Estrogen Receptor Alpha mRNA Research Articles

Alteration in Estrogen Receptor α mRNA Levels in Frontal Cortex and Hippocampus of Patients with Major Mental Illness

Gender differences have been described in major mental illnesses (MMI). The dorsolateral prefrontal cortex (DLPFC) and hippocampus are estrogen-sensitive brain regions structurally and functionally altered in patients with MMI. We hypothesized that gender-specific alterations in DLPFC and hippocampus estrogen receptor alpha (ERalpha) mRNA levels may exist in MMI patients. We used Northern blot analysis to survey the expression of ERalpha mRNA transcripts in brain and body, detected by our human ERalpha riboprobe and in situ hybridization, to examine the expression pattern and quantify ERalpha mRNA levels in DLPFC and anterior hippocampus of patients with major depressive disorder (MDD), schizophrenia, and bipolar disorder compared with normal control subjects. Northern blotting revealed brain-region-specific differences in expression levels of a 5 kb ERalpha mRNA transcript. By in situ hybridization, ERalpha mRNA was detected in all layers of DLPFC and all hippocampal subfields in all subjects. We detected greater DLPFC ERalpha mRNA expression in male compared with female MDD subjects and reduced ERalpha mRNA levels in the dentate gyrus of schizophrenics compared with control subjects. Our results suggest that alterations in ERalpha mRNA levels exist in distinct telencephalic regions in male and female MDD patients, and in both genders in schizophrenia.

17β-Estradiol Modulates hMT1 Melatonin Receptor Function

Estrogen modulates expression and function of G-protein-coupled receptors. The goal of this study was to assess the effect of 17β-estradiol (10 nM) exposure for 1 (E1) or 6 (E6) days on density and function of hMT₁ and hMT₂ melatonin receptors expressed in Chinese hamster ovary (CHO) cells (CHO-MT₁/CHO-MT₂ cells). This strain of CHO cells expressed both estrogen receptor alpha and beta mRNAs, as determined by RT-PCR amplification. 17β-Estradiol treatment did not modify the affinity of either receptor; however, it significantly increased the density of 2-[¹²⁵I]iodomelatonin-binding sites in CHO-MT₂ cells. 17β-Estradiol treatment (1–6 days) did not affect the potency of melatonin to inhibit forskolin stimulation of cAMP formation through activation of either MT₁ or MT₂ receptors; however, it significantly attenuated the maximal inhibition of forskolin-stimulated cAMP formation induced by melatonin (0.01–1 µM) in CHO-MT₁ cells. Melatonin stimulation of [³⁵S]GTPγS binding to CHO-MT₁ cell membranes was also attenuated following estradiol treatment. The inverse agonist luzindole reduced basal [³⁵S]GTPγS binding in estradiol-treated cells but not in control CHO-MT₁ cells, suggesting that estradiol promotes constitutive activity of MT₁ melatonin receptors. We suggest that 17β-estradiol differentially affects MT₁ and MT₂ melatonin receptor functions, attenuates melatonin responses through activation of MT₁ receptors, and increases the MT₂ receptors density.

Estrogen-Induced Abnormal Accumulation of Fat Cells in the Rat Penis and Associated Loss of Fertility Depends upon Estrogen Exposure during Critical Period of Penile Development

We previously reported that diethylstilbestrol (DES) or estradiol valerate (EV) exposure at a dose of 0.10-0.12 mg/kg, or higher, per day, on alternate days, from postnatal days 2-12, resulted in abnormal penis development and infertility (H. O. Goyal et al., 2005, J. Androl. 26, 32-43). The objective of this study was to identify a critical developmental period(s) during which EV exposure results in the observed penile abnormalities. Male pups received EV at a dose of 0.10-0.12 mg/kg on postnatal day(s) 1, 1-3, 4-6, 1-6, 7-12, 13-18, 19-24, or 25-30. Fertility was tested at 102-115 days of age and tissues were examined at 117-137 days. Both penile morphology and fertility were unaltered in rats treated with EV after 12 days of age. Conversely, except in rats treated on postnatal day 1 only, none of the males treated prior to 12 days of age sired pups, and all had abnormal penises, including varying degrees of abnormal accumulation of fat cells and loss of cavernous spaces and smooth muscle cells in the corpora cavernosa penis, which were maximal in the 1-6-day group. Also, the preputial sheath was partially released or its release was delayed, and the weight of the bulbospongiosus muscle was significantly reduced. Plasma testosterone (T) in the 1-6- and 4-6-day groups and intratesticular T in the 4-6-day group were significantly lower. The testosterone surge, characteristic of controls in the first week of life, was suppressed in the 1-3-day group. Estrogen receptor alpha mRNA expression was enhanced in the body of the penis in the 1-3-day group, but not in the 13-18-day group. Hence, EV exposure prior to 12 days of age (as short as 1-3 days postnatal), but not after 12 days of age, results in long-term abnormal penile morphology, characterized by abnormal accumulation of fat cells in the corpora cavernosa penis and, consequently, loss of fertility.

Estrogen-related receptor in reproductive organs.

Estrogen-related receptor (ERR) was studied in the placenta and uterine endometrium, especially endometrial cancers, among reproductive organs. In the placenta, the estrogen receptor (ER) alpha and beta mRNA levels increased from the first to the second trimester, and then decreased until normal term delivery. Estrogen-related receptor alpha, beta and gamma mRNA levels gradually increased up to the second trimester, and then comparatively rapidly increased until normal term delivery. In endometrial cancers, ER alpha and beta mRNA levels decreased with clinical stage, myometrial invasion and dedifferentiation. Estrogen-related receptor alpha levels increased with clinical stage and myometrial invasion, and the ERR gamma levels increased with myometrial invasion. Estrogen-related receptors can bind to the steroid receptor coactivator family without any ligands, and drive transcription activity of the target genes. The manner of ERR and ER gene expressions might show a competitive interaction associated with the use of common cofactors. It is speculated that the upregulation of ERR is related to the placental growth after the downregulation of ER from the second trimester until delivery, and that ERR alpha and gamma are candidates for prognostic factors in endometrial cancer, although ERR are not directly related to tumor growth and advancement of endometrial cancer. (Reprod Med Biol 2005; 4: 129-131).

Human articular chondrocytes exhibit sexual dimorphism in their responses to 17β-estradiol

The higher incidence of osteoarthritis in females suggests that there may be intrinsic sex-specific differences in human articular chondrocytes. 17beta-Estradiol (E2) regulates rat growth plate chondrocytes through traditional nuclear receptor mechanisms, but only female cells exhibit rapid membrane-associated effects mediated through protein kinase C (PKC) alpha. Here we demonstrate sexual dimorphism in the physiological response of human articular chondrocytes to E2. Articular chondrocytes were obtained at the time of autopsy from three male and three female donors between 16 and 39 years of age. Second passage cultures were treated with E2 for 24 h to assess the effects of the hormone on [3H]-thymidine incorporation, [35S]-sulfate incorporation, and alkaline phosphatase specific activity. In addition, the chondrocytes were treated for 3, 9, 90 or 270 min and PKC specific activity was determined. All chondrocytes were positive for aggrecan and estrogen receptor alpha mRNAs but were negative for type II collagen mRNA. Only cells from female donors responded to E2. DNA synthesis, sulfate incorporation and alkaline phosphatase activity were increased. E2 caused a rapid increase in PKC activity in the female cells within 9 min that was maximal at 90 min. Treatment with the PKC inhibitor chelerythrine blocked these effects. These results provide the first definitive evidence that normal human cells exhibit an intrinsic sex-specific response to E2 and suggest that sexual dimorphism may be an important variable in assessing the pathways that modulate cell behavior.

Estrogen-related receptor expression in placenta throughout gestation

Estrogen receptor (ER) alpha and beta mRNA levels increased from the first to the second trimester and then decreased until normal term delivery. Estrogen-related receptor (ERR) alpha, beta and gamma mRNA levels gradually increased up to the second trimester and then comparatively rapidly increased until normal term delivery. ERRs can bind to the steroid receptor coactivator family without any ligands and drive transcription activity of the target genes. The manner of ERR and ER gene expressions might show a competitive interaction associated with the use of common cofactors. It is speculated that the up-regulation of ERRs is related to placental growth after the down-regulation of ERs because of the remarkably high concentration of estrogens for ERs from the second trimester until delivery.

Distribution of estrogen receptor alpha mRNA in the brain and inner ear of a vocal fish with comparisons to sites of aromatase expression

Among vertebrates, teleost fish have the greatest capacity for estrogen production in the brain. Previously, we characterized the distribution of the estrogen-synthesizing enzyme aromatase in the brain of the midshipman fish. Here, we investigated the distribution of estrogen receptor alpha (ERalpha). A partial cDNA of ERalpha was cloned and used to generate midshipman-specific primers for RT and real-time PCR which identified transcripts in liver and ovary, the CNS, and the sensory epithelium of the main auditory endorgan (sacculus). In situ hybridization revealed abundant expression throughout the preoptic area, a vocal-acoustic site in the hypothalamus, amygdala homologs of the dorsal pallium, the pineal organ, the inner ear, the pituitary, and the ovary. Weaker expression was found in the midbrain's nucleus of the medial longitudinal fasciculus and in the dimorphic vocal motor nucleus. ERalpha expression in the pineal, gonad, and pituitary axis may function to time seasonal abiotic cues to reproductive state, while expression in the vocal motor and auditory systems support neurophysiological evidence for estrogen as a modulator of vocal motor and auditory encoding mechanisms in midshipman fish. While ERalpha is restricted to specific nuclei, aromatase expression is abundant in glial cells throughout the entire forebrain, and high in midbrain and hindbrain - spinal vocal regions. The only site of aromatase-containing neurons is in the peripheral auditory system, where it is localized to ganglion cells in the auditory nerve. Estrogen production proximal to ERalpha-positive neurons may provide for focal sites of estrogen effects on reproductive-, vocal-, and auditory-related neurons.

Expression of estrogen receptor alpha exon-deleted mRNA variants in the human and non-human primate frontal cortex

Although estrogen receptor alpha (ERα) mRNA has been detected in the primate frontal cortex, the types of ERα transcripts expressed, including exon-deleted variants (Δ), have not been determined in the monkey or human frontal cortex. Because the types of ERα mRNA expressed in brain could define neuronal responses to estrogens, we examined the transcript pool of ERα mRNAs expressed in normal adult and developing human and macaque frontal cortex. We reverse transcribed total RNA from the postmortem frontal cortex of 29 normal adult humans, 12 rhesus macaques, and 19 people ranging from infants to adults and employed two rounds of nested polymerase chain reaction (PCR) to generate ERα products spanning the coding domain. In a third nested PCR, we used primers specific for novel sequences of exon–exon junctions created when whole exons are missing. By sequencing PCR products, we detected 60 instances of 12 distinct ΔERα mRNAs in adult humans and 94 instances of 13 distinct ΔERα mRNAs in monkeys in differing patterns from one individual to another. In adult humans, 83% of individuals expressed at least 1 ΔERα mRNA variant, and 100% of the monkeys expressed at least 1 ΔERα mRNA variant. The single Δ2, Δ5, and Δ7 variants were frequently expressed in both human and monkey frontal cortex, Δ3 variants were rare in both species, and Δ6 variants were more frequently expressed in monkeys. In both species, we detected double, triple and quadruple Δs, but these were less common than single Δs. The pattern of human variant expression did not appear to change dramatically as a function of age. These findings imply the potential to produce different ERα proteins in frontal cortex, possibly with altered structure and function which may have physiological relevance for gene transcription by virtue of altered functional interactions with each other, other steroid hormone receptors, and genomic DNA.

Expression of estrogen receptor α mRNA in theca and granulosa layers of the ovary in relation to follicular growth in quail

Changes in expression of estrogen receptor alpha (ERa) mRNA were studied in special reference to follicular growth of the ovarian follicles in laying quail. Levels of mRNA were determined by RT-PCR in the ovarian stroma, each class of the ovarian follicles, oviductal parts and in the liver. Low levels of ERalpha mRNA were detected in stroma, the small white follicles, large white follicles and small yellow follicles and in the theca layer of the three largest preovulatoy follicles. Although the level in the granulosa layer of the F3 was also low, the level significantly increased in F2 and F1. Relatively higher levels were found in the liver and oviduct, and that in the magnum was the highest among all tissues examined. The level in the F1 granulosa layer was comparable to that in the liver which actively synthesises egg yolk proteins for the sake of estrogen and ER. The results of the present study demonstrate that (1) ERalpha mRNA is present in each compartment of the reproductive tissue in quail, (2) the marked expression of ERalpha mRNA in the granulosa layer of the largest follicle may indicate the involvement of estrogens in the biosynthesis of inhibin/activin, progesterone and yolk perivitelline layer protein, (3) very high expression of ERalpha in the oviductal tissues may be related to the role of estrogens in cell proliferation and protein synthesis in the oviduct.

Reduced glucocorticoid and estrogen receptor alpha messenger ribonucleic acid levels in the amygdala of patients with major mental illness

The amygdala is a limbic structure involved in the stress response and the regulation of emotional behaviors, both of which are disrupted in patients with neuropsychiatric illnesses. Because glucocorticoids are mediators of the stress response, we hypothesized that glucocorticoid receptor (GR) messenger ribonucleic acid (mRNA) levels might be altered in the amygdala. We also hypothesized that estrogen receptor alpha (ERalpha) mRNA expression might be altered in the amygdala, on the basis of observed gender differences in mental illness. Using quantitative film autoradiography after in situ hybridization with human GR and ERalpha probes, we measured mRNA levels on adjacent amygdala sections in four groups (n = 15 each of subjects with schizophrenia, major depressive disorder, and bipolar disorder, and unaffected control subjects) provided by the Stanley Consortium. We detected main effects of diagnosis and exposure to antidepressant medication on the levels of both mRNAs but no main effect of gender. Compared with control subjects, GR mRNA expression was reduced in the basolateral/lateral nuclei in schizophrenia and bipolar disorder. Estrogen receptor alpha mRNA levels were reduced in the basomedial nucleus in major depressive disorder and bipolar disorder. Our results support and extend previous findings describing a pattern of steroid hormone mRNA alterations that differs depending on which brain region is being examined in a given mental illness.

Upstream regions of the estrogen receptor alpha proximal promoter transcript regulate ER protein expression through a translational mechanism

Estrogen receptor alpha (ER) mRNA is primarily transcribed from two promoters, the two transcripts share identical sequence encoding the same ER protein but differ in upstream regions. The 5′ region of the two transcripts contain upstream open reading frames (uORFs) encoding potential peptides of 20 and 18 amino acids. The peptides have five C-terminal residues in common. These studies were undertaken to determine if the uORFs and encoded peptides differentially affected expression of ER. Expression of green fluorescent protein (GFP) reporter constructs containing upstream proximal promoter transcript sequences with the first 18 codons of ER fused to GFP was tested in HeLa cells. The cells expressed reduced levels of GFP as compared to the pEGFP-N1 parent vector; the effect was dependent on the presence of an intact proximal ER transcript uORF. Similar regulation by the uORF was seen in transfected MCF-7, MDA MB-231 and Ishikawa cells. Only protein expression was affected by eliminating the uORF; RNA levels were unchanged. This indicates the mechanism is translational rather than being an effect of the introduced point mutations on either mRNA stability or transcription. Eliminating the uORF did not significantly increase expression from similar distal promoter transcript ER-GFP constructs. However, study of in-frame fusions of GFP to the entire proximal and distal uORFs and to their translational start motifs showed that the translational start region of the distal uORF was inherently better at initiating translation than the AUG environment of the proximal promoter transcript uORF. The data indicate there are regulatory properties suppressing expression from the ER translation start which are specific to the unique regions of the ER proximal promoter transcript and these are likely associated with the proximal transcript uORF peptide product.

Induction of calbindin-D9k messenger RNA and protein by maternal exposure to alkylphenols during late pregnancy in maternal and neonatal uteri of rats.

Environmental chemicals are proposed to possess hormone-like properties, such as mimicking natural hormones, inhibiting the action of hormones, and inducing abnormal gene expression. Among environmental chemicals, the alkylphenol products (APs), octylphenol (OP) and nonylphenol (NP), are derived from alkylphenol ethoxylates and have been reported to be environmentally persistent. Thus, in the present study, we examined the effect of two APs, OP and NP, on the expression of Calbindin-D(9k) (CaBP-9k) following maternal exposure during late pregnancy in maternal and fetal uteri. Treatment with a high dose (600 mg/kg body weight [BW]) of OP and NP resulted in an induction of CaBP-9k mRNA at Day 5 of lactation, as did a single treatment with diethylstilbestrol (DES) and 17beta-estradiol (E2) in maternal uteri. The expression of CaBP-9k mRNA was also induced following treatment with a high dose (600 mg/kg BW) of OP, transferred from the mother, exposed to fetuses during late pregnancy, and persisted through Day 5 of lactation. It is of interest that treatments with high doses of OP (400 and 600 mg/kg BW) reduced the expression of maternal estrogen receptor alpha (ERalpha) mRNA, as E2 did. However, all doses of NP resulted in an inhibition of neonatal ERalpha, while only the high does of OP (600 mg/kg BW) induced the reduction of neonatal ERalpha mRNA expression, as E2 did. Parallel to mRNA, the expression of CaBP-9k protein was significantly induced by treatment with a high dose of OP and NP. In conclusion, maternal exposure to APs, OP and NP, during late pregnancy increased the expressions of CaBP-9k mRNA and protein in maternal and neonatal uteri. These results suggest that the absorption and distribution of environmental estrogenic compounds in maternal and neonatal uteri are extremely rapid, and these chemicals can easily pass though the placenta during pregnancy to affect functions of neonatal reproductive tissues.

Estrogen stimulates differentiation of megakaryocytes and modulates their expression of estrogen receptors alpha and beta.

Estrogen has multifunctional effects influencing growth, differentiation, and function in many tissues. High-dose estrogen has been shown to produce anabolic skeletal effects in the skeleton of postmenopausal women with increased megakaryocyte (MK) population in the bone marrow, suggesting a possible role for these cells in bone remodelling. To investigate if estrogen stimulates megakaryocytopoiesis and affects on estrogen receptor (ER) expression, CD34(+) cells were cultured for 6, 9, and 14 days plus or minus low-dose or high-dose 17 beta estradiol (E). Cells were immunolocalised for CD61, CD41, ER alpha and beta. ER mRNA expression was assessed by RT-PCR. Cells formed more CD61 positive MK colonies with low- and high-dose E treatment (P < 0.001) at 6 and 9 days. CD41 expression was increased dose-dependently in MK (3- and 5-fold P < 0.001) at 9 days. E-stimulated ER alpha expression at 6 days (P < 0.001) whilst ER beta was dose-dependently increased only at 9 days (P < 0.01). ER alpha mRNA was increased at 6 days but not at 14 days whilst ER beta mRNA expression was only increased at 14 days with E treatment. These results demonstrate that E stimulates the colony forming potential of CD34(+) cells to a more megakaryocytic phenotype in vitro. This finding together with the stimulation of ER protein and mRNA expression adds to the increasing evidence for a role for MKs in estrogen-induced bone formation.

Expression of estrogen receptors and enzymes involved in sex steroid metabolism in the rat tibia during sexual maturation.

Estrogens are essential for bone mass accrual but their role before sexual maturation has remained elusive. Using in situ hybridization and immunohistochemistry, we investigated the expression of both estrogen receptor (ER) alpha and beta mRNA and protein as well as several mRNAs coding for enzymes involved in sex steroid metabolism (aromatase, type I and II 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), steroid sulfatase (STS) and type I 5 alpha-reductase) on sections of tibial metaphyses before (1- and 4-week-old), during (7-week-old) and after (16-week-old) sexual maturation in female and male rats. ER alpha and ER beta mRNA and protein were detected in metaphyseal bone in lining cells, osteoblasts, osteoclasts and some osteocytes with no apparent differences in expression during development or between the sexes. In contrast, aromatase, type I and II 17 beta-HSD and type I 5 alpha-reductase mRNAs were first detected in osteoblasts, osteoclasts and occasionally in osteocytes from sexual maturation (7-week-old rat) and onwards. Only STS was present before sexual maturation. To study the significance of ER alpha and beta expression in bone before sexual maturation when circulating sex steroid levels are low, 26-day-old female and male rats underwent gonadectomy or 17 beta-estradiol (E(2)) supplementation (0.5 mg/21 days) during 3 weeks. Following gonadectomy, trabecular bone volume (TBV) was lower in males (P=0.03) and there was a trend towards reduction in females (P=0.057). E(2) supplementation increased tibial TBV compared with controls in both genders as assessed by Masson-Goldner staining. These data suggest that the presence of ERs in bone cells before sex maturation might be of significance for bone mass accrual. Furthermore, based on the mRNA expression of the crucial enzymes aromatase and type I 17 beta-HSD, we suggest that bone cells in the tibial metaphysis acquire the intrinsic capacity to metabolize sex steroids from sexual maturation onwards. This process may contribute to the beneficial effects of estrogen on bone mass accrual, possibly by intracrinology.

Effects of extracts from Bangladeshi medicinal plants on in vitro proliferation of human breast cancer cell lines and expression of estrogen receptor α gene

In this study we determined the activity of extracts from Bangladeshi medicinal plants (Emblica officinalis, Aegle marmelos, Vernonia anthelmintica, Oroxylum indicum, Argemone mexicana) on human breast tumor cell lines. Extracts from E. officinalis and O. indicum displayed anti-proliferative activity on MCF7 and MDA-MB-231 breast cancer cell lines, while extracts from A. mexicana were active on MCF7 cells, exhibiting on the contrary low antiproliferative effects on MDA-MB-231 cells. Extracts from A. marmelos and V. anthelmintica were antiproliferative on both cell lines, but at higher concentrations. The accumulation of estrogen receptor alpha (ERalpha) mRNA, a marker of neoplastic status, was analysed by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The data obtained demonstrated that only extracts from E. officinalis induce an increase of ERalpha mRNA in MCF7 cells. When MDA-MB-231 cell line was employed, extracts from E. officinalis, V. anthelmintica and A. mexicana were found to be inducers of the increase of ERalpha mRNA accumulation. Since activation of ERalpha gene expression could have clinical impact, our results suggest a possible use of extracts from medicinal plants to identify compounds of possible interest in the treatment of breast cancer.

Uterine androgen receptors: roles in estrogen-mediated gene expressionand DNA synthesis.

The localization of androgen receptors (AR) and their ligands in the uterine microenvironment at early pregnancy suggest a role for AR in uterine physiology. We have evaluated AR expression in the pig uterine endometrium and examined whether AR ligands modulate peri-implantation uterine gene expression. Northern blot analysis demonstrated the approximately 10.5 kilobase AR transcript in endometrium. Endometrial levels of AR mRNA and protein were greater at early than at mid- or late pregnancy. Estrogen receptor-alpha mRNA levels showed similar maximal expression at early pregnancy. Immunocytochemical analysis of endometrium at early pregnancy localized AR to nuclei of glandular epithelial (GE) and stromal (ST) cells. To evaluate a role for AR in uterine gene regulation, the levels of mRNAs for insulin-like growth factor-I (IGF-I), proliferative cell nuclear antigen (PCNA), and AR itself were assessed in uterine endometrial explant cultures treated with estradiol-17beta (E), testosterone (T), and 19-nortestosterone (N). Induction by E of AR mRNA abundance occurred in endometrium from Day 10 but not from Day 12 pregnant animals and this was partially blocked by coaddition of N or T, although neither androgen alone had any effect. Abundance of IGF-I and PCNA mRNAs was increased by E and inhibited by coaddition of either T or N in Day 10 pregnant pig endometrium. In endometrium from Day 12 pregnant animals, addition of either N or T with E increased IGF-I mRNA levels over that of controls, although E alone was without effect. In contrast, PCNA mRNA abundance was suppressed by all steroid treatments in these explants. DNA synthesis in primary cultures of GE cells from endometrium at Days 10 and 12 of pregnancy was increased by E and was suppressed by T, the latter only at Day 12. E did not affect DNA synthesis in ST cells from endometrium at either pregnancy day, although T inhibited this process in an E-dependent manner in ST cells from pregnancy Day 12. Results identify AR in the pig endometrium during the window of maternal receptivity for implantation and demonstrate the functional, albeit complex, interactions of androgens and estrogens in the regulation of uterine endometrial gene expression and cell growth in vitro. Further elucidation of the role of androgens and their receptor in early pregnancy events may be relevant to an understanding of peri-implantation embryo loss.

Estrogen receptor ? expression in mice kidney shows sex differences during aging

We have used semi quantitative RT-PCR and western blotting for the analysis of expression of estrogen receptor (ER) alpha and beta mRNA and protein in the kidney of adult and old mice of both sexes. Uterus, a well-known target for estrogen action, was used for comparison. As compared to adult, the expression of both ER alpha mRNA and protein of old mice decreased in male but increased in female. However, unlike uterus, neither ERbeta mRNA nor protein could be detected in the kidney of adult or old mice. Thus the present data reveal that the expression of ERalpha in mice kidney shows sex differences during aging.

Regulation of gene expression by 8-prenylnaringenin in uterus and liver of Wistar rats.

The potential estrogenic activity of 8-prenylnaringenin has been investigated using several in vitro test systems. 8-Prenylnaringenin is a natural secondary product of the female blossoms of hops. The aim of the present study was to characterize 8-prenylnaringenin for its estrogenic effects in vivo. A three day uterotrophic assay was carried out on ovariectomized young female rats. A single dose of 8-prenylnaringenin (10 mg/day/kg body mass) was administered subcutaneously. 17beta-Estradiol (0.03 mg/day/kg body mass; subcutaneous administration) was used as a positive control. Uterine wet weight, endometrial and vaginal epithelial height were determined by histological methods. Gene expression in uterus and in liver was assessed using realtime RT-PCR. Both estradiol and 8-prenylnaringenin significantly stimulated uterine wet weight accompanied by a proliferative response. The three day treatment resulted in a statistically significant increase of the uterine epithelial height as well as of the vaginal epithelial height, the latter being the more sensitive parameter. In the uterus of ovariectomized animals estrogen receptor-alpha and clusterin gene expression were down regulated following treatment with estradiol, whereas expression of complement C3 was up-regulated. In response to treatment with 8-prenylnaringenin the same gene expression pattern was detectable, but less pronounced. The levels of estrogen receptor-alpha mRNA in rat liver were very low and therefore could not be quantitatively assessed. Like in the uterine tissue, estradiol down regulated clusterin expression. The response to 8-prenylnaringenin was weaker but still significant. Conversely, 8-prenylnaringenin was found to be more potent than estradiol in inducing expression of IGFBP-1. In summary, the multiparametric assessment of the estrogenic activity of 8-prenylnaringenin provides overwhelming evidence that 8-prenylnaringenin has largely to be regarded as a pure estrogen agonist and is therefore a questionable candidate molecule for hormone replacement therapy.

Estradiol represses prolactin-induced expression of Na+/taurocholate cotransporting polypeptide in liver cells through estrogen receptor-alpha and signal transducers and activators of transcription 5a.

Na(+)/taurocholate cotransporting polypeptide (ntcp) mediates the uptake of bile salts from plasma across the basolateral domain of the hepatocyte. We have demonstrated that ntcp expression can be induced by prolactin (PRL) and placental lactogen via the PRL receptor and signal transducers and activators of transcription (Stat)5a pathway. However, elevated levels of placental lactogen do not increase the expression of ntcp in pregnant rats. Because plasma estradiol (E(2)) levels are also elevated in pregnancy, we investigated the inhibitory effects of E(2) on PRL-induced ntcp activation. E(2) treatment inhibited the PRL-induced increase in liver ntcp mRNA to the same levels as in rats treated with E(2) alone. Estrogen receptor-alpha (ERalpha) mRNA and protein expression in liver were increased 2.6-fold and 2.2-fold, respectively, in pregnancy relative to controls. In HepG2 cells, E(2) repressed PRL-induced ntcp reporter gene expression in a dose-dependent manner in the presence of cotransfected ERalpha. The ERalpha antagonist ICI 182,780 reversed E(2)-induced repression, indicating specificity of inhibition by E(2). Overexpression of coactivator p300 did not reverse the inhibitory effects of E(2) and ERalpha. Western and gel shift analysis revealed that E(2)-bound ERalpha decreased the tyrosine phosphorylation and DNA-binding activity of Stat5a, indicating that the inhibitory effect of E(2) was mediated, at least in part, by interfering with PRL-mediated signal transduction. The present studies demonstrate the physiological significance of cross-talk between ERalpha and Stat5a in liver, in which both proteins are expressed. These data also establish a novel mechanism by which expression of ntcp, an important hepatic bile acid transporter, can be regulated by multiple hormones.

Oestradiol-dependent and -independent modulation of tyrosine hydroxylase mRNA levels in subpopulations of A1 and A2 neurones with oestrogen receptor (ER)alpha and ER beta gene expression.

Oestradiol (E2) induces luteinizing hormone-releasing hormone (LHRH) hypersecretion, thereby triggering LH surge release in ovariectomized (OVX) rats. Neural signals responsible for the surge are marked by a morning increase in LHRH gene expression and an afternoon increase in LHRH release. Evidence suggests that subpopulations of noradrenergic neurones may be responsible for one or both of these signals. To further investigate this issue, we examined effects of E2 on the activity of A1 and A2 noradrenergic neurones, as reflected in changes in tyrosine hydroxylase (TH) mRNA expression, on the day of LH surge release. We then used dual-label in situ hybridization to determine whether E2-induced changes occurred primarily in A1 and A2 subdivisions wherein most noradrenergic neurones expressed oestrogen receptor (ER)alpha and/or ER beta mRNA. We found that in all subdivisions, levels of TH mRNA were higher in E2- than oil-treated rats at 12.00 h. These differences resulted from a decline in TH mRNA expression in oil-treated rats, as well as a rise in levels in E2-treated rats between 10.00 h and 12.00 h. During the afternoon, TH mRNA expression in most A1 and A2 subdivisions peaked at 14.00 h when LH surge release began. However, in all but the middle and caudal A2 subdivisons, levels were similar in E2-treated and control rats at this time. This was attributable to a widespread increase in TH mRNA expression between 12.00 h and 14.00 h in OVX rats. There was no evidence that E2 induced changes in TH mRNA expression preferentially in regions wherein most neurones contained ER alpha or ER beta mRNA. Our findings suggest that E2 activation of middle and caudal A2 neurones, in conjunction with the widespread E2-independent activation of noradrenergic neurones in other subdivisions, may play a role in the induction of LH surge release.