Biomedical subjects

Kenneth S Korach

Publications and source records attributed to Kenneth S Korach.

At least 55 records · Page 3Linked to original sources

PubMed2004

Critical in vivo roles for classical estrogen receptors in rapid estrogen actions on intracellular signaling in mouse brain.

Estrogen exerts classical genomic as well as rapid nongenomic actions on neurons. The mechanisms involved in rapid estrogen signaling are poorly defined, and the roles of the classical estrogen receptors (ERs alpha and beta) are unclear. We examined here the in vivo role of classical ERs in rapid estrogen actions by evaluating the estrogen-induced effects on two major signaling pathways within the brains of alphaER-, betaER-, and double alphabetaER-knockout (ERKO) ovariectomized female mice. Estrogen significantly (P < 0.05) increased the numbers of phospho-cAMP response element binding protein (phospho-CREB)-immunoreactive cells in specific brain regions of wild-type mice in a time-dependent manner beginning within 15 min. In brain areas that express predominantly ERbeta, this response was absent in betaERKO mice, whereas brain regions that express mostly ERalpha displayed no change in alphaERKO mice. In the medial preoptic nucleus (MPN), an area that expresses both ERs, the estrogen-induced phosphorylation of CREB was normal in both alphaERKO and betaERKO mice. However, estrogen had no effect on CREB phosphorylation in the MPN, or any other brain region, in double alphabetaERKO animals. Estrogen was also found to increase MAPK phosphorylation levels in a rapid (<15 min) manner within the MPN. In contrast to CREB signaling, this effect was lost in either alphaERKO or betaERKO mice. These data show that ERalpha and ERbeta play region- and pathway-specific roles in rapid estrogen actions throughout the brain. They further indicate an indispensable role for classical ERs in rapid estrogen actions in vivo and highlight the importance of ERs in coordinating both classical and rapid actions of estrogen.

Animals↗

PubMed2004

Estrogen receptor-alpha, sexual dimorphism and reduced-size liver ischemia and reperfusion injury in mice.

Estrogen (E(2)) exerts its effect on target organs principally by interacting with specific estrogen receptors (ER) such as ER-alpha or ER-beta. The role that these E(2) receptors play in mediating the protective effects observed in RSL+I/R induced injury remains to be defined. To study the role of ER-alpha, we anesthetized female and male wild type (wt; C57Bl/6) and ER-alpha-deficient (alphaERKO) mice and subjected them to 70% liver ischemia for 45 min followed by resection of the remaining 30% nonischemic lobes and reperfusion of the ischemic tissue. For some experiments, wt and alphaERKO male mice were injected with E(2). Survival was monitored on a daily basis while liver injury was assessed by quantifying serum alanine aminotransferase (ALT) levels and histopathology. Hepatic eNOS mRNA levels were evaluated using semi-quantitative RT-PCR. Our data showed that untreated females or males treated with E(2) survived RSL+I/R surgery indefinitely whereas all male mice given vehicle died within 3-5 days following surgery. This protective effect was diminished in alphaERKO female mice such that only 40% of alphaERKO females survived 7 d following RSL+I/R. Furthermore, liver injury was significantly higher in alphaERKO females compared with their wt counterparts and similar to those seen in wild type males and alphaERKO males. The protective effect observed in wild type females or E(2) treated males correlated well with increases in hepatic eNOS message whereas both male and female alphaERKO mice exhibited significantly lower levels of eNOS mRNA. We conclude that this protection may in part be due to the E(2)/ER-alpha-mediated activation of eNOS.

Animals↗

PubMed2004

Interaction of estrogen receptor alpha with protein kinase C alpha and c-Src in osteoblasts during differentiation.

In cultured osteoblasts, protein kinase C (PKC) activity increases and estrogen receptor alpha (ERalpha) binding capacity decreases upon confluence. We investigated potential interactions between ERalpha and PKC isoforms and their confluence-induced modulations in clonal ROS.SMER#14 cells and primary osteoblasts. In sub-confluent ROS.SMER#14 cells, which express an exogenous plus small amounts of the endogenous ERalpha gene, the receptor appeared as two main bands of approximately 66 and approximately 46 kDa. In over-confluent, more differentiated cells, the cytosolic approximately 66 kDa ERalpha appeared decreased and the approximately 46 kDa variant increased. Enhanced expression and/or membrane translocation of PKCalpha and PKCepsilon, but not PKCzeta, was evidenced at over-confluence, along with transient increases in expression and kinase activity of c-Src, accompanied by membrane translocation of the kinase-activated enzyme. In contrast, negligible membrane translocation of PKCalpha and/or activated c-Src was observed in parental ROS 17/2.8 cells, which express low levels of full-length ERalpha. PKCalpha from over-confluent cells phosphorylated p60c-Src in vitro, suggesting functional interaction between the two kinases. ERalpha co-immunoprecipitated c-Src and PKCalpha, mostly in its cleaved form (PKMalpha). An analogous interaction was observed in primary osteoblasts. However, in these cells, much more PKCalpha/PKMalpha was ERalpha-co-immunoprecipitated at over-confluence, a condition in which the shorter, approximately 46 kDa ERalpha variant is increased. This interaction was enhanced by estradiol treatment or PKC down-regulation, but was unaffected by c-Src inhibition. These data highlight direct PKCalpha-c-Src-ERalpha interactions, which may be crucial in the modulation of estrogen responsiveness and the differentiation process in osteoblasts.

Animals↗

PubMed2004

Endogenous estrogen receptor beta is transcriptionally active in primary ovarian cells from estrogen receptor knockout mice.

The estrogen receptor (ER) alpha is a hormone-inducible transcription factor that has a pivotal physiological role. Intriguingly, a clear and undisputed physiological function of the recently described ERbeta remains elusive, with the exception of the ovary where a cooperative role of ERalpha and ERbeta has been demonstrated. We have, therefore, investigated whether endogenous ERs, in particular ERbeta, act as ligand-inducible transcription factors in primary ovarian cells derived from wild-type, ERalpha or ERbeta knockout mice. Granulosa-enriched cell fractions naturally expressing ERbeta and thecal cell fractions that express ERalpha were analyzed in transactivation assays using the vitellogenin A2 consensus estrogen response element and potent ER agonists diethylstilbestrol and S-indenestrol A. We studied also the potency-selective ERbeta agonist R-indenestrol A, the pure ERalpha agonist and ERbeta antagonist R,R-diethyl-tetrahydrochrysene and the pure ERalpha agonist propylpyrazole-triol. Using ER subtype-specific physiological cell models and these ER subtype-specific structural probes, we analyzed trans-activation of ERalpha and ERbeta. This analysis revealed that endogenously expressed ERbeta is indeed functional as a transcription factor, that it responds to estrogens appropriately, and that the ligands used are true ER subtype-specific probes in primary ovarian cells. In conclusion, this study demonstrates that endogenously expressed ERbeta is capable of regulating gene transcription independent of ERalpha.

Animals↗

PubMed2004

Estrogen receptor knockout mice as a model for endocrine research.

The biological effects of estrogen in mammalian target tissues are important for multiple organ systems including the male and female reproductive tract and the neuroendocrine, skeletal, and cardiovascular systems. Numerous physiological effects of estradiol are modulated by the estrogen receptor (ER), a Class I member of the nuclear receptor superfamily. However, more recent studies have also implicated nongenomic effects of estrogen, which may involve a membrane-binding site. The two forms of the ER are the classical estrogen receptor-alpha (ERalpha) and the more recently discovered estrogen receptor-beta (ERbeta). Gene-targeting techniques were used to generate mice lacking either functional ERalpha (alphaERKO), ERbeta (betaERKO), or both ERs (alphabetaERKO) to provide a model for evaluating estrogen receptor action. These knockout models provide a unique tool to study the effects of estrogen in the context of the whole animal and to discern the role of each ER in various tissues. The reproductive phenotypes as well as some of the nonreproductive phenotypes of the different ERKO models are summarized.

Animals↗

PubMed2004

Regulation of mouse renal CYP2J5 expression by sex hormones.

Mouse CYP2J5 is abundant in kidney and active in the metabolism of arachidonic acid to epoxyeicosatrienoic acids. Western blots of microsomes prepared from mouse kidneys demonstrate that after puberty, CYP2J5 protein is present at higher levels in male mice than in female mice. Northern analysis reveals that CYP2J5 transcripts are more abundant in adult male versus female kidneys, indicating that gender differences in renal CYP2J5 expression are regulated at a pretranslational level. Castration of male mice results in decreased renal CYP2J5 expression, and treatment of castrated male mice or female mice with 5alpha-dihydrotestosterone increases expression to levels that approximate those in intact male mice. In contrast, treatment of ovariectomized female mice or castrated male mice with 17beta-estradiol causes a further reduction in CYP2J5 expression. Growth hormone-deficient (lit/lit) mice respond similarly to castration and 5alpha-dihydrotestosterone treatment, indicating that the androgen effects are not mediated by alterations in the growth hormone secretory pattern. Mice that lack a functional androgen receptor (Tfm hemizygous) have reduced levels of renal CYP2J5 and do not respond to 5alpha-dihydrotestosterone treatment. Similarly, wild-type male mice treated with flutamide, an androgen antagonist, exhibit reduced renal CYP2J5 levels. Female estrogen receptor-alpha knockout (alphaERKO) mice, which are known to have elevated circulating testosterone levels, have significantly increased renal CYP2J5 expression compared with wild-type female mice, and these differences are abrogated by ovariectomy or treatment with flutamide. Based on these data, we conclude that the renal expression of CYP2J5 is up-regulated by androgen and down-regulated by estrogen.

Androgens↗

PubMed2003

Octamethylcyclotetrasiloxane exhibits estrogenic activity in mice via ERalpha.

Octamethylcyclotetrasiloxane (D4) is a low molecular weight cyclic silicone used in the synthesis of larger silicone polymers and in the formulation of a variety of personal care products. The effects of oral D4 exposure in mice on serum estradiol levels, uterine wet weight, and uterine peroxidase activity were investigated. Additionally, in vitro estrogen receptor binding activity was evaluated. Serum estradiol levels decreased in a dose-dependent manner after exposure to 100 mg/kg to 1000 mg/kg D4. Studies with adrenalectomized animals demonstrated that the decreased serum estradiol levels were not due to elevated serum corticosterone levels. Uterine wet weights in ovariectomized mice were significantly increased in a dose-dependent manner by exposure to 250-1000 mg of D4/kg, but not by exposure to other silicone compounds tested (hexamethylcyclotrisiloxane, decamethylcyclopentasiloxane, decamethyltetrasiloxane, and octaphenylcyclotetrasiloxane). Uterine peroxidase activity, a marker for estrogenic activity, was also significantly increased in D4-exposed mice, but not in mice exposed to the other siloxanes. Pretreating mice with the estrogen receptor antagonist ICI 182,780 completely blocked the D4-induced increase in uterine weight, and ovariectomized estrogen receptor-alpha knockout mice showed no increases in uterine weights when orally exposed to D4 or estradiol. In an in vitro estrogen receptor binding assay, D4 showed significant competition with (3)H-estradiol for binding to estrogen receptor-alpha, but not estrogen receptor-beta. The data presented here indicate that D4 has weak estrogenic activity, and that these effects are mediated through estrogen receptor-alpha.

Adjuvants, Immunologic↗

PubMed2003

Estrogen receptor-dependent genomic responses in the uterus mirror the biphasic physiological response to estrogen.

The physiological responses of the rodent uterus to acute estrogen (E) dosing can be divided into early and late events. Examples of early responses include increased RNA transcription, hyperemia, and water imbibition 2 and 6 h following E administration respectively, whereas later responses include cycles of DNA synthesis and mitosis of epithelial cells beginning 10 and 16 h after E. The development of estrogen receptor (ER) knockout (ERKO) mice, combined with microarray technology, has allowed us to design a genomic approach to study the acute response of the rodent reproductive tract to E. To determine whether early and late biological responses are correlated with altered regulation of a single set of genes or distinct sets of genes characteristic of early and late responses, uterine RNA was obtained from ovariectomized mice that were treated with vehicle or with estradiol for 2 h (early) or 24 h (late). Samples were also prepared from identically treated mice that lacked either ERalpha (alphaERKO) or ERbeta (betaERKO) to address the relative contributions of the ERs in the uterine responses. Microarray analysis of the relative expression of 8700 mouse cDNAs indicated distinct clusters of genes that were regulated both positively and negatively by E in the early or late phases as well as clusters of genes regulated at both times. Both early and late responses by the betaERKO samples were indistinguishable from those of WT samples, whereas the alphaERKO showed little change in gene expression in response to E, indicating the predominant role for ERalpha in the genomic response. Further studies indicated that the genomic responses in samples from intermediate time points (6 h, 12 h) fall within the early or late clusters, rather than showing unique clusters regulated in the intermediary period. The use of this genomic approach has illustrated how physiological responses are reflected in genomic patterns. Furthermore, the identification of functional gene families that are regulated by E in the uterus combined with the utilization of genetically altered experimental animal models can help to uncover and define novel mechanisms of E action.

Animals↗

PubMed2003

Sexually dimorphic roles of steroid hormone receptor signaling in gonadal tumorigenesis.

Sex steroids control cellular phenotypes by binding to receptor proteins that in turn regulate downstream gene expression. They are important tropic factors in hormonally responsive tissues and have been implicated in the pathogenesis of both benign proliferations and malignancies at some of these sites. Knockout mice lacking inhibins, alpha:beta heterodimeric peptide hormones of the TGFbeta superfamily, develop gonadal tumors that produce sex steroids and depend on pituitary gonadotropin hormones. To better appreciate how sex steroid receptor signaling pathways contribute to the loss of granulosa/Sertoli cell proliferation in the ovary and testis of inhibin alpha (Inhalpha) knockout mice, we are using both pharmacologic and genetic approaches. Roles of androgens in testicular tumor development have been investigated in our previous studies using double-mutant mice lacking inhibins and carrying the null testicular feminization (tfm) mutation of the androgen receptor. Herein, we report that androgens also participate in the development of ovarian tumors, as tumor development is forestalled in mice treated with flutamide, a nonsteroidal inhibitor of androgen actions. Additionally, we generated double-mutant mice lacking estrogen receptor alpha (ERalpha) and Inhalpha or ERbeta and Inhalpha, as well as triple-mutant mice lacking ERalpha, ERbeta, and Inhalpha to determine the effects of individual and combined ER signaling pathways on tumor development. Although estrogens may have proliferative effects during follicle development and are important in specifying the granulosa cell phenotype, ERalpha and ERbeta signaling are not essential for timely granulosa cell tumor development or granulosa cell-like morphological features in ovarian tumors. However, redundant ER signaling through ERalpha and ERbeta in males is critical for testicular tumor formation, as triple-knockout, but not double-knockout, males are protected from early Sertoli cell tumorigenesis and death. Together, these studies indicate important and sexually dimorphic functions of estrogens and androgens in tumor development in this mouse model and indicate, for the first time, overlapping functions of ERalpha and ERbeta in Sertoli cell pathophysiology.

Androgens↗

PubMed2003

Estrogen receptor beta mediates rapid estrogen actions on gonadotropin-releasing hormone neurons in vivo.

The gonadal steroid estrogen exerts an important modulatory influence on the activity of multiple neuronal networks. In addition to classical genomic mechanisms of action, estrogen also exerts poorly understood rapid, nongenomic effects on neurons. To examine whether estrogen may exert rapid actions on intracellular signaling within gonadotropin-releasing hormone (GnRH) neurons in vivo,we examined the phosphorylation status of cAMP response element-binding protein (CREB) in these cells after the administration of 17-beta-estradiol to ovariectomized (OVX) mice. The percentage of GnRH neurons expressing phosphorylated CREB was increased more than sixfold (p < 0.05) in a time- and dose-dependent manner by estrogen, with the increase first observed 15 min after estrogen administration. A series of in vitro studies demonstrated that estrogen acted directly on native GnRH neurons to phosphorylate CREB, but that estrogen conjugated to bovine serum albumin was without effect. The role of classical estrogen receptors (ERs) was evaluated using ER knock-out mice in vivo. The effect of estrogen on CREB phosphorylation in GnRH neurons was normal in ERalpha knock-out mice but completely absent in ERbeta knock-out mice. Finally, studies in intact female mice revealed levels of CREB phosphorylation within GnRH neurons that were equivalent to those of estrogen-treated OVX mice. These observations demonstrate that ERbeta mediates the rapid, direct effects of estrogen on the GnRH neuronal phenotype, and that these actions persist under physiological conditions. They also provide the first evidence for a role of ERbeta in nongenomic estrogen signaling within the brain in vivo.

Animals↗

PubMed2003

An estrogen-dependent four-gene micronet regulating social recognition: a study with oxytocin and estrogen receptor-alpha and -beta knockout mice.

Estrogens control many physiological and behavioral processes, some of which are connected to reproduction. These include sexual and other social behaviors. Here we implicate four gene products in a micronet required for mammalian social recognition, through which an individual learns to recognize other individuals. Female mice whose genes for the neuropeptide oxytocin (OT) or the estrogen receptor (ER)-beta or ER-alpha had been selectively "knocked out" were deficient specifically in social recognition and social anxiety. There was a remarkable parallelism among results from three separate gene knockouts. The data strongly suggest the involvement in social recognition of the four genes coding for ER-alpha, ER-beta, OT, and the OT receptor. We thus propose here a four-gene micronet, which links hypothalamic and limbic forebrain neurons in the estrogen control over the OT regulation of social recognition. In our model, estrogens act on the OT system at two levels: through ER-beta, they regulate the production of OT in the hypothalamic paraventricular nucleus, and through ER-alpha, they drive the transcription of the OT receptor in the amygdala. The proper operation of a social recognition mechanism allows for the expression of appropriate social behaviors, aggressive or affiliative.

Animals↗

PubMed2003

Characterization of the hypothalamic-pituitary-gonadal axis in estrogen receptor (ER) Null mice reveals hypergonadism and endocrine sex reversal in females lacking ERalpha but not ERbeta.

To determine the role of each estrogen receptor (ER) form (ERalpha, ERbeta) in mediating the estrogen actions necessary to maintain proper function of the hypothalamic-pituitary-gonadal axis, we have characterized the hypothalamic-pituitary-gonadal axis in female ER knockout (ERKO) mice. Evaluation of pituitary function included gene expression assays for Gnrhr, Cga, Lhb, Fshb, and Prl. Evaluation of ovarian steroidogenic capacity included gene expression assays for the components necessary for estradiol synthesis: i.e. Star, Cyp11a, Cyp17, Cyp19, Hsd3b1, and Hsd17b1. These data were corroborated by assessing plasma levels of the respective peptide and steroid hormones. alphaERKO and alphabetaERKO females exhibited increased pituitary Cga and Lhb expression and increased plasma LH levels, whereas both were normal in betaERKO. Pituitary Fshb expression and plasma FSH were normal in all three ERKOs. In the ovary, all three ERKOs exhibited normal expression of Star, Cyp11a, and Hsd3b1. In contrast, Cyp17 and Cyp19 expression were elevated in alphaERKO but normal in betaERKO and alphabetaERKO. Plasma steroid levels in each ERKO mirrored the steroidogenic enzyme expression, with only the alphaERKO exhibiting elevated androstenedione and estradiol. Elevated plasma testosterone in alphaERKO and alphabetaERKO females was attributable to aberrant expression of Hsd17b3 in the ovary, representing a form of endocrine sex reversal, as this enzyme is unique to the testes. Enhanced steroidogenic capacity in alphaERKO ovaries was erased by treatment with a GnRH antagonist, indicating these phenotypes to be the indirect result of excess LH stimulation that follows the loss of ERalpha in the hypothalamic-pituitary axis. Overall, these findings indicate that ERalpha, but not ERbeta, is indispensable to the negative-feedback effects of estradiol that maintain proper LH secretion from the pituitary. The subsequent hypergonadism is illustrated as increased Cyp17, Cyp19, Hsd17b1, and ectopic Hsd17b3 expression in the ovary.

Animals↗

PubMed2003

Stromal cell-derived factor 1, a novel target of estrogen receptor action, mediates the mitogenic effects of estradiol in ovarian and breast cancer cells.

Recent clinical studies estimate that 60-70% of human ovarian and breast cancers overexpress the estrogen receptor (ER). However, despite the established mitogenic effects of estrogen in these tumors, proliferative markers of hormone action are limited. In the current study, we report that the growth stimulatory cytokine stromal cell-derived factor 1 (SDF-1) is a bona fide target of estrogen action in ERalpha-positive human ovarian and breast cancer cells. Notably, estradiol treatment of BG-1 (ovarian carcinoma) and MCF-7 (breast carcinoma) cells leads to rapid and robust induction of the SDF-1alpha and beta isoforms. This response is blocked by the pure ER antagonist ICI 182,780 and is not apparent in ER-negative ovarian cells, indicating that SDF-1 regulation is ERalpha mediated. Treatment with the protein synthesis inhibitor cycloheximide had no effect on estradiol induction of induction of SDF-1 mRNA levels mRNA levels, demonstrating that SDF-1 is a direct target of ERalpha. SDF-1 protein levels, although undetectable under basal conditions, were strikingly increased by hormone both intracellularly and in the media of cultured BG-1 and MCF-7 cells. In cell proliferation assays, the mitogenic effects of estradiol were neutralized by addition of an SDF-1 antibody and mimicked by the addition of exogenous SDF-1 protein, indicating that SDF-1 mediates the proliferative actions of hormone. Furthermore, activation of the SDF-1 receptor CXCR4 stimulated BG-1 and MCF-7 cell proliferation in a manner comparable to estradiol. Taken together, these results demonstrate a novel estrogen-mediated paracrine pathway for inducing cancer cell proliferation and suggest that SDF-1 and CXCR4 may represent novel therapeutic targets in ERalpha-positive ovarian and breast tumors.

Breast Neoplasms↗

PubMed2003

Estrogen receptor beta (ERbeta) protein levels in neurons depend on estrogen receptor alpha (ERalpha) gene expression and on its ligand in a brain region-specific manner.

Estrogen receptors (ERs) alpha and beta are very similar estrogen-binding proteins, perhaps gene duplication products, which act as ligand-dependent transcription factors. While the estrogenic regulation of ERalpha has been well documented, little is known about how estrogen regulates ERbeta and whether ERalpha plays a role in the expression and estrogenic regulation of ERbeta. In the present study, we examined the effects of gonadectomy and estrogen replacement on ERbeta immunoreactivity (ir) in wild-type (WT) and ERalpha knockout (alphaERKO) adult male mice in six brain regions, the medial preoptic area (MPOA), the bed nucleus of the stria terminalis (BNST), the paraventricular nucleus (PVN), the ventromedial nucleus of hypothalamus (VMH), the medial amygdala nucleus (MeAMY) and the dorsal raphe nucleus (DRN). Mice were divided into four different treatment groups: gonadally intact, gonadectomized (GDX), GDX+short-term treatment with estrogen (s.c. injection of estradiol benzoate (EB), 5 microg, at 48 h before perfusion) or GDX+long-term treatment with estrogen (implant of an EB pellet, 2.5 microg/day, for 10 days). In intact alphaERKO mice, the number of ERbeta expressing cells was significantly decreased in the MPOA and increased in the BNST, compared to WT mice. Both in the MPOA and BNST, steroid hormone regulation of ERbeta protein (an increase by GDX and a decline to intact levels by EB) was found only in WT, not in alphaERKO mice. In the VMH, GDX significantly increased the number of ERbeta ir expressing cells in both genotypes. EB treatment tended to decrease the number of ERbeta ir cells in WT mice, whereas EB treatment tended to increase ERbeta ir cell counts in alphaERKO mice. No effects of GDX or EB treatment were found in the DRN and MeAMY regardless of genotype. These results suggest that gonadal steroid hormones may regulate ERbeta protein in male mice and ERalpha may be involved in the expression and regulation of ERbeta in a region-specific manner.

Aggression↗

PubMed2003

Molecular determinants of the stereoselectivity of agonist activity of estrogen receptors (ER) alpha and beta.

The two known estrogen receptors, ERalpha and ERbeta, are hormone-inducible transcription factors that have distinct roles in regulating cell proliferation and differentiation. Previously, our laboratory demonstrated that ERalpha exhibits stereoselective ligand binding and transactivation for several structural derivatives and metabolites of the synthetic estrogen diethylstilbestrol. We have previously described the properties of indenestrol A (IA) enantiomers on ERalpha. In the study presented here, the estrogenic properties of the S and R enantiomers of IA, IA-S and IA-R, respectively, were expanded to examine the activity in different cell and promoter contexts using ERalpha and ERbeta. Using human cell lines stably expressing either ERalpha or ERbeta, we found that IA-S was a more potent activator of transcription than IA-R through ERalpha in human endometrial Ishikawa and breast MDA-MB 231 (MDA) cells. Interestingly, IA-R was more potent on ERbeta when compared with ERalpha in MDA, but not in Ishikawa cells, and IA-R exhibited equally low binding affinities to ERalpha and ERbeta in vitro in contrast to its cell line-dependent preferential activation of ERbeta. Alignment of the protein structures of the ligand-binding domains of ERalpha and ERbeta revealed one mismatched residue, Leu-384 in ERalpha and Met-283 in ERbeta, which may be responsible for making contact with the methyl substituent at the chiral carbon of IA-S and IA-R. Mutagenesis and exchange of this one residue showed that the binding of IA-R and IA-S was not affected by this mutation in ERalpha and ERbeta. However, in transactivation studies, IA-R showed higher potency in activating L384M-mutated ERalpha and wild-type ERbeta compared with wild-type ERalpha and M283L-mutated ERbeta in all cell and promoter contexts examined. Furthermore, IA-R-bound ERalpha L384M and wild-type ERbeta displayed enhanced interactions with the nuclear receptor interaction domains of the coactivators SRC-1 and GRIP1. These data demonstrate that a single residue in the ligand-binding domain determines the stereoselectivity of ERalpha and ERbeta for indenestrol ligands and that IA-R shows cell type selectivity through ERbeta.

Breast↗

PubMed2003

Update on animal models developed for analyses of estrogen receptor biological activity.

Targeted disruption of the different ER genes has generated experimental animal models that are very useful in evaluating the distinct and cooperative roles of the two estrogen receptors, ERalpha and ERbeta, in reproductive but also non-reproductive tissues of both sexes. Phenotypic analysis has provided definitive experimental findings for estrogen receptor mediated physiological actions, involving ERalpha in uterine, mammary gland and neuroendocrine sites. ERbeta is involved most dramatically in the ovary as is ERalpha. More detailed studies in combination with tissue specific or inducible ER knock outs will be important for future research.

Animals↗

PubMed2003

Effect of exogenous epidermal-like growth factors on mammary gland development and differentiation in the estrogen receptor-alpha knockout (ERKO) mouse.

The development of the mouse mammary gland requires the interaction between several different ovarian and pituitary hormones such as estrogen, progesterone and prolactin as well as several locally-derived growth factors in the mammary gland such as epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha), amphiregulin (AR) and heregulin (HRG). The focus of this study was to investigate the degree of mammary growth and differentiation in the adult, virgin mammary gland of wild type (wt) and estrogen receptor knockout (ERKO) females that lack estrogen receptor alpha (ERalpha) after reciprocal transplantation into the cleared mammary fat pad of virgin wt or ERKO mice. In addition, we assessed the local response of ERKO mammary tissue to TGFalpha or HRGbeta1 delivered from slow release-Elvax pellets. Our initial results indicated that when we transplanted virgin wt mammary tissue into ERKO mammary fat pads, mammary morphogenesis failed to occur. However, when transplanted virgin ERKO mammary tissue was transplanted into fat pads of virgin or pregnant wt mice, the development and differentiation of lobuloalveoli was readily observed. In addition, treatment of the virgin ERKO mammary gland with TGFalpha or HRGbeta1 stimulated ducts to undergo localized branching and growth and both growth factors induced secretory differentiation as evidenced by the production of milk proteins, caseins and/or whey acidic protein (WAP). The results from this study imply that in ERKO mammary tissue. ERKO ductal epithelium has the capacity to proliferate and differentiate in response to non-estrogenic, morphogenic stimuli.

Animals↗

PubMed2003

Critical role for estrogen receptor alpha in negative feedback regulation of gonadotropin-releasing hormone mRNA expression in the female mouse.

Estrogen exerts an important regulatory influence upon the functioning of the gonadotropin-releasing hormone (GnRH) neurons. Whether this is mediated by estrogen receptor alpha (ERalpha) or ERbeta or both ERs is presently unclear. Using female mice with targeted disruptions of ERalpha and ERbeta (alphaERKO and betaERKO, respectively) we have investigated the in vivo role of the two ERs in the negative feedback influence of estrogen upon GnRH mRNA expression. Compared with intact wild-type mice, plasma luteinizing hormone (LH) levels were substantially (p < 0.01) higher in intact alphaERKO females and increased modestly (p < 0.05) in intact betaERKO mice. Three weeks after ovariectomy, LH concentrations were elevated significantly in wild-type (p < 0.01) and betaERKO (p < 0.05) mice but not changed in alphaERKO females. Quantitative analysis of GnRH mRNA expression using in situ hybridization revealed that cellular GnRH mRNA content was greater (p < 0.05) in intact alphaERKO mice compared with intact wild-type and betaERKO mice. Following ovariectomy, GnRH mRNA expression was elevated in wild-type (p = 0.06) and betaERKO (p < 0.05) females but not alphaERKO mice. These data demonstrate that both ERalpha and ERbeta are involved in inhibiting LH levels at times of estrogen-negative feedback in vivo. However, only ERalpha appears to be critical for the estrogen-negative feedback suppression of GnRH mRNA expression in the female mouse.

Analysis of Variance↗