ERα In Mice Research Articles

Efficient in vivoknock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA

BackgroundAdenovirus (Ad) mediated gene transfer is a well-established tool to transiently express constructs in livers of mice in vivo. In the present study, we determined the specificity and efficiency of Ad vectors expressing short hairpin (sh) RNA constructs to knock-down the estrogen receptor α (ERα).ResultsTwo different shRNA constructs derived from the murine ERα coding sequence were designed (shERα). In vitro, transfection of three mouse cell lines with pSUPER-shERα constructs resulted in up to 80% reduction of endogenous ERα activity. A single mismatch in the target sequence eliminated the reduction of ERα activity, demonstrating the specificity of shERα. The subsequently generated Ad.shERα vectors were equally effective in vitro. In vivo, intravenous administration of Ad.shERα resulted in 70% reduced hepatic mouse ERα mRNA levels. Co-injection of Ad.shERα with an Ad vector containing a luciferase (luc) gene driven by an estrogen responsive element (ERE) containing promoter resulted in a significant (90% on day five) down-regulation of hepatic luciferase activity, as determined by non-invasive optical imaging. Down-regulation was sustained up to day seven post-injection.ConclusionAd mediated transfer of shERα expression constructs results in efficient and specific knockdown of endogenous ERα transcription both in vitro and in vivo.

Estrogen receptor α and β subtype expression and transactivation capacity are differentially affected by receptor-, hsp90- and immunophilin-ligands in human breast cancer cells

In MCF-7 (estrogen receptor (ER)+) and in MDA-MB-231 (ER−) cells stably transfected with either estrogen receptor α (ERα) or β (ERβ) subtype (MDA-MB-231 stably transfected with the mouse ERα cDNA (MERA) and MDA-MB-231 stably transfected with the human ERβ cDNA (HERB), respectively) N-term heat shock protein of 90 kDa (hsp90) ligands (geldanamycin and radicicol) and C-term hsp90 ligands (novobiocin) decrease the basal and estradiol (E 2)-induced transcription activity of ER on an estrogen responsive element (ERE)-LUC reporter construct concomitantly with or 1 h after E 2 treatment. All hsp90 ligands induced an E 2- and MG132-inhibited decrease of both ER cell content. However, the kinetics of these degradations are slower than those induced by the selective estrogen receptor down-regulator RU 58668 (RU). This suggests that inhibition of the hsp90 ATPase activity targets both ERs to the 26S proteasome and that hsp90 interacts with both ER subtypes. Rapamycin (Rapa) and cyclosporin A (CsA), ligands of immunophilins FK506 binding protein (FKBP52) and cyclophilin of 40 kDa (CYP40) interacting in separate ER–hsp90 complexes, both induced a proteasomal-mediated degradation of ERs but not of their cognate immunophilin. Moreover, they also decrease the E 2-induced luciferase transcription but weaker than RU and hsp90 ligands. Fluorescence activated cell sorter (FACS) analysis revealed a blockade of cell progression by RU and 4-hydroxy-tamoxifen at the G 1 phase of the cell cycle and an induction of apoptosis in MCF-7 cells. Rapa and mainly CsA (but not FK506) and hsp90 ligands promote by their own apoptosis in MCF-7, in MERA, and in HERB cells and in MDA-MB-231 ER-null cells. These data suggest that (1) hsp90, as for all steroid receptors, acts as a molecular chaperone for ERβ; (2) ER-ligands (except tamoxifen), hsp90- and immunophilin-ligands (except FK506) target the two ER subtypes to a proteasome-mediated proteolysis via different signalling pathways; (3) hsp90- and immunophilin-ligands Rapa and CsA, alone or in association with anti-estrogens such as RU, may constitute a potential therapeutic strategy for breast cancer treatment.

Plasma membrane estrogen receptors exist and functions as dimers.

A small pool of estrogen receptors (ERalpha and -beta) localize at the plasma membrane and rapidly signal to affect cellular physiology. Although nuclear ERs function mainly as homodimers, it is unknown whether membrane-localized ER exists or functions with similar requirements. We report that the endogenous ER isoforms at the plasma membrane of breast cancer or endothelial cells exist predominantly as homodimers in the presence of 17beta-estradiol (E2). Interestingly, in endothelial cells made from ERalpha /ERbeta homozygous double-knockout mice, membrane ERalpha or ERbeta are absent, indicating that the endogenous membrane receptors derive from the same gene(s) as the nuclear receptors. In ER-negative breast cancer cells or Chinese hamster ovary cells, we expressed and compared wild-type and dimer mutant mouse ERalpha. Only wild-type ERalpha supported the ability of E2 to rapidly activate ERK, cAMP, and phosphatidylinositol 3-kinase signaling. This resulted from E2 activating Gsalpha and Gqalpha at the membrane in cells expressing the wild-type, but not the dimer mutant, ERalpha. Intact, but not dimer mutant, ERalpha also supported E2-induced epidermal growth factor receptor transactivation and cell survival. We also confirmed the requirement of dimerization for membrane ER function using a second, less extensively mutated, human ERalpha. In summary, endogenous membrane ERs exist as dimers, a structural requirement that supports rapid signal transduction and affects cell physiology.

Genomic structure and identification of a truncated variant message of the mouse estrogen receptor α gene

Estrogen receptor alpha (ERα) is a ligand-dependent transcription factor that directs the transcription of a wide number of estrogen-regulated genes. ERα mediates the effects of 17-β-estradiol in both males and females, and was the first estrogen receptor identified. Despite the cloning of the mouse ERα cDNA over 15 years ago, the precise genomic organization of the mouse ERα gene has not yet been elucidated. In order to determine the structure of this gene, overlapping BAC and P1 clones containing partial genomic sequences of the mouse ERα cDNA were obtained from a mouse ES cell genomic library. Using standard restriction fragment analysis followed by Southern blotting, the mouse ERα gene was determined to be greater than 220 kb in length. The introns vary widely in size, from 1.8 to 60 kb in length. Sequencing of intron–exon boundaries shows that these boundaries are highly conserved between the human and mouse ERα genes. Additionally, we have identified a splice variant message of mouse ERα arising from a failure to properly splice at the 3′ end of exon 4; the resulting message is predicted to produce a protein lacking the ligand-binding domain. Variant message was detected by RT-PCR in several tissues, including uterus, ovary, mammary gland, placenta and testis.

Cloning and characterization of the functional promoter of mouse estrogen receptor β gene

The recently discovered estrogen receptor β (ERβ) exhibits some properties distinct from those of the classical estrogen receptor, ERα. To elucidate the mechanism underlying the regulation of ERβ gene expression, we cloned and characterized the 5′-flanking (promoter) region of the mouse ERβ (mERβ) gene. A TATA-like motif was found in the 5′-flanking region, and transcription initiation sites were mapped 24 bp and 27 bp downstream from the motif by primer extension analysis and 5′-rapid amplification of cDNA ends. The mERβ promoter contains several putative cis-acting elements, many of which are also found in the mouse ERα promoter. Therefore, the expression of both ERs may be partially regulated by a common mechanism. Luciferase assays revealed that the mERβ promoter activity paralleled the endogenous expression levels of mERβ in several cell lines. Successive 5′-deletion analyses showed that element(s) critical for its basal activity and negative regulatory element(s) are located in the sequences −90/+33 and −505/−372, respectively. The characterization of the mERβ promoter will allow further studies to investigate the transcriptional regulation of mERβ.

New Solutions to an Ancient Riddle: Defining the Differences between Adam and Eve

As our current knowledge of the mammalian sex-differentiation pathway expands, a connection of this process to the potential genetic regulation of sexual behavior and sex-specific development of the brain is natural. At least in invertebrate systems, a tangible genetic link has been forged between the sex-development pathway and normal courtship behavior. Two genes, fruitless and dissatisfaction, are now known to contribute to normal male courtship behavior, by specifying the identity of neurons required for execution of the reproductive behaviors. Additionally, these genes begin to define new branches of the Drosophila sex-determination pathway that are downstream of the transformer gene (tra) and apparently independent of one another (Finley et al. 1998xDissatisfaction encodes a tailless-like nuclear receptor expressed in a subset of CNS neurons controlling Drosophila sexual behavior. Finley, KD, Edeen, PT, Foss, M, Gross, E, Ghbeish, N, Palmer, RH, Taylor, BJ et al. Neuron. 1998; 21: 1363–1374Abstract | Full Text | Full Text PDF | PubMed | Scopus (70)See all References1998, and references therein). These findings offer an interesting new perspective for those contemplating the potential mechanisms governing sex differences in the mammalian brain.Previous studies have shown that steroids affect sex-specific brain development during critical periods before and after birth, with consequences for adult behavior (for review, see Breedlove 1992xSexual dimorphism in the vertebrate nervous system. Breedlove, SM. J Neurosci. 1992; 12: 4133–4142PubMedSee all References1992). For instance, perinatal exposure of female animals to sex steroids can masculinize adult behaviors, and androgens can alter the morphology of brain nuclei, such as in the case of the sexually dimorphic nucleus of the preoptic area of the hypothalamus. Defining the precise contributions of individual nuclei to adult sexual behavior has been difficult. Moreover, the effects of sex steroids on brain development are complex, because these steroids are locally metabolized, as in the case of androgens. One major metabolite of the aromatization of testosterone is estrogen, which is generally assumed to be required in male-specific brain development. The persistence of a masculine preoptic area in androgen-insensitive rats suggests that some developmental effects of testosterone occur after aromatization to estrogen (Breedlove 1992xSexual dimorphism in the vertebrate nervous system. Breedlove, SM. J Neurosci. 1992; 12: 4133–4142PubMedSee all References1992). Interestingly, deficiency for the estrogen-receptor subtype ERα in mice disrupts male-specific aggressive and mating behaviors but does not affect male sexual motivation (Ogawa et al. 1997xBehavioral effects of estrogen receptor gene disruption in male mice. Ogawa, S, Lubahn, DB, Korach, KS, and Pfaff, DW. Proc Natl Acad Sci USA. 1997; 94: 1476–1481Crossref | PubMed | Scopus (314)See all References1997). Moreover, male mice lacking aromatase are fully capable of breeding (Fisher et al. 1998xCharacterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene. Fisher, CR, Graves, KH, Parlow, AF, and Simpson, ER. Proc Natl Acad Sci USA. 1998; 95: 6965–6970Crossref | PubMed | Scopus (629)See all References1998). These recent studies underscore how little is understood regarding the molecular basis of sex steroids and their cognate nuclear receptors, in brain development and sex differences in the brain.Continued molecular and genetic investigations will be required to identify which steroids and what nuclei or brain regions are involved in sexually dimorphic brain development. These studies promise to be challenging, given the complexity of (a) steroid action on early brain development and modulation of adult behavior and (b) the indirect effects of steroid action on peripheral tissues. It will be of great interest to know whether mammals use a steroid-independent arm of the sex-determination cascade to dictate sex differences in the brain, as Drosophila does. During the next decade, the convergence of vertebrate genetics, behavior analyses, and genomics should elucidate the remaining mysteries of gonadal development and may begin to provide a genetic rationale for neurological and behavioral differences between men and women.