Estrogen Receptor Alpha Gene Expression Research Articles

The effect of moderate-intensity aerobic training on pulmonary function and estrogen receptor-alpha gene in postmenopausal women with vitamin D deficiency: A randomized control trial

BackgroundThe impact of aerobic training on pulmonary function by modulating gene expression of estrogen receptor-alpha (ERα), sex hormones and 25-hydroxy vitamin D (Vit D) in postmenopausal women (PMW) with Vit D deficiency is uncertain. ObjectiveThe purpose of this study was to examine the effect of 12 weeks of moderate-intensity aerobic training on pulmonary function, ERα gene expression, serum levels of sex hormones and Vit D in PMW with Vit D deficiency. MethodsTwenty-nine sedentary PMW with Vit D deficiency (aged 45-65 yrs) were randomized to exercise (EX, n = 15) and control (C, n = 14) groups. The EX group performed moderate-intensity aerobic training for 12 weeks (50-60 min/day, 3 days/week at 65-70% of maximal heart rate reserve), but the C group participated in no intervention and maintain their normal lifestyle during 12 weeks. The pulmonary function parameters, ERα gene expression, serum levels of sex hormones and Vit D were measured at baseline and week-12. ResultsAfter 12 weeks of aerobic exercise intervention, the increase in lymphocyte ERα gene expression (p = 0.005, estimate of effect size/Eta = 32.8%) and serum Vit D (p = 0.001, Eta = 54.7%) were significantly higher in the EX group compared to the C group, whereas pulmonary function parameters and sex hormones (17β-estradiol and progesterone) were not significantly different (P > 0.05). ConclusionsThe results suggested that 12 weeks of moderate-intensity aerobic training increased lymphocyte ERα gene expression as well as serum Vit D in sedentary PMW with Vit D deficiency although pulmonary function was not improved.

The isoflavone genistein enhances osteoblastogenesis: signaling pathways involved

Phytoestrogens have been proposed as a natural therapy for prevention of bone loss. In this work, we studied the mechanism of action of genistein on osteoblast differentiation. Primary cell cultures of calvarial osteoblasts isolated from female Wistar rats were in vitro exposed to genistein. Osteoblast differentiation markers were measured. Genistein stimulated osteoblast migration (71-257% above control). An earlier upregulation of estrogen receptor alpha gene expression and an enhancement of mRNA levels of the Runt-related transcription factor 2 were detected after 3days of culture. The isoflavone significantly increased osteocalcin expression, extracellular collagen deposition, and alkaline phosphatase activity. The mechanism displayed by genistein involved estrogen receptor and nitric oxide pathway participation, since cell preincubation with the estrogen receptor antagonist ICI 182780, or the nitric oxide synthase inhibitor L-NAME, suppressed the phytoestrogen action. Evidence of MAPK and PI3K transduction systems participation on the stimulatory action of genistein on extracellular collagen deposition and alkaline phosphatase activity was also obtained. Genistein favored monocyte adhesion to osteoblasts (77% above control) in an ER; NOS; and MAPK kinase-dependent and PI3K-dependent manner. Co-cultured osteoblast-monocyte long term exposed (21days) to genistein exhibited a high number of multinucleated and tartrate-resistant acid phosphatase-positive cells added to osteoblasts, suggesting that the phytoestrogen promotes osteoclast differentiation. In conclusion, genistein promoted osteoblastogenesis through the participation of ER and NOS pathways, and the contribution of ERK or PI3K signal transduction pathways, and also stimulates osteoclast differentiation from its mononuclear progenitor.

Association of Placental Tissue Estrogen Receptor Alpha Gene Expression with Postpartum Depression in Women at Risk: A Pilot Study

Association of Placental Tissue Estrogen Receptor Alpha Gene Expression with Postpartum Depression in Women at Risk: A Pilot Study

Estrogenic effects of fluorinated diiodine alkanes in MCF-7 cells, H295R cells and zebrafish embryo assays.

Fluorinated diiodine alkanes (FDIAs), important industrial intermediates in the synthesis of various perfluorinated compounds, which are distributed widely in wildlife and humans. Recent studies showed that FDIAs had in vitro estrogenic effects. However, to date, little information is available regarding the in vivo estrogenic effects of FDIAs and the mechanisms are unclear. In this study, a combination of in vitro and in vivo assays was used to investigate the estrogenic effects of FDIAs. We tested the in vitro estrogenic effects and estrogen receptor-related gene expression via MCF-7 cell assay. The hormone level of estradiol and the expression of estrogenic synthesis genes were measured in the H295R cell assay. Finally, the in vivo effects of FDIAs on development and estrogen-related gene expression were assessed in the zebrafish embryos assay. The results demonstrated that FDIAs could exhibit estrogenic activity through inducing cell proliferation (1.6-6.7-fold of the control) and estrogen receptor alpha gene expression (1.07-1.39-fold of the control), altering estradiol production (1.14-1.22-fold of the control) and the major estrogenic synthesis gene expression of CYP19 (1.22-1.31-fold of the control), disrupting the estrogen-related genes (esr1 and cyp19b) levels in zebrafish (1.52-2.99-fold and 2.95-5.00-fold of the control for esr1 and cyp19b, respectively). The current findings indicated the potential estrogenic effects of FDIAs and provided novel information for human risk assessment.

Phase I study of panobinostat (LBH589) and letrozole in post-menopausal women with metastatic breast cancer.

e13501 Background: Loss of estrogen receptor alpha gene expression has been associated with insensitivity to endocrine therapy in human breast cancer patients. Histone deacetylase (HDAC) inhibitors have recently been found to restore sensitivity to the estrogen receptor by modulation of the estrogen and progesterone receptors. This had been shown with both aromatase and tamoxifen refractory and in triple negative cell lines . We performed a Phase I study of the combination of LBH589 (panobinostat) and letrozole to evaluate safety and tolerability in patients with metastatic breast cancer prior to the performance of a phase II trial. Methods: We enrolled postmenopausal women with metastatic breast cancer, ECOG PS 0 or 1, ANC>1500/mm3, platelets>100,000/mm3, normal total bilirubin, and ALT/AST adequate laboratory tests were eligible. Letrozole dose was 2.5 mg/day orally. Dose of LBH589: Level 1, 20 mg orally three times weekly; Level 2, 30 mg orally three times a week. Results: 12 patients (dose level 1:6 patients, dose level 2: 6 patients) have been enrolled. 43 cycles of treatment have been given to these12 patients. Initial cohort of 3 patients at the 20 mg dose level had no dose limiting toxicity (DLT). At the 30 mg dose level 3/6 patients had DLT (thrombocytopenia grade 1: 2 pts; grade 3:1 pt; grade 4: 1 pt; and diarrhea grade 3: 1 pt. One pt at the 30 mg dose level has a confirmed partial response and remains on study after 6 cycles of treatment. Subsequent cohort of 3 patients had 1 dose DLT with doubling of the creatinine. The main DLT was thrombocytopenia in 3/6 pts at the 30 mg dose level. Conclusions: The recommended dose for phase II testing of LBH589 is 20 mg orally 3 times per week in combination with standard dose letrozole.

Regulation of estrogen receptor alpha gene expression in the mouse prefrontal cortex during early postnatal development

Estrogens have many functions in the developing rodent brain, and most of these depend on the presence of estrogen receptors. Understanding how expression of these receptors are regulated is crucial for understanding the roles of estradiol in the male and female brain during development In rodents, the prefrontal cortex (PFC) has been shown to be involved in working memory, attention, and behavioral inhibition. Many studies have demonstrated an effect of estradiol on sex difference in these functions attributed to differences in the PFC. We have previously demonstrated that estrogen receptor alpha (ERα) expression decreases in the isocortex across early postnatal development. This decrease corresponds with an increase in methylation of many sites along the ERα promoter. Here we have examined both ERα and ERβ mRNA expression in the PFC to determine if methylation also plays a role in this important brain region. We investigated expression of alternate promoters for ERα and methylation of CpG sites along two of these promoters. We found that the pattern of ERα mRNA expression in PFC was similar to the pattern of ERα expression in the isocortex and that there were no sex differences in the level of expression across development. We did, however, find subtle differences in promoter expression and methylation that may indicate a sex-specific difference in PFC during development resulting in a difference in adult response.

Development of a Bio-Assay for Estrogens using Estrogen Receptor Alpha Gene Expression by MCF7 Cells as Biomarker

Estrogenic endocrine disruptors (EDCs) have been identified in soil, food, air, and water, and may produce adverse health effects in both humans and wildlife. Various in vitro assays, including the E-screen that measures estrogen dependent proliferation of the MCF-7 human breast cancer cell line, have been developed and implemented to screen for environmental estrogenic EDCs. This study describes a new amendment to the well known E-screen. A direct ELISA to quantify ERα protein levels on MCF-7 cells cultured in a high through put 96-well format were validated as a biomarker for estrogenicity. The ELISA shows that there is an inverse correlation between ERα levels and 17β-estradiol (E2) concentration (R2 = 1). The detection range of the assay is between 1 and 1000 nM for E2. Results obtained with the ERα ELISA showed a good inverse correlation with total cellular LDH levels that is conventionally used to quantify MCF-7 cell proliferation. This ELISA was employed to assess environmental water extracts for estrogenicity.

Study of the CpG methylation status of ER alpha gene in estrogen receptor alpha-negative breast cancer cell lines and the role of hydralazine demethylation

To detect the 5'CpG island methylation of estrogen receptor (ER) alpha gene promotor region in ER alpha-negative human breast cancer cell lines (MDA-MB-231 and MDA-MB-435) and breast cancer tissues; and to investigate the possibility of hydralazine in restoring the expression of ER alpha gene through demethylation. The CpG island methylation status of ER alpha gene promotor region-A, B and C in ER alpha-negative human breast cancer cell lines (MDA-MB-231 and MDA-MB-435) and 20 cases of breast cancer tissues were analyzed using methylation-specific polymerase chain reaction (MSP). The mRNA expression profile of ER alpha isoforms (ERalpha-A, ERalpha-B and ERalpha-C) and coding region in ER alpha gene were analyzed by using RT-PCR, after hydralazine treatment. In the two cell lines studied, CpG island was methylated in ERalpha-A and ERalpha-B but not ERalpha-C. Among them, methylation of CpG island in ERalpha-A was obtained in 13 breast tumor cases; methylation of ERalpha-B seen in 10 tumor cases; methylation of both ERalpha-A and ERalpha-B seen in 9 cases; and methylation of ERalpha-C was obtained in only 1 case. The positivity rates were 65%, 50%, 45% and 5% respectively. The ER alpha gene non-expression in breast cancer is probably associated with CpG island methylation in ER alpha gene promotor region A and B, and the level of methylation is enhanced as advance of tumors in clinical stage. Hydralazine, served as a demethylating agent enables to restore the expression of ER alpha gene.

Effect of estradiol on estrogen receptor-alpha gene expression and activity can be modulated by the ErbB2/PI 3-K/Akt pathway.

Epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and heregulin-beta1 (HRG-beta1), can modulate the expression and activity of the estrogen receptor-alpha (ER-alpha) via the phosphatidylinositol 3-kinase (PI 3-K)/Akt pathway in the ER-alpha-positive breast cancer cell line, MCF-7. Estradiol can also rapidly activate PI 3-K/Akt in these cells (nongenomic effect). The recent study examines whether Akt is involved in the ER-alpha regulation by estradiol (genomic effect). Stable transfection of parental MCF-7 cells with a dominant-negative Akt mutant, as well as the PI 3-K inhibitors wortmannin and LY 294,002, blocked the effect of estradiol on ER-alpha expression and activity by 70-80 and 55-63%, respectively. Stable transfection of MCF-7 cells with a constitutively active Akt mimicked the effect of estradiol. The changes in ER-alpha expression and activity were abrogated in response to estradiol by an arginine to cysteine mutation in the pleckstrin homology (PH) domain of Akt (R25C), suggesting the involvement of this amino acid in the interaction between Akt and ER-alpha. Experiments employing selective ErbB inhibitors demonstrate that the effect of estradiol on ER-alpha expression and activity is mediated by ErbB2 and not by EGFR. Moreover, anchorage-dependent and -independent growth assays, cell cycle and membrane ruffling analyses showed that Akt exerts estrogen-like activity on cell growth and membrane ruffling and that a selective ErbB2 inhibitor, but not anti-ErbB2 antibodies directed to the extracellular domain, can block these effects. In the presence of constitutively active Akt, tamoxifen only partially inhibits cell growth. In contrast, in cells stably transfected with either a dominant-negative Akt or with R25C-Akt, as well as in parental cells in the presence of a selective ErbB2 inhibitor, the effect of estradiol on anchorage-dependent and -independent cell growth was inhibited by 50-75 and 100%, respectively. Dominant-negative Akt inhibited membrane ruffling by 54%; however, R25C-Akt did not have any effect, suggesting that kinase activity plays an important role in this process. Scatchard analysis demonstrated a 67% reduction in estrogen-binding capacity in cells transfected with constitutively active Akt. No change in binding affinity of estradiol to the receptor was observed upon transfection with either Akt mutant. Taken together, our results suggest that estradiol treatment results in binding to membrane ER-alpha and interaction with a heterodimer containing ErbB2, leading to tyrosine phosphorylation. This results in the activation of PI 3-K and Akt. Akt, in turn, may interact with nuclear ER-alpha, altering its expression and activity.

Studies on estrogenic activities of food additives with human breast cancer MCF-7 cells and mechanism of estrogenicity by BHA and OPP

Estrogenic activities of more than 90 chemicals including food additives, foodstuffs of plant origin, and some chemicals, which could be orally ingested, were examined by assaying estrogen receptor (ER)-dependent proliferation of MCF-7 cells. Among 66 food additives, 17 compounds stimulated the proliferation, but their concentrations giving maximal cell yield were higher than that of 17 beta-estradiol and their estrogenic activities were weak. Flavonoids had relatively strong estrogenic activities. In the assay of ER competitive binding to human ER alpha and ER beta in vitro, the antioxidant t-butylhydroxyanisole (BHA) had the capacity to compete with 17 beta-estradiol, while the capacity of o-phenyl phenol (OPP) was too small to calculate. Both BHA and OPP induced a decrease in gene expression of ER alpha and an increase in that of progesterone receptor in a time-dependent manner. These effects were similar to that of 17 beta-estradiol, a though much higher concentrations were required for these compounds than 17 beta-estradiol. These results may suggest that we should be careful not to ingest excessive food additives.

Heregulin-beta1 regulates the estrogen receptor-alpha gene expression and activity via the ErbB2/PI 3-K/Akt pathway.

This study examines whether the serine/threonine protein kinase, Akt, is involved in the crosstalk between the ErbB2 and estrogen receptor-alpha (ER-alpha) pathways. Treatment of MCF-7 cells with 10(-9) M heregulin-beta1 (HRG-beta1) resulted in a rapid phosphorylation of Akt and a 15-fold increase in Akt activity. Akt phosphorylation was blocked by inhibitors of phosphatidylinositol 3-kinase (PI 3-K), by antiestrogens, the protein tyrosine kinase inhibitor, genistein, and by AG825, a selective ErbB2 inhibitor; but not by AG30, a selective EGFR inhibitor. Akt phosphorylation by HRG-beta1 was abrogated by an arginine to cysteine mutation (R25C) in the pleckstrin homology (PH) domain of Akt, and HRG-beta1 did not induce Akt phosphorylation in the ER-negative variant of MCF-7, MCF-7/ADR. Transient transfection of ER-alpha into these cells restored Akt phosphorylation by HRG-beta1, suggesting the requirement of ER-alpha. HRG-beta1 did not activate Akt in MCF-7 cells stably transfected with an anti-ErbB2-targeted ribozyme, further confirming a role for ErbB2. Stable transfection of the cells with a dominant negative Akt or with the R25C-Akt mutant, as well as PI 3-K inhibitors, blocked the effect of HRG-beta1 on ER-alpha expression and activity and on the growth of MCF-7 cells. Stable transfection of MCF-7 cells with a constitutively active Akt mimicked the effect of HRG-beta1. Experiments employing selective ErbB inhibitors demonstrate that the effect of HRG-beta1 on ER-alpha expression and activity is also mediated by ErbB2 and not by EGFR, demonstrating that ErbB2 is the primary mediator of the effects of HRG-beta1 on ER-alpha regulation. Taken together, our data suggest that HRG-beta1, bound to the ErbB2 ErbB3 heterodimer, in the presence of membrane ER-alpha, interacts with and activates PI 3-K/Akt. Akt leads to nuclear ER-alpha phosphorylation, thereby altering its expression and transcriptional activity.

Modulation of gene expression in human osteoblasts by targeting a distal promoter region of human estrogen receptor-alpha gene.

Estrogen receptor (ER) alpha is expressed during osteoblast differentiation; however, both its functional role in bone metabolism and its involvement in osteoporotic pathogenesis caused by estrogen deficiency are not well understood. Loss of ER alpha gene expression could be one of the mechanisms leading to osteoporosis. Therefore, we investigated a possible modulation of ER alpha gene expression in a human osteoblastic cell line and in four primary osteoblast cultures by using a decoy strategy. Double stranded DNA molecules, mimicking a regulatory region of the ER alpha gene promoter (DNA-102) and acting as a 'silencer' in breast cancer cells, were introduced into osteoblasts as 'decoy' cis-elements to bind and functionally inactivate a putative negative transcription factor, and thus to induce ER alpha gene expression. We found that the DNA-102 molecule was able to specifically bind osteoblast nuclear proteins. Before decoy treatment, absence or variable low levels of ER alpha RNAs in the different cultures were detected. When the cells were transfected with the DNA-102 decoy, an increase in expression of ER alpha and osteoblastic markers, such as osteopontin, was observed, indicating a more differentiated osteoblastic phenotype both in the cell line and in primary cultures. These results showed that the DNA-102 sequence competes with endogenous specific negative transcription factors that may be critical for a decrease in or lack of ER alpha gene transcription. Therefore, osteoblastic transfection with the DNA-102 decoy molecule may be considered a tempting model in a putative therapeutic approach for those pathologies, such as osteoporosis, in which the decrease or loss of ER alpha expression plays a critical role in bone function.

Distinct mechanisms of loss of estrogen receptor alpha gene expression in human breast cancer: methylation of the gene and alteration of trans-acting factors.

We have previously shown that the distal promoter (promoter B) of the estrogen receptor alpha (ER alpha) gene is responsible for the enhanced expression of the ER alpha gene seen in human breast cancer and that a novel trans-acting factor, estrogen receptor promoter B associated factor 1 (ERBF-1), is required for transcription from promoter B in breast cancer cells. In development of breast cancer, loss of ER alpha gene expression is one of the most important steps in acquiring hormone resistance, though the mechanisms are poorly understood. Recent studies have reported that methylation of the ER alpha gene promoter A and exon 1 was inversely associated with ER alpha gene expression in human breast cancer and cell lines. The methylation status of the promoter B region, which is responsible for overexpression of ER alpha protein in cancer tissue, has not been investigated. In this report, we found that the methylation status of promoter B, as well as that of promoter A, was inversely associated with ER alpha gene expression in human breast cancer and cell lines. Specific methylation of ER alpha gene promoters in vitro directly decreased transcription of the ER alpha gene in a reporter assay. Demethylating treatment induced transcription of ER alpha mRNA from promoter B in ZR-75-1 cells, which showed no transcription from promoter B, despite weak ERBF-1 expression, but not in ER alpha-negative MDA-MB-231 and BT-20 cells, which lack ERBF-1. ZR-75-1 cells showed promoter activity equal to that of MCF-7 cells in a reporter assay. Our results indicate that methylation of promoter B of the ER alpha gene is important for loss of ER alpha gene expression in human breast cancer, and methylation of the promoters can directly modulate ER alpha gene expression. However, loss of critical transcriptional factors such as ERBF-1 may also be involved in some ER alpha-negative cases.

Regulation of estrogen receptor-alpha gene expression by epidermal growth factor.

The role of epidermal growth factor (EGF) in the regulation of estrogen receptor-alpha (ER-alpha) gene expression in the human breast cancer cell line MCF-7 was investigated. Treatment of cells with 0.4 ng/ml EGF resulted in an approximately 60% decrease in ER-alpha protein concentration by 6 h and the amount of receptor remained suppressed for 24 h. Ligand binding assays demonstrated that the decrease in ER-alpha protein corresponded to a similar decrease (approximately 50%) in estradiol binding sites. Although EGF treatment resulted in a decrease in the number of binding sites, it had no effect on the binding affinity of ER-alpha. The dissociation constant of the estradiol-ER-alpha complex in the presence or absence of EGF was the same (K(d)=2.3x10(-)(10) M in control cells versus K(d)=1.98x10(-)(10) M in EGF-treated cells). The decrease in ER-alpha protein concentration paralleled a decrease in the steady-state amount of ER-alpha mRNA. By 9 h there was an approximately 60% decrease in ER-alpha mRNA. The amount of ER-alpha mRNA remained suppressed for 48 h. Transcription run-on experiments demonstrated that there was a decrease of approximately 70% in ER-alpha gene transcription upon EGF treatment, suggesting that the mechanism by which EGF regulates ER-alpha gene expression is transcriptional. In addition to regulating the amount of ER-alpha, EGF affected the activity of the receptor. At high concentrations, EGF induced progesterone receptor. Estradiol and high concentrations of EGF had an additive effect on progesterone receptor. In contrast to high concentrations, low concentrations of EGF had no effect on progesterone receptor and blocked estradiol induction. The effects of EGF on ER-alpha expression were inhibited by tyrophostins and wortmannin, suggesting that the effects of the growth factor are mediated by the EGF receptor and protein kinase B. When the cells were placed in serum-free medium and then treated with EGF, there was no effect on ER-alpha protein concentration or activity. However, increasing concentrations of serum restored the effects of EGF on ER-alpha, suggesting that an additional serum factor was required for the EGF-mediated effect on the decrease in ER-alpha protein concentration.

Neonatal exposure to estrogen differentially alters estrogen receptor alpha and beta mRNA expression in rat testis during postnatal development.

The biological actions of estrogens on target cells are mediated by two nuclear receptors: the estrogen receptor (ER) alpha and the recently characterized ER beta. In the male rat, the physiological role of estrogens involves multiple actions, from masculinization of brain areas related to reproductive function and sexual behavior to regulation of testicular development and function. Paradoxically, however, administration of high doses of estrogen during the critical period of neonatal differentiation results in an array of defects in the reproductive axis that permanently disrupt male fertility. The focus of this study was to characterize the effects and mechanism(s) of action of neonatal estrogenization on the pattern of testicular ER alpha and beta gene expression during postnatal development. To this end, groups of male rats were treated at day 1 of age with estradiol benzoate (500 microg/rat), and testicular ER alpha and ER beta mRNA levels were assayed by semi-quantitative RT-PCR from the neonatal period until puberty (days 1-45 of age). Furthermore, the expression of androgen receptor (AR) mRNA was evaluated, given the partially overlapping pattern of tissue distribution of ER alpha, ER beta and AR messages in the developing rat testis. In addition, potential mechanisms for neonatal estrogen action were explored. Thus, to discriminate between direct effects and indirect actions through estrogen-induced suppression of serum gonadotropins, the effects of neonatal estrogenization were compared with those induced by blockade of gonadotropin secretion with a potent LHRH antagonist in the neonatal period. Our results indicate that neonatal exposure to estrogen differentially alters testicular expression of alpha and beta ER messages: ER alpha mRNA levels, as well as those of AR, were significantly decreased, whereas relative and total expression levels of ER beta mRNA increased during postnatal/prepubertal development after neonatal estrogen exposure, a phenomenon that was not mimicked by LHRH antagonist treatment. It is concluded that the effect of estrogen on the expression levels of ER alpha and beta mRNAs probably involves a direct action on the developing testis, and cannot be attributed to estrogen-induced suppression of gonadotropin secretion during the neonatal period.

Role of insulin-like growth factor-I in regulating estrogen receptor-alpha gene expression.

The role of insulin-like growth factor-I (IGF-I) in regulating estrogen receptor-alpha (ER-alpha) gene expression and activity was investigated in the human breast cancer cell line MCF-7. Treatment of cells with 40 ng/ml IGF-I resulted in a 60% decrease in ER-alpha protein concentration by 3 h, and the amount of ER-alpha remained suppressed for 24 h. A multiple-dose ligand-binding assay demonstrated that the decrease in ER-alpha protein corresponded to a similar decrease of 50% in estradiol-binding sites with no effect on the binding affinity of ER-alpha. The dissociation constant of the estradiol-ER-alpha complex in the absence of IGF-I (K(d) = 3 x 10(-10) +/- 0.5 x 10(-10) M) was similar to the dissociation constant in the presence of IGF-I (K(d) = 6 x 10(-10) +/- 0.3 x 10(-10) M). The decrease in ER-alpha protein concentration was paralleled by an 80% decrease in the steady-state amount of ER-alpha mRNA by 3 h. The IGF-I induced decrease in ER-alpha mRNA was due to the inhibition of ER-alpha gene transcription. When an 128-base pair ER-alpha-promoter-CAT construct was transfected into MCF-7 cells, treatment with IGF-I resulted in a 40% decrease in CAT activity. In contrast to the effects on ER-alpha, treatment with IGF-I induced two endogenous estrogen-regulated genes, progesterone receptor and pS2, by 4- and twofold, respectively. The pure antiestrogen ICI-164, 384 blocked this induction, suggesting that ER-alpha mediates the effects of IGF-I. Transient co-transfections of wild-type ER-alpha and an estrogen response element-CAT reporter into COS-1 cells demonstrated that IGF-I increased reporter gene activity. This effect was also blocked by ICI 164,384. Protein kinase A and phosphatidylinositol 3-kinase inhibitors blocked the IGF-I effects on ER-alpha expression and activity, suggesting that these kinases may be involved in the cross-talk between the IGF-I and ER-alpha pathways.

Chronic hypersecretion of luteinizing hormone in transgenic mice selectively alters responsiveness of the alpha-subunit gene to gonadotropin-releasing hormone and estrogens.

Steroid hormones can act either at the level of the hypothalamus or the pituitary to regulate gonadotropin subunit gene expression. However, their exact site of action remains controversial. Using the bovine gonadotropin alpha-subunit promoter linked to an expression cassette encoding the beta-subunit of LH, we have developed a transgenic mouse model where hypersecretion of LH occurs despite the presence of elevated ovarian steroids. We used this model to determine how hypersecretion of LH could occur when steroid levels are pathological. During transition from the neonatal period to adulthood, the endogenous LHbeta subunit gene becomes completely silent in these mice, whereas the alpha-directed transgene and endogenous alpha-subunit gene remain active. Interestingly, gonadectomy stimulates expression of the endogenous alpha and LHbeta subunit genes as well as the transgene; however, only the endogenous LHbeta gene retains responsiveness to 17beta-estradiol and GnRH. In contrast, LH levels remain responsive to negative regulation by androgen. Thus, alpha-subunit gene expression, as reflected by both the transgene and the endogenous gene, has become independent of GnRH regulation and, as a result, unresponsive to estradiol-negative feedback. This process is accompanied by a decrease in estrogen receptor alpha gene expression as well as an increase in the expression of transcription factors known to regulate the alpha-subunit promoter, such as cJun and P-LIM. These studies provide in vivo evidence that estrogen-negative feedback on alpha and LHbeta subunit gene expression requires GnRH input, reflecting an indirect mechanism of action of the steroid. In contrast, androgen suppresses alpha-subunit expression in both transgenic and nontransgenic mice. This suggests that androgens must regulate alpha-subunit promoter activity independently of GnRH. In addition to allowing the assessment of site of action of sex steroids on alpha-subunit gene expression, these studies also indicate that chronic exposure of the pituitary to LH-dependent ovarian hyperstimulation leads to a heretofore-undescribed pathological condition, whereby normal regulation of alpha, but not LHbeta, subunit gene expression becomes compromised.