Regulation of the uterine epidermal growth factor receptor by estrogen.
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Biomedical subjects
G M Stancel
Publications and source records attributed to G M Stancel.
Regulation of uterine epidermal growth factor (EGF) receptors by estrogen in the mature rat and during the estrous cycle.
Previous work has shown that the immature rat uterus contains epidermal growth factor (EGF) receptors and that tissue levels of this receptor are increased by the administration of exogenous estrogens. This study was undertaken to determine if estrogen administration also elevated EGF receptor levels in the mature animal and if the growth factor receptor levels varied in concert with endogenous estrogens throughout the estrous cycle. In the mature, castrate rat administration of estradiol, but not non-estrogenic steroids, causes a 2-3-fold elevation of uterine EGF receptors as judged by ligand binding. This increase is maximum in 18 h and is due to an increase in the number of binding sites. In cycling animals EGF receptor levels are low at metestrus, rise at diestrus, reach a maximum (approximately twice metestrus values) at proestrus, and then return at estrus to metestrus levels. These changes in EGF receptor levels parallel changes in plasma estrogens and occupied nuclear estrogen receptor reported by other workers. These results indicate that uterine EGF receptors are increased by exogenous estrogens in both mature and immature animals, and support a physiological role for estrogens in the regulation of this growth factor receptor.
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Blockade of epidermal growth factor-induced uterine contractions by indomethacin or nordihydroguaritic acid.
Epidermal growth factor (EGF) induces uterine contractions in an in vitro system. As an initial approach to elucidating the cellular mechanism for this action of EGF, we have characterized this effect pharmacologically in uteri from castrate, estrogenprimed mature rats. EGF-induced contractions are observed in both intact tissue and isolated myometrium, are dependent on extracellular calcium and are inhibited by nifedipine and chlorpromazine. Both indomethacin and nordihydroguaritic acid (NGA) inhibit EGF-induced uterine contractions. At the maximum doses which do not affect contractile responses to prostaglandin E2 or carbachol, indomethacin (10 microM) alone or NGA (1 microM) alone only decrease the effect of EGF by 60 to 80%, but indomethacin and NGA in combination abolish totally the response to the growth factor. Arachidonic acid also causes uterine contractions in this system. EGF increases uterine contractions in the presence of a submaximal (1 microM) but not a maximal (10 microM) dose of arachidonic acid. These findings suggest that EGF-induced contractions result from a mobilization of myometrial arachidonic acid, production of intermediates via both lipoxygenase and cyclooxygenase catalyzed pathways and a resultant influx of extracellular calcium.
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Autoradiographic localization of epidermal growth factor receptors to all major uterine cell types.
We have recently studied the structure and function of the uterine epidermal growth factor (EGF) receptor, its hormonal regulation, and its possible role in estrogen-induced uterine DNA synthesis. Since the uterus is composed of multiple cell types, we sought, in the work reported here, to localize EGF binding in this organ by autoradiography. Prior to the actual autoradiography, we performed a companion series of experiments to insure that EGF binding to uterine tissue in situ represented a true receptor interaction. Uteri from immature female rats were incubated in vitro with 125I-EGF at 25 degrees C. Tissue binding was maximal within 120 min and remained constant for at least an additional 120 min. This binding of labeled EGF was largely abolished by excess unlabeled EGF but not by other growth factors, indicating that binding was to specific receptors. The binding of 125I-EGF was saturable and reached a plateau at 4-8 nM; specific binding was half-maximal at 1-2 nM EGF. In situ cross-linking studies revealed that 125I-EGF was bound predominantly to a 170,000 MW EGF receptor similar to that seen in isolated uterine membranes. Incubation of uteri with 125I-EGF followed by autoradiography revealed binding to epithelial cells, stroma, and myometrium. These results provide evidence for the presence of specific EGF receptors in all major uterine cell types of the immature rat.
Animals↗
Regulation of deoxyribonucleic acid polymerase activity in uterine luminal epithelium after multiple doses of estrogen.
Estradiol (E2) exerts both inhibitory and stimulatory effects on DNA synthesis in the rat uterine luminal epithelium (LE). This inhibitory effect is due to a shift in the time course of DNA synthesis, i.e. in animals receiving a single injection of E2, a peak of DNA synthesis occurs 24 h after treatment, but in animals receiving multiple injections of E2, DNA synthesis is suppressed until 10-12 h after hormone treatment ceases. In these previous studies LE DNA synthesis was assessed by measuring tritiated thymidine incorporation. In the present study, we sought to determine if the molecular basis for this decrease in DNA synthesis was due to a suppression of DNA polymerase activity in LE nuclei. Animals receiving a single injection of E2 exhibit a peak of nuclear DNA polymerase activity 20-24 h later. Animals receiving multiple injections of E2 (0, 12, 15, and 18 h) show more than a 50% decrease in DNA polymerase activity at 20-24 h, due to a shift in the maximum increase in enzyme activity to 32-36 h after the initial treatment. The observed differences between these groups are not due to different levels of DNase activity or different degrees of leakage of the nuclear enzyme. The observed enzyme activity is due to DNA polymerase-alpha, since it requires ATP as well as deoxyribonucleoside triphosphates, and is aphidicolin sensitive. These results indicate that the inhibitory effect of E2 on LE DNA synthesis is due at least in part to a suppression of nuclear DNA polymerase-alpha activity.
Animals↗
Estrogen regulation of c-fos messenger ribonucleic acid.
Acute administration of 17 beta-estradiol to immature female rats elicits a rapid and striking increase in the size of the uterus. This increase in size to caused by both hypertrophy and hyperplasia in the epithelial, stromal, and myometrial cells in the uterus. Previous studies have shown that induction of mRNA for the epidermal growth factor receptor, the cellular homolog of the erb-B oncogene, occurs early during estrogen-stimulated uterine growth. We report here that estradiol causes a very rapid induction of the mRNA for the cellular oncogene c-fos in immature rat uterus. Steady state levels of c-fos mRNA reach a maximum 3 h after 17 beta-estradiol administration and slowly return to low basal levels in 15 h. Dexamethasone, progesterone, and 5 alpha-dihydrotestosterone had no effect on uterine c-fos mRNA expression. The induction of c-fos mRNA by estrogen was unaffected by the protein synthesis inhibitor puromycin but completely abolished by the RNA synthesis inhibitor actinomycin D.
Animals↗
Estrogen regulation of epidermal growth factor receptor messenger ribonucleic acid.
Previous studies have demonstrated that 17 beta-estradiol (E2) causes a 3-fold increase in epidermal growth factor (EGF) receptors in uterine membranes. We now report that the increase in uterine EGF receptor levels is due to an increase in the steady-state levels of EGF receptor mRNA. After a single E2 injection, EGF receptor mRNA levels, as determined by RNA blots, increase 3- to 4-fold between 1 and 3 h, remain elevated at 6 h, and decline between 12 and 18 h. The effect is specific for E2 since the nonestrogenic hormones progesterone, dexamethasone, 5 alpha-dihydrotestosterone, and the inactive stereoisomer of E2, 17 alpha-estradiol, are without effect. E2-Mediated increases in EGF receptor mRNA levels are blocked by actinomycin D but not by puromycin. Taken together, these results indicate that E2 regulates the level of EGF receptor by increasing the steady-state concentration of EGF receptor mRNA in vivo.
Animals↗
Interactions between estrogen and EGF in uterine growth and function.
The rat uterus contains specific, high-affinity EGF receptors which possess a tyrosine kinase activity. As demonstrated autoradiographically, these receptors are present in the epithelial, stromal and myometrial cells of the uterus. Estrogen treatment in vivo produces a 2-3-fold increase in EGF receptor levels in the immature rat, the immature mouse and the ovariectomized adult rat; furthermore, EGF receptor levels vary throughout the estrus cycle in concert with levels of occupied nuclear estrogen receptor. This estrogen-induced increase in EGF receptor is preceded by an increase in the level of EGF receptor mRNA as judged by Northern blot analysis. In general, there is a good correlation between estrogen-induced DNA synthesis and EGF receptor levels in the uterus, although in certain situations EGF receptor levels are elevated without a subsequent increase in DNA synthesis. These observations suggest that an increase in tissue EGF receptor levels is important in estrogen-induced uterine growth, but that this increase in receptor levels alone is not sufficient to stimulate DNA synthesis. In addition to its possible role in tissue growth, we have shown very recently that EGF causes contraction of myometrial smooth muscle in a completely in vitro organ bath system. The qualitative nature of this contractile response is distinct from that produced by other classical uterotonic agents. The physiological significance of this uterine response to EGF remains to be elucidated.
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Contractions of the isolated uterus stimulated by epidermal growth factor.
Epidermal growth factor (EGF) produces uterine contractions in tissues removed from immature and adult rats. EGF produces both an increase in resting tone and the development of rhythmic contractions, and this effect is abolished by treatment with antibodies against EGF. The ED50 for this response is observed at an EGF concentration of 3.5 nM. This in vitro effect of EGF requires treatment of the animals in vivo with estradiol for 24 h before removal of uterine tissue. This effect of estradiol does not appear to be due solely to an induction of tissue EGF receptors. These results demonstrate a new activity of EGF and suggest a previously unrecognized function for this peptide growth factor.
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Hormonal carcinogenesis: separation of estrogenicity from carcinogenicity.
Estrogens are known to induce tumors in several animal species. To understand the mechanism of hormonal carcinogenesis, estrogen-induced renal carcinoma in male Syrian hamsters was investigated using estradiol and 2-fluoroestradiol. The biological activities of the latter steroid were compared with those of the natural hormone, because of the reduced metabolic conversion of 2-fluoroestradiol to catechol estrogen metabolites. 2-Fluoroestradiol was administered to male Syrian hamsters at three times the dose (60 mg) of estradiol (20 mg, positive control) by s.c. implantation. After 7 months, 75% of the estradiol-treated hamsters had kidney tumors, while in animals exposed to 2-fluoroestradiol renal carcinoma could not be detected. The reduced tumor incidence by the fluorinated steroid is not due to a lack of estrogenic potency. In the test animals, pituitary LH concentrations matched those measured in estradiol-treated hamsters and the reduction in testes weights was comparable. Furthermore, in immature female rats, uterine wet weight increases illustrate that 2-fluoroestradiol is a potent estrogen. The observed increases in uterine weight were shown to be accompanied by increases in protein and DNA synthesis comparable to those observed in estradiol-treated animals. 2-Fluoroestradiol stimulated growth of H-301 cells in vivo. These cells are estrogen-dependent for growth and are derived from the primary hamster kidney tumor. The results indicate that hormonal activity and carcinogenicity of estrogens are separable properties.
Animals↗
Regulation of epidermal growth factor receptor by estrogen.
Administration of 17 beta-estradiol (E2) to immature female rats produces a 3-fold increase in 125I-epidermal growth factor (EGF) binding to uterine membranes with no change in the affinity of membrane receptors for EGF. E2 treatment also increases the EGF receptor visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after affinity labeling of uterine membranes and the EGF-stimulated receptor autophosphorylation activity. In addition, E2 administration stimulates EGF-dependent tyrosine kinase activity in an assay system using exogenous angiotensin II as substrate. Following hormone treatment, EGF receptor levels increase between 6 and 12 h, remain elevated at 18 h, and decline between 24 and 36 h. This stimulation of EGF receptor levels by E2 is specific, since the non-estrogenic hormones progesterone, dexamethasone, and dihydrotestosterone fail to elevate receptor levels. E2-stimulated increases in EGF receptor levels are also blocked by cycloheximide and actinomycin D, suggesting that the observed effect represents de novo synthesis of the EGF receptor and may be mediated by a transcriptional mechanism. These results demonstrate that estrogen can regulate acutely the levels of EGF receptor in vivo and raise the possibility that events coupled to this receptor may play a role in estrogen-stimulated growth.
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DDT supports the growth of an estrogen-responsive tumor.
MT2 cells, a clonal cell line of MTW9/PL cells originally obtained from a mammary adenocarcinoma, from estrogen-responsive tumors in Wistar-Furth rats. o,p'-DDT supports MT2 tumor growth at a rate similar to 17 beta-estradiol. This effect of o,p'-DDT is dose-dependent and specific, since the DDT congener p,p'-DDD, which does not bind to tumor estrogen receptors, does not support tumor growth. This is the first demonstration that DDT can support the growth of an estrogen-responsive tumor.
Adenocarcinoma↗
Estrogenic activity of DDT: estrogen-receptor profiles and the responses of individual uterine cell types following o,p'-DDT administration.
The administration of o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT) to immature rats stimulates DNA synthesis and cell division in the uterine luminal epithelium (LE), stroma (S), and myometrium (M). The time course of DNA synthesis/cell division in the S and M is similar following administration of o,p'-DDT or 17 beta-estradiol, but the maximum response following pesticide treatment is only 70% of that produced by the hormone. In the LE both compounds yield the same maximum response, but the time course of DNA synthesis/cell division is delayed following o,p'-DDT administration relative to 17 beta-estradiol treatment. The patterns of estrogen receptor retention in uterine nuclei following o,p'-DDT administration are prolonged relative to those observed after 17 beta-estradiol treatment. o,p'-DDT thus produces the uterine hyperplasia characteristic of estrogens, but the magnitude and timing of the response is dependent on the specific cell type observed and is different from that produced by estradiol.
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Receptors for epidermal growth factor in the rat uterus.
Uterine membranes contain high affinity [dissociation constant (Kd) = 0.36 nM], saturable binding sites for [125I]iodo-epidermal growth factor (EGF). The binding of [125I] iodo-EGF is specific since it is abolished by excess unlabeled EGF but not by excess unlabeled insulin, fibroblast growth factor, or multiplication-stimulating activity. Incubation of [125I] iodo-EGF with uterine membranes, followed by chemical cross-linking with disuccinimidyl suberate and detergent extraction reveals a major species of specifically bound EGF (mol wt = 170,000) and a minor species (mol wt = 150,000) visualized by autoradiography of sodium dodecyl sulfate gels after electrophoresis of the extracts. In detergent-solubilized preparations EGF also stimulates the phosphorylation of major (mol wt = 170,000) and minor (mol wt = 150,000) species of identical molecular weight. The increased phosphorylation produced by incubation of membrane extracts with EGF occurs largely at tyrosine residues, as indicated by phosphoamino acid analysis. These results indicate that the rat uterus contains high affinity EGF binding sites with the properties expected of EGF receptors.
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Effects of experimentally-induced diabetes on oestradiol-stimulated changes in the ultrastructure of the rat endometrium.
Alterations in endometrial cell morphology following oestradiol treatment of ovariectomized animals were examined in euglycaemic and streptozotocin-induced diabetic rats. Endometrial morphology was examined 18, 24, 36 and 48 h after oestradiol administration. Nuclei of endometrial cells from saline injected euglycaemic and diabetic rats exhibited pleomorphic nuclei with areas of heterochromatin dispersed throughout the nucleus. Nucleoli were homogeneous in appearance and endometrial cells showed no mitotic chromosome function or cell division. The cytoplasm of euglycaemic and diabetic cells contained few mitochondria. The mitochondria that were present contained a uniformly dense matrix and cristae that extended across the mitochondria. The formation of round to oval shaped nuclei with diffuse euchromatin was observed at 18, 24 and 36 h following oestradiol administration in euglycaemic animals. Alterations in nuclear morphology and the formation of euchromatin were absent or reduced at the same time intervals examined in diabetic animals. Nucleoli in oestradiol-treated euglycaemic animals at 18, 24 and 36 h post-injections became very prominent and displayed easily visible granules and fibrillar material. Comparable changes in nucleolar morphology in oestradiol-treated diabetic rats were absent. The development of oval to round mitochondria containing short cristae and an electron-lucent matrix became apparent at 18 h post-injection of oestradiol in euglycaemic control animals. Alterations in mitochondrial morphology did not appear in endometrial cells of oestradiol-treated diabetic animals at any of the time points examined. At 24 and 36 h post-injection of oestradiol in euglycaemic animals, mitotic figures were observed in mitochondrial cells. Cell division and mitotic figures were not observed in oestradiol-treated diabetic animals.(ABSTRACT TRUNCATED AT 250 WORDS)
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Effect of a diabetic state on myometrial ultrastructure and isolated uterine contractions in the rat.
Alterations in rat myometrial ultrastructure and in vivo uterine contractile responses to oxytocin were examined in estradiol-treated (40 micrograms/kg) euglycemic and streptozotocin-induced (85 mg/kg) diabetic rats. Myometrial morphology was examined 18, 24, and 36 hr after estradiol administration. At the time points examined, nuclei of myometrial cells from euglycemic and diabetic rats were pleomorphic and contained large areas of heterochromatin dispersed throughout the nuclei. Mitochondria were round to oval in shape and contained a dense matrix with cristae that extended across the mitochondria. Myofilaments were found in both euglycemic and diabetic cells but the relative number of myofilaments in diabetic cells appeared to be less than the number found in myometrial cells removed from euglycemic animals. The number of free cytoplasmic ribosomes in diabetic cells also appeared to be less than those found in euglycemic cells. In order to determine if apparent differences in the number of myofilament found in diabetic myometrial cells could be correlated with changes in uterine contractile responses to hormones, oxytocin dose-response curves (10(-8) to 10(-5) M) were examined in isolated uteri removed from saline-injected and estradiol-injected (24-hr pretreatments) euglycemic and diabetic rats. The maximal contractile responses (milligrams tension developed per milligrams tissue) in saline-injected euglycemic and diabetic rats were 49 +/- 5 and 36 +/- 4, respectively, while maximal contractile responses in estradiol-injected euglycemic and diabetic rats were 68 +/- 7 and 45 +/- 5, respectively. Maximal contractile responses induced by oxytocin in estradiol-treated diabetic uteri were significantly smaller than the contractile responses measured in euglycemic estradiol-treated uteri. This study demonstrates that estradiol-induced changes in both myometrial cell morphology and in vitro uterine contractile responses to oxytocin are altered in diabetic rats.
Actin Cytoskeleton↗
Binding and function of serotonin 2 receptors following chronic administration of imipramine.
Experiments were undertaken to determine whether alterations in the binding of 5-HT2 receptors in the cerebral cortex of rat brain, induced by antidepressant drugs, are related to behavioral changes observed in response to the administration of a precursor of serotonin. Chronic (5 days), but not acute, administration of an imipramine-yohimbine combination resulted in a significant decrease in the binding of 5-HT2 receptors in the frontal cortex and wet-dog shakes induced by 5-hydroxytryptophan (5-HTP). The decrease in the binding to 5-HT2 receptors was due entirely to a change in the concentration of binding sites with no modification in receptor affinity. The behavioral alteration resulted from a decrease in the maximal response to 5-HTP. Further experiments revealed that chronic (5 days) administration of imipramine alone altered the behavioral response to 5-HTP without changing the binding of 5-HT2 receptor binding and that changes in 5-HT2 binding in the cortex were not always accompanied by an alteration in the response to 5-HTP. These results suggest a lack of correlation between the drug-induced change in 5-HT2 binding in frontal cortex and the function of serotonin 2 receptors.
5-Hydroxytryptophan↗
The estrogenic activity of DDT: the in vitro induction of an estrogen-inducible protein by o,p'-DDT.
Induced protein is a commonly measured marker for estrogenic action. The induction of induced protein by o,p'-DDT was studied in an in vitro system. Nuclear levels of estrogen receptor translocated by o,p'-DDT correlated highly with induced protein induction, and the time course for induced protein induction was consistent with an estrogen receptor-mediated mechanism. While the maximum amount of induced protein produced by o,p'-DDT was less than after 17 beta-estradiol exposure, the induced protein formed by each compound was indistinguishable on nondenaturing and denaturing polyacrylamide gels. Also, it was shown that o,p'-DDT does not cause additional induction of induced protein over that seen with maximum levels of 17 beta-estradiol, further supporting the premise that these compounds share a common pathway in stimulating the synthesis of induced protein.
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