The high mobility group protein 1 enhances binding of the estrogen receptor DNA binding domain to the estrogen response element

We have examined the ability of the high-mobility group protein 1 (HMG1) to alter binding of the estrogen receptor DNA-binding domain (DBD) to the estrogen response element (ERE). HMG1 dramatically enhanced binding of purified, bacterially expressed DBD to the consensus vitellogenin A2 ERE in a dose-dependent manner. The ability of HMG1 to stabilize the DBD-ERE complex resulted in part from a decrease in the dissociation rate of the DBD from the ERE. Antibody supershift experiments demonstrated that HMG1 was also capable of forming a ternary complex with the ERE-bound DBD in the presence of HMG1-specific antibody. HMG1 did not substantially affect DBD-ERE contacts as assessed by methylation interference assays, nor did it alter the ability of the DBD to induce distortion in ERE-containing DNA fragments. Because HMG1 dramatically enhanced estrogen receptor DBD binding to the ERE, and the DBD is the most highly conserved region among the nuclear receptor superfamily members, HMG1 may function to enhance binding of other nuclear receptors to their respective response elements and act in concert with coactivator proteins to regulate expression of hormone-responsive genes.     

Endothelin-1 and endothelin receptors are present in the sheep uterus and conceptus at implantation

Previous studies have demonstrated that endothelin is present in the ovine endometrium and increases at around the expected time of implantation. To characterize further uterine endothelin at the time of establishment of pregnancy in sheep, endothelin was measured by radioimmunoassay in uterine flushings obtained during the oestrous cycle and in pregnant ewes up to the time of implantation (day 16). During the oestrous cycle, the highest amounts of endothelin were present in uterine flushings on day 14 (1.1 +/- 0.2 ng endothelin/uterus). During early pregnancy, basal levels of endothelin (0.5-0.6 ng endothelin/uterus) were present in uterine flushings for the first 10 days and then increased on day 14 to levels similar to those found at the equivalent stage of the oestrous cycle. On days 15 and 16 of pregnancy, endothelin content in the uterine lumen increased to significantly (P < 0.05) higher concentrations (2.9 +/- 0.4 ng endothelin/uterus) when compared with the non-fertile cycle. The principal isoform present in flushings at the time of implantation was endothelin-1, as determined by reverse-phase HPLC. Endothelin was released principally by purified endometrial epithelial cells in culture, with barely detectable amounts released by endometrial stromal cells or conceptus tissue, which is consistent with the epithelium being the principal source of endothelin in the uterine lumen. Endothelin binding sites were present in endometrium and myometrium, as demonstrated by specific binding of 125I-labelled endothelin-1, which was saturable and displaced by endothelin-1. Both endothelinA and B sub-types of receptors were present as demonstrated by the biphasic displacement of 125I-labelled endothelin-1 binding by the specific endothelinB agonist BQ3020. These were localised principally on luminal and glandular epithelium and in the vasculature of the endometrium and myometrium as shown by autoradiography. Endothelin receptors were also present on the conceptus obtained at the time of implantation. In the day 20 conceptus, endothelin immunostaining was localised principally in the heart, in trophoblast in uninucleate but not in binucleate cells, and in fetal membranes. This immunostaining of the conceptus may represent binding to receptor sites. It is concluded that endothelin-1 is present in the uterine lumen and may play an important role in the paracrine regulation of the conceptus and endometrium at the time of rapid embryo development, implantation and early placentation.     

DNA binding of hypothalamic nuclear proteins on estrogen response element and preproenkephalin promoter: modification by estrogen

Preproenkephalin (PPE) gene expression is specifically induced by estrogen in hypothalamus of ovariectomized (OVX) females, better than in male rats. To study estrogen actions on gene regulation, we have presently characterized protein-DNA interactions by use of a consensus estrogen response element (ERE) and a putative ERE from PPE gene, with nuclear extracts from hypothalamus. By use of the electrophoretic mobility shift assay (EMSA), ERE binding activity was detected in nuclear extracts from neuronal tissues including hypothalamus, hippocampus, striatum, cerebellum and frontal cortex, and non-neuronal tissues such as pituitary and uterus, but not lung of OVX female rats with a consensus ERE, as well as a 129-bp PCR fragment from PPE promoter and a hairpin oligonucleotide that contains a putative ERE of the rat PPE gene. The ERE binding was eliminated by the addition of specific ERE-containing oligonucleotide, but not control oligonucleotides. Protein and DNA associated and dissociated very rapidly. By use of supershift assay, interactions of estrogen receptor with ERE were demonstrated in hypothalamic nuclear extracts. The initial levels of specific ERE binding in the hypothalamic nuclear extracts were comparable between castrated male and OVX female rats. However, estrogen treatment, either estradiol or estradiol benzoate, produced a rapid and tissue-specific induction of a slow mobility complex of ERE binding in hypothalamic nuclear extracts from females, better than in male rats, presumably from other associated factors, or a conformational change or other posttranslational modifications. This estrogen-induced slow mobility complex of ERE binding in hypothalamus was not observed after treatment with progesterone or tamoxifen. These results suggest that specific ERE binding is present in rat hypothalamic nuclear proteins, which may contribute to the upregulation of PPE gene expression by estrogen, and that the sexually differentiated action of estrogen may be related to an estrogen-induced conformational change, but not to the initial level of ERE-binding activity.     

BRCA1 and BRCA2 mRNA levels are coordinately elevated in human breast cancer cells in response to estrogen

The steady state levels of BRCA1 and BRCA2 mRNAs were shown to be coordinately elevated by the steroid hormone estrogen but not progesterone in the human breast cancer cell lines BT-483 and MCF-7. Two different antiestrogens, trans 4'-hydroxytamoxifen and ICI 182,780, blocked the elevation of BRCA1 and BRCA2 mRNA levels, confirming that the effect was mediated through the estrogen receptor. In BT-483 cells, BRCA1 and BRCA2 mRNA levels were both elevated 18 to 24 h after estrogen stimulation, suggesting that the effect of estrogen was indirect. Cycloheximide blocked the estrogen effect implying that estrogen induces synthesis of an unidentified estrogen-responsive protein(s) that then result in the elevation of BRCA1 and BRCA2 mRNAs.     

Transcriptional regulation of lysosomal acid lipase in differentiating monocytes is mediated by transcription factors Sp1 and AP-2

The spectrin membrane skeleton is a ubiquitous cytoskeletal structure with several cellular roles, including the maintenance of cell integrity, determination of cell shape and as a contributor to cell polarity. We have isolated mutations in the gene encoding &bgr ;Heavy-spectrin in Drosophila, and have named this essential locus karst. karst mutant individuals have a pleiotropic phenotype characterized by extensive larval lethality and, in adult escapers, rough eyes, bent wings, tracheal defects and infertility. Within karst mutant eyes, a significant number of ommatidia specifically lack photoreceptor R7 alongside more complex morphological defects. Immunolocalization of betaHeavy-spectrin in wild-type eye-antennal and wing imaginal discs reveals that betaHeavy-spectrin is present in a restricted subdomain of the membrane skeleton that colocalizes with DE-cadherin. We propose a model where normal levels of Sevenless signaling are dependent on tight cell-cell adhesion facilitated by the betaHeavy-spectrin membrane skeleton. Immunolocalization of betaHeavy-spectrin in the adult and larval midgut indicates that it is a terminal web protein, but we see no gross morphological defects in the adult apical brush border in karst mutant flies. Rhodamine phalloidin staining of karst mutant ovaries similarly reveals no conspicuous defect in the actin cytoskeleton or cellular morphology in egg chambers. This is in contrast to mutations in alpha-spectrin, the molecular partner of betaHeavy-spectrin, which affect cellular structure in both the larval gut and adult ovaries. Our results emphasize the fundamental contribution of the spectrin membrane skeleton to normal development and reveals a critical interplay between the integrity of a cell's membrane skeleton, the structure of cell-cell contacts and cell signaling.     

Inhibition of gene expression by steroid hormone receptors via a negative glucocorticoid response element: evidence for the involvement of DNA-binding and agonistic effects of the antiglucocorticoid/antiprogestin RU486

We have used a negative glucocorticoid response element (nGRE) from the bovine prolactin promoter linked to the gene for chloramphenicol acetyltransferase (PRL3CAT) to study the inhibition of gene expression by steroid hormone receptors. This nGRE increased basal expression from a heterologous promoter in COS-7 cells. In the presence of cotransfected glucocorticoid (GR), androgen, or progesterone receptor (PR) expression vectors and their cognate ligands, the expression of PRL3CAT could be repressed, indicating that these steroid receptor subfamily members could function through the same negative response element. No repression was observed with the estrogen receptor, showing that the repressive effect was specific for members of the GR-subfamily. Mutation of three amino acids within the GR-DNA binding domain that determine the specificity of GR-GRE interaction abolished the ability of the GR to inhibit the expression of PRL3CAT, demonstrating the requirement for DNA binding of the GR in the mechanism of repression. The antiglucocorticoid/antiprogestin RU486 when bound to PR or GR also repressed the expression of the PRL3CAT, but higher concentrations of RU486 were required to obtain an effect with the GR when compared to the PR. RU486 was unable to antagonize the effect of progestins on PRL3CAT and only partially antagonized the glucocorticoid repression. Thus, regarding the repression of PRL3CAT, RU486 acted as an agonist when bound to the PR and as a partial agonist when bound to the GR.     

Pharmacological profiles of absence seizure-induced increases in CRE- and AP-1 DNA-binding activities in gamma-butyrolactone-treated mice

Absence seizures are characterised by a well-defined disturbance of thalamocortical function, and there is no spread to other systems. In this study, we continue our examination of the mechanisms underlying the increased nuclear cyclic AMP responsive element (CRE)- and activator protein 1 (AP-1) DNA-binding activities in a gamma-butyrolactone (GBL)-induced mouse model of absence seizure. The administration of GBL increased CRE- and AP-1 DNA-binding activities in the cerebral cortex and thalamus, but not in other regions such as the hippocampus, cerebellum or pons + medulla oblongata, at doses which induced absence seizures. Not only the absence-seizure behavior but also the increased CRE- and AP-1 DNA-binding activities in the thalamocortical regions were reversibly inhibited by ethosuximide, a typical anti-absence drug, and the GABAB antagonists CGP 35348 and CGP 46381. A gel-supershift assay revealed that the GBL-induced CRE-binding activity was supershifted by an anti-CRE-binding protein (CREB) antibody, and that AP-1 DNA-binding activity was blocked by anti-c-Jun and anti-c-Fos antibodies. These results suggest that increased CRE- and AP-1 DNA-binding activities in the cerebral cortex and thalamus are related to the pathogenesis of generalized absence seizures and that these increases in DNA-binding activity are related to ethosuximide- and GABAB antagonist-sensitive abnormal neuronal activity in the thalamocortical circuit.