Peptide growth factor cross-talk with the estrogen receptor requires the A/B domain and occurs independently of protein kinase C or estradiol.

Modulation of steroid receptor-dependent transcription by extra- cellular ligands represents a novel mechanism of steroid receptor regulation. We have assessed the effects of epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and insulin-like growth factor I (IGF-I) on transcription from consensus estrogen response elements (ERE) in estrogen receptor (ER)-positive BG-1 human ovarian adenocarcinoma calls. EGF, TGF alpha, IGF-I, and estradiol (E2) enhanced transcription in a dose-dependent manner using either a strong or a minimal promoter, and ICI 164,384, a specific ER antagonist, inhibited these responses. Combinations of E2 with TGF alpha or IGF-I induced synergistic activation of transcription from an ERE, whereas as additive response was observed with combinations of IGF-I and TGF alpha of EGF. Tetradecanoyl 12-phorbol 13-acetate (TPA), a protein kinase C (PKC) activator, stimulated ERE-mediated transcription, and this effect was inhibited by ICI 164,384. Bisindolylmaleimide, a relatively specific inhibitor of PKC, completely antagonized TPA-induced transcription, but did not affect the response to TGF alpha, IGF-I, or E2. The combination of TPA with E2 in transcriptional synergism was inhibited by ICI 164,384; conversely, the combination of TPA with either TGF alpha of IGF-I elicited a response only equal to the maximal TPA response. Thus, peptide growth factors elicit ER-dependent transcription independently of PFC; however, there may be a common mechanistic component, as saturation of response was observed. Finally, activation of ERE-dependent transcription in Chinese hamster ovary cells by IGF-I was observed in the presence of a mutant receptor that lacks estrogen-binding activity. The effect of both IGF-I and E2 were dependent on the ability of the ER to bind to DNA. IGF-I elicited only weak transcriptional activation in the presence of a deletion mutant that lacked the entire A/B domain; however, synergism between IGF-I and E2 was observed with this mutant. Therefore, ligand-independent activation of ER-dependent transcription by IGF-I is predominantly mediated through activation function I by a mechanism distinct from that of E2.

Cross talk between peptide growth factor and estrogen receptor signaling systems.

Epidermal growth factor reproduces many of the effects of estrogen on the murine female reproductive tract and may partially mediate estrogen-induced growth and differentiation. The mechanism by which the actions of estrogens and epidermal growth factor (EGF) converge is unknown. The studies described herein were performed to investigate the possibility that some of the actions of EGF may be mediated through the estrogen receptor. A specific estrogen receptor (ER) antagonist inhibited estrogenlike effects of EGF in the mouse uterus, specifically induction of DNA synthesis and phosphatidylinositol turnover. In addition, EGF elicited enhanced nuclear localization of uterine ER and formation of a unique nuclear form of ER that is present after estrogen treatment. These in vivo observations indicated that EGF may elicit some of its actions by activation of nuclear ER. Thus, the effect of peptide growth factors on activation of a consensus estrogen response element was assessed in Ishikawa human endometrial adenocarcinoma cells, which contain negligible ER levels, and in BG-1 human ovarian adenocarcinoma cells, which contain abundant ER. EGF and TGF alpha induced transcriptional activation of a consensus estrogen response element (ERE) in an ER-dependent manner in both cell types. In addition, insulinlike growth factor I (IGF-I) was as potent as 17 beta-estradiol in BG-1 cells. Synergism between growth factors and estrogen was observed in both cell types, although synergism was not observed between the different classes of growth factors [i.e., transforming growth factor alpha (TGF alpha) and IGF-I] in BG-1 cells. The most potent activator of ERE-dependent transcription was a protein kinase C activator (TPA), which acted synergistically with 17 beta-estradiol. A protein kinase C inhibitor abolished the effect of TPA but not that of 17 beta-estradiol, IGF-I, or TGF alpha. A protein kinase A activator elicited ER-dependent activation of transcription and did not synergize with estrogen or growth factors. In conclusion, some physiologic actions of peptide growth factors are dependent on ER. Indeed, growth factors are capable of eliciting ER-dependent activation of an ERE. Both the protein kinase A and protein kinase C pathways can elicit ER-dependent transcriptional activation; however, it is unlikely that these pathways mediate the effects of peptide growth factors on the ER in BG-1 cells.

Peptide growth factors elicit estrogen receptor-dependent transcriptional activation of an estrogen-responsive element.

Epidermal growth factor (EGF) elicits estrogen receptor (ER)-dependent physiological sequelae and estrogen-like biochemical effects on the ER in the mouse uterus. These in vivo observations indicate that EGF may elicit some of its actions by activation of the ER. The effect of peptide growth factors on activation of a consensus estrogen-responsive element was assessed in a strain of Ishikawa human endometrial adenocarcinoma cells with negligible levels of ERs, as determined by Western blot and [3H]estradiol binding, and in BG-1 human ovarian adenocarcinoma cells, which contain abundant ERs. EGF and transforming growth factor-alpha induced transcriptional activation of a consensus ERE in an ER-dependent manner in both cell types. Transcriptional activation by the growth factors was inhibited by ICI 164,384, an ER receptor antagonist, and neutralizing antibodies to the EGF receptor. Immunodetection of the ER in BG-1 cells demonstrated that receptor levels were not induced by transforming growth factor-alpha vs. untreated cells. ER deletion mutants containing amino acids 1-339 and 121-599 were transfected into Ishikawa cells. The 1-339 mutant was more active in inducing transcription after EGF treatment than the 121-599 mutant. Estrogen only stimulated transcription in the presence of the 121-599 mutant, while 1-339 was inactive. Interestingly, synergism between a physiological dose of estrogen and peptide growth factors was observed. The presence of cross-talk between EGF receptor and ER signaling pathways suggests that interactions between growth factors and steroid receptors may modulate hormonal activity influencing normal and aberrant function in mammalian cells.

Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor.

Epidermal growth factor (EGF) reproduces many of the effects of estrogen on the murine female reproductive tract and may partially mediate estrogen-induced growth and differentiation. This study was performed to investigate the mechanism by which EGF elicits estrogen-like actions in the whole animal. EGF was administered to adult ovariectomized mice by slow release pellets implanted under the kidney capsule. The induction of uterine DNA synthesis and phosphatidylinositol lipid turnover by EGF or administration of diethylstilbestrol (5 micrograms/kg), a potent estrogen, was attenuated by the estrogen receptor antagonist ICI 164,384. Furthermore, EGF mimicked the effects of estrogen on enhanced nuclear localization of the estrogen receptor and the formation of a unique form of the estrogen receptor found exclusively in the nucleus. These results suggest that EGF may induce effects similar to those of estrogen in the mouse uterus by an interaction between the EGF signaling pathway and the classical estrogen receptor. The demonstration of cross-talk between polypeptide growth factors and steroid hormone receptors may be of importance to our understanding of the regulation of normal growth and differentiation as well as the mechanisms of transmission of extracellular mitogen signals to the nucleus.

Diethylstilbestrol stimulates persistent phosphatidylinositol lipid turnover by an estrogen receptor-mediated mechanism in immature mouse uterus.

The effect of estrogen on phosphoinositide (PI) metabolism was evaluated in the immature mouse uterus, a tissue which undergoes estrogen-induced proliferation. Uteri isolated from untreated mice or from mice injected ip with diethylstilbestrol (DES) were incubated with [3H]myo-inositol and assessed for incorporation of label into PI lipids or inositol phosphate generation. DES administration elicited a rapid increase in [3H]myo-inositol incorporation, which persisted until at least 18 h post treatment. This effect could not be duplicated by incubation of uteri with DES in vitro, although [3H]myo-inositol incorporation in uteri removed from DES-treated mice remained elevated for 3 h of in vitro incubation. Stimulation of PI lipid metabolism by DES was blocked by ICI 164,384, a specific estrogen receptor antagonist. The effect of DES on PI metabolism consisted of a time-dependent increase in the specific activity of both phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate and a significant increase of inositol (1,4,5)-trisphosphate mass by 12 h post treatment. These changes occur before the onset of estrogen-induced DNA synthesis. The results indicate that estrogens rapidly modulate PI lipid turnover through an estrogen receptor-mediated mechanism. Since the metabolic products of PI lipids are important for signal transduction and cellular proliferation, altered metabolism of these lipids may play an integral role in estrogen-induced mitogenesis.

Localization of the estrogen receptor in uterine cells by affinity labeling with [3H]tamoxifen aziridine.

The possibility that estrogen receptors may exist in uterine plasma membranes was investigated by covalent labeling of estrogen receptors in mouse uterine cells with [3H]tamoxifen aziridine (TA). Isolated epithelial and stromal cells of immature mice were incubated with [3H]TA in the presence or absence of unlabeled tamoxifen, homogenized and separated into nuclear, cytosolic and microsomal fractions by differential centrifugation. These fractions were subjected to SDS-polyacrylamide gel electrophoresis and the proteins labeled covalently with TA were visualized by autoradiography. Proteins labeled specifically with [3H]TA were observed almost exclusively in the nuclear fraction of both epithelial and stromal cells. In contrast, very little labeled protein was detected in the cytosolic or microsomal fraction. Although these data do not preclude the possibility that estrogen binding sites are present in plasma membranes of uterine cells, this cellular fraction is definitely not labeled to a significant extent by [3H]TA. Thus, if membrane estrogen binding sites exist, their structural conformations may be different from that of nuclear estrogen receptors.