Norepinephrine modulates the zonally different hepatocyte proliferation through the regulation of transglutaminase activity

2010 ◽  
Vol 299 (1) ◽  
pp. G106-G114 ◽  
Author(s):  
Yosuke Ohtake ◽  
Tomonori Kobayashi ◽  
Akiko Maruko ◽  
Nao Oh-ishi ◽  
Fumihiko Yamamoto ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Egf Receptor ◽  
Transglutaminase 2 ◽  
Hepatocyte Proliferation ◽  
Scatchard Analysis ◽  
Binding Studies ◽  
Perfusion Technique ◽  
Egfr Activation ◽  
Epidermal Growth ◽  
Collagenase Perfusion

A neurotransmitter, norepinephrine (NE), amplifies the mitogenic effect of epidermal growth factor (EGF) in the liver by acting on the α1-adrenergic receptor coupled with G protein, Gαh. However, the molecular mechanism is not well understood. Gαh is known as a transglutaminase 2 (TG2), a cross-linking enzyme implicated in hepatocyte proliferation. We investigated the effect of NE on EGF-induced cell proliferation and TG2 activity using hepatocytes isolated in periportal and perivenous regions of the liver, which differ in proliferative capacity. Periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were isolated by the digitonin-collagenase perfusion technique. EGF or NE receptor binding was analyzed by Scatchard analysis. Changes in NE-induced DNA synthesis, EGF receptor (EGFR) dimerization and phosphorylation, and TG2 activity were measured. NE enhanced EGF-induced DNA synthesis, EGF-induced EGFR dimerization, and its phosphorylation in PVH but not in PPH. [3H]NE binding studies indicated that PVH was found to have a greater affinity and number of receptors than PPH. Furthermore, NE treatment decreased TG2 activity and increased phospholipase C activity in PVH although TG2 level showed no change. These results suggest that NE-induced amplification of EGF-induced DNA synthesis especially in PVH is caused by upregulation of EGFR activation through the switching of function from TG2 to Gαh.

Effect of aging on norepinephrine-related proliferative response in primary cultured periportal and perivenous hepatocytes

2012 ◽  
Vol 303 (7) ◽  
pp. G861-G869 ◽  
Author(s):  
Tomonori Kobayashi ◽  
Yohei Saito ◽  
Yosuke Ohtake ◽  
Akiko Maruko ◽  
Yumi Yamamoto ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
G Protein ◽  
Proliferative Response ◽  
Egf Receptor ◽  
Transglutaminase 2 ◽  
Scatchard Analysis ◽  
Binding Studies ◽  
Protein Activity ◽  
Age Related ◽  
Old Rats

Norepinephrine (NE) amplifies the mitogenic effect of EGF in a rat liver through the adrenergic receptor coupled with G protein, Ghα. Ghα is also known as a transglutaminase 2 (TG2), whose cross-linking activity is implicated in hepatocyte growth. Recently, we found that NE-induced amplification of EGF-induced DNA synthesis in hepatocytes obtained from perivenous regions of liver is caused by inhibiting the downregulation of EGF receptor (EGFR) by TG2. In the present study, we investigated the effect of aging on NE-related proliferative response. Hepatocytes were obtained from the liver of 7- and 90-wk-old rats. To examine this in detail, periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were isolated using the digitonin/collagenase perfusion technique. EGF or NE receptor binding was analyzed by Scatchard analysis. Changes in NE-induced DNA synthesis, G protein activity, and TG2 activity were measured. NE slightly potentiated [125I]EGF binding to EGFR, and EGF-induced DNA synthesis in PVH but not in PPH. [3H]NE binding studies indicated that PVH have a greater number of receptors than PPH, and that the number of receptors in both subpopulations increased with aging. NE-induced changes in G protein activity and TG2 activity in 90-wk-old rats were slight compared with 7-wk-old rats. These results suggest that NE results in a slight recovery effect on the age-related decline in EGF-induced DNA synthesis because of incomplete switching of the function from TG2 to Ghα.

scholarly journals Asbestos-induced phosphorylation of epidermal growth factor receptor is linked to c-fos and apoptosis

1999 ◽  
Vol 277 (4) ◽  
pp. L684-L693 ◽  
Author(s):  
Christine L. Zanella ◽  
Cynthia R. Timblin ◽  
Andrew Cummins ◽  
Michael Jung ◽  
Jonathan Goldberg ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Direct Interaction ◽  
Mrna Levels ◽  
Binding Studies ◽  
Therapeutic Implications ◽  
Asbestos Fibers ◽  
Epidermal Growth

We examined the mechanisms of interaction of crocidolite asbestos fibers with the epidermal growth factor (EGF) receptor (EGFR) and the role of the EGFR-extracellular signal-regulated kinase (ERK) signaling pathway in early-response protooncogene (c- fos/c- jun) expression and apoptosis induced by asbestos in rat pleural mesothelial (RPM) cells. Asbestos fibers, but not the nonfibrous analog riebeckite, abolished binding of EGF to the EGFR. This was not due to a direct interaction of fibers with ligand, inasmuch as binding studies using fibers and EGF in the absence of membranes showed that EGF did not adsorb to the surface of asbestos fibers. Exposure of RPM cells to asbestos caused a greater than twofold increase in steady-state message and protein levels of EGFR ( P < 0.05). The tyrphostin AG-1478, which inhibits the tyrosine kinase activity of the EGFR, but not the tyrphostin A-10, which does not affect EGFR activity, significantly ameliorated asbestos-induced increases in mRNA levels of c- fos but not of c- jun. Pretreatment of RPM cells with AG-1478 significantly reduced apoptosis in cells exposed to asbestos. Our findings suggest that asbestos-induced binding to EGFR initiates signaling pathways responsible for increased expression of the protooncogene c- fos and the development of apoptosis. The ability to block asbestos-induced elevations in c- fos mRNA levels and apoptosis by small-molecule inhibitors of EGFR phosphorylation may have therapeutic implications in asbestos-related diseases.

Eicosanoids enhance epidermal growth factor receptor activation and proliferation in glomerular epithelial cells

1992 ◽  
Vol 262 (4) ◽  
pp. F639-F646 ◽  
Author(s):  
A. V. Cybulsky ◽  
P. R. Goodyer ◽  
M. D. Cyr ◽  
A. J. McTavish
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Epithelial Cells ◽  
Egf Receptor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Receptor Activation ◽  
Scatchard Analysis ◽  
Glomerular Epithelial Cells ◽  
Epidermal Growth

Proliferation of glomerular epithelial cells (GEC) and release of prostaglandins (PG) and thromboxane (Tx) A2 may occur in glomerular injury. We studied the relationship of eicosanoids to epidermal growth factor (EGF)-induced proliferation of rat GEC in culture. After 48 h of serum-deprivation, EGF stimulated [3H]thymidine incorporation ninefold above serum-deprived cells. Inhibition of cyclooxygenase with indomethacin or of Txsynthase with OKY-046 decreased the proliferative effect of EGF by 50 and 38%, respectively. The effect of indomethacin was reversed by addition of PGE2. Synthesis of PGE2, PGF2 alpha, and TxA2 by serum-deprived GEC was not enhanced by EGF. Scatchard analysis of 125I-EGF binding to GEC demonstrated two populations of EGF receptors; the high-affinity site had a dissociation constant (Kd) of 444 pM and 24,864 receptors/cell. EGF receptor autophosphorylation (reflecting receptor activation) was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of GEC membrane proteins with anti-phosphotyrosine antibody. EGF increased phosphorylation of a protein of approximately 170 kDa, which comigrated with proteins immunoprecipitated from [35S]methionine-labeled GEC with antibodies to EGF receptor. Indomethacin and OKY-046 decreased the EGF-dependent phosphorylation of the 170-kDa protein, and this decrease was overcome by addition of PGE2. Indomethacin and OKY-046 did not, however, reduce 125I-EGF binding. Thus, in GEC, the basal synthesis of eicosanoids enhanced EGF-induced proliferation. This effect appears to be due to enhancement of EGF receptor activation.

Specific receptors for epidermal growth factor in rat intestinal microvillus membranes

1988 ◽  
Vol 254 (3) ◽  
pp. G429-G435 ◽  
Author(s):  
J. F. Thompson
Keyword(s):  
Small Intestine ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Sodium Dodecyl ◽  
Intestinal Lumen ◽  
Scatchard Analysis ◽  
Rat Small Intestine ◽  
Binding Studies ◽  
Epidermal Growth ◽  
Specific Receptors

Epidermal growth factor (EGF) is present in high concentrations in milk, salivary, and pancreaticobiliary secretions. EGF, delivered to the intestinal lumen by these fluids, appears to influence intestinal proliferation. Because EGF exerts its mitogenic effect through binding to specific membrane-bound receptors, binding studies of 125I-labeled EGF to purified microvillus membrane (MVM) preparations from fetal, newborn, and adult rat small intestine were performed. Using the membrane filter technique, binding of 125I-EGF to adult MVM was specific, saturable, and reversible. Adult and fetal MVM binding was rapid and reached a plateau after 30 min at both 20 and 37 degrees C. No binding was detected at 4 degrees C. Specific binding increased linearly from 0 to 75 micrograms MVM protein. Scatchard analysis revealed a single class of receptors in fetal and adult MVM with an association constant of 1.0 +/- 0.35 X 10(9) and 2.3 +/- 1.6 X 10(9) M-1, respectively. Binding capacity was 435.0 +/- 89 and 97.7 +/- 41.3 fmol 125I-EGF bound/mg MVM protein for fetal and adult MVM, respectively. Newborn MVM binding was negligible. After binding, cross-linking utilizing disuccinimidyl suberate, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography revealed a 170-kDa receptor. These data demonstrate specific receptors for EGF on MVM of rat small intestine and, thus, suggest a mechanism for the intraluminal regulation of enterocyte proliferation by EGF.

scholarly journals Insulin Growth Factor-I and Epidermal Growth Factor Receptors Recruit Distinct Upstream Signaling Molecules to Enhance AKT Activation in Mammary Epithelial Cells

Endocrinology ◽  
2006 ◽  
Vol 147 (12) ◽  
pp. 6027-6035 ◽  
Author(s):  
Jodie M. Fleming ◽  
Gwenaëlle Desury ◽  
Tiffany A. Polanco ◽  
Wendie S. Cohick
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Egf Receptor ◽  
Mammary Epithelial ◽  
Insulin Receptor Substrate ◽  
Phosphatidylinositol 3 Kinase ◽  
Mapk Activation ◽  
Igf I ◽  
Epidermal Growth

IGF-I and epidermal growth factor (EGF) stimulate both normal mammary epithelial cell (MEC) growth and tumorigenesis. Whereas both growth factors increase DNA synthesis in MECs, how they evoke a greater response in combination when they activate similar signaling pathways remains unknown. In the present study, we investigated the signaling pathways by which these mitogens act in concert to increase DNA synthesis. Only EGF activated the MAPK pathway, and no further increase in MAPK activation was observed when both mitogens were added together. Both growth factors activated the phosphatidylinositol-3 kinase pathway, and simultaneous treatment enhanced phosphorylation of both AKT and its downstream target, p70S6K. The enhanced activation of AKT was observed at multiple time points (5 and 15 min) and growth factor concentrations (2.5–100 ng/ml). IGF-I activated AKT via insulin receptor substrate-1 and p85, the regulatory subunit of phosphatidylinositol-3 kinase. Treatment with EGF had no effect on insulin receptor substrate-1; however, it activated the EGF receptor, SHC, and c-Src. EGF treatment caused the association of SHC with Grb2 and Gab2 with phospho-SHC, phospho-Gab1, Grb2, and p85. Interestingly, inhibition of Src activation blocked the ability of EGF, but not IGF-I, to activate AKT. This corresponded with a decrease in phosphorylation of the EGF receptor and its association with phospho-SHC as well as downstream signaling. Unexpectedly, inhibition of Src increased basal MAPK activation. This is the first study to show that EGF and IGF-I use separate upstream components within a given MEC line to enhance AKT phosphorylation, contributing to increased DNA synthesis.

scholarly journals EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

2018 ◽  
Author(s):  
Charlotte Fournier ◽  
Adam J. M. Wollman ◽  
Isabel Llorente-Garcia ◽  
Oliver Harriman ◽  
Djamila Ouarat ◽  
...  
Keyword(s):  
Single Molecule ◽  
Egf Receptor ◽  
Receptor Activation ◽  
Carcinoma Cells ◽  
Live Cells ◽  
Egfr Expression ◽  
Egfr Activation ◽  
Before And After ◽  
Epidermal Growth ◽  
Differentiation And Proliferation

AbstractEpidermal growth factor (EGF) signalling regulates cell growth, differentiation and proliferation in epithelium and EGF receptor (EGFR) overexpression has been reported in several carcinoma types. Structural and biochemical evidence suggests EGF binding stimulates EGFR monomer-dimer transitions, activating downstream signalling. However, mechanistic details of ligand binding to functional receptors in live cells remain contentious. We report real time single-molecule TIRF of human epithelial carcinoma cells with negligible native EGFR expression, transfected with GFP-tagged EGFR, before and after receptor activation with TMR-labelled EGF ligand. Fluorescently labelled EGFR and EGF are simultaneously tracked to 40nm precision to explore stoichiometry and spatiotemporal dynamics upon EGF binding. Using inhibitors that block binding to EGFR directly, or indirectly through HER2, our results indicate that pre-activated EGFR consists of preformed homoclusters, while larger heteroclusters including HER2 form upon activation. The relative stoichiometry of EGFR to EGF after binding peaks at 2, indicating negative cooperativity of EGFR activation.

scholarly journals Receptor-Based Antidote for Diphtheria

2002 ◽  
Vol 70 (5) ◽  
pp. 2344-2350 ◽  
Author(s):  
Jeong-Heon Cha ◽  
Joanna S. Brooke ◽  
Mee Young Chang ◽  
Leon Eidels
Keyword(s):  
Growth Factor ◽  
Egf Receptor ◽  
Binding Domain ◽  
Amino Acid Residues ◽  
Heparin Binding ◽  
Soluble Receptor ◽  
Binding Studies ◽  
Diphtheria Antitoxin ◽  
Heparin Binding Domain ◽  
Epidermal Growth

ABSTRACT Although equine diphtheria antitoxin may be an effective therapy for human diphtheria, its use often induces serum sickness. We describe here a strategy for developing an alternative treatment based on the human diphtheria toxin (DT) receptor/heparin-binding epidermal growth factor-like growth factor (HB-EGF) precursor. Recombinant mature human HB-EGF acts as a soluble receptor analog, binding radioiodinated DT and preventing its binding to the cellular DT receptor/HB-EGF precursor. However, the possibility existed that radioiodinated DT-HB-EGF complexes associate with cells due to the binding of the heparin-binding domain of recombinant HB-EGF to cell surface heparan sulfate proteoglycans. This possibility was confirmed by performing DT binding studies in the presence of heparin. A recombinant truncated HB-EGF (residues 106 to 149), which lacks most of the heparin-binding domain, showed an essentially heparin-independent binding of radioiodinated DT to cells. Furthermore, it was a more effective inhibitor of DT binding than was recombinant mature HB-EGF. Since mature HB-EGF is a known ligand for the EGF receptor and is thus highly mitogenic (tumorigenic), we then changed amino acid residues in the EGF-like domain of the recombinant truncated HB-EGF and demonstrated that this DT receptor analog (I117A/L148A) displayed a low mitogenic effect. The truncated (I117A/L148A) HB-EGF protein retained high DT binding affinity, as confirmed by using surface plasmon resonance. Our results suggest that the truncated (I117A/L148A) HB-EGF protein could be an effective, safe antidote for human diphtheria.

scholarly journals Transforming growth factor-α-induced DNA synthesis and c-myc expression in primary rat hepatocyte cultures is modulated by indomethacin

1992 ◽  
Vol 281 (3) ◽  
pp. 729-733 ◽  
Author(s):  
G G Skouteris ◽  
M McMenamin
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Phospholipase A2 ◽  
Culture Medium ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Receptor Gene ◽  
Hepatocyte Proliferation ◽  
Transforming Growth Factor Α

Primary hepatocytes stimulated with epidermal growth factor (EGF) secrete prostaglandins into the culture medium as soon as 1 h after the addition of the EGF. Transforming growth factor-alpha (TGF alpha), a potent hepatocyte mitogen, shares the same receptor with EGF, and its expression is increased after partial hepatectomy. TGF alpha is also secreted in culture. We have observed that TGF alpha induced hepatocyte DNA synthesis (30 h after addition) and at the same time stimulated the production of prostaglandins E2 and F2 alpha by the cultured hepatocytes. Indomethacin at 20-100 microM inhibited the TGF alpha-induced hepatocyte DNA synthesis, and this effect was specifically due to the inhibition of prostaglandin formation. Indomethacin also inhibited a TGF-alpha-induced increase in hepatocyte c-myc expression, indicating that prostaglandins mediate this increase, as previously shown for EGF. TGF alpha increased the expression of the EGF receptor gene, and this was prevented by the presence of an antibody against TGF alpha in the culture medium. We therefore suggest that TGF alpha induces hepatocyte proliferation either through coupling with its receptor (i.e. the EGF receptor) or by subsequent phosphorylation of lipocortin I. This leads to activation of phospholipase. A2, which seems to regulate the metabolism of arachidonic acid and the formation of prostaglandins. Thus hepatocyte proliferation in vitro appears to be controlled by a self-regulatory autocrine pathway involving activation of phospholipase A2 and secretion of prostaglandins and TGF alpha.

scholarly journals Bradykinin-induced growth inhibition of normal rat kidney (NRK) cells is paralleled by a decrease in epidermal-growth-factor receptor expression

1994 ◽  
Vol 298 (2) ◽  
pp. 335-340 ◽  
Author(s):  
E J J Van Zoelen ◽  
P H J Peters ◽  
G B Afink ◽  
S Van Genesen ◽  
A D G De Roos ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Rat Kidney ◽  
Receptor Expression ◽  
Binding Studies ◽  
Normal Rat ◽  
Epidermal Growth

Normal rat kidney fibroblasts, grown to density arrest in the presence of epidermal growth factor (EGF), can be induced to undergo phenotypic transformation by treatment with transforming growth factor beta or retinoic acid. Here we show that bradykinin blocks this growth-stimulus-induced loss of density-dependent growth arrest by a specific receptor-mediated mechanism. The effects of bradykinin are specific, and are not mimicked by other phosphoinositide-mobilizing agents such as prostaglandin F2 alpha. Northern-blot analysis and receptor-binding studies demonstrate that bradykinin also inhibits the retinoic acid-induced increase in EGF receptor levels in these cells. These studies provide additional evidence that EGF receptor levels modulate EGF-induced expression of the transformed phenotype in these cells.

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