Nuclear matrix acceptor binding sites for steroid hormone receptors: a candidate nuclear matrix acceptor protein.

Steroid/nuclear-hormone receptors are ligand-activated transcription factors that have been localized to the nuclear matrix. The classic model of hormone action suggests that, following activation, these receptors bind to specific "steroid response elements" on the DNA, then interact with other factors in the transcription initiation complex. However, evidence demonstrates the existence of specific chromatin proteins that act as accessory factors by facilitating the binding of the steroid receptors to the DNA. One such protein, the "receptor binding factor (RBF)-1", has been purified and shown to confer specific, high-affinity binding of the progesterone receptor to the DNA. Interestingly, the RBF-1 is localized to the nuclear matrix. Further, the RBF-1 binds specifically to a sequence of the c-myc proto-oncogene that has the appearance of a nuclear matrix attached region (MAR). These results, and other findings reviewed here, suggest that the nuclear matrix is involved intimately in steroid hormone-regulated gene expression.

Progesterone substitutes: cGMP mediation.

Over the past 20 years many investigators have shown that one can facilitate sexual receptivity in estrogen-primed rats either by giving progesterone or a drug which stimulates or inhibits a neurotransmitter system. Drugs which have been reported to substitute for progesterone include cholinergic agonists, serotonergic agonists and antagonists, dopaminergic agonists and antagonists, opiate antagonists, neurohormones, pituitary, ovarian and adrenal hormones and drugs that interact with cyclic nucleotide systems. Most of the drugs that are active are known to increase neural levels of cyclic GMP either by acting on guanylate cyclase or on phosphodiesterase. We propose that the cGMP system mediates the common behavioral effect of the wide variety of drugs that facilitate receptivity.

Estrogen receptor messenger RNA expression in rat hypothalamus as a function of genetic sex and estrogen dose.

Previously, we showed that estrogen receptor (ER) messenger RNA (mRNA) levels are decreased in cells of the mediobasal hypothalamus of ovariectomized (OVX) female rats following an acute estradiol treatment. Here, we examined whether the level of ER mRNA remains depressed in the continued long-term presence of estradiol, and questioned if there is a systematic relationship between the concentration of estradiol and the decrease in ER mRNA level. OVX female rats were implanted for 2 weeks with silastic capsules containing various concentrations of estradiol. Tissue sections were hybridized with a [3H] single-stranded DNA probe prepared from the region of the rat ER complementary DNA corresponding to the steroid binding domain, and relative mRNA level was assessed by counting grains over cells in specific hypothalamic nuclei. Estradiol induced a dose-dependent decrease in ER mRNA levels. Message levels declined in the ventrolateral aspect of the ventromedial nucleus (VLVM) by 57% and in the arcuate nucleus by 62% at the highest hormone concentrations used. Thus, ER mRNA down-regulation in female rat hypothalamus exhibits orderly dose dependence at a time following hormone treatment which ensures the system is at steady state. A second study determined if there exist differences in basal levels of ER mRNA expression between castrated (CAS) females and males, and if estradiol can down-regulate ER mRNA levels in male hypothalamus. CAS rats of both sexes were exposed acutely to estradiol benzoate (EB) for different periods of time. Again, in females, EB significantly decreased ER mRNA levels in VLVM by 55% (18 h) and in the arcuate nucleus by 74% (18 h). Interestingly, control CAS males had significantly lower basal ER mRNA levels than OVX females (52% lower than female levels in VLVM; 56% in arcuate), suggesting a sex difference in constitutive expression levels. Moreover, EB failed to down-regulate significantly ER message levels in males. There was no significant effect of sex or EB treatment on ER mRNA levels in medial amygdala. Thus, the second study shows sex differences and brain-region specificity in hormonal regulation of ER mRNA. These findings show that differences in basal levels and regulation of ER mRNA could be a substrate for sex differences in ER concentrations in the hypothalamus of the rat, and further raise the possibility of sex differences in concentrations of nuclear proteins related to the control of ER gene expression.

Bimodal regulation of epidermal growth factor receptor by estrogen in breast cancer cells.

In breast cancer, epidermal growth factor (EGF) receptor (EGFR) expression is inversely correlated with expression of estrogen receptor (ER) and predicts the prognosis and failure of endocrine therapy. We report here, for the first time, that in ER-positive breast cancer cell lines, MCF-7, T47D, and BT474, 17 beta-estradiol (E2) transiently induced EGFR messenger RNA (mRNA) levels 2- to 3-fold; this induction was prevented by the presence of the antiestrogen ICI 164,384 and was also reflected in the level of EGFR protein. Up-regulation of EGFR mRNA is most likely due to a direct effect of ER on the EGFR gene, with no involvement of protein synthesis, as it was not inhibited in the presence of cycloheximide; however, the subsequent down-regulation of EGFR required de novo protein synthesis. E2 had no effect on EGFR mRNA stability, and EGFR transcript levels were found to parallel EGFR mRNA levels, further supporting a direct transcriptional mechanism in the regulation of EGFR expression by estrogens. Additionally, sequencing of the EGFR promoter revealed putative imperfect estrogen-responsive elements that were capable of binding human ER. The transient nature of EGFR induction by E2, with a rapid return to a basal level that is dependent on protein synthesis, suggests that breast cancer cells possess active mechanisms to maintain low levels of EGFR expression in the presence of estrogen and a functional ER.

Estradiol induction of proenkephalin messenger RNA in hypothalamus: dose-response and relation to reproductive behavior in the female rat.

Hormone effects on proenkephalin (PE) mRNA allow an opportunity to compare a brain region-specific molecular change with a quantifiable behavior. Slot blots were used to measure PE mRNA levels in the ventromedial hypothalamus (VMN) and preoptic area (POA) as a function of the dose of estrogen administered to ovariectomized rats. Every rat used had been characterized for the ability to display lordosis behavior. Estradiol treatment led to a monotonic dose-dependent increase in PE mRNA level in VMN, while only a small effect was observed in POA at the higher estradiol doses. Lordosis behavior, assessed manually and in mating tests, also increased monotonically with estradiol dose. The data indicate that an apparent 'threshold' level of PE mRNA in VMN coincided wit display of behavior, and suggest further that high levels of PE mRNA, alone, are not sufficient for lordosis. While the exact relationship of the eventual product, Met-enkephalin, to female reproductive behavior remains to be determined, the parallel changes in PE mRNA level and behavior encourage further analysis.

Estradiol Regulation of Estrogen Receptor Messenger Ribonucleic Acid in Rat Mediobasal Hypothalamus: An in situ Hybridization Study.

Abstract We have used in situ hybridization to investigate estradiol regulation of estrogen receptor (ER) mRNA in regions of the mediobasal hypothalamus which contain ER and are related to specific neuroendocrine functions. Ovariectomized rats were treated with oil or 10 mug estradiol benzoate for 2, 4, 18 or 24 h. Brains were sectioned and hybridized with a [(3) H]single-stranded DNA probe prepared from the pORF cDNA of the human ER gene and exposed to autoradiographic emulsion for 4 months. Specificity of labeling was determined by counting the number of grains over cells in hypothalamic regions known to bind estradiol, compared to cells in the thalamus and cortex, and by comparing with sections pretreated with ribonuclease or hybridized with a [(3) H]single-stranded message-sense (control) probe. Labeling for ER mRNA was distributed differently than glucocorticoid and thyroid hormone receptor mRNAs, and was regulated by estrogen differently than progestin receptor mRNA. These differences indicated specific hybridization for ER mRNA. ER-expressing cells constituted 11.5% of the cells in the dorsomedial nucleus, 30% of the cells in the arcuate nucleus and 43% in the ventromedial nucleus, in close accordance with previous studies of ER autoradiography and binding. Quantitative analysis showed that the highest level of ER mRNA was present in the ovariectomized controls. ER mRNA levels fell 42% (ventromedial), 64% (arcuate), or remained unchanged (dorsomedial) 18 h following estradiol benzoate treatment. The pattern of decrease was similar for cells in the ventromedial nucleus and arcuate nucleus. These data show that estrogen regulation of ER mRNA in brain parallels that reported for MCF-7 cells and rat uterus. These results also demonstrate that in situ hybridization can be used to detect and measure the relative level of a low abundance mRNA in a heterogeneous tissue in which only 12% to 40% of the cells in limited regions express the message.

Gene expression for estrogen and progesterone receptor mRNAs in rat brain and possible relations to sexually dimorphic functions.

A clear neuroendocrine sex difference lies in the ability of the female rat to produce an ovulatory surge of luteinizing hormone. Preoptic neurons, as they respond to estrogen and progesterone, have been proven to be involved in this mechanism, with an emphasis on the possible participation of neurons in the anteroventral periventricular nucleus and the suprachiasmatic portion of the preoptic area (POA). Further, prominent morphological sex differences have been reported in the rat medial POA. To examine expression of the estrogen receptor (ER) and the progesterone receptor (PR) messenger RNAs (mRNAs) in these critical preoptic neurons, we have used in situ hybridization with tritiated single-stranded DNA probes complimentary for ER and PR mRNA. ER mRNA containing cells were found in the periventricular, suprachiasmatic and medical preoptic cell groups, in a manner which agrees with steroid hormone autoradiography. In the female rat, preoptic neurons expressing PR mRNA were distributed very similarly to those for ER mRNA. Moreover, in the male rat brain, all subsets of preoptic neurons which express the PR gene in the female were also detected in the male. Thus, the distribution of PR expressing cells was very similar between females and males. We conclude that the insensitivity to the male to progesterone, as regards the hormonal control of ovulation, cannot be due to a total failure of PR gene expression in a specific subset of POA neurons. Instead, male preoptic neurons must be less sensitive to neural or hormonal inducers in the physiological range or perhaps lack sufficient levels of a transcription factor linking progesterone responsive elements to the start sites of hormone-controlled genes.

Bethanechol-induced increase in hypothalamic estrogen receptor binding in female rats is related to capacity for estrogen-dependent reproductive behavior.

Neuroactive agents associated with different neurotransmitter systems can modulate the number of hypothalamic estrogen binding sites. It has been demonstrated previously that the muscarinic cholinergic agonist, bethanechol, administered 30 min prior to in vitro estrogen receptor assays increases the concentration of hypothalamic estrogen binding sites by 30-35% in female rats. Bethanechol was without effect on male hypothalamic preparations. In order to investigate further this sex difference and in an attempt to determine a relationship between the modulation of estrogen binding sites and a sexually differentiated function, bethanechol was given to female rats rendered either anovulatory and capable of displaying lordosis or anovulatory and behaviorally insensitive to estrogen. The results showed that bethanechol significantly increased the number of estrogen binding sites in females capable of displaying lordosis but not in females which did not show this estrogen-dependent behavior. It is possible that the capacity for drug-induced modulation of estrogen binding sites could be related functionally to the ability to display lordosis behavior.

Muscarinic cholinergic modulation of hypothalamic estrogen binding sites.

Studies from other laboratories have demonstrated that agents which interact with the dopaminergic and noradrenergic neurotransmitter systems alter the concentrations of cytosolic hypothalamic estrogen receptors. These results have led to the hypothesis that catecholamine systems are involved intimately with the regulation of brain estrogen receptors. The present study was undertaken to determine if agents from a different neurotransmitter system similarly affect [3H]estradiol binding. The data presented here show that the muscarinic cholinergic agonist, bethanechol, increases the number of cytosolic hypothalamic estradiol binding sites in ovariectomized female rats by as much as 38% above control values. Pretreatment with atropine sulfate, a highly specific muscarinic antagonist, blocked the bethanechol effect. Interestingly, bethanechol failed to alter the concentration of estradiol binding sites in castrated male rats. The results of the present experiments show not only that pharmacological modulation of cytosolic hypothalamic estradiol binding sites is not limited to drugs which interact with catecholaminergic systems, but that such effects may be sex-specific.

Atropine sulfate modulates estrogen binding by female, but not male, rat hypothalamus.

Studies have shown that pharmacological manipulation of the dopamine, norepinephrine and muscarinic cholinergic neurotransmitter systems modulates the number of neural estrogen binding sites. Previously, we reported that the muscarinic agonist, bethanechol, increased estrogen receptor binding by hypothalamic cytosols from female, but not male, rats. Moreover, pretreatment with atropine prevented the bethanechol-induced effect. The experiments reported here were executed with the expectation that atropine alone would either decrease or fail to alter estrogen binding. However, the present data show that atropine increases estrogen binding by female, but not male, hypothalamic cytosols. Thus, it appears that a muscarinic antagonist and agonist can similarly affect the concentration of estrogen binding sites in female rat hypothalamus.

Off-vertical rotation produces conditioned taste aversion and suppressed drinking in mice.

The effects of off-vertical rotation upon the intake of tap water immediately after rotation, and upon conditioned taste aversion, were assessed in mice with the tilt of the rotation axis varying from 5 to 20 degrees from the earth-vertical. Conditioned taste aversion occurred in all mice that were rotated, but the intake of tap water was suppressed only in mice that were rotated at 15 or 20 degrees of tilt. The greater suppression of tap water intake and the stronger conditioned aversion in the mouse as the angle of tilt was increased in this experiment are consistent with predictions from similar experiments with human subjects where motion sickness develops more rapidly as the angle of tilt is increased. It was suggested that off-vertical rotation may be a useful procedure for insuring experimental control over vestibular stimulation in animal studies of motion sickness.

Sex difference in estradiol regulation of progestin receptor mRNA in rat mediobasal hypothalamus as demonstrated by in situ hybridization.

Previous studies have shown that estrogen increases the level of progestin receptors (PR) to a greater extent in female than in male rat hypothalamus. In order to determine if sex-specific regulation of the PR protein might be attributable to estrogenic effects on the PR message, in situ hybridization was used to assess sex differences in levels of estrogen-inducible PR mRNA in specific brain nuclei. Here, we report a sexually differentiated pattern of estrogen-regulated PR gene expression. In female hypothalamus, estrogen administered to gonadectomized rats induced a 3.6- and a 3.3-fold increase in PR mRNA in the ventrolateral aspect of the ventromedial nucleus and arcuate nucleus, respectively, but failed to alter the level of PR mRNA in the same neuronal groups of the male. Hormone treatment did not affect the levels of PR mRNA in the dorsomedial or medial amygdaloid nuclei of either sex. These results lead towards a molecular explanation of sex differences in female reproductive behavior by revealing an estrogen-dependent up-regulation of the message for PR, a transcription factor, in a region- and sex-specific fashion.

A DNA-binding element for a steroid receptor-binding factor is flanked by dual nuclear matrix DNA attachment sites in the c-myc gene promoter.

The receptor-binding factor (RBF) for the avian oviduct progesterone (Pg) receptor (PR) has previously been shown to be a unique 10-kDa nuclear matrix protein that generates high affinity PR-binding sites on avian DNA. This paper describes the use of Southwestern blot and DNA gel shift analyses with RBF protein to identify a minimal 54-base pair RBF-binding element in the matrix-associated region (MAR) of the Pg-regulated c-myc gene promoter. This element contains a 5'-GC-rich domain and a 3'-AT-rich domain, the latter of which has a homopurine/homopyrimidine structure. The gel shift assays required the generation of an RBF-maltose fusion protein (RBF-MBP), which specifically binds this element and is supershifted when the anti-RBF polyclonal antibody is added. Computer analysis of the full-length amino acid sequence for RBF predicts a DNA-binding motif involving a beta-sheet structure at the N-terminal domain. Southern blot analyses using nuclear matrix DNA suggests that there are dual MAR sites in the c-myc promoter, which flank an intervening domain containing the RBF element. The co-transfection of this MAR sequence, containing the RBF element and cloned into a luciferase reporter vector, together with an RBF expression vector construct, into steroid treated human MCF-7 cells, results in a decrease of the c-myc promoter activity relative to control transfections containing only the parent vector of the RBF expression construct. These data suggest that a unique chromatin/nuclear matrix structure, composed of the RBF-DNA element complex which is flanked by nuclear matrix attachment sites, serves to bind the PR and repress the c-myc promoter.

Mechanisms of EGF receptor regulation in breast cancer cells.

Overexpression of the EGF receptor in breast cancer correlates with poor prognosis and failure on endocrine therapy for both ER-/EGFR+ and ER+/EGFR+ tumors, suggesting a role for EGFR in the progression to hormone independence. The identification of specific DNAse I hypersensitive site patterns for the EGFR gene in ER+ vs. ER- cells implicates regions of the EGFR first intron in up-regulation of EGFR, while estrogen regulation studies indicate the involvement of a repressor(s) in the maintenance of low levels of EGFR. Based on these findings, a multi-step model is proposed for the progression of breast cancer from a hormone-dependent, ER+/EGFR-phenotype to an aggressive, hormone-independent, ER-/EGFR+ stage.

A nuclear matrix acceptor site for the progesterone receptor in the avian c-myc gene promoter.

It has been the goal of this project to determine the location, composition, and biological function of the nuclear acceptor sites (i.e., the nuclear binding sites) for the avian oviduct progesterone (Pg) receptor (PR). Many laboratories have demonstrated a native-(in vivo) like cell-free binding of steroid receptor complexes to specific acceptor sites in nuclei/chromatin in a variety of target tissue systems. These sites appear to involve protein-DNA complexes and some of these have been shown to reside in the nuclear matrix, including the chromatin acceptor sites for the avian oviduct PR. We have purified a nuclear matrix "acceptor protein" for the avian PR. termed receptor binding factor-1 (RBF-1), based on its ability to generate specific, high-affinity PR binding on avian genomic DNA. This 10 kD nuclear matrix protein was found to be unique with minimal homology to a couple of other proteins. Using immunohistochemical techniques and antibodies against the purified RBF-1, the RBF-1 was localized to the nuclei of many avian and rat tissues. Co-localizations of RBF-1 and PR in select cell types in the avian oviduct and rat reproductive organs were also reported. A tissue specificity was found with regard to RBF-1 concentrations. The full length cDNA to RBF-1 has been isolated and used to identify a 0.7 kb mRNA whose levels in various avian tissues reflect the protein levels. Genomic sequences of RBF-1 have been isolated and characterized. Preliminary studies indicate that the over-expression of the RBF in human MCF-7 cells results in an inhibition of the c-myc gene promoter activity which is further inhibited by steroid hormone treatments of the cells. Past studies in our laboratory demonstrated that the c-myc mRNA steady state levels are rapidly (approximately 15 min) reduced by Pg and glucocorticoids in the avian oviduct. Further, partially purified fractions of RBF-1 were shown to generate specific PR binding sites only on the genomic DNAs of certain animal species and on the c-myc gene, but not ovalbumin gene. Using Southwestern blot analyses, the purified RBF-1 was shown to bind specifically to sequences in the 5' end of c-myc, c-jun proto-oncogenes but not to genomic sequences of the ovalbumin gene. A specific DNA binding element in the promoter region of the c-myc proto-oncogene has been identified as AT-rich domain of homopurine/pyrimidine stretches flanked by GC-rich sequences. Southern blot analyses using 200 bp matrix DNA fragments protected by the nuclear matrix structure indicate that the matrix is attached on either side of the RBF-1 binding element. A model is described for a nuclear matrix acceptor site attached to the c-myc promoter which may mediate the Pg-induced down-regulation of the c-myc gene expression.