E2-induced Changes Research Articles

Linkage of Rapid Estrogen Action to MAPK Activation by ERα-Shc Association and Shc Pathway Activation

E2 rapidly activates MAPK in breast cancer cells, and the mechanism for this effect has not been fully identified. Since growth factor-induced MAPK activation involves signaling via the adapter protein Shc (Src-homology and collagen homology) and its association with membrane receptors, we hypothesized that breast cancer cells utilize similar signaling mechanisms in response to E2. In the present study, we demonstrated that E2 rapidly induced Shc phosphorylation and Shc-Grb2 (growth factor receptor binding protein 2)-Sos (son of sevenless) complex formation in MCF-7 cells. Overexpression of dominant negative Shc blocked the effect of E2 on MAPK, indicating a critical role of Shc in E2 action. Using selective inhibitors, we also demonstrated that ERalpha and Src are upstream regulators of Shc. A rapid physical association between ERalpha and Shc upon E2 stimulation further evidenced the role of ERalpha on Shc activation. Mutagenesis studies showed that the phosphotyrosine binding and SH2 domains of Shc are required to interact with the activation function 1, but not activation function 2, domain of ERalpha. Using a glutathione-S-transferase-Shc pull-down assay, we demonstrated that this ERalpha-Shc association was direct. Biological consequences of this pathway were further investigated at the genomic and nongenomic levels. E2 stimulated MAPK-mediated Elk-1 transcriptional activity. Confocal microscopy studies showed that E2 rapidly induced formation of membrane ruffles, pseudopodia, and ERalpha membrane translocation. The E2-induced morphological changes were prevented by antiestrogen. Together our results demonstrate that ERalpha can mediate the rapid effects of E2 on Shc, MAPK, Elk-1, and morphological changes in breast cancer cells

Effects of progesterone on prostaglandin E(2)-induced changes in glycosaminoglycan synthesis by human cervical fibroblasts in culture.

Prostaglandins are known to induce cervical ripening and this effect may be mediated by an increase in glycosaminoglycan (GAG) concentration. The aim of this study was to assess the effects of progesterone on prostaglandin E(2) (PGE(2))-induced changes in GAG synthesis by human cervical cells in culture. Human cervical fibroblasts were obtained by cervical biopsies in hormonally active women and cultured. Cells were submitted to an incubation with progesterone or control medium. A second incubation was then performed with increasing concentrations of PGE(2). GAG synthesis by the cervical cells was assayed after extraction, by incorporation of [(3)H]-glucosamine and [(35)S]-sulphate into GAGs. It was found that progesterone alone induced a dose-dependent increase in GAG synthesis. After pre-incubation with progesterone, PGE(2) further increased [(3)H]-glucosamine and [(35)S]-sulphate uptake. However, when expressed as percentage of stimulation, the stimulatory effect of PGE(2) on GAG synthesis was inhibited at high progesterone concentrations. Therefore we concluded that, although high concentrations of progesterone increase the overall synthesis of GAG, they may also play a preventative role against PGE(2)-induced changes in GAG production during pregnancy.

Interaction between anterior pituitary prodynorphin and the secretion of luteinizing hormone (LH)

The purpose of present research was to investigate the possible involvement of prodynorphin (proDYN)-derived peptides acting locally within the anterior pituitary (AP) on the effects of estradiol-17 β (E2) and gonadotropin-releasing hormone (GnRH) on the release of luteinizing hormone (LH). Exposure of bovine AP cells in primary suspension cultures to E2 increased ( P<0.05) the spontaneous release of proDYN-derived peptides and also augmented ( P<0.05) the GnRH-induced release of LH. Both of these E2-induced responses required either high E2 dosages or prolonged exposure to produce significant changes, but there were a few cases in which the association between E2-induced changes in both parameters was absent. Therefore, it seems unlikely that proDYN-derived AP peptides mediate the effects of E2 on GnRH-induced LH release. Using another approach, cultured cells were exposed for 48 h to an antisense oligodeoxynucleotide (oligo) targeted against the translation initiation site of bovine proDYN. Compared with the two control treatments (scrambled oligo sequence or no oligo treatment), the antisense treatment decreased ( P<0.05) the quantity of LH released in response to challenge of the cells with 5 nM GnRH. There were no concurrent changes in cellular contents of proDYN-derived peptides or mRNA for LH- β, but the antisense treatment tended to decrease ( P<0.10) the relative abundance of proDYN mRNA. In summary, proDYN-derived peptides probably do not mediate direct intrapituitary effects of E2 on LH, but the antisense treatment interfered in an unknown way with GnRH-induced release of LH from cultured AP cells.

Examination of steroid-induced changes in LHRH gene transcription using 33P-and 35S-labeled probes specific for intron 2.

We previously demonstrated that estrogen, but not progesterone, induces a increase in LHRH messenger RNA levels between 0800 and 1200 h in numerous of the rostral preoptic area/organum vasculosum of the lamina terminalis (rPOA/OVLT). To determine whether these changes are a result of increased transcription, we used in situ hybridization histochemistry to examine the temporal effects of estradiol (E2) and progesterone on cellular levels of intron-2-containing LHRH heteronuclear RNA transcripts. To make our method sufficiently sensitive to detect changes in these levels, we used 33P/35S probes to minimize both background and grain stacking over labeled cell nuclei. We found that levels of LHRH heteronuclear RNA rose between 0800 and 1000 in OVX, E2-treated rats, remained elevated at 1200 h, and declined by 1400 h. Progesterone did not affect these E2-induced changes. Our results suggest that an E2-dependent signal initiates LHRH gene transcription in neurons of the rPOA/OVLT hours before LHRH release begins and that progesterone does not affect this signal.

Human vascular smooth muscle cells express an estrogen receptor isoform

In women, estrogen (E2) exerts a clinically relevant anti-atherogenic effect. The atheroprotective effects of E2 are mediated both by E2-induced changes in systemic factors and by direct effects of E2 on the blood vessel wall. In studies to characterize E2 signaling pathways in vascular smooth muscle cells (VSMC), we recently demonstrated that human VSMC express a functional estrogen receptor [1]. In the present study, we applied a reverse transcription/PCR-based strategy to identify isoforms of the E2 receptor in human VSMC. We now report that in addition to the classical E2 receptor, human VSMC derived from both mammary artery and saphenous vein express an estrogen receptor isoform containing an in-frame deletion of Exon 4 (ERΔ4). RNase protection assays confirm the presence of ERΔ4 message in VSMC and demonstrate it is nearly as abundant as the classical E2 receptor. Transient transfection experiments in VSMC and HeLa cells demonstrate that, in contrast to the classical 67 kDa nuclear-localized E2 receptor, ERΔ4: (a) is a 55 kDa protein that is widely distributed throughout the cell; (b) does not transactivate an E2 response element-driven reporter plasmid in response to E2; and (c) does not modulate transactivation of the ERE-reporter by the classical (wild type) estrogen receptor. Thus, human VSMC express an E2 receptor isoform that does not appear to alter gene transcription. The presence of a novel isoform of the E2 receptor may have important implications for studies of E2-mediated signaling in VSMC.

Human vascular smooth muscle cells contain functional estrogen receptor.

The decreased incidence of coronary artery disease observed in postmenopausal women given estrogen (E2) replacement demonstrates an atheroprotective effect of E2 that is generally believed to be mediated by indirect, E2-induced changes in cardiovascular risk factor profiles. We hypothesized that the atheroprotective effect of E2 may be in part mediated by a direct effect of E2 on vascular smooth muscle cells (VSMCs). Therefore, a series of experiments was performed to determine whether human VSMCs contain a competent E2 receptor, a ligand-activated transcription factor known to mediate E2-induced effects in nonvascular cells. Ribonuclease protection assays, with a probe derived from the human E2 receptor, were used to demonstrate E2-receptor mRNA in human saphenous vein VSMCs. To show that VSMCs contain E2-receptor protein as well as message, immunoblotting and immunofluorescence studies with a monoclonal anti-E2-receptor antibody were performed, and E2-receptor protein was detected by both methods. Transient transfection assays using a specific E2-responsive reporter system were used next to determine whether the VSMC E2 receptor is capable of E2-induced transcriptional transactivation. Initial studies using mammary artery-derived VSMCs resulted in a 2.4-fold increase in reporter activity in response to 10(-7) mol/L E2. Subsequent studies using saphenous vein VSMCs demonstrated increasing levels of reporter activation as the concentration of E2 was increased from 10(-9) mol/L (1.3-fold increase; SEM, 0.07; P = .05, n = 3) to 10(-7) mol/L (1.6-fold increase; SEM, 0.04; P = .002, n = 6). The specificity of the E2-induced transactivation of the reporter gene was shown by dose-dependent inhibition of transactivation by the pure E2 antagonist ICI 164,384 and by enhancement of the transactivation by simultaneous overexpression of the E2 receptor. We have demonstrated for the first time that human VSMCs express E2-receptor mRNA and protein and that the E2 receptor in VSMCs is capable of estrogen-dependent gene activation. These data suggest a mechanism by which estrogen may directly alter VSMC function.

Prostaglandin E2-induced changes in the phenotype, morphology, and lytic activity of IL-2-activated natural killer cells.

During pregnancy, many large granular NK lineage cells that are recognized by mAb 4H12 accumulate at uterine implantation sites. Similar large, granular, 4H12+ NK cells accumulate during the 2nd wk of culture of IL-2-activated NK cells. Inasmuch as uterine NK cell lytic activity is suppressed during pregnancy, apparently due to the effects of PGE2, we have analyzed the effect of PGE2 on the phenotype, morphology, and lytic activity of IL-2-activated NK cells as a model for NK cell differentiation at uterine implantation sites. When cultures of IL-2-activated NK cells were supplemented with 1 microM PGE2, the cells increased in size and granularity, accompanied by an increase in the number of cells expressing the 4H12 Ag. This effect was specific to PGE2 because PGF2 alpha at the same concentration had no effect. In addition, changes in morphology and up-regulation of 4H12 Ag expression could be augmented by 100 mM dibutyryl cAMP, but not 100 mM dibutyryl cGMP, suggesting that the PGE2 effects are mediated by changes in cytoplasmic cAMP levels. When indomethacin was added to cultures of IL-2-activated NK cells at the beginning of the culture period, 4H12 expression was markedly reduced. Indomethacin also increased the cytolytic capacity of NK cells and reduced the number of cells that developed the large granular morphology characteristic of 4H12+ cells. When activated NK cells were sorted into 4H12+ and 4H12- populations and tested for their ability to kill YAC-1 cells, we found that 4H12+ NK cells have lower lytic activity as compared to the 4H12- subset. Furthermore, when activated NK cells were labeled with [3H]TdR, combined with 4H12 labeling, we found that the 4H12+ subset was not proliferative. These results are likely to have direct relevance to NK cell differentiation at uterine implantation sites where NK cell activity is suppressed by PGE2, 4H12 expression is up-regulated and NK cells differentiate into large, granular and relatively nonlytic cells termed granulated metrial gland cells.

The anabolic action of 17β-estradiol (E 2) on rat trabecular bone is suppressed by (3-amino-1-hydroxypropylidene)-1-bisphosphonate (AHPrBP)

We have previously found that high doses of 17 beta-estradiol (E2), similar to those seen in late pregnancy, stimulate bone formation in adult rats. In this communication we tested the effects of a combination of E2 and (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP) on bone formation and bone volume in rat bone. E2 (4 mg/kg/day subcutaneously for 17 days) stimulated the bone formation rate to 6 times that of control rats. This was reduced by a single administration of AHPrBP (0.3 mg/kg subcutaneously) to 3 times control levels, and by similar daily injections of AHPrBP to levels not significantly different from those of untreated rats. Suppression of bone formation was effected predominantly through a reduction in the percentage of double-labelled surfaces, consistent with reduced osteoblast recruitment. We found only relatively minor effects of AHPrBP on the mineral apposition rate, suggesting that AHPrBP affected osteoblast function less than osteoblast recruitment. Suppression of histodynamic parameters of bone formation by AHPrBP was associated with suppression of the increase in bone volume otherwise induced by E2. The suppression by AHPrBP of the effect of E2 on bone formation contrasted with its lack of effect on other target tissues for E2, since AHPrBP did not affect the E2-induced changes in longitudinal bone growth or uterine weight. These results suggest that AHPrBP inhibits the anabolic effect of estrogen on rat trabecular bone.

Estradiol Modulates Protein Kinase C Activity in the Rat Pituitary in Vivo and in Vitro

17 beta-Estradiol (E2) alters different functions of pituitary cells, including cell sensitivity to several neurohormones such as LHRH, TRH, somatostatin, or dopamine, presumably by affecting receptor coupling mechanisms. Attempting to pinpoint the membrane processes underlying this modulation, we studied the effect of E2 on pituitary kinase-C (PKC) activity, a major signal transduction enzyme. The distribution of calcium- and phospholipid-dependent partially purified PKC (chromatography on DEAE-52 cellulose columns) was evaluated in membrane and cytosol fractions from anterior pituitaries of ovariectomized (OVX) or OVX plus E2-treated rats. E2 administration by implants to OVX animals increased significantly both soluble and particulate enzyme activity. The effect increased progressively from 24 h to 5 days after E2 treatment. Administration of 17 alpha-estradiol, an inactive stereoisomer of E2, was ineffective, pointing to stereospecific interaction. Total destruction of neural connections to the pituitary (complete hypothalamic lesions) did not modify the enzyme response to E2 administration, indicating a direct effect of the steroid on pituitary PKC activity. A direct E2 (10(-9) M) effect was confirmed in primary mixed cultures of pituitary cells; it was time dependent (15-96 h) and specific, and reflects a genomic E2 action. E2 treatment for shorter times had no effect on the enzyme levels or the membrane redistribution of PKC activity. In contrast, under the same experimental conditions phorbol esters (12-O-tertadecanoyl-phorbol-13-acetate (TPA] induced a rapid and sustained translocation of the enzyme. PKC activity was found in all pituitary cell types, with maximal activity in fractions of gonadotropes and thyrotropes, as evaluated in cultures enriched in certain types of pituitary cells separated by means of unit gravity gradient sedimentation. E2 treatment (10(-9) M; 72 h) significantly increased both soluble and particulate enzyme levels in all cell types. In addition, administration of E2 (10(-9) M; 72 h) to cell cultures strongly increased the TPA-evoked LH and PRL release. These results indicate that E2-induced changes in pituitary function include selective effects of the steroid on PKC activity involved at different levels in the coupling mechanisms.

Time Course of the Estradiol-Dependent Induction of Oxytocin Receptor Binding in the Ventromedial Hypothalamic Nucleus of the Rat*

Oxytocin (OT) transmission is involved in the steroid-dependent display of sexual receptivity in rats. One of the biochemical processes stimulated by the ovarian steroid 17 beta-estradiol (E2) that is relevant to reproduction is the induction of OT receptor binding in the ventromedial hypothalamic nucleus (VMN). The purpose of these experiments was to determine if E2-induced changes in OT receptor binding in the VMN occur within a time frame relevant to cyclic changes in ovarian steroid secretion. OT receptor binding was measured in the VMN of ovariectomized rats implanted for 0-96 h with E2-containing Silastic capsules. The rate of decay of OT receptor binding was measured in another group of animals 6-48 h after capsule removal. Receptors were labeled with the specific OT receptor antagonist [125I]d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT, and binding was measured with quantitative autoradiographic methods. In addition, plasma E2 levels and uterine weights were assessed in animals from each treatment condition. Significant increases in E2-dependent OT receptor binding and uterine weight occurred within 24 h of steroid treatment. After E2 withdrawal, OT receptor binding and uterine weight decreased significantly within 24 h. These results are consistent with the hypothesis that steroid modulation of OT receptor binding is necessary for the induction of sexual receptivity.

Estradiol activates methylating enzyme(s) involved in the conversion of phosphatidylethanolamine to phosphatidylcholine in rat pituitary membranes.

17 beta-Estradiol (E2) affects the sensitivity of pituitary cells to several neurohormones as LHRH, TRH, or dopamine, presumably by modulating receptor coupling mechanisms. We attempted to pinpoint the membrane processes underlying this modulation and studied the effect of E2 on pituitary membrane phospholipid methylation. Anterior pituitary membranes prepared from ovariectomized (ovx) or ovx plus E2-treated rats were assayed for phospholipid methylation. Methylated phospholipids were separated by TLC. Incorporation of [3H]methyl groups into phospholipids increased with membrane concentration and incubation time with S-adenosyl-L-methyl [3H]methionine; it was not Mg2+ dependent and was inhibited in a dose-dependent manner by S-adenosyl-L-homocysteine, methyltransferase inhibitor. pH was found to be critical. Formation of phosphatidyl-monoethanolamine, phosphatidyl-dimethylethanolamine, and phosphatidylcholine was markedly stimulated by treatment with E2. The effect increased progressively when animals were killed 15 h to 5 days after E2 implantation. The response involved a shift in the maximum velocity (Vmax) although there was no change in the available substrate for the methylating enzyme. This change in Vmax probably reflects changes in the amount of the methylating enzyme itself. Administration of 17 alpha-estradiol, an inactive stereoisomer of E2 was ineffective, pointing to a stereospecific interaction. After differential centrifugation of pituitary membranes, the highest specific methyltransferase activity was found in light mitochondrial (L) and microsomal (P) fractions and the lowest in nuclei (N) and the heavy mitochondrial (M) fractions. After sucrose density gradient centrifugation, methylated phospholipids were preferentially recovered from fractions corresponding to the endoplasmic reticulum and/or secretory granules. E2 treatment for 5 days did not modify the subcellular distribution of methyltransferase activity but stimulated it in all fractions; in contrast, it did not modify the activity of the other enzymes measured as fraction markers. Under the same experimental conditions, phospholipid methylation in membranes prepared from cortex, and anterior and mediobasal hypothalamic structures was not affected by the steroid, with the exception of a slight increment of [3H]methyl incorporation into mediobasal hypothalamic membrane phospholipids after 5 days of E2 treatment. These results indicate that E2-induced changes in pituitary responsiveness might be concomitant with selective effects of the steroid on specific membrane enzymatic activities involved in coupling mechanisms.

Dexamethasone inhibits the effects of oestrogen on the pituitary gland in rats.

The administration of glucocorticoids has been shown to be effective for the induction of ovulation in patients with anovulation. In the present study, we investigated the effects of a glucocorticoid on oestrogen-induced changes in the pituitary gland. A single ip injection of 10 micrograms oestradiol-17 beta (E2) in ovariectomized and adrenalectomized rats resulted in a significant increase in pituitary weight and progesterone receptor (PgR) concentration. In these animals, serum LH level was initially suppressed and restored to control level 24 h after E2 injection. However, 1 mg of dexamethasone (Dex) injected before E2, but not after E2 administration, completely inhibited both the increases in pituitary weight and PgR concentration. The restoration of serum LH level 24 h after E2 was also prevented. These antioestrogenic effects of Dex were blocked by ip administration of the synthetic antiglucocorticoid, RU486. Dex treatment alone did not have any effect on E2-induced changes in the dynamics of pituitary oestrogen receptor. Finally, E2-pellet implanted sc in ovariectomized and adrenalectomized rats for 7 days caused marked increases in pituitary weight and PgR concentration. A single ip injection of 250 micrograms clomiphene citrate (clomiphene) significantly reduced both the pituitary weight and PgR concentration in these animals, but 1 mg of Dex failed to have a similar effect. These results suggest that glucocorticoids antagonize E2 effects on the pituitary by a mechanism different from antioestrogens such as clomiphene. These antioestrogenic effects of glucocorticoid may be involved in induction of ovulation in anovulatory women.

Early estrogen‐induced nuclear changes in rat hypothalamic ventromedial neurons: An ultrastructural and morphometric analysis

Alterations in the fine structure of nuclei of ventromedial hypothalamic neurons in ovariectomized (OVX) rats after either a 2-hour exposure to estradiol (E2) or a discontinuous exposure (2 hours E2/7 hours off/2 hours E2), previously shown sufficient for female rat sexual behavior (Parsons et al., '82a), were examined with the electron microscope. Morphometric measurements of nucleolar, nuclear, and somal areas, and nuclear shape and perimeter were accomplished at the light microscope level. After 2 hours of E2, the appearance of the nucleoplasm was altered, with a decrease in the small, scattered clumps of heterochromatin. Nuclear shape appeared dramatically altered from nonspherical, invaginated profiles toward spherical, smooth profiles. Nucleolar, nuclear, and somal hypertrophy were evident. In addition, stacked rough endoplasmic reticulum was present more frequently in E2-treated than control OVX neurons. After the discontinuous (2 hours/7 hours/2 hours) E2 treatment, progressive loss of small clumps of heterochromatin along the nuclear envelope as well as in the nucleoplasm had occurred. Decrease in a large heterochromatin clump along the nuclear envelope was correlated with an increase in nucleolus-associated chromatin. As determined by a distribution analysis, these estrogen-induced nuclear changes co-occurred more frequently than predicted from mutual independence. These findings, the marked co-occurrence of E2-induced changes in 30% or more of the cells, and the differences between the 2-hour E2 and the 2-hour/7-hour/2-hour group are consistent with a cascade of cell nuclear changes in the first few hours after estrogen onset.

Induction of lesions in deoxyribonucleic acid by low molecular weight substances from normal and colicin E2-treated cells of Escherichia coli

A fraction containing a variety of low molecular weight substances was extracted into 80% aqueous acetone from both a colicin E2-treated cell culture of Escherichia coli and an untreated one. The extract was divided into five fractions by Sephadex G15 chromatography. One of them, Fraction B, was separated into three subfractions by Sephadex G10 chromatography. Two subfractions, Fraction BI and Fraction BII, were further fractionated by several chromatographic systems. DNA was incubated with an aliquot from each of these fractions and was then analyzed by sedimentation in an alkaline sucrose density gradient. The activity which caused a decrease in the sedimentation coefficient of the DNA was found in some of these fractions. The activity from colicin E2-treated cells was compared with that from untreated ones. It was revealed that colicin E2 induces some increases in the activity toward DNA in one of the subfractions, Fraction BI, and also causes the appearance of a new species in another fraction, Fraction BII, which potentiates the activity in Fraction BI. These colicin E2-induced changes appeared at 5 min after the addition of colicin E2. The possible significance of such reactions for the action of colicin E2 are discussed.