Estrogen Receptor mRNA Research Articles

Estrogen acutely activates prostacyclin synthesis in ovine fetal pulmonary artery endothelium.

Prostacyclin (PGI(2)) is a key mediator of pulmonary vasodilation during perinatal cardiopulmonary transition, at a time when fetal plasma estrogen levels are rising. We have previously shown that estradiol-17beta (E(2)) rapidly stimulates nitric oxide production by ovine fetal pulmonary artery endothelial cells (PAEC), and that this occurs through nongenomic mechanisms which are calcium- and tyrosine kinase-mitogen-activated protein (MAP) kinase-dependent. In the present study, we determined if E(2) acutely activates PGI(2) production in PAEC. E(2) (10(-8) M for 15 min) caused a 52% increase in PGI(2), the threshold concentration was 10(-10) M E(2), the effect occurred within 5 min, and it was not related to changes in cyclooxygenase type 1 (COX-1) or COX-2 abundance. Estrogen receptor (ER) alpha and ER beta proteins and mRNAs were found to be constitutively expressed in PAEC, and PGI(2) stimulation with E(2) was fully blocked by both ER antagonism with ICI 182,780, which is not selective for either ER isoform, and the ER beta-specific antagonist RR-tetrahydrochrysene. The rapid response to E(2) was also inhibited by calcium chelation, whereas genistein- or PD98059-induced inhibition of tyrosine kinase and MAP kinase kinase, respectively, had no effect. Thus, E(2) causes rapid stimulation of PGI(2) synthesis in fetal PAEC, this process is mediated by ER beta, and it is calcium-dependent and tyrosine kinase-MAP kinase-independent. These mechanisms may play a role in pulmonary vasodilation in the perinatal period.

Expression of estrogen receptors α and β in human lung tissue and cell lines

Epidemiological studies have indicated that females may be at greater risk of smoking associated lung cancer compared with males. Several lines of biochemical evidence support these observations. A possible role of circulating steroid hormones in the etiology of lung cancer has been hypothesized. In the present paper, we have studied the expression of the estrogen receptors (ER)-α and ERβ in histologically normal human lung tissue and lung tumor cell lines. Relative ER mRNA levels were measured by reverse transcriptase-PCR and normalized to the level of expression of the glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH). In lung tissue, an ERα transcript was found at various levels in 38 out of 46 cases (83%). ERβ was expressed in all cases. The ERs were expressed at similar levels in females and males, and the levels of ERα and ERβ mRNA were significantly related ( P<0.0001). Compared with the lung tissue, ER expression levels were lower in 16 human lung tumor cell lines and two immortalized human bronchial epithelial cell lines. Five of the tumor cell lines (31%) expressed detectable levels of ERα and both of the immortalized cell lines showed a weak ERα expression level. All cell lines expressed the ERβ. The lung cell lines BEAS-2B and DB354 showed significantly reduced cell proliferation in response to tamoxifen and a minor increased growth in response to 17β-estradiol. In conclusion, ER genes are abundantly expressed in both histologically normal human lung and lung tumor cell lines. This indicates a possible role of ERs in lung carcinogenesis.

Effects of estradiol and estradiol sulfamate on the liver of ovariectomized or ovariectomized and hypophysectomized rats

This study was performed to evaluate and compare the effects of estradiol sulfamate (J995) and estradiol (E2) on the hepatic levels of the estrogen receptor (ER) and its mRNA, in ovariectomized (OVX) and OVX+hypophysectomized (OVXHX) female rats and to study the effects on the liver-derived serum compounds angiotensin I, triglycerides, high-density lipoprotein (HDL) and cholesterol. ER concentrations were determined using ligand-binding assay (LBA) and enzyme immuno assay (EIA), and the mRNA levels using solution hybridization. The rats were treated orally (p.o.) or subcutaneously (s.c.) for 7 days, with treatments initiated 14 days after surgery. No differences were found in ER mRNA levels between J995 and E2 treated rats. The s.c. administered estrogens increased ER levels in OVX rats. Addition of GH+DEX to OVXHX rats restored the ER to levels above those seen in intact rats, whereas simultaneous oral treatment with E2 significantly decreased ER levels again. The s.c. treatment with either J995 or E2 limited the increase caused by addition of GH+DEX. After oral treatment angiotensin I levels were increased by E2, but not by J995, while triglycerides, HDL and cholesterol levels were decreased by oral E2, J995 showing a similar pattern but was less effective. In summary, these results on hepatic ER levels and estrogen dependent compounds produced by the liver showed that J995 has a lower impact on the normal liver functions after oral treatment than E2. Thus, J995 is a very promising substance for development of oral estrogen treatment with reduced hepatic side effects.

Molecular cloning of rainbow trout ( Oncorhynchus mykiss) eggshell zona radiata protein complementary DNA: mRNA expression in 17β-estradiol- and nonylphenol-treated fish

A complementary DNA (cDNA) encoding the eggshell zona radiata protein ( RbtZR: AF407574) has been cloned from the liver of estradiol-17β (E 2)-treated rainbow trout ( Oncorhynchus mykiss) by reverse-transcriptase polymerase chain reaction (RT-PCR). A set of degenerate primers homologous to the highly conserved cysteine-rich region of the zona radiata protein gene from salmon, winter flounder, medaka and carp were used for the initial RT-PCR. The resulting PCR product was cloned, sequenced and identified as the Zrp gene fragment based on amino acid sequence similarities. Based on the Zrp sequence from the initial PCR, a pair of gene-sequence primers was designed for 3′- and 5′- random amplification of cDNA ends (RACE). Cloning and sequencing of RACE products showed a 1349-bp Zrp gene encoding a 403-amino acid protein with a theoretical molecular mass of approximately 45 kDa. Alignment of the deduced amino acid sequence reveals that RbtZR is similar to piscine and mammalian zona pellucida proteins. The RbtZR gene, together with the estrogen receptor (ER) and vitellogenin (Vtg) genes, was further characterized and comparatively studied for transcriptional and translational expression in xenoestrogen- (nonylphenol, NP) and E 2-treated juvenile rainbow trout in a time-course experiment. Northern and slot blot analysis showed that the RbtZR mRNA was expressed, in parallel with the ER and Vtg mRNA, in both NP- and E 2-treated juvenile rainbow trout. Indirect enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody raised against Atlantic salmon Zrp indicated the translational expression of RbtZR protein in blood plasma samples from NP- and E 2-treated juvenile trout. The differential time-dependent transcriptional and translational expression and use of Zrp, ER and Vtg as sensitive biomarkers in environmental monitoring of endocrine disrupters in fish is discussed.

Estradiol and a selective estrogen receptor modulator affect steroid hormone receptor messenger RNA levels and turnover in explant cultures of sheep endometrium.

Estrogens upregulate estrogen receptor (ER) and progesterone receptor (PR) gene expression in endometrium immediately before ovulation to prepare it for nurturing embryos. Most in vitro model systems have lost the ability to upregulate expression of the ER gene in response to estradiol (E2) or the ability to express the ER gene at all. Here, we used explant cultures from control and E2-treated ewes and assessed expression of four genes (ER, PR, glyceraldehyde 3-phosphate dehydrogenase [GAPDH], and cyclophilin [CYC] genes) that are upregulated by E2 in vivo on Northern blots. In cultures from control and E2-treated ewes, ER and PR messenger ribonucleic acid (mRNA) levels dropped significantly during 24 h of culture in the absence of E2. Glyceraldehyde 3-phosphate dehydrogenase mRNA levels increased 300% in explants from control ewes to match the higher levels in the endometrium of the E2-treated ewe (in vivo and in explant culture). The only effect of E2 in the explant cultures was to prevent the decrease in PR mRNA. The new selective ER modulator, EM-800 (EM), decreased ER and PR mRNA levels in explants from control ewes but upregulated GAPDH and CYC mRNA levels. The EM treatment in vitro mimicked that of E2 by increasing the half-life of ER mRNA in endometrial explants. These data illustrate distinct, gene-specific effects of the explant culture process, E2, and EM on the expression of endometrial genes.

Maternal Exposure to Diesel Exhaust Decreases Expression of Steroidogenic Factor-1 and Muellerian Inhibiting Substance in the Murine Fetus.

for 8 hr per day between days 2 and 13 post coitum. Expression levels of mRNAs for steroidogenic cytochrome P450 genes regulated by Ad4BP/SF-1, especially 3β-hydroxysteroid dehydrogenase (3β-HSD) and aromatase, were also decreased by exposure to diesel exhaust. There were no significant differences in levels of estrogen receptor (ER) or androgen receptor (AR) mRNAs between control and exposed mice. The data indicate that exposure of pregnant mice to diesel exhaust affects the expression of genes essential in the early stages of embryonic development.

Uterine molecular responses to bisphenol A treatment before and after decidual induction in pseudopregnant rats

AbstractThe results of the study demonstrate that the weak estrogenic action of bisphenol A (a daily subcutaneous dose of 200mg/kg on 4 consecutive days, administered before or after decidual induction that occurs on day 4 of pseudopregnancy) on deciduoma growth in pseudopregnant Sprague-Dawley rats, was functionally associated with the hormonal status of the uterus. Whereas bisphenol A displayed uterotrophic action during the pre-decidual, estrogen related period, it inhibited decidual growth and progesterone secretion during the post-decidual, progesterone-dominated period. The estrogenic action of bisphenol A on uterine decidual growth was not correlated with the reduced levels of estrogen receptor binding sites and mRNA expression, nor the unchanged serum estradiol concentrations. BPA action appeared to be antagonized by progesterone.

Estrogen receptor, calcium mobilization and rat sperm motility.

Oral treatment with 0.4 mg/kg/day of tamoxifen citrate, an antiestrogen, has been reported to reduce the fertility of adult male rat, presumably through estrogen receptors expressed throughout the male reproductive tract. During the course of these studies, tamoxifen was observed to gradually alter the pattern of sperm motility in the cauda epididymides without reducing sperm counts. Studies were carried out to understand the mechanism involved in tamoxifen induced change in the sperm motility pattern. In order to study the direct effects of tamoxifen on motility, biochemical levels/activities of sperm calcium, cAMP, phosphodiesterase and dynein ATPase, normally implicated in sperm motility were studied In view of the fact that tamoxifen is a ligand of estrogen receptor, estrogen receptor alpha protein and transcript were localized on rat sperm membrane and the effect of tamoxifen studied. The present study demonstrated presence of estrogen receptor protein and mRNA in the rat sperm by immunofluorescence, western blotting and in situ hybridization respectively. Specificity of sperm estrogen receptors was confirmed by conventional binding studies using [3H]-estradiol. There was no effect of tamoxifen treatment on estrogen receptors in rat sperms. Biochemical analysis of the sperms from tamoxifen treated cauda epididymides revealed a significant increase in the levels of calcium and cAMP. A significant reduction was also apparent in the activity of dynein ATPase. Tamoxifen treatment did not alter phosphodiesterase activity. Estrogen receptors could be identified both in the control as well as tamoxifen treated rat sperms. It was concluded that tamoxifen treatment mobilized calcium from the intra- or extra-cellular pools with a concomitant increase in cAMP and presumably activation of PKA (protein kinase A). Tamoxifen altered the pattern of sperm motility through a calcium induced block in the activity of dynein ATPase, presumably through the activation of sperm phosphatase. The putative estrogen receptor mediated signal transduction pathway appears to be directly affected in the tamoxifen treated, sub-motile rat sperm.

Distribution of estrogen receptor subtypes, expression of their variant forms, and clinicopathological characteristics of human colorectal cancer.

We examined the expression of estrogen receptor (ER) messenger RNAs (ER-alpha, ER-beta, and ER-beta isoforms) in colorectal tumor samples and corresponding normal mucosa, paying particular attention exons 3 and 5 of both ER mRNA subtypes that likely suffer deletions, and may encode proteins that have lost either DNA- or ligand-binding moieties. Then we correlated these findings with the clinicopathological properties of the tumors. Our results demonstrated that in all patients the two ER subtype mRNAs were coexpressed in wild-type form. In 10% of the patients the ER-alpha mRNA was also present as an exon-5-deleted form that encoded any aberrant protein. Immunohistochemical analysis revealed that the ER-beta protein was present in tumor stroma, but not in infiltrating lymphocytes. ER-beta1 and ER-beta2, isoforms of ER-beta, were up-regulated in malignant tissues, whereas the ER-beta5 isoform, was found to be equally expressed, at very low levels, in the two tissue compartments. No correlations between ER levels and clinicopathological parameters were found. This suggests that the ER-beta mRNA levels are independent of the tumor characteristics.

Estrogen receptor α mRNA variant lacking exon 5 is co-expressed with the wild-type in endometrial adenocarcinoma

Background: Endometrial adenocarcinoma is a typical estrogen-dependent neoplasia. The molecular mechanisms underlying carcinogenesis in the endometrium are still largely unknown. Recently, estrogen receptor (ER) mRNA splicing variants have been investigated in several normal and neoplastic human tissues. It has been suggested that the variant receptors compete with the wild-type receptors and thereby modulate the effects of estrogens and related steroids. Objective: To investigate the possible expression of the ER α mRNA variant-type lacking exon 5 (ERΔ5) in endometrial adenocarcinoma and peritumoral tissues, non-neoplastic endometrium of healthy women served as control. Study design: The study included 16 patients divided in two groups. The first group ( n=6) was submitted to hysteroscopy and endometrial biopsy for metrorrhage, showing normal proliferative ( n=2) or secretory ( n=4) endometrium, the second group ( n=10) included patients submitted to hysterectomy for endometrial adenocarcinoma (stages Ib–IIIb). In this latter group, specimens from peritumoral tissues were also analyzed ( n=3). Characterization of the variant and wild-type α estrogen receptor transcripts was performed by RT-PCR with primers located in exons 4 and 6, followed by southern hybridization with probes directed to a specific 29 nucleotide sequence of exon 6, internal to the amplified fragments. Results: The ER α mRNA variant was co-expressed with the wild-type ER in five or six samples of non-neoplastic endometrium and in 10/10 cases of adenocarcinoma, with a more intense hybridization signal corresponding to the wild-type 439 bp band compared to the variant-type 300 bp band. Specimens from peritumoral tissue also expressed the variant ERΔ5 along with wild-type ER. Conclusion: The presence of α mRNA variant lacking exon 5 in both normal and endometrial adenocarcinoma do not support a major role of variant estrogens receptors in the biology of endometrial cancer.

Variation of ER status between primary and metastatic breast cancer and relationship to p53 expression*.

Estrogen-dependent growth of breast cancer can be blocked by anti-estrogens. Estrogen receptor (ER) presence in breast cancer implies responsiveness to endocrine therapy. However, for those patients who ultimately develop resistance to endocrine therapy, the mechanisms remain unclear. The present study attempted to compare the expression status of ER mRNA in a series of primary breast tumors with matched metastases and explored the relation between ER and mutant p53 expression. In situ hybridization using a digoxigenin-labeled estrogen receptor cDNA probe was employed to determine the expression of ER mRNA in 52 cases of primary tumors and their matched axillary lymph node metastases. Immunohistochemical staining using a monoclonal antibody against ER was also performed. ER expression was observed in 53.8% (28/52) of primary tumors and 48% (25/52) of metastases, while 57.7% (30/52) of primary tumors and 53.8% (28/52) of metastases showed ER mRNA positivity. There were variations in ER status between in situ hybridization and immunohistochemistry measurements and between primary tumors and metastases. Mutant p53 expression was inversely associated with ER-negative, high-grade tumors. In situ hybridization may be a more specific and sensitive method for determination of ER status than immunohistochemistry. It is possible that the biologic properties of ER change, and these changes may influence tumor response to endocrine therapy. In view of the ER variation, it was suggested that the ER status of metastatic tumors in addition to primary tumors should be taken into consideration in order to better determine the benefit of clinical endocrine therapy.

Estradiol and para-Chlorophenylalanine Downregulate the Expression of Brain Aromatase and Estrogen Receptor-α mRNA during the Critical Period of Feminization in Tilapia (Oreochromis mossambicus)

The period of maximal feminizing action of 17β-estradiol (E₂) upon sex ratio is before 10 days posthatching in tilapia (Oreochromis mossambicus). The effect of E₂ at this time is mimicked by para-chlorophenylalanine (p-CPA), a serotonin (5-hydroxytryptamine; 5-HT) synthesis inhibitor. The effect of E₂ on sexual differentiation may be mediated by the 5-HT system, which is consistent with the suggestion in mammals. The masculinizing actions of 17α-methyltestosterone (MT) are most potent later at up to day 20 of age, and may depend on MT induction of aromatase activity. In the present study, the effects of gonadal steroids and p-CPA on brain aromatase and estrogen receptor (ER) mRNA expression during the critical period of sexual differentiation were investigated. Treatment of tilapia with E₂ resulted in a significant decrease in the expression of brain aromatase and ERα between days 0 and 10, but not subsequently. The effect of E₂ at this time can be mimicked by p-CPA. Treatment of tilapia with MT, by contrast, resulted in a significant increase in brain aromatase, ERα and ERβ mRNA expression when given between days 10 and 20. The downregulation of brain aromatase and ERα mRNA expression by E₂ before 10 days of age and, in turn, the upregulation of brain aromatase and ERα and ERβ mRNA expression by MT at up to day 20 of age coincide with the period in which E₂ and MT have the maximal effect on gonadal feminization and masculinization, respectively.

Prevention of neonatal estrogen imprinting by vitamin A as indicated by estrogen receptor expression in the mouse vagina.

Treatment of female mice with estrogen during the neonatal period induces estrogen-independent persistent proliferation and cornification of the vaginal epithelium when the animals become adults. However, the occurrence of such irreversible vaginal changes is blocked by concurrent retinol acetate (RA) treatment. This study aimed to determine the expression pattern of estrogen receptor (ER) alpha and beta in the vaginas of ovariectomized 35-day-old mice treated neonatally with 17beta-estradiol (E(2)) and/or RA. The amounts of ERalpha and ERbeta mRNA molecules in the vaginal RNA samples were determined by competitive reverse transcription/polymerase chain reaction. The levels of both mRNAs were lower in ovariectomized mice that had been treated neonatally with E(2) but not in those treated with E(2) plus RA. Neonatal E(2) treatment caused a decrease in the percentage of ERalpha-immunoreactive cells in the vaginal stroma during adulthood, and concurrent RA treatment inhibited the decrease. The amount of each ER mRNA was also measured in the vaginas of mature mice treated with E(2) and RA; no inhibitory activity of RA was seen in the mature mice. Our studies indicate that, in mouse vagina, the irreversible effects of neonatal imprinting by estrogen might be prevented by the simultaneous administration of vitamin A through the inhibition of a decrease of the number of ER-expressing cells.

Changes in the expression and binding properties of the estrogen receptor in MCF-7 breast cancer cells during growth inhibition by tamoxifen and cisplatin.

Most mammary carcinomas contain estrogen receptors (ER), which are an important factor in diagnosis and prognosis, and in deciding on the type of therapy. ER-positive tumors are most commonly treated with the antiestrogen tamoxifen or with a combination of chemotherapeutic drugs. An important aspect for further treatment and anticipating possible side effects is the fate of the ER during the course of therapy. To study the effect of drug-induced growth inhibition on ER expression and binding properties. human breast cancer MCF-7 cells were treated with tamoxifen and cisplatin. and also estradiol (E2) for 5 days. Following this incubation, intact cells were incubated with [3H]E2 to determine the dissociation constant (KD) and maximal number of binding sites (Bmax) of the ER. The amount of ER protein per cell was quantified using anti-ER antibodies. For analysis of ER mRNA, total cellular RNA was subjected to Northern blotting. The 5-day treatment with E2 reduced Bmax and the amount of ER protein by about 70%, while the cellular level of ER mRNA was reduced by 40%. Treatment with E2 did not affect the subsequent growth inhibitory response to tamoxifen or cisplatin. In contrast, tamoxifen reduced the capacity for E2 binding; it caused about a 30-fold increase in the KD value. At the same time, Bmax and ER protein content were increased (about 3.5- and 2-fold, respectively), but the cellular level of ER mRNA was again reduced by 40%. The growth of tamoxifen-treated cells remained sensitive to subsequent treatment with estradiol, tamoxifen or cisplatin. Treatment of MCF-7 cells with cisplatin likewise reduced E2 binding due to a 20-fold increase in KD value. In this case, both Bmax and the amount of ER protein were decreased when calculated per milligram of protein, but were increased on a cellular basis due to an increase in cell size. The ER mRNA content was not altered in cisplatin-treated cells. Growth of these cells also remained sensitive to tamoxifen and cisplatin. In conclusion, drug-induced changes in ER expression and binding capacity do not necessarily indicate a loss of sensitivity of breast cancer cells to a subsequent chemotherapeutic treatment.

Microwave strategy for improving the simultaneous detection of estrogen receptor and galanin receptor mRNA in the rat hypothalamus.

In a attempt to improve the sensitivity of the simultaneous use of immunohistochemistry (IHC) with estrogen receptor (ER) and in situ hybridization (ISH) with a neuropeptide receptor, we first applied an existing microwave (MW) irradiation protocol for immunohistochemical detection of the estrogen receptor in frozen brain sections. Regions of interest were the preoptic area and the arcuate nucleus of the hypothalamus. ER signal was effective only after MW heating of sections in the two regions. Control sections without pretreatment exhibited no staining for ER. Second, the MW protocol was applied in a novel procedure that consists of evaluation of the expression of the galanin receptor mRNA with a radioactive riboprobe after MW pretreatment. The galanin receptor mRNA signal intensity obtained after heating was quantitatively at least as good or significantly increased according to the region, with no discernible loss of tissue morphology. Finally, we describe a novel application of MW pretreatment on the same frozen section processed with ER antibody and a radioactive galanin receptor riboprobe. The stainings for estrogen and galanin receptors were intense in many cells of the preoptic area, with very low background. These results show that both IHC and ISH can be significantly improved by subjecting frozen sections to MW heating before the double labeling. This approach may provide a potential method to answer the important question of whether or not estrogen has a direct action on the expression of a peptide receptor. (J Histochem Cytochem 49:901-910, 2001)

Localization of estrogen receptor alpha (ERα) in pyramidal neurons of the developing rat hippocampus

During development, estrogen has a variety of effects on morphological, biochemical and electrophysiological properties of hippocampal neurons. Correspondingly, estrogen receptor (ER) binding and mRNA increase transiently in the developing hippocampus. In this study, we used immunocytochemistry to determine the localization of the ERα subtype in the developing rat hippocampus. Nuclear staining was present in pyramidal cells and some interneurons of the CA1 and CA3 regions of the developing rat hippocampus. Little or no immunoreactivity was observed in postnatal day (P)0 animals (day of birth=P0), however, beginning on P4, ERα-immunoreactivity (ERα-ir) was visible and reached maximal levels by P10. These levels subsequently declined to low levels so that by P15, levels approximated those of adult females. Western blot analysis confirmed that this antibody recognized a 67 kDa protein, characteristic of the full-length ERα protein, in the hippocampus and pituitary. Furthermore, most of the ERα-immunopositive cells in the hippocampus were located in the pyramidal cell layer, and did not co-localize appreciably with γ-aminobutyric acid (GABA) at any age examined. We conclude, based on the immunocytochemical localization of ERα, that the effects of estrogen on biochemistry and morphology of the developing hippocampus may be direct through the ERα subtype in hippocampal pyramidal cells.

Expression of estrogen receptor-alpha and -beta mRNAs in the male reproductive system of the rat as revealed by in situ hybridization.

We mapped the cellular expression of estrogen receptor (ER) alpha and ERbeta mRNAs in the male reproductive system of the rat during development and adulthood by in situ hybridization. The expression patterns of ERalpha mRNA in the gonad, efferent duct and initial segment of the epididymis during the perinatal period were essentially similar to those of the adult: ERalpha mRNA signals were expressed most intensely in the epithelia of the efferent ducts and initial segment of the epididymis, and in the interstitial cells of the testis from the prenatal period to adulthood. However, ERalpha mRNA signals in the primordial epididymis and vas deferens during the prenatal period were confined to the outermost cellular layer of the ducts, whereas thereafter they were only expressed weakly in the epithelium and stroma of the epididymis and moderately in the muscle layer of the vas deferens. ERbeta signals were expressed intensely (1) in primordial germ and Sertoli cells only during the prenatal period, (2) in arterial walls in the adult testis, and (3) in the epithelium of the sex accessory glands from the perinatal period to adulthood. This report is the first to describe the cellular distribution of ER mRNA in the male reproductive organs during the perinatal period, and complements and confirms earlier data on its distribution in the adult. The broad expression of ERs in male reproductive organs suggests roles for estrogen in regulating tissue development and reproductive events.

Calmodulin Enhances the Stability of the Estrogen Receptor

The estrogen receptor mediates breast cell proliferation and is the principal target for chemotherapy of breast carcinoma. Previous studies have demonstrated that the estrogen receptor binds to calmodulin-Sepharose in vitro. However, the association of endogenous calmodulin with endogenous estrogen receptors in intact cells has not been reported, and the function of the interaction is obscure. Here we demonstrate by co-immunoprecipitation from MCF-7 human breast epithelial cells that endogenous estrogen receptors bind to endogenous calmodulin. Estradiol treatment of the cells had no significant effect on the interaction. However, incubation of the cells with tamoxifen enhanced by 5-10-fold the association of calmodulin with the estrogen receptor and increased the total cellular content of estrogen receptors by 1.5-2-fold. In contrast, the structurally distinct calmodulin antagonists trifluoperazine and CGS9343B attenuated the interaction between calmodulin and the estrogen receptor and dramatically reduced the number of estrogen receptors in the cell. Neither of these agents altered the amount of estrogen receptor mRNA, suggesting that calmodulin stabilizes the protein. This hypothesis is supported by the observation that, in the presence of Ca2+, calmodulin protected estrogen receptors from in vitro proteolysis by trypsin. Furthermore, overexpression of wild type calmodulin, but not a mutant calmodulin incapable of binding Ca2+, increased the concentration of estrogen receptors in MCF-7 cells, whereas transient expression of a calmodulin inhibitor peptide reduced the estrogen receptor concentration. These data demonstrate that calmodulin binds to the estrogen receptor in intact cells in a Ca2+-dependent, but estradiol-independent, manner, thereby modulating the stability and the steady state level of estrogen receptors.

Expression of Three Vitelline Envelope Protein Genes in Arctic Char

Several studies have shown effects of estrogenic substances on endocrine and reproductive systems in wildlife. Measurement of plasma vitellogenin (VTG) is a commonly used method to determine exposure to estrogenic substances in fish. There is, however, a growing need for additional sensitive and accurate methods to detect estrogenic substances in vivo. The vitelline envelope proteins (VEPs) have been suggested, in other studies, as suitable biomarkers for estrogenic substances. The present study investigates the induction of VEPs in juvenile Arctic char (Salvelinus alpinus). The results demonstrate that VEP mRNA exhibits earlier induction than estrogen receptor mRNA or VTG mRNA following injection of juvenile Arctic char with a single dose of 17β-estradiol (E2; 10 mg/kg bw). These results indicate that the VEPs have a higher sensitivity for E2 than VTG. However, an early and sex-independent expression of VEPβ in estrogen-unchallenged juvenile Arctic char was observed. These findings suggests that the regulatory mechanisms of VEPs might be more complex than previously thought, which in turn may have implications for the usage of VEPs as biomarkers for xenoestrogen exposure.

Alzheimer's disease and estrogen

The preventive effect of estrogen on Alzheimer's disease (AD) has become clear with epidemiological data. Therapeutic effects of estrogen have not yet been established. In this presentation, we report our new basic and clinical data. The estrogen receptor, (ER)α, and ERβ mRNA were investigated in rat brain. Estradiol-17β (E 2) treatment following OVX reduced the levels of ERα mRNA in the hypothalamus. In the substantia innominata (SI), the number of choline acetyltransferase immunoreacive cells increased significantly in the estrogen treatment rat. The neurons in SI projecting to the forebrain cortex contained ERα. Increasing amounts of intracellular calcium, peroxidation, and apoptosis with amyloid β were suppressed in neuronal cells from rat pheochromocytoma (PC12) cells with E 2. ERα cDNA transfected PC 12 cells elaborated more neurite-like processes with E 2. In clinics, we are currently preparing vaginal progesterone tablets, which essentially may concentrate in the endometrium to prevent endometrial cancer, with few general circulation of progesterone inviting less depression. The therapeutic effects of cyclic estrogen, such as its preventive effect, are suggested in these studies, at least on mild AD.