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.

scholarly journals Epidermal growth factor (EGF) stimulates association and kinase activity of Raf-1 with the EGF receptor.

1991 ◽  
Vol 11 (2) ◽  
pp. 913-919 ◽  
Author(s):  
H App ◽  
R Hazan ◽  
A Zilberstein ◽  
A Ullrich ◽  
J Schlessinger ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Sodium Dodecyl ◽  
Specific Protein ◽  
Kinase Assay ◽  
Downstream Effector ◽  
Epidermal Growth

Raf-1 serine- and threonine-specific protein kinase is transiently activated in cells expressing the epidermal growth factor (EGF) receptor upon treatment with EGF. The stimulated EGF receptor coimmunoprecipitates with Raf-1 kinase and mediates protein kinase C-independent phosphorylation of Raf-1 on serine residues. Hyperphosphorylated Raf-1 has lower mobility on sodium dodecyl sulfate gels and has sixfold-increased activity in immunocomplex kinase assay with histone H1 or Raf-1 sequence-derived peptide as a substrate. Raf-1 activation requires kinase-active EGF receptor; a point mutant lacking tyrosine kinase activity in inactive in Raf-1 coupling and association. It is noteworthy that tyrosine phosphorylation of c-Raf-1 induced by EGF was not detected in these cells. These observations suggest that Raf-1 kinase may act as an important downstream effector of EGF signal transduction.

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 Apical Epidermal Growth Factor Receptor Signaling: Regulation of Stretch-dependent Exocytosis in Bladder Umbrella Cells

2007 ◽  
Vol 18 (4) ◽  
pp. 1312-1323 ◽  
Author(s):  
Elena M. Balestreire ◽  
Gerard Apodaca
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Physiological Role ◽  
Receptor Activation ◽  
Mitogen Activated Protein Kinase ◽  
Apical Surface ◽  
Mechanical Stimuli ◽  
Epidermal Growth ◽  
Umbrella Cells

The apical surface of polarized epithelial cells receives input from mediators, growth factors, and mechanical stimuli. How these stimuli are coordinated to regulate complex cellular functions such as polarized membrane traffic is not understood. We analyzed the requirement for growth factor signaling and mechanical stimuli in umbrella cells, which line the mucosal surface of the bladder and dynamically insert and remove apical membrane in response to stretch. We observed that stretch-stimulated exocytosis required apical epidermal growth factor (EGF) receptor activation and that activation occurred in an autocrine manner downstream of heparin-binding EGF-like growth factor precursor cleavage. Long-term changes in apical exocytosis depended on protein synthesis, which occurred upon EGF receptor-dependent activation of mitogen-activated protein kinase signaling. Our results indicate a novel physiological role for the EGF receptor that couples upstream mechanical stimuli to downstream apical EGF receptor activation that may regulate apical surface area changes during bladder filling.

scholarly journals Isolation and partial characterization of canine epidermal growth factor/urogastrone

1985 ◽  
Vol 229 (3) ◽  
pp. 611-619 ◽  
Author(s):  
R Kobayashi ◽  
J R Reeve ◽  
J H Walsh
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Mouse Fibroblast ◽  
A431 Cells ◽  
Peptide Component ◽  
Epidermal Growth

Canine epidermal growth factor (EGF)/urogastrone was partially purified from dog urine by fractional precipitation with (NH4)2SO4, ion-exchange chromatography with DEAE-cellulose DE-52, gel filtration with Sephadex G-50, and a second DE-52 chromatography, to yield receptor-competing activity equivalent to 13 micrograms of standard mouse EGF/litre of starting urine. The purification was monitored by a competitive radioreceptor assay using fixed monolayers of A431 cells. The partially purified canine EGF/urogastrone demonstrated a growth-stimulating activity in 3T3 mouse fibroblast cells as potent as mouse EGF. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one major peptide component with an Mr similar to that of mouse EGF, and two minor peptides of slightly higher Mr. The major peptide component was isolated after reduction and its amino acid composition was determined.

Heparin-binding epidermal growth factor and Src family kinases in proliferation of renal epithelial cells

2008 ◽  
Vol 294 (3) ◽  
pp. F459-F468 ◽  
Author(s):  
Shougang Zhuang ◽  
Gilbert R. Kinsey ◽  
Kyle Rasbach ◽  
Rick G. Schnellmann
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Receptor Activation ◽  
Heparin Binding ◽  
Oxidant Injury ◽  
Mmp Inhibitor ◽  
Epidermal Growth ◽  
Renal Proximal Tubular Cells ◽  
Renal Proximal Tubular

Our recent studies have shown that proliferation of renal proximal tubular cells (RPTC) in the absence of growth factors requires activation of the epidermal growth factor (EGF) receptor. We sought to identify the endogenous EGF receptor ligand and investigate the mechanism(s) by which RPTC proliferate in different models. RPTC expressed both pro- and cleaved forms of heparin-binding epidermal growth factor (HB-EGF) and several metalloproteinases (MMP-2, -3, -9, and ADAM10, ADAM17) that have been reported to cleave HB-EGF. Treatment of RPTC with CRM 197, an inhibitor of HB-EGF binding to the EGF receptor, or downregulation of HB-EGF with small interfering RNA inhibited RPTC proliferation following plating. Furthermore, GM6001 (pan-MMP inhibitor), tumor-necrosis factor protease inhibitor-1 (TAPI-1; MMP and ADAM17 inhibitor), and GW280264X (ADAM10 and -17 inhibitor), but not GI254023X (ADAM10 inhibitor), attenuated the proliferation after plating. Although EGF receptor activation is required for RPTC proliferation after oxidant injury, CRM197, GM6001, and TAPI-1 did not block this response. In contrast, inhibition of Src with PP1 blocked EGF receptor activation and RPTC proliferation after oxidant injury. In addition, PP1 treatment attenuated HB-EGF-enhanced RPTC proliferation. We suggest that RPTC proliferation after plating is mediated by HB-EGF produced through an autocrine/paracrine mechanism and RPTC proliferation following oxidant injury is mediated by Src without involvement of HB-EGF.

scholarly journals COMPARISON OF EXTRACELLULAR SECRETION OF RECOMBINANT HUMAN EPIDERMAL GROWTH FACTOR USING TORA AND PELB SIGNAL PEPTIDES IN ESCHERICHIA COLI BL21 (DE3)

2019 ◽  
pp. 81-84 ◽  
Author(s):  
RIMA MELATI ◽  
ANNISA INDRIYANI ◽  
SHABARNI GAFFAR ◽  
SRIWIDODO ◽  
IMAN PERMANA MAKSUM
Keyword(s):  
Escherichia Coli ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Signal Peptides ◽  
Extracellular Expression ◽  
E Coli ◽  
Human Epidermal Growth Factor ◽  
Epidermal Growth

Objective: The objective of this study was to evaluate two signal peptides (TorA and PelB), representing the most common secretion pathways in Escherichia coli, for their ability to secrete recombinant human epidermal growth factor (rhEGF) protein in the extracellular expression. Methods: E. coli BL21 (DE3) as the host cell to be transformed using recombinant plasmid pD881-TorA the consensus already containing hEGF gene and the signal peptide TorA or PelB, then expressed by L-rhamnose induction. rhEGF purified by heat treatment and ion-exchange chromatography. The hEGF protein was characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ELISA. Results: The result showed that PelB was secreting more hEGF protein compared to TorA with protein expression results of 48.2 μg/L and purification results of 0.360 μg/L, with a purity level of 83%. Conclusion: The results of this study explain in extracellular expression of hEGF protein in E. coli, PelB helps hEGF protein secretion to culture media better than TorA.

Quantitative analysis of the EGF receptor autocrine system reveals cryptic regulation of cell response by ligand capture

2001 ◽  
Vol 114 (12) ◽  
pp. 2301-2313 ◽  
Author(s):  
Ann E. DeWitt ◽  
Jian Ying Dong ◽  
H. Steven Wiley ◽  
Douglas A. Lauffenburger
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Expression System ◽  
Receptor Occupancy ◽  
Receptor Activation ◽  
Quantitative Model ◽  
Receptor Expression ◽  
Extracellular Medium ◽  
Epidermal Growth

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 with receptor activation using a microphysiometer-based assay and analyzed our data using 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 (VLT) and the rate of receptor production (VR). At a VLT/VR ratio of <0.3, essentially no ligand was found in the extracellular medium, but a significant number of cell receptors (30-40%) were occupied. As the VLT/VR ratio increased from 0.3 towards unity, receptor occupancy increased and significant amounts of ligand appeared in the medium. Above a VLT/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 VLT/VR ratio of <5×10−4was sufficient to evoke >20% of a maximal proliferative response. This demonstrates that natural autocrine systems can be active even when no ligand appears in the extracellular medium.

scholarly journals Application of β-Galactosidase Enzyme Complementation Technology as a High Throughput Screening Format for Antagonists of the Epidermal Growth Factor Receptor

2001 ◽  
Vol 6 (6) ◽  
pp. 401-411
Author(s):  
Debbie L. Graham ◽  
Nicola Bevan ◽  
Peter N. Lowe ◽  
Michelle Palmer ◽  
Stephen Rees
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
High Throughput ◽  
False Positive ◽  
Mammalian Cells ◽  
Egf Receptor ◽  
Receptor Activation ◽  
C2c12 Cells ◽  
Hit Rate ◽  
Epidermal Growth

We have applied enzyme complementation technology to develop a screen for antagonists of the epidermal growth factor (EGF) receptor. Chimeric proteins containing two weakly complementing deletion mutants of Escherichia coli β-galactosidase (β-gal), each fused to the EGF receptor extracellular and transmembrane domains, have been stably expressed in C2C12 cells. In this cell line, formation of active β-gal is dependent on agonist-stimulated dimerization of the EGF receptor. We have developed a homogenous 384-well assay protocol and have applied this to characterize the pharmacology of the receptor and to develop a high throughput screen (HTS) for EGF receptor antagonists. The assay is tolerant to DMSO concentrations of up to 2% and, across 21 passages in culture, exhibits an EC50 for EGF of 5.4 ± 3.6 ng/ml (n = 11) and a Z' of 0.55 ± 0.13 (n = 11). A random set of 1,280 compounds was screened in duplicate at 11 μM to examine the robustness of enzyme complementation technology and to characterize the false-positive hit rate in the assay. Using a cutoff of 40% inhibition of EGF-promoted β-gal activity, the hit rate on day 1 was 2.5% and on day 2 was 1.9%. After retesting the active compounds, the hit rate was reduced to 0.4%, of which one of the compounds was identified as a β-gal inhibitor and the remainder appeared to be nonspecific inhibitors in the assay. This technology is amenable to automated screen workstations, there are highly sensitive chemiluminescent and fluorescent β-gal assay reagents amenable to detection in miniaturized plate formats, and the assay benefits from a low false-positive hit rate. Enzyme complementation technology may have wide application within the HTS environment for the detection of modulators of receptor activation or inhibitors of protein-protein interactions in mammalian cells.

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