EGF receptor activities in mammalian development

The receptor for epidermal growth factor (EGF) and its analog transforming growth factor alpha (TGF alpha) is ubiquitous, implying quite general roles for EGF/TGF alpha in cell viability and tissue maintenance in adult tissues. There is also evidence that the EGF receptor is active in promoting wound healing and tissue regeneration in adult organs, such as skin, liver, and intestinal epithelium. It is likely that EGF receptors have more specific roles during the gestation period. For example, we have detected EGF receptors on the 3.5-day blastocyst (trophectoderm) surface and since TGF alpha-like mRNA sequences and peptides have been detected at this time (Rappolee et al., Science 241:1823, 1988), there is a strong implication for autocrine stimulation in pre- and peri-implantation stage embryos. Paracrine stimulation between the embryo and maternal tissues is also likely since both receptors and TGF alpha are present in decidual cells. Therefore EGF receptors may take part in growth regulation of the early embryo and in the process of implantation. Other examples where EGF receptors may play specific roles during embryonic development are discussed.     

Expression of progesterone and oestrogen receptors by early intrauterine equine conceptuses

Progesterone and oestrogen play essential roles in the maintenance of pregnancy in eutherian mammals and are thought to exert their effects on the developing conceptus indirectly, via the endometrium. In some species, early embryos have themselves been shown to express steroid receptors, thereby suggesting that reproductive steroids may also influence embryonic development directly. The aim of this study was to determine whether early intrauterine equine conceptuses express either the classical intracellular progesterone (PR) and oestrogen receptors (ERalpha and ERbeta) or the more recently characterised membrane-bound progesterone receptors (PGRMC1 and mPR). Horse conceptuses recovered on days 7, 10 and 14 after ovulation (n=8 at each stage) were examined for steroid receptor mRNA expression using quantitative rtPCR. Where commercial antibodies were available (PR, ERbeta), receptor localisation was examined immunohistochemically in day 10, 12, 14, 15 and 16 conceptuses (n=2 at each stage). mRNA for PR, PGRMC1 and mPR was detected at all stages examined, but while PGRMC1 and mPR expression increased during the day 7-14 period, PR expression decreased. ERalpha mRNA was not detected at any stage examined, whereas ERbeta mRNA was detected in all day 14, some day 10 and no day 7 conceptuses. Immunoreactive ERbeta receptors were localised to the trophectoderm of day 14-16 conceptuses; PR were not detected immunohistochemically in conceptus tissue. In summary, this study demonstrates that equine conceptuses express mRNA and, in the case of ERbeta, protein for steroid hormone receptors during the period encompassing rapid conceptus growth, differentiation and maternal pregnancy recognition.     

Characterization of the epidermal growth factor receptor in preimplantation pig conceptuses.

Embryos recovered from sows on Days 9-13 of pregnancy (Day 0 = first day of estrus) exhibited saturable and time-dependent specific binding of 125I-epidermal growth factor (EGF). The specific binding (pg/mg protein) was greater (P less than 0.001) for Day 13 elongated conceptuses than for conceptuses of earlier stages. Scatchard analyses showed two classes of binding sites (Kd = 7.0 +/- 2.6 x 10(-11) M, Bmax = 6.2 +/- 1.4 fmol/mg protein and Kd = 3.4 +/- 0.2 x 10(-8) M, Bmax = 420 +/- 80 fmol/mg protein). The EGF receptor in Day 13 conceptus membranes is a 170-kDa protein and was phosphorylated in the presence of EGF and adenosine triphosphate. EGF stimulated protein tyrosine kinase activity about 1.6-fold over basal levels. The results show that the preimplantation pig conceptus possesses EGF-binding sites with the properties of functional EGF-receptors.     

The retinol-binding protein of the expanding pig blastocyst: molecular cloning and expression in trophectoderm and embryonic disc.

Retinol-binding protein (RBP) is a major secretory product of the porcine conceptus. Using an oligonucleotide probe corresponding to a highly conserved region of all known mammalian RBP, we have isolated an apparently full-length cDNA clone for porcine conceptus RBP from a cDNA library constructed from pig conceptuses collected between days 13-17 of pregnancy. The cDNA was 937 base-pairs in length and coded for a protein whose inferred amino-terminal sequence was identical to that reported for both porcine conceptus RBP and porcine serum RBP. Its length was consistent with the size (approximately 1 kilobase) of the RBP message in porcine conceptuses. Porcine conceptus RBP and human serum RBP share 91% amino acid sequence identity. The inferred differences in sequence were evenly distributed throughout the length of the polypeptide. RBP mRNA was detectable within the trophoblast of day 11 porcine conceptuses by in situ hybridization with a 618-basepair 35S-labeled probe corresponding to the 3' end of porcine RBP. Silver grain density was distributed relatively uniformly over the trophoblast and the inner cell mass. Western blot analysis of conceptus culture medium demonstrated that the conceptuses of cattle (on day 19) and sheep (on day 15) as well as pigs secrete RBP during early pregnancy. Secretion of large quantities of RBP by the trophoblast of preimplantation pig conceptuses suggests important roles for vitamin A and RBP near the time of conceptus elongation.     

Transforming growth factor alpha production and epidermal growth factor receptor expression in normal and oncogene transformed human mammary epithelial cells

We have characterized the expression of transforming growth factor alpha (TGF alpha) and its receptor, the epidermal growth factor receptor (EGF-R), in normal and malignantly transformed human mammary epithelial cells. Human mammary epithelial cells were derived from a reduction mammoplasty (184), immortalized by benzo-a-pyrene (184A 1N4), and further transformed by the oncogenes simian virus 40 T (SV40 T), v-Ha-ras, and v-mos alone or in combination using retroviral vectors. 184 and 184A 1N4 cells require EGF for anchorage-dependent clonal growth. In mass culture, they secrete TGF alpha at high concentrations and exhibit an attenuated requirement for exogenous EGF/TGF alpha. SV40 T transformed cells have 4-fold increased EGF-R, have acquired the ability to clone in soft agar with EGF/TGF alpha supplementation, but are not tumorigenic. Cells transformed by v-mos or v-Ha-ras are weakly tumorigenic and capable of both anchorage dependent and independent growth in the absence of EGF/TGF alpha. Cells transformed by both SV40 T and v-Ha-ras are highly tumorigenic, are refractory to EGF/TGF alpha, and clone with high efficiency in soft agar. The expression of v-Ha-ras is associated with a loss of the high (but not low) affinity binding component of the EGF-R. Malignant transformation and loss of TGF alpha/EGF responsiveness did not correlate with an increase in TGF alpha production. Thus, TGF alpha production does not appear to be a tumor specific marker for human mammary epithelial cells. Differential growth responses to EGF/TGF alpha, rather than enhanced production of TGF alpha, may determine the transition from normal to malignant human breast epithelium.     

Transforming growth factor alpha.

Transforming growth factor alpha (TGFα) is a close relative of epidermal growth factor (EGF), the first polypeptide mitogen discovered in 1962 (Cohen, 1962). TGFα, like EGF, exerts its effect on cells through binding to the EGF-Receptor (EGF-R). Here we review the molecular and cell biology of TGFα before proceeding to describe our own work on signaling molecules induced in response to activation of the EGF-R.     

Transforming growth factor-beta (TGF beta) inhibits TGF alpha expression in bovine anterior pituitary-derived cells.

Transforming growth factor-beta 1 (TGF beta 1) is a multifunctional regulator of cell growth and differentiation. We report here that TGF beta 1 decreased the proliferation of nontransformed bovine anterior pituitary-derived cells grown in culture. We have previously demonstrated that these cells express both TGF alpha and its receptor [the epidermal growth factor (EGF) receptor] and that expression can be stimulated by phorbol ester (TPA) and EGF. TGF beta 1 treatment over a 2-day period decreased the proliferation of pituitary cells. This decreased growth rate was accompanied by a decrease in the TGF alpha mRNA level. The effect of TGF beta 1 on TGF alpha mRNA down-regulation was both dose dependent (maximal effect observed at 1.0 ng/ml TGF beta 1) and time dependent (minimum of 2-day treatment with TGF beta 1 was required before a decrease in TGF alpha mRNA was observed). Studies on TGF alpha mRNA stability indicated that TGF beta 1 did not alter the TGF alpha mRNA half-life. Treatment of the TGF beta 1 down-regulated cells with EGF resulted in the stimulation of TGF alpha mRNA levels; thus, the TGF beta 1-treated cells remained responsive to EGF. The decreased proliferation in response to TGF beta 1 could be only partially reversed by simultaneous treatment of the cells with EGF (10(-9)M) and TGF beta 1 (3.0 ng/ml). Qualitatively, the TGF beta 1-induced reduction of TGF alpha mRNA content was independent of cell density. TGF beta 1 treatment of the anterior pituitary-derived cells also reduced the levels of c-myc and EGF receptor mRNA. These results represent the first demonstration of the down-regulation of TGF alpha synthesis by a polypeptide growth factor and suggest that TGF beta 1 may be a physiological regulator of TGF alpha production in vivo.     

Immunocytochemical localization of BP, a conceptus secretory protein, in trophectoderm during pig trophoblast elongation

Tissue sections of periimplantation pig conceptuses (days 9-15 of pregnancy) were incubated with antiserum to the basic protein (BP), a major secretory protein of filamentous pig blastocysts. Bound antibody was detected by the peroxidase-antiperoxidase method. BP was restricted to trophectoderm in conceptuses which had made the transition from a spherical to an ovoid shape having a diameter of greater than 5 mm (day 11). Tubular (day 11, 10-20 mm) and filamentous (day 11-15) conceptus trophectoderm contained BP. These results suggest that BP synthesis commences at the time of rapid trophoblast growth.     

Epidermal growth factor modulation of prostaglandins and nitrite biosynthesis in rat fetal membranes

The production of prostaglandins (PGs) and nitric oxide (NO) by amnion tissue may play a significant role in parturition. It is thought that epidermal growth factor (EGF) may be one of the fetal signals that governs the initiation of labor. The aim of the present study was to investigate the effect of EGF in vivo on the PGs and nitrite production of rat fetal membranes. We have evaluated the regulation of PGs and nitrite production in rat fetal membranes ex vivo. The intra-uterine administration of EGF 500 ng in day 21 of pregnancy induced increases in PGE(2) (P<0.001) and PGF(2alpha) (P<0.01) compared to the control fetal membranes from pregnant rats on day 22. Also, this dose of EGF diminished nitrate production significantly (P<0.01). We found that fetal membranes at term (days 18-22 of gestation) expressed EGF-R. The NO donor, nitroprussiate 300 and 600 microM, elicited an inhibitory effect on the PGE(2) and PGF(2alpha) stimulated synthesis. On the other hand, indomethacin 10(-6) and 10(-7)M, a non-selective cyclooxygenase inhibitor, reverted the inhibitory effect exerted by EGF. Hence, rat fetal membranes were found to express epidermal growth factor receptors and, under the effect of EGF, PGs and nitrites production pathways interact probably to prevent a toxic effect caused by an exacerbated synthesis of these mediators.     

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.     

Epidermal growth factor (EGF) in the goat uterus: immunohistochemical localization of EGF and EGF receptor and effect of EGF on uterine activity in vivo

This study examined the distribution of immunoreactive epidermal growth factor (EGF) and EGF receptor (EGF-R) in the uterus and the effects of EGF on uterine activity in goats. Immunohistochemistry of EGF and EGF-R in the uteri showed distinct staining in the luminal and glandular epithelium and slight to moderate staining in the stromal and myometrial cells. To examine possible roles of the EGF system in the regulation of uterine activity, pressure changes in the intrauterine balloon were determined after intraluminal infusion of EGF into the uterine horn. Either at estrus or diestrus (9 to 14 days after the first day of estrus), treatment with 1 or 5 microg of EGF gradually reduced uterine activity, whereas infusion of the vehicle alone had no effect. The maximum reduction in uterine activity was seen 4 h after the treatment with 1 microg of EGF (40% to 45% reduction in the area surrounded by the contraction curve and its baseline), and the activity slowly returned thereafter. These results suggest that EGF in the uterus may play a role in regulating uterine activity in goats.     

EGF receptor regulation in normal mouse mammary gland.

Estrogen (E), progesterone (P), and epidermal growth factor (EGF) are known to regulate growth and development of the normal mammary gland, and it is possible that EGF may interact with E and/or P. Estrogen (ER), progesterone (PR), and EGF receptors (EGF-R) have been detected in both mammary epithelial and stromal cells, and the relative roles of the various cells types in hormone-dependent growth regulation are not known. The present studies were undertaken to determine if E and/or P influence EGF action by exerting a regulatory effect on EGF-R levels and which cell types are affected. The comparative effects of ovariectomy and hormone treatments on EGF-R levels were examined in immature, pubertal 5-week-old and sexually mature 10-week-old female mice. EGF-R were characterized as a single class of high affinity sites and EGF-R concentration was 2-fold higher in glands of 5-week-old mice. Ovariectomy had no significant effect on EGF-R concentration in either age group, and treatment with E and/or P had no effect on EGF-R levels in either epithelial or stromal cells in 5-week-old mice. In contrast, E+P treatment caused a 2-fold increase in receptor concentration in 10-week-old mice in the mammary epithelium. Thus it appears that the developmental state of the gland may determine the nature and extent of the interaction of of EGF, E, and P.     

Abelson transformed fibroblasts lacking the EGF receptor are not tumourigenic in nude mice.

Cells transformed by the v-abl-oncogene produce large amounts of the tumour growth factor alpha TGF. alpha TGF is homologous to the epidermal growth factor (EGF) and stimulates cell growth via the EGF receptor pathway. To separate metabolic events in the v-abl-transformed cells mediated by alpha TGF as opposed to the v-abl-encoded protein-tyrosine kinase, we have employed the Swiss 3T3 variant cell line NR6 which lacks a functional EGF receptor. v-abl was found to transform efficiently NR6 cells in vitro. These transformed NR6 cells displayed a variety of in vitro properties which were indistinguishable from transformed wild-type fibroblast lines. However, in contrast to the wild-type lines, v-abl-transformed NR6 cells failed to form tumours when injected into athymic nude mice. These results imply an important function for alpha TGF and the EGF receptor in the establishment of the v-abl-induced fibrosarcomas.     

Prolactin inhibits epidermal growth factor (EGF)-stimulated signaling events in mouse mammary epithelial cells by altering EGF receptor function.

We have previously shown that lactogenic hormones stimulate epidermal growth factor (EGF) mRNA accumulation in mouse mammary glands in vivo and in mouse mammary epithelial cells (NMuMG line). However, our in vitro studies indicate that the lactogenic hormone prolactin (PRL) completely inhibits EGF-stimulated DNA synthesis. PRL does not alter cholera toxin or insulin-like growth factor-1-stimulated cell growth, thus the inhibition appears to be specific for EGF. Our current studies are designed to evaluate the effects of PRL on EGF-stimulated signaling events in the NMuMG cell line. Cells treated with PRL for 30 min demonstrated a loss of high affinity EGF-binding ability. After long-term PRL treatment (18 h) there was a decrease in EGF receptor (R) number, as determined by [125I]EGF binding. PRL treatment (8 h) also decreased EGF-R mRNA levels. An EGF-stimulated increase in EGF-R mRNA observed 2-4 h after treatment was decreased when PRL was added to the cultures. Furthermore, levels of EGF-stimulated tyrosine phosphorylation of the EGF-R (170 kDa) and phospholipase C gamma (145 kDa) are dramatically decreased in cells treated with PRL. Also of great interest was a decrease in EGF-stimulated c-myc mRNA in PRL-treated cells. We conclude that PRL is acting to down-regulate the EGF-R, thus limiting EGF-stimulated cell signaling in mammary tissue.     

EGF and amphiregulin differentially regulate Cbl recruitment to endosomes and EGF receptor fate

EGF-R [EGF (epidermal growth factor) receptor] ligands can promote or inhibit cell growth. The biological outcome of receptor activation is dictated, at least in part, by ligand-specified patterns of endocytic trafficking. EGF-R trafficking downstream of the ligands EGF and TGF-alpha (transforming growth factor-alpha) has been investigated extensively. However, less is known about EGF-R fates induced by the ligands BTC (betacellulin) and AR (amphiregulin). We undertook comparative analyses to identify ligand-specific molecular events that regulate EGF-R trafficking and degradation. EGF (17 nM) and BTC (8.5 nM) induced significant EGF-R degradation, with or without ectopic expression of the ubiquitin ligase Cbl. Human recombinant AR (17 nM) failed to affect receptor degradation in either case. Notably, levels of ligand-induced EGF-R ubiquitination did not correlate strictly with receptor degradation. Dose-response experiments revealed that AR at a saturating concentration was a partial agonist at the EGF-R, with approx. 40% efficacy (relative to EGF) at inducing receptor tyrosine phosphorylation, ubiquitination and association with Cbl. EGF-R down-regulation and degradation also were compromised upon cell stimulation with AR (136 nM). These outcomes correlated with decreased degradation of the Cbl substrate and internalization inhibitor hSprouty2. Downstream of the hSprouty2 checkpoint in AR-stimulated cells, Cbl-free EGF-R was incorporated into endosomes from which Cbl-EGF-R complexes were excluded. Our results suggest that the AR-specific EGF-R fate results from decreased hSprouty2 degradation and reduced Cbl recruitment to underphosphorylated EGF-R, two effects that impair EGF-R trafficking to lysosomes.     

Porcine conceptuses secrete an interferon during the preattachment period of early pregnancy

Maternal recognition of pregnancy in sheep and cattle is thought to be initiated by the conceptus secretory proteins ovine trophoblast protein-1 (oTP-1) and bovine trophoblast protein-1 (bTP-1), respectively. Recently, these proteins have been shown to be members of the interferon-alpha (IFN-alpha) family. In this study, we have examined whether pig conceptuses also produce IFN during early pregnancy. conceptuses were collected at Days 8, 10, 11, 12, 13, 15, and 17 of pregnancy and cultured in serum-free medium for 24 h. Day 11 conceptuses secreted a dominant 22,000-24,000 Mr cluster of acidic proteins (pI 5.2-5.4), that appeared to cross-react on immunoblots with antiserum against human IFN-alpha but not against oTP-1. Antiviral activity characteristic of an IFN was present in conceptus culture medium and uterine flushings from Day 11 through Day 17 of pregnancy, but was absent in flushings prior to Day 11 of pregnancy and in flushings from Day 12 nonpregnant gilts. The antiviral activity coeluted with a 22,000-24,000 Mr protein during partial purification through a gel filtration column. The activity was extremely labile, but could be restored by sequential protein denaturation, reduction, and renaturation. We conclude that production of IFN by early conceptuses is not restricted to ruminant species, and may therefore represent a more general phenomenon.     

Anti-sense transforming growth factor alpha oligonucleotides inhibit autocrine stimulated proliferation of a colon carcinoma cell line.

Many carcinoma cells secrete transforming growth factor alpha (TGF alpha). A 23 base anti-sense oligonucleotide that recognizes the TGF alpha mRNA inhibits both DNA synthesis and the proliferation of the colon carcinoma cell line LIM 1215. The effects of the anti-sense TGF alpha oligonucleotide are reversed by epidermal growth factor (EGF) at 20 ng/ml. When the LIM 1215 cells are grown under serum free conditions, the anti-sense TGF alpha oligonucleotides have their greatest effects at high cell density (2 x 10(5) cells/cm2), indicating that the secreted TGF alpha is acting as an exogenous growth stimulus. In addition, at higher cell densities, the kinase activity of the EGF receptor is activated and the receptor is down-modulated. The cell density dependent activation of the EGF receptor is inhibited by the application of the antisense TGF alpha oligonucleotides.