EGF Family Of Growth Factors Research Articles

Endogenous EGF-Family Growth Factors are Necessary for the Progression from the G1 to S Phase in Human Keratinocytes

Recently several endogenous epidermal growth factor (EGF)-family growth factors (transforming growth factor-alpha, amphiregulin, and heparin-binding EGF-like growth factor) have been identified in human keratinocytes. These factors are known to play an important role in the regulation of cell proliferation. Here we show that the interaction between these factors and EGF receptor are key factors in the progression from the G1 phase to the S phase (the G1/S progression) in human keratinocytes. In this study, human keratinocytes were cultured in serum-free MCDB153 medium and then partially synchronized by isoleucine deprivation. After synchronization, the number of S phase cells increased and reached a maximum after 18-24 h. The immediate addition of anti-EGF receptor blocking antibody (1 microg per ml) to synchronized cells decreased S phase cells by 42.5% compared with untreated keratinocytes at 18 h. By contrast, the addition of anti-EGF receptor antibodies at 12 h or later did not alter the percentage of S phase cells. Northern blot analysis of synchronized cells demonstrated that mRNA expression of transforming growth factor-alpha, amphiregulin, heparin-binding EGF-like growth factor, and EGF receptor reached a maximum within 0.5-3 h after synchronization, when many cells initiated progression from the G1 to the S phase. The results show that anti-EGF receptor antibodies block the G1/S progression and the rapid increase of mRNA expression of endogenous EGF-family growth factors and EGF receptor during G1/S progression. These findings indicate that growth factor binding and EGF receptor activation are involved in the G1/S cell cycle progression of human keratinocytes.

Tyrosine kinase inhibitors. 14. Structure-activity relationships for methylamino-substituted derivatives of 4-[(3-bromophenyl)amino]-6-(methylamino)-pyrido[3,4-d]pyrimidine (PD 158780), a potent and specific inhibitor of the tyrosine kinase activity of receptors for the EGF family of growth factors.

The 4-[(3-bromophenyl)amino]pyrido[3,4-d]pyrimidine PD 158780 is a very potent in vitro inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) (IC50 0.08 nM), and other members of the erbB family, by competitive binding at the ATP site of these signal transduction enzymes. A series of analogues of PD 158780 bearing solubilizing functions off the 6-methylamino substituent were prepared by reaction of the 6-fluoro derivatives with appropriate amine nucleophiles. These were evaluated for their ability to inhibit the tyrosine phosphorylating action of EGF-stimulated full-length EGFR enzyme and for inhibition of autophosphorylation of the EGFR in A431 human epidermoid carcinoma cells in culture. The most effective analogues were those bearing weakly basic substituents through a secondary amine linkage, which proved water-soluble (> 10 mM) and potent (IC50S generally < 1 nM). No clear SAR could be discerned for these compounds with respect to amine base strength or the distance of the cationic center from the chromophore, suggesting that 6-substituents are in a favorable area of bulk tolerance in the enzyme binding site. More distinct SAR emerged for the ability of the compounds to inhibit EGFR autophosphorylation in A431 cells, where analogues bearing lipophilic weak bases were preferred. Representative analogues were evaluated for antitumor effectiveness against four in vivo tumor models. Significant in vivo activity was observed in estrogen-dependent MCF-7 breast and A431 epidermoid tumors. Marginal activity was seen in an EGFR-transfected tumor model, suggesting that while this cell line requires EGF for clone formation in soft agar, other growth factors may be able to replace EGF in vivo. Also, no activity was seen against the SK-OV-3 ovarian cancer model, which is known to express other EGF receptor family members (although it is not clear whether these are absolutely required for growth in vivo). While substantial growth delays were seen in A431 and MCF-7 tumor models, the treated tumors remained approximately the same size throughout therapy, suggesting that the compounds are cytostatic rather than cytotoxic under these test conditions. It remains to be determined if more prolonged therapy has cytotoxic effects in vivo, resulting in net tumor cell kill.

Purification and Characterization of a Recombinant Human Cripto-1 Protein

Cripto-1 (CR-1) is a novel protein that contains a modified EGF-like motif and that does not directly bind to any of the known erb B type-1 receptor tyrosine kinase receptors. To more clearly define the biological effects of CR-1 and to more adequately compare the structure-function relationships of CR-1 with other members of the EGF family of growth factors, we have expressed a modified, full-length recombinant human CR-1 protein (rhCR-1) in E. coli and have devised a procedure for the solubilization, refolding and purification of a biologically active form of this protein. We have generated the mature form of hCR-1 from computer assisted predictions of potential signal peptide cleavage sites. Expression of the modified rhCR-1 protein in E. coli was limited to the inclusion bodies. The rhCR-1 protein was found to be expressed at high levels in bacterial cells when fused to a histidine-tag sequence. Refolding of rhCR-1 was found to be difficult because of the large number of cysteine residues in the protein which results in protein aggregation. By chemically modifying the cysteine residues in the rhCR-1 protein with 3-trimethyl-ammoniopropyl methanethiosulfonate, additional positive charges have been introduced into the protein by this disulfiding reagent. This modification facilitates solubilization of the protein when rhCR-1 is denatured. The solubilized, denatured protein was then purified by CM cation exchange and C4 reverse phase HPLC chromatography and refolded in a redox buffer. The refolded, modified rhCR-1 protein was found to be biologically active by its ability to inhibit β-casein expression, to stimulate the tyrosine phosphorylation of She and the activation of MAPK and by its capacity to facilitate branching growth of mouse mammary epithelial cells in type I collagen gels.

Differential expression of the full-length and truncated forms of the epidermal growth factor receptor in the preimplantation mouse uterus and blastocyst.

The present investigation examined the differential expression of the full-length (fl) and the truncated (tr) forms of the epidermal growth factor receptor (EGFr) in the preimplantation mouse uterus and blastocyst. Northern blot hybridization using a complementary RNA probe specific to the full-length form (EGFr-fl) detected a 6.5-kb transcript, whereas that of the truncated form (EGFr-tr) detected a 2.7-kb transcript in the preimplantation mouse uterus on days 1 and 4 of pregnancy (day 1 = vaginal plug). In situ hybridization using these probes detected the EGFr-fl transcripts only in the stroma and myometrium, but not in the epithelium, whereas EGFr-tr transcripts were detected in all major uterine cell-types. To confirm the results of in situ hybridization, RT-PCR was performed on RNA isolated from separated uterine cell-types on day 4 of pregnancy using sequence specific primers for the two forms of the receptor. The results concur that the EGFr-tr transcript is expressed in the epithelium, stroma and myometrium, whereas that of the EGFr-fl transcript is not expressed in the epithelium. In the preimplantation blastocyst, RT-PCR detected the EGFr-fl messenger RNA, but not the EGFr-tr messenger RNA. These results suggest that the blastocyst, not the uterine epithelium, is the target for EGF family of growth factors in embryo-uterine interaction during implantation.

Membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) and diphtheria toxin receptor-associated protein (DRAP27)/CD9 form a complex with integrin alpha 3 beta 1 at cell-cell contact sites.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family of growth factors, which interact with EGF receptor to exert mitogenic activity. The membrane-anchored form of HB-EGF, proHB-EGF, is biologically active, providing mitogenic stimulation to neighboring cells in a juxtacrine mode. ProHB-EGF forms a complex with diphtheria toxin receptor-associated protein (DRAP27)/CD9, a tetra membrane-spanning protein that upregulates the juxtacrine mitogenic activity of proHB-EGF. We explored whether other proteins associate with DRAP27/CD9 and proHB-EGF. Immunoprecipitation with anti-DRAP27/CD9 resulted in preferential coprecipitation of integrin alpha 3 beta 1 from Vero cell, A431 cell and MG63 cell lysates. Anti-integrin alpha 3 or anti-integrin beta 1 coprecipitated DRAP27/CD9 from the same cell lysates. Chemical cross-linking confirmed the physical association of DRAP27/CD9 and integrin alpha 3 beta 1. Using Vero-H cells, which overexpress HB-EGF, we also demonstrated the association of proHB-EGF with DRAP27/CD9 and integrin alpha 3 beta 1. Moreover, colocalization of proHB-EGF, DRAP27/CD9, and integrin alpha 3 beta 1 at cell-cell contact sites was observed by double-immunofluorescence staining. At cell-cell contact sites, DRAP27/CD9 was highly coincident with alpha-catenin and vinculin, suggesting that DRAP27/CD9, proHB-EGF, and integrin alpha 3 beta 1 are colocalized with adherence junction-locating proteins. These results indicate that direct interaction of growth factors and cell adhesion molecules may control cell proliferation during the cell-cell adhesion process.

Heparin-binding EGF-like growth factor gene is induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: a possible ligand for interaction with blastocyst EGF-receptor in implantation

Heparin-binding EGF-like growth factor (HB-EGF) is a newly discovered member of the EGF family of growth factors. HB-EGF can bind to two loci on cell surfaces, heparan sulphate proteoglycans and EGF-receptor (EGF-R), and either one or both of these interactions could play a role in cell-cell interactions. In the rodent, increased endometrial vascular permeability at the site of blastocyst apposition is considered to be an earliest discernible prerequisite event in the process of implantation and this event coincides with the initial attachment reaction between the blastocyst trophectoderm and uterine luminal epithelium. This investigation demonstrates that the HB-EGF gene is expressed in the mouse uterine luminal epithelium surrounding the blastocyst 6-7 hours before the attachment reaction that occurs at 2200-2300 hours on day 4 of pregnancy. It was further demonstrated that this gene is not expressed in the luminal epithelium at the site of the blastocyst apposition during the progesterone-maintained delayed implantation, but is readily induced in the luminal epithelium surrounding an activated blastocyst after termination of the delay by an estrogen injection. In vitro studies showed that HB-EGF induced blastocyst EGF-R autophosphorylation, and promoted blastocyst growth, zona-hatching and trophoblast outgrowth. These results suggest possible interactions between the uterine HB-EGF and blastocyst EGF-R very early in the process of implantation, earlier than any other embryo-uterine interactions defined to date at the molecular level.

Endogenous EGF-family growth factors are necessary for transition from G1 to S phase in human keratinocytes

Endogenous EGF-family growth factors are necessary for transition from G1 to S phase in human keratinocytes

Metabolism and Effects of Epidermal Growth Factor and Related Growth Factors in Mammals*

The initial observations of Stanley Cohen in the 1960s established that EGF induced in vivo effects such as precocious eyelid opening and tooth eruption. Subsequently the actions of EGF have been extensively explored in cell culture systems. The receptor for EGF was characterized as a prototype model for other growth factors and the now extensive in vitro data indicate multiple functions for EGF. Moreover, EGF and EGF receptors have been characterized in many tissues, and EGF has been identified in most body fluids of several mammalian species. Interestingly, neither EGF antibody administration to newborn animals nor passive immunization of pregnant rodents against EGF has caused major deleterious effects (except the delay in epidermal maturation events), as might be expected from the in vitro studies. This is in contrast to the effects of nerve growth factor antiserum in developing rodents. Also, to date, no pathological EGF deficiency disorder has been characterized. However, the EGF family of growth factors appears to be important in mammalian development and function, although the precise roles and significance are not yet clear. Members of the family may have a role in embryogenesis and fetal growth since receptors have been identified in fetal tissues. Available evidence suggests that TGF alpha subserves the growth factor family roles in fetal development. In the developing postnatal animal pro-EGF mRNA, immunoreactive EGF, immunoreactive TGF alpha, and EGF receptors are present in many tissues. EGF also is produced and secreted by the maternal mammary gland, and mammary derived EGF appears to be important in gut development in the neonatal rodent. There is now extensive data to indicate important hormone-EGF interactions. In the postnatal period, thyroid and steroid hormones including retinoic acid have been shown to modulate EGF and/or EGF receptors in several tissues. GH increases EGF binding in liver and increases urine EGF concentrations. Moreover, EGF stimulates secretion of several hypothalamic and pituitary hormones, increases placental production of hCG and human chorionic somatomammotropin, increases adrenal cortisol production, and inhibits testicular, ovarian, and thyroid hormone secretions. As summarized in this review EGF has been implicated in a number of developmental events including palate and skin differentiation, growth of hair follicles, eye opening and tooth eruption, lung maturation, gut and liver growth, and differentiation of neurons. These EGF actions probably are mediated via autocrine, paracrine, and endocrine routes. A role for salivary and urine EGF in the maintenance of adult stomal, gut, and urinary epithelial surface integrity seems likely, although not yet proven.(ABSTRACT TRUNCATED AT 400 WORDS)