Modulation of the epidermal growth factor receptor by basic fibroblast growth factor

Treatment of Swiss 3T3 fibroblasts with basic fibroblast growth factor (bFGF) lead to a rapid reduction in epidermal growth factor (EGF) binding and a slower inhibition of EGF receptor autophosphorylation. The reduction in binding was due to a complete loss of the highest affinity EGF binding sites and a reduction in the lower affinity binding sites. Neither the inhibition of EGF binding nor the inhibition of EGF receptor autophosphorylation required protein kinase C. Treatment of cells with bFGF stimulated the phosphorylation of the EGF receptor, which persisted for several hours. The inhibition of EGF receptor autophosphorylation by bFGF was reduced in the presence of cycloheximide. However, cycloheximide had no effect on the reduction of EGF binding by bFGF. In contrast to these results with Swiss 3T3 fibroblasts, treatment of PC12 cells with bFGF lead to a reduction in EGF binding but no inhibition of EGF receptor autophosphorylation. Thus inhibited of EGF receptor autophosphorylation and inhibition of EGF binding can be uncoupled. © 1993 Wiley-Liss, Inc.     

An epidermal growth factor receptor intron 1 polymorphism in healthy women in Poland

The frequency of CA allele combinations was assessed in healthy women from Poland and compared to previously published polymorphism data of individuals from Germany and a Caucasian reference group. There were close similarities between these three geographically and ethnically similar populations. By contrast, the distribution of these alleles in European and Asian (Japan) populations proved to be different. There might therefore be major ethnic differences in allelic frequencies of EGFR intron 1 polymorphism. Our results provide new data on EGFR microsatellite instability and may contribute to the understanding of EGFR gene expression regulation. The clinical relevance of these findings warrants further evaluation.     

Two polymorphic dinucleotide repeats in intron 44 of the dystrophin gene

Duchenne muscular dystrophy (DMD) is one of the most common and severe X-linked disorders with an incidence of approximately 1 in 3500 newborn males. In more than 60% of DMD patients, deletions of part or all of the dystrophin gene have been shown. Despite this, carrier detection still poses a problem in some cases, because of the enormous size of the gene and the lack of sufficient numbers of informative markers. Here, we report the isolation and characterization of two additional microsatellite markers (IVS44SK12 and IVS44SK21) in intron 44 of the dystrophin gene. Both markers are useful for carrier detection either by indirect DNA analysis or by direct proof of loss of heterozygosity.     

Highly polymorphic dinucleotide repeat at the NF2 gene

A highly polymorphic CA repeat was identified in a cosmid containing the 5 end of the NF2 tumour suppressor gene. This marker has proved useful in presymptomatic diagnosis in affected families.     

Modulation of arsenic-induced epidermal growth factor receptor pathway signalling by resveratrol

Arsenic (As) is both a human carcinogen and an effective anticancer drug. These aspects of arsenic toxicity develop as a consequence of arsenic-induced oxidative stress and modifications to signal pathway activity which alter gene expression. Resveratrol (RVL) a food antioxidant found in grapes and other fruits, exhibits anti-carcinogenic properties by reducing oxidative stress and restoring signal pathway control. This study investigated the impact of RVL on arsenite [As(III)]-induced cell signalling in HaCaT keratinocytes by assaying phosphorylation status of epidermal growth factor receptor (EGFR) signalling intermediates and measuring changes in expression of Phase II and DNA repair biomarkers. As(III) exposure produced dose-dependent toxicity which was associated with increased activation of EGFR pathway intermediates, cSrc, Rac1 and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Arsenic-mediated ERK1/2 activation negatively regulated DNA polymerase beta expression and up regulated heme-oxygenase-1 at toxic concentrations. RVL treatment modulated As(III)-mediated ERK1/2 activation by shifting the balance of cSrc regulatory domain phosphorylation. These effects significantly altered the response of the EGFR pathway to growth factor-induced stimulation. Our research provides evidence that treatment with pharmacologically relevant doses of RVL influences cellular responses to As(III), largely due to RVL-mediated changes to Src and ERK1/2 activation.     

Tenascin cytotactin epidermal growth factor-like repeat binds epidermal growth factor receptor with low affinity

Select epidermal growth factor (EGF)-like (EGFL) repeats of human tenascin cytotactin (tenascin C) can stimulate EGF receptor (EGFR) signaling, but activation requires micromolar concentrations of soluble EGFL repeats in contrast to subnanomolar concentrations of classical growth factors such as EGF. Using in silico homology modeling techniques, we generated a structure for one such repeat, the 14th EGFL repeat (Ten14). Ten14 assumes a tight EGF-like fold with truncated loops, consistent with circular dichroism studies. We generated bound structures for Ten14 with EGFR using two different approaches, resulting in two distinctly different conformations. Normal mode analysis of both structures indicated that the binding pocket of EGFR exhibits a significantly higher mobility in Ten14-EGFR complex compared to that of the EGF-EGFR complex; we hypothesized this may be attributed to loss of key high-affinity interactions within the Ten14-EGFR complex. We proved the efficacy of our in silico models by in vitro experiments. Surface plasmon resonance measurements yielded equilibrium constant K(D) of 74 microM for Ten14, approximately three orders of magnitude weaker than that of EGF. In accordance with our predicted bound models, Ten14 in monomeric form does not bind EGFR with sufficient stability so as to induce degradation of receptor, or undergo EGFR-mediated internalization over either the short (20 min) or long (48 h) term. This transient interaction with the receptor on the cell surface is in marked contrast to other EGFR ligands which cause EGFR transit through, and signaling from intracellular locales in addition to cell surface signaling.     

Modulation of transforming growth factor alpha-dependent expression of epidermal growth factor receptor gene by transforming growth factor beta, triiodothyronine, and retinoic acid

We have investigated the actions of transforming growth factor (TGF) type alpha on epidermal growth factor (EGF) receptor mRNA expression in MDA-468 human mammary carcinoma cells in serum-free media. We found that exposure of MDA-468 cells to TGF alpha results in elevated levels of EGF receptor mRNA. This increase in mRNA accumulation showed time and dose dependence. Addition of TGF beta 1 enhanced the accumulation of EGF receptor mRNA induced by TGF alpha in a time- and dose-dependent manner. We also found that triiodothyronine at physiological concentrations exerts synergistic control on the action of TGF alpha alone, or in association with TGF beta 1, on EGF receptor mRNA expression. Similarly, retinoic acid treatment also enhanced in a time- and dose-dependent manner the TGF alpha-dependent response of EGF receptor mRNA and acted synergistically with TGF beta 1. The results described here suggest that optimum regulation of EGF receptor gene expression by TGF alpha is a complex process involving synergistic interactions with heterologous growth factors and hormones.     

Regulation of epidermal growth factor receptor gene expression

Synthesis and metabolism of the epidermal growth factor (EGF) receptor are extensively regulated to modulate cellular responses to ligand. To study regulation of EGF receptor gene expression, the 5' region of the gene was isolated from a human placental genomic library. A 5' proximal 1.1-kilobase fragment (-1100 to -19 relative to the ATG translation start site) and subfragments of this were subcloned in both forward and reverse orientations into the luciferase expression vector pSVOAL delta 5' and transfected into human cell lines. Luciferase activity was stimulated by treatment of transfected HeLa cells with EGF, 12-O-tetradecanoylphorbol 13-acetate (TPA), (Bu)2 cAMP, retinoic acid, and dexamethasone. Deletion analysis indicated full retention of activity after removal of the -1100 to -485 region (-485 to -19 fragment), but a 5-fold reduction in activity on removal of the -485 to -153 region (-153 to -19 fragment). Despite a reduction in basal activity, the proximal 134-basepair fragment retained responses to all inducers. Additivity was observed in response to maximal concentrations of TPA plus retinoic acid and of TPA plus (Bu)2 cAMP; the response to a combination of four inducers exceeded that to the RSV-LTR strong promoter. Differences in stimulated responses were observed in various recipients, with hepatoma HepG2 cells lacking responses to (Bu)2 cAMP and glioblastoma T98G cells lacking responses to EGF and TPA. These results indicate that a 134-basepair DNA fragment closely adjacent to the translation start site contains elements responsible for directing basal and stimulated expression of the EGF receptor gene.     

Modulation of the epidermal growth factor receptor by brain-derived growth factor in Swiss mouse 3T3 cells

Incubation of Swiss mouse 3T3 cells at 37 degrees C with bovine brain-derived growth factor (BDGF) decrease the cell surface 125I-EGF binding activity of these cells by 70-80%. This down-modulation of the EGF receptor by BDGF was time, temperature, and dose dependent. Scatchard plot analysis indicated that BDGF binding led to a selective decrease in the number of high-affinity EGF receptors. The BDGF-induced down-modulation of the EGF receptor was completely blocked by protamine, a potent inhibitor of receptor binding and mitogenic activities of BDGF. BDGF down-modulated the EGF receptor in phorbol myristic acetate (PMA)-pretreated cells, as well as in control cells. Furthermore, PMA-pretreated cells responded mitogenically to BDGF, whereas PMA itself failed to stimulate the mitogenic response of PMA-pretreated cells. This BDGF-induced down-modulation of the EGF receptor in PMA-desensitized cells suggests that BDGF down-regulates the EGF receptor by a mechanism distinct from that of PMA. Incubation of cells with compounds which are known to inhibit pinocytosis blocked the down-modulation induced either by BDGF or by platelet-derived growth factor (PDGF) but had no effect on the PMA-induced down-modulation. Incubation of cells with inhibitors of receptor recycling enhanced the BDGF-induced down-modulation of the EGF receptor. These results suggest that BDGF and PDGF induce down-modulation of the EGF receptor by increasing the internalization of cell surface high-affinity receptors and that the internalization process may not be required for down-modulation induced by PMA.     

Transactivation of epidermal growth factor receptor by insulin-like growth factor 1 requires basal hydrogen peroxide

Insulin-like growth factor (IGF-1) plays an important role in prostate cancer development. Recent studies suggest that IGF-1 has mitogenic action through epidermal growth factor receptor (EGFR). However, the mechanism remains largely unknown. Here, we demonstrated in prostate cancer DU145 cells that IGF-1 induced EGFR transactivation, leading to ERK activation. Matrix metalloproteinase-mediated shedding of heparin-binding EGF is involved in this process. Antioxidants and catalase inhibited IGF-1-stimulated EGFR phosphorylation, indicating that H(2)O(2) is required for EGFR activation. However, exogenous H(2)O(2) did not activate EGFR or IGF-1R in DU145 cells. IGF-1 did not induced production of H(2)O(2) in DU145 cells. Our results suggest that transactivation of EGFR by IGF-1 requires basal intracellular H(2)O(2) in DU145 cells.