesis rates and decreased ( < 0.05) protein degradation rates when compared to control cultures. Treatment of fused BSC cultures with 10 n TBA in the presence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R suppressed ( < 0.05) TBA-mediated increases in protein synthesis rate. Alternatively, inhibition of GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R in the presence of 10 n TBA each had no ( > 0.05) effect on TBA-mediated decreases in protein degradation. However, inhibition of both EGFR and erbB2 in the presence of 10 n TBA resulted in decreased ( < 0.05) ability of TBA to decrease protein degradation rate. Additionally, fused BSC cultures treated with 10 n

  12. TCDD AND EGF AFFECT MAPK PATHWAY ACTIVATION IN MURINE EMBRYONIC PALATE

    EPA Science Inventory

    Palatal fusion occurs on GD 14-15 in the mouse, accompanied by a decrease in EGF receptor (EGFR) at the medial edge of the palatal shelves. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces cleft palate and maintains EGF and EGF receptor (EGFR) expression levels in the medial ed...

  13. Regulation of osteosarcoma EGF receptor affinity by phorbol ester and cyclic AMP

    SciTech Connect

    Borst, S.E.; Catherwood, B.D. )

    1989-04-01

    We studied the binding and degradation of 125I-labeled epidermal growth factor (EGF) by UMR-106 osteosarcoma cells and the regulation of EGF receptor affinity for EGF by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and by treatments that raise intracellular levels of cyclic AMP. Cell surface binding of (125I)EGF to A431 cells reached a plateau after a 30 minute incubation at 37 degrees C but was undetectable in UMR-106 cells. Degradation of (125I)EGF proceeded at a 50-fold higher rate in A431 cells on a per cell basis, but receptor-bound (125I)EGF was internalized and degraded at a 3.5-fold higher rate by UMR-106 cells on a per receptor basis. At 4 degrees C, (125I)EGF labeled a single class of surface binding sites in the UMR-106 cell. Treatment with TPA at 37 degrees C reduced subsequent cell surface binding of (125I)EGF at 4 degrees C a maximum of 80% with an IC50 of 1.25 ng/ml. Maximal TPA reduction of (125I)EGF binding was observed within 5-15 minutes and was due to a reduction in the affinity of cell surface receptors of (125I)EGF without a change in receptor density. Pretreatment of the cells for 4 h with 30 microM forskolin, 1 mM isobutylmethylxanthine (IBMX) plus 30 microM forskolin, or 1 mM IBMX plus 100 ng/ml parathyroid hormone (PTH) attenuated the loss in (125I)EGF binding caused by a subsequent dose of 10 ng/ml of TPA by 17% (p less than 0.0005), 39% (p less than 0.0002), and 35% (p less than 0.002), respectively.

  14. Malignant Peripheral Nerve Sheath Tumor Invasion Requires Aberrantly Expressed Epidermal Growth Factor (EGF) Receptors and is Variably Enhanced by Multiple EGF Family Ligands

    PubMed Central

    Byer, Stephanie J.; Brossier, Nicole M.; Peavler, Lafe T.; Eckert, Jenell M.; Watkins, Stacey; Roth, Kevin A.; Carroll, Steven L.

    2013-01-01

    Aberrant epidermal growth factor receptor (EGFR) expression promotes the pathogenesis of malignant peripheral nerve sheath tumors (MPNSTs), the most common malignancy associated with neurofibromatosis type 1, but the mechanisms by which EGFR expression promotes MPNST pathogenesis are poorly understood. We hypothesized that inappropriately expressed EGFRs promote MPNST invasion and found that these kinases are concentrated in MPNST invadopodia in vitro. EGFR knockdown inhibited the migration of unstimulated MPNST cells in vitro and exogenous EGF further enhanced MPNST migration in a substrate-specific manner, promoting migration on laminin and, to a lesser extent, collagen. Thus, in this setting, EGF acts as a chemotactic factor. We also found that the 7 known EGFR ligands (EGF, betacellulin, epiregulin, heparin-binding EGF, transforming growth factor α [TGFα], amphiregulin, and epigen) variably enhanced MPNST migration in a concentration-dependent manner, with TGFα being particularly potent. With the exception of epigen, these factors similarly promoted the migration of non-neoplastic Schwann cells. Although transcripts encoding all 7 EGFR ligands were detected in human MPNST cells and tumor tissues, only TGFα was consistently overexpressed and was found to colocalize with EGFR in situ. These data indicate that constitutive EGFR activation, potentially driven by autocrine or paracrine TGFα signaling, promotes the aggressive invasive behavior characteristic of MPNSTs. PMID:23399900

  15. Expression of functional human EGF receptor on murine bone marrow cells.

    PubMed Central

    von Rüden, T; Wagner, E F

    1988-01-01

    The human epidermal growth factor-receptor (EGF-R) was introduced into primary mouse bone marrow cells (BMC), utilizing retrovirus mediated gene transfer. Cultivation of infected BMC in the presence of interleukin-3 (IL-3) led to the outgrowth of IL-3 dependent myeloid cells, which efficiently expressed functional EGF-R, exhibiting its two characteristic affinity states. EGF acts on these cells synergistically with IL-3 in stimulating DNA synthesis and cell proliferation even under IL-3 saturation conditions. However, EGF was not sufficient to replace the requirement for IL-3. In contrast, EGF was able to maintain proliferation of a factor-dependent hemopoietic cell line (FDC-P1) infected with the EGF-R retrovirus in the absence of IL-3, but these cells did not respond to EGF in the presence of IL-3. No influence of EGF on IL-3 induced mast cell differentiation of BMC expressing the EGF-R could be observed by histological criteria. These data show that the expression of EGF-R alone is not sufficient to induce or maintain cell proliferation in IL-3 dependent bone marrow derived cells, although it can do so in established hemopoietic cell lines. Images PMID:3053164

  16. Catalytic control in the EGF Receptor and its connection to general kinase regulatory mechanisms

    PubMed Central

    Jura, Natalia; Zhang, Xuewu; Endres, Nicholas F.; Seeliger, Markus A.; Schindler, Thomas; Kuriyan, John

    2011-01-01

    Summary In contrast to the active conformations of protein kinases, which are essentially the same for all kinases, inactive kinase conformations are structurally diverse. Some inactive conformations are, however, observed repeatedly in different kinases, perhaps reflecting an important role in catalysis. In this review, we analyze one of these recurring conformations, first identified in CDK and Src kinases, which turned out to be central to understanding of how kinase domain of the EGF receptor is activated. This mechanism, which involves the stabilization of the active conformation of an α helix, has features in common with mechanisms operative in several other kinases. PMID:21474065

  17. The EGF receptor is an actin-binding protein

    PubMed Central

    1992-01-01

    In a number of recent studies it has been shown that in vivo part of the EGF receptor (EGFR) population is associated to the actin filament system. In this paper we demonstrate that the purified EGFR can be cosedimented with purified filamentous actin (F-actin) indicating a direct association between EGFR and actin. A truncated EGFR, previously shown not to be associated to the cytoskeleton, was used as a control and this receptor did not cosediment with actin filaments. Determination of the actin-binding domain of the EGFR was done by measuring competition of either a polyclonal antibody or synthetic peptides on EGFR cosedimentation with F-actin. A synthetic peptide was made homologous to amino acid residues 984-996 (HL-33) of the EGFR which shows high homology with the actin-binding domain of Acanthamoeba profilin. A polyclonal antibody raised against HL-33 was found to prevent cosedimentation of EGFR with F-actin. This peptide HL-33 was shown to bind directly to actin in contrast with a synthetic peptide homologous to residues 1001-1013 (HL-34). During cosedimentation, HL-33 competed for actin binding of the EGFR and HL-34 did not, indicating that the EGFR contains one actin-binding site. These results demonstrate that the EGFR is an actin-binding protein which binds to actin via a domain containing amino acids residues 984-996. PMID:1383230

  18. Delta/Notch-Like EGF-Related Receptor (DNER) Is Not a Notch Ligand.

    PubMed

    Greene, Maxwell; Lai, Yongjie; Pajcini, Kostandin; Bailis, Will; Pear, Warren S; Lancaster, Eric

    2016-01-01

    Delta/Notch-like EGF-related receptor (DNER) has been reported to act as a Notch ligand, despite lacking a Delta/Serrate/Lag (DSL) binding domain common to all other known ligands. The established Notch ligand Delta-like 1 (DLL1), but not DNER, activated Notch1 in a luciferase assay, prevented the differentiation of myoblasts through Notch signaling, and bound Notch-fc in a cell-based assay. DNER is not a Notch ligand and its true function remains unknown. PMID:27622512

  19. Delta/Notch-Like EGF-Related Receptor (DNER) Is Not a Notch Ligand.

    PubMed

    Greene, Maxwell; Lai, Yongjie; Pajcini, Kostandin; Bailis, Will; Pear, Warren S; Lancaster, Eric

    2016-01-01

    Delta/Notch-like EGF-related receptor (DNER) has been reported to act as a Notch ligand, despite lacking a Delta/Serrate/Lag (DSL) binding domain common to all other known ligands. The established Notch ligand Delta-like 1 (DLL1), but not DNER, activated Notch1 in a luciferase assay, prevented the differentiation of myoblasts through Notch signaling, and bound Notch-fc in a cell-based assay. DNER is not a Notch ligand and its true function remains unknown.

  20. Delta/Notch-Like EGF-Related Receptor (DNER) Is Not a Notch Ligand

    PubMed Central

    Lai, Yongjie; Pajcini, Kostandin; Bailis, Will; Pear, Warren S.; Lancaster, Eric

    2016-01-01

    Delta/Notch-like EGF-related receptor (DNER) has been reported to act as a Notch ligand, despite lacking a Delta/Serrate/Lag (DSL) binding domain common to all other known ligands. The established Notch ligand Delta-like 1 (DLL1), but not DNER, activated Notch1 in a luciferase assay, prevented the differentiation of myoblasts through Notch signaling, and bound Notch-fc in a cell-based assay. DNER is not a Notch ligand and its true function remains unknown. PMID:27622512

  1. A Structural Perspective on the Regulation of the EGF Receptor

    PubMed Central

    Kovacs, Erika; Zorn, Julie Anne; Huang, Yongjian; Barros, Tiago; Kuriyan, John

    2015-01-01

    The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a critical role in the pathogenesis of many cancers. EGFR is unique in that its ligand-induced dimerization is established solely by contacts between regions of the receptor that are occluded within the monomeric, unliganded state. Activation of EGFR depends on the formation of an asymmetric dimer of the intracellular module of two receptor molecules, a configuration observed in crystal structures of the EGFR kinase domain in the active state. Coupling between the extracellular and intracellular modules is achieved by a switch between alternative geometries of the transmembrane and juxtamembrane segments within the receptor dimer. As the structure of the full-length receptor is yet to be determined, here we review recent structural studies on isolated modules of EGFR and molecular dynamics simulations that have provided much of our current understanding of its signaling mechanism, including how its regulation is compromised by oncogenic mutations. PMID:25621509

  2. Intracellular trafficking by Star regulates cleavage of the Drosophila EGF receptor ligand Spitz

    PubMed Central

    Tsruya, Rachel; Schlesinger, Ayelet; Reich, Aderet; Gabay, Limor; Sapir, Amir; Shilo, Ben-Zion

    2002-01-01

    Spitz (Spi) is a TGFα homolog that is a cardinal ligand for the Drosophila EGF receptor throughout development. Cleavage of the ubiquitously expressed transmembrane form of Spi (mSpi) precedes EGF receptor activation. We show that the Star and Rhomboid (Rho) proteins are necessary for Spi cleavage in Drosophila cells. Complexes between the Spi and Star proteins, as well as between the Star and Rho proteins were identified, but no Spi–Star–Rho triple complex was detected. This observation suggests a sequential activity of Star and Rho in mSpi processing. The interactions between Spi and Star regulate the intracellular trafficking of Spi. The Spi precursor is retained in the periphery of the nucleus. Coexpression of Star promotes translocation of Spi to a compartment where Rho is present both in cells and in embryos. A Star deletion construct that maintains binding to Spi and Rho, but is unable to facilitate Spi translocation, lost biological activity. These results underscore the importance of regulated intracellular trafficking in processing of a TGFα family ligand. PMID:11799065

  3. Pancreatic cancer cells require an EGF receptor-mediated autocrine pathway for proliferation in serum-free conditions

    PubMed Central

    Murphy, L O; Cluck, M W; Lovas, S; Ötvös, F; Murphy, R F; Schally, A V; Permert, J; Larsson, J; Knezetic, J A; Adrian, T E

    2001-01-01

    In-vitro and in-vivo studies have shown that autocrine growth factors and receptors are frequently expressed in human malignancies. Few of these studies, however, provide evidence that the identified autocrine pathway is functional. In this study, a functional autocrine growth pathway in pancreatic cancer has been identified using an in-vitro cell culture system. When pancreatic cancer cells were grown without change of medium, proliferation was greater than when either medium was replaced frequently (HPAF, CAPAN-2, PANC-1 or SW1990) or cells were grown in the presence of the EGF receptor tyrosine kinase inhibitor AG1478 or the MEK inhibitor PD098059 (HPAF or CAPAN-2). Activity of extracellular-regulated kinases (ERK) 1 and 2 and c- jun and c- fos mRNA levels were significantly elevated in CAPAN-2 cells cultured continuously in serum-free medium. Collectively, the observations indicate that the EGF receptor and the ERK MAP kinase pathway mediate autocrine signals. In contrast to previous reports, the GRP and IGF-I receptors were shown not to be required for autocrine effects on pancreatic cancer cell proliferation. Autocrine stimulation of the EGF receptor can contribute to sustained mitogenic activity and proliferation of pancreatic cancer cells. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11286473

  4. EGF receptor specificity for phosphotyrosine-primed substrates provides signal integration with Src

    PubMed Central

    Begley, Michael J; Yun, Cai-hong; Gewinner, Christina A; Asara, John M; Johnson, Jared L; Coyle, Anthony J; Eck, Michael J; Apostolou, Irina; Cantley, Lewis C

    2016-01-01

    Aberrant activation of the EGF receptor (EGFR) contributes to many human cancers by activating the Ras-MAPK and other pathways. EGFR signaling is augmented by Src-family kinases, but the mechanism is poorly understood. Here, we show that human EGFR preferentially phosphorylates peptide substrates that are primed by a prior phosphorylation. Utilizing peptides based on the sequence of the adaptor protein Shc1, we show that Src mediates the priming phosphorylation, promoting subsequent phosphorylation by EGFR. Importantly, the doubly phosphorylated Shc1 peptide binds more tightly to the Ras activator Grb2, a key step in activating the Ras-MAPK pathway, than singly phosphorylated peptides. Finally, a crystal structure of EGFR in complex with a primed Shc1 peptide reveals the structural basis for EGFR substrate specificity. These results provide a molecular explanation for the integration of Src and EGFR signaling with downstream effectors such as Ras. PMID:26551075

  5. Endocytosis separates EGF receptors from endogenous fluorescently labeled HRas and diminishes receptor signaling to MAP kinases in endosomes.

    PubMed

    Pinilla-Macua, Itziar; Watkins, Simon C; Sorkin, Alexander

    2016-02-23

    Signaling from epidermal growth factor receptor (EGFR) to extracellular-stimuli-regulated protein kinase 1/2 (ERK1/2) is proposed to be transduced not only from the cell surface but also from endosomes, although the role of endocytosis in this signaling pathway is controversial. Ras is the only membrane-anchored component in the EGFR-ERK signaling axis, and therefore, its location determines intracellular sites of downstream signaling. Hence, we labeled endogenous H-Ras (HRas) with mVenus fluorescent protein using gene editing in HeLa cells. mVenus-HRas was primarily located at the plasma membrane, and in small amounts in tubular recycling endosomes and associated vesicles. EGF stimulation resulted in fast but transient activation of mVenus-HRas. Although EGF:EGFR complexes were rapidly accumulated in endosomes together with the Grb2 adaptor, very little, if any, mVenus-HRas was detected in these endosomes. Interestingly, the activities of MEK1/2 and ERK1/2 remained high beyond the point of the physical separation of HRas from EGF:EGFR complexes and down-regulation of Ras activity. Paradoxically, this sustained MEK1/2 and ERK1/2 activation was dependent on the active EGFR kinase. Cell surface biotinylation and selective inactivation of surface EGFRs suggested that a small fraction of active EGFRs remaining in the plasma membrane is responsible for continuous signaling to MEK1/2 and ERK1/2. We propose that, under physiological conditions of cell stimulation, EGFR endocytosis serves to spatially separate EGFR-Grb2 complexes and Ras, thus terminating Ras-mediated signaling. However, sustained minimal activation of Ras by a small pool of active EGFRs in the plasma membrane is sufficient for extending MEK1/2 and ERK1/2 activities.

  6. Endocytosis separates EGF receptors from endogenous fluorescently labeled HRas and diminishes receptor signaling to MAP kinases in endosomes

    PubMed Central

    Pinilla-Macua, Itziar; Watkins, Simon C.; Sorkin, Alexander

    2016-01-01

    Signaling from epidermal growth factor receptor (EGFR) to extracellular-stimuli–regulated protein kinase 1/2 (ERK1/2) is proposed to be transduced not only from the cell surface but also from endosomes, although the role of endocytosis in this signaling pathway is controversial. Ras is the only membrane-anchored component in the EGFR–ERK signaling axis, and therefore, its location determines intracellular sites of downstream signaling. Hence, we labeled endogenous H-Ras (HRas) with mVenus fluorescent protein using gene editing in HeLa cells. mVenus-HRas was primarily located at the plasma membrane, and in small amounts in tubular recycling endosomes and associated vesicles. EGF stimulation resulted in fast but transient activation of mVenus-HRas. Although EGF:EGFR complexes were rapidly accumulated in endosomes together with the Grb2 adaptor, very little, if any, mVenus-HRas was detected in these endosomes. Interestingly, the activities of MEK1/2 and ERK1/2 remained high beyond the point of the physical separation of HRas from EGF:EGFR complexes and down-regulation of Ras activity. Paradoxically, this sustained MEK1/2 and ERK1/2 activation was dependent on the active EGFR kinase. Cell surface biotinylation and selective inactivation of surface EGFRs suggested that a small fraction of active EGFRs remaining in the plasma membrane is responsible for continuous signaling to MEK1/2 and ERK1/2. We propose that, under physiological conditions of cell stimulation, EGFR endocytosis serves to spatially separate EGFR–Grb2 complexes and Ras, thus terminating Ras-mediated signaling. However, sustained minimal activation of Ras by a small pool of active EGFRs in the plasma membrane is sufficient for extending MEK1/2 and ERK1/2 activities. PMID:26858456

  7. Synthesis and characterization of N-parinaroyl analogs of ganglioside GM3 and de-N-acetyl GM3. Interactions with the EGF receptor kinase

    NASA Technical Reports Server (NTRS)

    Song, W.; Welti, R.; Hafner-Strauss, S.; Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    A specific plasma membrane glycosphingolipid, known as ganglioside GM3, can regulate the intrinsic tyrosyl kinase activity of the epidermal growth factor (EGF) receptor; this modulation is not associated with alterations in hormone binding to the receptor. GM3 inhibits EGF receptor tyrosyl kinase activity in detergent micelles, in plasma membrane vesicles, and in whole cells. In addition, immunoaffinity-purified EGF receptor preparations contain ganglioside GM3 (Hanai et al. (1988) J. Biol. Chem. 263, 10915-10921), implying that the glycosphingolipid is intimately associated with the receptor kinase in cell membranes. Both the nature of this association and the molecular mechanism of kinase inhibition remain to be elucidated. In this report, we describe the synthesis of a fluorescent analog of ganglioside GM3, in which the native fatty acid was replaced with trans-parinaric acid. This glycosphingolipid inhibited the receptor kinase activity in a manner similar to that of the native ganglioside. A modified fluorescent glycosphingolipid, N-trans-parinaroyl de-N-acetyl ganglioside GM3, was also prepared. This analog, like the nonfluorescent de-N-acetyl ganglioside GM3, had no effect on receptor kinase activity. Results from tryptophan fluorescence quenching and steady-state anisotropy measurements in membranes containing these fluorescent probes and the human EGF receptor were consistent with the notion that GM3, but not de-N-acetyl GM3, interacts specifically with the receptor in intact membranes.

  8. BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation.

    PubMed

    Tóth, József T; Gulyás, Gergő; Tóth, Dániel J; Balla, András; Hammond, Gerald R V; Hunyady, László; Balla, Tamás; Várnai, Péter

    2016-03-01

    Deciphering many roles played by inositol lipids in signal transduction and membrane function demands experimental approaches that can detect their dynamic accumulation with subcellular accuracy and exquisite sensitivity. The former criterion is met by imaging of fluorescence biosensors in living cells, whereas the latter is facilitated by biochemical measurements from populations. Here, we introduce BRET-based biosensors able to detect rapid changes in inositol lipids in cell populations with both high sensitivity and subcellular resolution in a single, convenient assay. We demonstrate robust and sensitive measurements of PtdIns4P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics, as well as changes in cytoplasmic Ins(1,4,5)P3 levels. Measurements were made during either experimental activation of lipid degradation, or PI 3-kinase and phospholipase C mediated signal transduction. Our results reveal a previously unappreciated synthesis of PtdIns4P that accompanies moderate activation of phospholipase C signaling downstream of both EGF and muscarinic M3 receptor activation. This signaling-induced PtdIns4P synthesis relies on protein kinase C, and implicates a feedback mechanism in the control of inositol lipid metabolism during signal transduction.

  9. EGF-receptor regulation of matrix metalloproteinases in epithelial ovarian carcinoma

    PubMed Central

    Hudson, Laurie G; Moss, Natalie M; Stack, M Sharon

    2009-01-01

    Ovarian carcinoma is most frequently detected when disease has already disseminated intra-abdominally, resulting in a 5-year survival rate of less than 20% owing to complications of metastasis. Peritoneal ascites is often present, establishing a unique microenvironmental niche comprised of tumor and inflammatory cells, along with a wide range of bioactive soluble factors, several of which stimulate the EGF-receptor (EGFR). Elevated EGFR is associated with less favorable disease outcome in ovarian cancer, related in part to EGFR activation of signaling cascades that lead to enhanced matrix metalloproteinase expression and/or function. The available data suggest that modulating the expression or activity of the EGFR and/or matrix metalloproteinases offers opportunity for targeted intervention in patients with metastatic disease. PMID:19374540

  10. PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response

    PubMed Central

    Liao, Hsin-Wei; Hsu, Jung-Mao; Xia, Weiya; Wang, Hung-Ling; Wang, Ying-Nai; Chang, Wei-Chao; Arold, Stefan T.; Chou, Chao-Kai; Tsou, Pei-Hsiang; Yamaguchi, Hirohito; Fang, Yueh-Fu; Lee, Hong-Jen; Lee, Heng-Huan; Tai, Shyh-Kuan; Yang, Mhu-Hwa; Morelli, Maria P.; Sen, Malabika; Ladbury, John E.; Chen, Chung-Hsuan; Grandis, Jennifer R.; Kopetz, Scott; Hung, Mien-Chie

    2015-01-01

    Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment. PMID:26571401

  11. Both clathrin-positive and -negative coats are involved in endosomal sorting of the EGF receptor

    SciTech Connect

    Myromslien, Froydis D.; Grovdal, Lene Melsaether; Raiborg, Camilla; Stenmark, Harald; Madshus, Inger Helene; Stang, Espen . E-mail: espen.stang@medisin.uio.no

    2006-10-01

    Sorting of endocytosed EGF receptor (EGFR) to internal vesicles of multivesicular bodies (MVBs) depends on sustained activation and ubiquitination of the EGFR. Ubiquitination of EGFR is mediated by the ubiquitin ligase Cbl, being recruited to the EGFR both directly and indirectly through association with Grb2. Endosomal sorting of ubiquitinated proteins further depends on interaction with ubiquitin binding adaptors like Hrs. Hrs localizes to flat, clathrin-coated domains on the limiting membrane of endosomes. In the present study, we have investigated the localization of EGFR, Cbl and Grb2 with respect to coated and non-coated domains of the endosomal membrane and to vesicles within MVBs. Both EGFR, Grb2, and Cbl were concentrated in coated domains of the limiting membrane before translocation to inner vesicles of MVBs. While almost all Hrs was in clathrin-positive coats, EGFR and Grb2 in coated domains only partially colocalized with Hrs and clathrin. The extent of colocalization of EGFR and Grb2 with Hrs and clathrin varied with time of incubation with EGF. These results demonstrate that both clathrin-positive and clathrin-negative electron dense coats exist on endosomes and are involved in endosomal sorting of the EGFR.

  12. An EGF receptor inhibitor induces RAR-beta expression in breast and ovarian cancer cells.

    PubMed

    Grunt, Thomas W; Puckmair, Klaudia; Tomek, Katharina; Kainz, Birgit; Gaiger, Alexander

    2005-04-22

    Inhibition of the epidermal growth factor (EGF)-receptor (EGFR) has become a promising anticancer treatment strategy. In addition, application of retinoids yields encouraging results for cancer prevention and therapy. Many tumors express no or low amounts of retinoic acid receptor-beta2 (RAR-beta2) due to epigenetic silencing via DNA hypermethylation. RAR-beta2 is the main mediator of the antiproliferative effect of retinoids. RAR-beta2 re-expression causes reversal of transformation, cell cycle arrest, and restoration of retinoid sensitivity. RAR-beta2 is thus a tumor suppressor. Western blotting, colorimetric in vitro cell proliferation assays, and reverse transcription-polymerase chain reaction demonstrated that the EGFR inhibitor PD153035 not only blocked activation of EGFR and inhibited cell growth, but also stimulated RAR-beta expression in MDA-MB-468 breast and OVCAR-3 ovarian carcinoma cells. Upregulation of RAR-beta by PD153035 was confirmed by real-time reverse transcription-polymerase chain reaction. In contrast, expression of other retinoid receptors and of estrogen receptor-alpha was not affected. PD153035-mediated re-induction of RAR-beta was associated with demethylation of the RAR-beta2 gene promoter P2 as demonstrated by methylation-specific polymerase chain reaction. These novel results on the ErbB/retinoid receptor cross-talk may be useful for designing future anticancer combination regimens.

  13. An EGF receptor inhibitor induces RAR-{beta} expression in breast and ovarian cancer cells

    SciTech Connect

    Grunt, Thomas W. . E-mail: thomas.grunt@meduniwien.ac.at; Puckmair, Klaudia; Tomek, Katharina; Kainz, Birgit; Gaiger, Alexander

    2005-04-22

    Inhibition of the epidermal growth factor (EGF)-receptor (EGFR) has become a promising anticancer treatment strategy. In addition, application of retinoids yields encouraging results for cancer prevention and therapy. Many tumors express no or low amounts of retinoic acid receptor-{beta}2 (RAR-{beta}2) due to epigenetic silencing via DNA hypermethylation. RAR-{beta}2 is the main mediator of the antiproliferative effect of retinoids. RAR-{beta}2 re-expression causes reversal of transformation, cell cycle arrest, and restoration of retinoid sensitivity. RAR-{beta}2 is thus a tumor suppressor. Western blotting, colorimetric in vitro cell proliferation assays, and reverse transcription-polymerase chain reaction demonstrated that the EGFR inhibitor PD153035 not only blocked activation of EGFR and inhibited cell growth, but also stimulated RAR-{beta} expression in MDA-MB-468 breast and OVCAR-3 ovarian carcinoma cells. Upregulation of RAR-{beta} by PD153035 was confirmed by real-time reverse transcription-polymerase chain reaction. In contrast, expression of other retinoid receptors and of estrogen receptor-{alpha} was not affected. PD153035-mediated re-induction of RAR-{beta} was associated with demethylation of the RAR-{beta}2 gene promoter P2 as demonstrated by methylation-specific polymerase chain reaction. These novel results on the ErbB/retinoid receptor cross-talk may be useful for designing future anticancer combination regimens.

  14. Urine acidification has no effect on peroxisome proliferator-activated receptor (PPAR) signaling or epidermal growth factor (EGF) expression in rat urinary bladder urothelium

    SciTech Connect

    Achanzar, William E. Moyer, Carolyn F.; Marthaler, Laura T.; Gullo, Russell; Chen, Shen-Jue; French, Michele H.; Watson, Linda M.; Rhodes, James W.; Kozlosky, John C.; White, Melvin R.; Foster, William R.; Burgun, James J.; Car, Bruce D.; Cosma, Gregory N.; Dominick, Mark A.

    2007-09-15

    We previously reported prevention of urolithiasis and associated rat urinary bladder tumors by urine acidification (via diet acidification) in male rats treated with the dual peroxisome proliferator-activated receptor (PPAR){alpha}/{gamma} agonist muraglitazar. Because urine acidification could potentially alter PPAR signaling and/or cellular proliferation in urothelium, we evaluated urothelial cell PPAR{alpha}, PPAR{delta}, PPAR{gamma}, and epidermal growth factor receptor (EGFR) expression, PPAR signaling, and urothelial cell proliferation in rats fed either a normal or an acidified diet for 5, 18, or 33 days. A subset of rats in the 18-day study also received 63 mg/kg of the PPAR{gamma} agonist pioglitazone daily for the final 3 days to directly assess the effects of diet acidification on responsiveness to PPAR{gamma} agonism. Urothelial cell PPAR{alpha} and {gamma} expression and signaling were evaluated in the 18- and 33-day studies by immunohistochemical assessment of PPAR protein (33-day study only) and quantitative real-time polymerase chain reaction (qRT-PCR) measurement of PPAR-regulated gene expression. In the 5-day study, EGFR expression and phosphorylation status were evaluated by immunohistochemical staining and egfr and akt2 mRNA levels were assessed by qRT-PCR. Diet acidification did not alter PPAR{alpha}, {delta}, or {gamma} mRNA or protein expression, PPAR{alpha}- or {gamma}-regulated gene expression, total or phosphorylated EGFR protein, egfr or akt2 gene expression, or proliferation in urothelium. Moreover, diet acidification had no effect on pioglitazone-induced changes in urothelial PPAR{gamma}-regulated gene expression. These results support the contention that urine acidification does not prevent PPAR{gamma} agonist-induced bladder tumors by altering PPAR{alpha}, {gamma}, or EGFR expression or PPAR signaling in rat bladder urothelium.

  15. Influence of biotransformation of luteolin, luteolin 7-O-glucoside, 3',4'-dihydroxyflavone and apigenin by cultured rat hepatocytes on antioxidative capacity and inhibition of EGF receptor tyrosine kinase activity.

    PubMed

    Schlupper, Doreen; Giesa, Sabine; Gebhardt, Rolf

    2006-06-01

    Flavonoids are known as biologically active compounds. Although this has been shown by several in vivo studies, it is still elusive whether their metabolites exert similar activities. Herein we investigated the biotransformation of four different flavonoids, 3',4'-dihydroxyflavone, apigenin, luteolin and luteolin 7-O-glucoside, by cultured rat hepatocytes using a combination of enzymatic deconjugation, HPLC separation and high-resolution mass spectrometry. These flavonoids were chosen because they are active components of many plants, e. g., artichokes. All flavonoids showed rather complex metabolite patterns dominated by phase II metabolites, mainly sulfates, methyl sulfates and methyl glucuronides, but also of combined glucuronide and sulfate conjugates. Phase I metabolism by hydroxylation was rendered likely only for apigenin to form luteolin. When culture media containing the flavonoids and their metabolites were assayed for antioxidative capacity by the DPPH assay, only compounds with hydroxy groups in position 3' and 4' of the B ring were active. Thus, during metabolism of (inactive) apigenin a strong increase in the antioxidative effect was observed while that of the other three flavonoids decreased with time. Determination of EGF receptor tyrosine kinase activity likewise revealed strong inhibition in the presence of a catechol group at ring B. However, in this case the situation was much more complex resulting in a significant increase of the inhibitory activity of 3',4'-dihydroxyflavone and apigenin, but not of luteolin and luteolin 7-O-glucoside during 22 h of incubation. These results show that the biotransformation of flavonoids is very complex and may result not only in a loss but also in a gain of biological activity depending on the individual structural features. PMID:16732514

  16. Detergent solubilization of the EGF receptor from A431 cells

    NASA Technical Reports Server (NTRS)

    Dayanidhi, R.; Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Functional reconstitution of purified preparations of human epidermal growth factor receptor (EGFR) requires dissociation of the protein from its plasma membrane lipid environment. Solubilization of membrane proteins in this manner requires the use of detergents, which are known to disrupt plasma membrane lipid/protein interactions. We have investigated the ability of three nonionic detergents to solubilize the human EGFR selectively, and have also analyzed the effect of these various treatments on the intrinsic tyrosyl kinase activity of the receptor. The nonionic detergent known as n-octyl glucoside (n-octyl beta-D-glucopyranoside) was found to give the best combination of selectivity, yield, and maintenance of enzymatic activity of the human EGFR.

  17. Understanding CELSRs - Cadherin EGF LAG seven-pass G-type receptors

    PubMed Central

    Wang, Xiao-Jing; Zhang, Dao-Lai; Xu, Zhi-Gang; Ma, Ming-Liang; Wang, Wen-Bo; Li, Lin-Lin; Han, Xiao-Lin; Huo, Yuqing; Yu, Xiao; Sun, Jin-Peng

    2014-01-01

    The cadherin EGF LAG seven-pass G-type receptors (CELSRs) are a special subgroup of adhesion G protein-coupled receptors (GPCRs), which are pivotal regulators of many biological processes such as neuronal/endocrine cell differentiation, vessel valve formation and the control of planar cell polarity during embryonic development. All three members of the CELSR family (CELSR1-3) have large ecto-domains that form homophilic interactions and encompass more than 2,000 amino acids. Mutations in the ecto-domain or other gene locations of CELSRs are associated with neural tube defects (NTDs) and other diseases in humans. Celsr knockout (KO) animals have many developmental defects. Therefore, specific agonists or antagonists of CELSR members may have therapeutic potential. Although significant progress has been made regarding the functions and biochemical properties of CELSRs, our knowledge of these receptors is still lacking, especially considering that they are broadly distributed but have few characterized functions in a limited number of tissues. The dynamic activation and inactivation of CELSRs and the presence of endogenous ligands beyond homophilic interactions remain elusive, as do the regulatory mechanisms and downstream signaling of these receptors. Given this motivation, future studies with more advanced cell biology or biochemical tools, such as conditional KO mice, may provide further insights into the mechanisms underlying CELSR function, laying the foundation for the design of new CELSR-targeted therapeutic reagents. PMID:25280249

  18. Immunological Visibility: Posttranscriptional Regulation of Human NKG2D Ligands by the EGF Receptor Pathway

    PubMed Central

    Vantourout, Pierre; Willcox, Carrie; Turner, Andrea; Swanson, Chad; Haque, Yasmin; Sobolev, Olga; Grigoriadis, Anita; Tutt, Andrew; Hayday, Adrian

    2014-01-01

    Human cytolytic T lymphocytes and NK cells can limit tumor growth and are being increasingly harnessed for tumor immunotherapy. One way cytolytic lymphocytes recognize tumor cells is by engagement of their activating receptor, NKG2D, by stress-antigens of the MICA/B and ULBP families. This study shows that surface upregulation of NKG2D ligands by human epithelial cells in response to ultraviolet irradiation, osmotic shock, oxidative stress, and growth factor provision, is attributable to activation of the EGF-receptor (EGFR). EGFR activation causes intracellular re-localisation of AUF1 proteins that ordinarily destabilise NKG2D ligand mRNAs by targeting an AU-rich element conserved within the 3′ ends of most human but not murine NKG2D ligand genes. Consistent with these findings, NKG2D ligand expression by primary human carcinomas positively correlated with EGFR expression that is commonly hyper-activated in such tumours, and was reduced by clinical EGFR inhibitors. Thus, stress-induced activation of EGFR not only regulates cell growth but concomitantly regulates the cells’ immunological visibility. Thus, therapeutics designed to limit cancer cell growth should also be considered in terms of their impact on immunosurveillance. PMID:24718859

  19. Dysregulation of Ack1 inhibits down-regulation of the EGF receptor

    SciTech Connect

    Grovdal, Lene Melsaether; Johannessen, Lene E.; Rodland, Marianne Skeie; Madshus, Inger Helene; Stang, Espen

    2008-04-01

    The protein tyrosine kinase Ack1 has been linked to cancer when over-expressed. Ack1 has also been suggested to function in clathrin-mediated endocytosis and in down-regulation of the epidermal growth factor (EGF) receptor (EGFR). We have studied the intracellular localization of over-expressed Ack1 and found that Ack1 co-localizes with the EGFR upon EGF-induced endocytosis in cells with moderate over-expression of Ack. This co-localization is mainly observed in early endosomes. Furthermore, we found that over-expression of Ack1 retained the EGFR at the limiting membrane of early endosomes, inhibiting sorting to inner vesicles of multivesicular bodies. Down-regulation of Ack1 in HeLa cells resulted in reduced rate of {sup 125}I-EGF internalization, whereas internalization of {sup 125}I-transferrin was not affected. In cells where Ack1 had been knocked down by siRNA, recycling of internalized {sup 125}I-EGF was increased, while degradation of {sup 125}I-EGF was inhibited. Together, these data suggest that Ack1 is involved in an early step of EGFR desensitization.

  20. G protein-coupled receptor 30 expression is up-regulated by EGF and TGF alpha in estrogen receptor alpha-positive cancer cells.

    PubMed

    Vivacqua, Adele; Lappano, Rosamaria; De Marco, Paola; Sisci, Diego; Aquila, Saveria; De Amicis, Francesca; Fuqua, Suzanne A W; Andò, Sebastiano; Maggiolini, Marcello

    2009-11-01

    In the present study, we evaluated the regulation of G protein-coupled receptor (GPR)30 expression in estrogen receptor (ER)-positive endometrial, ovarian, and estrogen-sensitive, as well as tamoxifen-resistant breast cancer cells. We demonstrate that epidermal growth factor (EGF) and TGF alpha transactivate the GPR30 promoter and accordingly up-regulate GPR30 mRNA and protein levels only in endometrial and tamoxifen-resistant breast cancer cells. These effects exerted by EGF and TGF alpha were dependent on EGF receptor (EGFR) expression and activation and involved phosphorylation of the Tyr(1045) and Tyr(1173) EGFR sites. Using gene-silencing experiments and specific pharmacological inhibitors, we have ascertained that EGF and TGF alpha induce GPR30 expression through the EGFR/ERK transduction pathway, and the recruitment of c-fos to the activator protein-1 site located within GPR30 promoter sequence. Interestingly, we show that functional cross talk of GPR30 with both activated EGFR and ER alpha relies on a physical interaction among these receptors, further extending the potential of estrogen to trigger a complex stimulatory signaling network in hormone-sensitive tumors. Given that EGFR/HER2 overexpression is associated with tamoxifen resistance, our data may suggest that ligand-activated EGFR could contribute to the failure of tamoxifen therapy also by up-regulating GPR30, which in turn could facilitates the action of estrogen. In addition, important for resistance is the ability of tamoxifen to bind to and activate GPR30, the expression of which is up-regulated by EGFR activation. Our results emphasize the need for new endocrine agents able to block widespread actions of estrogen without exerting any stimulatory activity on transduction pathways shared by the steroid and growth factor-signaling networks.

  1. Ammonia-induced Na,K-ATPase/ouabain-mediated EGF receptor transactivation, MAPK/ERK and PI3K/AKT signaling and ROS formation cause astrocyte swelling.

    PubMed

    Dai, Hongliang; Song, Dan; Xu, Junnan; Li, Baoman; Hertz, Leif; Peng, Liang

    2013-11-01

    Ammonia toxicity is clinically important and biologically poorly understood. We reported previously that 3mM ammonia chloride (ammonia), a relevant concentration for hepatic encephalopathy studies, increases production of endogenous ouabain and activity of Na,K-ATPase in astrocytes. In addition, ammonia-induced upregulation of gene expression of α2 isoform of Na,K-ATPase in astrocytes could be inhibited by AG1478, an inhibitor of the EGF receptor (EGFR), and by PP1, an inhibitor of Src, but not by GM6001, an inhibitor of metalloproteinase and shedding of growth factor, suggesting the involvement of endogenous ouabain-induced EGF receptor transactivation. In the present cell culture study, we investigated ammonia effects on phosphorylation of EGF receptor and its intracellular signal pathway towards MAPK/ERK1/2 and PI3K/AKT; interaction between EGF receptor, α1, and α2 isoforms of Na,K-ATPase, Src, ERK1/2, AKT and caveolin-1; and relevance of these signal pathways for ammonia-induced cell swelling, leading to brain edema, an often fatal complication of ammonia toxicity. We found that (i) ammonia increases EGF receptor phosphorylation at EGFR(845) and EGFR(1068); (ii) ammonia-induced ERK1/2 and AKT phosphorylation depends on the activity of EGF receptor and Src, but not on metalloproteinase; (iii) AKT phosphorylation occurs upstream of ERK1/2 phosphorylation; (iv) ammonia stimulates association between the α1 Na,K-ATPase isoform, Src, EGF receptor, ERK1/2, AKT and caveolin-1; (v) ammonia-induced ROS production might occur later than EGFR transactivation; (vi) both ammonia induced ERK phosphorylation and ROS production can be abolished by canrenone, an inhibitor of ouabain, and (vii) ammonia-induced cell swelling depends on signaling via the Na,K-ATPase/ouabain/Src/EGF receptor/PI3K-AKT/ERK1/2, but in response to 3mM ammonia it does not appear until after 12h. Based on literature data it is suggested that the delayed appearance of the ammonia-induced swelling at

  2. Mechanism of HSV infection through soluble adapter-mediated virus bridging to the EGF receptor

    SciTech Connect

    Nakano, Kenji; Kobayashi, Masatoshi; Nakamura, Kei-ichiro; Nakanishi, Takeshi; Asano, Ryutaro; Kumagai, Izumi; Tahara, Hideaki; Kuwano, Michihiko; Cohen, Justus B.; Glorioso, Joseph C.

    2011-04-25

    Herpes simplex virus entry into cells requires the binding of envelope glycoprotein D (gD) to an entry receptor. Depending on the cell, entry occurs by different mechanisms, including fusion at the cell surface or endocytosis. Here we examined the entry mechanism through a non-HSV receptor mediated by a soluble bi-specific adapter protein composed of recognition elements for gD and the EGF receptor (EGFR). Virus entered into endosomes using either EGF or an EGFR-specific single chain antibody (scFv) for receptor recognition. Infection was less efficient with the EGF adapter which could be attributed to its weaker binding to a viral gD. Infection mediated by the scFv adapter was pH sensitive, indicating that gD-EGFR bridging alone was insufficient for capsid release from endosomes. We also show that the scFv adapter enhanced infection of EGFR-expressing tumor tissue in vivo. Our results indicate that adapters may retarget HSV infection without drastically changing the entry mechanism.

  3. P38 AND EGF RECEPTOR KINASE-MEDIATED ACTIVATION OF THE PHOSPHATIDYLINOSITOL 3-KINASE/AKT PATHWAY IS REQUIRED FOR ZN2+INDUCED CYCLOOXYGENASE-2 EXPRESSION

    EPA Science Inventory

    Cyclooxygenase 2 (COX-2) expression is induced by physiological and inflammatory stimuli. Regulation of COX-2 expression is stimulus- and cell type-specific. Exposure to Zn2+ has been associated with activation of multiple intracellular signaling pathways as well as the induction...

  4. Quantum dots implementation as a label for analysis of early stages of EGF receptor endocytosis: a comparative study on cultured cells

    PubMed Central

    Salova, Anna V.; Belyaeva, Tatiana N.; Leontieva, Ekaterina A.; Zlobina, Maria V.; Kharchenko, Marianna V.; Kornilova, Elena S.

    2016-01-01

    EGF complexed to fluorescent photostable quantum dots by biotin-streptavidin system (bEGF-savQD) is attractive for both the basic research and therapeutic application such as targeted drug delivery in EGF-receptor (EGFR) expressing cancers. However, compared to native EGF, the large size of QD and its quasi-multivalency can have unpredictable effects on EGFR endocytosis changing the internalization portal and/or endosomal processing tightly bound to EGF signaling. We have found that bEGF-savQDs enter HeLa cells via the temperature-dependent clathrin-mediated EGF-receptor-specific pathway characteristic for native EGF. We also found that EGF-to-QD concentration ratios used for the complex preparation and the level of EGF receptor expression affect the number and integral densities of the formed endosomes. So, at EGF-to-QD ratio from 4:1 to 12:1 (at nanomolar bEGF concentrations) on average 100 bright endosomes per HeLa cell were formed 15 min after the complex addition, while 1:1 ratio resulted in formation of very few dim endosomes. However, in A431 cells overexpressing EGFR 1:1 ratio was effective. Using dynamin inhibition and Na-acidic washout we showed that bEGF-savQDs bind surface receptors and enter clathrin-coated pits slower than the same ligands without QD. Yet, the bEGF-savQD demonstrated similar to native EGF and bEGF-savCy3 co-localization dynamics with tethering protein EEA1 and HRS, the key component of sorting ESCRT0 complex. In conclusion, our comparative study reveals that in respect to entrapment into coated pits, endosomal recruitment, endosome fusions, and the initial steps of endosomal maturation, bEGF-savQD behaves like native EGF and QD implementation does not affect these important events. PMID:26716513

  5. Structural Basis for Negative Cooperativity in Growth Factor Binding to an EGF Receptor

    SciTech Connect

    Alvarado, Diego; Klein, Daryl E.; Lemmon, Mark A.

    2010-09-27

    Transmembrane signaling by the epidermal growth factor receptor (EGFR) involves ligand-induced dimerization and allosteric regulation of the intracellular tyrosine kinase domain. Crystallographic studies have shown how ligand binding induces dimerization of the EGFR extracellular region but cannot explain the high-affinity and low-affinity classes of cell-surface EGF-binding sites inferred from curved Scatchard plots. From a series of crystal structures of the Drosophila EGFR extracellular region, we show here how Scatchard plot curvature arises from negatively cooperative ligand binding. The first ligand-binding event induces formation of an asymmetric dimer with only one bound ligand. The unoccupied site in this dimer is structurally restrained, leading to reduced affinity for binding of the second ligand, and thus negative cooperativity. Our results explain the cell-surface binding characteristics of EGF receptors and suggest how individual EGFR ligands might stabilize distinct dimeric species with different signaling properties.

  6. Ligand regulation of a constitutively dimeric EGF receptor

    NASA Astrophysics Data System (ADS)

    Freed, Daniel M.; Alvarado, Diego; Lemmon, Mark A.

    2015-06-01

    Ligand-induced receptor dimerization has traditionally been viewed as the key event in transmembrane signalling by epidermal growth factor receptors (EGFRs). Here we show that the Caenorhabditis elegans EGFR orthologue LET-23 is constitutively dimeric, yet responds to its ligand LIN-3 without changing oligomerization state. SAXS and mutational analyses further reveal that the preformed dimer of the LET-23 extracellular region is mediated by its domain II dimerization arm and resembles other EGFR extracellular dimers seen in structural studies. Binding of LIN-3 induces only minor structural rearrangements in the LET-23 dimer to promote signalling. Our results therefore argue that EGFR can be regulated by allosteric changes within an existing receptor dimer--resembling signalling by insulin receptor family members, which share similar extracellular domain compositions but form covalent dimers.

  7. Alternative splicing of human papillomavirus type-16 E6/E6* early mRNA is coupled to EGF signaling via Erk1/2 activation

    PubMed Central

    Rosenberger, Simone; Arce, Johanna De-Castro; Langbein, Lutz; Steenbergen, Renske D. M.; Rösl, Frank

    2010-01-01

    Certain types of human papillomaviruses (HPVs) are etiologically linked to cervical cancer. Their transforming capacity is encoded by a polycistronic premRNA, where alternative splicing leads to the translation of functional distinct proteins such as E6, E6*, and E7. Here we show that splicing of HPV16 E6/E7 ORF cassette is regulated by the epidermal growth factor (EGF) pathway. The presence of EGF was coupled to preferential E6 expression, whereas depletion of EGF, or treatment with EGF receptor (EGFR) neutralizing antibodies or the EGFR inhibitor tyrphostin AG1478, resulted in E6 exon exclusion in favor of E6*. As a consequence, increased p53 levels and enhanced translation of E7 with a subsequent reduction of the retinoblastoma protein pRb could be discerned. E6 exon exclusion upon EGF depletion was independent from promoter usage, mRNA stability, or selective mRNA transport. Time-course experiments and incubation with cycloheximide demonstrated that E6 alternative splicing is a direct and reversible effect of EGF signal transduction, not depending on de novo protein synthesis. Within this process, Erk1/2-kinase activation was the critical event for E6 exon inclusion, mediated by the upstream MAP kinase MEK1/2. Moreover, siRNA knockdown experiments revealed an involvement of splicing factors hnRNPA1 and hnRNPA2 in E6 exon exclusion, whereas the splicing factors Brm and Sam68 were found to promote E6 exon inclusion. Because there is a natural gradient of EGF and EGF receptor expression in the stratified epithelium, it is reasonable to assume that EGF modulates E6/E7 splicing during the viral life cycle and transformation. PMID:20351270

  8. Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells.

    PubMed

    Kosheverova, Vera V; Kamentseva, Rimma S; Gonchar, Ilya V; Kharchenko, Marianna V; Kornilova, Elena S

    2016-04-22

    Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering.

  9. Anti-MUC1 antibody inhibits EGF receptor signaling in cancer cells

    SciTech Connect

    Hisatsune, Akinori; Nakayama, Hideki; Kawasaki, Mitsuru; Horie, Ichiro; Miyata, Takeshi; Isohama, Yoichiro; Kim, Kwang Chul; Katsuki, Hiroshi

    2011-02-18

    Research highlights: {yields} We identified changes in the expression and function of EGFR by anti-MUC1 antibody. {yields} An anti-MUC1 antibody GP1.4 decreased EGFR from cell surface by internalization. {yields} GP1.4 specifically inhibited ERK signaling triggered EGF-EGFR signaling pathway. {yields} Internalization of EGFR was dependent on the presence of MUC1 on cell surface. {yields} GP1.4 significantly inhibited EGF-dependent cancer cell proliferation and migration. -- Abstract: MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells. High expression of MUC1 is closely associated with cancer progression and metastasis, leading to poor prognosis. We previously reported that MUC1 is internalized by the binding of the anti-MUC1 antibody, from the cell surface to the intracellular region via the macropinocytotic pathway. Since MUC1 is closely associated with ErbBs, such as EGF receptor (EGFR) in cancer cells, we examined the effect of the anti-MUC1 antibody on EGFR trafficking. Our results show that: (1) anti-MUC1 antibody GP1.4, but not another anti-MUC1 antibody C595, triggered the internalization of EGFR in pancreatic cancer cells; (2) internalization of EGFR by GP1.4 resulted in the inhibition of ERK phosphorylation by EGF stimulation, in a MUC1 dependent manner; (3) inhibition of ERK phosphorylation by GP1.4 resulted in the suppression of proliferation and migration of pancreatic cancer cells. We conclude that the internalization of EGFR by anti-MUC1 antibody GP1.4 inhibits the progression of cancer cells via the inhibition of EGFR signaling.

  10. DEPENDENCE OF PPAR LIGAND-INDUCED MAPK SIGNALING ON EPIDERMAL GROWTH FACTOR RECEPTOR TRANSACTIVATION HEPARIN-BINDING EGF CLEAVAGE MEDIATES ZINC-INDUCED EGF RECEPTOR PHOSPHORYLATION

    EPA Science Inventory

    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that function as ligand-activated transcription factors regulating lipid metabolism and homeostasis. In addition to their ability to regulate PPAR-mediated gene transcription, PPARalpha and gamma li...

  11. Trefoil peptides as proangiogenic factors in vivo and in vitro: implication of cyclooxygenase-2 and EGF receptor signaling.

    PubMed

    Rodrigues, Sylvie; Van Aken, Elisabeth; Van Bocxlaer, Saskia; Attoub, Samir; Nguyen, Quang-Dé; Bruyneel, Erik; Westley, Bruce R; May, Felicity E B; Thim, Lars; Mareel, Marc; Gespach, Christian; Emami, Shahin

    2003-01-01

    We previously established that the trefoil peptides (TFFs) pS2, spasmolytic polypeptide, and intestinal trefoil factor are involved in cellular scattering and invasion in kidney and colonic cancer cells. Using the chorioallantoic membrane (CAM) assay and the formation of tube-like structures by human umbilical vein endothelial cells (HUVEC) plated on the Matrigel matrix substratum, we report here that TFFs are proangiogenic factors. Angiogenic activity of TFFs is comparable to that induced by vascular endothelial growth factor, leptin, and transforming growth factor-alpha. Stimulation of angiogenesis by pS2 in the CAM assay is blocked by pharmacological inhibitors of cyclooxygenase COX-2 (NS-398) and epidermal growth factor receptor (EGF-R) tyrosine kinase (ZD1839), but is independent of KDR/Flk-1 and thromboxane A2 receptors. In contrast, the morphogenic switch induced by pS2 in HUVEC cells could be inhibited by the specific KDR heptapeptide antagonist ATWLPPR and by inhibitors of COX-2 and EGF-R signaling. These results implicate TFFs in the formation of new blood vessels during normal and pathophysiological processes linked to wound healing, inflammation, and cancer progression in the digestive mucosa and other human solid tumors associated with aberrant expression of TFFs.

  12. Matrikine and matricellular regulators of EGF receptor signaling on cancer cell migration and invasion.

    PubMed

    Grahovac, Jelena; Wells, Alan

    2014-01-01

    Cancer invasion is a complex process requiring, among other events, extensive remodeling of the extracellular matrix including deposition of pro-migratory and pro-proliferative moieties. In recent years, it has been described that while invading through matrices cancer cells can change shape and adapt their migration strategies depending on the microenvironmental context. Although intracellular signaling pathways governing the mesenchymal to amoeboid migration shift and vice versa have been mostly elucidated, the extracellular signals promoting these shifts are largely unknown. In this review, we summarize findings that point to matrikines that bind specifically to the EGF receptor as matricellular molecules that enable cancer cell migrational plasticity and promote invasion.

  13. Engineered epidermal growth factor mutants with faster binding on-rates correlate with enhanced receptor activation

    PubMed Central

    Lahti, Jennifer L.; Lui, Bertrand H.; Beck, Stayce E.; Lee, Stephen S.; Ly, Daphne P.; Longaker, Michael T.; Yang, George P.; Cochran, Jennifer R.

    2011-01-01

    Receptor tyrosine kinases (RTKs) regulate critical cell signaling pathways, yet the properties of their cognate ligands that influence receptor activation are not fully understood. There is great interest in parsing these complex ligand-receptor relationships using engineered proteins with altered binding properties. Here we focus on the interaction between two engineered epidermal growth factor (EGF) mutants and the EGF receptor (EGFR), a model member of the RTK superfamily. We found that EGF mutants with faster kinetic on-rates stimulate increased EGFR activation compared to wild-type EGF. These findings support previous predictions that faster association rates correlate with enhanced receptor activity. PMID:21439278

  14. Quantitative Analysis of the EGF Receptor Autocrine System Reveals Cryptic Regulation of Cell Response by Ligand Capture

    SciTech Connect

    Dewitt, Ann E.; Dong, Jian Y.; Wiley, H S.; Lauffenburger, Douglas A.

    2001-06-15

    Autocrine signaling is important in normal tissue physiology as well as pathological conditions. It is difficult to analyze these systems, however, because they are both self-contained and recursive. To understand how parameters, such as ligand production and receptor expression influence autocrine activity, we investigated a human epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) loop engineered into mouse B82 fibroblasts. We varied the level of ligand production using the tet-off expression system and used metalloprotease inhibitors to modulate ligand release. Receptor expression was varied using antagonistic, blocking antibodies. We compared autocrine ligand release to receptor activation using a microphysiometer-based assay and analyzed our data with a quantitative model of ligand release and receptor dynamics. We found that the activity of our autocrine system could be described in terms of a simple ratio between the rate of ligand production (VL) and the rate of receptor production (VR). At a VL/VR ratio of < 0.3, essentially no ligand was found in the extracellular medium, but a significant number cell receptors (30-40%) were occupied. As the VL/VR ratio increased from 0.3 towards unity, receptor occupancy increased, and significant amounts of ligand now appeared in the medium. Above a VL/VR ratio of 1.0, receptor occupancy approached saturation and most of the released ligand was lost into the medium. Analysis of human mammary epithelial cells showed that a VL/VR ratio of < 5 x 10 -4 was sufficient to evoke >20% of a maximal proliferative response. This suggests that natural autocrine systems are active even when no ligand appears in the extracellular medium; i.e., they operate 'invisibly' to general detection.

  15. Cholesterol Dictates the Freedom of EGF Receptors and HER2 in the Plane of the Membrane

    SciTech Connect

    Orr, Galya; Hu, Dehong; Ozcelik, Serdar; Opresko, Lee; Wiley, H. S.; Colson, Steve D.

    2005-05-20

    The flow of information through the EGF receptor (EGFR) is shaped by molecular interactions in the plasma membrane. The EGFR is associated with lipid rafts, but their role in modulating receptor mobility and subsequent interactions is unclear. To investigate the role of nanoscale rafts in EGFR dynamics, we used single-molecule fluorescence imaging to track individual receptors and their dimerization partner, HER2, in the membrane of human mammary epithelial cells. We found that the motion of both receptors was interrupted by dwellings within nanodomains. EGFR was significantly less mobile than HER2. This difference was likely due to F-actin because its deploymerization led to similar diffusion patterns between the EGFR and HER2. Manipulations of membrane cholesterol content dramatically altered the diffusion pattern of both receptors. Cholesterol depletion led to almost complete confinement of the receptors, whereas cholesterol enrichment extended the boundaries of the restricted areas. Interestingly, F-actin deploymerization partially restored receptor mobility in cholesterol depleted membranes. Our observations suggest that membrane cholesterol provides a dynamic environment that facilitates the free motion of EGFR and HER2, possible by modulating the dynamic state of F-actin. The association of the receptors with lipid rafts could therefore promote their rapid interactions only upon ligand stimulation.

  16. [INHIBITORS OF MAP-KINASE PATHWAY U0126 AND PD98059 DIFFERENTLY AFFECT ORGANIZATION OF TUBULIN CYTOSKELETON AFTER STIMULATION OF EGF RECEPTOR ENDOCYTOSIS].

    PubMed

    Zlobina, M V; Steblyanko, Yu Yu; Shklyaeva, M A; Kharchenko, V V; Salova, A V; Kornilova, E S

    2015-01-01

    To confirm the hypothesis about the involvement of EGF-stimulated MAP-kinase ERK1/2 in the regulation of microtubule (MT) system, the influence of two widely used ERK1/2 inhibitors, U0126 and PD98059, on the organization of tubulin cytoskeleton in interphase HeLa cells during EGF receptor endocytosis has been investigated. We have found that addition of U0126 or PD98059 to not-stimulated with EGF ells for 30 min has no effect on radially organized MT system. However, in the case of U0126 addition before EGF endocytosis stimulation, the number of MT per cell decreased within 15 min after such stimulation and was followed by complete MT depolymerization by 60-90 min. Stimulation of EGF endocytosis in the presence of PD98059 resulted only in insignificant depolymerization of MT and it could be detected mainly from their minus-ends. At the same time, MT regions close to plasma membrane became stabilized, which was proved by increase in tubulin acetylation level. This situation was characteristic for all period of the experiment. It has been also found that the inhibitors affect endocytosis dynamics of EGF-receptor complexes. Quantitative analysis demonstrated that the stimulation of endocytosis in the presence of U0126 generated a greater number of endosomes compared to control cells, and their number did not change significantly during the experiment. All these endosomes were localized peripherally. Effect of PD98059 resulted in the formation of lower number of endosomes that in control, but they demonstrated very slow clusterization despite the presence of some intact MT. Both inhibitors decreased EGFR colocolization with early endosomal marker EEA1, which indicated a delay in endosome fusions and maturation. The inhibitors were also shown to affect differently phospho-ERK 1 and 2 forms: U0126 completely inhibited phospho-ERK1 and 2, white, in the presence of PD98059, the two ERK forms demonstrated sharp transient activation in 15 min after stimulation, but only

  17. A novel taspine derivative, HMQ1611, inhibits breast cancer cell growth via estrogen receptor α and EGF receptor signaling pathways.

    PubMed

    Zhan, Yingzhuan; Zhang, Yanmin; Liu, Cuicui; Zhang, Jie; Smith, Wanli W; Wang, Nan; Chen, Yinnan; Zheng, Lei; He, Langchong

    2012-06-01

    Breast cancer is a common cancer with a leading cause of cancer mortality in women. Currently, the chemotherapy for breast cancer is underdeveloped. Here, we report a novel taspine derivative, HMQ1611, which has anticancer effects using in vitro and in vivo breast cancer models. HMQ1611 reduced cancer cell proliferation in four human breast cancer cell lines including MDA-MB-231, SK-BR-3, ZR-75-30, and MCF-7. HMQ1611 more potently reduced growth of estrogen receptor α (ERα)-positive breast cancer cells (ZR-75-30 and MCF-7) than ERα-negative cells (MDA-MB-231 and SK-BR-3). Moreover, HMQ1611 arrested breast cancer cell cycle at S-phase. In vivo tumor xenograft model, treatment of HMQ1611 significantly reduced tumor size and weight compared with vehicles. We also found that HMQ1611 reduced ERα expression and inhibited membrane ERα-mediated mitogen-activated protein kinase (MAPK) signaling following the stimulation of cells with estrogen. Knockdown of ERα by siRNA transfection in ZR-75-30 cells attenuated HMQ1611 effects. In contrast, overexpression of ERα in MDA-MB-231 cells enhanced HMQ1611 effects, suggesting that ERα pathway mediated HMQ1611's inhibition of breast cancer cell growth in ERα-positive breast cancer. HMQ1611 also reduced phosphorylation of EGF receptor (EGFR) and its downstream signaling players extracellular signal-regulated kinase (ERK)1/2 and AKT activation both in ZR-75-30 and MDA-MB-231 cells. These results showed that the novel compound HMQ1611 had anticancer effects, and partially via ERα and/or EGFR signaling pathways, suggesting that HMQ1611 may be a potential novel candidate for human breast cancer intervention.

  18. Shoc2 is targeted to late endosomes and required for Erk1/2 activation in EGF-stimulated cells.

    PubMed

    Galperin, Emilia; Abdelmoti, Lina; Sorkin, Alexander

    2012-01-01

    Shoc2 is the putative scaffold protein that interacts with RAS and RAF, and positively regulates signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). To elucidate the mechanism by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor (EGF) receptor (EGFR), we studied subcellular localization of Shoc2. Upon EGFR activation, endogenous Shoc2 and red fluorescent protein tagged Shoc2 were translocated from the cytosol to a subset of late endosomes containing Rab7. The endosomal recruitment of Shoc2 was blocked by overexpression of a GDP-bound H-RAS (N17S) mutant and RNAi knockdown of clathrin, suggesting the requirement of RAS activity and clathrin-dependent endocytosis. RNAi depletion of Shoc2 strongly inhibited activation of ERK1/2 by low, physiological EGF concentrations, which was rescued by expression of wild-type recombinant Shoc2. In contrast, the Shoc2 (S2G) mutant, that is myristoylated and found in patients with the Noonan-like syndrome, did not rescue ERK1/2 activation in Shoc2-depleted cells. Shoc2 (S2G) was not located in late endosomes but was present on the plasma membrane and early endosomes. These data suggest that targeting of Shoc2 to late endosomes may facilitate EGFR-induced ERK activation under physiological conditions of cell stimulation by EGF, and therefore, may be involved in the spatiotemporal regulation of signaling through the RAS-RAF module. PMID:22606262

  19. Androgen receptor in Sertoli cells regulates DNA double-strand break repair and chromosomal synapsis of spermatocytes partially through intercellular EGF-EGFR signaling.

    PubMed

    Chen, Su-Ren; Hao, Xiao-Xia; Zhang, Yan; Deng, Shou-Long; Wang, Zhi-Peng; Wang, Yu-Qian; Wang, Xiu-Xia; Liu, Yi-Xun

    2016-04-01

    Spermatogenesis does not progress beyond the pachytene stages of meiosis in Sertoli cell-specific AR knockout (SCARKO) mice. However, further evidence of meiotic arrest and underlying paracrine signals in SCARKO testes is still lacking. We utilized co-immunostaining of meiotic surface spreads to examine the key events during meiotic prophase I. SCARKO spermatocytes exhibited a failure in chromosomal synapsis observed by SCP1/SCP3 double-staining and CREST foci quantification. In addition, DNA double-strand breaks (DSBs) were formed but were not repaired in the mutant spermatocytes, as revealed by γ-H2AX staining and DNA-dependent protein kinase (DNA-PK) activity examination. The later stages of DSB repair, such as the accumulation of the RAD51 strand exchange protein and the localization of mismatch repair protein MLH1, were correspondingly altered in SCARKO spermatocytes. Notably, the expression of factors that guide RAD51 loading onto sites of DSBs, including TEX15, BRCA1/2 and PALB2, was severely impaired when either AR was down-regulated or EGF was up-regulated. We observed that some ligands in the epidermal growth factor (EGF) family were over-expressed in SCARKO Sertoli cells and that some receptors in the EGF receptor (EGFR) family were ectopically activated in the mutant spermatocytes. When EGF-EGFR signaling was repressed to approximately normal by the specific inhibitor AG1478 in the cultured SCARKO testis tissues, the arrested meiosis was partially rescued, and functional haploid cells were generated. Based on these data, we propose that AR in Sertoli cells regulates DSB repair and chromosomal synapsis of spermatocytes partially through proper intercellular EGF-EGFR signaling.

  20. Androgen receptor in Sertoli cells regulates DNA double-strand break repair and chromosomal synapsis of spermatocytes partially through intercellular EGF-EGFR signaling.

    PubMed

    Chen, Su-Ren; Hao, Xiao-Xia; Zhang, Yan; Deng, Shou-Long; Wang, Zhi-Peng; Wang, Yu-Qian; Wang, Xiu-Xia; Liu, Yi-Xun

    2016-04-01

    Spermatogenesis does not progress beyond the pachytene stages of meiosis in Sertoli cell-specific AR knockout (SCARKO) mice. However, further evidence of meiotic arrest and underlying paracrine signals in SCARKO testes is still lacking. We utilized co-immunostaining of meiotic surface spreads to examine the key events during meiotic prophase I. SCARKO spermatocytes exhibited a failure in chromosomal synapsis observed by SCP1/SCP3 double-staining and CREST foci quantification. In addition, DNA double-strand breaks (DSBs) were formed but were not repaired in the mutant spermatocytes, as revealed by γ-H2AX staining and DNA-dependent protein kinase (DNA-PK) activity examination. The later stages of DSB repair, such as the accumulation of the RAD51 strand exchange protein and the localization of mismatch repair protein MLH1, were correspondingly altered in SCARKO spermatocytes. Notably, the expression of factors that guide RAD51 loading onto sites of DSBs, including TEX15, BRCA1/2 and PALB2, was severely impaired when either AR was down-regulated or EGF was up-regulated. We observed that some ligands in the epidermal growth factor (EGF) family were over-expressed in SCARKO Sertoli cells and that some receptors in the EGF receptor (EGFR) family were ectopically activated in the mutant spermatocytes. When EGF-EGFR signaling was repressed to approximately normal by the specific inhibitor AG1478 in the cultured SCARKO testis tissues, the arrested meiosis was partially rescued, and functional haploid cells were generated. Based on these data, we propose that AR in Sertoli cells regulates DSB repair and chromosomal synapsis of spermatocytes partially through proper intercellular EGF-EGFR signaling. PMID:26959739

  1. Pancreatic secretory trypsin inhibitor causes autocrine-mediated migration and invasion in bladder cancer and phosphorylates the EGF receptor, Akt2 and Akt3, and ERK1 and ERK2.

    PubMed

    Marchbank, Tania; Mahmood, Asif; Playford, Raymond J

    2013-08-01

    Pancreatic secretory trypsin inhibitor (PSTI) is expressed in most bladder carcinomas, where its pathophysiological relevance is unclear. Using recombinant normal sequence PSTI/tumor-associated trypsin inhibitor (TATI), a variant associated with familial pancreatitis (N34S), an active site-inactivated variant (R18/V19), and immunoneutralization and RNA interference-mediated knockdown techniques, we investigated the actions of PSTI/TATI on cell migration (wounding monolayers), collagen invasion (gel invasion assays), and proliferation (Alamar blue) on 253J, RT4, and HT1376 human bladder carcinoma cell lines. All three forms of PSTI/TATI stimulated migration twofold, and normal sequence PSTI/TATI showed synergistic promigratory effects when added with EGF. Addition of structurally unrelated soybean trypsin inhibitor had no promigratory activity. Similar results were seen using collagen invasion assays, although the active site mutated variant had no proinvasive activity, probably due to reduced Akt2 activation. PSTI/TATI did not stimulate proliferation despite acting, at least partially, through the EGF receptor, as effects of PSTI/TATI were truncated by the addition of an EGF receptor blocking antibody or the tyrosine kinase inhibitor tyrphostin. Cell lines produced endogenous PSTI/TATI, and PSTI/TATI RNA interference knockdown or the addition of PSTI/TATI, EGF receptor, or tyrphostin blocking agents reduced migration and invasion below baseline. PSTI/TATI induced phosphorylation of the EGF receptor, ERK1 and ERK2, Akt2 and Akt3, JNK1, MKK3, and ribosomal protein S6 kinase 1. This profile was more limited than that induced by EGF and did not include Akt1, probably explaining the lack of proproliferative activity. Our findings of autocrine stimulation and synergistic responses between EGF and PSTI/TATI at concentrations found in urine and tissue suggest that PSTI/TATI has pathophysiological relevance.

  2. Ezrin-radixin-moesin-binding phosphoprotein-50 regulates EGF-induced AKT activation through interaction with EGFR and PTEN.

    PubMed

    Zheng, Junfang; Dai, Yuanping; Yang, Zhiyu; Yang, Longyan; Peng, Zhiqiang; Meng, Ran; Xiong, Ying; He, Junqi

    2016-01-01

    Dysregulated epidermal growth factor receptor (EGFR) signaling, especially EGFR/AKT signaling, plays important roles in tumorigenesis and progression, the study on intracellular regulation of this signaling pathway has great clinical significance. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is an important antagonist of AKT activity. Its regulation of AKT activity can be enhanced by ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50)-mediated PTEN/EBP50/platelet-derived growth factor receptor (PDGFR) complex. EBP50 was reported to bind to EGFR, and that it may also mediate the formation of PTEN/EGFR complex to regulate EGFR/AKT signaling. In this study, experiments were performed to verify the hypothesis. Results showed that PTEN co-immunoprecipitated with EGFR, demonstrating PTEN/EGFR complex can form in tissue. Further studies showed that EBP50 knockdown decreased the amount of PTEN/EGFR complex by GST pull-down assay, and EBP50 overexpression increased the amount of PTEN/EGFR complex in a dose-dependent manner. While PTEN mutant (V403A), which can not bind with EBP50, only slightly mediated the formation of PTEN/EGFR complex, confirming that EBP50 specifically mediated the formation of the PTEN/EGFR complex. Both PTEN (V403A) and EGFR (L1043/1063F) mutants can not bind with EBP50. The expression of PTEN (V403A) or EGFR (L1043/1063F) mutant in cells resulted in higher AKT activation level than their respective wild-types by EGF stimulation, indicating that EBP50-mediated PTEN/EGFR complex can effectively inhibit EGF-induced AKT activation. EGF stimulation of siEBP50 cells induced higher AKT activation level compared with control cells, further confirming EBP50-mediated PTEN/EGFR complex can more effectively inhibit EGF-induced AKT activation. These results demonstrated the PTEN/EGFR complex formed under the mediation of EBP50, revealing a novel mechanism for negative regulation of EGF-induced AKT pathway, which may be an important molecular

  3. ANALYSIS OF ANDROGEN- AND EGF-RECEPTOR EXPRESSION IN THE FETAL RAT PHALLUS AFTER EXPOSURE TO VINCLOZOLIN

    EPA Science Inventory

    Analysis of Androgen- and EGF-Receptor Expression in the Fetal Rat Phallus After Exposure to Vinclozolin
    Cynthia Wolf1,2, Barbara Abbott1, Gerald A. LeBlanc2, and L. Earl Gray, Jr.1
    1USEPA, ORD, NHEERL, RTD, RTP, NC 27711, 2NCSU, Environmental and Molecular Toxicology, Ral...

  4. Models for the activation pathway of epidermal growth factor receptor protein-tyrosine kinase

    SciTech Connect

    Campion, S.R.; Niyogi, S.K. )

    1991-03-15

    Activation of the epidermal growth factor (EGF) receptor's intrinsic protein-tyrosine kinase activity, which occurs upon formation of the receptor-ligand complex, is the critical regulatory event affecting the subsequent EGF-dependent cellular responses leading to DNA synthesis and cell proliferation. The molecular mechanism by which EGF-dependent activation of receptor kinase activity takes place is not clearly understood. In this study, the growth factor-dependent activation of the EGF receptor tyrosine kinase was examined in vitro using detergent-solubilized, partially purified GEF receptors from A5431 human epidermoid carcinoma cells. Evaluation of the cooperativity observed in the EGF-dependent activation of soluble receptor tyrosine kinase would suggest a mechanism requiring the binding of the EGF peptide to both ligand binding sites on a receptor dimer to induce full receptor kinase activity. Equations describing potential cooperative kinase activation pathways have been examined. The theoretical system which best simulates the allosteric regulation observed in the experimental kinase activation data is that describing multiple essential activation. In addition, studies using mutant analogs of the EGF peptide ligand appear to confirm the requirement for an essential conformational change in the receptor-ligand complex to activate the receptor kinase activity. Several mutant growth factor analogues are able to occupy the ligand binding sites on the receptor without inducing the fully active receptor conformation.

  5. Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers

    PubMed Central

    Iwamoto, Ryo; Takagi, Mika; Akatsuka, Jun-ichi; Ono, Ken-ichiro; Kishi, Yoshiro

    2016-01-01

    Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF. PMID:26974561

  6. Alpha6beta4 integrin crosslinking induces EGFR clustering and promotes EGF-mediated Rho activation in breast cancer

    PubMed Central

    Gilcrease, Michael Z; Zhou, Xiao; Lu, Xiaolin; Woodward, Wendy A; Hall, Brian E; Morrissey, Phillip J

    2009-01-01

    Background The α6β4 integrin is overexpressed in the basal subtype of breast cancer and plays an important role in tumor cell motility and invasion. EGFR is also overexpressed in the basal subtype of breast cancer, and crosstalk between α6β4 integrin and EGFR appears to be important in tumor progression. Methods We evaluated the effects of α6β4 crosslinking on the distribution and function of EGFR in breast carcinoma cell line MDA-MB-231. Receptor distribution was evaluated by fluorescence microscopy and multispectral imaging flow cytometry, and ligand-mediated EGFR signaling was evaluated using Western blots and a Rho pull-down assay. Results Antibody-mediated crosslinking of α6β4 integrin was sufficient to induce cell-surface clustering of not only α6β4 but also EGFR in nonadherent cells. The induced clustering of EGFR was observed minimally after 5 min of integrin crosslinking but was more prominent after 15 min. EGFR clustering had minimal effect on the phosphorylation of Akt or Erk1,2 in response to EGF in suspended cells or in response to HB-EGF in adherent cells. However, EGFR clustering induced by crosslinking α6β4 had a marked effect on Rho activation in response to EGF. Conclusion Crosslinking α6β4 integrin in breast carcinoma cells induces EGFR clustering and preferentially promotes Rho activation in response to EGF. We hypothesize that this integrin-EGFR crosstalk may facilitate tumor cell cytoskeletal rearrangements important for tumor progression. PMID:19470173

  7. Use of SLAM and PVRL4 and identification of pro-HB-EGF as cell entry receptors for wild type phocine distemper virus.

    PubMed

    Melia, Mary M; Earle, John Philip; Abdullah, Haniah; Reaney, Katherine; Tangy, Frederic; Cosby, Sara Louise

    2014-01-01

    Signalling lymphocyte activation molecule (SLAM) has been identified as an immune cell receptor for the morbilliviruses, measles (MV), canine distemper (CDV), rinderpest and peste des petits ruminants (PPRV) viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4), also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV) remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt) MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG) and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF),for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO) cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation. PMID:25171206

  8. Use of SLAM and PVRL4 and identification of pro-HB-EGF as cell entry receptors for wild type phocine distemper virus.

    PubMed

    Melia, Mary M; Earle, John Philip; Abdullah, Haniah; Reaney, Katherine; Tangy, Frederic; Cosby, Sara Louise

    2014-01-01

    Signalling lymphocyte activation molecule (SLAM) has been identified as an immune cell receptor for the morbilliviruses, measles (MV), canine distemper (CDV), rinderpest and peste des petits ruminants (PPRV) viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4), also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV) remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt) MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG) and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF),for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO) cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation.

  9. Immunotherapy of human tumour xenografts overexpressing the EGF receptor with rat antibodies that block growth factor-receptor interaction.

    PubMed Central

    Modjtahedi, H.; Eccles, S.; Box, G.; Styles, J.; Dean, C.

    1993-01-01

    Athymic mice bearing xenografts of human tumours that overexpress the receptor (EGFR) for EGF and TGF alpha have been used to evaluate the therapeutic potential of three new rat monoclonal antibodies (mAbs) directed against two distinct epitopes on the extracellular domain of the human EGFR. The antibodies, ICR16 (IgG2a), ICR62 (IgG2b) and ICR64 (IgG1), have been shown (Modjtahedi et al., 1993) to be potent inhibitors of the growth in vitro of a number of human squamous cell carcinomas because they block receptor-ligand interaction. When given i.p. at 200 micrograms dose, the three antibodies were found to induce complete regression of xenografts of the HN5 tumour if treatment with antibody commenced at the time of tumour implantation (total doses: ICR16, 3.0 mg; ICR62, 1.2 mg; ICR64, 2.2 mg). More importantly when treatment was delayed until the tumours were established (mean diam. 0.5 cm) both ICR16 and ICR62 induced complete or almost complete regression of the tumours. Furthermore, treatment with a total dose of only 0.44 mg of ICR62 was found to induce complete remission of xenografts of the breast carcinoma MDA-MB 468, but ICR16 was less effective at this dose of antibody and only 4/8 tumours regressed completely. ICR16 and ICR62 were poor inhibitors of the growth in vitro of the vulval carcinoma A431, but both induced a substantial delay in the growth of xenografts of this tumour and 4/8 tumours regressed completely in the mice treated with ICR62 (total dose 2.2 mg). Although ICR16 and ICR64 were more effective than ICR62 as growth inhibitors in vitro, ICR62 was found to be substantially better at inducing regression of the tumour xenografts due perhaps to additional activation of host immune effector functions by the IgG2b antibody. We conclude that these antibodies may be useful therapeutic agents that can be used alone without conjugation to other cytotoxic moieties. PMID:7679281

  10. The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

    SciTech Connect

    Hoskins, J.T.; Zhou, Z.; Harding, P.A.

    2008-10-31

    A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser{sub 108/121}, HB-EGF-Cys/Ser{sub 116/132}, and HB-EGF-Cys/Ser{sub 134/143}) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with M{sub r} of 6.5, 21 and 24 kDa were observed from lysates of HB-EGF and each HB-EGF disulfide analogue. HB-EGF immunohistochemical analyses of each HB-EGF stable cell line demonstrated ubiquitous protein expression except HB-EGF-Cys/Ser{sub 108/121} and HB-EGF-Cys/Ser{sub 116/132} stable cell lines which exhibited accumulated expression immediately outside the nucleus. rHB-EGF, HB-EGF, and HB-EGF{sub 134/143} proteins competed with {sup 125}I-EGF in an A431 competitive binding assay, whereas HB-EGF-Cys/Ser{sub 108/121} and HB-EGF-Cys/Ser{sub 116/132} failed to compete. Each HB-EGF disulfide analogue lacked the ability to stimulate tyrosine phosphorylation of the 170 kDa EGFR. These results suggest that HB-EGF-Cys/Ser{sub 134/143} antagonizes EGFRs.

  11. Autocrine production of TGF-{beta} confers resistance to apoptosis after an epithelial-mesenchymal transition process in hepatocytes: Role of EGF receptor ligands

    SciTech Connect

    Castillo, Gaelle del; Murillo, Miguel M.; Bertran, Esther; Sanchez, Aranzazu; Fabregat, Isabel . E-mail: ifabregat@iro.es

    2006-09-10

    Transforming growth factor-beta (TGF-{beta}) induces apoptosis in fetal rat hepatocytes. However, a subpopulation of these cells survives, concomitant with changes in phenotype, reminiscent of an epithelial-mesenchymal transition (EMT). We have previously suggested that EMT might confer cell resistance to apoptosis (Valdes et al., Mol. Cancer Res., 1: 68-78, 2002). However, the molecular mechanisms responsible for this resistance are not explored yet. In this work, we have isolated and subcultured the population of hepatocytes that suffered the EMT process and are resistant to apoptosis (TGF-{beta}-treated fetal hepatocytes: T{beta}T-FH). We prove that they secrete mitogenic and survival factors, as analyzed by the proliferative and survival capacity of conditioned medium. Inhibition of the epidermal growth factor receptor (EGFR) sensitizes T{beta}T-FH to die after serum withdrawal. T{beta}T-FH expresses high levels of transforming growth factor-alpha (TGF-{alpha}) and heparin-binding EGF-like growth factor (HB-EGF) and shows constitutive activation of the EGFR pathway. A blocking anti-TGF-{alpha} antibody restores the capacity of cells to die. TGF-{beta}, which is expressed by T{beta}T-FH, mediates up-regulation of TGF-{alpha} and HB-EGF expression in those cells. In summary, results suggest that an autocrine loop of TGF-{beta} confers resistance to apoptosis after an EMT process in hepatocytes, through the increase in the expression of EGFR ligands.

  12. TEIF associated centrosome activity is regulated by EGF/PI3K/Akt signaling.

    PubMed

    Zhao, Jing; Zou, Yongxin; Liu, Haijing; Wang, Huali; Zhang, Hong; Hou, Wei; Li, Xin; Jia, Xinying; Zhang, Jing; Hou, Lin; Zhang, Bo

    2014-09-01

    Centrosome amplification, which is a characteristic of cancer cells, has been understood as a driving force of genetic instability in the development of cancer. In previous work, we demonstrated that TEIF (transcriptional element-interacting factor) distributes in the centrosomes and regulates centrosome status under both physiologic and pathologic conditions. Here we identify TEIF as a downstream effector in EGF/PI3K/Akt signaling. The addition of EGF or transfection of active Akt stimulates centrosome TEIF distribution, resulting in an increase of centrosome splitting and amplification, while inhibitors of either PI3K or Akt attenuate these changes in TEIF and the associated centrosome status. A consensus motif for Akt phosphorylation (RHRVLT) proved to be involved in centrosomal TEIF localization, and the 469-threonine of this motif may be phosphorylated by Akt both in vitro and in vivo. Elimination of this phosphorylated site on TEIF caused reduced centrosome distribution and centrosome splitting or amplification. Moreover, TEIF closely co-localized with C-NAP1 at the proximal ends of centrioles, and centriolar loading of TEIF stimulated by EGF/Akt could displace C-NAP1, resulting in centrosome splitting. These findings reveal linkage of the EGF/PI3K/Akt signaling pathway to regulation of centrosome status which may act as an oncogenic pathway and induce genetic instability in carcinogenesis. PMID:24769208

  13. Transgenic Soybean Production of Bioactive Human Epidermal Growth Factor (EGF)

    PubMed Central

    He, Yonghua; Schmidt, Monica A.; Erwin, Christopher; Guo, Jun; Sun, Raphael; Pendarvis, Ken; Warner, Brad W.; Herman, Eliot M.

    2016-01-01

    Necrotizing enterocolitis (NEC) is a devastating condition of premature infants that results from the gut microbiome invading immature intestinal tissues. This results in a life-threatening disease that is frequently treated with the surgical removal of diseased and dead tissues. Epidermal growth factor (EGF), typically found in bodily fluids, such as amniotic fluid, salvia and mother’s breast milk, is an intestinotrophic growth factor and may reduce the onset of NEC in premature infants. We have produced human EGF in soybean seeds to levels biologically relevant and demonstrated its comparable activity to commercially available EGF. Transgenic soybean seeds expressing a seed-specific codon optimized gene encoding of the human EGF protein with an added ER signal tag at the N’ terminal were produced. Seven independent lines were grown to homozygous and found to accumulate a range of 6.7 +/- 3.1 to 129.0 +/- 36.7 μg EGF/g of dry soybean seed. Proteomic and immunoblot analysis indicates that the inserted EGF is the same as the human EGF protein. Phosphorylation and immunohistochemical assays on the EGF receptor in HeLa cells indicate the EGF protein produced in soybean seed is bioactive and comparable to commercially available human EGF. This work demonstrates the feasibility of using soybean seeds as a biofactory to produce therapeutic agents in a soymilk delivery platform. PMID:27314851

  14. Transgenic Soybean Production of Bioactive Human Epidermal Growth Factor (EGF).

    PubMed

    He, Yonghua; Schmidt, Monica A; Erwin, Christopher; Guo, Jun; Sun, Raphael; Pendarvis, Ken; Warner, Brad W; Herman, Eliot M

    2016-01-01

    Necrotizing enterocolitis (NEC) is a devastating condition of premature infants that results from the gut microbiome invading immature intestinal tissues. This results in a life-threatening disease that is frequently treated with the surgical removal of diseased and dead tissues. Epidermal growth factor (EGF), typically found in bodily fluids, such as amniotic fluid, salvia and mother's breast milk, is an intestinotrophic growth factor and may reduce the onset of NEC in premature infants. We have produced human EGF in soybean seeds to levels biologically relevant and demonstrated its comparable activity to commercially available EGF. Transgenic soybean seeds expressing a seed-specific codon optimized gene encoding of the human EGF protein with an added ER signal tag at the N' terminal were produced. Seven independent lines were grown to homozygous and found to accumulate a range of 6.7 +/- 3.1 to 129.0 +/- 36.7 μg EGF/g of dry soybean seed. Proteomic and immunoblot analysis indicates that the inserted EGF is the same as the human EGF protein. Phosphorylation and immunohistochemical assays on the EGF receptor in HeLa cells indicate the EGF protein produced in soybean seed is bioactive and comparable to commercially available human EGF. This work demonstrates the feasibility of using soybean seeds as a biofactory to produce therapeutic agents in a soymilk delivery platform. PMID:27314851

  15. Egr-1 is a critical regulator of EGF-receptor-mediated expansion of subventricular zone neural stem cells and progenitors during recovery from hypoxia–hypoglycemia

    PubMed Central

    Alagappan, Dhivyaa; Balan, Murugabaskar; Jiang, Yuhui; Cohen, Rachel B.; Kotenko, Sergei V.; Levison, Steven W.

    2013-01-01

    We recently established that the EGF-R (epidermal growth factor receptor) (EGF-R) is an essential regulator of the reactive expansion of SVZ (subventricular zone) NPs (neural precursors) that occurs during recovery from hypoxic-ischemic brain injury. The purpose of the current studies was to identify the conditions and the transcription factor (s) responsible for inducing the EGF-R. Here, we show that the increase in EGF-R expression and the more rapid division of the NPs can be recapitulated in in vitro by exposing SVZ NPs to hypoxia and hypoglycemia simultaneously, but not separately. The EGF-R promoter has binding sites for multiple transcription factors that includes the zinc finger transcription factor, Egr-1. We show that Egr-1 expression increases in NPs, but not astrocytes, following hypoxia and hypoglycemia where it accumulates in the nucleus. To determine whether Egr-1 is necessary for EGF-R expression, we used SiRNAs (small interfering RNA) specific for Egr-1 to decrease Egr-1 expression. Knocking-down Egr-1 decreased basal levels of EGF-R and it abolished the stress-induced increase in EGF-R expression. By contrast, HIF-1 accumulation did not contribute to EGF-R expression and FGF-2 only modestly induced EGF-R. These studies establish a new role for Egr-1 in regulating the expression of the mitogenic EGF-R. They also provide new information into mechanisms that promote NP expansion and provide insights into strategies for amplifying the numbers of stem cells for CNS (central nervous system) regeneration. PMID:23763269

  16. An EGFR wild type-EGFRvIII-HB-EGF feed forward loop regulates the activation of EGFRvIII

    PubMed Central

    Li, Li; Chakraborty, Sharmistha; Yang, Chin-Rang; Hatanpaa, Kimmo J.; Cipher, Daisha J.; Puliyappadamba, Vineshkumar Thidil; Rehman, Alizeh; Jiwani, Ameena J.; Mickey, Bruce; Madden, Christopher; Raisanen, Jack; Burma, Sandeep; Saha, Debabrata; Wang, Zhixiang; Pingle, Sandeep C.; Kesari, Santosh; Boothman, David A.; Habib, Amyn A.

    2014-01-01

    EGFRvIII is a key oncogene in glioblastoma (GBM). EGFRvIII results from an in frame deletion in the extracellular domain of EGFR, does not bind ligand, and is thought to be constitutively active. While EGFRvIII dimerization is known to activate EGFRvIII, the factors that drive EGFRvIII dimerization and activation are not well understood. Here we present a new model of EGFRvIII activation and propose that oncogenic activation of EGFRvIII in glioma cells is driven by co-expressed activated EGFR wild type (EGFRwt). Increasing EGFRwt leads to a striking increase in EGFRvIII tyrosine phosphorylation and activation while silencing EGFRwt inhibits EGFRvIII activation. Both the dimerization arm and the kinase activity of EGFRwt are required for EGFRvIII activation. EGFRwt activates EGFRvIII by facilitating EGFRvIII dimerization. We have previously identified HB-EGF, a ligand for EGFRwt, as a gene induced specifically by EGFRvIII. In this study we show that HB-EGF, is induced by EGFRvIII only when EGFRwt is present. Remarkably, altering HB-EGF recapitulates the effect of EGFRwt on EGFRvIII activation. Thus, increasing HB-EGF leads to a striking increase in EGFRvIII tyrosine phosphorylation while silencing HB-EGF attenuates EGFRvIII phosphorylation, suggesting that an EGFRvIII-HB-EGF-EGFRwt feed forward loop regulates EGFRvIII activation. Silencing EGFRwt or HB-EGF leads to a striking inhibition of EGFRvIII induced tumorigenicity, while increasing EGFRwt or HB-EGF levels resulted in accelerated EGFRvIII mediated oncogenicity in an orthotopic mouse model. Furthermore, we demonstrate the existence of this loop in human GBM. Thus, our data demonstrate that oncogenic activation of EGFRvIII in GBM is likely maintained by a continuous EGFRwt-EGFRvIII-HBEGF loop, potentially an attractive target for therapeutic intervention. PMID:24077285

  17. Activation of the IGF1R pathway potentially mediates acquired resistance to mutant-selective 3rd-generation EGF receptor tyrosine kinase inhibitors in advanced non-small cell lung cancer

    PubMed Central

    Park, Ji Hyun; Choi, Yun Jung; Kim, Seon Ye; Lee, Jung-Eun; Sung, Ki Jung; Park, Sojung; Kim, Woo Sung; Song, Joon Seon; Choi, Chang-Min; Sung, Young Hoon; Rho, Jin Kyung; Lee, Jae Cheol

    2016-01-01

    Mutant-selective, 3rd-generation EGFR-TKIs were recently developed to control lung cancer cells harboring T790M-mediated resistance. However, the development of resistance to these novel drugs seems inevitable. Thus, we investigated the mechanism of acquired resistance to the mutant-selective EGFR-TKI WZ4002. We established five WZ4002-resistant cells, derived from cells harboring both EGFR and T790M mutations by long-term exposure to increasing doses of WZ4002. Compared with the parental cells, all resistant cells showed 10–100-folds higher resistance to WZ4002, as well as cross-resistance to other mutant-selective inhibitors. Among them, three resistant cells (HCC827/WR, PC-9/WR and H1975/WR) showed dependency on EGFR signaling, but two other cells (PC-9/GR/WR and PC-9/ER/WR) were not. Notably, insulin-like growth factor-1 receptor (IGF1R) was aberrantly activated in PC-9/GR/WR cells in phospho-receptor tyrosine kinase array, consistently accompanied by loss of IGF binding protein-3 (IGFBP3). Down-regulation of IGF1R by shRNA, as well as inhibition of IGF1R activity either by AG-1024 (a small molecule IGF1R inhibitor) or BI 836845 (a monoclonal anti-IGF1/2 blocking antibody), restored the sensitivity to WZ4002 both in vitro and xenograft. Taken together, these results suggest that activation of the IGF1R pathway associated with IGFBP3 loss can induce an acquired resistance to the mutant-selective EGFR-TKI, WZ4002. Therefore, a combined therapy of IGF1R inhibitors and mutant-selective EGFR-TKIs might be a viable treatment strategy for overcoming acquired resistance. PMID:26980747

  18. Induction of COX-2 protein expression by vanadate in A549 human lung carcinoma cell line through EGF receptor and p38 MAPK-mediated pathway

    SciTech Connect

    Chien, P.-S.; Mak, O.-T.; Huang, H.-J. . E-mail: haojen@mail.ncku.edu.tw

    2006-01-13

    Vanadate is a transition metal widely distributed in the environment. It has been reported that vanadate associated with air pollution particles can modify DNA synthesis, causing cell growth arrest, and apoptosis. Moreover, vanadium exposure was also found to cause the synthesis of inflammatory cytokines, such as interleukin-1, tumor necrosis factor-{alpha}, and prostaglandin E{sub 2}. Here, we found that exposure of A549 human lung carcinoma cells to vanadate led to extracellular signal-regulated kinase, c-Jun NH{sub 2}-terminal protein kinases (JNKs), p38 mitogen-activated protein kinase (p38) activation, and COX-2 protein expression in a dose-dependent manner. SB203580, a p38 MAPK inhibitor, but not PD098059 and SP600125, specific inhibitor of MKK1 and selective inhibitor of JNK, respectively, suppressed COX-2 expression. Furthermore, the epithelial growth factor (EGF) receptor specific inhibitor (PD153035) reduced vanadate-induced COX-2 expression. However, scavenging of vanadate-induced reactive oxygen species by catalase, a specific H{sub 2}O{sub 2} inhibitor, or DPI, an NADPH oxidase inhibitor, resulted in no inhibition on COX-2 expression. Together, we suggested that EGF receptor and p38 MAPK signaling pathway may be involved in vanadate-induced COX-2 protein expression in A549 human lung carcinoma cell line.

  19. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    SciTech Connect

    Berger, Christian; Madshus, Inger Helene; Stang, Espen

    2012-12-10

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: Black-Right-Pointing-Pointer Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. Black-Right-Pointing-Pointer Antibody combination causes internalization of EGFR by macropinocytosis. Black-Right-Pointing-Pointer Antibody-induced internalization of EGFR is independent of EGFR kinase activity. Black-Right-Pointing-Pointer Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  20. STAT3 is required but not sufficient for EGF receptor-mediated migration and invasion of human prostate carcinoma cell lines

    PubMed Central

    Zhou, W; Grandis, J R; Wells, A

    2006-01-01

    Growth factor-induced migration is a rate-limiting step in tumour invasiveness. The molecules that regulate this cellular behaviour would represent novel targets for limiting tumour cell progression. Epidermal growth factor (EGF) receptor (EGFR)-mediated motility, present in both autocrine and paracrine modes in prostate carcinomas, requires de novo transcription to persist over times greater than a few hours. Therefore, we sought to define specific signalling pathways that directly alter cellular transcription. Signal transducer and activator of transcription 3 (STAT3) is activated, as determined by electrophoretic motility shift assays, by EGFR in DU145 and PC3 human prostate carcinoma cells in addition to the motility model NR6 fibroblast cell line. Inhibition of STAT3 activity by antisense or siRNA downregulation or expression of a dominant-negative construct limited cell motility as determined by an in vitro wound healing assay and invasiveness through a extracellular matrix barrier. The expression of constitutively activated STAT3 did not increase the migration, which indicates that STAT3 is necessary but not sufficient for EGFR-mediated migration. These findings suggest that STAT3 signalling may be a new target for limiting prostate tumour cell invasion. In a microarray gene analysis of what transcription units are altered by EGF in a STAT3-dependent manner we found that the expression of motility-limiting VASP protein and the apoptosis nexus caspase 3 were both downregulated upon EGF exposure. These findings suggest a molecular basis for the STAT3 dependence of EGFR-mediated prostate tumour progression. PMID:16804520

  1. p53 protein, EGF receptor, and anti-p53 antibodies in serum from patients with occupationally derived lung cancer.

    PubMed

    Schneider, J; Presek, P; Braun, A; Bauer, P; Konietzko, N; Wiesner, B; Woitowitz, H J

    1999-08-01

    The oncogene product epidermal growth factor receptor (EGF-R), the tumour suppressor gene product p53 and anti-p53 antibodies are detectable in the serum of certain cancer patients. Increased levels of some of these products were reported in lung cancer patients after occupational asbestos exposure and after exposure to polycyclic aromatic hydrocarbons or vinylchloride. In the first step, this study investigated the possible diagnostic value of serum EGF-R, p53-protein and anti-p53 antibodies, measured by an enzyme-linked immunosorbent assay, in lung tumour patients. In addition to being investigated on a molecular epidemiological basis, these parameters were examined as biomarkers of carcinogenesis, especially with regard to asbestos incorporation effects or of radon-induced lung cancers. Also, a possible effect of cigarette smoking and age dependence were studied. A total of 116 male patients with lung or pleural tumours were examined. The histological classification was four small-cell cancers, six large-cell cancers, 32 adenocarcinomas, 47 squamous carcinomas, 12 mixed lung carcinomas, five diffuse malignant mesotheliomas and ten lung metastasis of extrapulmonary tumours. Twenty-two lung cancers and all mesotheliomas were related to asbestos, 22 lung cancers were related to ionizing radiation and 61 patients had cigarette smoke-related lung cancer. Besides these patients 50 male patients with non-malignant lung or pleural diseases were included; of the latter eight subjects suffered from asbestosis. Controls were 129 male subjects without any lung disease. No significantly elevated or decreased serum values for p53 protein, EGF-R, or anti-p53 antibodies as a function of histological tumour type, age, or degree and type of exposure (asbestos, smoking, ionizing radiation) could be found. The utility of p53-protein, EGF-R and anti-p53 antibodies as routine biomarkers for screening occupationally derived lung cancers is limited.

  2. Propranolol Restricts the Mobility of Single EGF-Receptors on the Cell Surface before Their Internalization

    PubMed Central

    Otero, Carolina; Linke, Max; Sanchez, Paula; González, Alfonso; Schaap, Iwan A. T.

    2013-01-01

    The epidermal growth factor receptor is involved in morphogenesis, proliferation and cell migration. Its up-regulation during tumorigenesis makes this receptor an interesting therapeutic target. In the absence of the ligand, the inhibition of phosphatidic acid phosphohydrolase activity by propranolol treatment leads to internalization of empty/inactive receptors. The molecular events involved in this endocytosis remain unknown. Here, we quantified the effects of propranolol on the mobility of single quantum-dot labelled receptors before the actual internalization took place. The single receptors showed a clear stop-and-go motion; their diffusive tracks were continuously interrupted by sub-second stalling events, presumably caused by transient clustering. In the presence of propranolol we found that: i) the diffusion rate reduced by 22 %, which indicates an increase in drag of the receptor. Atomic force microscopy measurements did not show an increase of the effective membrane tension, such that clustering of the receptor remains the likely mechanism for its reduced mobility. ii) The receptor got frequently stalled for longer periods of multiple seconds, which may signal the first step of the internalization process. PMID:24349439

  3. Parthenolide induces superoxide anion production by stimulating EGF receptor in MDA-MB-231 breast cancer cells.

    PubMed

    D'Anneo, A; Carlisi, D; Emanuele, S; Buttitta, G; Di Fiore, R; Vento, R; Tesoriere, G; Lauricella, M

    2013-12-01

    The sesquiterpene lactone parthenolide (PN) has recently attracted considerable attention because of its anti-microbial, anti-inflammatory and anticancer effects. However, the mechanism of its cytotoxic action on tumor cells remains scarcely defined. We recently provided evidence that the effect exerted by PN in MDA-MB-231 breast cancer cells was mediated by the production of reactive oxygen species (ROS). The present study shows that PN promoted the phosphorylation of EGF receptor (phospho-EGFR) at Tyr1173, an event which was observed already at 1 h of incubation with 25 µM PN and reached a peak at 8-16 h. This effect seemed to be a consequence of ROS production, because N-acetylcysteine (NAC), a powerful ROS scavenger, prevented the increment of phospho-EGFR levels. In addition fluorescence analyses performed using dihydroethidium demonstrated that PN stimulated the production of superoxide anion already at 2-3 h of incubation and the effect further increased prolonging the time of treatment, reaching a peak at 8-16 h. Superoxide anion production was markedly hampered by apocynin, a well known NADPH oxidase (NOX) inhibitor, suggesting that the effect was dependent on NOX activity. The finding that AG1478, an EGFR kinase inhibitor, substantially blocked both EGFR phosphorylation and superoxide anion production strongly suggested that phosphorylation of EGFR can be responsible for the activation of NOX with the consequent production of superoxide anion. Therefore, EGFR phosphorylation can exert a key role in the production of superoxide anion and ROS induced by PN in MDA-MB-231 cells.

  4. SB203580 promotes EGF-stimulated early morphological differentiation in PC12 cell through activating ERK pathway.

    PubMed

    New, L; Li, Y; Ge, B; Zhong, H; Mansbridge, J; Liu, K; Han, J

    2001-01-01

    MAP kinases have important role in PC12 cell differentiation, since the activities of both extracellular regulated protein kinase (ERK) and p38 have been indicated as necessary signal for PC12 cell differentiation. Epidermal growth factor (EGF) and NGF both activate ERK and p38 in PC12 cells, but only NGF trigger differentiation. It has been proposed that the duration of ERK activation determines the switch from proliferation to differentiation, since EGF causes more transient activation of ERK than NGF in PC12 cells. Here we report that treatment of PC12 cells with EGF in the presence of SB203580, a widely used p38 inhibitor, caused differentiation. The pro-differentiation effect of SB203580 in EGF-treated PC12 cells was found to be independent of its function of p38 inhibition but was through an effect on the ERK pathway that has been recently reported (Kalmes et al. [1999] FEBS Lett. 444: 71-74; Hall-Jackson et al. [1999] Onc. 18: 2047-2054). We found that SB203580 by itself did not affect the activity of ERK1/2 but significantly extended EGF-induced ERK activation in PC12 cells, which resulted in early morphological differentiation. Our data indicated that although both ERK and p38 are required for PC12 cell differentiation, activation of p38 is not required when ERK is superactivated. Our data provided further evidence for the threshold theory that differentiation is determined by the duration of ERK activation.

  5. An In Vivo EGF Receptor Localization Screen in C. elegans Identifies the Ezrin Homolog ERM-1 as a Temporal Regulator of Signaling

    PubMed Central

    Walser, Michael; Yang, Qiutan; Langouët, Maeva; Kradolfer, David; Fröhli, Erika; Herrmann, Christina J.; Hajnal, Alex; Escobar-Restrepo, Juan M.

    2014-01-01

    The subcellular localization of the epidermal growth factor receptor (EGFR) in polarized epithelial cells profoundly affects the activity of the intracellular signaling pathways activated after EGF ligand binding. Therefore, changes in EGFR localization and signaling are implicated in various human diseases, including different types of cancer. We have performed the first in vivo EGFR localization screen in an animal model by observing the expression of the EGFR ortholog LET-23 in the vulval epithelium of live C. elegans larvae. After systematically testing all genes known to produce an aberrant vulval phenotype, we have identified 81 genes regulating various aspects of EGFR localization and expression. In particular, we have found that ERM-1, the sole C. elegans Ezrin/Radixin/Moesin homolog, regulates EGFR localization and signaling in the vulval cells. ERM-1 interacts with the EGFR at the basolateral plasma membrane in a complex distinct from the previously identified LIN-2/LIN-7/LIN-10 receptor localization complex. We propose that ERM-1 binds to and sequesters basolateral LET-23 EGFR in an actin-rich inactive membrane compartment to restrict receptor mobility and signaling. In this manner, ERM-1 prevents the immediate activation of the entire pool of LET-23 EGFR and permits the generation of a long-lasting inductive signal. The regulation of receptor localization thus serves to fine-tune the temporal activation of intracellular signaling pathways. PMID:24785082

  6. Radiotherapy of non-small-cell lung cancer in the era of EGFR gene mutations and EGF receptor tyrosine kinase inhibitors.

    PubMed

    Moschini, Ilaria; Dell'Anna, Cristina; Losardo, Pier Luigi; Bordi, Paola; D'Abbiero, Nunziata; Tiseo, Marcello

    2015-01-01

    Non-small-cell lung cancer (NSCLC) occurs, approximately, in 80-85% of all cases of lung cancer. The majority of patients present locally advanced or metastatic disease when diagnosed, with poor prognosis. The discovery of activating mutations in the EGFR gene has started a new era of personalized treatment for NSCLC patients. To improve the treatment outcome in patients with unresectable NSCLC and, in particular, EGFR mutated, a combined strategy of radiotherapy and medical treatment can be undertaken. In this review we will discuss preclinical data regarding EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) and radiotherapy, available clinical trials investigating efficacy and toxicity of combined treatment (thoracic or whole brain radiotherapy and EGFR-TKIs) and, also, the role of local radiation in mutated EGFR patients who developed EGFR-TKI resistance.

  7. EGF receptor mutations in lung cancer: from humans to mice and maybe back to humans.

    PubMed

    Arteaga, Carlos L

    2006-06-01

    Deletions in exon 19 and nucleotide substitutions in exon 21 are the most common mutations of the EGFR (ErbB1) in NSCLC. These mutations endow the receptor with constitutive kinase activity. Most tumors expressing these mutants respond well to EGFR tyrosine kinase inhibitors, suggesting that they are dependent on mutant EGFR signaling. Two groups developed transgenic mice in which expression of these mutants is temporally induced in mouse lung. Mice expressing EGFR mutants develop bronchioloalveolar cancer and lung adenocarcinoma, which are highly sensitive to EGFR inhibitors. These mouse models provide important opportunities for studying the biology of NSCLC and the refinement of anti-EGFR therapies.

  8. Bypassing cellular EGF receptor dependence through epithelial-to-mesenchymal-like transitions

    PubMed Central

    Thomson, Stuart; Buck, Elizabeth; Russo, Suzanne; Petti, Filippo; Sujka-Kwok, Izabela; Eyzaguirre, Alexandra; Rosenfeld-Franklin, Maryland; Gibson, Neil W.; Miglarese, Mark; Epstein, David; Iwata, Kenneth K.; Haley, John D.

    2008-01-01

    Over 90% of all cancers are carcinomas, malignancies derived from cells of epithelial origin. As carcinomas progress, these tumors may lose epithelial morphology and acquire mesenchymal characteristics which contribute to metastatic potential. An epithelial-to-mesenchymal transition (EMT) similar to the process critical for embryonic development is thought to be an important mechanism for promoting cancer invasion and metastasis. Epithelial-to-mesenchymal transitions have been induced in vitro by transient or unregulated activation of receptor tyrosine kinase signaling pathways, oncogene signaling and disruption of homotypic cell adhesion. These cellular models attempt to mimic the complexity of human carcinomas which respond to autocrine and paracrine signals from both the tumor and its microenvironment. Activation of the epidermal growth factor receptor (EGFR) has been implicated in the neoplastic transformation of solid tumors and overexpression of EGFR has been shown to correlate with poor survival. Notably, epithelial tumor cells have been shown to be significantly more sensitive to EGFR inhibitors than tumor cells which have undergone an EMT-like transition and acquired mesenchymal characteristics, including non-small cell lung (NSCLC), head and neck (HN), bladder, colorectal, pancreas and breast carcinomas. EGFR blockade has also been shown to inhibit cellular migration, suggesting a role for EGFR inhibitors in the control of metastasis. The interaction between EGFR and the multiple signaling nodes which regulate EMT suggest that the combination of an EGFR inhibitor and other molecular targeted agents may offer a novel approach to controlling metastasis. PMID:18236164

  9. Opposing Regulation of the EGF Receptor: A Molecular Switch Controlling Cytomegalovirus Latency and Replication

    PubMed Central

    Zeltzer, Sebastian; Reitsma, Justin; Petrucelli, Alex; Umashankar, Mahadevaiah; Rak, Mike; Zagallo, Patricia; Schroeder, Joyce; Terhune, Scott; Goodrum, Felicia

    2016-01-01

    Herpesviruses persist indefinitely in their host through complex and poorly defined interactions that mediate latent, chronic or productive states of infection. Human cytomegalovirus (CMV or HCMV), a ubiquitous β-herpesvirus, coordinates the expression of two viral genes, UL135 and UL138, which have opposing roles in regulating viral replication. UL135 promotes reactivation from latency and virus replication, in part, by overcoming replication-suppressive effects of UL138. The mechanism by which UL135 and UL138 oppose one another is not known. We identified viral and host proteins interacting with UL138 protein (pUL138) to begin to define the mechanisms by which pUL135 and pUL138 function. We show that pUL135 and pUL138 regulate the viral cycle by targeting that same receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR). EGFR is a major homeostatic regulator involved in cellular proliferation, differentiation, and survival, making it an ideal target for viral manipulation during infection. pUL135 promotes internalization and turnover of EGFR from the cell surface, whereas pUL138 preserves surface expression and activation of EGFR. We show that activated EGFR is sequestered within the infection-induced, juxtanuclear viral assembly compartment and is unresponsive to stress. Intriguingly, these findings suggest that CMV insulates active EGFR in the cell and that pUL135 and pUL138 function to fine-tune EGFR levels at the cell surface to allow the infected cell to respond to extracellular cues. Consistent with the role of pUL135 in promoting replication, inhibition of EGFR or the downstream phosphoinositide 3-kinase (PI3K) favors reactivation from latency and replication. We propose a model whereby pUL135 and pUL138 together with EGFR comprise a molecular switch that regulates states of latency and replication in HCMV infection by regulating EGFR trafficking to fine tune EGFR signaling. PMID:27218650

  10. Opposing Regulation of the EGF Receptor: A Molecular Switch Controlling Cytomegalovirus Latency and Replication.

    PubMed

    Buehler, Jason; Zeltzer, Sebastian; Reitsma, Justin; Petrucelli, Alex; Umashankar, Mahadevaiah; Rak, Mike; Zagallo, Patricia; Schroeder, Joyce; Terhune, Scott; Goodrum, Felicia

    2016-05-01

    Herpesviruses persist indefinitely in their host through complex and poorly defined interactions that mediate latent, chronic or productive states of infection. Human cytomegalovirus (CMV or HCMV), a ubiquitous β-herpesvirus, coordinates the expression of two viral genes, UL135 and UL138, which have opposing roles in regulating viral replication. UL135 promotes reactivation from latency and virus replication, in part, by overcoming replication-suppressive effects of UL138. The mechanism by which UL135 and UL138 oppose one another is not known. We identified viral and host proteins interacting with UL138 protein (pUL138) to begin to define the mechanisms by which pUL135 and pUL138 function. We show that pUL135 and pUL138 regulate the viral cycle by targeting that same receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR). EGFR is a major homeostatic regulator involved in cellular proliferation, differentiation, and survival, making it an ideal target for viral manipulation during infection. pUL135 promotes internalization and turnover of EGFR from the cell surface, whereas pUL138 preserves surface expression and activation of EGFR. We show that activated EGFR is sequestered within the infection-induced, juxtanuclear viral assembly compartment and is unresponsive to stress. Intriguingly, these findings suggest that CMV insulates active EGFR in the cell and that pUL135 and pUL138 function to fine-tune EGFR levels at the cell surface to allow the infected cell to respond to extracellular cues. Consistent with the role of pUL135 in promoting replication, inhibition of EGFR or the downstream phosphoinositide 3-kinase (PI3K) favors reactivation from latency and replication. We propose a model whereby pUL135 and pUL138 together with EGFR comprise a molecular switch that regulates states of latency and replication in HCMV infection by regulating EGFR trafficking to fine tune EGFR signaling. PMID:27218650

  11. Stromal cell-derived factor-1{alpha} (SDF-1{alpha}/CXCL12) stimulates ovarian cancer cell growth through the EGF receptor transactivation

    SciTech Connect

    Porcile, Carola; Bajetto, Adriana . E-mail: bajetto@cba.unige.it; Barbieri, Federica; Barbero, Simone; Bonavia, Rudy; Biglieri, Marianna; Pirani, Paolo; Florio, Tullio . E-mail: florio@cba.unige.it; Schettini, Gennaro

    2005-08-15

    Ovarian cancer (OC) is the leading cause of death in gynecologic diseases in which there is evidence for a complex chemokine network. Chemokines are a family of proteins that play an important role in tumor progression influencing cell proliferation, angiogenic/angiostatic processes, cell migration and metastasis, and, finally, regulating the immune cells recruitment into the tumor mass. We previously demonstrated that astrocytes and glioblastoma cells express both the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1), and that SDF-1{alpha} treatment induced cell proliferation, supporting the hypothesis that chemokines may play an important role in tumor cells' growth in vitro. In the present study, we report that CXCR4 and SDF-1 are expressed in OC cell lines. We demonstrate that SDF-1{alpha} induces a dose-dependent proliferation in OC cells, by the specific interaction with CXCR4 and a biphasic activation of ERK1/2 and Akt kinases. Our results further indicate that CXCR4 activation induces EGF receptor (EGFR) phosphorylation that in turn was linked to the downstream intracellular kinases activation, ERK1/2 and Akt. In addition, we provide evidence for cytoplasmic tyrosine kinase (c-Src) involvement in the SDF-1/CXCR4-EGFR transactivation. These results suggest a possible important 'cross-talk' between SDF-1/CXCR4 and EGFR intracellular pathways that may link signals of cell proliferation in ovarian cancer.

  12. STRUCTURE OF THE EGF RECEPTOR TRANSACTIVATION CIRCUIT INTEGRATES MULTIPLE SIGNALS WITH CELL CONTEXT

    PubMed Central

    Joslin, Elizabeth J.; Shankaran, Harish; Opresko, Lee K.; Bollinger, Nikki; Lauffenburger, Douglas A.; Wiley, H. Steven

    2012-01-01

    Summary Transactivation of the epidermal growth factor receptor (EGFR) is thought to be a process by which a variety of cellular inputs can be integrated into a single signaling pathway through either stimulated proteolysis (shedding) of membrane-anchored EGFR ligands or by modification of the activity of the EGFR. As a first step towards building a predictive model of the EGFR transactivation circuit, we quantitatively defined how signals from multiple agonists were integrated both upstream and downstream of the EGFR to regulate extracellular signal regulated kinase (ERK) activity in human mammary epithelial cells. By using a “non-binding” reporter of ligand shedding, we found that transactivation triggers a positive feedback loop from ERK back to the EGFR such that ligand shedding drives EGFR-stimulated ERK that in turn drives further ligand shedding. Importantly, activated Ras and ERK levels were nearly linear functions of ligand shedding and the effect of multiple, sub-saturating inputs was additive. Simulations showed that ERK-mediated feedback through ligand shedding resulted in a stable steady-state level of activated ERK, but also showed that the extracellular environment can modulate the level of feedback. Our results suggest that the transactivation circuit acts as a context-dependent integrator and amplifier of multiple extracellular signals and that signal integration can effectively occur at multiple points in the EGFR pathway. PMID:20458382

  13. Targeting EGF-receptor-signalling in squamous cell carcinomas of the head and neck

    PubMed Central

    Reuter, C W M; Morgan, M A; Eckardt, A

    2007-01-01

    Despite significant advances in the use of surgery, chemotherapy and radiotherapy to treat squamous cell carcinoma of the head and neck (SCCHN), prognosis has improved little over the past 30 years. There is a clear need for novel, more effective therapies to prevent relapse, control metastases and improve overall survival. Improved understanding of SCCHN disease biology has led to the introduction of molecularly targeted treatment strategies in these cancers. The epidermal growth factor receptor (EGFR) is expressed at much higher levels in SCCHN tumours than in normal epithelial tissue, and EGFR expression correlates with poor prognosis. Therefore, much effort is currently directed toward targeting aberrant EGFR activity (e.g. cell signalling) in SCCHN. This review discusses the efficacy of novel therapies targeting the EGFR (e.g. anti-EGFR antibodies and EGFR tyrosine kinase inhibitors) that are currently tested in SCCHN patients. PMID:17224925

  14. Epithelial EGF receptor signaling mediates airway hyperreactivity and remodeling in a mouse model of chronic asthma.

    PubMed

    Le Cras, Timothy D; Acciani, Thomas H; Mushaben, Elizabeth M; Kramer, Elizabeth L; Pastura, Patricia A; Hardie, William D; Korfhagen, Thomas R; Sivaprasad, Umasundari; Ericksen, Mark; Gibson, Aaron M; Holtzman, Michael J; Whitsett, Jeffrey A; Hershey, Gurjit K Khurana

    2011-03-01

    Increases in the epidermal growth factor receptor (EGFR) have been associated with the severity of airway thickening in chronic asthmatic subjects, and EGFR signaling is induced by asthma-related cytokines and inflammation. The goal of this study was to determine the role of EGFR signaling in a chronic allergic model of asthma and specifically in epithelial cells, which are increasingly recognized as playing an important role in asthma. EGFR activation was assessed in mice treated with intranasal house dust mite (HDM) for 3 wk. EGFR signaling was inhibited in mice treated with HDM for 6 wk, by using either the drug erlotinib or a genetic approach that utilizes transgenic mice expressing a mutant dominant negative epidermal growth factor receptor in the lung epithelium (EGFR-M mice). Airway hyperreactivity (AHR) was assessed by use of a flexiVent system after increasing doses of nebulized methacholine. Airway smooth muscle (ASM) thickening was measured by morphometric analysis. Sensitization to HDM (IgG and IgE), inflammatory cells, and goblet cell changes were also assessed. Increased EGFR activation was detected in HDM-treated mice, including in bronchiolar epithelial cells. In mice exposed to HDM for 6 wk, AHR and ASM thickening were reduced after erlotinib treatment and in EGFR-M mice. Sensitization to HDM and inflammatory cell counts were similar in all groups, except neutrophil counts, which were lower in the EGFR-M mice. Goblet cell metaplasia with HDM treatment was reduced by erlotinib, but not in EGFR-M transgenic mice. This study demonstrates that EGFR signaling, especially in the airway epithelium, plays an important role in mediating AHR and remodeling in a chronic allergic asthma model.

  15. Cell Surface Epidermal Growth Factor Receptors Increase Src and c-Cbl Activity and Receptor Ubiquitylation*

    PubMed Central

    Parks, Eileen E.; Ceresa, Brian P.

    2014-01-01

    There is an established role for the endocytic pathway in regulation of epidermal growth factor receptor (EGFR) signaling to downstream effectors. However, because ligand-mediated EGFR endocytosis utilizes multiple “moving parts,” dissecting the spatial versus temporal contributions has been challenging. Blocking all endocytic trafficking can have unintended effects on other receptors as well as give rise to compensatory mechanisms, both of which impact interpretation of EGFR signaling. To overcome these limitations, we used epidermal growth factor (EGF) conjugated to polystyrene beads (EGF beads). EGF beads simultaneously activate the EGFR while blocking its endocytosis and allow analysis of EGFR signaling from the plasma membrane. Human telomerase immortalized corneal epithelial (hTCEpi) cells were used to model normal epithelial cell biology. In hTCEpi cells, both cell surface and intracellular EGFRs exhibited dose-dependent increases in effector activity after 15 min of ligand stimulation, but only the serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) was statistically significant when accounting for receptor phosphorylation. However, over time with physiological levels of receptor phosphorylation, cell surface receptors produced either enhanced or sustained mitogen-activated protein kinase kinase (MEK), Casitas B-lineage lymphoma (c-Cbl), and the pro-oncogene Src activity. These increases in effector communication by cell surface receptors resulted in an increase in EGFR ubiquitylation with sustained ligand incubation. Together, these data indicate that spatial regulation of EGFR signaling may be an important regulatory mechanism in receptor down-regulation. PMID:25074934

  16. Structure of the EGF receptor transactivation circuit integrates multiple signals with cell context

    SciTech Connect

    Joslin, Elizabeth J.; Shankaran, Harish; Opresko, Lee K.; Bollinger, Nikki; Lauffenburger, Douglas A.; Wiley, H. S.

    2010-05-10

    Transactivation of the epidermal growth factor receptor (EGFR) has been proposed to be a mechanism by which a variety of cellular inputs can be integrated into a single signaling pathway, but the regulatory topology of this important system is unclear. To understand the transactivation circuit, we first created a “non-binding” reporter for ligand shedding. We then quantitatively defined how signals from multiple agonists were integrated both upstream and downstream of the EGFR into the extracellular signal regulated kinase (ERK) cascade in human mammary epithelial cells. We found that transactivation is mediated by a recursive autocrine circuit where ligand shedding drives EGFR-stimulated ERK that in turn drives further ligand shedding. The time from shedding to ERK activation is fast (<5 min) whereas the recursive feedback is slow (>15 min). Simulations showed that this delay in positive feedback greatly enhanced system stability and robustness. Our results indicate that the transactivation circuit is constructed so that the magnitude of ERK signaling is governed by the sum of multiple direct inputs, while recursive, autocrine ligand shedding controls signal duration.

  17. The NMR solution structure of human epidermal growth factor (hEGF) at physiological pH and its interactions with suramin

    SciTech Connect

    Huang, Hsiao-Wen; Mohan, Sepuru K.; Yu, C.

    2010-11-26

    Research highlights: {yields} The solution structure of hEGF at pH 6.8 was determined. {yields} hEGF contains a unique hydrophobic core around its C-terminus. {yields} The conformational change happens once hEGF binds to EGFR. {yields} The interaction between hEGF and suramin is dominated by van der Waals contacts. {yields} Suramin blocks the conformational change of hEGF which is crucial in binding to its receptor. -- Abstract: Human epidermal growth factor (hEGF) induces the proliferation, differentiation and survival of various cell types including tumor-derived cells. Generally, hEGF performs its biological function by binding to a specific receptor (hEGFR) on the cell surface, thereby inducing signal transduction. Suramin, a polysulfonated naphthylurea that acts as a growth factor blocker, exhibits antiproliferative activity against non-small cell lung cancer (NSCLC) cells that overexpress EGFR on the cell surface. We determined the solution structure of hEGF under physiological conditions and investigated the interaction of suramin with hEGF using isothermal titration calorimetry and NMR spectroscopy techniques. The solution structure of hEGF presented in this paper is different from the bound form of hEGF present in the crystal structure of the 2:2 EGF-EGFR complex because its C-tail contains a hydrophobic core. This conformational difference supports the hypothesis that hEGF undergoes a conformational change when it binds to hEGFR and subsequently induces signal transduction. Based on the docking structure of the hEGF-suramin complex, we demonstrated how suramin blocks hEGF by binding to its receptor binding site (the C-terminal region around Arg45) and inhibits the crucial conformational change.

  18. Signaling pathways used by EGF to stimulate conjunctival goblet cell secretion.

    PubMed

    Hodges, Robin R; Bair, Jeffrey A; Carozza, Richard B; Li, Dayu; Shatos, Marie A; Dartt, Darlene A

    2012-10-01

    The purpose of this study was to identify the signaling pathways that epidermal growth factor (EGF) uses to stimulate mucin secretion from cultured rat conjunctival goblet cells and to compare the pathways used by EGF with those used by the known secretagogue muscarinic, cholinergic agonists. To this end, goblet cells from rat conjunctiva were grown in culture using RPMI media. For immunofluorescence experiments, antibodies against EGF receptor (EGFR) and ERK 2 as well as muscarinic receptors (M(1)AchR, M(2)AchR, and M(3)AchR) were used, and the cells viewed by fluorescence microscopy. Intracellular [Ca(2+)] ([Ca(2+)](i)) was measured using fura 2/AM. Glycoconjugate secretion was determined after cultured goblet cells were preincubated with inhibitors, and then stimulated with EGF or the cholinergic agonist carbachol (Cch). Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-I or ELISA for MUC5AC. In cultured goblet cells EGF stimulated an increase in [Ca(2+)](i) in a concentration-dependent manner. EGF-stimulated increase in [Ca(2+)](i) was blocked by inhibitors of the EGF receptor and removal of extracellular Ca(2+). Inhibitors against the EGFR and ERK 1/2 blocked EGF-stimulated mucin secretion. In addition, cultured goblet cells expressed M(1)AchR, M(2)AchR, and M(3)AchRs. Cch-stimulated increase in [Ca(2+)](i) was blocked by inhibitors for the M(1)AchRs, matrix metalloproteinases, and EGF receptors. Inhibitors against the EGF receptor and ERK 1/2 also blocked Cch-stimulated mucin secretion. We conclude that in conjunctival goblet cells, EGF itself increases [Ca(2+)](i) and activates ERK 1/2 to stimulate mucin secretion. EGF-stimulated secretion is dependent on extracellular Ca(2+). This mechanism of action is similar to cholinergic agonists that use muscarinic receptors to transactivate the EGF receptor, increase [Ca(2+)](i), and activate ERK 1/2 leading to an increase in mucin secretion.

  19. Free Energy Landscape of Lipid Interactions with Regulatory Binding Sites on the Transmembrane Domain of the EGF Receptor.

    PubMed

    Hedger, George; Shorthouse, David; Koldsø, Heidi; Sansom, Mark S P

    2016-08-25

    Lipid molecules can bind to specific sites on integral membrane proteins, modulating their structure and function. We have undertaken coarse-grained simulations to calculate free energy profiles for glycolipids and phospholipids interacting with modulatory sites on the transmembrane helix dimer of the EGF receptor within a lipid bilayer environment. We identify lipid interaction sites at each end of the transmembrane domain and compute interaction free energy profiles for lipids with these sites. Interaction free energies ranged from ca. -40 to -4 kJ/mol for different lipid species. Those lipids (glycolipid GM3 and phosphoinositide PIP2) known to modulate EGFR function exhibit the strongest binding to interaction sites on the EGFR, and we are able to reproduce the preference for interaction with GM3 over other glycolipids suggested by experiment. Mutation of amino acid residues essential for EGFR function reduce the binding free energy of these key lipid species. The residues interacting with the lipids in the simulations are in agreement with those suggested by experimental (mutational) studies. This approach provides a generalizable tool for characterizing the interactions of lipids that bind to specific sites on integral membrane proteins.

  20. Free Energy Landscape of Lipid Interactions with Regulatory Binding Sites on the Transmembrane Domain of the EGF Receptor

    PubMed Central

    2016-01-01

    Lipid molecules can bind to specific sites on integral membrane proteins, modulating their structure and function. We have undertaken coarse-grained simulations to calculate free energy profiles for glycolipids and phospholipids interacting with modulatory sites on the transmembrane helix dimer of the EGF receptor within a lipid bilayer environment. We identify lipid interaction sites at each end of the transmembrane domain and compute interaction free energy profiles for lipids with these sites. Interaction free energies ranged from ca. −40 to −4 kJ/mol for different lipid species. Those lipids (glycolipid GM3 and phosphoinositide PIP2) known to modulate EGFR function exhibit the strongest binding to interaction sites on the EGFR, and we are able to reproduce the preference for interaction with GM3 over other glycolipids suggested by experiment. Mutation of amino acid residues essential for EGFR function reduce the binding free energy of these key lipid species. The residues interacting with the lipids in the simulations are in agreement with those suggested by experimental (mutational) studies. This approach provides a generalizable tool for characterizing the interactions of lipids that bind to specific sites on integral membrane proteins. PMID:27109430

  1. Evaluation of adhesion force and binding affinity of phytohemagglutinin erythroagglutinating to EGF receptor on human lung cancer cells.

    PubMed

    Kuo, W-T; Dong, G-C; Yao, C-H; Huang, J-Y; Lin, F-H

    2013-01-01

    PHA-E is a natural product extracted from red kidney beans, and it has been reported to induce cell apoptosis by blocking EGFR in lung cancer cells. Because EGF is the major in vivo competitor to PHA-E in clinical application, PHA-E must be proved that has better affinity to EGFR than EGF. This study would focus on how PHA-E tightly bind to EGFR and the results would compare with EGF. The adhesion force, measured by AFM, between EGFR and PHA-E was 207.14±74.42 pN that was higher than EGF (183.65±86.93 pN). The equilibrium dissociation constant of PHA-E and EGF to EGFR was 2.4 10(-9)±1.4 10(-9) and 7.3 10(-8)±2.7 10(-8), respectively, that could evaluate binding affinity. The result showed that binding affinity of PHA-E to EGFR was one order higher than EGF to EGFR. In the results of flow cytometer and confocal microscope, we found binding efficiency of EGF to EGFR was decrease as the concentration of PHA-E increased. In the analysis of Western blot, treatment of A-549 cells with PHA-E resulted in a dose-dependent decrease in EGFR phosphorylation. In conclusion, we found that PHA-E had better adhesion force and binding affinity to EGFR than that of the EGF. The interaction between PHA-E and EGFR could block EGF binding and then inhibit EGFR phosphorylation. PHA-E could be developed into a new target molecule for lung cancer treatment that could be immobilized on the drug carrier to guide therapeutic particles to the tumor site. PMID:23394551

  2. Ultraviolet B-induced activated protein-1 activation does not require epidermal growth factor receptor but is blocked by a dominant negative PKClambda/iota.

    PubMed

    Huang, C; Ma, W y; Bowden, G T; Dong, Z

    1996-12-01

    The exposure of mammalian cells to UV irradiation leads to the activation of transcription factors such as activated protein-1 (AP-1) and NFkappaB. It is postulated that epidermal growth factor (EGF) receptor, but not protein kinase C (PKC), is the major membrane mediator in UV-induced signal transduction. Since UVB is responsible for most of the carcinogenic effects of sun exposure, we investigated the role of EGF receptors and PKC in UVB-induced AP-1 activation. Our results indicated that while the down-regulation of novel PKC (nPKC) and conventional PKC (cPKC) by pretreatment of cells with 12-O-tetradecanoyl phorbol-13-acetate cannot block UVB-induced AP-1 activity, it can block 12-O-tetradecanoyl phorbol-13-acetate-induced AP-1 activity. Further, the dominant negative mutant PKClambda/iota blocked UVB-induced AP-1 activity in all doses and time courses studied. In contrast, UVB-induced AP-1 activity from cells devoid of EGF receptor (B82) was not significantly different from that of the stable transfectants with a kinase-deficient EGF receptor (B82M721) or those with a wild-type EGF receptor (B82L) at all UVB irradiation doses and time courses studied. All of this evidence indicated that aPKC, but not EGF receptor, is involved in UVB-induced AP-1 activation. PMID:8940130

  3. Ectodomain cleavage of the EGF ligands HB-EGF, neuregulin1-beta, and TGF-alpha is specifically triggered by different stimuli and involves different PKC isoenzymes.

    PubMed

    Herrlich, Andreas; Klinman, Eva; Fu, Jonathan; Sadegh, Cameron; Lodish, Harvey

    2008-12-01

    Metalloproteinase cleavage of transmembrane proteins (ectodomain cleavage), including the epidermal growth factor (EGF) ligands heparin-binding EGF-like growth factor (HB-EGF), neuregulin (NRG), and transforming growth factor-alpha (TGF-alpha), is important in many cellular signaling pathways and is disregulated in many diseases. It is largely unknown how physiological stimuli of ectodomain cleavage--hypertonic stress, phorbol ester, or activation of G-protein-coupled receptors [e.g., by lysophosphatidic acid (LPA)]--are molecularly connected to metalloproteinase activation. To study this question, we developed a fluorescence-activated cell sorting (FACS)- based assay that measures cleavage of EGF ligands in single living cells. EGF ligands expressed in mouse lung epithelial cells are differentially and specifically cleaved depending on the stimulus. Inhibition of protein kinase C (PKC) isoenzymes or metalloproteinase inhibition by batimastat (BB94) showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. For example, hypertonic stress led to strong cleavage of HB-EGF and NRG but only moderate cleavage of TGF-alpha. HB-EGF, NRG, and TGF-alpha cleavage was not dependent on PKC, and only HB-EGF and NRG cleavage were inhibited by BB94. In contrast, phorbol 12-myristate-13-acetate (TPA) -induced cleavage of HB-EGF, NRG, and TGF-alpha was dependent on PKC and sensitive to BB94 inhibition. LPA led to significant cleavage of only NRG and TGF-alpha and was inhibited by BB94; only LPA-induced NRG cleavage required PKC. Surprisingly, specific inhibition of atypical PKCs zeta and iota [not activated by diacylglycerol (DAG) and calcium] significantly enhanced TPA-induced NRG cleavage. Employed in a high-throughput cloning strategy, our cleavage assay should allow the identification of candidate proteins involved in signal transduction of different

  4. Effect of epidermal growth factor (EGF) on the phosphorylation of mitogen-activated protein kinase (MAPK) in the bovine oviduct in vitro: Alteration by heat stress

    PubMed Central

    WIJAYAGUNAWARDANE, Missaka P. B.; HAMBRUCH, Nina; HAEGER, Jan-Dirk; PFARRER, Christiane

    2015-01-01

    Epidermal growth factor (EGF) has been shown to be involved in control of the oviductal microenvironment. To elucidate the potential mechanisms responsible for the detrimental effect of heat stress and to identify the relation with the endocrine status, the effects of EGF on the level of phosphorylated mitogen-activated-protein kinase (MAPK) and proliferation of bovine oviductal epithelial cells (OECs) exposed to different cyclic ovarian steroidal environments (luteal phase (LP), follicular phase (FP) and postovulatory phase (PO)) and temperatures (mild heat stress (40 C) and severe heat stress (43 C)) were investigated. Western blot was performed to evaluate phosphorylated MAPK, while proliferation was analyzed by MTT assay. Stimulation of OECs with EGF alone or with EGF in the PO and FP environments significantly increased the amount of phosphorylated MAPK, with MAPK 44 phosphorylation being highest during exposure to PO conditions. These effects were not observed in the LP. Heat treatment completely blocked effects of EGF on phosphorylated MAPK. Additionally, severe heat stress led to a significantly lower basal level of phosphorylated MAPK. PD98059 (MAPK inhibitor) completely abolished EGF-stimulated MAPK phosphorylation and OECs proliferation. Overall the results indicate that EGF has the potential to increase the amount of phosphorylated MAPK in OECs and therefore could be involved in regulation of the bovine oviductal microenvironment. However, these regulatory mechanisms may be compromised in the presence of heat stress (high ambient temperature), leading to low fertility rates and impaired embryo survival. PMID:26050642

  5. Development of Anti-EGF Receptor Peptidomimetics (AERP) as Tumor Imaging Agent

    PubMed Central

    Ponde, Datta E.; Su, ZiFen; Berezov, Alan; Zhang, Hongtao; Alavi, Abbas; Greene, Mark I.; Murali, Ramachandran

    2011-01-01

    EGFR is over-expressed in several solid tumors including breast, prostate, pancreas and lung cancers and is correlated to the metastasic potential of the tumor. Anti-EGFR receptor-binding peptidomimetics (AERP) were examined to assess the small molecule's potential use as tumor-specific imaging agents. The aim of this work was to design and characterize the binding specificity of the radiolabeled peptidomimetics to EGFR over-expressing cell lysate and to A431 xenograft tumors. Our newly designed peptidomimetic, AERP, was conjugated to DTPA and labeled with 99mTc. The in vivo tumor accumulation of [99mTc] DTPA-AERP-2 was 1.6 ± 0.1 %ID/g and tumor to muscle ratio was 5.5. Our studies suggest that this novel peptidomimetic, AERP-2, warrants further development as an EGFR-specific tumor-imaging agent. PMID:21392985

  6. Validation of a Flow Cytometry Based Binding Assay for Evaluation of Monoclonal Antibody Recognizing EGF Receptor

    PubMed Central

    Cedeño-Arias, Mercedes; Sánchez-Ramírez, Javier; Blanco-Santana, Rancés; Rengifo-Calzado, Enrique

    2011-01-01

    An ideal test used to characterize a product must be appropriate for the measurement of product quality, manufacturing consistency, product stability, and comparability studies. Flow cytometry has been successfully applied to the examination of antibodies and receptors on membrane surfaces; however, to date, the analytical validation of cytometry based assays is limited. Here we report on the validation of a flow cytometry-based assay used in the evaluation of nimotuzumab binding to cells over-expressing EGFR on cell surface. The assay was validated by examining, assay robustness, specificity, repeatability and intermediate precision. The assay was highly specific, robust for all studied factors except for cell fixation with 1% paraformaldehyde and met criteria for precision with RSD < 2%. In addition the assay has stability-indicating properties evidenced by the ability to detect changes in mAb degraded samples. Most importantly, the assay demonstrated to be useful for its intended use. PMID:21886904

  7. Structural basis for EGF receptor inhibition by the therapeutic antibody IMC-11F8.

    PubMed

    Li, Shiqing; Kussie, Paul; Ferguson, Kathryn M

    2008-02-01

    Therapeutic anticancer strategies that target and inactivate the epidermal growth factor receptor (EGFR) are under intense study in the clinic. Here we describe the mechanism of EGFR inhibition by an antibody drug IMC-11F8. IMC-11F8 is a fully human antibody that has similar antitumor potency as the chimeric cetuximab/Erbitux and might represent a safer therapeutic alternative. We report the X-ray crystal structure of the Fab fragment of IMC-11F8 (Fab11F8) in complex with the entire extracellular region and with isolated domain III of EGFR. We compare this to our previous study of the cetuximab/EGFR interaction. Fab11F8 interacts with a remarkably similar epitope, but through a completely different set of interactions. Both the similarities and differences in binding of these two antibodies have important implications for the development of inhibitors that could exploit this same mechanism of EGFR inhibition.

  8. The role of peroxisome proliferator-activated receptor-{beta}/{delta} in epidermal growth factor-induced HaCaT cell proliferation

    SciTech Connect

    Liang Pengfei; Jiang Bimei; Yang Xinghua; Xiao Xianzhong Huang Xu; Long Jianhong; Zhang Pihong; Zhang Minghua; Xiao Muzhang; Xie Tinghong; Huang Xiaoyuan

    2008-10-15

    Epidermal growth factor (EGF) has been shown to be a potent mitogen for epidermal cells both in vitro and in vivo, thus contributing to the development of an organism. It has recently become clear that peroxisome proliferator-activated receptor-{beta}/{delta} (PPAR{beta}/{delta}) expression and activation is involved in the cell proliferation. However, little is known about the role of PPAR{beta}/{delta} in EGF-induced proliferation of HaCaT keratinocytes. In this study, HaCaT cells were cultured in the presence and absence of EGF and we identified that EGF induced an increase of PPAR{beta}/{delta} mRNA and protein level expression in time-dependent and dose-dependent manner, and AG1487, an EGF receptor (EGFR) special inhibitor, caused attenuation of PPAR{beta}/{delta} protein expression. Electrophoretic mobility shift assay (EMSA) revealed that EGF significantly increased PPAR{beta}/{delta} binding activity in HaCaT keratinocytes. Antisense phosphorothioate oligonucleotides (asODNs) against PPAR{beta}/{delta} caused selectively inhibition of PPAR{beta}/{delta} protein content induced by EGF and significantly attenuated EGF-mediated cell proliferation. Treatment of the cells with L165041, a specific synthetic ligand for PPAR{beta}/{delta}, significantly enhanced EGF-mediated cell proliferation. Finally, c-Jun ablation inhibited PPAR{beta}/{delta} up-regulation induced by EGF, and chromatin immunoprecipitation (ChIP) showed that c-Jun bound to the PPAR{beta}/{delta} promoter and the binding increased in EGF-stimulated cells. These results demonstrate that EGF induces PPAR{beta}/{delta} expression in a c-Jun-dependent manner and PPAR{beta}/{delta} plays a vital role in EGF-stimulated proliferation of HaCaT cells.

  9. EBP50 inhibits EGF-induced breast cancer cell proliferation by blocking EGFR phosphorylation.

    PubMed

    Yao, Wenfang; Feng, Duiping; Bian, Weihua; Yang, Longyan; Li, Yang; Yang, Zhiyu; Xiong, Ying; Zheng, Junfang; Zhai, Renyou; He, Junqi

    2012-11-01

    Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) suppresses breast cancer cell proliferation, potentially through its regulatory effect on epidermal growth factor receptor (EGFR) signaling, although the mechanism by which this occurs remains unknown. Thus in our studies, we aimed to determine the effect of EBP50 expression on EGF-induced cell proliferation and activation of EGFR signaling in the breast cancer cell lines, MDA-MB-231 and MCF-7. In MDA-MB-231 cells, which express low levels of EBP50, EBP50 overexpression inhibited EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. In MCF-7 cells, which express high levels of EBP50, EBP50 knockdown promoted EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. Knockdown of EBP50 in EBP50-overexpressed MDA-MB-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation and restoration of EBP50 expression in EBP50-knockdown MCF-7 cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation, further confirming that the activation of EGF-induced downstream molecules could be specifically inhibited by EBP50 expression. Since EGFR signaling was triggered by EGF ligands via EGFR phosphorylation, we further detected the phosphorylation status of EGFR in the presence or absence of EBP50 expression. Overexpression of EBP50 in MDA-MB-231 cells inhibited EGF-stimulated EGFR phosphorylation, whereas knockdown of EBP50 in MCF-7 cells enhanced EGF-stimulated EGFR phosphorylation. Meanwhile, total expression levels of EGFR were unaffected during EGF stimulation. Taken together, our data shows that EBP50 can suppress EGF-induced proliferation of breast cancer cells by inhibiting EGFR phosphorylation and blocking EGFR downstream signaling in breast cancer cells. These results provide further insight into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer.

  10. EGF Functionalized Polymer-Coated Gold Nanoparticles Promote EGF Photostability and EGFR Internalization for Photothermal Therapy

    PubMed Central

    Silva, Catarina Oliveira; Petersen, Steffen B.; Reis, Catarina Pinto; Rijo, Patrícia; Molpeceres, Jesús; Fernandes, Ana Sofia; Gonçalves, Odete; Gomes, Andreia C.; Correia, Isabel; Vorum, Henrik; Neves-Petersen, Maria Teresa

    2016-01-01

    The application of functionalized nanocarriers on photothermal therapy for cancer ablation has wide interest. The success of this application depends on the therapeutic efficiency and biocompatibility of the system, but also on the stability and biorecognition of the conjugated protein. This study aims at investigating the hypothesis that EGF functionalized polymer-coated gold nanoparticles promote EGF photostability and EGFR internalization, making these conjugated particles suitable for photothermal therapy. The conjugated gold nanoparticles (100–200 nm) showed a plasmon absorption band located within the near-infrared range (650–900 nm), optimal for photothermal therapy applications. The effects of temperature, of polymer-coated gold nanoparticles and of UVB light (295nm) on the fluorescence properties of EGF have been investigated with steady-state and time-resolved fluorescence spectroscopy. The fluorescence properties of EGF, including the formation of Trp and Tyr photoproducts, is modulated by temperature and by the intensity of the excitation light. The presence of polymeric-coated gold nanoparticles reduced or even avoided the formation of Trp and Tyr photoproducts when EGF is exposed to UVB light, protecting this way the structure and function of EGF. Cytotoxicity studies of conjugated nanoparticles carried out in normal-like human keratinocytes showed small, concentration dependent decreases in cell viability (0–25%). Moreover, conjugated nanoparticles could activate and induce the internalization of overexpressed Epidermal Growth Factor Receptor in human lung carcinoma cells. In conclusion, the gold nanoparticles conjugated with Epidermal Growth Factor and coated with biopolymers developed in this work, show a potential application for near infrared photothermal therapy, which may efficiently destroy solid tumours, reducing the damage of the healthy tissue. PMID:27788212

  11. Effect of antigen-dependent clearance on pharmacokinetics of anti-heparin-binding EGF-like growth factor (HB-EGF) monoclonal antibody.

    PubMed

    Kasai, Noriyuki; Yoshikawa, Yukitaka; Enokizono, Junichi

    2014-01-01

    Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family and is an important therapeutic target in some types of human cancers. KM3566 is a mouse anti-HB-EGF monoclonal antibody that neutralizes HB-EGF activity by inhibiting the binding of HB-EGF to its receptors. Based on the results of our pharmacokinetics study, a humanized derivative antibody, KHK2866, is rapidly cleared from serum and shows nonlinear pharmacokinetics in cynomolgus monkeys. In this study, we examined the antigen-dependent clearance of an anti-HB-EGF monoclonal antibody in vivo and in vitro in order to pharmacokinetically explain the rapid elimination of KHK2866. We revealed tumor size-dependent clearance of KM3566 in in vivo studies and obtained good fits between the observed and simulated concentrations of KM3566 based on the two-compartment with a saturable route of clearance model. Furthermore, in vivo imaging analyses demonstrated tumor-specific distribution of KM3566. We then confirmed rapid internalization and distribution to lysosome of KM3566 at a cellular level. Moreover, we revealed that the amounts of HB-EGF on cell surface membrane were maintained even while HB-EGF was internalized with KM3566. Recycled or newly synthesized HB-EGF, therefore, may contribute to a consecutive clearance of KM3566, which could explain a rapid clearance from serum. These data suggested that the rapid elimination in pharmacokinetics of KM3566 is due to antigen-dependent clearance. Given that its antigen is expressed in a wide range of normal tissue, it is estimated that the rapid elimination of KHK2866 from cynomolgus monkey serum is caused by antigen-dependent clearance. PMID:25517307

  12. SMOC Binds to Pro-EGF, but Does Not Induce Erk Phosphorylation via the EGFR

    PubMed Central

    Thomas, J. Terrig; Chhuy-Hy, Lina; Andrykovich, Kristin R.; Moos, Malcolm

    2016-01-01

    In an attempt to identify the cell-associated protein(s) through which SMOC (Secreted Modular Calcium binding protein) induces mitogen-activated protein kinase (MAPK) signaling, the epidermal growth factor receptor (EGFR) became a candidate. However, although in 32D/EGFR cells, the EGFR was phosphorylated in the presence of a commercially available human SMOC-1 (hSMOC-1), only minimal phosphorylation was observed in the presence of Xenopus SMOC-1 (XSMOC-1) or human SMOC-2. Analysis of the commercial hSMOC-1 product demonstrated the presence of pro-EGF as an impurity. When the pro-EGF was removed, only minimal EGFR activation was observed, indicating that SMOC does not signal primarily through EGFR and its receptor remains unidentified. Investigation of SMOC/pro-EGF binding affinity revealed a strong interaction that does not require the C-terminal extracellular calcium-binding (EC) domain of SMOC or the EGF domain of pro-EGF. SMOC does not appear to potentiate or inhibit MAPK signaling in response to pro-EGF, but the interaction could provide a mechanism for retaining soluble pro-EGF at the cell surface. PMID:27101391

  13. Lipopolysaccharide disrupts the directional persistence of alveolar myofibroblast migration through EGF receptor.

    PubMed

    Li, Huiping; Yuan, Xiaobing; Tang, Jun; Zhang, Yongjun

    2012-03-15

    Bronchopulmonary dysplasia (BPD) is characterized by alveolar simplification with decreased alveolar number and increased airspace size. Formation of alveoli involves a process known as secondary septation triggered by myofibroblasts. This study investigated the underlying mechanisms of altered lung morphogenesis in a rat model of BPD induced by intra-amniotic injection of lipopolysaccharide (LPS). Results showed that LPS disrupted alveolar morphology and led to abnormal localization of myofibroblasts in the lung of newborn rats, mostly in primary septa with few in secondary septa. To identify potential mechanisms, in vitro experiments were carried out to observe the migration behavior of myofibroblasts. The migration speed of lung myofibroblasts increased with LPS treatment, whereas the directional persistence decreased. We found that LPS induced activation of EGFR and overexpression of its ligand, TGF-α in myofibroblasts. AG1478, an EGFR inhibitor, abrogated the enhanced locomotivity of myofibroblasts by LPS and also increased the directional persistence of myofibroblast migration. Myofibroblasts showed a high asymmetry of phospho-EGFR localization, which was absent after LPS treatment. Application of rhTGF-α to myofibroblasts decreased the directional persistence. Our findings indicated that asymmetry of phospho-EGFR localization in myofibroblasts was important for cell migration and its directional persistence. We speculate that LPS exposure disrupts the asymmetric localization of phospho-EGFR, leading to decreased stability of cell polarity and final abnormal location of myofibroblasts in vivo, which is critical to secondary septation and may contribute to the arrested alveolar development in BPD.

  14. Enhanced Antitumor Activity of EGFP-EGF1-Conjugated Nanoparticles by a Multitargeting Strategy.

    PubMed

    Zhang, Bo; Jiang, Ting; Ling, Li; Cao, Zhonglian; Zhao, Jingjing; Tuo, Yanyan; She, Xiaojian; Shen, Shun; Jiang, Xinguo; Hu, Yu; Pang, Zhiqing

    2016-04-13

    Tumor stromal cells have been increasingly recognized to interact with tumor parenchyma cells and promote tumor growth. Therefore, we speculated that therapeutics delivery to both parenchyma cells and stromal cells simultaneously might treat a tumor more effectively. Tissue factor (TF) was shown to be extensively located in a tumor and was abundantly sited in both tumor parenchyma cells and stromal cells including neo-vascular cells, tumor-associated fibroblasts, and tumor-associated macrophages, indicating it might function as a favorable target for drug delivery to multiple cell types simultaneously. EGFP-EGF1 is a fusion protein derived from factor VII, the natural ligand of TF. It retains the specific TF binding capability but does not cause coagulation. In the present study, a nanoparticle modified with EGFP-EGF1 (ENP) was constructed as a multitargeting drug delivery system. The protein binding experiment showed EGFP-EGF1 could bind well to A549 tumor cells and other stromal cells including neo-vascular cells, tumor-associated fibroblasts, and tumor-associated macrophages. Compared with unmodified nanoparticles (NP), ENP uptake by A549 cells and those stromal cells was significantly enhanced but inhibited by excessive free EGFP-EGF1. In addition, ENP induced more A549 tumor cell apoptosis than Taxol and NP when paclitaxel (PTX) was loaded. In vivo, ENP accumulated more specially in TF-overexpressed A549 tumors by in vivo imaging, mainly regions unoccupied by factor VII and targeted tumor parenchyma cells as well as different types of stromal cells by immunofluorescence staining. Treatment with PTX-loaded ENP (ENP-PTX) significantly reduced the A549 tumor growth in nude mice while NP-PTX- and Taxol-treated mice had lower response to the therapy. Furthermore, H&E and TUNEL staining revealed that ENP-PTX induced more severe tumor necrosis and more extensive cell apoptosis. Altogether, the present study demonstrated that ENP could target multiple key cell types

  15. Olig2-Dependent Reciprocal Shift in PDGF and EGF Receptor Signaling Regulates Tumor Phenotype and Mitotic Growth in Malignant Glioma.

    PubMed

    Lu, Fanghui; Chen, Ying; Zhao, Chuntao; Wang, Haibo; He, Danyang; Xu, Lingli; Wang, Jincheng; He, Xuelian; Deng, Yaqi; Lu, Ellen E; Liu, Xue; Verma, Ravinder; Bu, Hong; Drissi, Rachid; Fouladi, Maryam; Stemmer-Rachamimov, Anat O; Burns, Dennis; Xin, Mei; Rubin, Joshua B; Bahassi, El Mustapha; Canoll, Peter; Holland, Eric C; Lu, Q Richard

    2016-05-01

    Malignant gliomas exhibit extensive heterogeneity and poor prognosis. Here we identify mitotic Olig2-expressing cells as tumor-propagating cells in proneural gliomas, elimination of which blocks tumor initiation and progression. Intriguingly, deletion of Olig2 resulted in tumors that grow, albeit at a decelerated rate. Genome occupancy and expression profiling analyses reveal that Olig2 directly activates cell-proliferation machinery to promote tumorigenesis. Olig2 deletion causes a tumor phenotypic shift from an oligodendrocyte precursor-correlated proneural toward an astroglia-associated gene expression pattern, manifest in downregulation of platelet-derived growth factor receptor-α and reciprocal upregulation of epidermal growth factor receptor (EGFR). Olig2 deletion further sensitizes glioma cells to EGFR inhibitors and extends the lifespan of animals. Thus, Olig2-orchestrated receptor signaling drives mitotic growth and regulates glioma phenotypic plasticity. Targeting Olig2 may circumvent resistance to EGFR-targeted drugs. PMID:27165742

  16. 9-Hydroxystearic acid interferes with EGF signalling in a human colon adenocarcinoma

    SciTech Connect

    Calonghi, Natalia; Pagnotta, Eleonora; Parolin, Carola; Tognoli, Cristina; Boga, Carla; Masotti, Lanfranco . E-mail: natalia.calonghi@unibo.it

    2006-04-07

    The epidermal growth factor has long been known to be strictly correlated with the highly proliferating activities of cancer cells and primary tumors. Moreover, in the nucleus, the epidermal growth factor/epidermal growth factor receptor complex (EGF/EGFR) functions as a transcriptional regulator that activates the cyclin D1 gene. 9-hydroxystearic acid (9-HSA) induces cell proliferation arrest and differentiation in HT29 colon cancer cells by inhibiting histone deacetylase 1 (HDAC1). 9-HSA-treated HT29, when stimulated with EGF, are not responsive and surprisingly undergo a further arrest. In order to understand the mechanisms of this effect, we analyzed the degree of internalization of the EGF/EGFR complex and its interactions with HDAC1. It appears that HDAC1, as modified by 9-HSA, is unable to associate with cyclin D1, interfering with the cell proliferation program, and sequesters the EGF/EGFR complex interrupting the transduction of the mitogenic signal.

  17. Clinical significance of proliferation, apoptosis and senescence of nasopharyngeal cells by the simultaneously blocking EGF, IGF-1 receptors and Bcl-xl genes

    SciTech Connect

    Dai, Guodong; Peng, Tao; Zhou, Xuhong; Zhu, Jun; Kong, Zhihua; Ma, Li; Xiong, Zhi; Yuan, Yulin

    2013-11-01

    Highlight: •Construction of shRNA segments expression vectors is valid by the investigation of RT-PCR for IGF1R, EGFR and Bcl-xl mRNA and protein expression. •Studies have suggested that the vectors in blocking these genes of the growth factor receptors and anti- apoptosis is capable of breaking the balance of tumor growth so that tumor trend apoptosis and senescence. •Simultaneously blocking multiple genes that are abnormally expressed may be more effective in treating cancer cells than silencing a single gene. -- Abstract: Background: In previous work, we constructed short hairpin RNA (shRNA) expression plasmids that targeted human EGF and IGF-1 receptors messenger RNA, respectively, and demonstrated that these vectors could induce apoptosis of human nasopharyngeal cell lines (CNE2) and inhibit ligand-induced pAkt and pErk activation. Method: We have constructed multiple shRNA expression vectors of targeting EGFR, IGF1R and Bcl-xl, which were transfected to the CNE2 cells. The mRNA expression was assessed by RT-PCR. The growth of the cells, cell cycle progression, apoptosis of the cells, senescent tumor cells and the proteins of EGFR, IGF1R and Bcl-xl were analyzed by MTT, flow cytometry, cytochemical therapy or Western blot. Results: In group of simultaneously blocking EGFR, IGF1R and Bcl-xl genes, the mRNA of EGFR, IGF1R and Bcl-xl expression was decreased by (66.66 ± 3.42)%, (73.97 ± 2.83)% and (64.79 ± 2.83)%, and the protein expressions was diminished to (67.69 ± 4.02)%, (74.32 ± 2.30)%, and (60.00 ± 3.34)%, respectively. Meanwhile, the cell apoptosis increased by 65.32 ± 0.18%, 65.16 ± 0.25% and 55.47 ± 0.45%, and senescent cells increased by 1.42 ± 0.15%, 2.26 ± 0.15% and 3.22 ± 0.15% in the second, third and fourth day cultures, respectively. Conclusions: Simultaneously blocking EGFR, IGF1R and Bcl-xl genes is capable of altering the balance between proliferating versus apoptotic and senescent cells in the favor of both of apoptosis and

  18. Differential phosphorylation of the progesterone receptor by insulin, epidermal growth factor, and platelet-derived growth factor receptor tyrosine protein kinases.

    PubMed

    Woo, D D; Fay, S P; Griest, R; Coty, W; Goldfine, I; Fox, C F

    1986-01-01

    Purified preparations of insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) receptors were compared for their abilities to phosphorylate purified hen oviduct progesterone receptors. The specific activities of all three peptide hormone-induced receptor kinases were first defined using a synthetic tridecapeptide tyrosine protein kinase substrate. Next, equivalent ligand-activated activities of the three receptor kinases were tested for their abilities to phosphorylate hen oviduct progesterone receptor. Both the insulin and EGF receptors phosphorylated progesterone receptor at high affinity, exclusively at tyrosine residues and with maximal stoichiometries that were near unity. In contrast, the PDGF receptor did not recognize progesterone receptor as a substrate. Insulin decreased the Km of the insulin receptor for progesterone receptor subunits as substrates, but had no significant effect on Vmax values. On the other hand, EGF increased the Vmax of the EGF receptor for progesterone receptor subunits as substrates. Phosphorylation of progesterone receptor by the insulin and EGF receptor kinases differed in two additional ways. 1) EGF-activated receptor phosphorylated the 80- and 105-kDa progesterone receptor subunits to an equal extent, whereas insulin-activated receptor preferentially phosphorylated the 80-kDa subunit. 2) Phosphopeptide fingerprinting analyses revealed that while insulin and EGF receptors phosphorylated one identical major site on both progesterone receptor subunits, they differed in their specificities for other sites. PMID:3001059

  19. The F-BAR Protein PACSIN2 Regulates Epidermal Growth Factor Receptor Internalization

    PubMed Central

    de Kreuk, Bart-Jan; Anthony, Eloise C.; Geerts, Dirk; Hordijk, Peter L.

    2012-01-01

    Signaling via growth factor receptors, including the epidermal growth factor (EGF) receptor, is key to various cellular processes, such as proliferation, cell survival, and cell migration. In a variety of human diseases such as cancer, aberrant expression and activation of growth factor receptors can lead to disturbed signaling. Intracellular trafficking is crucial for proper signaling of growth factor receptors. As a result, the level of cell surface expression of growth factor receptors is an important determinant for the outcome of downstream signaling. BAR domain-containing proteins represent an important family of proteins that regulate membrane dynamics. In this study, we identify a novel role for the F-BAR protein PACSIN2 in the regulation of EGF receptor signaling. We show that internalized EGF as well as the (activated) EGF receptor translocated to PACSIN2-positive endosomes. Furthermore, loss of PACSIN2 increased plasma membrane expression of the EGF receptor in resting cells and increased EGF-induced phosphorylation of the EGF receptor. As a consequence, EGF-induced activation of Erk and Akt as well as cell proliferation were enhanced in PACSIN2-depleted cells. In conclusion, this study identifies a novel role for the F-BAR-domain protein PACSIN2 in regulating EGF receptor surface levels and EGF-induced downstream signaling. PMID:23129763

  20. TGFβ loss activates ADAMTS-1-mediated EGF-dependent invasion in a model of esophageal cell invasion

    SciTech Connect

    Le Bras, Grégoire F.; Taylor, Chase; Koumangoye, Rainelli B.; Revetta, Frank; Loomans, Holli A.; Andl, Claudia D.

    2015-01-01

    The TGFβ signaling pathway is essential to epithelial homeostasis and is often inhibited during progression of esophageal squamous cell carcinoma. Recently, an important role for TGFβ signaling has been described in the crosstalk between epithelial and stromal cells regulating squamous tumor cell invasion in mouse models of head-and-neck squamous cell carcinoma (HNSCC). Loss of TGFβ signaling, in either compartment, leads to HNSCC however, the mechanisms involved are not well understood. Using organotypic reconstruct cultures (OTC) to model the interaction between epithelial and stromal cells that occur in dysplastic lesions, we show that loss of TGFβ signaling promotes an invasive phenotype in both fibroblast and epithelial compartments. Employing immortalized esophageal keratinocytes established to reproduce common mutations of esophageal squamous cell carcinoma, we show that treatment of OTC with inhibitors of TGFβ signaling (A83-01 or SB431542) enhances invasion of epithelial cells into a fibroblast-embedded Matrigel/collagen I matrix. Invasion induced by A83-01 is independent of proliferation but relies on protease activity and expression of ADAMTS-1 and can be altered by matrix density. This invasion was associated with increased expression of pro-inflammatory cytokines, IL1 and EGFR ligands HB-EGF and TGFα. Altering EGF signaling prevented or induced epithelial cell invasion in this model. Loss of expression of the TGFβ target gene ROBO1 suggested that chemorepulsion may regulate keratinocyte invasion. Taken together, our data show increased invasion through inhibition of TGFβ signaling altered epithelial-fibroblasts interactions, repressing markers of activated fibroblasts, and altering integrin-fibronectin interactions. These results suggest that inhibition of TGFβ signaling modulates an array of pathways that combined promote multiple aspects of tumor invasion. - Highlights: • Chemical inhibition of TGFβ signaling advances collective invasion

  1. Insulin, concanavalin A, EGF, IFG-I and vanadate activate de novo phosphatidic acid and diacylglycerol synthesis, C-kinase, and glucose transport in BC3H-1 myocytes

    SciTech Connect

    Cooper, D.R.; Hernandez, H.; Konda, T.S.; Standaert, M.S.; Pollet, R.J.; Farese, R.V.

    1987-05-01

    The authors have reported that insulin stimulates de novo synthesis of phosphatidic acid (PA) which is metabolized directly to diacylglycerol (DG) in BS3H-1 myocytes; this is accompanied by increases in C-kinase activity in membrane and cytosolic extracts. This pathway may be involved in stimulating glucose transport and other metabolic processes. In this study, the authors have compared the effects of concanavalin A, EGF, IGF-I and sodium orthovanadate to insulin on PA/DG synthesis, C-kinase activity and glucose transport. All were found to be effective in stimulating glucose transport. Additionally, all activators rapidly increased the incorporation of (/sup 3/H)glycerol into DG and total glycerolipids, although none were as effective as insulin, which increased (/sup 3/H)DG 400% in 1 minute. Increased incorporation into phospholipids and triacylglycerols and to a lesser extent monoacylglycerol was also noted. They examined effects of concanavalin A and EGF on C-kinase activity and found that both agonists, like insulin, increase C-kinase activity in cytosolic and/or membrane fractions. Their findings raise the possibility that activation of receptors having associated tyrosine kinase activity may provoke some cellular responses through de novo PA/GD synthesis and C-kinase activation.

  2. Targeting autocrine HB-EGF signaling with specific ADAM12 inhibition using recombinant ADAM12 prodomain

    NASA Astrophysics Data System (ADS)

    Miller, Miles A.; Moss, Marcia L.; Powell, Gary; Petrovich, Robert; Edwards, Lori; Meyer, Aaron S.; Griffith, Linda G.; Lauffenburger, Douglas A.

    2015-10-01

    Dysregulation of ErbB-family signaling underlies numerous pathologies and has been therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands. For the latter, “decoy” antibodies have been developed to sequester ligands including heparin-binding epidermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has been difficult. Here, we hypothesized that this strategy depends on properties such as ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands. Guided by computational modeling, we found that high-affinity ligands such as HB-EGF are more difficult to target with decoy antibodies compared to low-affinity ligands such as amphiregulin (AREG). To address this issue, we developed an alternative method for inhibiting HB-EGF activity by targeting its cleavage from the cell surface. In a model of the invasive disease endometriosis, we identified A Disintegrin and Metalloproteinase 12 (ADAM12) as a protease implicated in HB-EGF shedding. We designed a specific inhibitor of ADAM12 based on its recombinant prodomain (PA12), which selectively inhibits ADAM12 but not ADAM10 or ADAM17. In endometriotic cells, PA12 significantly reduced HB-EGF shedding and resultant cellular migration. Overall, specific inhibition of ligand shedding represents a possible alternative to decoy antibodies, especially for ligands such as HB-EGF that exhibit high binding affinity and localized signaling.

  3. Progesterone receptor subunits are high-affinity substrates for phosphorylation by epidermal growth factor receptor.

    PubMed Central

    Ghosh-Dastidar, P; Coty, W A; Griest, R E; Woo, D D; Fox, C F

    1984-01-01

    Purified preparations of epidermal growth factor (EGF) receptor were used to test hen oviduct progesterone receptor subunits as substrates for phosphorylation catalyzed by EGF receptor. Both the 80-kilodalton (kDa) (A) and the 105-kDa (B) progesterone receptor subunits were phosphorylated in a reaction that required EGF and EGF receptor. No phosphorylation of progesterone receptor subunits was observed in the absence of EGF receptor, even when Ca2+ was substituted for Mg2+ and Mn2+. Phospho amino acid analysis revealed phosphorylation at tyrosine residues, with no phosphorylation detectable at serine or threonine residues. Two-dimensional maps of phosphopeptides generated from phosphorylated 80- or 105-kDa subunits by tryptic digestion revealed similar patterns, with resolution of two major, several minor, and a number of very minor phosphopeptides. The Km of progesterone receptor for phosphorylation by EGF-activated EGF receptor was 100 nM and the Vmax was 2.5 nmol/min per mg of EGF receptor protein at 0 degrees C. The stoichiometry of phosphorylation/hormone binding for progesterone receptor subunits was 0.31 at ice-bath temperature and approximately 1.0 at 22 degrees C. Images PMID:6200881

  4. Phosphorylation of Thr{sup 654} but not Thr{sup 669} within the juxtamembrane domain of the EGF receptor inhibits calmodulin binding

    SciTech Connect

    Aifa, Sami; Frikha, Fakher; Miled, Nabil; Johansen, Knut; Lundstroem, Ingemar; Svensson, Samuel P.S. . E-mail: samuel.svensson@imv.liu.se

    2006-08-25

    Calcium-calmodulin (CaM) binding to the epidermal growth factor receptor (EGFR) has been shown to both inhibit and stimulate receptor activity. CaM binds to the intracellular juxtamembrane (JM) domain (Met{sup 645}-Phe{sup 688}) of EGFR. Protein kinase C (PKC) mediated phosphorylation of Thr{sup 654} occurs within this domain. CaM binding to the JM domain inhibits PKC phosphorylation and conversely PKC mediated phosphorylation of Thr{sup 654} or Glu substitution of Thr{sup 654} inhibits CaM binding. A second threonine residue (Thr{sup 669}) within the JM domain is phosphorylated by the mitogen-activated protein kinase (MAPK). Previous results have shown that CaM interferes with EGFR-induced MAPK activation. If and how phosphorylation of Thr{sup 669} affects CaM-EGFR interaction is however not known.In the present study we have used surface plasmon resonance (BIAcore) to study the influence of Thr{sup 669} phosphorylation on real time interactions between the intracellular juxtamembrane (JM) domain of EGFR and CaM. The EGFR-JM was expressed as GST fusion proteins in Escherichia coli and phosphorylation was mimicked by generating Glu substitutions of either Thr{sup 654} or Thr{sup 669}. Purified proteins were coupled to immobilized anti-GST antibodies at the sensor surface and increasing concentration of CaM was applied. When mutating Thr{sup 654} to Glu{sup 654} no specific CaM binding could be detected. However, neither single substitutions of Thr{sup 669} (Gly{sup 669} or Glu{sup 669}) nor double mutants Gly{sup 654}/Gly{sup 669} or Gly{sup 654}/Glu{sup 669} influenced the binding of CaM to the EGFR-JM. This clearly shows that PKC may regulate EGF-mediated CaM signalling through phosphorylation of Thr{sup 654} whereas phosphorylation of Thr{sup 669} seems to play a CaM independent regulatory role. The role of both residues in the EGFR-calmodulin interaction was also studied in silico. Our modelling work supports a scenario where Thr{sup 654} from the JM domain

  5. Cross-talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells

    PubMed Central

    Liu, Xing; Qin, Jiaqiang; Luo, Qing; Bi, Yang; Zhu, Gaohui; Jiang, Wei; Kim, Stephanie H; Li, Mi; Su, Yuxi; Nan, Guoxin; Cui, Jing; Zhang, Wenwen; Li, Ruidong; Chen, Xiang; Kong, Yuhan; Zhang, Jiye; Wang, Jinhua; Rogers, Mary Rose; Zhang, Hongyu; Shui, Wei; Zhao, Chen; Wang, Ning; Liang, Xi; Wu, Ningning; He, Yunfeng; Luu, Hue H; Haydon, Rex C; Shi, Lewis L; Li, Tingyu; He, Tong-Chuan; Li, Ming

    2013-01-01

    Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross-talks with BMP9 and regulates BMP9-induced osteogenic differentiation. We find that EGF potentiates BMP9-induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG-1478 and AG-494 in a dose- and time-dependent manner. Furthermore, EGF significantly augments BMP9-induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9-induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up-regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross-talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine. PMID:23844832

  6. Development of Cu-64 labeled EGF for In Vivo PET Imaging of EGFR Expression

    SciTech Connect

    Backer, Joseph M.

    2009-07-12

    In this project we proposed to establish feasibility of the development of targeted tracers for radionuclide imaging of epidermal growth factor receptors (EGFR) in cancer patients. The significance and impact of the proposed radiotracers are determined by the crucial role that EGFR plays in many cancers and by the rapid entrance of EGFR-inhibiting drugs into clinic. Clinical experience, however, revealed that only 10-25% of patients that are defined as EGFR-positive by immunohistochemical analysis respond to EGFR-directed therapeutics and there is poor correlation between EGFR immunohistochemistry and treatment. Therefore, for more efficacious use of EGFR-targeting therapeutics, there is a need for information about EGFR activity in patients. We hypothesized that radionuclide imaging of functionally active EGFR will provide such information and would allow for 1) rational patient stratification, 2) rapid monitoring of responses to therapy, and 3) development of personalized treatment regimens. We hypothesized that tracers based epidermal growth factor (EGF), a natural EGFR ligand, as a targeting vector would be particularly advantageous. First, only functionally active and therefore critical for disease progression EGFRs will bind and internalize an EGF-based tracer. Second, continuous internalization of EGF-based tracers by recyclable EGFR would lead to intracellular accumulation of radionuclide and improved signal-to-background ratio. Third, small size of EGF relative to antibodies would facilitate tumor penetration with vastly better non-specific soft tissue and blood clearance rates. Fourth, as a human protein, EGF is not expected to be immunogenic. Finally, at the beginning of this project, we have already engineered and expressed functionally active EGF with an N-terminal Cys-tag for site-specific conjugation of various payloads, including radionuclide chelators. In the Phase I of this project, in collaboration with Dr. Blankenberg’s group at Stanford

  7. A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo1[S

    PubMed Central

    Ni, Yan G.; Di Marco, Stefania; Condra, Jon H.; Peterson, Laurence B.; Wang, Weirong; Wang, Fubao; Pandit, Shilpa; Hammond, Holly A.; Rosa, Ray; Cummings, Richard T.; Wood, Dana D.; Liu, Xiaomei; Bottomley, Matthew J.; Shen, Xun; Cubbon, Rose M.; Wang, Sheng-ping; Johns, Douglas G.; Volpari, Cinzia; Hamuro, Lora; Chin, Jayne; Huang, Lingyi; Zhao, Jing Zhang; Vitelli, Salvatore; Haytko, Peter; Wisniewski, Douglas; Mitnaul, Lyndon J.; Sparrow, Carl P.; Hubbard, Brian; Carfí, Andrea; Sitlani, Ayesha

    2011-01-01

    Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%–50% for over 2 weeks, despite its relatively short terminal half-life (t1/2 = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr. PMID:20959675

  8. Unconventional EGF-induced ERK1/2-mediated Kv1.3 endocytosis.

    PubMed

    Martínez-Mármol, Ramón; Comes, Núria; Styrczewska, Katarzyna; Pérez-Verdaguer, Mireia; Vicente, Rubén; Pujadas, Lluís; Soriano, Eduardo; Sorkin, Alexander; Felipe, Antonio

    2016-04-01

    The potassium channel Kv1.3 plays roles in immunity, neuronal development and sensory discrimination. Regulation of Kv1.3 by kinase signaling has been studied. In this context, EGF binds to specific receptors (EGFR) and triggers tyrosine kinase-dependent signaling, which down-regulates Kv1.3 currents. We show that Kv1.3 undergoes EGF-dependent endocytosis. This EGF-mediated mechanism is relevant because is involved in adult neural stem cell fate determination. We demonstrated that changes in Kv1.3 subcellular distribution upon EGFR activation were due to Kv1.3 clathrin-dependent endocytosis, which targets the Kv1.3 channels to the lysosomal degradative pathway. Interestingly, our results further revealed that relevant tyrosines and other interacting motifs, such as PDZ and SH3 domains, were not involved in the EGF-dependent Kv1.3 internalization. However, a new, and yet undescribed mechanism, of ERK1/2-mediated threonine phosphorylation is crucial for the EGF-mediated Kv1.3 endocytosis. Our results demonstrate that EGF triggers the down-regulation of Kv1.3 activity and its expression at the cell surface, which is important for the development and migration of adult neural progenitors.

  9. 5-Fluorocytosine combined with Fcy-hEGF fusion protein targets EGFR-expressing cancer cells

    SciTech Connect

    Lan, Keng-Hsueh; Shih, Yi-Sheng; Chang, Cheng Allen; Yen, Sang-Hue; Lan, Keng-Li

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer EGFR-expressing epithelial cancers account for significant portion of cancer deaths. Black-Right-Pointing-Pointer EGF-EGFR signaling pathway is validated as an important anticancer drug target. Black-Right-Pointing-Pointer EGF and Fcy fusion protein (Fcy-hEGF) can bind to EGFR and convert 5-FC to 5-FU. Black-Right-Pointing-Pointer Fcy-hEGF combined with 5-FC preferentially inhibits EGFR-expressing cells viability. -- Abstract: Human epithelial cancers account for approximately 50% of all cancer deaths. This type of cancer is characterized by excessive activation and expression of the epidermal growth factor receptor (EGFR). The EGFR pathway is critical for cancer cell proliferation, survival, metastasis and angiogenesis. The EGF-EGFR signaling pathway has been validated as an important anticancer drug target. Increasing numbers of targeted therapies against this pathway have been either approved or are currently under development. Here, we adopted a prodrug system that uses 5-fluorocytosine (5-FC) and human EGF (hEGF) fused with yeast cytosine deaminase (Fcy) to target EGFR-overexpressing cancer cells and to convert 5-FC to a significantly more toxic chemotherapeutic, 5-fluorouracil (5-FU). We cloned and purified the Fcy-hEGF fusion protein from Pichia pastoris yeast. This fusion protein specifically binds to EGFR with a similar affinity as hEGF, approximately 10 nM. Fcy-hEGF binds tightly to A431 and MDA-MB-468 cells, which overexpress EGFR, but it binds with a lower affinity to MDA-MB-231 and MCF-7, which express lower levels of EGFR. Similarly, the viability of EGFR-expressing cells was suppressed by Fcy-hEGF in the presence of increasing concentrations of 5-FC, and the IC{sub 50} values for A431 and MDA-MB-468 were approximately 10-fold lower than those of MDA-MB-231 and MCF-7. This novel prodrug system, Fcy-hEGF/5-FC, might represent a promising addition to the available class of inhibitors that specifically target EGFR

  10. Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes

    PubMed Central

    Ockenga, Wymke; Kühne, Sina; Bocksberger, Simone; Banning, Antje; Tikkanen, Ritva

    2014-01-01

    Non-neuronal acetylcholine plays a substantial role in the human skin by influencing adhesion, migration, proliferation and differentiation of keratinocytes. These processes are regulated by the Mitogen-Activated Protein (MAP) kinase cascade. Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to activation of the MAP kinase extracellular signal regulated kinase, together with the protein kinase Akt. The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR), which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells. The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation. Furthermore, phosphorylation, ubiquitination and endocytosis of the EGF receptor after cholinergic transactivation are different from that induced by a direct stimulation with EGF, suggesting that ligands other than EGF itself mediate the cholinergic transactivation. PMID:25421240

  11. Altered (/sup 125/I)epidermal growth factor binding and receptor distribution in psoriasis

    SciTech Connect

    Nanney, L.B.; Stoscheck, C.M.; Magid, M.; King, L.E. Jr.

    1986-03-01

    Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that (/sup 125/I)EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers.

  12. Developmental defects in zebrafish for classification of EGF pathway inhibitors

    SciTech Connect

    Pruvot, Benoist; Curé, Yoann; Djiotsa, Joachim; Voncken, Audrey; Muller, Marc

    2014-01-15

    One of the major challenges when testing drug candidates targeted at a specific pathway in whole animals is the discrimination between specific effects and unwanted, off-target effects. Here we used the zebrafish to define several developmental defects caused by impairment of Egf signaling, a major pathway of interest in tumor biology. We inactivated Egf signaling by genetically blocking Egf expression or using specific inhibitors of the Egf receptor function. We show that the combined occurrence of defects in cartilage formation, disturbance of blood flow in the trunk and a decrease of myelin basic protein expression represent good indicators for impairment of Egf signaling. Finally, we present a classification of known tyrosine kinase inhibitors according to their specificity for the Egf pathway. In conclusion, we show that developmental indicators can help to discriminate between specific effects on the target pathway from off-target effects in molecularly targeted drug screening experiments in whole animal systems. - Highlights: • We analyze the functions of Egf signaling on zebrafish development. • Genetic blocking of Egf expression causes cartilage, myelin and circulatory defects. • Chemical inhibition of Egf receptor function causes similar defects. • Developmental defects can reveal the specificity of Egf pathway inhibitors.

  13. Non-linear antigenic regions in epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) studied by EGF-TGF alpha chimaeras.

    PubMed

    van de Poll, M L; van Rotterdam, W; Gadellaa, M M; Stortelers, C; van Vugt, M J; van Zoelen, E J

    2000-07-01

    With the help of 16 chimaeras between human epidermal growth factor (hEGF) and human transforming growth factor alpha (hTGF alpha), a detailed analysis was performed on the epitope recognized by two polyclonal antibodies raised against hEGF, and one polyclonal antibody raised against hTGF alpha. All three antibodies recognized essentially the same antigenic site, a non-linear and conformation-dependent sequence that is located near the second and fourth disulphide-bonded cysteines and that includes the start of the B-loop beta-sheet. The epitope recognized by the anti-hEGF antibodies was further characterized using 8 chimaeras between hEGF and an EGF-repeat from Drosophila Notch and was found to include Met(21), Ala(30) and Asn(32). All three polyclonal antibodies were able to neutralize the biological activity of the respective growth factor when tested on 32D murine haematopoietic progenitor cells transfected with ErbB-1, indicating that the receptor binding domain is shielded upon binding of the antibody.

  14. Non-linear antigenic regions in epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) studied by EGF-TGF alpha chimaeras.

    PubMed Central

    van de Poll, M L; van Rotterdam , W; Gadellaa, M M; Stortelers, C; van Vugt , M J; van Zoelen , E J

    2000-01-01

    With the help of 16 chimaeras between human epidermal growth factor (hEGF) and human transforming growth factor alpha (hTGF alpha), a detailed analysis was performed on the epitope recognized by two polyclonal antibodies raised against hEGF, and one polyclonal antibody raised against hTGF alpha. All three antibodies recognized essentially the same antigenic site, a non-linear and conformation-dependent sequence that is located near the second and fourth disulphide-bonded cysteines and that includes the start of the B-loop beta-sheet. The epitope recognized by the anti-hEGF antibodies was further characterized using 8 chimaeras between hEGF and an EGF-repeat from Drosophila Notch and was found to include Met(21), Ala(30) and Asn(32). All three polyclonal antibodies were able to neutralize the biological activity of the respective growth factor when tested on 32D murine haematopoietic progenitor cells transfected with ErbB-1, indicating that the receptor binding domain is shielded upon binding of the antibody. PMID:10861238

  15. Activation of the Ras/Mitogen-Activated Protein Kinase Pathway by Kinase-Defective Epidermal Growth Factor Receptors Results in Cell Survival but Not Proliferation

    PubMed Central

    Walker, Francesca; Kato, Akiko; Gonez, L. Jorge; Hibbs, Margaret L.; Pouliot, Normand; Levitzki, Alexander; Burgess, Antony W.

    1998-01-01

    Signalling by the epidermal growth factor (EGF) receptor (EGFR) has been studied intensively, but for most cell types the analysis is complicated by the fact that EGFR not only homodimerizes but can also form heterodimers with other EGFR family members. Heterodimerization is a particular problem in the study of EGFR mutants, where the true phenotype of the mutants is confounded by the contribution of the heterodimer partner to signal transduction. We have made use of the murine hemopoietic cell line BaF/3, which does not express EGFR family members, to express wild-type (WT) EGFR, three kinase-defective EGFR mutants (V741G, Y740F, and K721R), or a C-terminally truncated EGFR (CT957) and have measured their responses to EGF. We found that under the appropriate conditions EGF can stimulate cell proliferation of BaF/3 cells expressing WT or CT957 EGFRs but not that of cells expressing the kinase-defective mutants. However, EGF promotes the survival of BaF/3 cells expressing either of the kinase-defective receptors (V741G and Y740F), indicating that these receptors can still transmit a survival signal. Analysis of the early signalling events by the WT, V741G, and Y740F mutant EGF receptors indicated that EGF stimulates comparable levels of Shc phosphorylation, Shc–GRB-2 association, and activation of Ras, B-Raf, and Erk-1. Blocking the mitogen-activated protein kinase (MAPK) signalling pathway with the specific inhibitor PD98059 abrogates completely the EGF-dependent survival of cells expressing the kinase-defective EGFR mutants but has no effect on the EGF-dependent proliferation mediated by WT and CT957 EGFRs. Similarly, the Src family kinase inhibitor PP1 abrogates EGF-dependent survival without affecting proliferation. However blocking phosphatidylinositol-3-kinase or JAK-2 kinase with specific inhibitors does arrest growth factor-dependent cell proliferation. Thus, EGFR-mediated mitogenic signalling in BaF/3 cells requires an intact EGFR tyrosine kinase activity

  16. Activated Protein C Enhances Human Keratinocyte Barrier Integrity via Sequential Activation of Epidermal Growth Factor Receptor and Tie2*

    PubMed Central

    Xue, Meilang; Chow, Shu-Oi; Dervish, Suat; Chan, Yee-Ka Agnes; Julovi, Sohel M.; Jackson, Christopher J.

    2011-01-01

    Keratinocytes play a critical role in maintaining epidermal barrier function. Activated protein C (APC), a natural anticoagulant with anti-inflammatory and endothelial barrier protective properties, significantly increased the barrier impedance of keratinocyte monolayers, measured by electric cell substrate impedance sensing and FITC-dextran flux. In response to APC, Tie2, a tyrosine kinase receptor, was rapidly activated within 30 min, and relocated to cell-cell contacts. APC also increased junction proteins zona occludens, claudin-1 and VE-cadherin. Inhibition of Tie2 by its peptide inhibitor or small interfering RNA abolished the barrier protective effect of APC. Interestingly, APC did not activate Tie2 through its major ligand, angiopoietin-1, but instead acted by binding to endothelial protein C receptor, cleaving protease-activated receptor-1 and transactivating EGF receptor. Furthermore, when activation of Akt, but not ERK, was inhibited, the barrier protective effect of APC on keratinocytes was abolished. Thus, APC activates Tie2, via a mechanism requiring, in sequential order, the receptors, endothelial protein C receptor, protease-activated receptor-1, and EGF receptor, which selectively enhances the PI3K/Akt signaling to enhance junctional complexes and reduce keratinocyte permeability. PMID:21173154

  17. Cardiotoxin III suppresses MDA-MB-231 cell metastasis through the inhibition of EGF/EGFR-mediated signaling pathway.

    PubMed

    Tsai, Pei-Chien; Hsieh, Chi-Ying; Chiu, Chien-Chih; Wang, Chih-Kuang; Chang, Long-Sen; Lin, Shinne-Ren

    2012-10-01

    Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. Epidermal growth factor (EGF) and its receptor, EGFR, play roles in cancer metastasis in various tumors. We use EGF as a metastatic inducer of MDA-MB-231 cells to investigate the effect of CTX III on cell migration. CTX III inhibited the EGF-induced activation of matrix metalloproteinase-9 (MMP-9), and further suppressed cell invasion and migration without obvious cellular cytotoxicity. CTX III suppressed EGF-induced nuclear factor-kappaB (NF-κB) nuclear translocation and also abrogated the EGF-induced phosphorylation of EGFR, phosphatidylinositol 3-kinase (PI3K)/Akt, and extracellular regulated kinase (ERK)1/2. In addition, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an up-stream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by EGF. Furthermore, the EGFR inhibitor AG1478 inhibited EGF-induced MMP-9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occur downstream of EGFR activation. These findings suggest that CTX III inhibited the EGF-induced invasion and migration of MDA-MB-231 cells via EGFR-dependent PI3K/Akt, ERK1/2, and NF-κB signaling, leading to the down-regulation of MMP-9 expression. These results provide a novel mechanism to explain the role of CTX III as a potent anti-metastatic agent in MDA-MB-231 cells.

  18. Association of EGF Receptor and NLRs signaling with Cardiac Inflammation and Fibrosis in Mice Exposed to Fine Particulate Matter.

    PubMed

    Jin, Yuefei; Wu, Zhaoke; Wang, Na; Duan, Shuyin; Wu, Yongjun; Wang, Jing; Wu, Weidong; Feng, Feifei

    2016-09-01

    ЄAmbient fine particulate matter (PM2.5 ) could induce cardiovascular diseases (CVD), but the mechanism remains unknown. To investigate the roles of epidermal growth factor receptor (EGFR) and NOD-like receptors (NLRs) in PM2.5 -induced cardiac injury, we set up a BALB/c mice model of PM2.5 -induced cardiac inflammation and fibrosis with intratracheal instillation of PM2.5 suspension (4.0 mg/kg b.w.) for 5 consecutive days (once per day). After exposure, we found that mRNA levels of CXCL1, interleukin (IL)-6, and IL-18 were elevated, but interestingly, mRNA level of NLRP12 was significant decreased in heart tissue from PM2.5 -induced mice compared with those of saline-treated mice using real-time PCR. Protein levels of phospho-EGFR (Tyr1068), phospho-Akt (Thr308), NLRP3, NF-κB-p52/p100, and NF-κB-p65 in heart tissue of PM2.5 -exposed mice were all significantly increased using immunohistochemistry or Western blotting. Therefore, PM2.5 exposure could induce cardiac inflammatory injury in mice, which may be involved with EGFR/Akt signaling, NLRP3, and NLRP12.

  19. Activation of Epidermal Growth Factor Receptor in Macrophages Mediates Feedback Inhibition of M2 Polarization and Gastrointestinal Tumor Cell Growth.

    PubMed

    Zhao, Gang; Liu, Liping; Peek, Richard M; Hao, Xishan; Polk, D Brent; Li, Hui; Yan, Fang

    2016-09-23

    EGF receptor (EGFR) in tumor cells serves as a tumor promoter. However, information about EGFR activation in macrophages in regulating M2 polarization and tumor development is limited. This study aimed to investigate the effects of EGFR activation in macrophages on M2 polarization and development of gastrointestinal tumors. IL-4, a cytokine to elicit M2 polarization, stimulated release of an EGFR ligand, HB-EGF, and transactivation and down-regulation of EGFR in Raw 264.7 cells and peritoneal macrophages from WT mice. Knockdown of HB-EGF in macrophages inhibited EGFR transactivation by IL-4. IL-4-stimulated STAT6 activation, Arg1 and YM1 gene expression, and HB-EGF production were further enhanced by inhibition of EGFR activity in Raw 264.7 cells using an EGFR kinase inhibitor and in peritoneal macrophages from Egfr(wa5) mice with kinase inactive EGFR and by knockdown of EGFR in peritoneal macrophages from Egfr(fl/fl) LysM-Cre mice with myeloid cell-specific EGFR deletion. Chitin induced a higher level of M2 polarization in peritoneal macrophages in Egfr(fl/fl) LysM-Cre mice than that in Egfr(fl/fl) mice. Accordingly, IL-4-conditioned medium stimulated growth and epithelial-to-mesenchymal transition in gastric epithelial and colonic tumor cells, which were suppressed by that from Raw 264.7 cells with HB-EGF knockdown but promoted by that from Egfr(wa5) and Egfr(fl/fl) LysM-Cre peritoneal macrophages. Clinical assessment revealed that the number of macrophages with EGFR expression became less, indicating decreased inhibitory effects on M2 polarization, in late stage of human gastric cancers. Thus, IL-4-stimulated HB-EGF-dependent transactivation of EGFR in macrophages may mediate inhibitory feedback for M2 polarization and HB-EGF production, thereby inhibiting gastrointestinal tumor growth.

  20. Proteinase-activated receptors 1 and 2 and the regulation of porcine coronary artery contractility: a role for distinct tyrosine kinase pathways

    PubMed Central

    El-Daly, Mahmoud; Saifeddine, Mahmoud; Mihara, Koichiro; Ramachandran, Rithwik; Triggle, Christopher R; Hollenberg, Morley D

    2014-01-01

    Background and Purpose Because angiotensin-II-mediated porcine coronary artery (PCA) vasoconstriction is blocked by protein tyrosine kinase (PYK) inhibitors, we hypothesized that proteinase-activated receptors (PARs), known to regulate vascular tension, like angiotensin-II, would also cause PCA contractions via PYK-dependent signalling pathways. Experimental Approach Contractions of intact and endothelium-free isolated PCA rings, stimulated by PAR1/PAR2-activating peptides, angiotensin-II, PGF2α, EGF, PDGF and KCl, were monitored with/without multiple signalling pathway inhibitors, including AG-tyrphostins AG18 (non-specific PYKs), AG1478 (EGF-receptor kinase), AG1296 (PDGF receptor kinase), PP1 (Src kinase), U0126 and PD98059 (MEK/MAPKinase kinase), indomethacin/SC-560/NS-398 (COX-1/2) and L-NAME (NOS). Key Results AG18 inhibited the contractions induced by all the agonists except KCl, whereas U0126 attenuated contractions induced by PAR1/PAR2 agonists, EGF and angiotensin-II, but not by PGF2α, the COX-produced metabolites of arachidonate and KCl. PP1 only affected the responses to PAR1/PAR2-activating peptides and angiotensin-II. The EGF-kinase inhibitor, AG1478, attenuated contractions initiated by the PARs (PAR2 >> PAR1) and EGF itself, but not by angiotensin-II, PGF2α or KCl. COX-1/2 inhibitors blocked the contractions induced by all the agonists, except KCl and PGF2α. Conclusion and Implications PAR1/2-mediated contractions of the PCA are dependent on Src and MAPKinase and, in part, involve EGF-receptor-kinase transactivation and the generation of a COX-derived contractile agonist. However, the PYK signalling pathways used by PARs are distinct from each other and from those triggered by angiotensin-II and EGF. These signalling pathways may be therapeutic targets for managing coagulation-proteinase-induced coronary vasospasm. PMID:24506284

  1. HB-EGF-Promoted Airway Smooth Muscle Cells and Their Progenitor Migration Contribute to Airway Smooth Muscle Remodeling in Asthmatic Mouse.

    PubMed

    Wang, Qing; Li, Hequan; Yao, Yinan; Lu, Guohua; Wang, Yuehong; Xia, Dajing; Zhou, Jianying

    2016-03-01

    The airway smooth muscle (ASM) cells' proliferation, migration, and their progenitor's migration are currently regarded as causative factors for ASM remodeling in asthma. Heparin-binding epidermal growth factor (HB-EGF), a potent mitogen and chemotactic factor, could promote ASM cell proliferation through MAPK pathways. In this study, we obtained primary ASM cells and their progenitors from C57BL/6 mice and went on to explore the role of HB-EGF in these cells migration and the underlying mechanisms. We found that recombinant HB-EGF (rHB-EGF) intratracheal instillation accelerated ASM layer thickening in an OVA-induced asthmatic mouse. Modified Boyden chamber assay revealed that rHB-EGF facilitate ASM cell migration in a dose-dependent manner and ASM cells from asthmatic mice had a greater migration ability than that from normal counterparts. rHB-EGF could stimulate the phosphorylation of ERK1/2 and p38 in ASM cells but further migration assay showed that only epidermal growth factor receptor inhibitor (AG1478) or p38 inhibitor (SB203580), but not ERK1/2 inhibitor (PD98059), could inhibit rHB-EGF-mediated ASM cells migration. Actin cytoskeleton experiments exhibited that rHB-EGF could cause actin stress fibers disassembly and focal adhesions formation of ASM cells through the activation of p38. Finally, airway instillation of rHB-EGF promoted the recruitment of bone marrow-derived smooth muscle progenitor cells, which were transferred via caudal vein, migrating into the airway from the circulation. These observations demonstrated that ASM remodeling in asthma might have resulted from HB-EGF-mediated ASM cells and their progenitor cells migration, via p38 MAPK-dependent actin cytoskeleton remodeling.

  2. EGF-R small inhibitors and anti-EGF-R antibodies: advantages and limits of a new avenue in anticancer therapy.

    PubMed

    Caraglia, Michele; Marra, Monica; Meo, Giuseppina; Addeo, Santolo R; Tagliaferri, Pierosandro; Budillon, Alfredo

    2006-06-01

    Cellular receptors for the Epidermal Growth Factor (EGF-R) are members of the ErbB receptor family and are considered important targets for the experimental treatment of human cancer. Monoclonal antibodies as well as small tyrosine kinase inhibitors (TKIs) have been developed and have undergone extensive evaluation in preclinical and clinical studies based on the general idea that EGF-R plays a critical role on the growth and survival of human tumors. This assumption has been derived by the successful development of BCR/ABL tyrosine kinase inhibitors in human chronic myeloid leukemia as well as on the activity of therapy with monoclonal antibodies (mAb) in breast cancer and lymphoproliferative diseases. It is now becoming clear that factors regulating sensitivity to kinase inhibitors may differ from monoclonal antibodies and that the molecules targeted by interfering drugs must be prioritaire for growth and survival of those specific tumors in order to achieve valuable results. In this article, we will describe the signal transduction pathways regulated by EGF-R and the principal pharmacological and biotechnological agents directed against EGF-R. We will discuss the significance of targeting the EGF-R driven survival pathways and the compensatory intracellular survival mechanisms that counteract the specific EGF-R inhibition and are the cause of the poor clinical results derived from study based on the use of these agents. We will describe new multipotent TKIs that target also other members of ErbB family (i.e. ErbB2) blocking one of the compensatory mechanism that can be triggered in cancer cells. Moreover, we will report new patent on bispecific mAbs that bind EGF-R and immune effectors in order to increase the immunological function of this agent that could be the basis of the different clinical results achieved with the use of TKI and mAbs. Finally, we will propose a pharmacological model able to make cancer cells dependent on EGF-R for their survival and

  3. Epidermal growth factor receptor distribution in burn wounds. Implications for growth factor-mediated repair.

    PubMed Central

    Wenczak, B A; Lynch, J B; Nanney, L B

    1992-01-01

    Epidermal growth factor (EGF) along with several related peptide growth factors has been shown both in vivo and in vitro to accelerate events associated with epidermal wound repair. EGF and transforming growth factor alpha act by binding to a common EGF receptor tyrosine kinase thereby initiating a series of events which ultimately regulate cell proliferation. This study examined the immunohistochemical localization of EGF receptor (EGF-R) in burn wound margins, adjacent proliferating epithelium, and closely associated sweat ducts, sebaceous glands, and hair follicles. Tissue specimens removed during surgical debridement were obtained from full and partial thickness burn wounds in 32 patients with total body surface area burns ranging from 2 to 88%. In the early postburn period (days 2-4), prominent staining for EGF-R was found in undifferentiated, marginal keratinocytes, adjacent proliferating, hypertrophic epithelium, and both marginal and nonmarginal hair follicles, sweat ducts, and sebaceous glands. During the late postburn period (days 5-16), EGF-R was depleted along leading epithelial margins; however, immunoreactive EGF-R remained intensely positive in the hypertrophic epithelium and all skin appendages. Increased detection of immunoreactive EGF-R and the presence of [125I]EGF binding in the hypertrophic epithelium correlated positively with proliferating cell nuclear antigen distributions. Thus, the presence of EGF-R in the appropriate keratinocyte populations suggests a functional role for this receptor during wound repair. Dynamic modulation in EGF receptor distribution during the temporal sequence of repair provides further evidence that an EGF/transforming growth factor alpha/EGF-R-mediated pathway is activated during human wound repair. Images PMID:1361495

  4. The effects of sialoadenectomy and exogenous EGF on taste bud morphology and maintenance.

    PubMed

    Morris-Wiman, J; Sego, R; Brinkley, L; Dolce, C

    2000-02-01

    Taste buds on the dorsal tongue surface are continually bathed in saliva rich in epidermal growth factor (EGF). In the following experiment, taste bud number and morphology were monitored following submandibular and sublingual salivary gland removal (sialoadenectomy), to determine if EGF plays a role in the maintenance and formation of taste buds. Adult male rats were divided into four groups: sialoadenectomized (SX, n = 4); sialoadenectomized with EGF replacement (SX + EGF, n = 5); sham-operated (SH, n = 4); and sham-operated with exogenous EGF (SH + EGF, n = 5). After a 3 week recovery, SX + EGF and SH + EGF animals were given 50 microg/day EGF in their drinking water for 14 days. At day 14, saliva was collected, the animals were killed and the presence of EGF determined by radioligand-binding assay. Tongues were removed and histologically examined for the presence and morphology of taste buds on fungiform and circumvallate papillae, or immunostained for the presence of EGF, TGFalpha (transforming growth factor alpha) and EGFR (EGF receptor). The removal of submandibular and sublingual salivary glands resulted in the loss of fungiform taste buds and normal fungiform papillae morphology. These effects were reversed by EGF supplementation, indicating a role for EGF in fungiform taste bud maintenance. In addition, supplementation of EGF to sham-operated animals increased the size of fungiform taste buds. In contrast, removal of salivary glands had no effect on the size, numbers, or morphology of circumvallate taste buds, suggesting that the formation and maintenance of taste buds in fungiform and circumvallate papillae may involve different and distinct processes. EGF, TGFalpha and EGFR were localized to distinct layers of the dorsal epithelium and to within both fungiform and circumvallate taste buds. Their expression within the epithelium or taste buds was not altered with sialoadenectomy, indicating that the actions of endogenous EGF and TGFalpha are distinct and

  5. Expression of human tyrosine kinase-negative epidermal growth factor receptor amplifies signaling through endogenous murine epidermal growth factor receptor.

    PubMed

    Hack, N; Sue-A-Quan, A; Mills, G B; Skorecki, K L

    1993-12-15

    Recent findings have suggested that certain ligand-dependent responses to EGF may be propagated in a manner that is not dependent on the intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGF-R, Campos-Gonzalez, R., and Glenney, J. R., Jr. (1992) J. Biol. Chem. 267, 14535-14538) or, alternatively, that these responses may occur through the interaction of the human tyrosine kinase-deficient EGF-R with an as yet unidentified kinase (Selva, E., Raden, D. L., and Davis, R. J. (1993) J. Biol. Chem. 268, 2250-2254). These conclusions represent a significant departure from our current understanding of signal transduction by receptor tyrosine kinases. Therefore we examined the effect of expression of tyrosine kinase-negative human EGF receptor in murine NIH-3T3-2.2 cells on the EGF-dependent phosphorylation of mitogen-activated protein (MAP-2) kinase. In parental cells (NIH-3T3-2.2) that express low levels of endogenous murine EGF-R, there was no demonstrable EGF-dependent coupling to MAP-2 kinase. In NIH-3T3-2.2 cells transfected with tyrosine kinase-negative human EGF-R, there was unexpected EGF-dependent phosphorylation of MAP-2 kinase. Analysis of the tyrosine kinase-negative human EGF-R in these cells revealed significant tyrosine phosphorylation of the EGF-R. A low level of endogenous murine EGF-R present in these cells were also phosphorylated on tyrosine residues and displayed autokinase activity. Similar results were obtained using an unrelated cell line (B82L cells), in which EGF-dependent phosphorylation of MAP-2 kinase was previously attributed to signal propagation through a tyrosine kinase-negative human EGF-R (Campos-Gonzalez, R., and Glenney, J. R., Jr. (1992) J. Biol. Chem. 267, 14535-14538). Taken together, these results suggest that the tyrosine kinase-negative human EGF-R are able to amplify the response to activation of low levels of endogenous murine EGF-R, thus leading to EGF-dependent phosphorylation of MAP-2 kinase in cells

  6. Shh/Ptch and EGF/ErbB cooperatively regulate branching morphogenesis of fetal mouse submandibular glands.

    PubMed

    Mizukoshi, Kenji; Koyama, Noriko; Hayashi, Toru; Zheng, Liguang; Matsuura, Sachiko; Kashimata, Masanori

    2016-04-15

    The hedgehog family includes Sonic hedgehog (Shh), Desert hedgehog, and Indian hedgehog, which are well known as a morphogens that play many important roles during development of numerous organs such as the tongue, pancreas, kidney, cartilage, teeth and salivary glands (SMG). In Shh null mice, abnormal development of the salivary gland is seen after embryonic day 14 (E14). Shh also induced lobule formation and lumen formation in acini-like structures in cultured E14 SMG. In this study, we investigated the relationship between Shh and epidermal growth factor (EGF)/ErbB signaling in developing fetal mouse SMG. Administration of Shh to cultured E13 SMG stimulated branching morphogenesis (BrM) and induced synthesis of mRNAs for EGF ligands and receptors of the ErbB family. Shh also stimulated activation of ErbB signaling system such as ERK1/2. AG1478, a specific inhibitor of ErbB receptors, completely suppressed BrM and activation of EGF/ErbB/ERK1/2 cascade in E13 SMGs cultured with Shh. The expressions of mRNA for Egf in mesenchyme and mRNA for Erbb1, Erbb2 and Erbb3 in epithelium of E13 SMG were specifically induced by administration of Shh. These results show that Shh stimulates BrM of fetal mouse SMG, at least in part, through activation of the EGF/ErbB/ERK1/2 signaling system. PMID:26930157

  7. Lung adenocarcinomas induced in mice by mutant EGF receptors found in human lung cancers respond to a tyrosine kinase inhibitor or to down-regulation of the receptors.

    PubMed

    Politi, Katerina; Zakowski, Maureen F; Fan, Pang-Dian; Schonfeld, Emily A; Pao, William; Varmus, Harold E

    2006-06-01

    Somatic mutations in exons encoding the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene are found in human lung adenocarcinomas and are associated with sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib. Nearly 90% of the EGFR mutations are either short, in-frame deletions in exon 19 or point mutations that result in substitution of arginine for leucine at amino acid 858 (L858R). To study further the role of these mutations in the initiation and maintenance of lung cancer, we have developed transgenic mice that express an exon 19 deletion mutant (EGFR(DeltaL747-S752)) or the L858R mutant (EGFR(L858R)) in type II pneumocytes under the control of doxycycline. Expression of either EGFR mutant leads to the development of lung adenocarcinomas. Two weeks after induction with doxycycline, mice that express the EGFR(L858R) allele show diffuse lung cancer highly reminiscent of human bronchioloalveolar carcinoma and later develop interspersed multifocal adenocarcinomas. In contrast, mice expressing EGFR(DeltaL747-S752) develop multifocal tumors embedded in normal lung parenchyma with a longer latency. With mice carrying either EGFR allele, withdrawal of doxycycline (to reduce expression of the transgene) or treatment with erlotinib (to inhibit kinase activity) causes rapid tumor regression, as assessed by magnetic resonance imaging and histopathology, demonstrating that mutant EGFR is required for tumor maintenance. These models may be useful for developing improved therapies for patients with lung cancers bearing EGFR mutations.

  8. Nanoconjugation prolongs endosomal signaling of the epidermal growth factor receptor and enhances apoptosis

    NASA Astrophysics Data System (ADS)

    Wu, L.; Xu, F.; Reinhard, B. M.

    2016-07-01

    It is becoming increasingly clear that intracellular signaling can be subject to strict spatial control. As the covalent attachment of a signaling ligand to a nanoparticle (NP) impacts ligand-receptor binding, uptake, and trafficking, nanoconjugation provides new opportunities for manipulating intracellular signaling in a controlled fashion. To establish the effect of nanoconjugation on epidermal growth factor (EGF) mediated signaling, we investigate here the intracellular fate of nanoconjugated EGF (NP-EGF) and its bound receptor (EGFR) by quantitative correlated darkfield/fluorescence microscopy and density-based endosomal fractionation. We demonstrate that nanoconjugation prolongs the dwell time of phosphorylated receptors in the early endosomes and that the retention of activated EGFR in the early endosomes is accompanied by an EGF mediated apoptosis at effective concentrations that do not induce apoptosis in the case of free EGF. Overall, these findings indicate nanoconjugation as a rational strategy for modifying signaling that acts by modulating the temporo-spatial distribution of the activated EGF-EGFR ligand-receptor complex.It is becoming increasingly clear that intracellular signaling can be subject to strict spatial control. As the covalent attachment of a signaling ligand to a nanoparticle (NP) impacts ligand-receptor binding, uptake, and trafficking, nanoconjugation provides new opportunities for manipulating intracellular signaling in a controlled fashion. To establish the effect of nanoconjugation on epidermal growth factor (EGF) mediated signaling, we investigate here the intracellular fate of nanoconjugated EGF (NP-EGF) and its bound receptor (EGFR) by quantitative correlated darkfield/fluorescence microscopy and density-based endosomal fractionation. We demonstrate that nanoconjugation prolongs the dwell time of phosphorylated receptors in the early endosomes and that the retention of activated EGFR in the early endosomes is accompanied by an EGF

  9. Disconnecting the Yin and Yang Relation of Epidermal Growth Factor Receptor (EGFR)-Mediated Delivery: A Fully Synthetic, EGFR-Targeted Gene Transfer System Avoiding Receptor Activation

    PubMed Central

    Schäfer, A.; Pahnke, A.; Schaffert, D.; van Weerden, W.M.; de Ridder, C.M.A.; Rödl, W.; Vetter, A.; Spitzweg, C.; Kraaij, R.; Wagner, E.

    2011-01-01

    Abstract The epidermal growth factor receptor (EGFR) is upregulated within a high percentage of solid tumors and hence is an attractive target for tumor-targeted therapies including gene therapy. The natural EGFR ligand epidermal growth factor (EGF) has been used for this purpose, despite the risk of mitogenic effects due to EGFR activation. We have developed a fully synthetic, EGFR-targeted gene delivery system based on PEGylated linear polyethylenimine (LPEI), allowing evaluation of different EGFR-binding peptides in terms of transfection efficiency and EGFR activation. Peptide sequences directly derived from the human EGF molecule enhanced transfection efficiency with concomitant EGFR activation. Only the EGFR-binding peptide GE11, which has been identified by phage display technique, showed specific enhancement of transfection on EGFR-overexpressing tumor cells including glioblastoma and hepatoma, but without EGFR activation. EGFR targeting led to high levels of cell association of fluorescently labeled polyplexes after only 30 min of incubation. EGF pretreatment of cells induced enhanced cellular internalization of all polyplex types tested, pointing at generally enhanced macropinocytosis. EGF polyplexes diminished cell surface expression of EGFR for up to 4 hr, whereas GE11 polyplexes did not. In a clinically relevant orthotopic prostate cancer model, intratumorally injected GE11 polyplexes were superior in inducing transgene expression when compared with untargeted polyplexes. PMID:21644815

  10. Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation

    PubMed Central

    1995-01-01

    The aggregation states of the epidermal growth factor receptor (EGFR) on single A431 human epidermoid carcinoma cells were assessed with two new techniques for determining fluorescence resonance energy transfer: donor photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence lifetime imaging microscopy (FLIM). Fluorescein-(donor) and rhodamine-(acceptor) labeled EGF were bound to the cells and the extent of oligomerization was monitored by the spatially resolved FRET efficiency as a function of the donor/acceptor ratio and treatment conditions. An average FRET efficiency of 5% was determined after a low temperature (4 degrees C) incubation with the fluorescent EGF analogs for 40 min. A subsequent elevation of the temperature for 5 min caused a substantial increase of the average FRET efficiency to 14% at 20 degrees C and 31% at 37 degrees C. In the context of a two-state (monomer/dimer) model for the EGFR, these FRET efficiencies were consistent with minimal average receptor dimerizations of 13, 36, and 69% at 4, 20, and 37 degrees C, respectively. A431 cells were pretreated with the monoclonal antibody mAb 2E9 that specifically blocks EGF binding to the predominant population of low affinity EGFR (15). The average FRET efficiency increased dramatically to 28% at 4 degrees C, indicative of a minimal receptor dimerization of 65% for the subpopulation of high affinity receptors. These results are in accordance with prior studies indicating that binding of EGF leads to a fast and temperature- dependent microclustering of EGFR, but suggest in addition that the high affinity functional subclass of receptors on quiescent A431 cells are present in a predimerized or oligomerized state. We propose that the transmission of the external ligand-binding signal to the cytoplasmic domain is effected by a concerted relative rotational rearrangement of the monomeric units comprising the dimeric receptor, thereby potentiating a mutual activation of

  11. A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: Activation of proteinase-activated receptor 1 and epidermal growth factor receptor

    SciTech Connect

    Gao, Lin; Chao, Lee; Chao, Julie

    2010-02-01

    Biological functions of tissue kallikrein (TK, KLK1) are mainly mediated by kinin generation and subsequent kinin B2 receptor activation. In this study, we investigated the potential role of TK and its signaling pathways in cultured human keratinocyte migration and in a rat skin wound healing model. Herein, we show that TK promoted cell migration and proliferation in a concentration- and time-dependent manner. Inactive TK or kinin had no significant effect on cell migration. Interestingly, cell migration induced by active TK was not blocked by icatibant or L-NAME, indicating an event independent of kinin B2 receptor and nitric oxide formation. TK's stimulatory effect on cell migration was inhibited by small interfering RNA for proteinase-activated receptor 1 (PAR{sub 1}), and by PAR{sub 1} inhibitor. TK-induced migration was associated with increased phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), which was blocked by inhibition of protein kinase C (PKC), Src, EGFR and ERK. TK-induced cell migration and EGFR phosphorylation were blocked by metalloproteinase (MMP) inhibitor, heparin, and antibodies against EGFR external domain, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR). Local application of TK promoted skin wound healing in rats, whereas icatibant and EGFR inhibitor blocked TK's effect. Skin wound healing was further delayed by aprotinin and neutralizing TK antibody. This study demonstrates a novel role of TK in skin wound healing and uncovers new signaling pathways mediated by TK in promoting keratinocyte migration through activation of the PAR{sub 1}-PKC-Src-MMP pathway and HB-EGF/AR shedding-dependent EGFR transactivation.

  12. Immunohistochemical Characterization of Three Monoclonal Antibodies Raised against the Epidermal Growth Factor and Its Receptor in Non-Small-Cell Lung Cancer: Their Potential Use in the Selection of Patients for Immunotherapy

    PubMed Central

    Rengifo, Charles E.; Blanco, Rancés; Blanco, Damián; Cedeño, Mercedes; Frómeta, Milagros; Calzado, Enrique Rengifo

    2013-01-01

    Adequate methods to identify which lung cancer patients are most likely to benefit from the targeted drugs against both epidermal growth factor receptor/epidermal growth factor (EGFR/EGF) are needed. For this reason, we evaluated both the tissue reactivity of ior egf/r3 monoclonal antibody (Mab) in human lung carcinomas and its biological activity in NCI-H125 cells. Additionally, we assessed the tissue expression of EGF using two Mabs, CB-EGF1 and CB-EGF2. The overexpression of EGFR was detected in 33.33% and 62.71% of small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC), respectively. The ability of ior egf/r3 Mab to bind the extracellular domain of EGFR inhibiting cell proliferation and inducing apoptosis in NCI-H125 cells was also demonstrated. The EGF expression was observed in about 17% and 70% of SCLC and NSCLC, respectively. However, differences in the reactivity of CB-EGF1 and CB-EGF2 were evidenced. A dual expression of EGFR and EGF was observed in 16.67% and 57.63% of SCLC and NSCLC patients, respectively. But, a correlation between them was only obtained in NSCLC. Our results permit to recommend the development of diagnostic kits using ior egf/r3 and/or CB-EGF1 Mabs in order to achieve a better selection of patients to EGFR/EGF-targeting treatment. PMID:26317020

  13. Immunohistochemical Characterization of Three Monoclonal Antibodies Raised against the Epidermal Growth Factor and Its Receptor in Non-Small-Cell Lung Cancer: Their Potential Use in the Selection of Patients for Immunotherapy.

    PubMed

    Rengifo, Charles E; Blanco, Rancés; Blanco, Damián; Cedeño, Mercedes; Frómeta, Milagros; Calzado, Enrique Rengifo

    2013-01-01

    Adequate methods to identify which lung cancer patients are most likely to benefit from the targeted drugs against both epidermal growth factor receptor/epidermal growth factor (EGFR/EGF) are needed. For this reason, we evaluated both the tissue reactivity of ior egf/r3 monoclonal antibody (Mab) in human lung carcinomas and its biological activity in NCI-H125 cells. Additionally, we assessed the tissue expression of EGF using two Mabs, CB-EGF1 and CB-EGF2. The overexpression of EGFR was detected in 33.33% and 62.71% of small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC), respectively. The ability of ior egf/r3 Mab to bind the extracellular domain of EGFR inhibiting cell proliferation and inducing apoptosis in NCI-H125 cells was also demonstrated. The EGF expression was observed in about 17% and 70% of SCLC and NSCLC, respectively. However, differences in the reactivity of CB-EGF1 and CB-EGF2 were evidenced. A dual expression of EGFR and EGF was observed in 16.67% and 57.63% of SCLC and NSCLC patients, respectively. But, a correlation between them was only obtained in NSCLC. Our results permit to recommend the development of diagnostic kits using ior egf/r3 and/or CB-EGF1 Mabs in order to achieve a better selection of patients to EGFR/EGF-targeting treatment.

  14. MAPK interacts with occludin and mediates EGF-induced prevention of tight junction disruption by hydrogen peroxide.

    PubMed

    Basuroy, Shyamali; Seth, Ankur; Elias, Bertha; Naren, Anjaparavanda P; Rao, Radhakrishna

    2006-01-01

    The MAPK (mitogen-activated protein kinase) pathway is a major intracellular signalling pathway involved in EGF (epithelial growth factor) receptor-mediated cell growth and differentiation. A novel function of MAPK activity in the mechanism of EGF-mediated protection of TJs (tight junctions) from H2O2 was examined in Caco-2 cell monolayers. EGF-mediated prevention of H2O2-induced increase in paracellular permeability was associated with the prevention of H2O2-induced Tyr-phosphorylation, Thr-dephosphorylation and cellular redistribution of occludin and ZO-1 (zonula occludin-1). EGF also prevented H2O2-induced disruption of the actin cytoskeleton and the dissociation of occludin and ZO-1 from the actin-rich detergent-insoluble fractions. MEK (MAPK/ERK kinase, where ERK stands for extracellular signal related kinase) inhibitors, PD98059 and U0126, completely blocked these protective effects of EGF on TJs. EGF rapidly increased the levels of phosphorylated MEK (p-MEK) in detergent-soluble fractions and phosphorylated ERK (p-ERK) in detergent-insoluble fractions. p-ERK was colocalized and co-immunoprecipitated with occludin. GST (glutathione S-transferase) pull-down assay showed that the C-terminal tail of occludin binds to p-ERK in Caco-2 cell extracts. Pair-wise binding studies using recombinant proteins demonstrated that ERK1 directly interacts with the C-terminal tail of occludin. Therefore the present study shows that ERK interacts with the C-terminal region of occludin and mediates the prevention of H2O2-induced disruption of TJs by EGF. PMID:16134968

  15. Insulin induces a transcriptional activation of epiregulin, HB-EGF and amphiregulin, by a PI3K-dependent mechanism: Identification of a specific insulin-responsive promoter element

    SciTech Connect

    Ornskov, Dorthe; Nexo, Ebba; Sorensen, Boe S. . E-mail: boess@as.aaa.dk

    2007-03-23

    Previously we have shown that insulin-stimulation of RT4 bladder cancer cells leads to increased proliferation, which require HER1 activation, and is accompanied by increased mRNA expression of the EGF-ligands heparin-binding EGF-like growth factor (HB-EGF), amphiregulin (AR), and epiregulin (EPI) [D. Ornskov, E. Nexo, B.S. Sorensen, Insulin-induced proliferation of bladder cancer cells is mediated through activation of the epidermal growth factor system, FEBS J. 273 (2006) 5479-5489]. In the present paper, we have investigated the molecular mechanism leading to this insulin-induced expression. We monitored the decay of mRNA after inhibiting transcription with Actinomycin D and demonstrated that the insulin-mediated increase was not caused by enhanced mRNA stability. In untreated cells, HB-EGF mRNA was the least stable, whereas AR and EPI mRNA decayed with slower kinetics. However, promoter analysis of HB-EGF and EPI demonstrated that insulin stimulated transcription. Studies on the EPI promoter identified the insulin-responsive element to be located in the region -564 to -365 bp. This region contains potential binding sites for the transcription factors SP1, AP1, and NF-{kappa}B. Interestingly, all three transcription factors can be activated by PI3K. We demonstrate that the insulin-induced expression of HB-EGF, AR, and EPI mRNA is completely prevented by the specific PI3K inhibitor Wortmannin, suggesting an involvement of the PI3K.

  16. NHERF-1 regulation of EGF and neurotensin signalling in HT-29 epithelial cells

    SciTech Connect

    Kruger, Wade A.; Monteith, Gregory R.; Poronnik, Philip

    2013-03-22

    Highlights: ► NHERF-1 expression was abundant throughout HT-29 cells consistent with a cancerous phenotype. ► Knockdown of NHERF-1 lead to a significant reduction in cell proliferation. ► EGF and neurotensin-mediated proliferation was inhibited by knockdown of NHERF-1. ► Neurotensin-mediated Ca{sup 2+} response was abolished by knockdown of NHERF-1. -- Abstract: Neurotensin receptors (NT-R) and the epidermal growth factor receptors (EGF-R) are commonly overexpressed in many epithelial origin tumours. In addition to their role as mitogenic mediators through specific cell signalling, recent studies indicate that the activity/expression of scaffold proteins responsible for the assembly and coordination of the signalling complexes may also have central roles in epithelial transformation. In particular, the “epithelial” PSD-95/Dlg/Zo-1 (PDZ) scaffold/adapter protein, Na{sup +}/H{sup +} exchanger regulatory factor isoform one (NHERF-1), has been identified as a potential regulator of cellular transformation. NHERF-1 is a known regulator of EGF-R function and plays numerous roles in G-protein-coupled receptor signalling. Because of the synergistic signalling between these two potent mitogens, we investigated a potential role for NHERF-1 in the molecular mechanism linking the aberrant proliferative phenotype initiated by some G-Protein-coupled receptor activators in the colon adenocarcinoma HT-29 cell line. Knockdown (80%) of endogenous NHERF-1 leads to significant reduction in proliferation rate; an effect that could not be recovered by exogenous application of either NT or EGF. Inhibition of the EGF-R with AG1487 also inhibited proliferation and this effect could not be recovered with NT. Knockdown of NHERF-1 significantly altered the expression of the EGF-R, and almost completely abolished the NT-mediated increases in intracellular free Ca{sup 2+}. Knockdown of NHERF-1 also attenuated UTP-mediated purinergic Ca{sup 2+} signalling. Taken together, these data

  17. Epidermal growth factor (EGF) as a potential targeting agent for delivery of boron to malignant gliomas

    SciTech Connect

    Capala, J.; Barth, R.F.; Adams, D.M.; Bailey, M.Q.; Soloway, A.H.; Carlsson, J.

    1994-12-31

    The majority of high grade gliomas express an amplified epidermal growth factor receptor (EGFR) gene, and this often is associated with an increase in cell surface receptor expression. The rapid internalization and degradation of EGF-EGFR complexes, as well as their high affinity make EGF a potential targeting agent for delivery of {sup 10}B to tumor cells with an amplified number of EGFR. Human glioma cells can expresses as many as 10{sup 5} {minus}10{sup 6} EGF receptors per cell, and if these could be saturated with boronated EGF, then > 10{sup 8} boron atoms would be delivered per cell. Since EGF has a comparatively low molecular weight ({approximately} 6 kD), this has allowed us to construct relatively small bioconjugates containing {approximately} 900 boron atoms per EGF molecule{sup 3}, which also had high affinity for EGFR on tumor cells. In the present study, the feasibility of using EGF receptors as a potential target for therapy of gliomas was investigated by in vivo scintigraphic studies using {sup 131}I{minus} or {sup 99m}{Tc}-labeled EGF in a rat brain tumor model. Our results indicate that intratumorally delivered boron- EGF conjugates might be useful for targeting EGFR on glioma cells if the boron containing moiety of the conjugates persisted intracellularly. Further studies are required, however, to determine if this approach can be used for BNCT of the rat glioma.

  18. Platelet-activating factor induces ovine fetal pulmonary venous smooth muscle cell proliferation: role of epidermal growth factor receptor transactivation.

    PubMed

    Zhou, Weilin; Ibe, Basil O; Raj, J Usha

    2007-06-01

    We have previously reported that platelet-activating factor (PAF) is present in very high levels in the ovine fetal lung and circulation and that PAF serves as an important physiological vasoconstrictor of the pulmonary circulation in utero. However, it is not known whether PAF stimulates pulmonary vascular smooth muscle cell (SMC) proliferation. In this study, we used ovine fetal pulmonary venous SMCs as our model system to study the effects and mechanisms of action of PAF on SMC proliferation. We found that PAF induced SMC proliferation in a dose-dependent manner. PAF also stimulated activation of both ERK and p38 but not c-Jun NH(2) terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. PAF (10 nM) induced phosphorylation of epidermal growth factor receptor (EGFR). Specific inhibition of EGFR by AG-1478 and by the expression of a dominant-negative EGFR mutant in SMCs attenuated PAF-stimulated cell proliferation. Inhibition of heparin-binding EGF-like growth factor (HB-EGF) release by CRM-197 and inhibition of matrix metalloproteinases (MMP) by GM-6001 abolished PAF-induced MAP kinase activation and cell proliferation. Increased alkaline phosphatase (AP) activity after PAF treatment in AP-HB-EGF fusion construct-transfected SMCs indicated that PAF induced the release of HB-EGF within 1 min. Gelatin zymography data showed that PAF stimulated MMP-2 activity and MMP-9 activity within 1 min. These results suggest that PAF promotes pulmonary vascular SMC proliferation via transactivation of EGFR through MMP activation and HB-EGF, resulting in p38 and ERK activation and that EGFR transactivation is essential for the mitogenic effect of PAF in pulmonary venous SMC. PMID:17322418

  19. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1.

    PubMed

    Kim, Chloe S; Mitchell, Isaiah P; Desotell, Anthony W; Kreeger, Pamela K; Masters, Kristyn S

    2016-07-01

    Epidermal growth factor (EGF) is a critical element in dermal repair, but EGF-containing wound dressings have not been successful clinically. However, these dressings have delivered only soluble EGF, and the native environment provides both soluble and matrix-bound EGF. To address our hypothesis that tethered EGF can stimulate cell behaviors not achievable with soluble EGF, we examined single-cell movement and signaling in human immortalized HaCaT keratinocytes treated with soluble or immobilized EGF. Although both EGF treatments increased collective sheet displacement and individual cell speed, only cells treated with immobilized EGF exhibited directed migration, as well as 2-fold greater persistence compared with soluble EGF. Immunofluorescence showed altered EGF receptor (EGFR) trafficking, where EGFR remained membrane-localized in the immobilized EGF condition. Cells treated with soluble EGF demonstrated higher phosphorylated ERK1/2, and cells on immobilized EGF exhibited higher pPLCγ1, which was localized at the leading edge. Treatment with U0126 inhibited migration in both conditions, demonstrating that ERK1/2 activity was necessary but not responsible for the observed differences. In contrast, PLCγ1 inhibition with U73122 significantly decreased persistence on immobilized EGF. Combined, these results suggest that immobilized EGF increases collective keratinocyte displacement via an increase in single-cell migration persistence resulting from altered EGFR trafficking and PLCγ1 activation.-Kim, C. S., Mitchell, I. P., Desotell, A. W., Kreeger, P. K., Masters, K. S. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1.

  20. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1.

    PubMed

    Kim, Chloe S; Mitchell, Isaiah P; Desotell, Anthony W; Kreeger, Pamela K; Masters, Kristyn S

    2016-07-01

    Epidermal growth factor (EGF) is a critical element in dermal repair, but EGF-containing wound dressings have not been successful clinically. However, these dressings have delivered only soluble EGF, and the native environment provides both soluble and matrix-bound EGF. To address our hypothesis that tethered EGF can stimulate cell behaviors not achievable with soluble EGF, we examined single-cell movement and signaling in human immortalized HaCaT keratinocytes treated with soluble or immobilized EGF. Although both EGF treatments increased collective sheet displacement and individual cell speed, only cells treated with immobilized EGF exhibited directed migration, as well as 2-fold greater persistence compared with soluble EGF. Immunofluorescence showed altered EGF receptor (EGFR) trafficking, where EGFR remained membrane-localized in the immobilized EGF condition. Cells treated with soluble EGF demonstrated higher phosphorylated ERK1/2, and cells on immobilized EGF exhibited higher pPLCγ1, which was localized at the leading edge. Treatment with U0126 inhibited migration in both conditions, demonstrating that ERK1/2 activity was necessary but not responsible for the observed differences. In contrast, PLCγ1 inhibition with U73122 significantly decreased persistence on immobilized EGF. Combined, these results suggest that immobilized EGF increases collective keratinocyte displacement via an increase in single-cell migration persistence resulting from altered EGFR trafficking and PLCγ1 activation.-Kim, C. S., Mitchell, I. P., Desotell, A. W., Kreeger, P. K., Masters, K. S. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1. PMID:27025961

  1. Hepatocyte uptake and nuclear binding of epidermal growth factor (EGF)

    SciTech Connect

    Moriarity, D.M.; Underwood, T.

    1987-05-01

    The internalization of /sup 125/I-EGF and its cell-membrane receptor by target cells suggests a possible intracellular role for EGF and/or its receptor. They have examined the uptake of /sup 125/I-EGF by primary cultures of adult rat hepatocytes after 1, 24 and 48 hours of incubation in the presence of the growth factor. A significant increase in the association of radioactivity with various nuclear fractions was observed between 1 and 24 hours incubation. After 1 hour approximately 2% of the total specific binding was associated with both the nuclear sap proteins extractable with 0.14 M NaCl and with the residual nucleoplasm, while about 1% or less was associated with the nuclear membrane and the chromatin fractions. After 24 hours the percentage associated with the nuclear membrane and chromatin fractions increased 2-4 fold. Binding of /sup 125/I-EGF to isolated nuclei from intact livers of adult rats followed by fractionation of the nuclei after incubation with /sup 125/I-EGF indicated that after 60 min at 37/sup 0/C there was a substantial amount of specific binding associated with the nucleoplasm, nuclear membranes and chromatin fractions. These data indicate that specific interactions of EGF with nuclear components occur in both intact normal hepatocytes and in isolated nuclei from intact liver.

  2. Androgen Receptor Expression in Prostate Cancer Cells Is Suppressed by Activation of Epidermal Growth Factor Receptor and ErbB2

    PubMed Central

    Cai, Changmeng; Portnoy, David C.; Wang, Hongyun; Jiang, Xinnong; Chen, Shaoyong; Balk, Steven P.

    2016-01-01

    Prostate cancers (PCa) that relapse after androgen deprivation therapies [castration-resistant PCa (CRPC)] express high levels of androgen receptor (AR) and androgen-regulated genes, and evidence from several groups indicates that ErbB family receptor tyrosine kinases [epidermal growth factor (EGF) receptor (EGFR) and ErbB2] may contribute to enhancing this AR activity. We found that activation of these kinases with EGF and heregulin-β1 rapidly (within 8 hours) decreased expression of endogenous AR and androgen-regulated PSA in LNCaP PCa cells. AR expression was similarly decreased in LAPC4 and C4-2 cells, but not in the CWR22Rv1 PCa cell line. The rapid decrease in AR was not due to increased AR protein degradation and was not blocked by phosphatidylinositol 3-kinase (LY294002) or MEK (UO126) inhibitors. Significantly, AR mRNA levels in LNCaP cells were markedly decreased by EGF and heregulin-β1, and experiments with actinomycin D to block new mRNA synthesis showed that AR mRNA degradation was increased. AR mRNA levels were still markedly decreased by EGF and heregulin-β1 in LNCaP cells adapted to growth in androgen-depleted medium, although AR protein levels did not decline due to increased AR protein stability. These findings show that EGFR and ErbB2 can negatively regulate AR mRNA and may provide an approach to suppress AR expression in CRPC. PMID:19491261

  3. Pharmacological effects of epidermal growth factor (EGF) with focus on the urinary and gastrointestinal tracts.

    PubMed

    Vinter-Jensen, L

    1999-01-01

    Epidermal growth factor (EGF) belongs to a family of growth factor ligands and receptors. At present, five ligands have been recognized which as EGF exert their effects via binding to the same EGF receptor. The family has three other receptors erbB2, erbB3, and erbB4, which have their own ligands (the heregulins). The system is ubiquitously distributed in mammals, and has important roles in normal development, and in regenerative and neoplastic growth. Mouse and human EGF were discovered in 1962 and 1975 by Stanley Cohen and Harry Gregory, respectively, due to EGFs potent systemic effects. EGF accelerated eyelid opening in newborn mice and inhibited gastric acid secretion in humans. Already in the late thirties, a factor in human urine was recognized which prevented or accelerated healing of experimental damage in the gastrointestinal tract. This factor appeared to be EGF. Around 1980, an effect of commercial interest was described-EGF caused shedding of the fleece in sheep. In line with the original observations, several studies have examined effects of EGF on developmental processes. Amongst other effects, EGF accelerates lung and intestinal maturation before birth and in newborn mammals. Due to the possible use of EGF in the wool industry, it was mandatory to know more about EGF. Amongst other effects in mature sheep and other animals are haemodynamic changes, changes in electrolyte homeostasis, and endocrinological changes. In relation to experimental damage, the therapeutic potential of systemic EGF has been demonstrated in all parts of the gastrointestinal tract, in the kidneys, in the liver and in the trachea. EGF has even been tried in humans in gastric ulcer healing and in necrotising enterocolitis. Studies on prolonged treatment with EGF have first recently appeared. We described effects of 4-5 weeks of treatment in Goettingen minipigs and in rats, and two other groups described effects in monkeys and in rats. In summary, species differences were observed

  4. Wnt and EGF pathways act together to induce C. elegans male hook development

    PubMed Central

    Yu, Hui; Seah, Adeline; Herman, Michael A.; Ferguson, Edwin L.; Horvitz, H. Robert; Sternberg, Paul W.

    2009-01-01

    Comparative studies of vulva development between Caenorhabditis elegans and other nematode species have provided some insight into the evolution of patterning networks. However, molecular genetic details are available only in C. elegans and Pristionchus pacificus. To extend our knowledge on the evolution of patterning networks, we studied the C. elegans male hook competence group (HCG), an equivalence group that has similar developmental origins to the vulval precursor cells (VPCs), which generate the vulva in the hermaphrodite. Similar to VPC fate specification, each HCG cell adopts one of three fates (1°, 2°, 3°), and 2°HCG fate specification is mediated by LIN-12/Notch. We show that 2° HCG specification depends on the presence of a cell with the 1° fate. We also provide evidence that Wnt signaling via the Frizzled-like Wnt receptor LIN-17 act to specify the 1° and 2° HCG fate. A requirement for EGF signaling during 1° fate specification is seen only when LIN-17 activity is compromised. In addition, activation of the EGF pathway decreases dependence on LIN-17 and causes ectopic hook development. Our results suggest that WNT plays a more significant role than EGF signaling in specifying HCG fates, whereas in VPC specification EGF signaling is the major inductive signal. Nonetheless, the overall logic is similar in the VPCs and the HCG: EGF and/or WNT induce a 1° lineage, and LIN-12/NOTCH induces a 2° lineage. Wnt signaling is also required for execution of the 1° and 2°HCG lineages. lin-17 and bar-1/β-catenin are preferentially expressed in the presumptive 1° cell P11.p. The dynamic subcellular localization of BAR-1–GFP in P11.p is concordant with the timing of HCG fate determination. PMID:19154732

  5. Expression of epidermal growth factor receptor in the preimplantation uterus and blastocyst of the western spotted skunk.

    PubMed

    Paria, B C; Das, S K; Mead, R A; Dey, S K

    1994-08-01

    The western spotted skunk is unique in that its blastocysts undergo a 180-220-day period of arrested development before implantation. We investigated the potential role of epidermal growth factor (EGF)-related growth factors in regulating uterine and embryonic development in this species by studying the status of EGF receptor (EGF-R) in these tissues during delayed implantation and resumption of embryonic development. The cell-specific distribution of EGF binding sites and the expression of EGF-R mRNA were assayed by autoradiography and Northern blot analysis, respectively. The size of EGF-R was determined by affinity cross-linking studies, and its bioactivity was examined by determining EGF-dependent subcellular protein tyrosine kinase (PTK) activity. EGF binding sites were localized in the uterine luminal and glandular epithelium, endometrial stroma, myometrium, and blood vessels during both stages of pregnancy. As examined by Northern blot hybridization, a cRNA probe specific to mouse EGF-R hybridized to poly(A)+ RNA of skunk uteri. Transcripts similar to those of mouse uterine EGF-R were identified. [125I]-EGF was cross-linked to a 170-kDa protein both in the uterus and in blastocysts collected during the delayed implantation and periimplantation periods. However, EGF-induced PTK activity was significantly elevated above background levels during the period of renewed embryonic development, but not during arrested embryonic development. The results suggest that EGF-related growth factors may play an important role in regulating embryonic development in this species and that a change in the number and/or functional status of the EGF-R may be a prerequisite for blastocyst activation and implantation in the spotted skunk.

  6. Molecular cloning and expression of an additional epidermal growth factor receptor-related gene.

    PubMed Central

    Plowman, G D; Whitney, G S; Neubauer, M G; Green, J M; McDonald, V L; Todaro, G J; Shoyab, M

    1990-01-01

    Epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and amphiregulin are structurally and functionally related growth regulatory proteins. These secreted polypeptides all bind to the 170-kDa cell-surface EGF receptor, activating its intrinsic kinase activity. However, amphiregulin exhibits different activities than EGF and TGF-alpha in a number of biological assays. Amphiregulin only partially competes with EGF for binding EGF receptor, and amphiregulin does not induce anchorage-independent growth of normal rat kidney cells (NRK) in the presence of TGF-beta. Amphiregulin also appears to abrogate the stimulatory effect of TGF-alpha on the growth of several aggressive epithelial carcinomas that overexpress EGF receptor. These findings suggest that amphiregulin may interact with a separate receptor in certain cell types. Here we report the cloning of another member of the human EGF receptor (HER) family of receptor tyrosine kinases, which we have named "HER3/ERRB3." The cDNA was isolated from a human carcinoma cell line, and its 6-kilobase transcript was identified in various human tissues. We have generated peptide-specific antisera that recognizes the 160-kDa HER3 protein when transiently expressed in COS cells. These reagents will allow us to determine whether HER3 binds amphiregulin or other growth regulatory proteins and what role HER3 protein plays in the regulation of cell growth. Images PMID:2164210

  7. Transcription-dependent epidermal growth factor receptor activation by hepatocyte growth factor.

    PubMed

    Reznik, Thomas E; Sang, Yingying; Ma, Yongxian; Abounader, Roger; Rosen, Eliot M; Xia, Shuli; Laterra, John

    2008-01-01

    The mechanisms and biological implications of coordinated receptor tyrosine kinase coactivation remain poorly appreciated. Epidermal growth factor receptor (EGFR) and c-Met are frequently coexpressed in cancers, including those associated with hepatocyte growth factor (HGF) overexpression, such as malignant astrocytoma. In a previous analysis of the HGF-induced transcriptome, we found that two EGFR agonists, transforming growth factor-alpha and heparin-binding epidermal growth factor-like growth factor (HB-EGF), are prominently up-regulated by HGF in human glioma cells. We now report that stimulating human glioblastoma cells with recombinant HGF induces biologically relevant EGFR activation. EGFR phosphorylation at Tyr(845) and Tyr(1068) increased 6 to 24 h after cell stimulation with HGF and temporally coincided with the induction of transforming growth factor-alpha (~5-fold) and HB-EGF (~23-fold) expression. Tyr(845) and Tyr(1068) phosphorylation, in response to HGF, was inhibited by cycloheximide and actinomycin D, consistent with a requirement for DNA transcription and RNA translation. Specifically, blocking HB-EGF binding to EGFR with the antagonist CRM197 inhibited HGF-induced EGFR phosphorylation by 60% to 80% and inhibited HGF-induced S-G(2)-M transition. CRM197 also inhibited HGF-induced anchorage-dependent cell proliferation but had no effect on HGF-mediated cytoprotection. These findings establish that EGFR can be activated with functional consequences by HGF as a result of EGFR ligand expression. This transcription-dependent cross-talk between the HGF receptor c-Met and EGFR expands our understanding of receptor tyrosine kinase signaling networks and may have considerable consequences for oncogenic mechanisms and cancer therapeutics.

  8. Smurf1 plays a role in EGF inhibition of BMP2-induced osteogenic differentiation

    SciTech Connect

    Lee, Hye-Lim; Park, Hyun-Jung; Kwon, Arang; Baek, Kyunghwa; Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik; Baek, Jeong-Hwa

    2014-05-01

    It has been demonstrated that epidermal growth factor (EGF) plays a role in supporting the proliferation of bone marrow stromal cells in bone but inhibits their osteogenic differentiation. However, the mechanism underlying EGF inhibition of osteoblast differentiation remains unclear. Smurf1 is an E3 ubiquitin ligase that targets Smad1/5 and Runx2, which are critical transcription factors for bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation. In this study, we investigated the effect of EGF on the expression of Smurf1, and the role of Smurf1 in EGF inhibition of osteogenic differentiation using C2C12 cells, a murine myoblast cell line. EGF increased Smurf1 expression, which was blocked by inhibiting the activity of either JNK or ERK. Chromatin immunoprecipitation and Smurf1 promoter assays demonstrated that c-Jun and Runx2 play roles in the EGF induction of Smurf1 transcription. EGF suppressed BMP2-induced expression of osteogenic marker genes, which were rescued by Smurf1 knockdown. EGF downregulated the protein levels of Runx2 and Smad1 in a proteasome-dependent manner. EGF decreased the transcriptional activity of Runx2 and Smurf1, which was partially rescued by Smurf1 silencing. Taken together, these results suggest that EGF increases Smurf1 expression via the activation of JNK and ERK and the subsequent binding of c-Jun and Runx2 to the Smurf1 promoter and that Smurf1 mediates the inhibitory effect of EGF on BMP2-induced osteoblast differentiation. - Highlights: • EGF increases the expression level of Smurf1 in mesenchymal precursor cells. • EGF reduces the protein levels and transcriptional activity of Runx2 and Smad1. • EGF suppresses BMP2-induced osteogenic differentiation, which is rescued by Smurf1 knockdown.

  9. EGF-induced centrosome separation promotes mitotic progression and cell survival.

    PubMed

    Mardin, Balca R; Isokane, Mayumi; Cosenza, Marco R; Krämer, Alwin; Ellenberg, Jan; Fry, Andrew M; Schiebel, Elmar

    2013-05-13

    Timely and accurate assembly of the mitotic spindle is critical for the faithful segregation of chromosomes, and centrosome separation is a key step in this process. The timing of centrosome separation varies dramatically between cell types; however, the mechanisms responsible for these differences and its significance are unclear. Here, we show that activation of epidermal growth factor receptor (EGFR) signaling determines the timing of centrosome separation. Premature separation of centrosomes decreases the requirement for the major mitotic kinesin Eg5 for spindle assembly, accelerates mitosis, and decreases the rate of chromosome missegregation. Importantly, EGF stimulation impacts upon centrosome separation and mitotic progression to different degrees in different cell lines. Cells with high EGFR levels fail to arrest in mitosis upon Eg5 inhibition. This has important implications for cancer therapy because cells with high centrosomal response to EGF are more susceptible to combinatorial inhibition of EGFR and Eg5. PMID:23643362

  10. Effect of blockade of the EGF system on wound healing in patients vaccinated with CIMAvax® EGF

    PubMed Central

    2013-01-01

    Background The epidermal growth factor receptor (EGFR) signaling system is frequently unbalanced in human malignancies due to increased ligand production, receptor overexpression, receptor mutations, and/or cross-talk with other receptor systems. For this reason, the EGFR is an attractive target for anticancer therapy. The epidermal growth factor also plays an important role in regulating multiple facets of cutaneous wound healing, including inflammation, wound contraction, proliferation, migration, and angiogenesis. In the Center of Molecular Immunology, a cancer vaccine is produced (CIMAvax® EGF) that blocks the binding of EGF to its receptor. This blockade causes a significant inverse association between the anti-EGF antibody titers and EGF concentration. Around 1,500 patients with non-small cell lung cancer have been treated, showing that this vaccine is safe, immunogenic, increases survival and improves quality of life. Taking into account the therapeutic benefits of CIMAvax® EGF vaccination and the role of EGF-EGFR system in the wound healing process, we decided to conduct a retrospective research with the aim of determining the effect to the CIMAvax® EGF vaccine on the wound healing process in patients undergoing surgical treatment. Methods Medical records of 452 vaccinated patients were reviewed and only six patients receiving surgical treatment were identified. Further information about these six patients was obtained from source documents, including medical records and operative reports using an observational list that included different variables. Post-surgical wound healing complications were identified using the National Cancer Institute Common Toxicity Criteria for Adverse Events (NCI-CTC) version 3.0. Results None of the six patients operated on presented adverse events related to the wound healing, that is to say, no wound dehiscence, wound infection, delayed wound healing, fistula formation, abscess formation or hemorrhage/bleeding associated

  11. Recombinant human epidermal growth factor precursor is a glycosylated membrane protein with biological activity.

    PubMed Central

    Mroczkowski, B; Reich, M; Chen, K; Bell, G I; Cohen, S

    1989-01-01

    NIH 3T3 cells were transfected with cDNA corresponding to human kidney prepro-epidermal growth factor (preproEGF) under control of the inducible mouse metallothionein promoter. The synthesis of recombinant human EGF precursor by these cells has provided us with a model system for analysis of the structure and activity of this precursor. In transfected cells, the precursor was present as an intrinsic 170-kilodalton membrane protein as well as a soluble protein in the extracellular medium; both forms were N glycosylated. Glycosylation of the EGF precursor was determined by (i) the direct incorporation of [3H]mannose and [3H]glucosamine, (ii) metabolic labeling in the presence or absence of glycosylation inhibitors, (iii) enzymatic cleavage of the precursor by N-glycanase or endoglycosidase II, and (iv) lectin chromatography. Recombinant human preproEGF was purified by affinity chromatography, using wheat germ lectin and antibodies to human EGF. The intact precursor was biologically active. Purified preparations of preproEGF (i) competed with 125I-labeled EGF for binding to the EGF receptor in intact fibroblast cells, (ii) activated the intrinsic tyrosine kinase activity of the EGF receptor in membrane preparations, and (iii) sustained the growth of a mouse keratinocyte cell line that is dependent on EGF for growth. These results suggest that proteolytic processing of the precursor may not be essential for its biological function. Images PMID:2789334

  12. Specificity in the synergism between retinoic acid and EGF on the growth of adult human skin fibroblasts

    SciTech Connect

    Harper, R.A. )

    1988-10-01

    Vitamin A (retinol) and five retinoids were tested for their ability to enhance epidermal growth factor (EGF) stimulation of adult human skin fibroblast growth in vitro. The retinoids utilized in this study were RO-1-5488 (all-trans-retinoic acid), RO-4-3780 (13-cis-retinoic acid), RO-10-9359, RO-10-1670, and RO-21-6583. Retinol and each retinoid were capable of stimulating fibroblast growth alone (0-86%), while 13-cis and all-trans-retinoic acid were the most potent in potentiating the EGF promotion of fibroblast growth. Since retinoic acid might enhance the EGF stimulation of cell growth by increasing either EGF receptor number or binding affinity, the binding of {sup 125}I-labeled EGF was carried out in the presence of retinoic acid and the data were subjected to a Scatchard-type analysis. No change in EGF receptor number or affinity was seen in the presence of retinoic acid. The data indicate a specific interaction between retinoid acid and EGF which results in the potentiation of the EGF-stimulated cell growth. Furthermore, the mechanism of this interaction does not seem to involve the initial binding of EGF to its plasma membrane receptor or the available number of EGF receptors located on the cell surface.

  13. Oocyte induction of EGF responsiveness in somatic cells is associated with the acquisition of porcine oocyte developmental competence.

    PubMed

    Ritter, Lesley J; Sugimura, Satoshi; Gilchrist, Robert B

    2015-06-01

    Oocytes progressively acquire the competence to support embryo development as oogenesis proceeds with ovarian folliculogenesis. The objectives of this study were to investigate oocyte-secreted factor (OSF) participation in the development of somatic cell epidermal growth factor (EGF) responsiveness associated with oocyte developmental competence. A well-established porcine model was employed using oocytes from small (<4 mm) vs medium sized (>4 mm) antral follicles, representing low vs moderate developmental competence, respectively. Cumulus-oocyte complexes (COCs) were treated in vitro with inducers of oocyte maturation, and cumulus cell functions and oocyte developmental competence were assessed. COCs from small follicles responded to FSH but, unlike COCs from larger follicles, were incapable of responding to EGF family growth factors known to mediate oocyte maturation in vivo, exhibiting perturbed cumulus expansion and expression of associated transcripts (HAS2 and TNFAIP6). Low and moderate competence COCs expressed equivalent levels of EGF receptor (EGFR) mRNA; however, the former had less total EGFR protein leading to failed activation of phospho-EGFR and phospho-ERK1/2, despite equivalent total ERK1/2 protein levels. Native OSFs from moderate, but not from low, competence oocytes established EGF responsiveness in low competence COCs. Four candidate recombinant OSFs failed to mimic the actions of native OSFs in regulating cumulus expansion. Treatment with OSFs and EGF enhanced oocyte competence but only of the low competence COCs. These data suggest that developmental acquisition by the oocyte of capacity to regulate EGF responsiveness in the oocyte's somatic cells is a major milestone in the oocyte's developmental program and contributes to coordinated oocyte and somatic cell development.

  14. EGF-dependent re-routing of vesicular recycling switches spontaneous phosphorylation suppression to EGFR signaling

    PubMed Central

    Baumdick, Martin; Brüggemann, Yannick; Schmick, Malte; Xouri, Georgia; Sabet, Ola; Davis, Lloyd; Chin, Jason W; Bastiaens, Philippe IH

    2015-01-01

    Autocatalytic activation of epidermal growth factor receptor (EGFR) coupled to dephosphorylating activity of protein tyrosine phosphatases (PTPs) ensures robust yet diverse responses to extracellular stimuli. The inevitable tradeoff of this plasticity is spontaneous receptor activation and spurious signaling. We show that a ligand-mediated switch in EGFR trafficking enables suppression of spontaneous activation while maintaining EGFR’s capacity to transduce extracellular signals. Autocatalytic phosphorylation of tyrosine 845 on unliganded EGFR monomers is suppressed by vesicular recycling through perinuclear areas with high PTP1B activity. Ligand-binding results in phosphorylation of the c-Cbl docking tyrosine and ubiquitination of the receptor. This secondary signal relies on EGF-induced EGFR self-association and switches suppressive recycling to directional trafficking. The re-routing regulates EGFR signaling response by the transit-time to late endosomes where it is switched-off by high PTP1B activity. This ubiquitin-mediated switch in EGFR trafficking is a uniquely suited solution to suppress spontaneous activation while maintaining responsiveness to EGF. DOI: http://dx.doi.org/10.7554/eLife.12223.001 PMID:26609808

  15. G protein coupling and second messenger generation are indispensable for metalloprotease-dependent, heparin-binding epidermal growth factor shedding through angiotensin II type-1 receptor.

    PubMed

    Mifune, Mizuo; Ohtsu, Haruhiko; Suzuki, Hiroyuki; Nakashima, Hidekatsu; Brailoiu, Eugen; Dun, Nae J; Frank, Gerald D; Inagami, Tadashi; Higashiyama, Shigeki; Thomas, Walter G; Eckhart, Andrea D; Dempsey, Peter J; Eguchi, Satoru

    2005-07-15

    A G protein-coupled receptor agonist, angiotensin II (AngII), induces epidermal growth factor (EGF) receptor (EGFR) transactivation possibly through metalloprotease-dependent, heparin-binding EGF (HB-EGF) shedding. Here, we have investigated signal transduction of this process by using COS7 cells expressing an AngII receptor, AT1. In these cells AngII-induced EGFR transactivation was completely inhibited by pretreatment with a selective HB-EGF inhibitor, or with a metalloprotease inhibitor. We also developed a COS7 cell line permanently expressing a HB-EGF construct tagged with alkaline phosphatase, which enabled us to measure HB-EGF shedding quantitatively. In the COS7 cell line AngII stimulated release of HB-EGF. This effect was mimicked by treatment either with a phospholipase C activator, a Ca2+ ionophore, a metalloprotease activator, or H2O2. Conversely, pretreatment with an intracellular Ca2+ antagonist or an antioxidant blocked AngII-induced HB-EGF shedding. Moreover, infection of an adenovirus encoding an inhibitor of G(q) markedly reduced EGFR transactivation and HB-EGF shedding through AT1. In this regard, AngII-stimulated HB-EGF shedding was abolished in an AT1 mutant that lacks G(q) protein coupling. However, in cells expressing AT1 mutants that retain G(q) protein coupling, AngII is still able to induce HB-EGF shedding. Finally, the AngII-induced EGFR transactivation was attenuated in COS7 cells overexpressing a catalytically inactive mutant of ADAM17. From these data we conclude that AngII stimulates a metalloprotease ADAM17-dependent HB-EGF shedding through AT1/G(q)/phospholipase C-mediated elevation of intracellular Ca2+ and reactive oxygen species production, representing a key mechanism indispensable for EGFR transactivation.

  16. JAK2 inhibitor TG101348 overcomes erlotinib-resistance in non-small cell lung carcinoma cells with mutated EGF receptor.

    PubMed

    Zhang, Fu-quan; Yang, Wen-tao; Duan, Shan-zhou; Xia, Ying-chen; Zhu, Rong-ying; Chen, Yong-bing

    2015-06-10

    Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations are responsive to EGFR-tyrosine kinase inhibitor (EGFR-TKI). However, NSCLC patients with secondary somatic EGFR mutations are resistant to EGFR-TKI treatment. In this study, we investigated the effect of TG101348 (a JAK2 inhibitor) on the tumor growth of erlotinib-resistant NSCLC cells. Cell proliferation, apoptosis, gene expression and tumor growth were evaluated by diphenyltetrazolium bromide (MTT) assay, flow cytometry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, Western Blot and a xenograft mouse model, respectively. Results showed that erlotinib had a stronger impact on the induction of apoptosis in erlotinib-sensitive PC-9 cells but had a weaker effect on erlotinib-resistant H1975 and H1650 cells than TG101348. TG101348 significantly enhanced the cytotoxicity of erlotinib to erlotinib-resistant NSCLC cells, stimulated erlotinib-induced apoptosis and downregulated the expressions of EGFR, p-EGFR, p-STAT3, Bcl-xL and survivin in erlotinib-resistant NSCLC cells. Moreover, the combined treatment of TG101348 and erlotinib induced apoptosis, inhibited the activation of p-EGFR and p-STAT3, and inhibited tumor growth of erlotinib-resistant NSCLC cells in vivo. Our results indicate that TG101348 is a potential adjuvant for NSCLC patients during erlotinib treatment.

  17. The heparin-binding domain of HB-EGF mediates localization to sites of cell-cell contact and prevents HB-EGF proteolytic release

    SciTech Connect

    Prince, Robin N.; Schreiter, Eric R.; Zou, Peng; Wiley, H. S.; Ting, Alice Y.; Lee, Richard T.; Lauffenburger, Douglas A.

    2010-07-01

    Heparin-binding EGF-like growth factor (HB-EGF) is a ligand for EGF receptor (EGFR) and possesses the ability to signal in juxtacrine, autocrine and/or paracrine mode, with these alternatives being governed by the degree of proteolytic release of the ligand. Although the spatial range of diffusion of released HB-EGF is restricted by binding heparan-sulfate proteoglycans (HSPGs) in the extracellular matrix and/or cellular glycocalyx, ascertaining mechanisms governing non-released HB-EGF localization is also important for understanding its effects. We have employed a new method for independently tracking the localization of the extracellular EGFlike domain of HB-EGF and the cytoplasmic C-terminus. A striking observation was the absence of the HB-EGF transmembrane proform from the leading edge of COS-7 cells in a wound-closure assay; instead, this protein localized in regions of cell-cell contact. A battery of detailed experiments found that this localization derives from a trans interaction between extracellular HSPGs and the HBEGF heparin-binding domain, and that disruption of this interaction leads to increased release of soluble ligand and a switch in cell phenotype from juxtacrine-induced growth inhibition to autocrine-induced proliferation. Our results indicate that extracellular HSPGs serve to sequester the transmembrane pro-form of HB-EGF at the point of cell-cell contact, and that this plays a role in governing the balance between juxtacrine versus autocrine and paracrine signaling.

  18. Aldosterone induces myofibroblast EGF secretion to regulate epithelial colonic permeability.

    PubMed

    Miró, Lluïsa; Pérez-Bosque, Anna; Maijó, Mònica; Amat, Concepció; Naftalin, Richard J; Moretó, Miquel

    2013-05-01

    In vivo studies show that raised aldosterone (Aldo) during low-Na adaptation regulates the growth of pericryptal myofibroblasts and reduces the permeability of the colonic epithelium. The aim of this study was to reproduce in vitro the in vivo condition of increased Aldo using human CCD-18Co myofibroblasts and T84 colonic epithelial cells to measure myofibroblast and epithelial proliferation and the expression of intercellular junction proteins. Proliferation was quantified by measuring 5-bromo-2'-deoxyuridine incorporation. The myofibroblast expression of EGF, VEGFa, and transforming growth factor-β1 (TGF-β1) was measured by real-time PCR and the expression of junctional complex proteins by Western blot. Aldo stimulated the proliferation of myofibroblasts by 70% (P < 0.05) and increased EGF mRNA expression by 30% (P < 0.05) without affecting VEGFa and TGF-β1. EGF concentration in the incubation medium increased by 30% (P < 0.05) 24 h after Aldo addition, and these effects were prevented by the addition of spironolactone. Myofibroblast proliferation in response to Aldo was mediated by EGF receptor (EGFR) and involved both MAPKK and phosphatidylinositol 3-kinase pathways. When T84 cells were incubated with medium from myofibroblasts stimulated with Aldo (conditioned medium), the expression of β-catenin and claudin IV was increased by 30% (P < 0.05) and proliferation by 40% (P < 0.05). T84 proliferation decreased when α-EGF, or the EGFR antagonist AG1478, was present. Results in vivo indicate that rats fed a low-salt diet showed an increased expression of EGF and EGFR in the colonic mucosa. These results support the view that changes in colonic permeability during low-Na adaptation are mediated by the EGF secreted by myofibroblasts in response to raised Aldo. PMID:23467299

  19. Activation of p38 Mitogen-Activated Protein Kinase Promotes Epidermal Growth Factor Receptor Internalization

    PubMed Central

    Vergarajauregui, Silvia; Miguel, Anitza San; Puertollano, Rosa

    2006-01-01

    Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions. PMID:16683917

  20. Heparin-binding EGF-like Growth Factor Protects Pericytes from Injury

    PubMed Central

    Yu, Xiaoyi; Radulescu, Andrei; Chen, Chun-Liang; James, Iyore O.; Besner, Gail E.

    2010-01-01

    Background We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) promotes angiogenesis and preserves mesenteric microvascular blood flow in several models of intestinal injury. The current study was designed to evaluate the effect of HB-EGF on pericytes, since these cells function to regulate capillary blood flow and new capillary growth. Materials and Methods C3H/10T1/2 mouse mesenchymal cells were differentiated into pericyte-like cells in vitro using transforming growth factor- β1 (TGF-β1). In addition, primary pericyte cultures were established from rat brain. The effect of HB-EGF on pericyte proliferation was assessed. In addition, cells were stressed by exposure to anoxia, and apoptosis determined. In vivo, we examined the effect of HB-EGF on pericytes in a model of intestinal I/R injury based on superior mesenteric artery occlusion (SMAO) in mice. Results Differentiated C3H/10T1/2 cells (pericyte-like cells) demonstrated morphologic characteristics of pericytes, and expressed pericyte specific markers. Addition of HB-EGF led to significant cell proliferation in differentiated pericyte-like cells, even under conditions of anoxic stress. Addition of the EGF receptor inhibitor AG 1478 led to complete inhibition of the proliferative effects of HB-EGF on pericyte-like cells. In addition, HB-EGF protected pericyte-like cells from anoxia-induced apoptosis. In addition, HB-EGF promoted cell proliferation in primary pericyte cultures. In vivo, administration of HB-EGF to mice subjected to intestinal I/R injury led to protection of pericytes from injury. Conclusions These results suggest that HB-EGF may function as a microcirculatory blood flow regulator, at least in part, via its effects on pericytes. PMID:20863525

  1. Breast milk protects against the development of necrotizing enterocolitis through inhibition of Toll Like Receptor 4 in the intestinal epithelium via activation of the epidermal growth factor receptor

    PubMed Central

    Good, Misty; Sodhi, Chhinder P.; Egan, Charlotte E.; Afrazi, Amin; Jia, Hongpeng; Yamaguchi, Yukihiro; Lu, Peng; Branca, Maria F.; Ma, Congrong; Prindle, Thomas; Mielo, Samantha; Pompa, Anthony; Hodzic, Zerina; Ozolek, John A.; Hackam, David J.

    2015-01-01

    Breast milk is the most effective strategy to protect infants against necrotizing enterocolitis (NEC), a devastating disease which is characterized by severe intestinal necrosis. Previous studies have demonstrated that the lipopolysaccharide receptor toll-like receptor 4 (TLR4) plays a critical role in NEC development via deleterious effects on mucosal injury and repair. We now hypothesize that breast milk protects against NEC by inhibiting TLR4 within the intestinal epithelium, and sought to determine the mechanisms involved. Breast milk protected against NEC and reduced TLR4 signaling in wild-type neonatal mice, but not in mice lacking the epidermal growth factor receptor (EGFR), while selective removal of EGF from breast milk reduced its protective properties, indicating that breast milk inhibits NEC and attenuates TLR4 signaling via EGF/EGFR activation. Over-expression of TLR4 in the intestinal epithelium reversed the protective effects of breast milk. The protective effects of breast milk occurred via inhibition of enterocyte apoptosis and restoration of enterocyte proliferation. Importantly, in IEC-6 enterocytes, breast milk inhibited TLR4 signaling via inhibition of GSK3β. Taken together, these findings offer mechanistic insights into the protective role for breast milk in NEC, and support a link between growth factor and innate immune receptors in NEC pathogenesis. PMID:25899687

  2. Epidermal growth factor induces WISP-2/CCN5 expression in estrogen receptor-alpha-positive breast tumor cells through multiple molecular cross-talks.

    PubMed

    Banerjee, Snigdha; Sengupta, Krishanu; Saxena, Neela K; Dhar, Kakali; Banerjee, Sushanta K

    2005-03-01

    Epidermal growth factor (EGF) is a mitogen for estrogen receptor (ER)-positive breast tumor cells, and it has been proven that EGF occasionally mimicked estrogen action and cross-talks with ER-alpha to exert its activity. Therefore, the present study was undertaken to explore whether EGF is able to modulate the expression of Wnt-1-induced signaling protein-2/connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed 5 (WISP-2/CCN5), an estrogen-responsive gene, in normal and transformed cell lines of the human breast and, if so, whether this induction is critical for EGF mitogenesis and what downstream signaling pathways are associated with this event. Here, we show that EGF-induced WISP-2 expression in ER- and EGF receptor-positive noninvasive MCF-7 breast tumor cells was dose and time dependent and that expression was modulated at transcription level. A synergism was seen in combination with estrogen. Moreover, small interfering RNA-mediated inhibition of WISP-2/CCN5 activity in MCF-7 cells resulted in abrogation of proliferation by EGF. The multiple molecular cross-talks, including the interactions between phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase signaling pathways and two diverse receptors (i.e., ER-alpha and EGFR), were essential in the event of EGF-induced WISP-2/CCN5 up-regulation in MCF-7 cells. Moreover, EGF action on WISP-2/CCN5 is restricted to ER- and EGFR-positive noninvasive breast tumor cells, and this effect of EGF cannot be instigated in ER-alpha-negative and EGFR-positive normal or invasive breast tumor cells by introducing ER-alpha. Finally, regulation of phosphorylation of ER-alpha and EGFR may play critical roles in EGF-induced transcriptional activation of WISP-2 gene in breast tumor cells.

  3. Expression, purification, and refolding of active recombinant human E-selectin lectin and EGF domains in Escherichia coli.

    PubMed

    Kawano, Susumu; Iyaguchi, Daisuke; Okada, Chiaki; Sasaki, Yusuke; Toyota, Eiko

    2013-06-01

    Attempts to obtain active E-selectin from Escherichia coli (E. coli) have not yet been successful. In this study, we succeeded in expressing the recombinant lectin and epidermal growth factor domain fragments of human E-selectin (rh-ESLE) in E. coli on a large-scale. The rh-ESLE protein was expressed as an inactive form in the inclusion bodies. The inactive form of rh-ESLE was denatured and solubilized by 6 M guanidine hydrochloride and then purified by Ni(2+) affinity chromatography under denaturing conditions. Denatured rh-ESLE was then refolded by a rapid-dilution method using a large amount of refolding buffer, which contained arginine and cysteine/cystine. The refolded rh-ESLE showed binding affinity for sLe(X) (K(d) = 321 nM, B(max) = 1.9 pmol/μg protein). This result suggests that the refolded rh-ESLE recovered its native and functional structure.

  4. Stimulation of neurite outgrowth in PC12 cells by EGF and KCl depolarization: a Ca(2+)-independent phenomenon

    PubMed Central

    1995-01-01

    MAP kinase activity is necessary for growth factor induction of neurite outgrowth in PC12 cells. Although NGF and EGF both stimulate MAP kinase activity, EGF does not stimulate neurite extension. We report that EGF, in combination with KCl, stimulates neurite outgrowth in PC12 cells. This phenomenon was independent of intracellular Ca2+ increases and not due to enhancement of MAP kinase activity over that seen with EGF alone. However, EGF plus KCl increased intracellular cAMP, and other cAMP elevating agents acted synergistically with EGF to promote neurite outgrowth. Stimulation of neurite outgrowth by cAMP and EGF was blocked by inhibitors of transcription suggesting that synergistic regulation of transcription by the cAMP and MAP kinase pathways may stimulate neurite growth. PMID:7622569

  5. Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation.

    PubMed

    Faria, Jerusa A Q A; de Andrade, Carolina; Goes, Alfredo M; Rodrigues, Michele A; Gomes, Dawidson A

    2016-09-01

    The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. PMID:27462018

  6. Mechanism for the activation of glutamate receptors

    Cancer.gov

    Scientists at the NIH have used a technique called cryo-electron microscopy to determine a molecular mechanism for the activation and desensitization of ionotropic glutamate receptors, a prominent class of neurotransmitter receptors in the brain and spina

  7. Arsenite and insulin exhibit opposing effects on epidermal growth factor receptor and keratinocyte proliferative potential

    SciTech Connect

    Patterson, Timothy J.; Rice, Robert H. . E-mail: rhrice@ucdavis.edu

    2007-05-15

    Previous work has suggested that arsenic exposure contributes to skin carcinogenesis by preserving the proliferative potential of human epidermal keratinocytes, thereby slowing the exit of putative target stem cells into the differentiation pathway. To find a molecular basis for this action, present work has explored the influence of arsenite on keratinocyte responses to epidermal growth factor (EGF). The ability of cultured keratinocytes to found colonies upon passaging several days after confluence was preserved by arsenite and EGF in an additive fashion, but neither was effective when the receptor tyrosine kinase activity was inhibited. Arsenite prevented the loss of EGF receptor protein and phosphorylation of tyrosine 1173, preserving its capability to signal. The level of nuclear {beta}-catenin was higher in cells treated with arsenite and EGF in parallel to elevated colony forming ability, and expression of a dominant negative {beta}-catenin suppressed the increase in both colony forming ability and yield of putative stem cells induced by arsenite and EGF. As judged by expression of three genes regulated by {beta}-catenin, this transcription factor had substantially higher activity in the arsenite/EGF-treated cells. Trivalent antimony exhibited the same effects as arsenite. A novel finding is that insulin in the medium induced the loss of EGF receptor protein, which was largely prevented by arsenite exposure.

  8. Epidermal growth factor (EGF) ligand release by substrate-specific a disintegrin and metalloproteases (ADAMs) involves different protein kinase C (PKC) isoenzymes depending on the stimulus.

    PubMed

    Dang, Michelle; Dubbin, Karen; D'Aiello, Antonio; Hartmann, Monika; Lodish, Harvey; Herrlich, Andreas

    2011-05-20

    The dysregulation of EGF family ligand cleavage has severe consequences for the developing as well as the adult organism. Therefore, their production is highly regulated. The limiting step is the ectodomain cleavage of membrane-bound precursors by one of several a disintegrin and metalloprotease (ADAM) metalloproteases, and understanding the regulation of cleavage is an important goal of current research. We have previously reported that in mouse lung epithelial cells, the pro-EGF ligands TGFα, neuregulin 1β (NRG), and heparin-binding EGF are differentially cleaved depending on the cleavage stimulus (Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) FASEB J.). In this study in mouse embryonic fibroblasts that lack different ADAMs, we show that induced cleavage of EGF ligands can involve the same substrate-specific metalloprotease but does require different stimulus-dependent signaling pathways. Cleavage was stimulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA), a mimic of diacylglycerol and PKC activator), hypertonic stress, lysophosphatidic acid (LPA)-induced G protein-coupled receptor activation, or by ionomycin-induced intracellular calcium release. Although ADAMs showed substrate preference (ADAM17, TGFα and heparin-binding EGF; and ADAM9, NRG), substrate cleavage differed substantially with the stimulus, and cleavage of the same substrate depended on the presence of different, sometimes multiple, PKC isoforms. For instance, classical PKC was required for TPA-induced but not hypertonic stress-induced cleavage of all EGF family ligands. Inhibition of PKCζ enhanced NRG release upon TPA stimulation, but it blocked NRG release in response to hypertonic stress. Our results suggest a model in which substantial regulation of ectodomain cleavage occurs not only on the metalloprotease level but also on the level of the substrate or of a third protein.

  9. Epidermal growth factor receptor in breast cancer: storage conditions affecting measurement, and relationship to steroid receptors.

    PubMed

    McLeay, W R; Horsfall, D J; Seshadri, R; Morrison, D A; Saccone, G T

    1992-01-01

    This study investigates the effect of freezing and storage of tissue and subcellular fractions on the measurement of epidermal growth factor receptors (EGF-r); compares competition binding and single saturating dose assays (SSD) for quantitating EGF-r levels; investigates several tissues as potential quality control; and examines the relationship between EGF-r and hormone receptor expression in human breast cancers. Mouse and calf uterine cell membranes were preferred sources of quality control tissue with similar levels of high affinity EGF-r to human breast cancer tissue (less than 150-200 fmol/mg membrane protein). Studies using pooled mouse uterine tissues indicated a loss of 40% in EGF-r activity following a single-20 degrees C freeze/thaw cycle, while a breast cancer tissue showed a 75% loss, independent of storage temperature (liquid nitrogen, -70 degrees C, -20 degrees C). A single freeze/thaw cycle of mouse uterine broken cell pellets (nuclei plus membrane fraction) again indicated a loss of EGF-r irrespective of storage temperature (43% loss at -70 degrees C, 52% loss at -20 degrees C). In most cases irrespective of the tissue type or tissue fraction being stored, the length of storage had little impact on the extent of the loss in activity. A second freeze/thaw cycle of intact tissue, or freezing of broken cell pellets from a previously-frozen tissue, led to a further major or total loss of the remaining EGF-r. Overall these results are commensurate with the published effects of freezing and storage on estrogen receptor measurement. In addition, our studies suggest that the most suitable procedure for assaying frozen breast cancer specimens for EGF-r levels in conjunction with steroid receptor quantitation is to prepare and assay both cytosol and membrane fractions for their respective receptor content without further storage. A concordance of 86% was found in 44 breast cancers assayed for EGF-4 by saturation analysis and SSD. Statistically significant

  10. ASBESTOS-INDUCED ACTIVATION OF CELL SIGNALING PATHWAYS IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Using respiratory epithelial cells transfected with either superoxide dismutase (SOD) or catalase, the authors tested the hypothesis that the activation of the epidermal growth factor (EGF) receptor signal pathway after asbestos exposure involves an oxidative stress. Western blot...

  11. Heparin-binding EGF-like growth factor contributes to reduced glomerular filtration rate during glomerulonephritis in rats

    PubMed Central

    Feng, Lili; Garcia, Gabriela E.; Yang, Young; Xia, Yiyang; Gabbai, Francis B.; Peterson, Orjan W.; Abraham, Judith A.; Blantz, Roland C.; Wilson, Curtis B.

    2000-01-01

    Heparin-binding epidermal growth factor–like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, is expressed during inflammatory and pathological conditions. We have cloned the rat HB-EGF and followed the expression of HB-EGF in rat kidneys treated with anti– glomerular basement membrane (anti–GBM) antibody (Ab) to induce glomerulonephritis (GN). We observed glomerular HB-EGF mRNA and protein within 30 minutes of Ab administration and showed by in situ hybridization that glomerular HB-EGF mRNA expression was predominantly in mesangial and epithelial cells. Expression of HB-EGF correlated with the onset of decreased renal function in this model. To test the direct effect of HB-EGF on renal function, we infused the renal cortex with active rHB-EGF, prepared from transfected Drosophila melanogaster cells. This treatment induced a significant decrease in single nephron GFR (SNGFR), single nephron plasma flow, and glomerular ultrafiltration coefficient and an increase in the glomerular capillary hydrostatic pressure gradient. In addition, anti–HB-EGF Ab administered just before anti-GBM Ab blocked the fall in SNGFR and GFR at 90 minutes without any change in the glomerular histologic response. These studies suggest that HB-EGF expressed early in the anti-GBM Ab GN model contributes to the observed acute glomerular hemodynamic alterations. PMID:10675360

  12. The Membrane-anchoring Domain of Epidermal Growth Factor Receptor Ligands Dictates Their Ability to Operate in Juxtacrine Mode

    SciTech Connect

    Dong, Jianying; Opresko, Lee; Chrisler, William B.; Orr, Galya; Quesenberry, Ryan D.; Lauffenburger, Douglas A.; Wiley, H S.

    2005-06-01

    All ligands of the epidermal growth factor receptor (EGFR) are synthesized as membrane-anchored precursors. Previous work has suggested that some ligands, such as EGF, must be proteolytically released to be active, whereas others, such as heparin binding EGF-like growth factor (HB-EGF) can function while still anchored to the membrane (i.e., juxtacrine signaling). To explore the structural basis for these differences in ligand activity, we engineered a series of membrane-anchored ligands in which the core, receptor-binding domain of EGF was combined with different domains of both EGF and HB-EGF. We found that ligands having the N-terminal extension of EGF could not bind to the EGFR, even when released from the membrane. Ligands lacking an N-terminal extension, but possessing the membrane-anchoring domain of EGF still required proteolytic release for activity, whereas ligands with the membrane anchoring domain of HB-EGF could elicit full biological activity while still membrane anchored. Ligands containing the HB-EGF membrane anchor, but lacking an N-terminal extension, activated EGFR during their transit through the Golgi apparatus . However, cell-mixing experiments and fluorescence resonance energy transfer (FRET) studies showed that juxtacrine signaling typically occurred in trans at the cell surface, at points of cell-cell contact. Our data suggest that the membrane-anchoring domain of ligands selectively controls their ability to participate in juxtacrine signaling and thus, only a subclass of EGFR ligands can act in a juxtacrine mode.

  13. New platform for controlled and sustained delivery of the EGF receptor tyrosine kinase inhibitor AG1478 using poly(lactic-co-glycolic acid) microspheres

    PubMed Central

    Robinson, Rebecca; Bertram, James P.; Reiter, Jill L.; Lavik, Erin B.

    2015-01-01

    Inhibition of the epidermal growth factor receptor (EGFR) has been shown to reduce tumor growth and metastases and promote axon regeneration in the central nervous system. Current strategies for inhibiting EGFR include the administration of reversible or irreversible small-molecule tyrosine kinase inhibitors (TKIs). However, to be effective in vivo constant and sustained delivery is required. This study explored the feasibility of encapsulating the tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) in poly(lactic-co-glycolic acid) (PLGA) microspheres to achieve sustained delivery of the TKI. We characterized microspheres prepared using three different emulsion methods: solid-in-oil-in-water, oil-in-water, and oil-in-water with co-solvent. Addition of a co-solvent increased the loading and release of AG1478, and significantly (P<0.001) decreased the size of the microspheres which facilitates administration of the spheres. On average, sustained delivery of AG1478 from microspheres was achieved for six months. However, the addition of a co-solvent prolonged release for over nine months (266 days). In addition, AG1478 retained its bioactivity upon delivery, and inhibited EGFR in both immortalized rat fibroblasts and in EGFR-amplified human carcinoma cells. These results demonstrate that AG1478 can be encapsulated in PLGA and retain bioactivity; thereby providing a new platform for controlled administration of EGFR TKIs. PMID:20055747

  14. EGF stimulates Mg{sup 2+} influx in mammary epithelial cells

    SciTech Connect

    Trapani, Valentina; Arduini, Daniela; Luongo, Francesca; Wolf, Federica I.

    2014-11-28

    Highlights: • EGF stimulation potentiates Mg{sup 2+} influx into epithelial cells. • EGF-induced Mg{sup 2+} influx does not depend on the concomitantly induced Ca{sup 2+} signal. • EGF-induced Ca{sup 2+} signal is dependent on the presence of extracellular Mg{sup 2+}. • New players in EGF-mediated signaling might be exploited as therapeutic targets. - Abstract: Magnesium is well established as a fundamental factor that regulates cell proliferation. However, the molecular mechanisms linking mitogenic signals, extracellular magnesium availability and intracellular effectors are still largely unknown. In the present study we sought to determine whether EGF regulates magnesium homeostasis in normal HC11 mammary epithelial cells. To this end, we measured Mg{sup 2+} and Ca{sup 2+} fluxes by confocal imaging in live cells loaded with specific fluorescent ion indicators (Mag-Fluo-4 and Fluo-4, respectively). EGF stimulation induces a rapid and sustained increase in intracellular Mg{sup 2+}, concomitantly with a rise in intracellular calcium. The increase in intracellular Mg{sup 2+} derives from an influx from the extracellular compartment, and does not depend on Ca{sup 2+}. On the contrary, the increase in intracellular Ca{sup 2+} derives from intracellular stores, and is impaired in the absence of extracellular magnesium. Inhibition of the EGF receptor tyrosine kinase by Tyrphostin AG1478 markedly inhibits EGF-induced Mg{sup 2+} and Ca{sup 2+} signals. These findings demonstrate that not only does Mg{sup 2+} influx represent an important step in the physiological response of epithelial cells to EGF, but unexpectedly the EGF-induced Mg{sup 2+} influx is essential for the Ca{sup 2+} signal to occur.

  15. SNP analyses of growth factor genes EGF, TGF{beta}-1, and HGF reveal haplotypic association of EGF with autism

    SciTech Connect

    Toyoda, Takao; Thanseem, Ismail; Kawai, Masayoshi; Sekine, Yoshimoto; Nakamura, Kazuhiko; Anitha, Ayyappan; Suda, Shiro . E-mail: nakamura@hama-med.ac.jp; Yamada, Kazuo; Tsujii, Masatsugu |; Iwayama, Yoshimi; Hattori, Eiji; Toyota, Tomoko; Yoshikawa, Takeo; Miyachi, Taishi; Tsuchiya, Kenji; Sugihara, Gen-ichi; Matsuzaki, Hideo; Iwata, Yasuhide; Suzuki, Katsuaki; Mori, Norio |; Ouchi, Yasuomi |; Sugiyama, Toshiro; Takei, Nori

    2007-09-07

    Autism is a pervasive neurodevelopmental disorder diagnosed in early childhood. Growth factors have been found to play a key role in the cellular differentiation and proliferation of the central and peripheral nervous systems. Epidermal growth factor (EGF) is detected in several regions of the developing and adult brain, where, it enhances the differentiation, maturation, and survival of a variety of neurons. Transforming growth factor-{beta} (TGF{beta}) isoforms play an important role in neuronal survival, and the hepatocyte growth factor (HGF) has been shown to exhibit neurotrophic activity. We examined the association of EGF, TGF{beta}1, and HGF genes with autism, in a trio association study, using DNA samples from families recruited to the Autism Genetic Resource Exchange; 252 trios with a male offspring scored for autism were selected for the study. Transmission disequilibrium test revealed significant haplotypic association of EGF with autism. No significant SNP or haplotypic associations were observed for TGF{beta}1 or HGF. Given the role of EGF in brain and neuronal development, we suggest a possible role of EGF in the pathogenesis of autism.

  16. A point mutation in the EGF-4 domain of β3 integrin is responsible for the formation of the Seca platelet alloantigen and affects receptor function

    PubMed Central

    Sachs, Ulrich J.; Bakchoul, Tamam; Eva, Olga; Giptner, Astrid; Bein, Gregor; Aster, Richard H.; Gitter, Maria; Peterson, Julie; Santoso, Sentot

    2013-01-01

    Summary Neonatal alloimmune thrombocytopenia (NAIT) is caused by fetomaternal platelet incompatibility with maternal antibodies crossing the placenta and destroying fetal platelets. Antibodies against human platelet antigen-1a (HPA-1a) and HPA-5b are responsible for the majority of NAIT cases. We observed a suspected NAIT in a newborn with a platelet count of 25 G/l and petechial haemorrhages. Serological analysis of maternal serum revealed an immunisation against αIIbβ3 on paternal platelets only, indicating the presence of an antibody against a new rare alloantigen (Seca) residing on αIIbβ3. The location of Seca on αIIbβ3 was confirmed by immunoprecipitation. Nucleotide sequence analysis of paternal β3 revealed a single nucleotide exchange (G1818T) in exon 11 of the β3 gene (ITGB3), changing Lys580 (wild-type) to Asn580 (Seca). Two additional members of the family Sec were typed Seca positive, but none of 300 blood donors. Chinese hamster ovary cells expressing Asn580, but not Lys580 αIIbβ3, bound anti-Seca, which was corroborated by immunoprecipitation. Adhesion of transfected cells onto immobilised fibrinogen showed reduced binding of the Asn580 variant compared to wild-type αIIbβ3. Analysis of transfected cells with anti-LIBS and PAC-1 antibody showed reduced binding when compared to the wild-type. No such effects were observed with Seca positive platelets, which, however, are heterozygous for the Lys580Asn mutation. In this study, we describe a NAIT case caused by maternal alloimmunisation against a new antigen on αIIbβ3. Analysis with mutant transfected cells showed that the Lys580Asn mutation responsible for the formation of the Seca antigenic determinant affects αIIbβ3 receptor function. PMID:22116617

  17. Signal transduction activated by cannabinoid receptors.

    PubMed

    Díaz-Laviada, Inés; Ruiz-Llorente, Lidia

    2005-07-01

    Since the discovery that cannabinoids exert biological actions through binding to specific receptors, signal mechanisms triggered by these receptors have been focus of extensive study. This review summarizes the current knowledge of the signalling events produced by cannabinoids from membrane receptors to downstream regulators. Two types of cannabinoid receptors have been identified to date: CB(1) and CB(2) both belonging to the heptahelichoidal receptor family but with different tissue distribution and signalling mechanisms. Coupling to inhibitory guanine nucleotide-binding protein and thus inhibition of adenylyl cyclase has been observed in both receptors but other signal transduction pathways that are regulated or not by these G proteins are differently activated upon ligand-receptor binding including ion channels, sphingomyelin hydrolysis, ceramide generation, phospholipases activation and downstream targets as MAP kinase cascade, PI3K, FAK or NOS regulation. Cannabinoids may also act independently of CB(1)or CB(2) receptors. The existence of new unidentified putative cannabinoid receptors has been claimed by many investigators. Endocannabinoids activate vanilloid TRPV1 receptors that may mediate some of the cannabinoid effects. Other actions of cannabinoids can occur through non-receptor-mediated mechanisms.

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

    PubMed Central

    Michailidis, Ioannis E.; Rusinova, Radda; Georgakopoulos, Anastasios; Chen, Yibang; Iyengar, Ravi; Robakis, Nikolaos K.; Logothetis, Diomedes E.; Baki, Lia

    2012-01-01

    Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2 or PIP2] 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 PIP2 and other phosphoinositides. Here, we report a novel requirement for PIP2 in the activation of the epidermal growth factor receptor (EGFR). Down-regulation of PIP2 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 PIP2 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 PIP2 and PIP2-dependent regulation of EGFR activation. A peptide mimicking the EGFR juxtamembrane domain that was assayed by surface plasmon resonance displayed strong binding to PIP2. Neutralization of positively charged amino acids abolished EGFR/PIP2 interaction in the context of this peptide and down-regulated epidermal growth factor (EGF)-induced EGFR autophosphorylation 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 PIP2 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 PIP2 may serve as a general modulator of the activity of many diverse eukaryotic transmembrane proteins through their basic JDs. PMID:21107857

  19. EGF-conjugated near-infrared quantum dots as nanoprobes for in-vivo imaging of EGFR expression

    NASA Astrophysics Data System (ADS)

    Diagaradjane, Parmeswaran; Orenstein-Cardona, Jacobo M.; Colón-Casasnovas, Norman E.; Deorukhkar, Amit; Shentu, Shujun; Kuno, Norihito; Schwartz, David L.; Gelovani, Juri G.; Krishnan, Sunil

    2008-02-01

    Noninvasive imaging of epidermal growth factor (EGF) receptor (EGFR) expression can provide valuable molecular information that could aid diagnostic and therapeutic decisions, particularly with targeted cancer therapies utilizing anti-EGFR antibodies. In this study we report on the development and validation of a nanoprobe for in-vivo imaging and discrimination of EGFR-overexpressing tumors from surrounding normal tissues that also expresses EGFR. Near-infrared quantum dots (QDs) were coupled to EGF using thiol-maleimide conjugation to create EGF-QD nanoprobes. These nanoprobes demonstrated excellent in-vitro and in-vivo binding affinity. In-vivo imaging demonstrated three distinct phases of tumor influx (~3min), clearance (~60min) and accumulation (1-6hrs) of EGF-QD nanoprobes. Both QD and EGF-QD demonstrated non-specific rapid tumor influx and clearance followed by an apparent dynamic equilibrium at ~60min. Subsequently (1-6hrs), while QD concentration gradually decreased in tumors, EGF-QDs progressively accumulated in tumors. At 24hrs, tumor fluorescence decreased to near baseline levels for both QD and EGF-QD. Ex vivo whole-organ, tissue-homogenate fluorescence, confocal microscopy and immunofluorescence staining confirmed tumor-specific accumulation of EGF-QD nanoprobes at an early time-point (4hrs). The favorable pharmacokinetics, the ability to discriminate EGFR-overexpressing tumors from surrounding normal tissues using low concentration (10-pmol) of EGF-QD nanoprobe underscores the clinical relevance of this probe to evaluate therapeutic intervention.

  20. PDGF-induced receptor phosphorylation and phosphoinositide hydrolysis are unaffected by protein kinase C activation in mouse swiss 3T3 and human skin fibroblasts

    SciTech Connect

    Sturani, E.; Vicentini, L.M.; Zippel, R.; Toschi, L.; Pandiella-Alonso, A.; Comoglio, P.M.; Meldolesi, J.

    1986-05-29

    Short (1-10 min) pretreatment of intact cells with activators of protein kinase C (e.g. phorbol-12 myristate, 13-acetate, PMA) affects the activity of a variety of surface receptors (for growth factors, hormones and neurotransmitters), with inhibition of transmembrane signal generation. In two types of fibroblasts it is demonstrated that the PDGF receptor is unaffected by PMA. Exposure to PMA at concentrations up to 100 nM for 10 min failed to inhibit either one of the agonist-induced, receptor-coupled responses of PDGF: the autophosphorylation of receptor molecules at tyrosine residues, and the hydrolysis of membrane polyphosphoinositides. In contrast, the EGF receptor autophosphorylation (in A 431 cells) and the bombesin-induced phosphoinositide hydrolysis were readily inhibited by PMA.

  1. Eicosopentaneoic Acid and Other Free Fatty Acid Receptor Agonists Inhibit Lysophosphatidic Acid- and Epidermal Growth Factor-Induced Proliferation of Human Breast Cancer Cells

    PubMed Central

    Hopkins, Mandi M.; Zhang, Zhihong; Liu, Ze; Meier, Kathryn E.

    2016-01-01

    Many key actions of ω-3 (n-3) fatty acids have recently been shown to be mediated by two G protein-coupled receptors (GPCRs) in the free fatty acid receptor (FFAR) family, FFA1 (GPR40) and FFA4 (GPR120). n-3 Fatty acids inhibit proliferation of human breast cancer cells in culture and in animals. In the current study, the roles of FFA1 and FFA4 were investigated. In addition, the role of cross-talk between GPCRs activated by lysophosphatidic acid (LPA), and the tyrosine kinase receptor activated by epidermal growth factor (EGF), was examined. In MCF-7 and MDA-MB-231 human breast cancer cell lines, both LPA and EGF stimulated proliferation, Erk activation, Akt activation, and CCN1 induction. LPA antagonists blocked effects of LPA and EGF on proliferation in MCF-7 and MDA-MB-231, and on cell migration in MCF-7. The n-3 fatty acid eicosopentaneoic acid inhibited LPA- and EGF-induced proliferation in both cell lines. Two synthetic FFAR agonists, GW9508 and TUG-891, likewise inhibited LPA- and EGF-induced proliferation. The data suggest a major role for FFA1, which was expressed by both cell lines. The results indicate that n-3 fatty acids inhibit breast cancer cell proliferation via FFARs, and suggest a mechanism involving negative cross-talk between FFARS, LPA receptors, and EGF receptor. PMID:26821052

  2. Enterocyte apoptosis after enterectomy in mice is activated independent of the extrinsic death receptor pathway.

    PubMed

    Knott, Andrew W; O'Brien, David P; Juno, Russell J; Zhang, Yufang; Williams, Jodi L; Erwin, Christopher R; Warner, Brad W

    2003-08-01

    Intestinal adaptation following small bowel resection (SBR) is associated with greater rates of enterocyte apoptosis by unknown mechanism(s). Because postresection adaptation is associated with increased translocation of luminal bacteria, we sought to characterize the role for the extrinsic, death receptor pathway for the activation of enterocyte apoptosis after massive SBR. We first performed SBR or sham operations in mice, and the temporal expression of caspases 8, 9, and 3, death receptors tumor necrosis factor receptor-1 (TNFR1) and Fas and corresponding ligands (TNF and Fas ligand) was determined in the remnant intestine at various postoperative time points. Ileal TNFR1 and Fas expression were then measured after SBR in the setting of increased (waved-2 mice) or decreased (exogenous EGF administration) apoptosis. Finally, intestinal adaptation and apoptosis were recorded in the remnant ileum after SBR in TNFR1-null and Fas-null mice. The expression of death receptor family proteins and caspases demonstrated only modest changes after SBR and did not correlate with the histological appearance of apoptosis. In the setting of accelerated apoptosis, TNFR1 and Fas expression were paradoxically decreased. Apoptotic and adaptive responses were preserved in both TNFR1-null and Fas-null mice. These results suggest that the mechanism for increased enterocyte apoptosis following massive SBR does not appear to involve the extrinsic, death receptor-mediated pathway.

  3. ACTIVATION OF THE EGF RECEPTOR SIGNALING PATHWAY IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO UTAH VALLEY PARTICULATE MATTER

    EPA Science Inventory

    Exposure to ambient particulate matter (PM) in the Utah Valley (UV) has previously been associated with a variety of adverse health effects. To investigate intracellular signaling mechanisms for pulmonary responses to UV PM inhalation, human primary airway epithelial cells (NHBE)...

  4. Towards predicting the lung fibrogenic activity of MWCNT: Key role of endocytosis, kinase receptors and ERK 1/2 signaling.

    PubMed

    Vietti, Giulia; Ibouraadaten, Saloua; Palmai-Pallag, Mihaly; Yakoub, Yousof; Piret, Jean-Pascal; Marbaix, Etienne; Lison, Dominique; van den Brule, Sybille

    2016-01-01

    Carbon nanotubes (CNT) have been reported to induce lung inflammation and fibrosis in rodents. We investigated the direct and indirect cellular mechanisms mediating the fibrogenic activity of multi-wall (MW) CNT on fibroblasts. We showed that MWCNT indirectly stimulate lung fibroblast (MLg) differentiation, via epithelial cells and macrophages, whereas no direct effect of MWCNT on fibroblast differentiation or collagen production was detected. MWCNT directly stimulated the proliferation of fibroblasts primed with low concentrations of growth factors, such as PDGF, TGF-β or EGF. MWCNT prolonged ERK 1/2 phosphorylation induced by low concentrations of PDGF or TGF-β in fibroblasts. This phenomenon and the proliferative activity of MWCNT on fibroblasts was abrogated by the inhibitors of ERK 1/2, PDGF-, TGF-β- and EGF-receptors. This activity was also reduced by amiloride, an endocytosis inhibitor. Finally, the lung fibrotic response to several MWCNT samples (different in length and diameter) correlated with their in vitro capacity to stimulate the proliferation of fibroblasts and to prolong ERK 1/2 signaling in these cells. Our findings point to a crosstalk between MWCNT, kinase receptors, ERK 1/2 signaling and endocytosis which stimulates the proliferation of fibroblasts. The mechanisms of action identified in this study contribute to predict the fibrogenic potential of MWCNT.

  5. Arrestin-3 is essential for the activation of Fyn by the luteinizing hormone receptor (LHR) in MA-10 cells*

    PubMed Central

    Galet, Colette; Ascoli, Mario

    2008-01-01

    Recent studies showed that Fyn is a mediator of the LHR-induced activation of the ERK1/2 cascade in MA-10 cells. Since the LHR is a G protein-coupled receptor and the Src family of kinases can be activated by some Gα subunits and by the non-visual arrestins we investigated the role of these signaling molecules in the LHR-provoked activation of Fyn. Small interfering RNAs (siRNAs) that target two Gα subunits that participate in LHR signaling (Gαs and Gα11) and one that targets arrestin-3 were co-transfected with the hLHR in MA-10 cells. We then determined the effects of these siRNAs on the LHR-provoked activation of Fyn, the phosphorylation of FAK (a prominent Fyn substrate) and the release of EGF-like growth factors (a Fyn-mediated process). Expression of the siRNA against Gαs decreased the level of Gαs and LHR-stimulated cAMP production by ~50% but did not affect LHR-stimulated Fyn activation or FAK phosphorylation. Likewise, expression of the siRNA against Gα11 decreased the level of Gα11 and LHR-stimulated inositol phosphate production by ~50% but did not affect LHR-stimulated Fyn activation or FAK phosphorylation. Expression of the siRNA against arrestin-3 decreased the level of arrestin-3 and the rate of internalization of hCG by ~50% and it also inhibited the LHR-provoked stimulation of Fyn, the phosphorylation of FAK and the release of EGF-like growth factors. These results show that, in MA-10 cells, the hLHR activates Fyn through and arrestin-3-dependent pathway and that this pathway is a mediator of the hLHR-provoked release of EGF-like growth factors. PMID:18647647

  6. Oxidative stress increases gene expression of heparin-binding EGF-like growth factor and amphiregulin in cultured rat gastric epithelial cells.

    PubMed

    Miyazaki, Y; Shinomura, Y; Tsutsui, S; Yasunaga, Y; Zushi, S; Higashiyama, S; Taniguchi, N; Matsuzawa, Y

    1996-09-13

    We investigated the effects of oxidative stress on mRNA levels of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and amphiregulin (AR) in rat gastric epithelial RGM1 cells. In response to stimulation with hydrogen peroxide (100-400 microM), gene expression of HB-EGF and AR increased in a dose-dependent manner, peaked at 3 h, and returned to the base line at 7 h. Hydrogen peroxide-induced HB-EGF and AR gene expression was blocked by pretreatment with an antioxidant N-acetyl-cysteine. In addition, it was significantly inhibited by pretreatment with EGF receptor-specific tyrphostin AG1478, but not by depletion of protein kinase C. These data indicate that oxidative stress upregulates expression of EGF-related polypeptides and the possible involvement of EGF receptor in this process.

  7. Blocking EGFR in the liver improves the tumor-to-liver uptake ratio of radiolabeled EGF.

    PubMed

    Kareem, Heewa; Sandström, Karl; Elia, Ronny; Gedda, Lars; Anniko, Matti; Lundqvist, Hans; Nestor, Marika

    2010-04-01

    Overexpression of epidermal growth factor receptor (EGFR) in several types of malignant tumors correlates with disease progression. EGFR could, therefore, be an excellent candidate for targeted radionuclide diagnostics. However, the high natural expression of EGFR in the liver may be problematic. The aim of this study was to improve the tumor-to-liver ratio of radiolabeled epidermal growth factor (EGF) by blocking its uptake by the liver with a nonradiolabeled EGFR-targeting molecule in tumor-bearing mice. Intraperitoneally injected nonradiolabeled EGF was first evaluated as a blocking agent, preadministered at various time intervals before intravenous injection of (125)I-labeled EGF. The anti-EGFR Affibody molecule (Z(EGFR:955))(2) was then assessed as a blocking agent of (111)In-labeled EGF in a dual isotope study (50, 100, and 200 microg, preadministered 30 or 60 min before (111)In-EGF). The 30-min preadministration of nonradiolabeled EGF significantly decreased (125)I-EGF uptake in the liver, whereas uptake in the tumor remained unchanged. Furthermore, preadministration of only 50 microg (Z(EGFR:955))(2) as a blocking agent 30 min before the (111)In-EGF decreased the uptake of (111)In-EGF by the liver and increased its uptake by the tumor, thereby increasing the tumor-to-liver ratio sixfold. We conclude that the Affibody molecule (Z(EGFR:955))(2) shows promise as a blocking agent that could enhance the outcome of radionuclide-based EGFR-expressing tumor diagnostics and imaging. PMID:20358420

  8. Embryonic Poly(A)-Binding Protein (EPAB) Is Required for Granulosa Cell EGF Signaling and Cumulus Expansion in Female Mice.

    PubMed

    Yang, Cai-Rong; Lowther, Katie M; Lalioti, Maria D; Seli, Emre

    2016-01-01

    Embryonic poly(A)-binding protein (EPAB) is the predominant poly(A)-binding protein in Xenopus, mouse, and human oocytes and early embryos before zygotic genome activation. EPAB is required for translational activation of maternally stored mRNAs in the oocyte and Epab(-/-) female mice are infertile due to impaired oocyte maturation, cumulus expansion, and ovulation. The aim of this study was to characterize the mechanism of follicular somatic cell dysfunction in Epab(-/-) mice. Using a coculture system of oocytectomized cumulus oophorus complexes (OOXs) with denuded oocytes, we found that when wild-type OOXs were cocultured with Epab(-/-) oocytes, or when Epab(-/-) OOXs were cocultured with WT oocytes, cumulus expansion failed to occur in response to epidermal growth factor (EGF). This finding suggests that oocytes and cumulus cells (CCs) from Epab(-/-) mice fail to send and receive the necessary signals required for cumulus expansion. The abnormalities in Epab(-/-) CCs are not due to lower expression of the oocyte-derived factors growth differentiation factor 9 or bone morphogenetic protein 15, because Epab(-/-) oocytes express these proteins at comparable levels with WT. Epab(-/-) granulosa cells (GCs) exhibit decreased levels of phosphorylated MEK1/2, ERK1/2, and p90 ribosomal S6 kinase in response to lutenizing hormone and EGF treatment, as well as decreased phosphorylation of the EGF receptor. In conclusion, EPAB, which is oocyte specific, is required for the ability of CCs and GCs to become responsive to LH and EGF signaling. These results emphasize the importance of oocyte-somatic communication for GC and CC function.

  9. Cross-linking of epidermal growth factor receptors in intact cells: detection of initial stages of receptor clustering and determination of molecular weight of high-affinity receptors

    SciTech Connect

    Fanger, B.O.; Austin, K.S.; Earp, H.S.; Cidlowski, J.A.

    1986-10-21

    A method was developed to label epidermal growth factor (EGF) receptors with /sup 125/I-EGF in whole cells using chemical cross-linking reagents. Polyacrylamide gel electrophoresis resolved an M/sub r/ approx. 180,000 EGF-receptor complex and larger M/sub r/ greater than or equal to 360,000 aggregates. The formation of the larger complexes was timed and temperature dependent and appeared to represent the initial events of EGF receptor clustering. Alteration of the ratio of /sup 125/I-EGF-labeled high- and low- affinity complexes by competition with unlabeled EGF or by induction of additional high-affinity sites with dexamethasone suggested that both sites were represented by the M/sub r/ approx. 180,000 /sup 125/I-EGF-receptor complexes. Digestion of cells before cross-linking detected a small population of trypsin-resistant M/sub r/ approx. 180,000 receptors, which could represent previously described cryptic and/or high-affinity receptors. Few of the M/sub r/ approx. 360,000 receptors were trypsin resistant. Glucocorticoid induction of high-affinity EGF receptors failed to induce detectable changes in the microclustering of EGF receptors but did result in a 50% increase in EGF-induced receptor phosphorylation in HeLa S/sub 3/ cell membranes at 4/sup 0/C. Thus, glucocorticoids increase high-affinity EGF binding sites, EGF-induced receptor phosphorylation, and cell growth.

  10. Apoptosis inducer NGFI-B is degraded by the proteasome and stabilized by treatment with EGF

    SciTech Connect

    Strom, Bjorn O.; Paulsen, Ragnhild E.

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer NGFI-B is a molecular target for some anti-cancer drugs. Black-Right-Pointing-Pointer NGFI-B turnover may be important for their anti-cancer action. Black-Right-Pointing-Pointer NGFI-B is degraded by the proteasome. Black-Right-Pointing-Pointer NGFI-B is stabilized by treatment with EGF. Black-Right-Pointing-Pointer Mimicking the EGF-induced phosphorylation also stabilizes the protein. -- Abstract: NGFI-B is a nuclear receptor and immediate early gene that is upregulated in many different tumour cell lines. As it is involved in cell death and survival, it has been suggested as a target for anti-cancer drugs. The protein level of NGFI-B is important for its functions and may be regulated through induction or stabilization. NGFI-B protein stability was studied using the protein synthesis inhibitor cycloheximide in CV1 cells transiently transfected with NGFI-B. Inhibiting the proteasome with MG132 stabilized NGFI-B, indicating that the proteasome is responsible for break-down of NGFI-B, as it is for many nuclear receptors. In order to determine regions responsible for the break-down of NGFI-B two N-terminal regions with high PEST-scores were deleted. Deletion of amino acids 122-195 containing a PEST-sequence which includes an ERK2 phosphorylation target, gave a more stable protein. In addition, treatment of the cells with the ERK2 activator EGF increased the stability of wild type NGFI-B. We then tested whether a mutation at threonine 142 influenced the stability of NGFI-B. We found that the phosphorylation-mimicking mutant NGFI-B T142E had an increased stability, while the non-phosphorylable mutant (T142A) showed similar stability to the wild type. Thus, EGF-stimulation of cells may be a mechanism for priming the cells for effects of NGFI-B by increasing its stability.

  11. Epidermal growth factor receptor signaling in tissue

    SciTech Connect

    Shvartsman, Stanislav; Wiley, H. S.; Lauffenburger, Douglas A.

    2004-08-01

    Abstract: A peptide purified from the salivary gland of a mouse was shown few years ago to accelerate incisor eruption and eyelid opening in newborn mice, and was named epidermal growth factor (EGF). The members of this family of peptide growth factors had been identified in numerous physiological and pathological contexts. EGF binds to a cell surface EGF receptor, which induces a biochemical modification (phosphorylation) of the receptor's cytoplasmic tail. There is a growing consensus in the research community that, in addition to cellular and molecular studies, the dynamics of the EGFR network and its operation must be examined in tissues. A key challenge is to integrate the existing molecular and cellular information into a system-level description of the EGFR network at the tissue and organism level. In this paper, the two examples of EGFR signaling in tissues are described, and the recent efforts to model EGFR autocrine loops, which is a predominant mode of EGFR activation in vivo, are summarized.

  12. Identification of a novel EGF-sensitive cell cycle checkpoint

    SciTech Connect

    Walker, Francesca . E-mail: francesca.walker@ludwig.edu.au; Zhang Huihua; Burgess, Antony W.

    2007-02-01

    The site of action of growth factors on mammalian cell cycle has been assigned to the boundary between the G1 and S phases. We show here that Epidermal Growth Factor (EGF) is also required for mitosis. BaF/3 cells expressing the EGFR (BaF/wtEGFR) synthesize DNA in response to EGF, but arrest in S-phase. We have generated a cell line (BaF/ERX) with defective downregulation of the EGFR and sustained activation of EGFR signalling pathways: these cells undergo mitosis in an EGF-dependent manner. The transit of BaF/ERX cells through G2/M strictly requires activation of EGFR and is abolished by AG1478. This phenotype is mimicked by co-expression of ErbB2 in BaF/wtEGFR cells, and abolished by inhibition of the EGFR kinase, suggesting that sustained signalling of the EGFR, through impaired downregulation of the EGFR or heterodimerization, is required for completion of the cycle. We have confirmed the role of EGFR signalling in the G2/M phase of the cell cycle using a human tumor cell line which overexpresses the EGFR and is dependent on EGFR signalling for growth. These findings unmask an EGF-sensitive checkpoint, helping to understand the link between sustained EGFR signalling, proliferation and the acquisition of a radioresistant phenotype in cancer cells.

  13. Heparin-Binding Epidermal Growth Factor and Its Receptors Mediate Decidualization and Potentiate Survival of Human Endometrial Stromal Cells

    PubMed Central

    Chobotova, Katya; Karpovich, Natalia; Carver, Janet; Manek, Sanjiv; Gullick, William J.; Barlow, David H.; Mardon, Helen J.

    2006-01-01

    Heparin-binding epidermal growth factor (HB-EGF) has pleiotropic biological functions in many tissues, including those of the female reproductive tract. It facilitates embryo development and mediates implantation and is thought to have a function in endometrial receptivity and maturation. The mature HB-EGF molecule manifests its activity as either a soluble factor (sol-HB-EGF) or a transmembrane precursor (tm-HB-EGF) and can bind two receptors, EGFR and ErbB4/HER4. In this study, we identify factors that modulate expression of HB-EGF, EGFR, and ErbB4 in endometrial stromal cells in vitro. We demonstrate that levels of sol- and tm-HB-EGF, EGFR, and ErbB4 are increased by cAMP, a potent inducer of decidualization of the endometrial stroma. We also show that production of sol- and tm-HB-EGF is differentially modulated by TNFα and TGFβ. Our data suggest that HB-EGF has a function in endometrial maturation in mediating decidualization and attenuating TNFα- and TGFβ-induced apoptosis of endometrial stromal cells. PMID:15562026

  14. PYK2 integrates growth factor and cytokine receptors signaling and potentiates breast cancer invasion via a positive feedback loop.

    PubMed

    Selitrennik, Michael; Lev, Sima

    2015-09-01

    The involvement of ErbB family members in breast cancer progression and metastasis has been demonstrated by many studies. However, the downstream effectors that mediate their migratory and invasive responses have not been fully explored. In this study, we show that the non-receptor tyrosine kinase PYK2 is a key effector of EGFR and HER2 signaling in human breast carcinoma. We found that PYK2 is activated by both EGF and heregulin (HRG) in breast cancer cells, and positively regulates EGF/HRG-induced cell spreading, migration and invasion. PYK2 depletion markedly affects ERK1/2 and STAT3 phosphorylation in response to EGF/HRG as well as to IL8 treatment. Importantly, PYK2 depletion also reduced EGF/HRG-induced MMP9 and IL8 transcription, while IL8 inhibition abrogated EGF-induced MMP9 transcription and attenuated cell invasion. IL8, which is transcriptionally regulated by STAT3 and induces PYK2 activation, prolonged EGF-induced PYK2, STAT3 and ERK1/2 phosphorylation suggesting that IL8 acts through an autocrine loop to reinforce EGF-induced signals. Collectively our studies suggest that PYK2 is a common downstream effector of ErbB and IL8 receptors, and that PYK2 integrates their signaling pathways through a positive feedback loop to potentiate breast cancer invasion. Hence, PYK2 could be a potential therapeutic target for a subset of breast cancer patients.

  15. Opposing roles of TGF-β and EGF in the regulation of TRAIL-induced apoptosis in human breast epithelial cells.

    PubMed

    Cano-González, Ana; López-Rivas, Abelardo

    2016-08-01

    Transforming growth factor-beta (TGF-β) induces the epithelial to mesenchymal transition (EMT) in breast epithelial cells and plays an important role in mammary morphogenesis and breast cancer. In non-transformed breast epithelial cells TGF-β antagonizes epidermal growth factor (EGF) action and induces growth inhibition. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to participate in lumen formation during morphogenesis of human breast epithelial cells. Our previous work indicated that sensitivity of human breast epithelial cells to TRAIL can be modulated through the activation of the epidermal growth factor receptor-1 (EGFR). Here, we show that TGF-β opposes EGF-mediated sensitization to TRAIL-induced caspase-8 activation and apoptosis in non-transformed breast epithelial cells. Death-inducing signalling complex (DISC) formation by TRAIL was significantly reduced in cells treated with TGF-β. TGF-β treatment activates cytoprotective autophagy and down-regulates TRAIL-R2 expression at the cell surface by promoting the intracellular accumulation of this receptor. Lastly, we demonstrate that EMT is not involved in the inhibitory effect of TGF-β on apoptosis by TRAIL. Together, the data reveal a fine regulation by EGF and TGF-β of sensitivity of human breast epithelial cells to TRAIL which may be relevant during morphogenesis.

  16. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes

    PubMed Central

    Nakase, Ikuhiko; Kobayashi, Nahoko Bailey; Takatani-Nakase, Tomoka; Yoshida, Tetsuhiko

    2015-01-01

    Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems. PMID:26036864

  17. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes.

    PubMed

    Nakase, Ikuhiko; Kobayashi, Nahoko Bailey; Takatani-Nakase, Tomoka; Yoshida, Tetsuhiko

    2015-06-03

    Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems.

  18. MIG6 is MEK-regulated and affects EGF-induced migration in mutant NRAS melanoma

    PubMed Central

    Vu, Ha Linh; Rosenbaum, Sheera; Capparelli, Claudia; Purwin, Timothy J.; Davies, Michael A.; Berger, Adam C.; Aplin, Andrew E.

    2015-01-01

    Activating mutations in NRAS are frequent driver events in cutaneous melanoma. NRAS is a GTP-binding protein, whose most well-characterized downstream effector is RAF leading to activation of MEK-ERK1/2 signaling. While there are no FDA-approved targeted therapies for melanoma patients with a primary mutation in NRAS, one form of targeted therapy that has been explored is MEK inhibition. In clinical trials, MEK inhibitors have shown disappointing efficacy in mutant NRAS patients, the reasons for which are unclear. To explore the effects of MEK inhibitors in mutant NRAS melanoma, we utilized a high-throughput reverse-phase protein array (RPPA) platform to identify signaling alterations. RPPA analysis of phospho-proteomic changes in mutant NRAS melanoma in response to trametinib indicated a compensatory increase in AKT signaling and decreased expression of mitogen-inducible gene 6 (MIG6), a negative regulator of EGFR/ERBB receptors. MIG6 expression did not alter the growth or survival properties of mutant NRAS melanoma cells. Rather, we identified a role for MIG6 as a negative regulator of EGF-induced signaling and cell migration and invasion. In MEK inhibited cells, further depletion of MIG6 increased migration and invasion, whereas MIG6 expression decreased these properties. Therefore, a decrease in MIG6 may promote the migration and invasiveness of MEK-inhibited mutant NRAS melanoma especially in response to EGF stimulation. PMID:26967478

  19. Localization of transforming growth factor alpha and its receptor in gastric mucosal cells. Implications for a regulatory role in acid secretion and mucosal renewal.

    PubMed Central

    Beauchamp, R D; Barnard, J A; McCutchen, C M; Cherner, J A; Coffey, R J

    1989-01-01

    Transforming growth factor alpha (TGF alpha) shares with epidermal growth factor (EGF) structural homology (35%), a common cell-surface membrane receptor (TGF alpha/EGF receptor), and a nearly identical spectrum of biological activity, including inhibition of gastric acid secretion. Herein, we report expression of TGF alpha mRNA in normal gastric mucosa of the adult guinea pig, rat, and dog. TGF alpha mRNA was also detected in matched surgically resected gastric mucosa and adjacent gastric carcinoma from 10 patients, and in gastric mucosa adjacent to a benign ulcer from an additional patient. TGF alpha protein was quantitated by radioimmunoassay and was present in tumor and adjacent mucosa. TGF alpha/EGF receptor mRNA was also detected in gastric mucosa from all species studied. Localization of TGF alpha and TGF alpha/EGF receptor mRNA expression was examined in samples of unfractionated guinea pig gastric mucosa and from chief cell-enriched and parietal cell-enriched fractions. All samples exhibited TGF alpha and TGF alpha/EGF receptor expression. The TGF alpha signal was greatest in the parietal cell fraction (5.8-fold increase), but was also enhanced in the chief cell fraction (1.9-fold increase) relative to the unfractionated gastric mucosa. Like TGF alpha expression, TGF alpha/EGF receptor mRNA expression was most intense in the parietal cell-enriched fraction (7.8-fold increase), but was also increased in the chief cell-enriched fraction (2.7-fold increase) relative to the unfractionated guinea pig gastric mucosa. We conclude that TGF alpha and TGF alpha/EGF receptor genes are expressed in normal adult mammalian gastric mucosa. These findings, when interpreted in light of described actions of TGF alpha and EGF, provide evidence that local production of TGF alpha could play an important role in the regulation of acid secretion and mucosal renewal in the stomach. Images PMID:2760208

  20. The cell-penetrating peptide domain from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) has anti-inflammatory activity in vitro and in vivo

    SciTech Connect

    Lee, Jue-Yeon; Seo, Yoo-Na; Park, Hyun-Jung; Park, Yoon-Jeong; Chung, Chong-Pyoung

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer HBP sequence identified from HB-EGF has cell penetration activity. Black-Right-Pointing-Pointer HBP inhibits the NF-{kappa}B dependent inflammatory responses. Black-Right-Pointing-Pointer HBP directly blocks phosphorylation and degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer HBP inhibits nuclear translocation of NF-{kappa}B p65 subunit. -- Abstract: A heparin-binding peptide (HBP) sequence from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified and was shown to exhibit cell penetration activity. This cell penetration induced an anti-inflammatory reaction in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. HBP penetrated the cell membrane during the 10 min treatment and reduced the LPS-induced production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cytokines (TNF-{alpha} and IL-6) in a concentration-dependent manner. Additionally, HBP inhibited the LPS-induced upregulation of cytokines, including TNF-{alpha} and IL-6, and decreased the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. HBP inhibited NF-{kappa}B-dependent inflammatory responses by directly blocking the phosphorylation and degradation of I{kappa}B{alpha} and by subsequently inhibiting the nuclear translocation of the p65 subunit of NF-{kappa}B. Taken together, this novel HBP may be potentially useful candidate for anti-inflammatory treatments and can be combined with other drugs of interest to transport attached molecules into cells.

  1. A comparative study of affibody, panitumumab, and EGF for near-infrared fluorescence imaging of EGFR- and EGFRvIII-expressing tumors.

    PubMed

    Gong, Haibiao; Kovar, Joy L; Cheung, Lael; Rosenthal, Eben L; Olive, D Michael

    2014-02-01

    Aberrant overexpression and/or activation of epidermal growth factor receptor (EGFR) is associated with many types of cancers. EGFR variant III (EGFRvIII) is a common in-frame deletion mutant, which lacks a large part of the extracellular portion (exons 2-7), including components of the ligand-binding domain. Although EGFR has been extensively studied as a molecular imaging target, information about EGFRvIII-targeted molecular imaging is lacking. In this study, the EGFR-specific affibody, therapeutic antibody panitumumab, and ligand EGF were labeled with IRDye 800CW (Ex/Em: 774/789 nm), yielding Aff800, Pan800, and EGF800, respectively. The binding affinities of the labeled agents were compared in cell-based assays using a rat glioma cell line F98 parental (F98-p) lacking EGFR expression, and 2 F98-derived transgenic cell lines expressing EGFR or EGFRvIII (designated as F98-EGFR and F98-vIII, respectively). Results showed that all agents could bind to F98-EGFR, with Pan800 having the highest binding affinity, followed by Aff800 and EGF800. Pan800 and Aff800, but not EGF800, also bound to F98-vIII. In vivo animal imaging demonstrated that compared with F98-p tumors, F98-EGFR tumors generated higher signals with all three agents. However, in the case of F98-vIII, only Pan800 and Aff800 signals were higher. Analysis of tissue lysates showed that a large portion of Pan800 was degraded into small fragments in F98-EGFR and F98-vIII tumors, possibly due to proteolytic digestion after its specific binding and internalization. In conclusion, Pan800 and Aff800 could be used as imaging agents for both wild-type EGFR and EGFRvIII, whereas EGF800 only targets wild-type EGFR. PMID:24100437

  2. INSULIN INDUCED EPIDERMAL GROWTH FACTOR ACTIVATION IN VASCULAR SMOOTH MUSCLE CELLS IS ADAM-DEPENDENT

    PubMed Central

    Roztocil, Elisa; Nicholl, Suzanne M.; Davies, Mark G.

    2008-01-01

    Background With the rise in metabolic syndrome, understanding the role of insulin signaling within the cells of vasculature has become more important but yet remains poorly defined. The study examines the role of insulin actions on a pivotal cross-talk receptor, Epidermal Growth Factor Receptor (EGFR). EGFR is transactivated by both G-protein-coupled receptors and receptor linked tyrosine kinases and is key to many of their responses. Objective To determine the pathway of EGFR transactivation by insulin in human coronary smooth muscle cells (VSMC) Methods VSMC were cultured in vitro. Assays of EGFR phosphorylation were examined in response to insulin in the presence and absence of the plasmin inhibitors (e-aminocaproic acid and aprotinin) matrix metalloprotease (MMP) inhibitor GM6001, the ADAM (A Disintegrin And Metalloproteinase Domain) inhibitors TAPI-0 and TAPI-1, Heparin binding epidermal growth factor (HB-EGF) inhibitor, CRM197, HB-EGF inhibitory antibodies, EGF inhibitory antibodies and the EGFR inhibitor AG1478. Results Insulin induced time-dependent EGFR phosphorylation, which was inhibited by AG1478 in a concentration dependent manner. Application of the plasmin inhibitors did not block the response. EGFR phosphorylation by insulin was blocked by inhibition of MMP activity and the ligand HB-EGF. The presence of the ADAM inhibitors, TAPI-0 and TAPI-1 significantly decreased EGFR activation. EGFR phosphorylation by EGF was not interrupted by inhibition of plasmin, MMPs TAPIs, or HB-EGF. Direct blockade of the EGFR prevented activation by both insulin and EGF. Conclusion Insulin can induce transactivation of EGFR by an ADAM-mediated, HB-EGF dependent process. This is the first description of crosstalk via ADAM between insulin and EGFR in vascular SMC. Targeting a pivotal cross-talk receptor such as EGFR, which can be transactivated by both G-protein-coupled receptors and receptor tyrosine kinases is an attractive molecular target. PMID:18656632

  3. Cypermethrin Stimulates GSK3β-Dependent Aβ and p-tau Proteins and Cognitive Loss in Young Rats: Reduced HB-EGF Signaling and Downstream Neuroinflammation as Critical Regulators.

    PubMed

    Maurya, Shailendra Kumar; Mishra, Juhi; Abbas, Sabiya; Bandyopadhyay, Sanghamitra

    2016-03-01

    Pesticide exposure is recognized as a risk factor for Alzheimer's disease (AD). We investigated early signs of AD-like pathology upon exposure to a pyrethroid pesticide, cypermethrin, reported to impair neurodevelopment. We treated weanling rats with cypermethrin (10 and 25 mg/kg) and detected dose-dependent increase in the key proteins of AD, amyloid beta (Aβ), and phospho-tau, in frontal cortex and hippocampus as early as postnatal day 45. Upregulation of Aβ pathway involved an increase in amyloid precursor protein (APP) and its pro-amyloidogenic processing through beta-secretase (BACE) and gamma-secretase. Tau pathway entailed elevation in tau and glycogen-synthase kinase-3-beta (GSK3β)-dependent, phospho-tau. GSK3β emerged as a molecular link between the two pathways, evident from reduction in phospho-tau as well as BACE upon treating GSK3β inhibitor, lithium chloride. Exploring the mechanism revealed an attenuated heparin-binding epidermal growth factor (HB-EGF) signaling and downstream astrogliosis-mediated neuroinflammation to be responsible for inducing Aβ and phospho-tau. Cypermethrin caused a proximal reduction in HB-EGF, which promoted astrocytic nuclear factor kappa B signaling and astroglial activation close to Aβ and phospho-tau. Glial activation stimulated generation of interleukin-1 (IL-1), which upregulated GSK3β, and APP and tau as well, resulting in co-localization of Aβ and phospho-tau with IL-1 receptor. Intracerebral insertion of exogenous HB-EGF restored its own signaling and suppressed neuroinflammation and thereby Aβ and phospho-tau in cypermethrin-exposed rats, proving a central role of reduced HB-EGF signaling in cypermethrin-mediated neurodegeneration. Furthermore, cypermethrin stimulated cognitive impairments, which could be prevented by exogenous HB-EGF. Our data demonstrate that cypermethrin induces premature upregulation of GSK3β-dependent Aβ and tau pathways, where HB-EGF signaling and neuroinflammation serve as

  4. Differential Expression Patterns of EGF, EGFR, and ERBB4 in Nasal Polyp Epithelium

    PubMed Central

    Zhao, Li; Subramaniam, Somasundaram; Yu, Xue Min; Li, Ying Ying; Chen, De Hua; Li, Tian Ying; Shen, Liang; Shi, Li; Wang, De Yun

    2016-01-01

    Epidermal growth factor receptors play an important role in airway epithelial cell growth and differentiation. The current study investigates the expression profiles of EGF, EGFR and ERBB4 in patients with nasal polyps (NP), and their response to glucocorticosteroid (GC) treatment. Fifty patients with NP (40 without GC treatment and 10 with oral GC) and 20 control subjects with septal deviation were recruited into the study. Protein levels of EGF, EGFR, and ERBB4 were evaluated by immune-staining. In healthy nasal epithelium, EGF and EGFR localized within p63+ basal cells, while ERBB4 localized within ciliated cells. GC-naïve NP epithelium showed weak expression of EGF in 90% of samples versus 5% of controls. EGFR was significantly increased in the epithelium with basal cell hyperplasia from GC-naïve NPs (78%, 31/40) compared to controls (23%, 4/17). EGFR was also found in some degranulating goblet cells. ERBB4 expression was significantly higher in hyperplastic epithelium from GC-naïve NPs (65%, 26/40) than in controls (6%, 1/17). GC treatment restored the EGF expression and normalized the EGFR and ERBB4 expression in NPs. Differential expression patterns of EGF, EGFR, and ERBB4 are essential in epithelial restitution and remodeling in nasal epithelium. PMID:27285994

  5. Superparamagnetic iron oxide nanoparticles conjugated with epidermal growth factor (SPION–EGF) for targeting brain tumors

    PubMed Central

    Shevtsov, Maxim A; Nikolaev, Boris P; Yakovleva, Ludmila Y; Marchenko, Yaroslav Y; Dobrodumov, Anatolii V; Mikhrina, Anastasiya L; Martynova, Marina G; Bystrova, Olga A; Yakovenko, Igor V; Ischenko, Alexander M

    2014-01-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) conjugated with recombinant human epidermal growth factor (SPION–EGF) were studied as a potential agent for magnetic resonance imaging contrast enhancement of malignant brain tumors. Synthesized conjugates were characterized by transmission electron microscopy, dynamic light scattering, and nuclear magnetic resonance relaxometry. The interaction of SPION–EGF conjugates with cells was analyzed in a C6 glioma cell culture. The distribution of the nanoparticles and their accumulation in tumors were assessed by magnetic resonance imaging in an orthotopic model of C6 gliomas. SPION–EGF nanosuspensions had the properties of a negative contrast agent with high coefficients of relaxation efficiency. In vitro studies of SPION–EGF nanoparticles showed high intracellular incorporation and the absence of a toxic influence on C6 cell viability and proliferation. Intravenous administration of SPION–EGF conjugates in animals provided receptor-mediated targeted delivery across the blood–brain barrier and tumor retention of the nanoparticles; this was more efficient than with unconjugated SPIONs. The accumulation of conjugates in the glioma was revealed as hypotensive zones on T2-weighted images with a twofold reduction in T2 relaxation time in comparison to unconjugated SPIONs (P<0.001). SPION–EGF conjugates provide targeted delivery and efficient magnetic resonance contrast enhancement of EGFR-overexpressing C6 gliomas. PMID:24421639

  6. Combined effect of gefitinib ('Iressa', ZD1839) and targeted radiotherapy with 211At-EGF.

    PubMed

    Sundberg, Asa Liljegren; Almqvist, Ylva; Orlova, Anna; Blomquist, Erik; Jensen, Holger J; Gedda, Lars; Tolmachev, Vladimir; Carlsson, Jörgen

    2003-10-01

    The EGFR-TKI (epidermal growth factor receptor tyrosine kinase inhibitor) gefitinib ['Iressa' (trademark of the AstraZeneca group of companies), ZD1839] increases the cellular uptake of radiolabelled epidermal growth factor (EGF). We investigated gefitinib treatment combined with astatine-211 EGF targeting in vitro using two cell lines expressing high levels of EGFR: A431 (sensitive to gefitinib) and U343MGaCl2:1 (resistant to gefitinib). In both cell lines, the uptake of 211At-EGF was markedly increased by concomitant treatment with gefitinib. Survival was investigated using both a clonogenic survival assay and a cell growth assay. Combined gefitinib and 211At-EGF treatment reduced the survival of U343 cells 3.5-fold compared with 211At-EGF alone. In A431 cells, 211At-EGF treatment resulted in very low survival, but combined treatment with gefitinib increased the survival by about 20-fold. These results indicate that combined treatment with gefitinib might increase the effect of ligand-mediated radionuclide therapy in gefitinib-resistant tumours and decrease the effect of such therapy in gefitinib-sensitive tumours. PMID:12937952

  7. Constitutive asymmetric dimerization drives oncogenic activation of epidermal growth factor receptor carboxyl-terminal deletion mutants

    PubMed Central

    Park, Angela K.J.; Francis, Joshua M.; Park, Woong-Yang; Park, Joon-Oh; Cho, Jeonghee

    2015-01-01

    Genomic alterations targeting the Epidermal Growth Factor Receptor (EGFR) gene have been strongly associated with cancer pathogenesis. The clinical effectiveness of EGFR targeted therapies, including small molecules directed against the kinase domain such as gefitinib, erlotinib and afatinib, have been proven successful in treating non-small cell lung cancer patients with tumors harboring EGFR kinase domain mutations. Recent large-scale genomic studies in glioblastoma and lung cancer have identified an additional class of oncogenic mutations caused by the intragenic deletion of carboxy-terminal coding regions. Here, we report that combinations of exonic deletions of exon 25 to 28 lead to the oncogenic activation of EGF receptor in the absence of ligand and consequent cellular transformation, indicating a significant role of C-terminal domain in modulating EGFR activation. Furthermore, we show that the oncogenic activity of the resulting C-terminal deletion mutants are efficiently inhibited by EGFR-targeted drugs including erlotinib, afatinib, dacomitinib as well as cetuximab, expanding the therapeutic rationale of cancer genome-based EGFR targeted approaches. Finally, in vivo and in vitro preclinical studies demonstrate that constitutive asymmetric dimerization in mutant EGFR is a key mechanism for oncogenic activation and tumorigenesis by C-terminal deletion mutants. Therefore, our data provide compelling evidence for oncogenic activation of C-terminal deletion mutants at the molecular level and we propose that C-terminal deletion status of EGFR can be considered as a potential genomic marker for EGFR-targeted therapy. PMID:25826094

  8. Using Nuclear Receptor Activity to Stratify Hepatocarcinogens

    PubMed Central

    Shah, Imran; Houck, Keith; Judson, Richard S.; Kavlock, Robert J.; Martin, Matthew T.; Reif, David M.; Wambaugh, John; Dix, David J.

    2011-01-01

    Background Nuclear receptors (NR) are a superfamily of ligand-activated transcription factors that control a range of cellular processes. Persistent stimulation of some NR is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. Here we report on a systematic analysis of new in vitro human NR activity data on 309 environmental chemicals in relationship to their liver cancer-related chronic outcomes in rodents. Results The effects of 309 environmental chemicals on human constitutive androstane receptors (CAR/NR1I3), pregnane X receptor (PXR/NR1I2), aryl hydrocarbon receptor (AhR), peroxisome proliferator-activated receptors (PPAR/NR1C), liver X receptors (LXR/NR1H), retinoic X receptors (RXR/NR2B) and steroid receptors (SR/NR3) were determined using in vitro data. Hepatic histopathology, observed in rodents after two years of chronic treatment for 171 of the 309 chemicals, was summarized by a cancer lesion progression grade. Chemicals that caused proliferative liver lesions in both rat and mouse were generally more active for the human receptors, relative to the compounds that only affected one rodent species, and these changes were significant for PPAR (p0.001), PXR (p0.01) and CAR (p0.05). Though most chemicals exhibited receptor promiscuity, multivariate analysis clustered them into relatively few NR activity combinations. The human NR activity pattern of chemicals weakly associated with the severity of rodent liver cancer lesion progression (p0.05). Conclusions The rodent carcinogens had higher in vitro potency for human NR relative to non-carcinogens. Structurally diverse chemicals with similar NR promiscuity patterns weakly associated with the severity of rodent liver cancer progression. While these results do not prove the role of NR activation in human liver cancer, they do have implications for nuclear receptor chemical biology and provide insights into putative toxicity pathways. More importantly, these findings suggest the

  9. The non-receptor tyrosine kinase Ack1 regulates the fate of activated EGFR by inducing trafficking to the p62/NBR1 pre-autophagosome.

    PubMed

    Jones, Sylwia; Cunningham, Debbie L; Rappoport, Joshua Z; Heath, John K

    2014-03-01

    Growth factor signalling regulates multiple cellular functions and its misregulation has been linked to the development and progression of cancer. Ack1 (activated Cdc42-associated kinase 1, also known as TNK2) is a non-receptor tyrosine kinase that has been implicated in trafficking and degradation of epidermal growth factor receptor (EGFR), yet its precise functions remain elusive. In this report, we investigate the role of Ack1 in EGFR trafficking and show that Ack1 partially colocalises to Atg16L-positive structures upon stimulation with EGF. These structures are proposed to be the isolation membranes that arise during formation of autophagosomes. In addition, we find that Ack1 colocalises and interacts with sequestosome 1 (p62/SQSTM1), a receptor for selective autophagy, through a ubiquitin-associated domain, and this interaction decreases upon treatment with EGF, thus suggesting that Ack1 moves away from p62/SQSTM1 compartments. Furthermore, Ack1 interacts and colocalises with NBR1, another autophagic receptor, and this colocalisation is enhanced in the presence of ectopically expressed p62/SQSTM1. Finally, knockdown of Ack1 results in accelerated localisation of EGFR to lysosomes upon treatment with EGF. Structure-function analyses of a panel of Ack1 deletion mutants revealed key mechanistic aspects of these relationships. The Mig6-homology domain and clathrin-binding domain both contribute to colocalisation with EGFR, whereas the UBA domain is essential for colocalisation with p62/SQSTM1, but not NBR1. Taken together, our studies demonstrate a novel role for Ack1 in diverting activated EGFR into a non-canonical degradative pathway, marked by association with p62/SQSTM1, NBR1 and Atg16L.

  10. TERATOGENICITY OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN MICE LACKING THE EXPRESSION OF EGF AND/OR TGFALPHA

    EPA Science Inventory

    Abstract
    2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure produces hydronephrosis and cleft palate in mice. These responses are correlated with disruption of expression of epidermal growth factor (EGF) receptor ligands, primarily EGF and transforming growth factor-alpha ...

  11. Graphene Enhances Cellular Proliferation through Activating the Epidermal Growth Factor Receptor.

    PubMed

    Liu, Wei; Sun, Cheng; Liao, Chunyang; Cui, Lin; Li, Haishan; Qu, Guangbo; Yu, Wenlian; Song, Naining; Cui, Yuan; Wang, Zheng; Xie, Wenping; Chen, Huiming; Zhou, Qunfang

    2016-07-27

    Graphene has promising applications in food packaging, water purification, and detective sensors for contamination monitoring. However, the biological effects of graphene are not fully understood. It is necessary to clarify the potential risks of graphene exposure to humans through diverse routes, such as foods. In the present study, graphene, as the model nanomaterial, was used to test its potential effects on the cell proliferation based on multiple representative cell lines, including HepG2, A549, MCF-7, and HeLa cells. Graphene was characterized by Raman spectroscopy, particle size analysis, atomic force microscopy, and transmission electron microscopy. The cellular responses to graphene exposure were evaluated using flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and alamarBlue assays. Rat cerebral astrocyte cultures, as the non-cancer cells, were used to assess the potential cytotoxicity of graphene as well. The results showed that graphene stimulation enhanced cell proliferation in all tested cell cultures and the highest elevation in cell growth was up to 60%. A western blot assay showed that the expression of epidermal growth factor (EGF) was upregulated upon graphene treatment. The phosphorylation of EGF receptor (EGFR) and the downstream proteins, ShC and extracellular regulating kinase (ERK), were remarkably induced, indicating that the activation of the mitogen-activated protein kinase (MAPK)/ERK signaling pathway was triggered. The activation of PI3 kinase p85 and AKT showed that the PI3K/AKT signaling pathway was also involved in graphene-induced cell proliferation, causing the increase of cell ratios in the G2/M phase. No influences on cell apoptosis were observed in graphene-treated cells when compared to the negative controls, proving the low cytotoxicity of this emerging nanomaterial. The findings in this study revealed the potential cellular biological effect of graphene, which may give useful hints on its biosafety

  12. Protein-tyrosine-phosphatase 2C is phosphorylated and inhibited by 44-kDa mitogen-activated protein kinase.

    PubMed Central

    Peraldi, P; Zhao, Z; Filloux, C; Fischer, E H; Van Obberghen, E

    1994-01-01

    Protein-tyrosine-phosphatase 2C (PTP2C, also named SHPTP2, SHPTP3, or PTP1D) is a cytosolic enzyme with two Src homology 2 domains. We have investigated its regulation by phosphorylation in PC12 rat pheochromocytoma cells. In untreated cells, PTP2C was phosphorylated predominantly on serine residues. A 5-min treatment with epidermal growth factor (EGF) induced an increase in phosphorylation on threonine and, to a lesser degree, on serine. After 45 min of exposure to EGF, PTP2C phosphorylation returned to basal levels. Using an in vitro kinase assay, we found that the 44-kDa mitogen-activated protein kinase, p44mapk, phosphorylated PTP2C on serine and threonine residues. This phosphorylation resulted in a pronounced inhibition of PTP2C enzyme activity measured with phosphorylated EGF receptors as substrate. Moreover, in intact PC12 cells, PTP2C was also inhibited following a short EGF treatment, but its activity returned to normal when the exposure to EGF was maintained for 45 min. The profile of this response to EGF can be inversely correlated to that of the stimulatory action of EGF on p44mapk. These data suggest that the EGF-induced regulation of PTP2C activity is mediated by p44mapk. These findings provide evidence for an additional role of the mitogen-activated protein kinase cascade--namely, the regulation of a PTP. Images PMID:8197172

  13. THE CONSUMPTION OF ACAI PULP CHANGES THE CONCENTRATIONS OF PLASMINOGEN ACTIVATOR INHIBITOR-1 AND EPIDERMAL GROWTH FACTOR (EGF) IN APPARENTLY HEALTHY WOMEN.

    PubMed

    de Sousa Pereira, Izabelle; Moreira Cançado Mascarenhas Pontes, Tereza Cristina; Lima Vieira, Renata Adrielle; de Freitas Folly, Gilce Andrezza; Cacilda Silva, Fernanda; Pereira de Oliveira, Fernando Luiz; Ferreira do Amaral, Joana; Nascimento de Freitas, Renata; Pinheiro Volp, Ana Carolina

    2015-08-01

    Introducción: la obesidad, que se caracteriza por el exceso de adiposidad, se asocia con disfunción endotelial y posible estado inflamatorio con liberación de citoquinas que determinan la función endotelial y pueden desencadenar enfermedades crónicas. El patrón de dieta está asociado con la síntesis de estas citoquinas. Los frutos de el acai, que es rico en flavonoides, tienen un efecto directo y positivo en el control de este proceso inflamatorio a través de los ejercicios de la capacidad antioxidante. Objetivo: evaluar el efecto del consumo de pulpa de acai en los marcadores inflamatorios, las medidas antropométricas, la composición corporal y los parámetros bioquímicos y dietéticos en mujeres sanas. Métodos: cuarenta mujeres fueron divididas en 25 eutróficas y 15 con sobrepeso. Se las adeministró 200 g de pulpa de acai durante 4 semanas. Antes y después de la intervención se evaluaron: medidas antropométricas, composición corporal, marcadores inflamatorios, datos bioquímicos, ingesta dietética y antioxidantes en la dieta. Resultados y discusión: después de la intervención, hubo un aumento significativo de EGF (p = 0,021) y PAI-1 (p = 0,011) en las mujeres con sobrepeso. Por otra parte, en las mujeres eutróficas hubo aumento del peso corporal (p = 0,031), el índice de masa corporal (p = 0,028), el porcentaje de grasa del tronco (p = 0,003) y el espesor del pliegue cutáneo del tríceps (p = 0,046). Sin embargo, el espesor del pliegue cutáneo (p = 0,018) y la grasa corporal total (p = 0,016) se redujeron en las mujeres con sobrepeso. Hubo una reducción de la proteína total (p = 0,049) debida a la disminución de globulina (p = 0,005), pero el estado nutricional se mantuvo en el grupo eutrófico. Conclusión: la ingesta de 200 g de pulpa de acai modula el EGF y PAI-1 de expresión, posiblemente por la modulación del acai en los parámetros de la composición corporal, la dieta, clínicos, bioquímicos e inflamatorios, lo que dio

  14. Conditional loss of heparin-binding EGF-like growth factor results in enhanced liver fibrosis after bile duct ligation in mice

    SciTech Connect

    Takemura, Takayo; Yoshida, Yuichi; Kiso, Shinichi; Kizu, Takashi; Furuta, Kunimaro; Ezaki, Hisao; Hamano, Mina; Egawa, Mayumi; Chatani, Norihiro; Kamada, Yoshihiro; Imai, Yasuharu; Higashiyama, Shigeki; Iwamoto, Ryo; Mekada, Eisuke; Takehara, Tetsuo

    2013-07-26

    Highlights: •HB-EGF expression was increased during the development of liver fibrosis. •Conditional HB-EGF knockout mouse showed enhanced experimental liver fibrosis. •HB-EGF antagonized TGF-β-induced activation of hepatic stellate cells. •We report a possible protective role of HB-EGF in cholestatic liver fibrosis. -- Abstract: Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.

  15. A matricellular protein and EGF-like repeat signalling in the social amoebozoan Dictyostelium discoideum.

    PubMed

    Huber, Robert J; O'Day, Danton H

    2012-12-01

    Matricellular proteins interact with the extracellular matrix (ECM) and modulate cellular processes by binding to cell surface receptors and initiating intracellular signal transduction. Their association with the ECM and the ability of some members of this protein family to regulate cell motility have opened up new avenues of research to investigate their functions in normal and diseased cells. In this review, we summarize the research on CyrA, an ECM calmodulin-binding protein in Dictyostelium. CyrA is proteolytically cleaved into smaller EGF-like (EGFL) repeat containing cleavage products during development. The first EGFL repeat of CyrA binds to the cell surface and activates a novel signalling pathway that modulates cell motility in this model organism. The similarity of CyrA to the most well-characterized matricellular proteins in mammals allows it to be designated as the first matricellular protein identified in Dictyostelium. PMID:22782112

  16. A matricellular protein and EGF-like repeat signalling in the social amoebozoan Dictyostelium discoideum.

    PubMed

    Huber, Robert J; O'Day, Danton H

    2012-12-01

    Matricellular proteins interact with the extracellular matrix (ECM) and modulate cellular processes by binding to cell surface receptors and initiating intracellular signal transduction. Their association with the ECM and the ability of some members of this protein family to regulate cell motility have opened up new avenues of research to investigate their functions in normal and diseased cells. In this review, we summarize the research on CyrA, an ECM calmodulin-binding protein in Dictyostelium. CyrA is proteolytically cleaved into smaller EGF-like (EGFL) repeat containing cleavage products during development. The first EGFL repeat of CyrA binds to the cell surface and activates a novel signalling pathway that modulates cell motility in this model organism. The similarity of CyrA to the most well-characterized matricellular proteins in mammals allows it to be designated as the first matricellular protein identified in Dictyostelium.

  17. Activation of Neuropeptide FF Receptors by Kisspeptin Receptor Ligands.

    PubMed

    Oishi, Shinya; Misu, Ryosuke; Tomita, Kenji; Setsuda, Shohei; Masuda, Ryo; Ohno, Hiroaki; Naniwa, Yousuke; Ieda, Nahoko; Inoue, Naoko; Ohkura, Satoshi; Uenoyama, Yoshihisa; Tsukamura, Hiroko; Maeda, Kei-Ichiro; Hirasawa, Akira; Tsujimoto, Gozoh; Fujii, Nobutaka

    2011-01-13

    Kisspeptin is a member of the RFamide neuropeptide family that is implicated in gonadotropin secretion. Because kisspeptin-GPR54 signaling is implicated in the neuroendocrine regulation of reproduction, GPR54 ligands represent promising therapeutic agents against endocrine secretion disorders. In the present study, the selectivity profiles of GPR54 agonist peptides were investigated for several GPCRs, including RFamide receptors. Kisspeptin-10 exhibited potent binding and activation of neuropeptide FF receptors (NPFFR1 and NPFFR2). In contrast, short peptide agonists bound with much lower affinity to NPFFRs while showing relatively high selectivity toward GPR54. The possible localization of secondary kisspeptin targets was also demonstrated by variation in the levels of GnRH release from the median eminence and the type of GPR54 agonists used. Negligible affinity of the reported NPFFR ligands to GPR54 was observed and indicates the unidirectional cross-reactivity between both ligands.

  18. Mechanism of FGF receptor dimerization and activation

    PubMed Central

    Sarabipour, Sarvenaz; Hristova, Kalina

    2016-01-01

    Fibroblast growth factors (fgfs) are widely believed to activate their receptors by mediating receptor dimerization. Here we show, however, that the FGF receptors form dimers in the absence of ligand, and that these unliganded dimers are phosphorylated. We further show that ligand binding triggers structural changes in the FGFR dimers, which increase FGFR phosphorylation. The observed effects due to the ligands fgf1 and fgf2 are very different. The fgf2-bound dimer structure ensures the smallest separation between the transmembrane (TM) domains and the highest possible phosphorylation, a conclusion that is supported by a strong correlation between TM helix separation in the dimer and kinase phosphorylation. The pathogenic A391E mutation in FGFR3 TM domain emulates the action of fgf2, trapping the FGFR3 dimer in its most active state. This study establishes the existence of multiple active ligand-bound states, and uncovers a novel molecular mechanism through which FGFR-linked pathologies can arise. PMID:26725515

  19. Synergistic and multidimensional regulation of plasminogen activator inhibitor type 1 expression by transforming growth factor type β and epidermal growth factor

    SciTech Connect

    Song, Xiaoling; Thalacker, F.W.; Nilsen-Hamilton, Marit

    2012-04-06

    The major physiological inhibitor of plasminogen activator, type I plasminogen activator inhibitor (PAI-1), controls blood clotting and tissue remodeling events that involve cell migration. Transforming growth factor type β (TGFβ) and epidermal growth factor (EGF) interact synergistically to increase PAI-1 mRNA and protein levels in human HepG2 and mink Mv1Lu cells. Other growth factors that activate tyrosine kinase receptors can substitute for EGF. EGF and TGFβ regulate PAI-1 by synergistically activating transcription, which is further amplified by a decrease in the rate of mRNA degradation, the latter being regulated only by EGF. The combined effect of transcriptional activation and mRNA stabilization results in a rapid 2-order of magnitude increase in the level of PAI-1. TGFβ also increases the sensitivity of the cells to EGF, thereby recruiting the cooperation of EGF at lower than normally effective concentrations. The contribution of EGF to the regulation of PAI-1 involves the MAPK pathway, and the synergistic interface with the TGFβ pathway is downstream of MEK1/2 and involves phosphorylation of neither ERK1/2 nor Smad2/3. Synergism requires the presence of both Smad and AP-1 recognition sites in the promoter. This work demonstrates the existence of a multidimensional cellular mechanism by which EGF and TGFβ are able to promote large and rapid changes in PAI-1 expression.

  20. Interference by 2,3,7,8-tetrachlorodibenzo-p-dioxin with cultured mouse submandibular gland branching morphogenesis involves reduced epidermal growth factor receptor signaling

    SciTech Connect

    Kiukkonen, Anu . E-mail: anummela@mappi.helsinki.fi; Sahlberg, Carin; Partanen, Anna-Maija; Alaluusua, Satu; Pohjanvirta, Raimo; Tuomisto, Jouko; Lukinmaa, Pirjo-Liisa

    2006-05-01

    Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to mouse embryonic teeth, sharing features of early development with salivary glands in common, involves enhanced apoptosis and depends on the expression of epidermal growth factor (EGF) receptor. EGF receptor signaling, on the other hand, is essential for salivary gland branching morphogenesis. To see if TCDD impairs salivary gland morphogenesis and if the impairment is associated with EGF receptor signaling, we cultured mouse (NMRI) E13 submandibular glands with TCDD or TCDD in combination with EGF or fibronectin (FN), both previously found to enhance branching morphogenesis. Explants were examined stereomicroscopically and processed to paraffin sections. TCDD exposure impaired epithelial branching and cleft formation, resulting in enlarged buds. The glands were smaller than normal. EGF and FN alone concentration-dependently stimulated or inhibited branching morphogenesis but when co-administered with TCDD, failed to compensate for its effect. TCDD induced cytochrome P4501A1 expression in the glandular epithelium, indicating activation of the aryl hydrocarbon receptor. TCDD somewhat increased epithelial apoptosis as observed by terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labeling method but the increase could not be correlated with morphological changes. The frequency of proliferating cells was not altered. Corresponding to the reduced cleft sites in TCDD-exposed explants, FN immunoreactivity in the epithelium was reduced. The results show that TCDD, comparably with EGF and FN at morphogenesis-inhibiting concentrations, impaired salivary gland branching morphogenesis in vitro. Together with the failure of EGF and FN at morphogenesis-stimulating concentrations to compensate for the effect of TCDD this implies that TCDD toxicity to developing salivary gland involves reduced EGF receptor signaling.

  1. Role of the 6-20 disulfide bridge in the structure and activity of epidermal growth factor.

    PubMed Central

    Barnham, K. J.; Torres, A. M.; Alewood, D.; Alewood, P. F.; Domagala, T.; Nice, E. C.; Norton, R. S.

    1998-01-01

    Two synthetic analogues of murine epidermal growth factor, [Abu6, 20] mEGF4-48 (where Abu denotes amino-butyric acid) and [G1, M3, K21, H40] mEGF1-48, have been investigated by NMR spectroscopy. [Abu6, 20] mEGF4-48 was designed to determine the contribution of the 6-20 disulfide bridge to the structure and function of mEGF. The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. Significant structural differences were observed near the N-terminus, however, with the direction of the polypeptide chain between residues four and nine being altered such that these residues were now located on the opposite face of the main beta-sheet from their position in native mEGF. Thermal denaturation experiments also showed that the structure of [Abu6, 20] mEGF4-48 was less stable than that of mEGF. Removal of this disulfide bridge resulted in a significant loss of both mitogenic activity in Balb/c 3T3 cells and receptor binding on A431 cells compared with native mEGF and mEGF4-48, implying that the structural changes in [Abu6, 20] mEGF4-48, although limited to the N-terminus, were sufficient to interfere with receptor binding. The loss of binding affinity probably arose mainly from steric interactions of the dislocated N-terminal region with part of the receptor binding surface of EGF. [G1, M3, K21, H40] mEGF1-48 was also synthesized in order to compare the synthetic polypeptide with the corresponding product of recombinant expression. Its mitogenic activity in Balb/c 3T3 cells was similar to that of native mEGF and analysis of its 1H chemical shifts suggested that its structure was also very similar to native. PMID:10082370

  2. Coagulation, Protease Activated Receptors and Viral Myocarditis

    PubMed Central

    Antoniak, Silvio; Mackman, Nigel

    2013-01-01

    The coagulation protease cascade plays an essential role in hemostasis. In addition, a clot contributes to host defense by limiting the spread of pathogens. Coagulation proteases induce intracellular signaling by cleavage of cell surface receptors called protease-activated receptors (PARs). These receptors allow cells to sense changes in the extracellular environment, such as infection. Viruses activate the coagulation cascade by inducing tissue factor expression and by disrupting the endothelium. Virus infection of the heart can cause myocarditis, cardiac remodeling and heart failure. Recent studies using a mouse model have shown that tissue factor, thrombin and PAR-1 signaling all positively regulate the innate immune during viral myocarditis. In contrast, PAR-2 signaling was found to inhibit interferon-β expression and the innate immune response. These observations suggest that anticoagulants may impair the innate immune response to viral infection and that inhibition of PAR-2 may be a new target to reduce viral myocarditis.. PMID:24203054

  3. Modulators of androgen and estrogen receptor activity.

    PubMed

    Clarke, Bart L; Khosla, Sundeep

    2010-01-01

    This review focuses on significant recent findings regarding modulators of androgen and estrogen receptor activity. Selective androgen receptor modulators (SARMs) interact with androgen receptors (ARs), and selective estrogen receptor modulators (SERMs) interact with estrogen receptors (ERs), with variable tissue selectivity. SERMs, which interact with both ERб and ERв in a tissue-specific manner to produce diverse outcomes in multiple tissues, continue to generate significant interest for clinical application. Development of SARMs for clinical application has been slower to date because of potential adverse effects, but these diverse compounds continue to be investigated for use in disorders in which modulation of the AR is important. SARMs have been investigated mostly at the basic and preclinical level to date, with few human clinical trials published. These compounds have been evaluated mostly for application in different stages of prostate cancer to date, but they hold promise for multiple other applications. Publication of the large STAR and RUTH clinical trials demonstrated that the SERMs tamoxifen and raloxifene have interesting similarities and differences in tissues that contain ERs. Lasofoxifene, bazedoxifene, and arzoxifene are newer SERMs that have been demonstrated in clinical trials to more potently increase bone mineral density and lower serum cholesterol values than tamoxifen or raloxifene. Both SARMs and SERMs hold great promise for therapeutic use in multiple disorders in which tissue-specific effects are mediated by their respective receptors.

  4. EFFECTS OF EPIDERMAL GROWTH FACTOR (EGF), TRANSFORMING GROWTH FACTOR- (TGF), AND 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN ON FUSION OF EMBRYONIC PALATES IN SERUM-FREE ORGAN CULTURE USING WILD-TYPE, EGF KNOCKOUT, AND TGF KNOCKOUT MOUSE STRAINS

    EPA Science Inventory

    Backround: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in mice, producing cleft palate (CP). TCDD exposure disrupts expression of epidermal growth factor (EGF) receptor, EGF, and transforming growth factor- (TGF) in the palate and affects proliferation and different...

  5. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

    SciTech Connect

    Nagata, Yosuke Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

    2014-08-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its

  6. Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells.

    PubMed Central

    Pietrzkowski, Z; Sell, C; Lammers, R; Ullrich, A; Baserga, R

    1992-01-01

    BALB/c3T3 cells are exquisitely growth regulated and require platelet-derived growth factor, epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1) for growth. When BALB/c3T3 cells are transfected with plasmids constitutively expressing both EGF and the human IGF-1 receptor mRNAs, the cells are capable of growing in serum-free medium without the addition of any exogenous growth factor. These cells, called p5 cells, can grow for prolonged periods in serum-free medium. BALB/c3T3 cells transfected with only the IGF-1 receptor expression plasmid (p6 cells) do not grow in serum-free medium but do grow if IGF-1 (or insulin in supraphysiological concentrations) is added. p6 cells also grow in response to EGF, confirming that the combination of EGF and an overexpressed IGF-1 receptor is sufficient for the growth of 3T3 cells. We have found that in EGF-stimulated p6 cells there is an increase in the expression of IGF-1 mRNA, that IGF-1 is secreted into the medium, and that the growth of p5 cells and EGF-stimulated p6 cells is inhibited by exposure to antisense oligodeoxynucleotides to IGF-1 receptor RNA. Finally, while cells constitutively expressing both EGF and EGF receptor RNAs grow, albeit modestly, in serum-free medium, their growth is also inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA. In contrast, in cells overexpressing the IGF-1 receptor, IGF-1-mediated cell growth occurs independently of the platelet-derived growth factor and EGF receptors (Z. Pietrzkowski, R. Lammers, G. Carpenter, A. M. Soderquist, M. Limardo, P. D. Phillips, A. Ullrich, and R. Baserga, Cell Growth Differ. 3:199-205, 1992, and this paper). These data indicate that an important role for EGF is participation in the activation of an autocrine loop based on the IGF-1-IGF-1 receptor interaction, which is obligatory for the proliferation of 3T3 cells. Images PMID:1324408

  7. Skeletal muscle stem cells express anti-apoptotic ErbB receptors during activation from quiescence

    SciTech Connect

    Golding, Jon P. . E-mail: j.p.golding@open.ac.uk; Calderbank, Emma; Partridge, Terence A.; Beauchamp, Jonathan R.

    2007-01-15

    To be effective for tissue repair, satellite cells (the stem cells of adult muscle) must survive the initial activation from quiescence. Using an in vitro model of satellite cell activation, we show that erbB1, erbB2 and erbB3, members of the EGF receptor tyrosine kinase family, appear on satellite cells within 6 h of activation. We show that signalling via erbB2 provides an anti-apoptotic survival mechanism for satellite cells during the first 24 h, as they progress to a proliferative state. Inhibition of erbB2 signalling with AG825 reduced satellite cell numbers, concomitant with elevated caspase-8 activation and TUNEL labelling of apoptotic satellite cells. In serum-free conditions, satellite cell apoptosis could be largely prevented by a mixture of erbB1, erbB3 and erbB4 ligand growth factors, but not by neuregulin alone (erbB3/erbB4 ligand). Furthermore, using inhibitors specific to discrete intracellular signalling pathways, we identify MEK as a pro-apoptotic mediator, and the erbB-regulated factor STAT3 as an anti-apoptotic mediator during satellite cell activation. These results implicate erbB2 signalling in the preservation of a full compliment of satellite cells as they activate in the context of a damaged muscle.

  8. Using Nuclear Receptor Activity to Stratify Hepatocarcinogens

    EPA Science Inventory

    Nuclear receptors (NR) are a superfamily of ligand-activated transcription factors that control a range of cellular processes. Persistent stimulation of some NR is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. Here we report on a systematic an...

  9. Epidermal growth factor stimulates the disruption of gap junctional communication and connexin43 phosphorylation independent of 12-0-tetradecanoylphorbol 13-acetate-sensitive protein kinase C: the possible involvement of mitogen-activated protein kinase.

    PubMed

    Kanemitsu, M Y; Lau, A F

    1993-08-01

    We previously reported that epidermal growth factor (EGF) induced the disruption of gap junctional communication (gjc) and serine phosphorylation of connexin43 (Cx43) in T51B rat liver epithelial cells. However, the cascade of events linking EGF receptor activation to these particular responses have not been fully characterized. Furthermore, the serine kinase(s) acting directly on Cx43 remain unidentified. In the current study, we demonstrate that downmodulation of 12-0-tetradecanoylphorbol 13-acetate (TPA)-sensitive protein kinase C (PKC) activity does not affect EGF's ability to reduce junctional permeability or phosphorylate Cx43 in T51B cells. EGF in the presence or absence of chronic TPA treatment stimulated marked increases in Cx43 phosphorylation on numerous sites as determined by two-dimensional tryptic phosphopeptide mapping. Computer-assisted sequence analysis of Cx43 identified several protein kinase phosphorylation consensus sites including two sites for mitogen-activated protein (MAP) kinase. EGF stimulated activation of MAP kinase in a time- and dose-dependent manner where the kinetics of kinase activity corroborated its possible involvement in mediating EGF's effects. Moreover, purified MAP kinase directly phosphorylated Cx43 on serine residues in vitro. Two-dimensional tryptic and chymotryptic phosphopeptide mapping demonstrated that the in vitro phosphopeptides represented a specific subset of the in vivo phosphopeptides produced in response to EGF after chronic TPA treatment. Therefore, EGF-induced disruption of gjc and phosphorylation of Cx43 may be mediated in part by MAP kinase in vivo.

  10. The Characteristics Variation of Hepatic Progenitors after TGF-β1-Induced Transition and EGF-Induced Reversion.

    PubMed

    Wang, Ping; Cong, Min; Liu, Tianhui; Yang, Aiting; Sun, Guangyong; Zhang, Dong; Huang, Jian; Sun, Shujie; Mao, Jia; Ma, Hong; Jia, Jidong; You, Hong

    2016-01-01

    Profibrogenesis cytokine, transforming growth factor- (TGF-) β1, induces hepatic progenitors experiencing epithelial to mesenchymal transition (EMT) to matrix synthesis cells, even tumor initiating cells. Our previous data found that epidermal growth factor (EGF) blocks and reverses TGF-β1-induced transition. The aim of this study is to determine the characteristic changes of hepatic progenitors after TGF-β1-induced transition and EGF-induced reversion. Hepatic oval cells, rat hepatic progenitors, were isolated from rats fed a choline-deficient diet supplemented with ethionine. TGF-β1-containing medium was used for inducing EMT, while EGF-containing medium was used for reversing EMT. During TGF-β1-induced transition and EGF-induced reversion, hepatic oval cells sustained their progenitor cell marker expression, including α-fetoprotein, albumin, and cytokeratin-19. The proliferation ability and differentiation potential of these cells were suppressed by TGF-β1, while EGF resumed these capacities to the level similar to the control cells. RNA microarray analysis showed that most of the genes with significant changes after TGF-β1 incubation were recovered by EGF. Signal pathway analysis revealed that TGF-β1 impaired the pathways of cell cycle and cytochrome P450 detoxification, and EGF reverted TGF-β1 effects through activating MAPK and PI3K-Akt pathway. EGF reverses the characteristics impaired by TGF-β1 in hepatic oval cells, serving as a protective cytokine to hepatic progenitors. PMID:26955393

  11. Linking γ-aminobutyric acid A receptor to epidermal growth factor receptor pathways activation in human prostate cancer.

    PubMed

    Wu, Weijuan; Yang, Qing; Fung, Kar-Ming; Humphreys, Mitchell R; Brame, Lacy S; Cao, Amy; Fang, Yu-Ting; Shih, Pin-Tsen; Kropp, Bradley P; Lin, Hsueh-Kung

    2014-03-01

    Neuroendocrine (NE) differentiation has been attributed to the progression of castration-resistant prostate cancer (CRPC). Growth factor pathways including the epidermal growth factor receptor (EGFR) signaling have been implicated in the development of NE features and progression to a castration-resistant phenotype. However, upstream molecules that regulate the growth factor pathway remain largely unknown. Using androgen-insensitive bone metastasis PC-3 cells and androgen-sensitive lymph node metastasis LNCaP cells derived from human prostate cancer (PCa) patients, we demonstrated that γ-aminobutyric acid A receptor (GABA(A)R) ligand (GABA) and agonist (isoguvacine) stimulate cell proliferation, enhance EGF family members expression, and activate EGFR and a downstream signaling molecule, Src, in both PC-3 and LNCaP cells. Inclusion of a GABA(A)R antagonist, picrotoxin, or an EGFR tyrosine kinase inhibitor, Gefitinib (ZD1839 or Iressa), blocked isoguvacine and GABA-stimulated cell growth, trans-phospohorylation of EGFR, and tyrosyl phosphorylation of Src in both PCa cell lines. Spatial distributions of GABAAR α₁ and phosphorylated Src (Tyr416) were studied in human prostate tissues by immunohistochemistry. In contrast to extremely low or absence of GABA(A)R α₁-positive immunoreactivity in normal prostate epithelium, elevated GABA(A)R α₁ immunoreactivity was detected in prostate carcinomatous glands. Similarly, immunoreactivity of phospho-Src (Tyr416) was specifically localized and limited to the nucleoli of all invasive prostate carcinoma cells, but negative in normal tissues. Strong GABAAR α₁ immunoreactivity was spatially adjacent to the neoplastic glands where strong phospho-Src (Tyr416)-positive immunoreactivity was demonstrated, but not in adjacent to normal glands. These results suggest that the GABA signaling is linked to the EGFR pathway and may work through autocrine or paracine mechanism to promote CRPC progression.

  12. Hormone-induced expression of tumor necrosis factor alpha-converting enzyme/A disintegrin and metalloprotease-17 impacts porcine cumulus cell oocyte complex expansion and meiotic maturation via ligand activation of the epidermal growth factor receptor.

    PubMed

    Yamashita, Yasuhisa; Kawashima, Ikkou; Yanai, Yoshiari; Nishibori, Masahide; Richards, Joanne S; Shimada, Masayuki

    2007-12-01

    The epidermal growth factor (EGF)-like growth factors, amphiregulin (AREG) and epiregulin (EREG), are expressed in murine cumulus oocyte complexes (COCs) where they impact the function of cumulus cells and oocyte maturation during LH-mediated ovulation. Because TNFalpha-converting enzyme (TACE)/a disintegrin and metalloprotease-17 (ADAM17) is essential for ectodomain shedding of AREG and EREG from the surface of other cell types, the expression and function of TACE/ADAM17 was analyzed in a porcine COC culture system in which FSH- and LH-mediated expansion and oocyte meiotic maturation have been well characterized and shown to occur between 20 and 40 h. In this model, Areg, Ereg, and Tace/Adam17 mRNAs increased significantly with maximal levels observed between 5 and 20 h of culture with FSH plus LH. TACE/ADAM17 protein and protease activity were up-regulated markedly at 10 h and maintained to 40 h. Treatment of COCs with the TACE/ADAM17-selective inhibitor TNFalpha-processing inhibitor-2 (TAPI-2) significantly suppressed in a time-dependent manner downstream targets of EGF receptor activation such as ERK1/2 phosphorylation, Ptgs2, Has2, and Tnfaip6 mRNA expression, hormone-induced COC expansion, and meiotic maturation of the oocytes. Addition of EGF to COCs cultured in the presence of FSH/LH reversed the inhibitory effects of TAPI-2 on these ovulation-related processes. Gonadotropin-induced phosphorylation of ERK1/2 was also inhibited in rat granulosa cells treated with TAPI-2 or after transfection with Tace/Adam17 small interfering RNA. Induced expression of Tnfaip6 mRNA was also reduced by Tace/Adam17 small interfering RNA. Thus, TACE/ADAM17 is induced and the activity is involved in porcine COC expansion as well as oocyte meiotic maturation through the activation of EGF receptor in cumulus cells.

  13. Phenobarbital and Insulin Reciprocate Activation of the Nuclear Receptor Constitutive Androstane Receptor through the Insulin Receptor

    PubMed Central

    Yasujima, Tomoya; Saito, Kosuke; Moore, Rick

    2016-01-01

    Phenobarbital (PB) antagonized insulin to inactivate the insulin receptor and attenuated the insulin receptor downstream protein kinase B (AKT)–forkhead box protein O1 and extracellular signal-regulated kinase 1/2 signals in mouse primary hepatocytes and HepG2 cells. Hepatic AKT began dephosphorylation in an early stage of PB treatment, and blood glucose levels transiently increased in both wild-type and constitutive androstane receptor (CAR) knockout (KO) mice. On the other hand, blood glucose levels increased in wild-type mice, but not KO mice, in later stages of PB treatment. As a result, PB, acting as an insulin receptor antagonist, elicited CAR-independent increases and CAR-dependent decreases of blood glucose levels at these different stages of treatment, respectively. Reciprocally, insulin activation of the insulin receptor repressed CAR activation and induction of its target CYP2B6 gene in HepG2 cells. Thus, PB and insulin cross-talk through the insulin receptor to regulate glucose and drug metabolism reciprocally. PMID:26994072

  14. Phenobarbital and Insulin Reciprocate Activation of the Nuclear Receptor Constitutive Androstane Receptor through the Insulin Receptor.

    PubMed

    Yasujima, Tomoya; Saito, Kosuke; Moore, Rick; Negishi, Masahiko

    2016-05-01

    Phenobarbital (PB) antagonized insulin to inactivate the insulin receptor and attenuated the insulin receptor downstream protein kinase B (AKT)-forkhead box protein O1 and extracellular signal-regulated kinase 1/2 signals in mouse primary hepatocytes and HepG2 cells. Hepatic AKT began dephosphorylation in an early stage of PB treatment, and blood glucose levels transiently increased in both wild-type and constitutive androstane receptor (CAR) knockout (KO) mice. On the other hand, blood glucose levels increased in wild-type mice, but not KO mice, in later stages of PB treatment. As a result, PB, acting as an insulin receptor antagonist, elicited CAR-independent increases and CAR-dependent decreases of blood glucose levels at these different stages of treatment, respectively. Reciprocally, insulin activation of the insulin receptor repressed CAR activation and induction of its target CYP2B6 gene in HepG2 cells. Thus, PB and insulin cross-talk through the insulin receptor to regulate glucose and drug metabolism reciprocally.

  15. Insulin receptor activation in solitary fibrous tumours.

    PubMed

    Li, Y; Chang, Q; Rubin, B P; Fletcher, C D M; Morgan, T W; Mentzer, S J; Sugarbaker, D J; Fletcher, J A; Xiao, S

    2007-04-01

    Solitary fibrous tumours (SFTs) are known to overexpress insulin-like growth factor 2 (IGF-2). The down-stream oncogenic pathways of IGF-2, however, are not clear. Here we report uniform activation of the insulin receptor (IR) pathway in SFTs, which are mesenchymal tumours frequently associated with hypoglycaemia. Whereas the IR and its downstream signalling pathways were constitutively activated in SFTs, insulin-like growth factor 1 receptor (IGF-1R) was not expressed in these tumours. We also find that SFT cells secrete IGF-2 and proliferate in serum-free medium, consistent with an IGF-2/IR autocrine loop. The aetiological relevance of IGF-2 is supported by expression of IR-A, the IR isoform with high affinity for IGF-2, in all SFTs. Our studies suggest that IR activation plays an oncogenic role in SFTs.

  16. Recurrent exposure to nicotine differentiates human bronchial epithelial cells via epidermal growth factor receptor activation

    SciTech Connect

    Martinez-Garcia, Eva; Irigoyen, Marta; Anso, Elena; Martinez-Irujo, Juan Jose; Rouzaut, Ana

    2008-05-01

    Cigarette smoking is the major preventable cause of lung cancer in developed countries. Nicotine (3-(1-methyl-2-pyrrolidinyl)-pyridine) is one of the major alkaloids present in tobacco. Besides its addictive properties, its effects have been described in panoply of cell types. In fact, recent studies have shown that nicotine behaves as a tumor promoter in transformed epithelial cells. This research focuses on the effects of acute repetitive nicotine exposure on normal human bronchial epithelial cells (NHBE cells). Here we show that treatment of NHBE cells with recurrent doses of nicotine up to 500 {mu}M triggered cell differentiation towards a neuronal-like phenotype: cells emitted filopodia and expressed neuronal markers such as neuronal cell adhesion molecule, neurofilament-M and the transcription factors neuronal N and Pax-3. We also demonstrate that nicotine treatment induced NF-kB translocation to the nucleus, phosphorylation of the epidermal growth factor receptor (EGFR), and accumulation of heparin binding-EGF in the extracellular medium. Moreover, addition of AG1478, an inhibitor of EGFR tyrosine phosphorylation, or cetuximab, a monoclonal antibody that precludes ligand binding to the same receptor, prevented cell differentiation by nicotine. Lastly, we show that differentiated cells increased their adhesion to the extracellular matrix and their protease activity. Given that several lung pathologies are strongly related to tobacco consumption, these results may help to better understand the damaging consequences of nicotine exposure.

  17. Identification and super-resolution imaging of ligand-activated receptor dimers in live cells

    NASA Astrophysics Data System (ADS)

    Winckler, Pascale; Lartigue, Lydia; Giannone, Gregory; de Giorgi, Francesca; Ichas, François; Sibarita, Jean-Baptiste; Lounis, Brahim; Cognet, Laurent

    2013-08-01

    Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensions and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Förster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-localization. This methodology which is specifically devoted to the study of molecules in interaction, may find other applications in biological systems where understanding of molecular organization is crucial.

  18. Biased Signaling of Protease-Activated Receptors

    PubMed Central

    Zhao, Peishen; Metcalf, Matthew; Bunnett, Nigel W.

    2014-01-01

    In addition to their role in protein degradation and digestion, proteases can also function as hormone-like signaling molecules that regulate vital patho-physiological processes, including inflammation, hemostasis, pain, and repair mechanisms. Certain proteases can signal to cells by cleaving protease-activated receptors (PARs), a family of four G protein-coupled receptors. PARs are expressed by almost all cell types, control important physiological and disease-relevant processes, and are an emerging therapeutic target for major diseases. Most information about PAR activation and function derives from studies of a few proteases, for example thrombin in the case of PAR1, PAR3, and PAR4, and trypsin in the case of PAR2 and PAR4. These proteases cleave PARs at established sites with the extracellular N-terminal domains, and expose tethered ligands that stabilize conformations of the cleaved receptors that activate the canonical pathways of G protein- and/or β-arrestin-dependent signaling. However, a growing number of proteases have been identified that cleave PARs at divergent sites to activate distinct patterns of receptor signaling and trafficking. The capacity of these proteases to trigger distinct signaling pathways is referred to as biased signaling, and can lead to unique patho-physiological outcomes. Given that a different repertoire of proteases are activated in various patho-physiological conditions that may activate PARs by different mechanisms, signaling bias may account for the divergent actions of proteases and PARs. Moreover, therapies that target disease-relevant biased signaling pathways may be more effective and selective approaches for the treatment of protease- and PAR-driven diseases. Thus, rather than mediating the actions of a few proteases, PARs may integrate the biological actions of a wide spectrum of proteases in different patho-physiological conditions. PMID:24860547

  19. Equivalent Activities of Repulsive Axon Guidance Receptors

    PubMed Central

    Long, Hong; Yoshikawa, Shingo

    2016-01-01

    Receptors on the growth cone at the leading edge of elongating axons play critical guidance roles by recognizing cues via their extracellular domains and transducing signals via their intracellular domains, resulting in changes in direction of growth. An important concept to have emerged in the axon guidance field is the importance of repulsion as a major guidance mechanism. Given the number and variety of different repulsive receptors, it is generally thought that there are likely to be qualitative differences in the signals they transduce. However, the nature of these possible differences is unknown. By creating chimeras using the extracellular and intracellular domains of three different Drosophila repulsive receptors, Unc5, Roundabout (Robo), and Derailed (Drl) and expressing them in defined cells within the embryonic nervous system, we examined the responses elicited by their intracellular domains systematically. Surprisingly, we found no qualitative differences in growth cone response or axon growth, suggesting that, despite their highly diverged sequences, each intracellular domain elicits repulsion via a common pathway. In terms of the signaling pathway(s) used by the repulsive receptors, mutations in the guanine nucleotide exchange factor Trio strongly enhance the repulsive activity of all three intracellular domains, suggesting that repulsion by Unc5, Robo, and Drl, and perhaps repulsion in general, involves Trio activity. SIGNIFICANCE STATEMENT A prevailing concept that has emerged in the axon guidance field is the importance of repulsion as a guidance mechanism for steering axons to their appropriate targets. Given the number and variety of different repulsive receptors, it is generally thought that there are differences in the signals that they transduce. However, this has never been tested directly. We have used the advanced genetics of Drosophila to compare directly the outputs of different repulsive receptors. Surprisingly, we found no qualitative

  20. Combination of MS protein identification and bioassay of chromatographic fractions to identify biologically active substances from complex protein sources.

    PubMed

    Kuromitsu, Sadao; Yokota, Hiroyuki; Hiramoto, Masashi; Yuri, Masatoshi; Naitou, Masanori; Nakamura, Naoto; Kawabata, Shigeki; Kobori, Masato; Katoh, Masao; Furuchi, Kiyoshi; Mita, Haruhisa; Yamada, Tetsuo

    2009-06-01

    Purification of biologically active proteins from complex biological sources is a difficult task, usually requiring large amounts of sample and many separation steps. We found an active substance in a serum response element-dependent luciferase reporter gene bioassay in interstitial cystitis urine that we attempted to purify with column chromatography and the bioassay. With anion-exchange Mono Q and C4 reversed-phase columns, apparently sharp active peaks were obtained. However, more than 20 kinds of proteins were identified from the active fractions with MS, indicating that the purification was not complete. As further purification was difficult, we chose a candidate molecule by means of studying the correlation between MS protein identification scores and bioassay responses of chromatographic fractions near the active peaks. As a result, epidermal growth factor (EGF) was nominated as a candidate molecule among the identified proteins because the elution profile of EGF was consistent with that of the bioassay, and the correlation coefficient of EGF between MS protein identification scores and bioassay responses was the highest among all the identified proteins. With recombinant EGF and anti-EGF and anti-EGF receptor antibodies, EGF was confirmed to be the desired substance in interstitial cystitis urine. This approach required only 20 ml of urine sample and two column chromatographic steps. The combination of MS protein identification and bioassay of chromatographic fractions may be useful for identifying biologically active substances from complex protein sources.

  1. Brain injury expands the numbers of neural stem cells and progenitors in the SVZ by enhancing their responsiveness to EGF

    PubMed Central

    Alagappan, Dhivyaa; Lazzarino, Deborah A; Felling, Ryan J; Balan, Murugabaskar; Kotenko, Sergei V; Levison, Steven W

    2009-01-01

    There is an increase in the numbers of neural precursors in the SVZ (subventricular zone) after moderate ischaemic injuries, but the extent of stem cell expansion and the resultant cell regeneration is modest. Therefore our studies have focused on understanding the signals that regulate these processes towards achieving a more robust amplification of the stem/progenitor cell pool. The goal of the present study was to evaluate the role of the EGFR [EGF (epidermal growth factor) receptor] in the regenerative response of the neonatal SVZ to hypoxic/ischaemic injury. We show that injury recruits quiescent cells in the SVZ to proliferate, that they divide more rapidly and that there is increased EGFR expression on both putative stem cells and progenitors. With the amplification of the precursors in the SVZ after injury there is enhanced sensitivity to EGF, but not to FGF (fibroblast growth factor)-2. EGF-dependent SVZ precursor expansion, as measured using the neurosphere assay, is lost when the EGFR is pharmacologically inhibited, and forced expression of a constitutively active EGFR is sufficient to recapitulate the exaggerated proliferation of the neural stem/progenitors that is induced by hypoxic/ischaemic brain injury. Cumulatively, our results reveal that increased EGFR signalling precedes that increase in the abundance of the putative neural stem cells and our studies implicate the EGFR as a key regulator of the expansion of SVZ precursors in response to brain injury. Thus modulating EGFR signalling represents a potential target for therapies to enhance brain repair from endogenous neural precursors following hypoxic/ischaemic and other brain injuries. PMID:19570028

  2. Peroxisome proliferator-activated receptors and angiogenesis.

    PubMed

    Biscetti, F; Straface, G; Pitocco, D; Zaccardi, F; Ghirlanda, G; Flex, A

    2009-12-01

    The peroxisome proliferator-activated receptors (PPARs) are a group of three nuclear receptor isoforms, PPARalpha, PPARgamma and PPARdelta, encoded by different genes, and they form a subfamily of the nuclear receptor superfamily. The clinical interest in PPARs originates with fibrates and thiazolidinediones, which, respectively, act on PPARalpha and PPARgamma and are used to ameliorate hyperlipidaemia and hyperglycaemia in subjects with type 2 diabetes mellitus (T2DM). PPARs play a central role in these patients due to their ability to regulate the expression of numerous genes involved in glycaemic control, lipid metabolism, vascular tone and inflammation. Abnormal angiogenesis is implicated in several of the long-term complications of diabetes mellitus, characterized by vasculopathy associated with aberrant growth of new blood vessels. This pathological process plays a crucial role in diabetic retinopathy, nephropathy and neuropathy, impaired wound healing and impaired coronary collateral vessel development. In recent years, there has been increasing appreciation of the fact that PPARs might be involved in the molecular mechanisms that regulate angiogenesis through the action of growth factors and cytokines that stimulate migration, proliferation and survival of endothelial cells. During the last few years direct comparative analyses have been performed, using selective PPARs agonists, to clarify the angiogenic properties of the different members of the PPAR family. Lately, the findings provide new information to order to understand the biological, clinical and therapeutic effects of PPARs, and the role of these nuclear receptors in angiogenesis, with potentially important implications for the management of subjects affected by T2DM. PMID:19628379

  3. Proteinase-activated receptors (PARs) – focus on receptor-receptor-interactions and their physiological and pathophysiological impact

    PubMed Central

    2013-01-01

    Proteinase-activated receptors (PARs) are a subfamily of G protein-coupled receptors (GPCRs) with four members, PAR1, PAR2, PAR3 and PAR4, playing critical functions in hemostasis, thrombosis, embryonic development, wound healing, inflammation and cancer progression. PARs are characterized by a unique activation mechanism involving receptor cleavage by different proteinases at specific sites within the extracellular amino-terminus and the exposure of amino-terminal “tethered ligand“ domains that bind to and activate the cleaved receptors. After activation, the PAR family members are able to stimulate complex intracellular signalling networks via classical G protein-mediated pathways and beta-arrestin signalling. In addition, different receptor crosstalk mechanisms critically contribute to a high diversity of PAR signal transduction and receptor-trafficking processes that result in multiple physiological effects. In this review, we summarize current information about PAR-initiated physical and functional receptor interactions and their physiological and pathological roles. We focus especially on PAR homo- and heterodimerization, transactivation of receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RSTKs), communication with other GPCRs, toll-like receptors and NOD-like receptors, ion channel receptors, and on PAR association with cargo receptors. In addition, we discuss the suitability of these receptor interaction mechanisms as targets for modulating PAR signalling in disease. PMID:24215724

  4. Epidermal growth factor-like repeats of tenascin-C-induced constriction of cerebral arteries via activation of epidermal growth factor receptors in rats.

    PubMed

    Fujimoto, Masashi; Shiba, Masato; Kawakita, Fumihiro; Liu, Lei; Nakasaki, Asuka; Shimojo, Naoshi; Imanaka-Yoshida, Kyoko; Yoshida, Toshimichi; Suzuki, Hidenori

    2016-07-01

    Tenascin-C (TNC), one of matricellular proteins, has been suggested to be involved in cerebral vasospasm after aneurysmal subarachnoid hemorrhage. However, the mechanisms of how TNC constricts cerebral arteries remain unclear. The aim of this study was to examine if epidermal growth factor (EGF)-like repeats of TNC is involved in TNC-induced constriction of cerebral arteries in rats via EGF receptor (EGFR) activation. Two dosages of recombinant TNC (r-TNC) consisting of the EGF-like repeats was administered intracisternally to healthy rats, and its vasoconstrictor effects were evaluated by neurobehavioral tests and India-ink angiography at 24, 48, and 72 hours after the administration. Western blotting and immunohistochemistry were performed to explore the underlying mechanisms on constricted cerebral arteries after 24 hours. The effects of a selective EGFR tyrosine kinase inhibitor (AG1478) on r-TNC-induced vasoconstriction were evaluated by neurobehavioral tests, India-ink angiography and immunohistochemistry at 24 hours after the administration. A higher dosage of r-TNC induced cerebral arterial constriction more severely, which continued for 48 hours. The effects were associated with the activation of EGFR and extracellular signal-regulated kinase (ERK)1/2 in the smooth muscle cell layer of the constricted cerebral artery, while c-Jun N-terminal kinase and p38 were not activated. AG1478 blocked r-TNC-induced vasoconstrictive effects, as well as activation of EGFR and ERK1/2. These findings demonstrate that TNC induces constriction of cerebral arteries via activation of EGFR and ERK1/2.

  5. Epidermal growth factor–stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis

    PubMed Central

    Garay, Camilo; Judge, Gurjeet; Lucarelli, Stefanie; Bautista, Stephen; Pandey, Rohan; Singh, Tanveer; Antonescu, Costin N.

    2015-01-01

    Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits—membrane structures containing clathrin and other proteins—eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation. PMID:26246598

  6. Global analysis of transcription in castration-resistant prostate cancer cells uncovers active enhancers and direct androgen receptor targets

    PubMed Central

    Toropainen, Sari; Niskanen, Einari A.; Malinen, Marjo; Sutinen, Päivi; Kaikkonen, Minna U.; Palvimo, Jorma J.

    2016-01-01

    Androgen receptor (AR) is a male sex steroid-activated transcription factor (TF) that plays a critical role in prostate cancers, including castration-resistant prostate cancers (CRPC) that typically express amplified levels of the AR. CRPC-derived VCaP cells display an excessive number of chromatin AR-binding sites (ARBs) most of which localize to distal inter- or intragenic regions. Here, we analyzed direct transcription programs of the AR in VCaP cells using global nuclear run-on sequencing (GRO-seq) and integrated the GRO-seq data with the ARB and VCaP cell-specific TF-binding data. Androgen immediately activated transcription of hundreds of protein-coding genes, including IGF-1 receptor and EGF receptor. Androgen also simultaneously repressed transcription of a large number of genes, including MYC. As functional enhancers have been postulated to produce enhancer-templated non-coding RNAs (eRNAs), we also analyzed the eRNAs, which revealed that only a fraction of the ARBs reside at functional enhancers. Activation of these enhancers was most pronounced at the sites that also bound PIAS1, ERG and HDAC3, whereas binding of HDAC3 and PIAS1 decreased at androgen-repressed enhancers. In summary, our genome-wide data of androgen-regulated enhancers and primary target genes provide new insights how the AR can directly regulate cellular growth and control signaling pathways in CPRC cells. PMID:27641228

  7. Global analysis of transcription in castration-resistant prostate cancer cells uncovers active enhancers and direct androgen receptor targets.

    PubMed

    Toropainen, Sari; Niskanen, Einari A; Malinen, Marjo; Sutinen, Päivi; Kaikkonen, Minna U; Palvimo, Jorma J

    2016-01-01

    Androgen receptor (AR) is a male sex steroid-activated transcription factor (TF) that plays a critical role in prostate cancers, including castration-resistant prostate cancers (CRPC) that typically express amplified levels of the AR. CRPC-derived VCaP cells display an excessive number of chromatin AR-binding sites (ARBs) most of which localize to distal inter- or intragenic regions. Here, we analyzed direct transcription programs of the AR in VCaP cells using global nuclear run-on sequencing (GRO-seq) and integrated the GRO-seq data with the ARB and VCaP cell-specific TF-binding data. Androgen immediately activated transcription of hundreds of protein-coding genes, including IGF-1 receptor and EGF receptor. Androgen also simultaneously repressed transcription of a large number of genes, including MYC. As functional enhancers have been postulated to produce enhancer-templated non-coding RNAs (eRNAs), we also analyzed the eRNAs, which revealed that only a fraction of the ARBs reside at functional enhancers. Activation of these enhancers was most pronounced at the sites that also bound PIAS1, ERG and HDAC3, whereas binding of HDAC3 and PIAS1 decreased at androgen-repressed enhancers. In summary, our genome-wide data of androgen-regulated enhancers and primary target genes provide new insights how the AR can directly regulate cellular growth and control signaling pathways in CPRC cells. PMID:27641228

  8. ErbB/EGF signaling and EMT in mammary development and breast cancer.

    PubMed

    Hardy, Katharine M; Booth, Brian W; Hendrix, Mary J C; Salomon, David S; Strizzi, Luigi

    2010-06-01

    Activation of the ErbB family of receptor tyrosine kinases via cognate Epidermal Growth Factor (EGF)-like peptide ligands constitutes a major group of related signaling pathways that control proliferation, survival, angiogenesis and metastasis of breast cancer. In this respect, clinical trials with various ErbB receptor blocking antibodies and specific tyrosine kinase inhibitors have proven to be partially efficacious in the treatment of this heterogeneous disease. Induction of an embryonic program of epithelial-to-mesenchymal transition (EMT) in breast cancer, whereupon epithelial tumor cells convert to a more mesenchymal-like phenotype, facilitates the migration, intravasation, and extravasation of tumor cells during metastasis. Breast cancers which exhibit properties of EMT are highly aggressive and resistant to therapy. Activation of ErbB signaling can regulate EMT-associated invasion and migration in normal and malignant mammary epithelial cells, as well as modulating discrete stages of mammary gland development. The purpose of this review is to summarize current information regarding the role of ErbB signaling in aspects of EMT that influence epithelial cell plasticity during mammary gland development and tumorigenesis. How this information may contribute to the improvement of therapeutic approaches in breast cancer will also be addressed.

  9. Radiotherapy-induced miR-223 prevents relapse of breast cancer by targeting the EGF pathway

    PubMed Central

    Fabris, L; Berton, S; Citron, F; D'Andrea, S; Segatto, I; Nicoloso, M S; Massarut, S; Armenia, J; Zafarana, G; Rossi, S; Ivan, C; Perin, T; Vaidya, J S; Avanzo, M; Roncadin, M; Schiappacassi, M; Bristow, R G; Calin, G; Baldassarre, G; Belletti, B

    2016-01-01

    In breast cancer (BC) patients, local recurrences often arise in proximity of the surgical scar, suggesting that response to surgery may have a causative role. Radiotherapy (RT) after lumpectomy significantly reduces the risk of recurrence. We investigated the direct effects of surgery and of RT delivered intraoperatively (IORT), by collecting irradiated and non-irradiated breast tissues from BC patients, after tumor removal. These breast tissue specimens have been profiled for their microRNA (miR) expression, in search of differentially expressed miR among patients treated or not with IORT. Our results demonstrate that IORT elicits effects that go beyond the direct killing of residual tumor cells. IORT altered the wound response, inducing the expression of miR-223 in the peri-tumoral breast tissue. miR-223 downregulated the local expression of epidermal growth factor (EGF), leading to decreased activation of EGF receptor (EGFR) on target cells and, eventually, dampening a positive EGF–EGFR autocrine/paracrine stimulation loop induced by the post-surgical wound-healing response. Accordingly, both RT-induced miR-223 and peri-operative inhibition of EGFR efficiently prevented BC cell growth and reduced recurrence formation in mouse models of BC. Our study uncovers unknown effects of RT delivered on a wounded tissue and prompts to the use of anti-EGFR treatments, in a peri-operative treatment schedule, aimed to timely treat BC patients and restrain recurrence formation. PMID:26876200

  10. Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2)*

    PubMed Central

    Chiluiza, David; Krishna, Sneha; Schumacher, Valérie A.; Schlöndorff, Johannes

    2013-01-01

    Gain-of-function mutations in the canonical transient receptor potential 6 (TRPC6) gene are a cause of autosomal dominant focal segmental glomerulosclerosis (FSGS). The mechanisms whereby abnormal TRPC6 activity results in proteinuria remain unknown. The ERK1/2 MAPKs are activated in glomeruli and podocytes in several proteinuric disease models. We therefore examined whether FSGS-associated mutations in TRPC6 result in activation of these kinases. In 293T cells and cultured podocytes, overexpression of gain-of-function TRPC6 mutants resulted in increased ERK1/2 phosphorylation, an effect dependent upon channel function. Pharmacologic inhibitor studies implicated several signaling mediators, including calmodulin and calcineurin, supporting the importance of TRPC6-mediated calcium influx in this process. Through medium transfer experiments, we uncovered two distinct mechanisms for ERK activation by mutant TRPC6, a cell-autonomous, EGF receptor-independent mechanism and a non-cell-autonomous mechanism involving metalloprotease-mediated release of a presumed EGF receptor ligand. The inhibitors KN-92 and H89 were able to block both pathways in mutant TRPC6 expressing cells as well as the prolonged elevation of intracellular calcium levels upon carbachol stimulation seen in these cells. However, these effects appear to be independent of their effects on calcium/calmodulin-dependent protein kinase II and PKA, respectively. Phosphorylation of Thr-70, Ser-282, and Tyr-31/285 were not necessary for ERK activation by mutant TRPC6, although a phosphomimetic TRPC6 S282E mutant was capable of ERK activation. Taken together, these results identify two pathways downstream of mutant TRPC6 leading to ERK activation that may play a role in the development of FSGS. PMID:23645677

  11. Transactivation of epidermal growth factor receptor in vascular and renal systems in rats with experimental hyperleptinemia: role in leptin-induced hypertension.

    PubMed

    Jamroz-Wiśniewska, Anna; Wójcicka, Grazyna; Łowicka, Ewelina; Ksiazek, Marta; Bełtowski, Jerzy

    2008-04-15

    We examined the role of epidermal growth factor (EGF) receptor in the pathogenesis of leptin-induced hypertension in the rat. Leptin, administered in increasing doses (0.1-0.5 mg/kg/day) for 10 days, increased phosphorylation levels of non-receptor tyrosine kinase, c-Src, EGF receptor and extracellular signal-regulated kinases (ERK) in aorta and kidney, which was accompanied by the increase in plasma concentration and urinary excretion of isoprostanes and H2O2. Blood pressure and renal Na+,K+-ATPase activity were higher, whereas urinary sodium excretion was lower in animals receiving leptin. The effects of leptin on renal Na+,K+-ATPase, natriuresis and blood pressure were abolished by NADPH oxidase inhibitor, apocynin, Src kinase inhibitor, PP2, EGF receptor inhibitor, AG1478, protein farnesyltransferase inhibitor, manumycin A, and ERK inhibitor, PD98059. In contrast, inhibitors of insulin-like growth factor-1 and platelet-derived growth factor receptors, AG1024 and AG1295, respectively, only slightly reduced ERK phosphorylation and had no effect on blood pressure in rats receiving leptin. These data indicate that: (1) experimental hyperleptinemia is associated with oxidative stress and c-Src-dependent transactivation of the EGF receptor, which stimulates ERK in vascular wall and the kidney, (2) overactivity of EGF receptor-ERK pathway contributes to leptin-induced hypertension by stimulating renal Na+,K+-ATPase and reducing sodium excretion, (3) inhibitors of c-Src, EGF receptor and ERK may be considered as a novel therapy for hypertension associated with hyperleptinemia, e.g. in patients with obesity and metabolic syndrome.

  12. CERAPP: Collaborative Estrogen Receptor Activity Prediction Project

    PubMed Central

    Mansouri, Kamel; Abdelaziz, Ahmed; Rybacka, Aleksandra; Roncaglioni, Alessandra; Tropsha, Alexander; Varnek, Alexandre; Zakharov, Alexey; Worth, Andrew; Richard, Ann M.; Grulke, Christopher M.; Trisciuzzi, Daniela; Fourches, Denis; Horvath, Dragos; Benfenati, Emilio; Muratov, Eugene; Wedebye, Eva Bay; Grisoni, Francesca; Mangiatordi, Giuseppe F.; Incisivo, Giuseppina M.; Hong, Huixiao; Ng, Hui W.; Tetko, Igor V.; Balabin, Ilya; Kancherla, Jayaram; Shen, Jie; Burton, Julien; Nicklaus, Marc; Cassotti, Matteo; Nikolov, Nikolai G.; Nicolotti, Orazio; Andersson, Patrik L.; Zang, Qingda; Politi, Regina; Beger, Richard D.; Todeschini, Roberto; Huang, Ruili; Farag, Sherif; Rosenberg, Sine A.; Slavov, Svetoslav; Hu, Xin; Judson, Richard S.

    2016-01-01

    Background: Humans are exposed to thousands of man-made chemicals in the environment. Some chemicals mimic natural endocrine hormones and, thus, have the potential to be endocrine disruptors. Most of these chemicals have never been tested for their ability to interact with the estrogen receptor (ER). Risk assessors need tools to prioritize chemicals for evaluation in costly in vivo tests, for instance, within the U.S. EPA Endocrine Disruptor Screening Program. Objectives: We describe a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project) and demonstrate the efficacy of using predictive computational models trained on high-throughput screening data to evaluate thousands of chemicals for ER-related activity and prioritize them for further testing. Methods: CERAPP combined multiple models developed in collaboration with 17 groups in the United States and Europe to predict ER activity of a common set of 32,464 chemical structures. Quantitative structure–activity relationship models and docking approaches were employed, mostly using a common training set of 1,677 chemical structures provided by the U.S. EPA, to build a total of 40 categorical and 8 continuous models for binding, agonist, and antagonist ER activity. All predictions were evaluated on a set of 7,522 chemicals curated from the literature. To overcome the limitations of single models, a consensus was built by weighting models on scores based on their evaluated accuracies. Results: Individual model scores ranged from 0.69 to 0.85, showing high prediction reliabilities. Out of the 32,464 chemicals, the consensus model predicted 4,001 chemicals (12.3%) as high priority actives and 6,742 potential actives (20.8%) to be considered for further testing. Conclusion: This project demonstrated the possibility to screen large libraries of chemicals using a consensus of different in silico approaches. This concept will be applied in future projects related to other

  13. Dopamine receptor activation promotes adult neurogenesis in an acute Parkinson model

    PubMed Central

    Winner, Beate; Desplats, Paula; Hagl, Christian; Klucken, Jochen; Aigner, Robert; Ploetz, Sonja; Laemke, Jörn; Karl, Alexandra; Aigner, Ludwig; Masliah, Eliezer; Buerger, Erich; Winkler, Jürgen

    2016-01-01

    Cell proliferation of neural progenitors in the subventricular zone (SVZ) of Parkinson disease (PD) patients and animal models is decreased. It was previously demonstrated that the neurotransmitter dopamine modulates cell proliferation in the embryonic brain. The aim of the present study was to analyze whether oral treatment with the dopamine receptor agonist pramipexole (PPX) modulates adult neurogenesis in the SVZ/ olfactory bulb system in a dopaminergic lesion model. 6-Hydroxydopamine (6-OHDA) lesioned adult rats received either PPX (1,0 mg/kg) or PBS orally twice daily and bromodeoxyuridine (BrdU, a cell proliferation marker) for 10 days and were perfused immediately after treatment or 4 weeks after PPX withdrawal. Stereological analysis revealed a significant augmentation in SVZ proliferation by PPX. Consecutively, enhanced neuronal differentiation and more new neurons were present in the olfactory bulb 4 weeks after PPX withdrawal. In addition, dopaminergic neurogenesis was increased in the olfactory bulb after PPX treatment. Motor activity as assessed by using an open field paradigm was permanently increased even after long term PPX withdrawal. In addition, we demonstrate that D2 and D3 receptors are present on adult rat SVZ derived neural progenitors in vitro, and PPX specifically increased mRNA levels of epidermal growth factor receptor (EGF-R) and paired box gene 6 (Pax6). Oral PPX treatment selectively increases adult neurogenesis in the SVZ-olfactory bulb system by increasing proliferation and cell survival of newly generated neurons. Analyzing the neurogenic fate decisions mediated by D2/D3 signaling pathways may lead to new avenues to induce neural repair in the adult brain. PMID:19619535

  14. How IGF-1 activates its receptor

    PubMed Central

    Kavran, Jennifer M; McCabe, Jacqueline M; Byrne, Patrick O; Connacher, Mary Katherine; Wang, Zhihong; Ramek, Alexander; Sarabipour, Sarvenaz; Shan, Yibing; Shaw, David E; Hristova, Kalina; Cole, Philip A; Leahy, Daniel J

    2014-01-01

    The type I insulin-like growth factor receptor (IGF1R) is involved in growth and survival of normal and neoplastic cells. A ligand-dependent conformational change is thought to regulate IGF1R activity, but the nature of this change is unclear. We point out an underappreciated dimer in the crystal structure of the related Insulin Receptor (IR) with Insulin bound that allows direct comparison with unliganded IR and suggests a mechanism by which ligand regulates IR/IGF1R activity. We test this mechanism in a series of biochemical and biophysical assays and find the IGF1R ectodomain maintains an autoinhibited state in which the TMs are held apart. Ligand binding releases this constraint, allowing TM association and unleashing an intrinsic propensity of the intracellular regions to autophosphorylate. Enzymatic studies of full-length and kinase-containing fragments show phosphorylated IGF1R is fully active independent of ligand and the extracellular-TM regions. The key step triggered by ligand binding is thus autophosphorylation. DOI: http://dx.doi.org/10.7554/eLife.03772.001 PMID:25255214

  15. Spongian diterpenoids inhibit androgen receptor activity

    PubMed Central

    Yang, Yu Chi; Meimetis, Labros G; Tien, Amy H; Mawji, Nasrin R; Carr, Gavin; Wang, Jun; Andersen, Raymond J; Sadar, Marianne D

    2013-01-01

    Androgen receptor (AR) is a ligand-activated transcription factor and a validated drug target for all stages of prostate cancer. Antiandrogens compete with physiological ligands for AR ligand-binding domain (LBD). High-throughput screening of a marine natural product library for small molecules that inhibit AR transcriptional activity yielded the furanoditerpenoid spongia-13(16),-14-dien-19-oic acid, designated terpene 1 (T1). Characterization of T1 and the structurally related semi-synthetic analogues (T2 and T3) revealed that these diterpenoids have antiandrogen properties that include inhibition of both androgen-dependent proliferation and AR transcriptional activity by a mechanism that involved competing with androgen for AR LBD and blocking essential N/C interactions required for androgen-induced AR transcriptional activity. Structure activity relationship analyses revealed some chemical features of T1 that are associated with activity and yielded T3 as the most potent analogue. In vivo, T3 significantly reduced the weight of seminal vesicles, which are an androgen-dependent tissue, thereby confirming T3’s on-target activity. The ability to create analogues of diterpenoids that have varying antiandrogen activity represents a novel class of chemical compounds for the analysis of AR ligand-binding properties and therapeutic development. PMID:23443807

  16. The prognostic value of epidermal growth factor receptor mRNA expression in primary ovarian cancer.

    PubMed Central

    Bartlett, J. M.; Langdon, S. P.; Simpson, B. J.; Stewart, M.; Katsaros, D.; Sismondi, P.; Love, S.; Scott, W. N.; Williams, A. R.; Lessells, A. M.; Macleod, K. G.; Smyth, J. F.; Miller, W. R.

    1996-01-01

    The expression of mRNA for the epidermal growth factor (EGF) receptor, EGF and transforming growth factor alpha (TGF-alpha) was determined in 76 malignant, six borderline and 15 benign primary ovarian tumours using the reverse transcriptase-polymerase chain reaction and related to clinical and pathological parameters. Of the malignant tumours, 70% (53/76) expressed EGF receptor mRNA, 31% (23/75) expressed EGF mRNA and 35% (26/75) expressed TGF-alpha mRNA. For the borderline tumours, four of six (67%) expressed EGF receptor mRNA, 1/6 (17%) expressed TGF-alpha mRNA and none expressed EGF mRNA. Finally, 33% (5/15) of the benign tumours expressed EGF receptor mRNA, whereas 40% (6/15) expressed EGF mRNA and 7% (1/15) expressed TGF-alpha mRNA. The presence of the EGF receptor in malignant tumours was associated with that of TGF-alpha (P = 0.0015) but not with EGF (P = 1.00), whereas there was no relationship between the presence of EGF and TGF-alpha (P = 1.00). EGF receptor mRNA expression was significantly and positively associated with serous histology (P = 0.006) but not with stage or grade. Neither EGF nor TGF-alpha showed any link with histological subtype or stage. The survival of patients with malignant tumours possessing EGF receptor mRNA was significantly reduced compared with that of patients whose tumours were negative (P = 0.030 for all malignant tumours; P = 0.007 for malignant epithelial tumours only). In contrast, neither the expression of TGF-alpha nor EGF was related to survival. These data suggest that the presence of EGF receptor mRNA is associated with poor prognosis in primary ovarian cancer. Images Figure 1 PMID:8562334

  17. Intrinsic relative activities of κ opioid agonists in activating Gα proteins and internalizing receptor: Differences between human and mouse receptors.

    PubMed

    DiMattio, Kelly M; Ehlert, Frederick J; Liu-Chen, Lee-Yuan

    2015-08-15

    Several investigators recently identified biased κ opioid receptor (KOP receptor) agonists. However, no comprehensive study of the functional selectivity of available KOP receptor agonists at the human and mouse KOP receptors (hKOP receptor and mKOP receptor, respectively) has been published. Here we examined the ability of over 20 KOP receptor agonists to activate G proteins and to internalize the receptor. Clonal neuro-2a mouse neuroblastoma (N2a) cells stably transfected with the hKOP receptor or mKOP receptor were used. We employed agonist-induced [(35)S]GTPγS binding and KOP receptor internalization as measures of activation of G protein and β-arrestin pathways, respectively. The method of Ehlert and colleagues was used to quantify intrinsic relative activities at G protein activation (RAi-G) and receptor internalization (RAi-I) and the degree of functional selectivity between the two [Log RAi-G - logRAi-I, RAi-G/RAi-I and bias factor]. The parameter, RAi, represents a relative estimate of agonist affinity for the active receptor state that elicits a given response. The endogenous ligand dynorphin A (1-17) was designated as the balanced ligand with a bias factor of 1. Interestingly, we found that there were species differences in functional selectivity. The most striking differences were for 12-epi-salvinorin A, U69,593, and ICI-199,441. 12-Epi-salvinorin A was highly internalization-biased at the mKOP receptor, but apparently G protein-biased at hKOP receptor. U69,593 was much more internalization-biased at mKOP receptor than hKOP receptor. ICI199,441 showed internalization-biased at the mKOP receptor and G protein-biased at the hKOP receptor. Possible mechanisms for the observed species differences are discussed.

  18. Intrinsic Relative Activities of Opioid Agonists in Activating Gα proteins and Internalizing Receptor: Differences between Human and Mouse Receptors

    PubMed Central

    DiMattio, Kelly M.; Ehlert, Frederick J.; Liu-Chen, Lee-Yuan

    2015-01-01

    Several investigators recently identified biased opioid receptor (KOP receptor) agonists. However, no comprehensive study of the functional selectivity of available KOP receptor agonists at the human and mouse KOP receptors (hKOP receptor and mKOP receptor, respectively) has been published. Here we examined the ability of over 20 KOP receptor agonists to activate G proteins and to internalize the receptor. Clonal neuro-2a mouse neuroblastoma (N2a) cells stably transfected with the hKOP receptor or mKOP receptor were used. We employed agonist-induced [35S]GTPγS binding and KOP receptor internalization as measures of activation of G protein and β-arrestin pathways, respectively. The method of Ehlert and colleagues was used to quantify intrinsic relative activities at G protein activation (RAi−G) and receptor internalization (RAi−I) and the degree of functional selectivity between the two [Log RAi−G − Log RAi−I, RAi−G/RAi−I and bias factor]. The parameter, RAi, represents a relative estimate of agonist affinity for the active receptor state that elicits a given response. The endogenous ligand dynorphin A (1–17) was designated as the balanced ligand with a bias factor of 1. Interestingly, we found that there were species differences in functional selectivity. The most striking differences were for 12-epi-salvinorin A, U69,593, and ICI-199,441. 12-Epi-salvinorin A was highly internalization-biased at the mKOP receptor, but apparently G protein-biased at hKOP receptor. U69,593 was much more internalization-biased at mKOP receptor than hKOP receptor. ICI199,441 showed internalization-biased at the mKOP receptor and G protein-biased at the hKOP receptor. Possible mechanisms for the observed species differences are discussed. PMID:26057692

  19. Rapidly activated epidermal growth factor receptor mediates lipopolysaccharide-triggered migration of microglia.

    PubMed

    Qu, Wen-Sheng; Liu, Jun-Li; Li, Chun-Yu; Li, Xiao; Xie, Min-Jie; Wang, Wei; Tian, Dai-Shi

    2015-11-01

    Previous reports have suggested that epidermal growth factor receptor (EGFR) is involved in microglia activation characterized by cell morphology changes, cytokine production and cell migration; and the biochemical regulation of the microglia migration is a potential therapeutic target following CNS inflammatory damages. However, the role of EGFR in microglia motility after inflammatory stimulation remains unknown. In the present study, lipopolysaccharide (LPS) was found to trigger rapid EGFR phosphorylation within 10 min, which was sustained during long-term stimulation in both primary microglial cells and the cultured BV2 microglial cells, furthermore, blocking EGFR phosphorylation by AG1478 significantly attenuated the LPS-induced chemotactic and chemokinetic migration of microglia. In addition, LPS could initiate calcium oscillation in microglia during live-cell recording, however, an intracellular calcium chelator and a selective inhibitor of calcium/calmodulin-dependent protein kinase II, but not an extracellular calcium chelator, remarkably suppressed the LPS-induced EGFR phosphorylation in BV2 microglia cells. As EGFR is not a traditional receptor for LPS, these findings suggest that the rapid phosphorylation of EGFR is attributed to the LPS-triggered intracellular calcium mobilization. By examining the downstream signals of EGFR, we further proved that extracellular signal-regulated kinase (ERK) is essential for EGFR-mediated microglia migration, because ERK inhibition attenuated the chemotactic and chemokinetic migration of microglia that had been induced by either LPS or EGF. Collectively, these results suggest that LPS could trigger the rapid phosphorylation of EGFR and subsequent ERK activation through mobilizing calcium activity, which underlies the microglia migration in an inflammatory condition.

  20. Signaling pathways engaged by NK cell receptors: double concerto for activating receptors, inhibitory receptors and NK cells.

    PubMed

    Tomasello, E; Bléry, M; Vély, F; Vivier, E

    2000-04-01

    Despite the absence of antigen-specific receptors at their surface, NK cells can selectively eliminate virus-infected cells, tumor cells and allogenic cells. A dynamic and precisely coordinated balance between activating and inhibitory receptors governs NK cell activation programs. Multiple activating and inhibitory NK cell surface molecules have been described, a group of them acting as receptors for MHC class I molecules. In spite of their heterogeneity, activating NK cell receptors present remarkable structural and functional homologies with T cell- and B cell-antigen receptors. Inhibitory NK cell receptors operate at early stages of activating cascades by recruiting protein tyrosine phosphatases via intra- cytoplasmic motifs (ITIM), a strategy which is widely conserved in hematopoietic and non-hematopoietic cells.

  1. Toxicological evaluation of u-hEGF.

    PubMed

    Maraschin, R; Bussi, R; Conz, A; Orlando, L; Pirovano, R; Nyska, A

    1995-01-01

    The toxicological evaluation of urinary human epidermal growth factor (u-hEGF) included mutagenicity, single and repeated dose general toxicity, and teratogenicity studies in various animal species. The mutagenic potential of u-hEGF was tested in vitro (Ames test, chromosome aberration in human lymphocytes, unscheduled DNA synthesis in HeLa cells) and in vivo (chromosome aberration in Chinese hamster bone marrow and micronucleus test in rat bone marrow). No mutagenic or clastogenic effects were found. The acute toxicity of u-hEGF was evaluated in mice and rats, using single subcutaneous (sc) or intravenous (i.v.) injection of 15 mg/kg. No toxic effects were observed Four-week i.v. daily administration of u-hEGF at the doses of 0.3, o.9, and 3 mg/kg in the SD rat followed by 2 wk of compound withdrawal induced pronounced and generally dose-related effects (i.e., epithelial hyperplasia) in a wide range of tissues and organs, at all doses. However, these effects were not apparently detrimental to the general health of the rats. The repeated sc administration of u-hEGF to cynomolgus monkeys for 4 wk at the same doses as used in the rat study resulted in lethality after about 7 days of treatment in the 2 higher dose groups or after 14 days at the lowest dose. The main clinical signs observed were gastrointestinal effects, respiratory distress, sedation, marked loss of body weight, and cutaneous desquamation. At histology, hyperplasia of most epithelia was seen in all groups. In addition, atrophy of the ovarian follicles and necrosis of the uterine endometrium were noted. Changes considered secondary to physical distress were atrophy of the hemopoietic and lymphatic system and hepatic steatosis. The embryofetal toxicity and teratogenicity of u-hEGF was tested, using the i.v. route in the SD rat and the i.v. and sc routes in the New Zealand White rabbit. In both species, the compound was administered at the doses of 0, 0.3, 0.9, and 3 mg/kg/day, from day 6 to 15 of

  2. PLC-gamma1 and Rac1 coregulate EGF-induced cytoskeleton remodeling and cell migration.

    PubMed

    Li, Siwei; Wang, Qian; Wang, Yi; Chen, Xinmei; Wang, Zhixiang

    2009-06-01

    It is well established that epidermal growth factor (EGF) induces the cytoskeleton reorganization and cell migration through two major signaling cascades: phospholipase C-gamma1 (PLC-gamma1) and Rho GTPases. However, little is known about the cross talk between PLC-gamma1 and Rho GTPases. Here we showed that PLC-gamma1 forms a complex with Rac1 in response to EGF. This interaction is direct and mediated by PLC-gamma1 Src homology 3 (SH3) domain and Rac1 (106)PNTP(109) motif. This interaction is critical for EGF-induced Rac1 activation in vivo, and PLC-gamma1 SH3 domain is actually a potent and specific Rac1 guanine nucleotide exchange factor in vitro. We have also demonstrated that the interaction between PLC-gamma1 SH3 domain and Rac1 play a significant role in EGF-induced F-actin formation and cell migration. We conclude that PLC-gamma1 and Rac1 coregulate EGF-induced cell cytoskeleton remodeling and cell migration by a direct functional interaction.

  3. Distinct structural specificities for functional coupling of the epidermal growth factor receptor to calcium-signalling versus phospholipase A2 responses.

    PubMed

    Hack, N; Margolis, B L; Ullrich, A; Schlessinger, J; Skorecki, K L

    1991-05-01

    Activation of phospholipase C (PLC), leading to a rise in cytosolic Ca2+, and of phospholipase A2 (PLA2) leading to a release of arachidonic acid, are among the early transmembrane signalling events that have been demonstrated in response to occupancy of the epidermal growth factor (EGF) receptor. The tyrosine kinase activity of the receptor has been shown to be necessary for both of these responses. This requirement for the tyrosine kinase activity could conceivably implicate a role for receptor autophosphorylation in the activation of PLA2. We now demonstrate that coupling of the EGF receptor to PLA2 was not impaired in a deletion mutant (CD126) devoid of the 126 amino acids from the C-terminus which include four major autophosphorylation sites. Functional coupling of the EGF receptor to PLA2 was demonstrated using three different experimental designs: (1) release of [14C]arachidonic acid from prelabelled intact cells. (2) release of [3H]arachidonic acid from prelabelled cells permeabilized with glass beads, and (3) direct measurement of PLA2 enzymic activity in cell-free extracts using an 'in vitro' assay employing exogenous phospholipid substrate. Functional coupling of the EGF receptor to PLA2 occurred despite the absence of a demonstrable Ca(2+)-signalling response and the detection of diminished but persistent PLC-gamma phosphorylation on tyrosine residues in the CD126 deletion mutants. These results point to a clear distinction in the biochemical mechanism and role for receptor autophosphorylation in functional coupling of the EGF receptor to PLA2 activation versus Ca2+ signalling.

  4. Principles of antibody-mediated TNF receptor activation

    PubMed Central

    Wajant, H

    2015-01-01

    From the beginning of research on receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF), agonistic antibodies have been used to stimulate TNFRSF receptors in vitro and in vivo. Indeed, CD95, one of the first cloned TNFRSF receptors, was solely identified as the target of cell death-inducing antibodies. Early on, it became evident from in vitro studies that valency and Fcγ receptor (FcγR) binding of antibodies targeting TNFRSF receptors can be of crucial relevance for agonistic activity. TNFRSF receptor-specific antibodies of the IgM subclass and secondary cross-linked or aggregation prone dimeric antibodies typically display superior agonistic activity compared with dimeric antibodies. Likewise, anchoring of antibodies to cell surface-expressed FcγRs potentiate their ability to trigger TNFRSF receptor signaling. However, only recently has the relevance of oligomerization and FcγR binding for the in vivo activity of antibody-induced TNFRSF receptor activation been straightforwardly demonstrated in vivo. This review discusses the crucial role of oligomerization and/or FcγR binding for antibody-mediated TNFRSF receptor stimulation in light of current models of TNFRSF receptor activation and especially the overwhelming relevance of these issues for the rational development of therapeutic TNFRSF receptor-targeting antibodies. PMID:26292758

  5. Directional Persistence of EGF-Induced Cell Migration Is Associated with Stabilization of Lamellipodial Protrusions

    PubMed Central

    Harms, Brian D.; Bassi, Gina M.; Horwitz, Alan Rick; Lauffenburger, Douglas A.

    2005-01-01

    Migrating cells can sustain a relatively constant direction of lamellipodial protrusion and locomotion over timescales ranging from minutes to hours. However, individual waves of lamellipodial extension occur over much shorter characteristic times. Little understanding exists regarding how cells might integrate biophysical processes across these disparate timescales to control the directional persistence of locomotion. We address this issue by examining the effects of epidermal growth factor (EGF) stimulation on long-timescale directional persistence and short-timescale lamellipodial dynamics of EGF receptor-transfected Chinese hamster ovary cells migrating on fibronectin-coated substrata. Addition of EGF increased persistence, with the magnitude of increase correlating with fibronectin coating concentration. Kymographic analysis of EGF-stimulated lamellipodial dynamics revealed that the temporal stability of lamellipodial protrusions similarly increased with fibronectin concentration. A soluble RGD peptide competitor reduced both the persistence of long-timescale cell paths and the stability of short-timescale membrane protrusions, indicating that cell-substratum adhesion concomitantly influences lamellipodial dynamics and directional persistence. These results reveal the importance of adhesion strength in regulating the directional motility of cells and suggest that the short-timescale kinetics of adhesion complex formation may play a key role in modulating directional persistence over much longer timescales. PMID:15713602

  6. Helicobacter pylori culture supernatant interferes with epidermal growth factor-activated signal transduction in human gastric KATO III cells.

    PubMed Central

    Pai, R.; Wyle, F. A.; Cover, T. L.; Itani, R. M.; Domek, M. J.; Tarnawski, A. S.

    1998-01-01

    The mechanisms by which Helicobacter pylori infection leads to gastroduodenal ulceration remain poorly understood. Previous studies have shown that H. pylori vacuolating cytotoxin (VacA) inhibits proliferation of gastric epithelial cells, which suggests that H pylori may interfere with gastric mucosal repair mechanisms. In this study, we investigated the effects of H. pylori broth culture supernatants on epidermal growth factor (EGF)-mediated signal transduction pathways in a gastric carcinoma cell line (KATO III). Exposure of these cells to EGF resulted in increased expression and phosphorylation of the EGF receptor (EGF-R), increased ERK2 activity and phosphorylation, and increased c-fos protein levels. Preincubation of cells with broth culture supernatant from VacA (+) H. pylori strain 60190 inhibited the capacity of EGF to induce each of these effects. In contrast, preincubation of cells with broth culture supernatant from an isogenic VacA-mutant strain (H. pylori 60190-v1) failed to inhibit the effects of EGF. These results suggest that the H. pylori vacuolating cytotoxin interferes with EGF-activated signal transduction pathways, which are known to be essential for cell proliferation and ulcer healing. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:9626065

  7. Helicobacter pylori culture supernatant interferes with epidermal growth factor-activated signal transduction in human gastric KATO III cells.

    PubMed

    Pai, R; Wyle, F A; Cover, T L; Itani, R M; Domek, M J; Tarnawski, A S

    1998-06-01

    The mechanisms by which Helicobacter pylori infection leads to gastroduodenal ulceration remain poorly understood. Previous studies have shown that H. pylori vacuolating cytotoxin (VacA) inhibits proliferation of gastric epithelial cells, which suggests that H pylori may interfere with gastric mucosal repair mechanisms. In this study, we investigated the effects of H. pylori broth culture supernatants on epidermal growth factor (EGF)-mediated signal transduction pathways in a gastric carcinoma cell line (KATO III). Exposure of these cells to EGF resulted in increased expression and phosphorylation of the EGF receptor (EGF-R), increased ERK2 activity and phosphorylation, and increased c-fos protein levels. Preincubation of cells with broth culture supernatant from VacA (+) H. pylori strain 60190 inhibited the capacity of EGF to induce each of these effects. In contrast, preincubation of cells with broth culture supernatant from an isogenic VacA-mutant strain (H. pylori 60190-v1) failed to inhibit the effects of EGF. These results suggest that the H. pylori vacuolating cytotoxin interferes with EGF-activated signal transduction pathways, which are known to be essential for cell proliferation and ulcer healing. PMID:9626065

  8. Identification of Gene Markers for Activation of the Nuclear Receptor Pregnane X Receptor

    EPA Science Inventory

    Many environmentally-relevant chemicals and drugs activate the nuclear receptor pregnane X receptor (PXR). Activation of PXR in the mouse liver can lead to increases in liver weight in part through increased hepatocyte replication similar to chemicals that activate other nuclear ...

  9. Epidermal growth factor binding and receptor distribution in the mouse reproductive tract during development

    SciTech Connect

    Bossert, N.L.; Nelson, K.G.; Ross, K.A.; Takahashi, T.; McLachlan, J.A. )

    1990-11-01

    The ontogeny of the epidermal growth factor (EGF) receptor in the different cell types in the neonatal and immature mouse uterus and vagina was examined. Immunohistochemical examination of prenatal and neonatal reproductive tracts with a polyclonal antibody to the EGF receptor shows immunoreactive EGF receptors as early as Day 13 of gestation. Autoradiographic analysis of tissue sections at 3 to 17 days of age (the day of birth is Day 1) demonstrates that both uterine and vaginal epithelial and stromal cells are capable of binding 125I-labeled EGF. Both the 125I-labeled EGF autoradiography and immunohistochemistry in whole tissue show higher EGF receptor levels in the uterine epithelium than the uterine stroma. The presence of EGF receptors was also confirmed by affinity labeling and Scatchard analysis of isolated uterine cell types at 7 and/or 17 days of age. However, in contrast to the autoradiography and immunohistochemistry data of intact tissue, the affinity labeling and Scatchard data of isolated cells indicate that the uterine stroma contains higher levels of EGF receptor than that of the uterine epithelium. The reason for this discrepancy between the different techniques is, as yet, unknown. Regardless of the differences in the actual numbers of EGF receptors obtained, our data demonstrate that the developing mouse reproductive tract contains immunoreactive EGF receptors that are capable of binding 125I-labeled EGF.

  10. Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

    SciTech Connect

    Brown, Richard J.; Adams, Julian J.; Pelekanos, Rebecca A.; Wan, Yu; McKinstry, William J.; Palethorpe, Kathryn; Seeber, Ruth M.; Monks, Thea A.; Eidne, Karin A.; Parker, Michael W.; Waters, Michael J.

    2010-07-13

    Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

  11. Agonist induced constitutive receptor activation as a novel regulatory mechanism. Mu receptor regulation.

    PubMed

    Sadée, W; Wang, Z

    1995-01-01

    We propose the hypothesis that certain G protein coupled receptors can become constitutively activated during agonist stimulation so that the receptor remains active even after the agonist is removed. This new paradigm of receptor regulation may account for some long term effects of neurotransmitters and hormones. We have tested the hypothesis that constitutive mu receptor activation represents a crucial step driving narcotic tolerance and dependence. Our results indeed support the conversion of mu to a constitutively active state, mu*, observed in neuroblastoma SK-N-SH and SH-SY5Y tissue culture, in U293 cells transfected with the mu receptor gene, and in vivo. Constitutive mu activation may result from receptor phosphorylation to yield mu*, and further, in vivo studies indicate that formation of mu* could account for narcotic tolerance and dependence.

  12. HB-EGF Improves Intestinal Microcirculation after Hemorrhagic Shock

    PubMed Central

    Zhang, Hong-yi; Radulescu, Andrei; Chen, Yan; Besner, Gail E.

    2010-01-01

    Background The goal of this study was to determine the role of heparin-binding epidermal growth factor-like growth factor (HB-EGF) as a mediator of gut microcirculation after hemorrhagic shock and resuscitation (HS/R) in mice. Materials and methods HS/R was induced in HB-EGF knockout (KO) and wild type (WT) mice. Ink-gelatin injection and vascular corrosion casting were performed to visualize the gut microvasculature. The degree of gut microcirculatory injury was graded using five patterns of injury (1–5) according to the severity of microvascular hypoperfusion. Statistical analyses were performed using linear mixed models with p<0.05 considered statistically significant. Results HB-EGF KO mice subjected to HS/R had significantly decreased perfusion of the gut microvasculature compared to WT mice subjected to HS/R (p=0.0001). HB-EGF KO mice subjected to HS/R and treated with exogenous HB-EGF had significantly increased gut microvascular perfusion compared to non-HB-EGF treated KO mice (p=0.01). Lastly, WT mice subjected to HS/R and treated with HB-EGF had significantly increased gut microvascular perfusion compared to non-HB-EGF-treated WT mice (p=0.04). Conclusions HB-EGF improves gut microcirculation after HS/R. These findings support the clinical use of HB-EGF in protection of the intestines from disease states associated with intestinal hypoperfusion injury. PMID:20421109

  13. Involvement of aquaporin-3 in epidermal growth factor receptor signaling via hydrogen peroxide transport in cancer cells.

    PubMed

    Hara-Chikuma, Mariko; Watanabe, Sachiko; Satooka, Hiroki

    2016-03-18

    Aquaporin 3 (AQP3), a water/glycerol channel protein, is capable of transporting hydrogen peroxide (H2O2). Here, we show that AQP3-mediated intracellular H2O2 is involved in epidermal growth factor (EGF)-induced cell signaling and its dependent cell function in the EGF receptor (EGFR)-positive cancer cell lines A431 and H1666. AQP3 knockdown suppressed the transport into the cells of extracellular H2O2 produced in response to EGF in A431 and H1666 cells. EGF-induced Erk and Akt activation, which occurred through SHP2 and/or PTEN modulation, was impaired by AQP3 knockdown. Cell growth and migration induced by EGF stimulation were attenuated in AQP3 knockdown cells compared with those in control cells. Coincidentally, tumor growth of A431 cell xenografts in immunodeficient mice was decreased by AQP3 knockdown. Accordingly, a xenograft with AQP3 knockdown A431 cells significantly enhanced the survival of recipient mice compared with the transplantation with control cells. In addition, AQP3 associated with EGFR and NADPH oxidase 2, which we propose is linked to AQP3 producing a localized increase in intracellular H2O2 to function as a second messenger during EGFR cell signaling. Therefore, our findings suggest that AQP3 is required for EGF-EGFR cell signaling in cancer cells and is a therapeutic target for cancer progression.

  14. Parsing ERK Activation Reveals Quantitatively Equivalent Contributions From Epidermal Growth Factor Receptor and HER2 In Human Mammary Epithelial Cells

    SciTech Connect

    Hendriks, Bart S.; Orr, Galya; Wells, Alan H.; Wiley, H. S.; Lauffenburger, Douglas A.

    2005-02-18

    HER2, a member of the EGFR tyrosine kinase family, functions as an accessory EGFR signaling component and alters EGFR trafficking by heterodimerization. HER2 overexpression leads to aberrant cell behavior including enhanced proliferation and motility. Here we apply a combination of computational modeling and quantitative experimental studies of the dynamic interactions between EGFR and HER2, and their downstream activation of extracellular signal-related kinase (ERK) to understand this complex signaling system. Using cells expressing different levels of HER2 relative to the EGFR, we can separate relative contributions of EGFR and HER2 to signaling amplitude and duration. Based on our model calculations, we demonstrate that, in contrast with previous suggestions in the literature, the intrinsic capabilities of EGFR and HER2 to activated ERK are quantitatively equivalent . We find that HER2-mediated effects on EGFR dimerization and trafficking are sufficient to explain the detected HER2-mediated amplification of EGF-induced ERK signaling. Our model suggests that transient amplification of ERK activity by HER2 arises predominantly from the 2-to-1 stoichiometry of receptor kinase to bound ligand in EGFR/HER2 heterodimers compared to the 1-to-1 stoichiometry of the EGFR homodimer, but alterations in receptor trafficking, with resultant EGFR sparing, cause the sustained HER2-mediated enhancement of ERK signaling.

  15. Activation of the neu tyrosine kinase induces the fos/jun transcription factor complex, the glucose transporter and ornithine decarboxylase

    PubMed Central

    1989-01-01

    We have studied the ability of the neu tyrosine kinase to induce a signal for the activation of cell growth-regulated genes. Serum-starved NIH 3T3 cells expressing an epidermal growth factor receptor (EGF- R)/neu construct encoding a hybrid receptor protein were stimulated with EGF and the activation of the neu tyrosine kinase and stimulation of growth factor inducible genes were followed at the mRNA, protein, and activity levels, and compared to the corresponding responses in the neu proto-oncogene and oncogene expressing cells. Induction of the expression of jun mRNAs was an immediate early effect of EGF stimulation, followed by a marked increase in the biosynthesis of the fos/jun transcription factor complex and an increased transcription factor activity as measured by a recombinant transcription unit using chloramphenicol acetyltransferase assays. In distinction, elevated AP- 1/PEA-1 activity in the absence of a significant increase in jun and fos expression was characteristic of the neu oncogene-expressing cells. The glucose transporter mRNA increased at 2 h of EGF stimulation and was associated with enhanced glucose transport of the EGF-treated cells. An increase of ornithine decarboxylase (ODC) mRNA and activity followed these changes. In contrast, serum-starved, EGF-treated neu proto-oncogene- and oncogene-expressing cells showed constitutively low and high glucose transporter and ODC activities, respectively. These findings demonstrate that the chimeric EGF-R/neu receptor is capable of activating the expression of both immediate early genes and biochemical activities associated with cell growth stimulation. PMID:2572601

  16. Activation of EGFR, HER2 and HER3 by neurotensin/neurotensin receptor 1 renders breast tumors aggressive yet highly responsive to lapatinib and metformin in mice

    PubMed Central

    Mourra, Najat; Liu, Jin; De Wever, Olivier; Llorca, Frédérique Penault; Cayre, Anne; Kouchkar, Amal; Gompel, Anne; Forgez, Patricia

    2014-01-01

    A present challenge in breast oncology research is to identify therapeutical targets which could impact tumor progression. Neurotensin (NTS) and its high affinity receptor (NTSR1) are up regulated in 20% of breast cancers, and NTSR1 overexpression was shown to predict a poor prognosis for 5 year overall survival in invasive breast carcinomas. Interactions between NTS and NTSR1 induce pro-oncogenic biological effects associated with neoplastic processes and tumor progression. Here, we depict the cellular mechanisms activated by NTS, and contributing to breast cancer cell aggressiveness. We show that neurotensin (NTS) and its high affinity receptor (NTSR1) contribute to the enhancement of experimental tumor growth and metastasis emergence in an experimental mice model. This effect ensued following EGFR, HER2, and HER3 over-expression and autocrine activation and was associated with an increase of metalloproteinase MMP9, HB-EGF and Neuregulin 2 in the culture media. EGFR over expression ensued in a more intense response to EGF on cellular migration and invasion. Accordingly, lapatinib, an EGFR/HER2 tyrosine kinase inhibitor, as well as metformin, reduced the tumor growth of cells overexpressing NTS and NTSR1. All cellular effects, such as adherence, migration, invasion, altered by NTS/NTSR1 were abolished by a specific NTSR1 antagonist. A strong statistical correlation between NTS-NTSR1-and HER3 (p< 0.0001) as well as NTS-NTSR1-and HER3- HER2 (p< 0.001) expression was found in human breast tumors. Expression of NTS/NTSR1 on breast tumoral cells creates a cellular context associated with cancer aggressiveness by enhancing epidermal growth factor receptor activity. We propose the use of labeled NTS/NTSR1 complexes to enlarge the population eligible for therapy targeting HERs tyrosine kinase inhibitor or HER2 overexpression. PMID:25249538

  17. The ontogeny of epidermal growth factor receptors during mouse development

    SciTech Connect

    Adamson, E.D.; Meek, J.

    1984-05-01

    In an attempt to understand the role(s) of epidermal growth factor (EGF) in vivo during murine development, we have examined the /sup 125/I-EGF binding characteristics of EGF-receptors in membrane preparations of tissues from the 12th day of gestation to parturition. Using autoradiography, the earliest time that we could detect EGF-receptors was on trophoblast cells cultured for 3 days as blastocyst outgrowths. Trophoblast eventually forms a large portion of the placenta, where EGF-receptors have long been recognized. We measured the number and affinity of EGF-receptors on tissues dissected from conceptuses from the 12th day of gestation in order to identify a stage when tissues may be most sensitive to EGF. Whereas the number of EGF receptors increases during gestation for all tissues examined, the affinity of the receptors declines for carcass and placenta and remains relatively unchanged for brain and liver. This suggests that EGF may function differently throughout development. Our hypothesis is that EGF (or its embryonic equivalent) initially stimulates proliferation in embryonic cells and then stimulates differentiation as the tissues mature. In the adult, its main role could be to stimulate tissue repair after damage.

  18. Modulation of Glucagon Receptor Pharmacology by Receptor Activity-modifying Protein-2 (RAMP2)*

    PubMed Central

    Weston, Cathryn; Lu, Jing; Li, Naichang; Barkan, Kerry; Richards, Gareth O.; Roberts, David J.; Skerry, Timothy M.; Poyner, David; Pardamwar, Meenakshi; Reynolds, Christopher A.; Dowell, Simon J.; Willars, Gary B.; Ladds, Graham

    2015-01-01

    The glucagon and glucagon-like peptide-1 (GLP-1) receptors play important, opposing roles in regulating blood glucose levels. Consequently, these receptors have been identified as targets for novel diabetes treatments. However, drugs acting at the GLP-1 receptor, although having clinical efficacy, have been associated with severe adverse side-effects, and targeting of the glucagon receptor has yet to be successful. Here we use a combination of yeast reporter assays and mammalian systems to provide a more complete understanding of glucagon receptor signaling, considering the effect of multiple ligands, association with the receptor-interacting protein receptor activity-modifying protein-2 (RAMP2), and the role of individual G protein α-subunits. We demonstrate that RAMP2 alters both ligand selectivity and G protein preference of the glucagon receptor. Importantly, we also uncover novel cross-reactivity of therapeutically used GLP-1 receptor ligands at the glucagon receptor that is abolished by RAMP2 interaction. This study reveals the glucagon receptor as a previously unidentified target for GLP-1 receptor agonists and highlights a role for RAMP2 in regulating its pharmacology. Such previously unrecognized functions of RAMPs highlight the need to consider all receptor-interacting proteins in future drug development. PMID:26198634

  19. Autoradiographic localization of epidermal growth factor receptors to all major uterine cell types

    SciTech Connect

    Lin, T.H.; Mukku, V.R.; Verner, G.; Kirkland, J.L.; Stancel, G.M.

    1988-03-01

    We have recently studied the structure and function of the uterine epidermal growth factor (EGF) receptor, its hormonal regulation, and its possible role in estrogen-induced uterine DNA synthesis. Since the uterus is composed of multiple cell types, we sought, in the work reported here, to localize EGF binding in this organ by autoradiography. Prior to the actual autoradiography, we performed a companion series of experiments to insure that EGF binding to uterine tissue in situ represented a true receptor interaction. Uteri from immature female rats were incubated in vitro with 125I-EGF at 25 degrees C. Tissue binding was maximal within 120 min and remained constant for at least an additional 120 min. This binding of labeled EGF was largely abolished by excess unlabeled EGF but not by other growth factors, indicating that binding was to specific receptors. The binding of 125I-EGF was saturable and reached a plateau at 4-8 nM; specific binding was half-maximal at 1-2 nM EGF. In situ cross-linking studies revealed that 125I-EGF was bound predominantly to a 170,000 MW EGF receptor similar to that seen in isolated uterine membranes. Incubation of uteri with 125I-EGF followed by autoradiography revealed binding to epithelial cells, stroma, and myometrium. These results provide evidence for the presence of specific EGF receptors in all major uterine cell types of the immature rat.

  20. Albumin-stimulated DNA synthesis is mediated by Ca2+/PKC as well as EGF receptor-dependent p44/42 MAPK and NF-kappaB signal pathways in renal proximal tubule cells.

    PubMed

    Lee, Yu Jin; Han, Ho Jae

    2008-03-01

    It is now recognized that significant tubular reabsorption of albumin occurs under physiological conditions that may play an important role in maintaining proximal tubular integrity and function. Therefore, this study examined the effect of bovine serum albumin (BSA) on DNA synthesis and its related signal molecules in primary cultured rabbit renal proximal tubule cells (PTCs). BSA increased the level of [(3)H]thymidine incorporation in a dose (> or =3 mg/ml)- and time (> or =3 h)-dependent manner, intracellular Ca(2+) concentration, and the level of protein kinase C (PKC) phosphorylation and stimulated the phosphorylation of the epidermal growth factor receptor (EGFR), which was inhibited by EGTA (extracellular Ca(2+) chelator), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM, intracellular Ca(2+) chelator), or PKC inhibitors (staurosporine or bisindolylmaleimide I). In addition, the PKC inhibitors or an EGFR inhibitor (AG-1478) blocked the BSA-induced phosphorylation of p44/42 mitogen-activated protein kinases (MAPKs). BSA also increased the level of nuclear factor-kappaB (NF-kappaB) and inhibitor of NF-kappaB (IkappaB) phosphorylation, which was blocked by staurosporine, AG-1478, or PD-98059 (p44/42 MAPK inhibitor). Inhibition of Ca(2+), PKC, EGFR, p44/42 MAPK, or NF-kappaB signal pathways blocked the BSA-induced incorporation of [(3)H]thymidine. Consequently, the inhibition of Ca(2+), PKC, EGFR, p44/42 MAPKs, or NF-kappaB blocked the BSA-induced increases in cyclin D1, cyclin-dependent kinase (CDK)4, cyclin E, or CDK2 and restored the BSA-induced inhibition of p21(WAF/Cip1) and p27(Kip1) expression. In conclusion, BSA stimulates DNA synthesis that is mediated by Ca(2+)/PKC as well as the EGFR-dependent p44/42 MAPK and NF-kappaB signal pathways in PTCs.

  1. The tumor suppressor PTEN inhibits EGF-induced TSP-1 and TIMP-1 expression in FTC-133 thyroid carcinoma cells

    SciTech Connect

    Soula-Rothhut, Mahdhia; Coissard, Cyrille; Sartelet, Herve; Boudot, Cedric; Bellon, Georges; Martiny, Laurent; Rothhut, Bernard . E-mail: bernard.rothhut@univ-reims.fr

    2005-03-10

    Thrombospondin-1 (TSP-1) is a multidomain extracellular macromolecule that was first identified as natural modulator of angiogenesis and tumor growth. In the present study, we found that epidermal growth factor (EGF) up-regulated TSP-1 expression in FTC-133 (primary tumor) but not in FTC-238 (lung metastasis) thyroid cancer cells. Both EGF and TSP-1 induced expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In FTC-133 cells, EGF induced proliferation in a TSP-1- and TIMP-1-dependent manner. In addition, we determined that re-expression of the tumor suppressor protein PTEN induced cell death, an effect that correlated with a block of Akt kinase phosphorylation. EGF-induced TSP-1 and TIMP-1 promoter activity and protein expression were inhibited in FTC-133 cells stably expressing wtPTEN but not in cells expressing mutant PTEN. Furthermore, we found that wtPTEN inhibited EGF-but not TSP-1-stimulated FTC-133 cell migration and also inhibited invasion induced by EGF and by TSP-1. Finally, an antibody against TSP-1 reversed EGF-stimulated FTC-133 cell invasion as well as the constitutive invasive potential of FTC-238 cells. Overall, our results suggest that PTEN can function as an important modulator of extracellular matrix proteins in thyroid cancer. Therefore, analyzing differential regulation of TSP-1 by growth factors such as EGF can be helpful in understanding thyroid cancer development.

  2. Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties*

    PubMed Central

    Watkins, Harriet A.; Chakravarthy, Madhuri; Abhayawardana, Rekhati S.; Gingell, Joseph J.; Garelja, Michael; Pardamwar, Meenakshi; McElhinney, James M. W. R.; Lathbridge, Alex; Constantine, Arran; Harris, Paul W. R.; Yuen, Tsz-Ying; Brimble, Margaret A.; Barwell, James; Poyner, David R.; Woolley, Michael J.; Conner, Alex C.; Pioszak, Augen A.; Reynolds, Christopher A.

    2016-01-01

    Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function. PMID:27013657

  3. A widely used retinoic acid receptor antagonist induces peroxisome proliferator-activated receptor-gamma activity.

    PubMed

    Schupp, Michael; Curtin, Joshua C; Kim, Roy J; Billin, Andrew N; Lazar, Mitchell A

    2007-05-01

    Nuclear receptors (NRs) are transcription factors whose activity is regulated by the binding of small lipophilic ligands, including hormones, vitamins, and metabolites. Pharmacological NR ligands serve as important therapeutic agents; for example, all-trans retinoic acid, an activating ligand for retinoic acid receptor alpha (RARalpha), is used to treat leukemia. Another RARalpha ligand, (E)-S,S-dioxide-4-(2-(7-(heptyloxy)-3,4-dihydro-4,4-dimethyl-2H-1-benzothiopyran-6-yl)-1-propenyl)-benzoic acid (Ro 41-5253), is a potent antagonist that has been a useful and purportedly specific probe of RARalpha function. Here, we report that Ro 41-5253 also activates the peroxisome proliferator-activated receptor gamma (PPARgamma), a master regulator of adipocyte differentiation and target of widely prescribed antidiabetic thiazolidinediones (TZDs). Ro 41-5253 enhanced differentiation of mouse and human preadipocytes and activated PPARgamma target genes in mature adipocytes. Like the TZDs, Ro 41-5253 also down-regulated PPARgamma protein expression in adipocytes. In addition, Ro 41-5253 activated the PPARgamma-ligand binding domain in transiently transfected HEK293T cells. These effects were not prevented by a potent RARalpha agonist or by depleting cells of RARalpha, indicating that PPARgamma activation was not related to RARalpha antagonism. Indeed, Ro 41-5253 was able to compete with TZD ligands for binding to PPARgamma, suggesting that Ro 41-5253 directly affects PPAR activity. These results vividly demonstrate that pharmacological NR ligands may have "off-target" effects on other NRs. Ro 41-5253 is a PPARgamma agonist as well as an RARalpha antagonist whose pleiotropic effects on NRs may signify a unique spectrum of biological responses.

  4. A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes

    SciTech Connect

    Itoh, Reina E.; Kurokawa, Kazuo; Fujioka, Aki; Sharma, Alok; Mayer, Bruce J.; Matsuda, Michiyuki . E-mail: matsudam@biken.osaka-u.ac.jp

    2005-07-01

    Epidermal growth factor (EGF) receptor plays a pivotal role in a variety of cellular functions, such as proliferation, differentiation, and migration. To monitor the EGF receptor (EGFR) activity in living cells, we developed a probe for EGFR activity based on the principle of fluorescence resonance energy transfer (FRET). Previously, we developed a probe designated as Picchu (Phosphorylation indicator of the CrkII chimeric unit), which detects the tyrosine phosphorylation of the CrkII adaptor protein. We used a pair of synthetic amphipathic helixes, WinZipA2 and WinZipB1, to bind Picchu non-covalently to the carboxyl-terminus of the EGFR. Using this modified probe named Picchu-Z, the activity of EGFR was followed in EGF-stimulated Cos7 cells. We found that a high level of tyrosine phosphorylation of Picchu-Z probe remained after endocytosis until the point when the EGFR was translocated to the perinuclear region. These findings are in agreement with the previously reported 'signaling endosome' model. Furthermore, by pulse stimulation with EGF and by acute ablation of EGFR activity with AG1478, it was suggested that the phosphorylation of Picchu-Z probe, and probably the phosphorylation of EGFR also, underwent a rapid equilibrium ({tau} {sub 1/2} < 2 min) between the phosphorylated and dephosphorylated states in the presence of EGF.

  5. Proteasome inhibition blocks ligand-induced dynamic processing and internalization of epidermal growth factor receptor via altered receptor ubiquitination and phosphorylation.

    PubMed

    Kesarwala, Aparna H; Samrakandi, Mustapha M; Piwnica-Worms, David

    2009-02-01

    Epidermal growth factor (EGF) receptor (EGFR), a member of the EGF superfamily of receptor tyrosine kinases, is a critical regulator of cell growth and an important target for single agent and combination anticancer therapeutics. To further investigate the dynamics of ligand-induced EGFR processing and regulation noninvasively, we developed a chimeric EGFR-firefly luciferase (FLuc) fusion reporter to directly monitor processing of EGFR in real-time. In a stable HeLa cell line expressing the reporter at physiologically relevant levels, bioluminescence imaging continuously monitored reporter dynamics, correlating with the ligand-induced response of endogenous EGFR as determined by Western blot, subcellular localization of an EGFR-green fluorescent protein (GFP) fusion protein, and validated pharmacologic responses. The signaling competency of the reporter was confirmed by gene rescue experiments in EGFR-null cells. Bioluminescence analysis further showed that proteasome inhibition with bortezomib or MG132 attenuated overall ligand-induced degradation of EGFR. In cells expressing EGFR-GFP, pretreatment with proteasome inhibitors trapped essentially all of the receptor at the cell membrane both before and after ligand-induced activation with EGF. Furthermore, proteasome inhibition enhanced receptor ubiquitination in both the basal and ligand-activated states as well as delayed the processing of ligand-activated phosphorylation of the receptor, kinetically correlating with attenuated receptor degradation. These observations point to a potential mechanism for the synergistic therapeutic effects of combination EGFR- and proteasome-targeted therapies.

  6. Integrin α5/β1 Mediates Fibronectin-dependent Epithelial Cell Proliferation through Epidermal Growth Factor Receptor Activation

    PubMed Central

    Kuwada, Scott K.; Li, Xiufen

    2000-01-01

    Human integrin α5 was transfected into the integrin α5/β1–negative intestinal epithelial cell line Caco-2 to study EGF receptor (EGFR) and integrin α5/β1 signaling interactions involved in epithelial cell proliferation. On uncoated or fibronectin-coated plastic, the integrin α5 and control (vector only) transfectants grew at similar rates. In the presence of the EGFR antagonistic mAb 225, the integrin α5 transfectants and controls were significantly growth inhibited on plastic. However, when cultured on fibronectin, the integrin α5 transfectants were not growth inhibited by mAb 225. The reversal of mAb 225–mediated growth inhibition on fibronectin for the integrin α5 transfectants correlated with activation of the EGFR, activation of MAPK, and expression of proliferating cell nuclear antigen. EGFR kinase activity was necessary for both MAPK activation and integrin α5/β1–mediated cell proliferation. Although EGFR activation occurred when either the integrin α5–transfected or control cells were cultured on fibronectin, coprecipitation of the EGFR with SHC could be demonstrated only in the integrin α5–transfected cells. These results suggest that integrin α5/β1 mediates fibronectin-induced epithelial cell proliferation through activation of the EGFR. PMID:10888683

  7. Mineralocorticoid receptor activation in obesity hypertension.

    PubMed

    Nagase, Miki; Fujita, Toshiro

    2009-08-01

    Obesity hypertension and metabolic syndrome have become major public health concerns. Nowadays, aldosterone is recognized as an important mediator of cardiovascular and renal damage. In the kidney, aldosterone injures glomerular visceral epithelial cells (podocytes), the final filtration barrier to plasma macromolecules, leading to proteinuria and glomerulosclerosis. Mineralocorticoid receptor (MR) antagonists effectively ameliorate proteinuria in patients or in animal models of hypertension, diabetes mellitus and chronic kidney disease (CKD), as well as in patients who experience 'aldosterone breakthrough.' Recently, clinical and experimental studies have shown that plasma aldosterone concentration is associated with obesity hypertension and metabolic syndrome. We showed that spontaneously hypertensive rats (SHR)/cp, an experimental model of obesity hypertension and metabolic syndrome, are prone to glomerular podocyte injury, proteinuria and left ventricular diastolic dysfunction, especially when the animals are fed a high-salt diet. Inappropriate activation of the aldosterone/MR system underlies the renal and cardiac injuries. Adipocyte-derived aldosterone-releasing factors (ARFs), although still unidentified, may account for aldosterone excess and the resultant target organ complication in SHR/cp. On the other hand, recent studies have shown that MR activation triggers target organ disease even in normal or low aldosterone states. We identified a small GTP (guanosine triphosphate)-binding protein, Rac1, as a novel activator of MR, and showed that this ligand-independent MR activation by Rac1 contributes to the nephropathy of several CKD models. We expect that ARFs and Rac1 can be novel therapeutic targets for metabolic syndrome and CKD. Future large-scale clinical trials are awaited to prove the efficacy of MR blockade in patients with obesity hypertension and metabolic syndrome.

  8. RELAXIN ACTIVATES PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA

    PubMed Central

    Singh, Sudhir; Bennett, Robert G

    2009-01-01

    SUMMARY Relaxin is a polypeptide hormone that triggers multiple signaling pathways through its receptor RXFP1. Many of relaxin’s functions, including vascular and antifibrotic effects, are similar to those induced by activation of PPARγ. In this study, we tested the hypothesis that relaxin signaling through RXFP1 would activate PPARγ activity. In cells overexpressing RXFP1 (HEK-RXFP1), relaxin increased transcriptional activity through a PPAR response element (PPRE) in a concentration-dependent manner. In cells lacking RXFP1, relaxin had no effect. Relaxin increased both the baseline activity and the response to the PPARγ agonists rosiglitazone and 15d-PGJ2, but not to agonists of PPARα or PPARδ. In HEK-RXFP1 cells infected with adenovirus expressing PPARγ, relaxin increased transcriptional activity through PPRE, and this effect was blocked with an adenovirus expressing a dominant-negative PPARγ. Knockdown of PPARγ using siRNA resulted in a decrease in the response to both relaxin and rosiglitazone. Both relaxin and rosiglitazone increased expression of the PPARγ target genes CD36 and LXRα in HEK-RXFP1 and in THP-1 cells naturally expressing RXFP1. Relaxin did not increase PPARγ mRNA or protein levels. Treatment of cells with GW9662, an inhibitor of PPARγ ligand binding, effectively blocked rosiglitazone-induced PPARγ activation, but had no effect on relaxin activation of PPARγ. These results suggest that relaxin activates PPARγ activity, and increases the overall response in the presence PPARγ agonists. This activation is dependent on the presence of RXFP1. Furthermore, relaxin activates PPARγ via a ligand-independent mechanism. These studies represent the first report that relaxin can activate the transcriptional activity of PPARγ. PMID:19712722

  9. Serotonin increases ERK1/2 phosphorylation in astrocytes by stimulation of 5-HT2B and 5-HT2C receptors.

    PubMed

    Li, Baoman; Zhang, Shiquen; Li, Min; Hertz, Leif; Peng, Liang

    2010-11-01

    We have previously shown that fluoxetine causes ERK(1/2) phosphorylation in cultured mouse astrocytes mediated exclusively by stimulation of 5-HT(2B) receptors (Li et al., 2008b). This raises the question whether this is also the case for serotonin (5-HT) itself. In the present study serotonin was found to induce ERK(1/2) phosphorylation by stimulation of 5-HT(2B) receptors with high affinity (EC(50): 20-30 pM), and by stimulation of 5-HT(2C) receptor with low affinity (EC(50): 1 microM or higher). ERK(1/2) phosphorylation induced by stimulation of either 5-HT(2B) or 5-HT(2C) receptors was mediated by epidermal growth factor (EGF) receptor transactivation (Peng et al., this issue), shown by the inhibitory effect of AG1478, an inhibitor of the EGF receptor tyrosine kinase, and GM6001, an inhibitor of Zn-dependent metalloproteinases, and thus of 5-HT(2B) receptor-mediated EGF receptor agonist release. It is discussed that the high potency of the 5-HT(2B)-mediated effect is consistent with literature data for binding affinity of serotonin to cloned human 5-HT(2B) receptors and with observations of low extracellular concentrations of serotonin in brain, which would allow a demonstrated moderate and modality-dependent increase in specific brain areas to activate 5-HT(2B) receptors. In contrast the relevance of the observed 5-HT(2C) receptors on astrocytes is questioned.

  10. Epidermal growth factor and its receptors in human pancreatic carcinoma

    SciTech Connect

    Chen, Y.F.; Pan, G.Z.; Hou, X.; Liu, T.H.; Chen, J.; Yanaihara, C.; Yanaihara, N. )

    1990-05-01

    The role of epidermal growth factor (EGF) in oncogenesis and progression of malignant tumors is a subject of vast interest. In this study, radioimmunoassay and radioreceptor assay of EGF were established. EGF contents in malignant and benign pancreatic tumors, in normal pancreas tissue, and in culture media of a human pancreatic carcinoma cell line were determined. EGF receptor binding studies were performed. It was shown that EGF contents in pancreatic carcinomas were significantly higher than those in normal pancreas or benign pancreatic tumors. EGF was also detected in the culture medium of a pancreatic carcinoma cell line. The binding of 125I-EGF to the pancreatic carcinoma cells was time and temperature dependent, reversible, competitive, and specific. Scatchard analysis showed that the dissociation constant of EGF receptor was 2.1 X 10(-9) M, number of binding sites was 1.3 X 10(5) cell. These results indicate that there is an over-expression of EGF/EGF receptors in pancreatic carcinomas, and that an autocrine regulatory mechanism may exist in the growth-promoting effect of EGF on tumor cells.

  11. Cell death sensitization of leukemia cells by opioid receptor activation

    PubMed Central

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich

    2013-01-01

    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

  12. Neuropeptide Y receptor mediates activation of ERK1/2 via transactivation of the IGF receptor.

    PubMed

    Lecat, Sandra; Belemnaba, Lazare; Galzi, Jean-Luc; Bucher, Bernard

    2015-07-01

    Neuropeptide Y binds to G-protein coupled receptors whose action results in inhibition of adenylyl cyclase activity. Using HEK293 cells stably expressing the native neuropeptide Y Y1 receptors, we found that the NPY agonist elicits a transient phosphorylation of the extracellular signal-regulated kinases (ERK1/2). We first show that ERK1/2 activation following Y1 receptor stimulation is dependent on heterotrimeric Gi/o since it is completely inhibited by pre-treatment with pertussis toxin. In addition, ERK1/2 activation is internalization-independent since mutant Y1 receptors unable to recruit β-arrestins, can still activate ERK signaling to the same extent as wild-type receptors. We next show that this activation of the MAPK pathway is inhibited by the MEK inhibitor U0126, is not dependent on calcium signaling at the Y1 receptor (no effect upon inhibition of phospholipase C, protein kinase C or protein kinase D) but instead dependent on Gβ/γ and associated signaling pathways that activate PI3-kinase. Although inhibition of the epidermal-growth factor receptor tyrosine kinase did not influence NPY-induced ERK1/2 activation, we show that the inhibition of insulin growth factor receptor IGFR by AG1024 completely blocks activation of ERK1/2 by the Y1 receptor. This Gβ/γ-PI3K-AG1024-sensitive pathway does not involve activation of IGFR through the release of a soluble ligand by metalloproteinases since it is not affected by the metalloproteinase inhibitor marimastat. Finally, we found that a similar pathway, sensitive to wortmannin-AG1024 but insensitive to marimastat, is implicated in activation of ERK signaling in HEK293 cells by endogenously expressed GPCRs coupled to Gq-protein (muscarinic M3 receptors) or coupled to Gs-protein (endothelin ETB receptors). Our analysis is the first to show that β-arrestin recruitment to the NPY Y1 receptor is not necessary for MAPK activation by this receptor but that transactivation of the IGFR receptor is required.

  13. Cellular localization of the activated EGFR determines its effect on cell growth in MDA-MB-468 cells

    SciTech Connect

    Hyatt, Dustin C.; Ceresa, Brian P.

    2008-11-01

    The epidermal growth factor (EGF) receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase that regulates diverse cell functions that are dependent upon cell type, the presence of downstream effectors, and receptor density. In addition to activating biochemical pathways, ligand stimulation causes the EGFR to enter the cell via clathrin-coated pits. Endocytic trafficking influences receptor signaling by controlling the duration of EGFR phosphorylation and coordinating the receptor's association with downstream effectors. To better understand the individual contributions of cell surface and cytosolic EGFRs on cell physiology, we used EGF that was conjugated to 900 nm polystyrene beads (EGF-beads). EGF-beads can stimulate the EGFR and retain the activated receptor at the plasma membrane. In MDA-MB-468 cells, a breast cancer cell line that over-expresses the EGFR, only internalized, activated EGFRs stimulate caspase-3 and induce cell death. Conversely, signaling cascades triggered from activated EGFR retained at the cell surface inhibit caspase-3 and promote cell proliferation. Thus, through endocytosis, the activated EGFR can differentially regulate cell growth in MDA-MB-468 cells.

  14. The growth hormone receptor: mechanism of activation and clinical implications.

    PubMed

    Brooks, Andrew J; Waters, Michael J

    2010-09-01

    Growth hormone is widely used clinically to promote growth and anabolism and for other purposes. Its actions are mediated via the growth hormone receptor, both directly by tyrosine kinase activation and indirectly by induction of insulin-like growth factor 1 (IGF-1). Insensitivity to growth hormone (Laron syndrome) can result from mutations in the growth hormone receptor and can be treated with IGF-1. This treatment is, however, not fully effective owing to the loss of the direct actions of growth hormone and altered availability of exogenous IGF-1. Excessive activation of the growth hormone receptor by circulating growth hormone results in gigantism and acromegaly, whereas cell transformation and cancer can occur in response to autocrine activation of the receptor. Advances in understanding the mechanism of receptor activation have led to a model in which the growth hormone receptor exists as a constitutive dimer. Binding of the hormone realigns the subunits by rotation and closer apposition, resulting in juxtaposition of the catalytic domains of the associated tyrosine-protein kinase JAK2 below the cell membrane. This change results in activation of JAK2 by transphosphorylation, then phosphorylation of receptor tyrosines in the cytoplasmic domain, which enables binding of adaptor proteins, as well as direct phosphorylation of target proteins. This model is discussed in the light of salient information from closely related class 1 cytokine receptors, such as the erythropoietin, prolactin and thrombopoietin receptors. PMID:20664532

  15. beta-Arrestin mediates beta1-adrenergic receptor-epidermal growth factor receptor interaction and downstream signaling.

    PubMed

    Tilley, Douglas G; Kim, Il-Man; Patel, Priyesh A; Violin, Jonathan D; Rockman, Howard A

    2009-07-24

    beta1-Adrenergic receptor (beta1AR) stimulation confers cardioprotection via beta-arrestin-de pend ent transactivation of epidermal growth factor receptors (EGFRs), however, the precise mechanism for this salutary process is unknown. We tested the hypothesis that the beta1AR and EGFR form a complex that differentially directs intracellular signaling pathways. beta1AR stimulation and EGF ligand can each induce equivalent EGFR phosphorylation, internalization, and downstream activation of ERK1/2, but only EGF ligand causes translocation of activated ERK to the nucleus, whereas beta1AR-stimulated/EGFR-transactivated ERK is restricted to the cytoplasm. beta1AR and EGFR are shown to interact as a receptor complex both in cell culture and endogenously in human heart, an interaction that is selective and undergoes dynamic regulation by ligand stimulation. Although catecholamine stimulation mediates the retention of beta1AR-EGFR interaction throughout receptor internalization, direct EGF ligand stimulation initiates the internalization of EGFR alone. Continued interaction of beta1AR with EGFR following activation is dependent upon C-terminal tail GRK phosphorylation sites of the beta1AR and recruitment of beta-arrestin. These data reveal a new signaling paradigm in which beta-arrestin is required for the maintenance of a beta1AR-EGFR interaction that can direct cytosolic targeting of ERK in response to catecholamine stimulation.

  16. AP-1-mediated invasion requires increased expression of the hyaluronan receptor CD44.

    PubMed Central

    Lamb, R F; Hennigan, R F; Turnbull, K; Katsanakis, K D; MacKenzie, E D; Birnie, G D; Ozanne, B W

    1997-01-01

    Fibroblasts transformed by Fos oncogenes display increased expression of a number of genes implicated in tumor cell invasion and metastasis. In contrast to normal 208F rat fibroblasts, Fos-transformed 208F fibroblasts are growth factor independent for invasion. We demonstrate that invasion of v-Fos- or epidermal growth factor (EGF)-transformed cells requires AP-1 activity. v-Fos-transformed cell invasion is inhibited by c-jun antisense oligonucleotides and by expression of a c-jun dominant negative mutant, TAM-67. EGF-induced invasion is inhibited by both c-fos and c-jun antisense oligonucleotides. CD44s, the standard form of a transmembrane receptor for hyaluronan, is implicated in tumor cell invasion and metastasis. We demonstrate that increased expression of CD44 in Fos- and EGF-transformed cells is dependent upon AP-1. CD44 antisense oligonucleotides reduce expression of CD44 in v-Fos- or EGF-transformed cells and inhibit invasion but not migration. Expression of a fusion protein between human CD44s and Aequorea victoria green fluorescent protein (GFP) in 208F cells complements the inhibition of invasion by the rat-specific CD44 antisense oligonucleotide. We further show that both v-Fos and EGF transformations result in a concentration of endogenous CD44 or exogenous CD44-GFP at the ends of pseudopodial cell extensions. These results support the hypothesis that one role of AP-1 in transformation is to activate a multigenic invasion program. PMID:9001250

  17. Tethering of Epidermal Growth Factor (EGF) to Beta Tricalcium Phosphate (βTCP) via Fusion to a High Affinity, Multimeric βTCP-Binding Peptide: Effects on Human Multipotent Stromal Cells/Connective Tissue Progenitors

    PubMed Central

    Stockdale, Linda; Saini, Sunil; Lee, Richard T.; Griffith, Linda G.

    2015-01-01

    Transplantation of freshly-aspirated autologous bone marrow, together with a scaffold, is a promising clinical alternative to harvest and transplantation of autologous bone for treatment of large defects. However, survival proliferation, and osteogenic differentiation of the marrow-resident stem and progenitor cells with osteogenic potential can be limited in large defects by the inflammatory microenvironment. Previous studies using EGF tethered to synthetic polymer substrates have demonstrated that surface-tethered EGF can protect human bone marrow-derived osteogenic stem and progenitor cells from pro-death inflammatory cues and enhance their proliferation without detriment to subsequent osteogenic differentiation. The objective of this study was to identify a facile means of tethering EGF to clinically-relevant βTCP scaffolds and to demonstrate the bioactivity of EGF tethered to βTCP using stimulation of the proliferative response of human bone-marrow derived mesenchymal stem cells (hBMSC) as a phenotypic metric. We used a phage display library and panned against βTCP and composites of βTCP with a degradable polyester biomaterial, together with orthogonal blocking schemes, to identify a 12-amino acid consensus binding peptide sequence, LLADTTHHRPWT, with high affinity for βTCP. When a single copy of this βTCP-binding peptide sequence was fused to EGF via a flexible peptide tether domain and expressed recombinantly in E. coli together with a maltose-binding domain to aid purification, the resulting fusion protein exhibited modest affinity for βTCP. However, a fusion protein containing a linear concatamer containing 10 repeats of the binding motif the resulting fusion protein showed high affinity stable binding to βTCP, with only 25% of the protein released after 7 days at 37oC. The fusion protein was bioactive, as assessed by its abilities to activate kinase signaling pathways downstream of the EGF receptor when presented in soluble form, and to enhance

  18. The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor

    SciTech Connect

    Zhou, X. Edward; Suino-Powell, Kelly M.; Xu, Yong; Chan, Cee-Wah; Tanabe, Osamu; Kruse, Schoen W.; Reynolds, Ross; Engel, James Douglas; Xu, H. Eric

    2015-11-30

    Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

  19. Gq protein mediates UVB-induced cyclooxygenase-2 expression by stimulating HB-EGF secretion from HaCaT human keratinocytes

    SciTech Connect

    Seo, MiRan; Juhnn, Yong-Sung

    2010-03-05

    Ultraviolet (UV) radiation induces cyclooxygenase-2 expression to produce cellular responses including aging and carcinogenesis in skin. We hypothesised that heterotrimeric G proteins mediate UV-induced COX-2 expression by stimulating secretion of soluble HB-EGF (sHB-EGF). In this study, we aimed to elucidate the role and underlying mechanism of the {alpha} subunit of Gq protein (G{alpha}q) in UVB-induced HB-EGF secretion and COX-2 induction. We found that expression of constitutively active G{alpha}q (G{alpha}qQL) augmented UVB-induced HB-EGF secretion, which was abolished by knockdown of G{alpha}q with shRNA in HaCaT human keratinocytes. G{alpha}q was found to mediate the UVB-induced HB-EGF secretion by sequential activation of phospholipase C (PLC), protein kinase C{delta} (PKC{delta}), and matrix metaloprotease-2 (MMP-2). Moreover, G{alpha}qQL mediated UVB-induced COX-2 expression in an HB-EGF-, EGFR-, and p38-dependent manner. From these results, we concluded that G{alpha}q mediates UV-induced COX-2 expression through activation of EGFR by HB-EGF, of which ectodomain shedding was stimulated through sequential activation of PLC, PKC{delta} and MMP-2 in HaCaT cells.

  20. A lactobacillus rhamnosus GG-derived soluble protein, p40, stimulates ligand release from intestinal epithelial cells to transactivate epidermal growth factor receptor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis and preserves barrier function by activation of EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study was to determine the mechanisms by which p40...

  1. The Mitogenic Potential of Heparin-Binding Epidermal Growth Factor in the Human Endometrium Is Mediated by the Epidermal Growth Factor Receptor and Is Modulated by Tumor Necrosis Factor-α

    PubMed Central

    CHOBOTOVA, KATYA; MUCHMORE, MARY-ELIZABETH; CARVER, JANET; YOO, HYUNG-J; MANEK, SANJIV; GULLICK, WILLIAM J.; BARLOW, DAVID H.; MARDON, HELEN J.

    2006-01-01

    Heparin-binding epidermal growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, is implicated in a variety of biological processes, including reproduction. Previous studies describe increased levels of HB-EGF in the human endometrium during the midsecretory stage of the menstrual cycle, suggesting a function for HB-EGF in implantation of the human blastocyst. Here we have investigated the expression and function of the soluble and transmembrane forms of HB-EGF in the human endometrium. We show that the expression of the transmembrane form of HB-EGF in the human endometrium is modulated according to the stage of the menstrual cycle. We present data demonstrating that both the soluble and transmembrane forms of HB-EGF induce DNA synthesis in human endometrial stromal cells. Furthermore, TNFα has a cooperative effect on HB-EGF, EGF, TGFα, and betacellulin-induced DNA synthesis in stromal cells, suggesting roles for the EGF family and TNFα in regeneration and maturation of human endometrium. Induction of DNA synthesis by HB-EGF and its modulation by TNFα in endometrial stromal cells are mediated by the EGF receptor and not the HB-EGF receptor ErbB4. Our data suggest key functions for HB-EGF, TNFα, and the EGF receptor in endometrial maturation, via autocrine/paracrine and juxtacrine pathways, in preparation for embryo implantation. PMID:12466384

  2. Cloning and biological activity of epigen, a novel member of the epidermal growth factor superfamily.

    PubMed

    Strachan, L; Murison, J G; Prestidge, R L; Sleeman, M A; Watson, J D; Kumble, K D

    2001-05-25

    High throughput sequencing of a mouse keratinocyte library was used to identify an expressed sequence tag with homology to the epidermal growth factor (EGF) family of growth factors. We have named the protein encoded by this expressed sequence tag Epigen, for epithelial mitogen. Epigen encodes a protein of 152 amino acids that contains features characteristic of the EGF superfamily. Two hydrophobic regions, corresponding to a putative signal sequence and transmembrane domain, flank a core of amino acids encompassing six cysteine residues and two putative N-linked glycosylation sites. Epigen shows 24-37% identity to members of the EGF superfamily including EGF, transforming growth factor alpha, and Epiregulin. Northern blotting of several adult mouse tissues indicated that Epigen was present in testis, heart, and liver. Recombinant Epigen was synthesized in Escherichia coli and refolded, and its biological activity was compared with that of EGF and transforming growth factor alpha in several assays. In epithelial cells, Epigen stimulated the phosphorylation of c-erbB-1 and mitogen-activated protein kinases and also activated a reporter gene containing enhancer sequences present in the c-fos promoter. Epigen also stimulated the proliferation of HaCaT cells, and this proliferation was blocked by an antibody to the extracellular domain of the receptor tyrosine kinase c-erbB-1. Thus, Epigen is the newest member of the EGF superfamily and, with its ability to promote the growth of epithelial cells, may constitute a novel molecular target for wound-healing therapy. PMID:11278323

  3. Flavonoids with epidermal growth factor-receptor tyrosine kinase inhibitory activity stimulate PEPT1-mediated cefixime uptake into human intestinal epithelial cells.

    PubMed

    Wenzel, U; Kuntz, S; Daniel, H

    2001-10-01

    We have tested 33 flavonoids, occurring ubiquitously in foods of plant origin, for their ability to alter the transport of the beta-lactam antibiotic cefixime via the H+-coupled intestinal peptide transporter PEPT1 in the human intestinal epithelial cell line Caco-2. Of the flavonoids tested, quercetin, genistein, naringin, diosmin, acacetin, and chrysin increased uptake of [14C]cefixime dose dependently by up to 60%. All other flavonoids were either without effect or decreased the absorption of cefixime. Quercetin was shown to increase the Vmax of cefixime influx without changing the apparent Km for transport. However, the expected concomitant increase in intracellular acidification due to PEPT1-mediated cefixime/H+-cotransport was less pronounced in the presence of quercetin. This suggested that pH regulatory systems such as apical Na+/H+-exchange could be activated by quercetin and maintain the proton-motive driving force for cefixime uptake. Since quercetin and genistein have been shown to inhibit epidermal growth factor (EGF)-receptor tyrosine kinases, we applied tyrphostin 25 to prove whether such an inhibition could explain the stimulatory effects seen on cefixime uptake. It was found that tyrphostin 25 simulated the effects of quercetin by increasing cefixime absorption due to maintenance of the transmembrane pH gradient. In conclusion, our studies show that flavonoids with EGF-receptor tyrosine kinase inhibitory activities enhance the intestinal absorption of the beta-lactam antibiotic cefixime in Caco-2 cells by activation of apical Na+/H+-exchange and a concomitant increase of the driving force for PEPT1.

  4. NEU3 Sialidase Is Activated under Hypoxia and Protects Skeletal Muscle Cells from Apoptosis through the Activation of the Epidermal Growth Factor Receptor Signaling Pathway and the Hypoxia-inducible Factor (HIF)-1α

    PubMed Central

    Scaringi, Raffaella; Piccoli, Marco; Papini, Nadia; Cirillo, Federica; Conforti, Erika; Bergante, Sonia; Tringali, Cristina; Garatti, Andrea; Gelfi, Cecilia; Venerando, Bruno; Menicanti, Lorenzo; Tettamanti, Guido; Anastasia, Luigi

    2013-01-01

    NEU3 sialidase, a key enzyme in ganglioside metabolism, is activated under hypoxic conditions in cultured skeletal muscle cells (C2C12). NEU3 up-regulation stimulates the EGF receptor signaling pathway, which in turn activates the hypoxia-inducible factor (HIF-1α), resulting in a final increase of cell survival and proliferation. In the same cells, stable overexpression of sialidase NEU3 significantly enhances cell resistance to hypoxia, whereas stable silencing of the enzyme renders cells more susceptible to apoptosis. These data support the working hypothesis of a physiological role played by NEU3 sialidase in protecting cells from hypoxic stress and may suggest new directions in the development of therapeutic strategies against ischemic diseases, particularly of the cerebro-cardiovascular system. PMID:23209287

  5. Temperature-induced unfolding of epidermal growth factor (EGF): insight from molecular dynamics simulation.

    PubMed

    Yan, Chunli; Pattani, Varun; Tunnell, James W; Ren, Pengyu

    2010-08-24

    Thermal disruption of protein structure and function is a potentially powerful therapeutic vehicle. With the emerging nanoparticle-targeting and femtosecond laser technology, it is possible to deliver heating locally to specific molecules. It is therefore important to understand how fast a protein can unfold or lose its function at high temperatures, such as near the water boiling point. In this study, the thermal damage of EGF was investigated by combining the replica exchange (136 replicas) and conventional molecular dynamics simulations. The REMD simulation was employed to rigorously explore the free-energy landscape of EGF unfolding. Interestingly, besides the native and unfolded states, we also observed a distinct molten globule (MG) state that retained substantial amount of native contacts. Based on the understanding that which the unfolding of EGF is a three-state process, we have examined the unfolding kinetics of EGF (N-->MG and MG-->D) with multiple 20-ns conventional MD simulations. The Arrhenius prefactors and activation energy barriers determined from the simulation are within the range of previously studied proteins. In contrast to the thermal damage of cells and tissues which take place on the time scale of seconds to hours at relatively low temperatures, the denaturation of proteins occur in nanoseconds when the temperature of heat bath approaches the boiling point. PMID:20466569

  6. Conserved structure and adjacent location of the thrombin receptor and protease-activated receptor 2 genes define a protease-activated receptor gene cluster.

    PubMed Central

    Kahn, M.; Ishii, K.; Kuo, W. L.; Piper, M.; Connolly, A.; Shi, Y. P.; Wu, R.; Lin, C. C.; Coughlin, S. R.

    1996-01-01

    BACKGROUND: Thrombin is a serine protease that elicits a variety of cellular responses. Molecular cloning of a thrombin receptor revealed a G protein-coupled receptor that is activated by a novel proteolytic mechanism. Recently, a second protease-activated receptor was discovered and dubbed PAR2. PAR2 is highly related to the thrombin receptor by sequence and, like the thrombin receptor, is activated by cleavage of its amino terminal exodomain. Also like the thrombin receptor, PAR2 can be activated by the hexapeptide corresponding to its tethered ligand sequence independent of receptor cleavage. Thus, functionally, the thrombin receptor and PAR2 constitute a fledgling receptor family that shares a novel proteolytic activation mechanism. To further explore the relatedness of the two known protease-activated receptors and to examine the possibility that a protease-activated gene cluster might exist, we have compared the structure and chromosomal locations of the thrombin receptor and PAR2 genes. MATERIALS AND METHODS: The genomic structures of the two protease-activated receptor genes were determined by analysis of lambda phage, P1 bacteriophage, and bacterial artificial chromosome (BAC) genomic clones. Chromosomal location was determined with fluorescent in situ hybridization (FISH) on metaphase chromosomes, and the relative distance separating the two genes was evaluated both by means of two-color FISH and analysis of YACs and BACs containing both genes. RESULTS: Analysis of genomic clones revealed that the two protease-activated receptor genes share a two-exon genomic structure in which the first exon encodes 5'-untranslated sequence and signal peptide, and the second exon encodes the mature receptor protein and 3'-untranslated sequence. The two receptor genes also share a common locus with the two human genes located at 5q13 and the two mouse genes at 13D2, a syntenic region of the mouse genome. These techniques also suggest that the physical distance separating

  7. Regulation of epidermal-growth-factor-receptor signal transduction by cis-unsaturated fatty acids. Evidence for a protein kinase C-independent mechanism.

    PubMed Central

    Casabiell, X; Pandiella, A; Casanueva, F F

    1991-01-01

    The effect of acute treatment with non-esterified fatty acids (NEFA) on transmembrane signalling has been investigated in three different cell lines. In EGFR T17 cells, pretreatment with cis-unsaturated (oleic and palmitoleic acids) NEFA, but not with saturated or trans-unsaturated NEFA, inhibited the epidermal-growth-factor (EGF)-induced increases in cytosolic [Ca2+], membrane potential and Ins(1,4,5)P3 generation. The blocking effect was found to be time- and dose-dependent and rapidly reversible after washout. However, oleic acid treatment did not block either binding of 125I-EGF to its receptor or EGF-induced autophosphorylation of the EGF receptor. The mechanism of action of NEFA could not be attributed to protein kinase C activation, since (i) down-regulation of the enzyme by long-term treatment with phorbol esters did not prevent blockade by oleic acid, and (ii) the effects of acutely administered phorbol ester and oleic acid were additive. In this cell line, signalling at bradykinin and bombesin receptors was also impaired by oleic acid. In A431 cells, oleic acid also blocked signal transduction at the EGF and B2 bradykinin receptors. Finally, in PC12 cells, oleic acid blocked the Ca2+ influx mediated by the activation of B2 bradykinin receptors. In conclusion: (1) NEFA block signal transduction by interfering with receptor-phospholipase C or phospholipase C-substrate interaction without preventing ligand binding; (2) NEFA do not act by a protein kinase C-mediated mechanism; (3) the effect of NEFA is dependent on their configuration rather than hydrophobicity or chain length; (4) this effect is evident in several different cell lines and receptor systems. Images Fig. 4. PMID:1898356

  8. Prothymosin alpha selectively enhances estrogen receptor transcriptional activity by interacting with a repressor of estrogen receptor activity.

    PubMed

    Martini, P G; Delage-Mourroux, R; Kraichely, D M; Katzenellenbogen, B S

    2000-09-01

    We find that prothymosin alpha (PTalpha) selectively enhances transcriptional activation by the estrogen receptor (ER) but not transcriptional activity of other nuclear hormone receptors. This selectivity for ER is explained by PTalpha interaction not with ER, but with a 37-kDa protein denoted REA, for repressor of estrogen receptor activity, a protein that we have previously shown binds to ER, blocking coactivator binding to ER. We isolated PTalpha, known to be a chromatin-remodeling protein associated with cell proliferation, using REA as bait in a yeast two-hybrid screen with a cDNA library from MCF-7 human breast cancer cells. PTalpha increases the magnitude of ERalpha transcriptional activity three- to fourfold. It shows lesser enhancement of ERbeta transcriptional activity and has no influence on the transcriptional activity of other nuclear hormone receptors (progesterone receptor, glucocorticoid receptor, thyroid hormone receptor, or retinoic acid receptor) or on the basal activity of ERs. In contrast, the steroid receptor coactivator SRC-1 increases transcriptional activity of all of these receptors. Cotransfection of PTalpha or SRC-1 with increasing amounts of REA, as well as competitive glutathione S-transferase pulldown and mammalian two-hybrid studies, show that REA competes with PTalpha (or SRC-1) for regulation of ER transcriptional activity and suppresses the ER stimulation by PTalpha or SRC-1, indicating that REA can function as an anticoactivator in cells. Our data support a model in which PTalpha, which does not interact with ER, selectively enhances the transcriptional activity of the ER but not that of other nuclear receptors by recruiting the repressive REA protein away from ER, thereby allowing effective coactivation of ER with SRC-1 or other coregulators. The ability of PTalpha to directly interact in vitro and in vivo with REA, a selective coregulator of the ER, thereby enabling the interaction of ER with coactivators, appears to explain

  9. Activating Receptor Signals Drive Receptor Diversity in Developing Natural Killer Cells

    PubMed Central

    Freund, Jacquelyn; May, Rebecca M.; Li, Hongchuan; McCullen, Matthew; Zhang, Bin; Lenvik, Todd; Cichocki, Frank; Anderson, Stephen K.; Kambayashi, Taku

    2016-01-01

    It has recently been appreciated that NK cells exhibit many features reminiscent of adaptive immune cells. Considerable heterogeneity exists with respect to the ligand specificity of individual NK cells and as such, a subset of NK cells can respond, expand, and differentiate into memory-like cells in a ligand-specific manner. MHC I-binding inhibitory receptors, including those belonging to the Ly49 and KIR families, are expressed in a variegated manner, which creates ligand-specific diversity within the NK cell pool. However, how NK cells determine which inhibitory receptors to express on their cell surface during a narrow window of development is largely unknown. In this manuscript, we demonstrate that signals from activating receptors are critical for induction of Ly49 and KIR receptors during NK cell development; activating receptor-derived signals increased the probability of the Ly49 bidirectional Pro1 promoter to transcribe in the forward versus the reverse direction, leading to stable expression of Ly49 receptors in mature NK cells. Our data support a model where the balance of activating and inhibitory receptor signaling in NK cells selects for the induction of appropriate inhibitory receptors during development, which NK cells use to create a diverse pool of ligand-specific NK cells. PMID:27500644

  10. Activating Receptor Signals Drive Receptor Diversity in Developing Natural Killer Cells.

    PubMed

    Freund, Jacquelyn; May, Rebecca M; Yang, Enjun; Li, Hongchuan; McCullen, Matthew; Zhang, Bin; Lenvik, Todd; Cichocki, Frank; Anderson, Stephen K; Kambayashi, Taku

    2016-08-01

    It has recently been appreciated that NK cells exhibit many features reminiscent of adaptive immune cells. Considerable heterogeneity exists with respect to the ligand specificity of individual NK cells and as such, a subset of NK cells can respond, expand, and differentiate into memory-like cells in a ligand-specific manner. MHC I-binding inhibitory receptors, including those belonging to the Ly49 and KIR families, are expressed in a variegated manner, which creates ligand-specific diversity within the NK cell pool. However, how NK cells determine which inhibitory receptors to express on their cell surface during a narrow window of development is largely unknown. In this manuscript, we demonstrate that signals from activating receptors are critical for induction of Ly49 and KIR receptors during NK cell development; activating receptor-derived signals increased the probability of the Ly49 bidirectional Pro1 promoter to transcribe in the forward versus the reverse direction, leading to stable expression of Ly49 receptors in mature NK cells. Our data support a model where the balance of activating and inhibitory receptor signaling in NK cells selects for the induction of appropriate inhibitory receptors during development, which NK cells use to create a diverse pool of ligand-specific NK cells.

  11. Biological activity of a polypeptide modulator of TRPV1 receptor.

    PubMed

    Dyachenko, I A; Andreev, Ya A; Logashina, Yu A; Murashev, A N; Grishin, E V

    2015-11-01

    This paper presents data on the activity of a new APHC2 polypeptide modulator of TRPV1 receptors, which was isolated from the sea anemone Heteractis crispa. It has been shown that APHC2 has an analgesic activity, does not impair normal motor activity, and does not change body temperature of experimental animals, which has a great practical value for design of potent analgesics of a new generation. Further study of the characteristics of binding of the polypeptide to the TRPV1 receptor may show approaches to the development of other antagonists of this receptor that do not influence the body temperature. PMID:26725234

  12. Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG.

    PubMed

    Kyakumoto, S; Kurokawa, R; Ota, M

    1990-07-12

    Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor

  13. The activation of the nicotinic acetylcholine receptor by the transmitter.

    PubMed

    Taylor, D B; Spivak, C E

    1985-02-01

    Experimental evidence has been published from isolated guinea pig muscle in vitro, and from direct ligand binding to receptors from T. californica, indicating that two agonist ions react with the nicotinic receptor by exchanging for one magnesium ion. It is the basis of the ion exchange receptor pair model, in which two acetylcholine ions exchange for one magnesium ion in contact with and between a pair of negatively charged receptor groups about 4 A apart. In the resting state the electrostatic attraction between the negatively charged receptor groups and the Mg2+ ion exerts a binding force. This binding force is opposed by the quantum mechanical repulsions of the electron clouds of the charged groups and ions in contact, together with the mutual repulsion of the pair of receptor oxyanions. When the Mg2+ ion is replaced by two acetylcholine ions the quaternary heads of the latter are positioned so that they form two mutually repelling ACh+ receptor group dipoles. As the Mg2+ ion leaves, its rehydration energy contributes to the sum of the electron cloud repulsions and the ACh+ receptor group dipole repulsions, causing the receptor groups to be forced apart activating the receptor macromolecule. The subsequent decrease in ACh+ concentration results in the reestablishment of the resting state. The coulombic electrostatic energy, the Born repulsion energy, the London attraction energy and the oxyanion ACh+ dipole repulsion energies have been calculated and shown to be consistent with the model. The displacement of the Mg2+ by two ACh+ ions makes several hundred kcals of energy available for receptor group separation and receptor activation.

  14. Characterization of peroxisome proliferator-activiated receptor alpha (PPARalpha)-independent effects of PPARalpha activators in the rodent liver: Di(2-ethylehexyl) phthalate activates the constitutive activated receptor

    EPA Science Inventory

    Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha). Recent studies indicate that the plasticizer di-2-ethylhexyl ph...

  15. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation

    PubMed Central

    Samarajeewa, Anshula; Goldemann, Lolita; Vasefi, Maryam S.; Ahmed, Nawaz; Gondora, Nyasha; Khanderia, Chandni; Mielke, John G.; Beazely, Michael A.

    2014-01-01

    The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands. PMID:25426041

  16. Tie2 and Eph Receptor Tyrosine Kinase Activation and Signaling

    PubMed Central

    Barton, William A.; Dalton, Annamarie C.; Seegar, Tom C.M.; Himanen, Juha P.

    2014-01-01

    The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition. PMID:24478383

  17. Althaea rosea Cavanil and Plantago major L. suppress neoplastic cell transformation through the inhibition of epidermal growth factor receptor kinase.

    PubMed

    Choi, Eun-Sun; Cho, Sung-Dae; Shin, Ji-Ae; Kwon, Ki Han; Cho, Nam-Pyo; Shim, Jung-Hyun

    2012-10-01

    For thousands of years in Asia, Althaea rosea Cavanil (ARC) and Plantago major L. (PML) have been used as powerful non-toxic therapeutic agents that inhibit inflammation. However, the anticancer mechanisms and molecular targets of ARC and PML are poorly understood, particularly in epidermal growth factor (EGF)-induced neoplastic cell transformation. The aim of this study was to evaluate the chemopreventive effects and mechanisms of the methanol extracts from ARC (MARC) and PML (MPML) in EGF-induced neoplastic cell transformation of JB6 P+ mouse epidermal cells using an MTS assay, anchorage-independent cell transformation assay and western blotting. Our results showed that MARC and MPML significantly suppressed neoplastic cell transformation by inhibiting the kinase activity of the EGF receptor (EGFR). The activation of EGFR by EGF was suppressed by MARC and MPML treatment in EGFR(+/+) cells, but not in EGFR(-/-) cells. In addition, MARC and MPML inhibited EGF-induced cell proliferation in EGFR-expressing murine embryonic fibroblasts (EGFR(+/+)). These results strongly indicate that EGFR targeting by MARC and MPML may be a good strategy for chemopreventive or chemotherapeutic applications. PMID:22767187

  18. Althaea rosea Cavanil and Plantago major L. suppress neoplastic cell transformation through the inhibition of epidermal growth factor receptor kinase.

    PubMed

    Choi, Eun-Sun; Cho, Sung-Dae; Shin, Ji-Ae; Kwon, Ki Han; Cho, Nam-Pyo; Shim, Jung-Hyun

    2012-10-01

    For thousands of years in Asia, Althaea rosea Cavanil (ARC) and Plantago major L. (PML) have been used as powerful non-toxic therapeutic agents that inhibit inflammation. However, the anticancer mechanisms and molecular targets of ARC and PML are poorly understood, particularly in epidermal growth factor (EGF)-induced neoplastic cell transformation. The aim of this study was to evaluate the chemopreventive effects and mechanisms of the methanol extracts from ARC (MARC) and PML (MPML) in EGF-induced neoplastic cell transformation of JB6 P+ mouse epidermal cells using an MTS assay, anchorage-independent cell transformation assay and western blotting. Our results showed that MARC and MPML significantly suppressed neoplastic cell transformation by inhibiting the kinase activity of the EGF receptor (EGFR). The activation of EGFR by EGF was suppressed by MARC and MPML treatment in EGFR(+/+) cells, but not in EGFR(-/-) cells. In addition, MARC and MPML inhibited EGF-induced cell proliferation in EGFR-expressing murine embryonic fibroblasts (EGFR(+/+)). These results strongly indicate that EGFR targeting by MARC and MPML may be a good strategy for chemopreventive or chemotherapeutic applications.

  19. Egf Signaling Directs Neoblast Repopulation by Regulating Asymmetric Cell Division in Planarians.

    PubMed

    Lei, Kai; Thi-Kim Vu, Hanh; Mohan, Ryan D; McKinney, Sean A; Seidel, Chris W; Alexander, Richard; Gotting, Kirsten; Workman, Jerry L; Sánchez Alvarado, Alejandro

    2016-08-22

    A large population of proliferative stem cells (neoblasts) is required for physiological tissue homeostasis and post-injury regeneration in planarians. Recent studies indicate that survival of a few neoblasts after sublethal irradiation results in the clonal expansion of the surviving stem cells and the eventual restoration of tissue homeostasis and regenerative capacity. However, the precise mechanisms regulating the population dynamics of neoblasts remain largely unknown. Here, we uncovered a central role for epidermal growth factor (EGF) signaling during in vivo neoblast expansion mediated by Smed-egfr-3 (egfr-3) and its putative ligand Smed-neuregulin-7 (nrg-7). Furthermore, the EGF receptor-3 protein localizes asymmetrically on the cytoplasmic membrane of neoblasts, and the ratio of asymmetric to symmetric cell divisions decreases significantly in egfr-3(RNAi) worms. Our results not only provide the first molecular evidence of asymmetric stem cell divisions in planarians, but also demonstrate that EGF signaling likely functions as an essential regulator of neoblast clonal expansion.

  20. Egf Signaling Directs Neoblast Repopulation by Regulating Asymmetric Cell Division in Planarians.

    PubMed

    Lei, Kai; Thi-Kim Vu, Hanh; Mohan, Ryan D; McKinney, Sean A; Seidel, Chris W; Alexander, Richard; Gotting, Kirsten; Workman, Jerry L; Sánchez Alvarado, Alejandro

    2016-08-22

    A large population of proliferative stem cells (neoblasts) is required for physiological tissue homeostasis and post-injury regeneration in planarians. Recent studies indicate that survival of a few neoblasts after sublethal irradiation results in the clonal expansion of the surviving stem cells and the eventual restoration of tissue homeostasis and regenerative capacity. However, the precise mechanisms regulating the population dynamics of neoblasts remain largely unknown. Here, we uncovered a central role for epidermal growth factor (EGF) signaling during in vivo neoblast expansion mediated by Smed-egfr-3 (egfr-3) and its putative ligand Smed-neuregulin-7 (nrg-7). Furthermore, the EGF receptor-3 protein localizes asymmetrically on the cytoplasmic membrane of neoblasts, and the ratio of asymmetric to symmetric cell divisions decreases significantly in egfr-3(RNAi) worms. Our results not only provide the first molecular evidence of asymmetric stem cell divisions in planarians, but also demonstrate that EGF signaling likely functions as an essential regulator of neoblast clonal expansion. PMID:27523733

  1. Mechanism of Activation and Inhibition of the HER4/ErbB4 Kinase

    SciTech Connect

    Qiu,C.; Tarrant, M.; Choi, S.; Sathyamurthy, A.; Bose, R.; Banjade, S.; Pal, A.; Bornmann, W.; Lemmon, M.; et al

    2008-01-01

    HER4/ErbB4 is a ubiquitously expressed member of the EGF/ErbB family of receptor tyrosine kinases that is essential for normal development of the heart, nervous system, and mammary gland. We report here crystal structures of the ErbB4 kinase domain in active and lapatinib-inhibited forms. Active ErbB4 kinase adopts an asymmetric dimer conformation essentially identical to that observed to be important for activation of the EGF receptor/ErbB1 kinase. Mutagenesis studies of intact ErbB4 in Ba/F3 cells confirm the importance of this asymmetric dimer for activation of intact ErbB4. Lapatinib binds to an inactive form of the ErbB4 kinase in a mode equivalent to its interaction with the EGF receptor. All ErbB4 residues contacted by lapatinib are conserved in the EGF receptor and HER2/ErbB2, which lapatinib also targets. These results demonstrate that key elements of kinase activation and inhibition are conserved among ErbB family members.

  2. Activation of muscarinic acetylcholine receptors via their allosteric binding sites.

    PubMed Central

    Jakubík, J; Bacáková, L; Lisá, V; el-Fakahany, E E; Tucek, S

    1996-01-01

    Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation. PMID:8710935

  3. Post-translational acquisition of ligand binding- and tyrosine kinase-domain function by the epidermal growth factor and insulin receptors.

    PubMed

    Lane, M D; Slieker, L J; Olson, T S; Martensen, T M

    1987-01-01

    The epidermal growth factor receptor (EGFR) and insulin receptor undergo slow post-translational modification by which they acquire hormone binding and tyrosine kinase (EGFR) function. The half-time for acquisition of EGF or insulin binding activity is 30-40 min and of tyrosine kinase activity (EGFR), is 10-15 min. Tunicamycin, an inhibitor of N-linked oligosaccharide addition, blocks acquisition of both EGF and insulin binding activity. With EGFR, activation precedes acquisition of resistance to endoglucosaminidase H (t1/2 approximately equal to 75 min), a medial Golgi event. Treatment of active high mannose receptor with endo H generates fully active aglyco-receptor; thus, core oligosaccharide addition is a prerequisite for activation, but not for EGF binding per se. EGFR is activated in and translocated from the endoplasmic reticulum (ER) slowly (t1/2 approximately equal to 75 min). Since translocation rate equals the rate for acquisition of endo H resistance, transit from the ER is rate limiting for EGFR maturation. Tunicamycin inhibits exit from the ER parallel to its effect on acquisition of binding activity. Insulin proreceptor, a 210 kDa high-mannose glycopolypeptide, acquires insulin binding function (t1/2 approximately equal to 45 min) then is proteolytically cleaved (t1/2 approximately equal to 3 hr) into subunits of the mature alpha 2 beta 2 receptor. Modification giving rise to insulin binding activity is due to a conformational change in the binding domain, since human autoimmune antibody recognizes only the active species, while rabbit polyclonal antibody recognizes all forms. Newly-translated EGF proreceptor lacks a functional tyrosine domain capable of autophosphorylation; 30-40 min after translation, while still in the ER, tyrosine kinase activity is acquired. Since the kinase domain is cytoplasmic, the receptor may become phosphorylated on tyrosine before reaching the plasma membrane. PMID:3305909

  4. Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins.

    PubMed

    Hay, Debbie L; Walker, Christopher S; Gingell, Joseph J; Ladds, Graham; Reynolds, Christopher A; Poyner, David R

    2016-04-15

    Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine. PMID:27068971

  5. Synthetic Human NOTCH1 EGF Modules Unraveled Molecular Mechanisms for the Structural and Functional Roles of Calcium Ions and O-Glycans in the Ligand-Binding Region.

    PubMed

    Hayakawa, Shun; Koide, Ryosuke; Hinou, Hiroshi; Nishimura, Shin-Ichiro

    2016-02-01

    The Notch signaling pathway is an evolutionarily highly conserved mechanism that operates across multicellular organisms and is critical for cell-fate decisions during development and homeostasis in most tissues. Notch signaling is modified by posttranslational glycosylations of the Notch extracellular EGF-like domain. To evaluate the structural and functional roles of various glycoforms at multiple EGF domains in the human Notch transmembrane receptor, we established a universal method for the construction of NOTCH1 EGF modules displaying the desired O-glycans at the designated glycosylation sites. The versatility of this strategy was demonstrated by the rapid and highly efficient synthesis of NOTCH1 EGF12 concurrently having a β-D-glucopyranose-initiated glycan (Xylα1 → 3Xylα1 → 3Glcβ1 →) at Ser458 and α-L-fucopyranose-initiated glycan (Neu5Acα2 → 3Galβ1 → 4GlcNAcβ1 → 3Fucα1 →) at Thr466. The efficiency of the proper folding of the glycosylated EGF12 was markedly enhanced in the presence of 5 mM CaCl2. A nuclear magnetic resonance study revealed the existence of strong nuclear Overhauser effects between key sugar moieties and neighboring amino acid residues, indicating that both O-glycans contribute independently to the intramolecular stabilization of the antiparallel β-sheet structure in the ligand-binding region of EGF12. A preliminary test using synthetic human NOTCH1 EGF modules showed significant inhibitory effects on the proliferation and adhesiveness of human breast cancer cell line MCF-7 and lung adenocarcinoma epithelial cell line A549, demonstrating for the first time evidence that exogenously applied synthetic EGF modules have the ability to interact with intrinsic Notch ligands on the surface of cancer cells.

  6. Activation of G protein by opioid receptors: role of receptor number and G-protein concentration.

    PubMed

    Remmers, A E; Clark, M J; Alt, A; Medzihradsky, F; Woods, J H; Traynor, J R

    2000-05-19

    The collision-coupling model for receptor-G-protein interaction predicts that the rate of G-protein activation is dependent on receptor density, but not G-protein levels. C6 cells expressing mu- or delta-opioid receptors, or SH-SY5Y cells, were treated with beta-funaltrexamine (mu) or naltrindole-5'-isothiocyanate (delta) to decrease receptor number. The time course of full or partial agonist-stimulated ¿35SGTPgammaS binding did not vary in C6 cell membranes containing <1-25 pmol/mg mu-opioid receptor, or 1. 4-4.3 pmol/mg delta-opioid receptor, or in SHSY5Y cells containing 0. 16-0.39 pmol/mg receptor. The association of ¿35SGTPgammaS binding was faster in membranes from C6mu cells than from C6delta cells. A 10-fold reduction in functional G-protein, following pertussis toxin treatment, lowered the maximal level of ¿35SGTPgammaS binding but not the association rate. These data indicate a compartmentalization of opioid receptors and G protein at the cell membrane. PMID:10822058

  7. Contribution of the NH2-terminal EGF-domain of factor IXa to the specificity of intrinsic tenase.

    PubMed

    Qureshi, Shabir H; Yang, Likui; Rezaie, Alireza R

    2012-12-01

    Factor IXa (FIXa) is a vitamin K-dependent coagulation serine protease which binds to factor VIIIa (FVIIIa) on negatively charged phospholipid vesicles (PCPS) to catalyse the activation of factor X (FX) to factor Xa (FXa) in the intrinsic pathway. Fluorescence resonance energy transfer (FRET) studies have indicated that the Gla-domain-dependent interaction of FIXa and FX with PCPS in the presence of FVIIIa positions the active-site of the protease at an appropriate height above the membrane surface to optimise the catalytic reaction. In this study, we investigated the contribution of the NH2-terminal EGF-domain (EGF1) of FIXa to the recognition specificity of intrinsic tenase by constructing an EGF1 deletion mutant of FIXa (FIXa-desEGF1) and characterising the properties of the mutant in kinetic, direct binding and FRET assays. The results of direct binding and kinetic studies demonstrated that the binding affinity of the mutant for interaction with FVIIIa on PCPS has been impaired greater than 10-fold and the catalytic efficiency of the mutant protease-FVIIIa-PCPS complex in the activation of FX has been decreased ~100-fold. By contrast, the mutant protease exhibited a normal activity toward FX in the absence of the protein cofactor. FRET measurements revealed that the distance of the active-site of the mutant FIXa relative to PCPS vesicles has been decreased 10 Å from 75 ± 2 Å for FIXa to 65 ± 2 Å for FIXa-desEGF1 independent of FVIIIa. These results suggest that the NH2-terminal EGF-domain of FIXa provides a binding-site for FVIIIa and plays an essential spacer function in the intrinsic tenase complex.

  8. Retinoic Acid-mediated Nuclear Receptor Activation and Hepatocyte Proliferation

    PubMed Central

    Bushue, Nathan; Wan, Yu-Jui Yvonne

    2016-01-01

    Due to their well-known differentiation and apoptosis-inducing abilities, retinoic acid (RA) and its analogs have strong anti-cancer efficacy in human cancers. However, in vivo RA is a liver mitogen. While speculation has persisted that RA-mediated signaling is likely involved in hepatocyte proliferation during liver regeneration, direct evidence is still required. Findings in support of this proposition include observations that a release of retinyl palmitate (the precursor of RA) occurs in liver stellate cells following liver injury. Nevertheless, the biological action of this released vitamin A is virtually unknown. More likely is that the released vitamin A is converted to RA, the biological form, and then bound to a specific receptor (retinoid x receptor; RXRα), which is most abundantly expressed in the liver. Considering the mitogenic effects of RA, the RA-activated RXRα would likely then influence hepatocyte proliferation and liver tissue repair. At present, the mechanism by which RA stimulates hepatocyte proliferation is largely unknown. This review summarizes the activation of nuclear receptors (peroxisome proliferator activated receptor-α, pregnane x receptor, constitutive androstane receptor, and farnesoid x receptor) in an RXRα dependent manner to induce hepatocyte proliferation, providing a link between RA and its proliferative role.

  9. An activation switch in the rhodopsin family of G protein-coupled receptors: the thyrotropin receptor.

    PubMed

    Urizar, Eneko; Claeysen, Sylvie; Deupí, Xavier; Govaerts, Cedric; Costagliola, Sabine; Vassart, Gilbert; Pardo, Leonardo

    2005-04-29

    We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche+ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of approximately 14 and approximately 10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of approximately 13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.

  10. Influence of epidermal growth factor (EGF) and hydrocortisone on the co-culture of mature adipocytes and endothelial cells for vascularized adipose tissue engineering.

    PubMed

    Huber, Birgit; Czaja, Alina Maria; Kluger, Petra Juliane

    2016-05-01

    The composition of vascularized adipose tissue is still an ongoing challenge as no culture medium is available to supply adipocytes and endothelial cells appropriately. Endothelial cell medium is typically supplemented with epidermal growth factor (EGF) as well as hydrocortisone (HC). The effect of EGF on adipocytes is discussed controversially. Some studies say it inhibits adipocyte differentiation while others reported of improved adipocyte lipogenesis. HC is known to have lipolytic activities, which might result in mature adipocyte dedifferentiation. In this study, we evaluated the influence of EGF and HC on the co-culture of endothelial cells and mature adipocytes regarding their cell morphology and functionality. We showed in mono-culture that high levels of HC promoted dedifferentiation and proliferation of mature adipocytes, whereas EGF seemed to have no negative influence. Endothelial cells kept their typical cobblestone morphology and showed a proliferation rate comparable to the control independent of EGF and HC concentration. In co-culture, HC promoted dedifferentiation of mature adipocytes, which was shown by a higher glycerol release. EGF had no negative impact on adipocyte morphology. No negative impact on endothelial cell morphology and functionality could be seen with reduced EGF and HC supplementation in co-culture with mature adipocytes. Taken together, our results demonstrate that reduced levels of HC are needed for co-culturing mature adipocytes and endothelial cells. In co-culture, EGF had no influence on mature adipocytes. Therefore, for the composition of vascularized adipose tissue constructs, the media with low levels of HC and high or low levels of EGF can be used.

  11. Influence of epidermal growth factor (EGF) and hydrocortisone on the co-culture of mature adipocytes and endothelial cells for vascularized adipose tissue engineering.

    PubMed

    Huber, Birgit; Czaja, Alina Maria; Kluger, Petra Juliane

    2016-05-01

    The composition of vascularized adipose tissue is still an ongoing challenge as no culture medium is available to supply adipocytes and endothelial cells appropriately. Endothelial cell medium is typically supplemented with epidermal growth factor (EGF) as well as hydrocortisone (HC). The effect of EGF on adipocytes is discussed controversially. Some studies say it inhibits adipocyte differentiation while others reported of improved adipocyte lipogenesis. HC is known to have lipolytic activities, which might result in mature adipocyte dedifferentiation. In this study, we evaluated the influence of EGF and HC on the co-culture of endothelial cells and mature adipocytes regarding their cell morphology and functionality. We showed in mono-culture that high levels of HC promoted dedifferentiation and proliferation of mature adipocytes, whereas EGF seemed to have no negative influence. Endothelial cells kept their typical cobblestone morphology and showed a proliferation rate comparable to the control independent of EGF and HC concentration. In co-culture, HC promoted dedifferentiation of mature adipocytes, which was shown by a higher glycerol release. EGF had no negative impact on adipocyte morphology. No negative impact on endothelial cell morphology and functionality could be seen with reduced EGF and HC supplementation in co-culture with mature adipocytes. Taken together, our results demonstrate that reduced levels of HC are needed for co-culturing mature adipocytes and endothelial cells. In co-culture, EGF had no influence on mature adipocytes. Therefore, for the composition of vascularized adipose tissue constructs, the media with low levels of HC and high or low levels of EGF can be used. PMID:26888598

  12. EGF and PGE2: effects on corneal endothelial cell migration and monolayer spreading during wound repair in vitro.

    PubMed

    Joyce, N C; Joyce, S J; Powell, S M; Meklir, B

    1995-07-01

    In vivo repair of the adult human corneal endothelium occurs mainly by movement of cells into the wound defect rather than by cell division. Two forms of cell movement contribute to endothelial wound repair: migration of individual cells into the defect and spreading of the confluent monolayer into the wound area. This laboratory has developed a tissue culture model using rabbit corneal endothelial cells pretreated with the mitotic inhibitor 5-fluorouracil to mimic the relatively amitotic state of human corneal endothelium in vivo. This model permits study of the effects of growth factors and other agents on individual cell migration and monolayer spreading in response to wounding. mRNA for epidermal growth factor (EGF) and its receptor has been detected in cultured corneal endothelial cells and EGF receptors have been detected on human corneal endothelial cells in situ, suggesting that this growth factor may act in an autocrine manner. Prostaglandin E2 (PGE2) is synthesized by cultured corneal endothelial cells and is present in relatively high quantity in aqueous humor in response to corneal wounding and to inflammation in the anterior chamber. Although corneal endothelial cells may be exposed to both EGF and PGE2, little is known about their effects on monolayer repair. The current study compared the effects of PGE2 alone, EGF alone, and both agents in combination on individual cell migration and monolayer spreading using the wound model system and also determined the effect of EGF on PGE2 secretion using a commercial immunoassay. A 15 min exposure of wounded cultures to exogenous PGE2 stimulated individual cell migration and suppressed monolayer spreading.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7587307

  13. Clinically used selective oestrogen receptor modulators increase LDL receptor activity in primary human lymphocytes

    PubMed Central

    Cerrato, F; Fernández-Suárez, M E; Alonso, R; Alonso, M; Vázquez, C; Pastor, O; Mata, P; Lasunción, M A; Gómez-Coronado, D

    2015-01-01

    Background and Purpose Treatment with selective oestrogen receptor modulators (SERMs) reduces low-density lipoprotein (LDL) cholesterol levels. We assessed the effect of tamoxifen, raloxifene and toremifene and their combinations with lovastatin on LDL receptor activity in lymphocytes from normolipidaemic and familial hypercholesterolaemic (FH) subjects, and human HepG2 hepatocytes and MOLT-4 lymphoblasts. Experimental Approach Lymphocytes were isolated from peripheral blood, treated with different compounds, and 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanine perchlorate (DiI)-labelled LDL uptake was analysed by flow cytometry. Key Results Tamoxifen, toremifene and raloxifene, in this order, stimulated DiI-LDL uptake by lymphocytes by inhibiting LDL-derived cholesterol trafficking and subsequent down-regulation of LDL receptor expression. Differently to what occurred in HepG2 and MOLT-4 cells, only tamoxifen consistently displayed a potentiating effect with lovastatin in primary lymphocytes. The SERM-mediated increase in LDL receptor activity was not altered by the anti-oestrogen ICI 182 780 nor was it reproduced by 17β-oestradiol. However, the tamoxifen-active metabolite endoxifen was equally effective as tamoxifen. The SERMs produced similar effects on LDL receptor activity in heterozygous FH lymphocytes as in normal lymphocytes, although none of them had a potentiating effect with lovastatin in heterozygous FH lymphocytes. The SERMs had no effect in homozygous FH lymphocytes. Conclusions and Implications Clinically used SERMs up-regulate LDL receptors in primary human lymphocytes. There is a mild enhancement between SERMs and lovastatin of lymphocyte LDLR activity, the potentiation being greater in HepG2 and MOLT-4 cells. The effect of SERMs is independent of oestrogen receptors but is preserved in the tamoxifen-active metabolite endoxifen. This mechanism may contribute to the cholesterol-lowering action of SERMs. PMID:25395200

  14. Transcriptional activation of nuclear estrogen receptor and progesterone receptor and its regulation.

    PubMed

    Xin, Qi-Liang; Qiu, Jing-Tao; Cui, Sheng; Xia, Guo-Liang; Wang, Hai-Bin

    2016-08-25

    Estrogen receptor (ER) and progesterone receptor (PR) are two important members of steroid receptors family, an evolutionarily conserved family of transcription factors. Upon binding to their ligands, ER and PR enter cell nucleus to interact with specific DNA element in the context of chromatin to initiate the transcription of diverse target genes, which largely depends on the timely recruitment of a wide range of cofactors. Moreover, the interactions between steroid hormones and their respective receptors also trigger post-translational modifications on these receptors to fine-tune their transcriptional activities. Besides the well-known phosphorylation modifications on tyrosine and serine/threonine residues, recent studies have identified several other covalent modifications, such as ubiquitylation and sumoylation. These post-translational modifications of steroid receptors affect its stability, subcellular localization, and/or cofactor recruitment; eventually influence the duration and extent of transcriptional activation. This review is to focus on the recent research progress on the transcriptional activation of nuclear ER and PR as well as their physiological functions in early pregnancy, which may help us to better understand related female reproductive diseases. PMID:27546504

  15. Overcoming acquired drug resistance in colorectal cancer cells by targeted delivery of 5-FU with EGF grafted hollow mesoporous silica nanoparticles

    NASA Astrophysics Data System (ADS)

    Chen, Lijue; She, Xiaodong; Wang, Tao; He, Li; Shigdar, Sarah; Duan, Wei; Kong, Lingxue

    2015-08-01

    Acquired drug resistance (ADR) can be developed in colorectal cancer cells after 5-fluorouracil (5-FU) treatment and diminish the effectiveness of chemotherapy. In this work, acquired 5-FU resistance in the colorectal cancer cell line SW480 was obtained with the up-regulation of dihydropyrimidine dehydrogenase (DPYD) gene expression which can convert 5-FU to its inactive metabolite. To overcome ADR in colorectal cancer, hollow mesoporous silica nanoparticles (HMSNs) grafted with epidermal growth factor (EGF) were used as nanocarriers to deliver 5-FU to colorectal cancer cells with acquired drug resistance. The effect and mechanism of 5-FU loaded EGF grafted HMSNs (EGF-HMSNs-5-FU) in overcoming acquired drug resistance in SW480/ADR cells were studied. The EGF-HMSNs were demonstrated to be specifically internalized in EGFR overexpressed SW480/ADR cells via a receptor-mediated endocytosis and can escape from endo-lysosomes. The EGF-HMSNs-5-FU exhibited much higher cytotoxicity on SW480/ADR cells than HMSNs-5-FU and free 5-FU while the plain HMSNs did not show significant cytotoxicity. The mechanism of EGF-HMSNs-5-FU in overcoming drug resistance in SW480/ADR cells could be attributed to the specific internalization of EGF-HMSNs-5-FU in EGFR overexpressed cells which can lead to high intracellular drug accumulation and cause cell death through S phase arrest.Acquired drug resistance (ADR) can be developed in colorectal cancer cells after 5-fluorouracil (5-FU) treatment and diminish the effectiveness of chemotherapy. In this work, acquired 5-FU resistance in the colorectal cancer cell line SW480 was obtained with the up-regulation of dihydropyrimidine dehydrogenase (DPYD) gene expression which can convert 5-FU to its inactive metabolite. To overcome ADR in colorectal cancer, hollow mesoporous silica nanoparticles (HMSNs) grafted with epidermal growth factor (EGF) were used as nanocarriers to deliver 5-FU to colorectal cancer cells with acquired drug resistance. The

  16. Immunohistochemical quantitation of oestrogen receptors and proliferative activity in oestrogen receptor positive breast cancer.

    PubMed Central

    Jensen, V; Ladekarl, M

    1995-01-01

    AIM--To evaluate the effect of the duration of formalin fixation and of tumour heterogeneity on quantitative estimates of oestrogen receptor content (oestrogen receptor index) and proliferative activity (MIB-1 index) in breast cancer. METHODS--Two monoclonal antibodies, MIB-1 and oestrogen receptor, were applied to formalin fixed, paraffin wax embedded tissue from 25 prospectively collected oestrogen receptor positive breast carcinomas, using a microwave antigen retrieval method. Tumour tissue was allocated systematically to different periods of fixation to ensure minimal intraspecimen variation. The percentages of MIB-1 positive and oestrogen receptor positive nuclei were estimated in fields of vision sampled systematically from the entire specimen and from the whole tumour area of one "representative" cross-section. RESULTS--No correlation was found between the oestrogen receptor and MIB-1 indices and the duration of formalin fixation. The estimated MIB-1 and oestrogen receptor indices in tissue sampled systematically from the entire tumour were closely correlated with estimates obtained in a "representative" section. The intra- and interobserver correlation of the MIB-1 index was good, although a slight systematical error at the second assessment of the intraobserver study was noted. CONCLUSION--Quantitative estimates of oestrogen receptor content and proliferative activity are not significantly influenced by the period of fixation in formalin, varying from less than four hours to more than 48 hours. The MIB-1 and the oestrogen receptor indices obtained in a "representative" section do not deviate significantly from average indices determined in tissue samples from the entire tumour. Finally, the estimation of MIB-1 index is reproducible, justifying its routine use. PMID:7629289

  17. Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid-Activated Receptor

    SciTech Connect

    Kruse, Schoen W; Suino-Powell, Kelly; Zhou, X Edward; Kretschman, Jennifer E; Reynolds, Ross; Vonrhein, Clemens; Xu, Yong; Wang, Liliang; Tsai, Sophia Y; Tsai, Ming-Jer; Xu, H Eric

    2010-01-12

    The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 {angstrom} crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix {alpha}10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.

  18. Insulin and rabbit anti-insulin receptor antibodies stimulate additively the intrinsic receptor kinase activity.

    PubMed Central

    Ponzio, G; Dolais-Kitabgi, J; Louvard, D; Gautier, N; Rossi, B

    1987-01-01

    This paper describes the properties of rabbit polyclonal antibodies directed against purified human insulin receptor which strongly stimulate the intrinsic tyrosine kinase activity. The stimulatory effect of the antibodies on the kinase activity was obtained on the insulin receptor autophosphorylation as well as on the kinase activity towards a synthetic substrate. This stimulation is additive to that induced by insulin. Moreover, rabbit antibodies do not impair insulin binding. These data strongly suggest that antibodies and insulin act through separate pathways. This conclusion is reinforced by the differences observed on the phosphopeptide maps of the receptor's beta subunit whose phosphorylation was performed either in the presence of insulin or rabbit antibodies. Interestingly, these polyclonal antibodies can also induce an activation of the receptor autophosphorylation by interacting only with extracellular determinants. The anti-insulin receptor antibodies mimic insulin in their stimulatory effect on amino acid (AIB) uptake, but they have a different effect to that found on the kinase activity; the simultaneous addition of the antiserum and insulin failed to stimulate this amino acid transport over the level induced by a saturating concentration of hormone. Images Fig. 1. Fig. 3. Fig. 4. Fig. 6. Fig. 7. PMID:3034584

  19. High-throughput de novo screening of receptor agonists with an automated single-cell analysis and isolation system.

    PubMed

    Yoshimoto, Nobuo; Tatematsu, Kenji; Iijima, Masumi; Niimi, Tomoaki; Maturana, Andrés D; Fujii, Ikuo; Kondo, Akihiko; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

    2014-01-01

    Reconstitution of signaling pathways involving single mammalian transmembrane receptors has not been accomplished in yeast cells. In this study, intact EGF receptor (EGFR) and a cell wall-anchored form of EGF were co-expressed on the yeast cell surface, which led to autophosphorylation of the EGFR in an EGF-dependent autocrine manner. After changing from EGF to a conformationally constrained peptide library, cells were fluorescently labeled with an anti-phospho-EGFR antibody. Each cell was subjected to an automated single-cell analysis and isolation system that analyzed the fluorescent intensity of each cell and automatically retrieved each cell with the highest fluorescence. In ~3.2 × 10(6) peptide library, we isolated six novel peptides with agonistic activity of the EGFR in human squamous carcinoma A431 cells. The combination of yeast cells expressing mammalian receptors, a cell wall-anchored peptide library, and an automated single-cell analysis and isolation system might facilitate a rational approach for de novo drug screening. PMID:24577528

  20. High-throughput de novo screening of receptor agonists with an automated single-cell analysis and isolation system.

    PubMed

    Yoshimoto, Nobuo; Tatematsu, Kenji; Iijima, Masumi; Niimi, Tomoaki; Maturana, Andrés D; Fujii, Ikuo; Kondo, Akihiko; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

    2014-01-01

    Reconstitution of signaling pathways involving single mammalian transmembrane receptors has not been accomplished in yeast cells. In this study, intact EGF receptor (EGFR) and a cell wall-anchored form of EGF were co-expressed on the yeast cell surface, which led to autophosphorylation of the EGFR in an EGF-dependent autocrine manner. After changing from EGF to a conformationally constrained peptide library, cells were fluorescently labeled with an anti-phospho-EGFR antibody. Each cell was subjected to an automated single-cell analysis and isolation system that analyzed the fluorescent intensity of each cell and automatically retrieved each cell with the highest fluorescence. In ~3.2 × 10(6) peptide library, we isolated six novel peptides with agonistic activity of the EGFR in human squamous carcinoma A431 cells. The combination of yeast cells expressing mammalian receptors, a cell wall-anchored peptide library, and an automated single-cell analysis and isolation system might facilitate a rational approach for de novo drug screening.

  1. Monitoring leptin activity using the chicken leptin receptor.

    PubMed

    Hen, Gideon; Yosefi, Sera; Ronin, Ana; Einat, Paz; Rosenblum, Charles I; Denver, Robert J; Friedman-Einat, Miriam

    2008-05-01

    We report on the construction of a leptin bioassay based on the activation of chicken leptin receptor in cultured cells. A human embryonic kidney (HEK)-293 cell line, stably transfected with the full-length cDNA of chicken leptin receptor together with a STAT3-responsive reporter gene specifically responded to recombinant human and Xenopus leptins. The observed higher sensitivity of chicken leptin receptor to the former is in agreement with the degree of sequence similarity among these species (about 60 and 38% identical amino acids between humans and chickens, and between humans and Xenopus respectively). The specific activation of signal transduction through the chicken leptin receptor, shown here for the first time, suggests that the transition of Gln269 (implicated in the Gln-to-Pro Zucker fatty mutation in rats) to Glu in chickens does not impair its activity. Analysis of leptin-like activity in human serum samples of obese and lean subjects coincided well with leptin levels determined by RIA. Serum samples of pre- and post partum cows showed a tight correlation with the degree of adiposity. However, specific activation of the chicken leptin receptor in this assay was not observed with serum samples from broiler or layer chickens (representing fat and lean phenotypes respectively) or with those from turkey. Similar leptin receptor activation profiles were observed with cells transfected with human leptin receptor. Further work is needed to determine whether the lack of leptin-like activity in the chicken serum samples is due to a lack of leptin in this species or simply to a serum level of leptin that is below the detection threshold.

  2. Monitoring leptin activity using the chicken leptin receptor.

    PubMed

    Hen, Gideon; Yosefi, Sera; Ronin, Ana; Einat, Paz; Rosenblum, Charles I; Denver, Robert J; Friedman-Einat, Miriam

    2008-05-01

    We report on the construction of a leptin bioassay based on the activation of chicken leptin receptor in cultured cells. A human embryonic kidney (HEK)-293 cell line, stably transfected with the full-length cDNA of chicken leptin receptor together with a STAT3-responsive reporter gene specifically responded to recombinant human and Xenopus leptins. The observed higher sensitivity of chicken leptin receptor to the former is in agreement with the degree of sequence similarity among these species (about 60 and 38% identical amino acids between humans and chickens, and between humans and Xenopus respectively). The specific activation of signal transduction through the chicken leptin receptor, shown here for the first time, suggests that the transition of Gln269 (implicated in the Gln-to-Pro Zucker fatty mutation in rats) to Glu in chickens does not impair its activity. Analysis of leptin-like activity in human serum samples of obese and lean subjects coincided well with leptin levels determined by RIA. Serum samples of pre- and post partum cows showed a tight correlation with the degree of adiposity. However, specific activation of the chicken leptin receptor in this assay was not observed with serum samples from broiler or layer chickens (representing fat and lean phenotypes respectively) or with those from turkey. Similar leptin receptor activation profiles were observed with cells transfected with human leptin receptor. Further work is needed to determine whether the lack of leptin-like activity in the chicken serum samples is due to a lack of leptin in this species or simply to a serum level of leptin that is below the detection threshold. PMID:18434362

  3. Neurotensin (NTS) and its receptor (NTSR1) causes EGFR, HER2 and HER3 over-expression and their autocrine/paracrine activation in lung tumors, confirming responsiveness to erlotinib

    PubMed Central

    Lupo, Audrey Mansuet; Mourra, Najat; Takahashi, Takashi; Fléjou, Jean François; Trédaniel, Jean; Régnard, Jean François; Damotte, Diane; Alifano, Marco; Forgez, Patricia

    2014-01-01

    Alterations in the signaling pathways of epidermal growth factor receptors (HERs) are associated with tumor aggressiveness. Neurotensin (NTS) and its high affinity receptor (NTSR1) are up regulated in 60% of lung cancers. In a previous clinical study, NTSR1 overexpression was shown to predict a poor prognosis for 5 year overall survival in a selected population of stage I lung adenocarcinomas treated by surgery alone. In a second study, shown here, the frequent and high expression of NTSR1 was correlated with a pejorative prognosis in 389 patients with stage I to III lung adenocarcinoma, and was an independent prognosis marker. Interactions between NTS and NTSR1 induce pro-oncogenic biological effects associated with neoplastic processes and tumor progression. Here we highlight the cellular mechanisms activated by Neurotensin (NTS) and its high affinity receptor (NTSR1) contributing to lung cancer cell aggressiveness. We show that the NTS autocrine and/or paracrine regulation causes EGFR, HER2, and HER3 over-expression and activation in lung tumor cells. The EGFR and HER3 autocrine activation is mediated by MMP1 activation and EGF “like” ligands (HB-EGF, Neuregulin 1) release. By establishing autocrine and/or paracrine NTS regulation, we show that tumor growth is modulated according to NTS expression, with a low growth rate in those tumors that do not express NTS. Accordingly, xenografted tumors expressing NTS and NTSR1 showed a positive response to erlotinib, whereas tumors void of NTSR1 expression had no detectable response. This is consistent with the presence of a NTS autocrine loop, leading to the sustained activation of EGFR and responsible for cancer aggressiveness. We propose the use of NTS/NTSR1 tumor expression, as a biomarker for the use of EGFR tyrosine kinase inhibitors in patients lacking EGFR mutation. PMID:25249545

  4. Neurotensin (NTS) and its receptor (NTSR1) causes EGFR, HER2 and HER3 over-expression and their autocrine/paracrine activation in lung tumors, confirming responsiveness to erlotinib.

    PubMed

    Younes, Mohamad; Wu, Zherui; Dupouy, Sandra; Lupo, Audrey Mansuet; Mourra, Najat; Takahashi, Takashi; Fléjou, Jean François; Trédaniel, Jean; Régnard, Jean François; Damotte, Diane; Alifano, Marco; Forgez, Patricia

    2014-09-30

    Alterations in the signaling pathways of epidermal growth factor receptors (HERs) are associated with tumor aggressiveness. Neurotensin (NTS) and its high affinity receptor (NTSR1) are up regulated in 60% of lung cancers. In a previous clinical study, NTSR1 overexpression was shown to predict a poor prognosis for 5 year overall survival in a selected population of stage I lung adenocarcinomas treated by surgery alone. In a second study, shown here, the frequent and high expression of NTSR1 was correlated with a pejorative prognosis in 389 patients with stage I to III lung adenocarcinoma, and was an independent prognosis marker. Interactions between NTS and NTSR1 induce pro-oncogenic biological effects associated with neoplastic processes and tumor progression. Here we highlight the cellular mechanisms activated by Neurotensin (NTS) and its high affinity receptor (NTSR1) contributing to lung cancer cell aggressiveness. We show that the NTS autocrine and/or paracrine regulation causes EGFR, HER2, and HER3 over-expression and activation in lung tumor cells. The EGFR and HER3 autocrine activation is mediated by MMP1 activation and EGF "like" ligands (HB-EGF, Neuregulin 1) release. By establishing autocrine and/or paracrine NTS regulation, we show that tumor growth is modulated according to NTS expression, with a low growth rate in those tumors that do not express NTS. Accordingly, xenografted tumors expressing NTS and NTSR1 showed a positive response to erlotinib, whereas tumors void of NTSR1 expression had no detectable response. This is consistent with the presence of a NTS autocrine loop, leading to the sustained activation of EGFR and responsible for cancer aggressiveness. We propose the use of NTS/NTSR1 tumor expression, as a biomarker for the use of EGFR tyrosine kinase inhibitors in patients lacking EGFR mutation.

  5. Flavonoids as dietary regulators of nuclear receptor activity

    PubMed Central

    Avior, Yishai; Bomze, David; Ramon, Ory

    2013-01-01

    Metabolic diseases such as obesity, type II diabetes, and dyslipidemia are a rising cause of mortality worldwide. The progression of many metabolic diseases is fundamentally regulated on the transcriptional level by a family of ligand-activated transcription factors, called nuclear receptors, which detect and respond to metabolic changes. Their role in maintaining metabolic homeostasis makes nuclear receptors an important pharmaceutical and dietary target. This review will present the growing evidence that flavonoids, natural secondary plant metabolites, are important regulators of nuclear receptor activity. Structural similarities between flavonoids and cholesterol derivatives combined with the promiscuous nature of most nuclear receptors provide a wealth of possibilities for pharmaceutical and dietary modulation of metabolism. While the challenges of bringing flavonoid-derived therapeutics to the market are significant, we consider this rapidly growing field to be an essential aspect of the functional food initiative and an important mine for pharmaceutical compounds. PMID:23598551

  6. Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs.

    PubMed

    Mi, Li-Zhi; Grey, Michael J; Nishida, Noritaka; Walz, Thomas; Lu, Chafen; Springer, Timothy A

    2008-09-30

    Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase domains have suggested mechanisms for growth factor-mediated receptor dimerization and allosteric kinase domain activation, understanding how the transmembrane and juxtamembrane domains contribute to transmembrane signaling requires structural studies on intact receptor molecules. In this report, recombinant EGFR constructs containing the extracellular, transmembrane, juxtamembrane, and kinase domains are overexpressed and purified from human embryonic kidney 293 cell cultures. The oligomerization state, overall structure, and functional stability of the purified EGF-bound receptor are characterized in detergent micelles and phospholipid bilayers. In the presence of EGF, catalytically active EGFR dimers can be isolated by gel filtration in dodecyl maltoside. Visualization of the dimeric species by negative stain electron microscopy and single particle averaging reveals an overall structure of the extracellular domain that is similar to previously published crystal structures and is consistent with the C-termini of domain IV being juxtaposed against one another as they enter the transmembrane domain. Although detergent-soluble preparations of EGFR are stable as dimers in the presence of EGF, they exhibit differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane and amphipathic juxtamembrane domains is important for stabilizing the tyrosine kinase activity in vitro.

  7. Characterization of epidermal growth factor receptors on plasma membranes isolated from rat gastric mucosa

    SciTech Connect

    Hori, R.; Nomura, H.; Iwakawa, S.; Okumura, K. )

    1990-06-01

    The binding of human epidermal growth factor (hEGF), beta-urogastrone, to plasma membranes isolated from rat gastric mucosa was studied to characterize gastric EGF receptors. The binding of ({sup 125}I)hEGF was temperature dependent, reversible, and saturable. A single class of binding sites for EGF with a dissociation constant of 0.42 nM and maximal binding capacity of 42 fmol/mg protein was suggested. There was little change in the binding of ({sup 125}I)hEGF upon addition of peptide hormones (secretin, insulin), antiulcer drugs (cimetidine), or an ulcer-inducing reagent (aspirin). Cross-linking of ({sup 125}I)hEGF to gastric plasma membranes with the use of disuccinimidyl suberate resulted in the labeling of a protein of 150 kDa. These results indicate the presence of EGF receptors on plasma membranes of rat gastric mucosa.

  8. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation.

    PubMed

    Nagata, Yosuke; Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

    2014-08-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor.

  9. Notch-mediated lateral inhibition regulates proneural wave propagation when combined with EGF-mediated reaction diffusion.

    PubMed

    Sato, Makoto; Yasugi, Tetsuo; Minami, Yoshiaki; Miura, Takashi; Nagayama, Masaharu

    2016-08-30

    Notch-mediated lateral inhibition regulates binary cell fate choice, resulting in salt and pepper patterns during various developmental processes. However, how Notch signaling behaves in combination with other signaling systems remains elusive. The wave of differentiation in the Drosophila visual center or "proneural wave" accompanies Notch activity that is propagated without the formation of a salt and pepper pattern, implying that Notch does not form a feedback loop of lateral inhibition during this process. However, mathematical modeling and genetic analysis clearly showed that Notch-mediated lateral inhibition is implemented within the proneural wave. Because partial reduction in EGF signaling causes the formation of the salt and pepper pattern, it is most likely that EGF diffusion cancels salt and pepper pattern formation in silico and in vivo. Moreover, the combination of Notch-mediated lateral inhibition and EGF-mediated reaction diffusion enables a function of Notch signaling that regulates propagation of the wave of differentiation. PMID:27535937

  10. Notch-mediated lateral inhibition regulates proneural wave propagation when combined with EGF-mediated reaction diffusion

    PubMed Central

    Sato, Makoto; Yasugi, Tetsuo; Minami, Yoshiaki; Miura, Takashi; Nagayama, Masaharu

    2016-01-01

    Notch-mediated lateral inhibition regulates binary cell fate choice, resulting in salt and pepper patterns during various developmental processes. However, how Notch signaling behaves in combination with other signaling systems remains elusive. The wave of differentiation in the Drosophila visual center or “proneural wave” accompanies Notch activity that is propagated without the formation of a salt and pepper pattern, implying that Notch does not form a feedback loop of lateral inhibition during this process. However, mathematical modeling and genetic analysis clearly showed that Notch-mediated lateral inhibition is implemented within the proneural wave. Because partial reduction in EGF signaling causes the formation of the salt and pepper pattern, it is most likely that EGF diffusion cancels salt and pepper pattern formation in silico and in vivo. Moreover, the combination of Notch-mediated lateral inhibition and EGF-mediated reaction diffusion enables a function of Notch signaling that regulates propagation of the wave of differentiation. PMID:27535937

  11. Extended Synaptotagmin Interaction with the Fibroblast Growth Factor Receptor Depends on Receptor Conformation, Not Catalytic Activity.

    PubMed

    Tremblay, Michel G; Herdman, Chelsea; Guillou, François; Mishra, Prakash K; Baril, Joëlle; Bellenfant, Sabrina; Moss, Tom

    2015-06-26

    We previously demonstrated that ESyt2 interacts specifically with the activated FGF receptor and is required for a rapid phase of receptor internalization and for functional signaling via the ERK pathway in early Xenopus embryos. ESyt2 is one of the three-member family of Extended Synaptotagmins that were recently shown to be implicated in the formation of endoplasmic reticulum (ER)-plasma membrane (PM) junctions and in the Ca(2+) dependent regulation of these junctions. Here we show that ESyt2 is directed to the ER by its putative transmembrane domain, that the ESyts hetero- and homodimerize, and that ESyt2 homodimerization in vivo requires a TM adjacent sequence but not the SMP domain. ESyt2 and ESyt3, but not ESyt1, selectively interact in vivo with activated FGFR1. In the case of ESyt2, this interaction requires a short TM adjacent sequence and is independent of receptor autophosphorylation, but dependent on receptor conformation. The data show that ESyt2 recognizes a site in the upper kinase lobe of FGFR1 that is revealed by displacement of the kinase domain activation loop during receptor activation.

  12. Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics*

    PubMed Central

    Zhang, Ping; Leger, Andrew J.; Baleja, James D.; Rana, Rajashree; Corlin, Tiffany; Nguyen, Nga; Koukos, Georgios; Bohm, Andrew; Covic, Lidija; Kuliopulos, Athan

    2015-01-01

    G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class. PMID:25934391

  13. Redefining the concept of protease-activated receptors: cathepsin S evokes itch via activation of Mrgprs

    PubMed Central

    Reddy, Vemuri B.; Sun, Shuohao; Azimi, Ehsan; Elmariah, Sarina B.; Dong, Xinzhong; Lerner, Ethan A.

    2015-01-01

    Sensory neurons expressing Mas-related G protein coupled receptors (Mrgprs) mediate histamine-independent itch. We show that the cysteine protease cathepsin S activates MrgprC11 and evokes receptor-dependent scratching in mice. In contrast to its activation of conventional protease-activated receptors, cathepsin S mediated activation of MrgprC11 did not involve the generation of a tethered ligand. We demonstrate further that different cysteine proteases selectively activate specific mouse and human Mrgpr family members. This expansion of our understanding by which proteases interact with GPCRs redefines the concept of what constitutes a protease-activated receptor. The findings also implicate proteases as ligands to members of this orphan receptor family while providing new insights into how cysteine proteases contribute to itch. PMID:26216096

  14. Assessment of redox changes to hydrogen peroxide-sensitive proteins during EGF signaling.

    PubMed

    Cuddihy, Sarah L; Winterbourn, Christine C; Hampton, Mark B

    2011-07-01

    Hydrogen peroxide acts as a second messenger in growth factor signaling where it can oxidize and modify the function of redox-sensitive proteins. While selective thiol oxidation has been measured, there has been no global assessment of protein oxidation following growth factor activation. Significant changes to the abundant and widely distributed redox sensitive thiol proteins were observed in A431 epidermoid carcinoma cells exposed to hydrogen peroxide, but no changes were observed following treatment with epidermal growth factor (EGF). This included members of the peroxiredoxin family, which were also monitored in the presence of the thioredoxin reductase inhibitor auranofin to limit their capacity to recycle to the reduced form. We conclude that widespread thiol oxidation does not occur in cells during EGF signaling, and that hydrogen peroxide must act in a highly localized or selective manner.

  15. Steroid signaling activation and intracellular localization of sex steroid receptors.

    PubMed

    Giraldi, Tiziana; Giovannelli, Pia; Di Donato, Marzia; Castoria, Gabriella; Migliaccio, Antimo; Auricchio, Ferdinando

    2010-12-01

    In addition to stimulating gene transcription, sex steroids trigger rapid, non-genomic responses in the extra-nuclear compartment of target cells. These events take place within seconds or minutes after hormone administration and do not require transcriptional activity of sex steroid receptors. Depending on cell systems, activation of extra-nuclear signaling pathways by sex steroids fosters cell cycle progression, prevents apoptosis, leads to epigenetic modifications and increases cell migration through cytoskeleton changes. These findings have raised the question of intracellular localization of sex steroid receptors mediating these responses. During the past years, increasing evidence has shown that classical sex steroid receptors localized in the extra-nuclear compartment or close to membranes of target cells induce these events. The emerging picture is that a process of bidirectional control between signaling activation and sex steroid receptor localization regulates the outcome of hormonal responses in target cells. This mechanism ensures cell cycle progression in estradiol-treated breast cancer cells, and its derangement might occur in progression of human proliferative diseases. These findings will be reviewed here together with unexpected examples of the relationship between sex steroid receptor localization, signaling activation and biological responses in target cells. We apologize to scientists whose reports are not mentioned or extensively discussed owing to space limitations.

  16. Glycine Potentiates AMPA Receptor Function through Metabotropic Activation of GluN2A-Containing NMDA Receptors

    PubMed Central

    Li, Li-Jun; Hu, Rong; Lujan, Brendan; Chen, Juan; Zhang, Jian-Jian; Nakano, Yasuko; Cui, Tian-Yuan; Liao, Ming-Xia; Chen, Jin-Cao; Man, Heng-Ye; Feng, Hua; Wan, Qi

    2016-01-01

    NMDA receptors are Ca2+-permeable ion channels. The activation of NMDA receptors requires agonist glutamate and co-agonist glycine. Recent evidence indicates that NMDA receptor also has metabotropic function. Here we report that in cultured mouse hippocampal neurons, glycine increases AMPA receptor-mediated currents independent of the channel activity of NMDA receptors and the activation of glycine receptors. The potentiation of AMPA receptor function by glycine is antagonized by the inhibition of ERK1/2. In the hippocampal neurons and in the HEK293 cells transfected with different combinations of NMDA receptors, glycine preferentially acts on GluN2A-containing NMDA receptors (GluN2ARs), but not GluN2B-containing NMDA receptors (GluN2BRs), to enhance ERK1/2 phosphorylation independent of the channel activity of GluN2ARs. Without requiring the channel activity of GluN2ARs, glycine increases AMPA receptor-mediated currents through GluN2ARs. Thus, these results reveal a metabotropic function of GluN2ARs in mediating glycine-induced potentiation of AMPA receptor function via ERK1/2 activation. PMID:27807405

  17. Overexpression and activation of epidermal growth factor receptor in hemangioblastomas

    PubMed Central

    Chen, Gregory J.; Karajannis, Matthias A.; Newcomb, Elizabeth W.

    2010-01-01

    Hemangioblastomas frequently develop in patients with von Hippel-Lindau (VHL) disease, an autosomal dominant genetic disorder. The tumors are characterized by a dense network of blood capillaries, often in association with cysts. Although activation of receptor tyrosine kinase (RTK) signaling, including epidermal growth factor receptor (EGFR) has been implicated in the development of malignant brain tumors such as high-grade gliomas, little is known about the role of RTK signaling in hemangioblastomas. To address this issue, we examined hemangioblastoma tumor specimens using receptor tyrosine kinase (RTK) activation profiling and immunohistochemistry. Six human hemangioblastomas were analyzed with a phospho-RTK antibody array, revealing EGFR phosphorylation in all tumors. EGFR expression was confirmed by immunohistochemistry in all tumors analyzed and downstream effector pathway activation was demonstrated by positive staining for phospho-AKT. Our findings suggest that, in primary hemangioblastomas, RTK upregulation and signaling predominantly involves EGFR, providing an attractive molecular target for therapeutic intervention. PMID:20730556

  18. Interfering with mineralocorticoid receptor activation: the past, present, and future

    PubMed Central

    2014-01-01

    Aldosterone is a potent mineralocorticoid produced by the adrenal gland. Aldosterone binds to and activates the mineralocorticoid receptor (MR) in a plethora of tissues, but the cardiovascular actions of aldosterone are of primary interest clinically. Although MR antagonists were developed as antihypertensive agents, they are now considered to be important therapeutic options for patients with heart failure. Specifically, blocking only the MR has proven to be a difficult task because of its similarity to other steroid receptors, including the androgen and progesterone receptors. This lack of specificity caused the use of the first-generation mineralocorticoid receptor antagonists to be fraught with difficulty because of the side effects produced by drug administration. However, in recent years, several advances have been made that could potentially increase the clinical use of agents that inhibit the actions of aldosterone. These will be discussed here along with some examples of the beneficial effects of these new therapeutic agents. PMID:25165560

  19. Histamine 3 receptor activation reduces the expression of neuronal angiotensin II type 1 receptors in the heart.

    PubMed

    Hashikawa-Hobara, Narumi; Chan, Noel Yan-Ki; Levi, Roberto

    2012-01-01

    In severe myocardial ischemia, histamine 3 (H₃) receptor activation affords cardioprotection by preventing excessive norepinephrine release and arrhythmias; pivotal to this action is the inhibition of neuronal Na⁺/H⁺ exchanger (NHE). Conversely, angiotensin II, formed locally by mast cell-derived renin, stimulates NHE via angiotensin II type 1 (AT₁) receptors, facilitating norepinephrine release and arrhythmias. Thus, ischemic dysfunction may depend on a balance between the NHE-modulating effects of H₃ receptors and AT₁ receptors. The purpose of this investigation was therefore to elucidate the H₃/AT₁ receptor interaction in myocardial ischemia/reperfusion. We found that H₃ receptor blockade with clobenpropit increased norepinephrine overflow and arrhythmias in Langendorff-perfused guinea pig hearts subjected to ischemia/reperfusion. This coincided with increased neuronal AT₁ receptor expression. NHE inhibition with cariporide prevented both increases in norepinephrine release and AT₁ receptor expression. Moreover, norepinephrine release and AT₁ receptor expression were increased by the nitric oxide (NO) synthase inhibitor N(G)-methyl-L-arginine and the protein kinase C activator phorbol myristate acetate. H₃ receptor activation in differentiated sympathetic neuron-like PC12 cells permanently transfected with H₃ receptor cDNA caused a decrease in protein kinase C activity and AT₁ receptor protein abundance. Collectively, our findings suggest that neuronal H₃ receptor activation inhibits NHE by diminishing protein kinase C activity. Reduced NHE activity sequentially causes intracellular acidification, increased NO synthesis, and diminished AT₁ receptor expression. Thus, H₃ receptor-mediated NHE inhibition in ischemia/reperfusion not only opposes the angiotensin II-induced stimulation of NHE in cardiac sympathetic neurons, but also down-regulates AT₁ receptor expression. Cardioprotection ultimately results from the combined

  20. Histamine 3 Receptor