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.

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

2005 ◽  
Vol 16 (6) ◽  
pp. 2984-2998 ◽  
Author(s):  
Jianying Dong ◽  
Lee K. Opresko ◽  
William Chrisler ◽  
Galya Orr ◽  
Ryan D. Quesenberry ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Resonance Energy ◽  
Structural Basis ◽  
Cell Contact ◽  
Heparin Binding ◽  
Epidermal Growth ◽  
Membrane Anchoring

All ligands of the epidermal growth factor (EGF) 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 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.

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.

scholarly journals Epidermal growth factor receptor activation is localized within low-buoyant density, non-caveolar membrane domains

1999 ◽  
Vol 337 (3) ◽  
pp. 591-597 ◽  
Author(s):  
Mark G. WAUGH ◽  
Durward LAWSON ◽  
J. Justin HSUAN
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Receptor Activation ◽  
Cell Fractionation ◽  
Membrane Domains ◽  
Egf Receptors ◽  
Cell Extracts ◽  
Epidermal Growth

Increasing evidence for the organization of cell-surface proteins and lipids into different detergent-insoluble rafts led us to investigate epidermal growth factor (EGF) receptor activation in the plasma membranes of A431 carcinoma cells, using a combination of cell fractionation and immunoprecipitation techniques. Density-gradient centrifugation of sodium carbonate cell extracts revealed that the vast majority of both stimulated and unstimulated EGF receptors were concentrated in a caveolin-rich light membrane (CLM) fraction, with the biochemical characteristics of detergent-insoluble glycolipid-rich domains (DIGs). However, ultrastructural analysis of the CLM fraction revealed that it contained a heterogeneous collection of vesicles, some with sizes greater than that expected for individual caveolae. Experiments with detergent-solubilized cells and isolated CLMs indicated that, in contrast with caveolin, EGF receptors were unlikely to be localized to DIG domains. Furthermore, immunoisolation of caveolin from CLMs revealed that EGF receptor activation occurs in a compartment distinct from caveolae. Similarly, using an anti-(EGF receptor) antibody, the bulk of the cellular caveolin was not co-immunoprecipitated from CLMs, thereby confirming that these two proteins reside in separate membrane domains. The deduction that caveolar signalling and EGF receptor activation occur in separable rafts argues for a multiplicity of signal transduction compartments within the plasma membrane. In addition, by demonstrating that EGF receptor activation is compartmentalized within low-density, non-caveolar regions of the plasma membrane, it is also shown that the co-localization of proteins in a CLM fraction is insufficient to prove caveolar localization.

scholarly journals Physiological functions of keratinocyte epidermal growth factor receptors and their role in the development of skin toxicity during targeted cancer therapy

2021 ◽  
Vol 24 (2) ◽  
pp. 111-118
Author(s):  
Ekaterina V. Orlova
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Pancreatic Head ◽  
Skin Toxicity ◽  
Receptor Activation ◽  
Tyrosine Kinase Receptor ◽  
Long Term Treatment ◽  
Epidermal Growth

The binding of epidermal growth factor (EGFR) receptors is a good target for the treatment of lung, colon, pancreatic, head and neck cancers. The adverse events that develop as a result of therapy, in the form of lesions of the skin and mucous membranes, is a serious problem for the doctor to choose a long-term treatment strategy. The developing symptoms of skin toxicity, as skin problems in patients are often called, are worrisome and often affect the quality of life and compliance with the treatment regimen. Thus, it is important for doctors to know the prerequisites and ways to manage skin toxicity associated with the use of tyrosine kinase receptor inhibitors of epidermal growth factor. The mechanism and consequences of EGF receptor activation are described to explain the development of undesirable skin toxicity associated with inhibition of the epidermal growth factor receptor.

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