Glutamine synthetase induction in embryonic neural retina. Interactions of receptor-hydrocortisone complexes with cell nuclei.

In the neural retina of the chick embryo, hydrocortisone (HC) elicits differential gene expression resulting in the induction of glutamine synthetase (GS), which is an enzyme marker of differentiation in the retina. The relationship between nuclear binding of receptor-hydrocortisone (R-HC) complexes and GS induction was investigated in cultures of retina tissue from 12-day chick embryos. The number of HC binding sites in the cytoplasm was estimated as 1650+/-200 per retina cell; there are approximately 1500+/-100 acceptor sites for R-HC per retina nucleus. GS induction in the retina became detectable only after R-HC bound to more than 40% of the nuclear acceptors sites; increased binding coincided with higher induction levels, until complete site saturation was attained; Proflavine, which blocks preferentially and completely GS induction in the retina by interfering in the nucleus with the enzyme-inducing action of the hormone, reduced nuclear binding of R-HC by only 20%; thus, only part of the R-HC that binds in the nucleus appears to be directly involved in eliciting the induction of GS. Within one hour after exposure of the retina to an inducing dose of HC, there was translocation of HC and HC-receptors (as R-HC complexes) from the cytoplasm into the nucleus and saturation of nuclear accepegan to decline; in 12 h, it was reduced to 50% of the initial saturation level. Since, during this time, the enzyme activity to increase, persistence of the induced state depends on association of the hormone with only a portion of the sites in the nucleus to which it can bind. The decrease in the amount of bound HC in the nuclei of induced cells was accompanied by an increase in the level of HC receptors in the cytoplasm. About 50% of this increase could be prevented by cycloheximide; this suggests that the reappearance of HC receptors in the cell cytoplasm may be due, at least in part, to de novo synthesis of HC receptors.     

Binding of receptor-hydrocortisone complexes to isolated nuclei from embryonic neural retina cells

Hydrocortisone (HC) induces glutamine synthetase in the embryonic chick neural retina. The binding of cytoplasmic receptor-hydrocortisone (R-HC) complexes to isolated retina nuclei has been studied in a cell-free system. Optimal conditions, specificity and quantitative aspects of binding were determined. The isolated nuclei retained binding specificity for the R-HC complex prepared from retina cytosol. Free HC, estradiol-receptor complexes from retina cytosol and HC-receptor complexes from mouse brain cytosol or from chick serum did not bind to the nuclei. Assuming monovalency of the binding sites, the number of nuclear acceptor sites per retina cell for the R-HC complex was estimated to be in the range of 1500. These sites were resistant to RNAse but sensitive to DNAse.     

Interaction of the calf uterine estrogen receptor with acceptor sites in heterologous chicken target cell nuclei

Estrogen receptor (ER) from chicken liver and calf uterus were used to study the capacity and the characteristics of the receptor binding sites (acceptor sites) in chicken target cell nuclei. Binding studies were performed at a physiological salt concentration of 0.15 M KCl. Binding of liver ER to liver nuclei was temperature-dependent, showing a 9-fold increase between 0 and 28 degrees C. The maximal number of acceptor sites measured in this cell-free system (280 sites/nucleus) was considerably lower than measured in nuclei after in vivo administration of estrogen (820 sites/nucleus). Moreover incubation of nuclei with the liver ER preparation resulted in a substantial breakdown of nuclear DNA, making this ER less suitable for DNA binding studies. The temperature-activated calf uterine receptor bound to liver nuclei at 0 degrees C, at which temperature no DNA degradation was measured. To all chicken cell nuclei tested, the receptor bound with a high affinity (Kd = 0.4-1.0 nM). Nuclear binding displayed tissue specificity: oviduct greater than heart, liver greater than spleen greater than erythrocytes and was salt dependent. Calf uterine ER binding in liver nuclei ranged from 3000-6000 acceptor sites per nucleus when assayed under conditions of a constant protein or a constant DNA concentration. Nuclei isolated from estrogen-treated cockerels bound a 2-fold lower number of calf uterine ER complexes when compared to control nuclei. Incubation of nuclei with a fixed concentration of [3H]ER from liver and increasing concentrations of uterine non-radioactive-ER also resulted in a reduced binding of the liver receptor. Both types of experiments suggest that liver and uterine ER compete for a common nuclear acceptor site. Our data demonstrate that the ER from calf uterus is very useful as a probe to examine the nature of the acceptor sites in heterologous chicken target cell nuclei. The assay system functions at 0 degrees C, a temperature at which no DNA degradation occurs.     

Interaction of calf uterine estrogen receptors with chicken target cell nuclei

The calf uterine estrogen receptor (ER) was used to study the capacity and the characteristics of the acceptor sites in chicken target cell nuclei. The temperature-activated ER is bound at 0 degrees C with a high affinity to all chicken cell nuclei tested (Kd = 0.4-1.0 nM). The nuclear binding displayed tissue specificity: oviduct greater than liver, heart greater than spleen greater than erythrocytes and was salt-dependent. ER binding to liver nuclei measured in 0.15 M KCl varied between 3000 and 6000 acceptor sites per nucleus. Liver nuclei isolated from estrogen-treated cockerels showed a 2-fold lower binding capacity than nuclei from non-treated chickens. When nuclei were incubated with [3H]ER from embryo liver and increasing concentrations of uterine non-radioactive-ER a progressive inhibition of the binding of the liver ER was found. These experiments suggest that liver and uterine ER compete for a common acceptor site. Liver nuclei charged in vitro with calf uterine ER were digested at 0 degree C with DNAase I and micrococcal nuclease. Both enzymes excised the ER in the form of a chromatin-ER complex. A considerable portion was associated with nucleosomal subunits and a minor fraction was associated with a nuclease-sensitive, protein-poor fraction of the chromatin.     

Binding of glucocorticoids to liver nuclei and chromatin of fetal,immature and adult rats

The interaction of dexamethasone with nuclei and chromatin was investigated following incubation of liver slices from fetal, immature (6-day-old) and adult rats with the labeled steroid at 37°. The number of specific binding sites for dexamethasone in purified liver nuclei increases with the age of the animal in a manner similar to that previously reported for the cytoplasmic receptor. The high affinity nuclear binding approaches saturation at 40 and 500 nM dexamethasone in fetal and adult liver, respectively. In comparison with dexamethasone, the relative efficiency of corticosterone to accumulate in the nucleus is 9 percent in fetal liver and only 1 percent in adult liver.Specifically bound dexamethasone in adult nuclei exists in at least three forms; a Tris-soluble, a KCl-soluble, and a residual (non-extractable with KC1 or DNase) form. Part of the Tris-soluble steroid is associated with macromolecules sedimenting at about 4 S both in the presence and absence of 0.4 M KCl. This form of the receptor was not detected in fetal liver nuclei. In liver chromatin, bound dexamethasone exists in a KCl-soluble and a residual form, the latter comprising the major fraction of steroid associated with chromatin from both fetal and adult tissue (60 and 75 percent, respectively). Treatment with Triton X-100 releases about 20 percent of the radioactivity in adult liver nuclei, but has no effect on fetal liver nuclei.In contrast with the above observations in the intact tissue, the major fraction of steroid bound to chromatin in cell-free systems is KCl- and DNase-soluble, only 30 percent remaining in the residual pellet.     

Studies on the regulation of the concentration of androgens and androgen receptors in nuclei of prostatic cells.

Experiments were performed to assess the effect of intracellular androgen metabolism and the availability of cytoplasmic receptors on the concentration of androgens and androgen receptors in nuclei of prostatic cells. It was found that androgens are incorporated into the nucleus by a regulated, selective process which appears to limit the type and amount of androgen transported across the nuclear membrane. The metabolic conversion of testosterone to dihydrotestosterone which takes place in cytoplasm does not reduce transport and, very likely, affects only the ratio of testosterone and dihydrotestosterone transferred into the nucleus. In vivo, when the intranuclear concentration of androgens approaches 250 nM (8 pmol per mg DNA), an apparent concentration ceiling is reached even in the presence of a downward concentration gradient that would be expected to promote further transport across the nuclear membrane. This finding strongly suggests that in vivo the nuclear membrane acts as a barrier to the passage of androgens and, therefore, mitigates against the possibility that passive diffusion is an important mechanism of afferent transport of androgens into the nucleus. The ability of the nucleus to concentrate testosterone and dihydrotestosterone was clearly demonstrated in vivo when cytoplasmic concentrations of androgens of approximately 20 nM were accompanied by intranuclear concentrations in the vicinity of 250 nM. Since the measured concentration of testosterone and dihydrotestosterone in prostate of several species fall within the 5-20 nM range, it is evident that androgen concentrations in the nucleus as high as 250 nM may be typical of the physiological steady state. At the latter concentration the nucleus contains 60 000 androgen molecules: in approximate terms one third of this total is bound to a large molecular weight component of the nucleus, one third is bound to a 3.3 S receptor and one third is free or loosely bound. Since 60 000 androgen molecules and 20 000 receptor molecules appear in the nucleus before transport stops, it seems that the quantity of 4.4 S cytoplasmic receptor estimated at 174 plus or minus 24 pmol per mg protein (equivalent to about 8000 molecules per cell) is insufficient to account for the total influx of androgens and androgen receptors into the nucleus. Thus, although these results support the view that cytoplasmic receptors and the capacity to transport androgens are closely linked phenotypic markers of intracellular steroid hormone action, they suggest that the control of androgen concentration in the nucleus is achieved in a more intricate fashion than simply through a dependence on the presumed translocation of 4.4 S androgen-receptor complex into the nucleus.     

Existence of receptors bound to endogenous estradiol in breast cancers of premenopausal and postmenopausal women

The amounts of free and occupied estrogen receptors were determined in cytosols and cellular (cytoplasmic + nuclear) extracts of 23 human breast cancers. $\text{In vivo}$ undersaturation of the receptors was observed in all tumors. A positive correlation was found between the degree of saturation and the concentration of circulating estrogens in premenopausal women. The saturation of the sites was higher in cellular extracts than in cytosols, pointing to the existence of receptor-estradiol complexes bound to nuclei. Our results suggest that the absolute number of occupied receptors depends upon the level of available cytoplasmic receptors as well as upon the level of tissue estradiol.     

Cortisol receptors and inducibility of glutamine synthetase in embryonic retina

Glutamine synthetase (GS) is a marker enzyme for Müller glia cells in neural retina. In chick embryo retina GS begins to increase sharply on the 16th day of development, but can be precociously induced by premature supply of the inducer, cortisol, already on the 8th day. At this stage GS inducibility is low, but it increases progressively with embryonic age. We investigated whether there was a corresponding age-dependent increase of cortisol-binding molecules (cortisol receptors) and found that their level is highest in the early retina and decreases with development. In light of this inverse relationship, we examined whether functional characteristics of these receptors change with age, but detected no differences. In in vitro tests, receptors from older retina translocated cortisol into nuclei from young retina, and vice versa, with similar effectiveness. Also, cortisol receptors from liver cells (which differ from retina receptors) can translocate the hormone into retina nuclei, and vice versa. These findings indicate that translocation of cortisol receptors is neither tissue-specific or age-dependent, nor is it conditional on the total amount of receptors normally present in cells. Therefore, the age-dependent increase of GS inducibility in embryonic retina cannot be directly related to quantitative or functional differences of cortisol receptors and is evidently controlled primarily at the gene level. The very large amount of cortisol-binding molecules in early embryonic retina raises the possibility that they play some role in early differentiation of retina cells unrelated to hormone binding.     

Progesterone-binding components of chick oviduct. IX. The kinetics of nuclear binding.

A cell-free system was used to study the kinetics of progesterone-receptor interaction with purified nuclei prepared from estrogen-primed chick oviducts. The binding process was a saturable phenomenon in both target and nontarget tissues. More nuclear acceptor sites were available to target tissue (similar to 9000 sites per oviduct nucleus) than in nontarget tissues (similar to 1000 to 3000 sites per nucleus), but the binding constant was essentially the same (Kd similar to 10-8 M). A second much smaller class of higher affinity sites (Kd similar to 10-11M) may exist. Its presence was detected by Scatchard plot nonlinearity at very low concentrations of added receptor-hormone complex (similar to 10-10 to 10-12M). The current study focused on the prevalent class of acceptor sites which was more readily detectable. Receptor binding to these sites was highly sensitive to salt. More sites were exposed at 25 degrees than at 0 degrees. Binding to these sites was inhibited in a nonselective fashion by the addition of protein. Although receptors may be activated by temperature or conditions of high ionic strength, these conditions could not capacitate more than 30 to 40% of the progesterone-receptor proteins for binding. Rate studies suggested that temperature plays a minimal role in nuclear uptake of activated receptors. Such a finding is consistent with a diffusion-limited uptake process.     

Interaction of glucocorticoid receptor-steroid complexes with acceptor sites.

The binding of the "activated" receptor-glucocorticoid complexes of cultured rat hepatoma cells to nuclei, chromatin, and DNA has been studied under cell-free conditions. A critical factor in determining the shape of the binding curve is shown to be an inhibitory material which is present in crude cytosol and which can be removed without destroying the receptor-steroid complex. These and other results argue that the apparent saturation observed in earlier experiments may have been due to the inhibitors. Thus, the actual number of acceptor sites in hepatoma tissue culture cell nuclei is much larger than previously estimated and their affinity for the complex is lower. Nuclear binding experiments indicate that the inhibitory material interacts with the receptor-steroid complex. The inhibitors appear to be macromolecular; but their effects cannot be mimicked by albumin or hemoglobin. The acceptor capacity at low ionic strength for binding receptor-glucocorticoid complexes increases when proceeding from nuclei to DNA. An analysis of the kinetics of association and dissociation and of the relative binding behavior of nuclei and DNA argues that the affinity of complex for nuclei is much greater than for DNA. DNA-associated histones reduce the amount of complex that binds to DNA. These and perhaps other chromosomal proteins may be responsible for the ordering of acceptor capacity. Evidence is presented that the difference in affinities of nuclear and DNA acceptors could also be due to chromosomal proteins. In nuclei, these proteins may thus both reduce the amount of complex binding by rendering regions of DNA less accessible and increase the binding affinity of some, or all, of those DNA binding sites which remain exposed.     

Estrogen dependent induction of low affinity binding sites in the nuclear fraction of rat uterus

Type II estradiol binding sites characterized by lower affinity and higher capacity than type I receptor sites have been described in rat uterine nuclei. These sites appeared to be dependent on estrogen stimulation. Reducing agents prevented estradiol binding to these sites. In the present study, the situation prevailing in adult rats (Ad) was studied and compared to ovariectomized (Ox) and ovariectomized estrogen prestimulated rats (OxPS). Nuclear precipitate from Ad, Ox and OxPS rats were incubated with tritiated estradiol (E2(3)H) in the presence and in the absence of mercaptoethanol as reducing agent. In the presence of mercaptoethanol, saturation was attained at E2(3)H concentrations above 16 nM. In the absence of reducing agents, a secondary binding was observed in Ad and OxPS which was not saturated at E2(3)H levels up to 80 nM. Non-specific binding obtained with paired aliquots containing 100-fold excess of DES as competitor was not linear but showed a saturation profile, distorting the saturation curve of the specific sites, obtained by subtracting non-specific from total E2(3)H binding. Increasing DES concentrations up to 10,000 nM did not allow to reach complete exchange with E3(3)H ligand bound to specific sites, preventing measurement of binding sites concentration. Incubation of nuclear fractions with increasing concentrations of E2(3)H (up to 6,000 nM) gave a saturation curve with a linear kinetics above 1-2,000 nM, which represented saturation concentration of the specific sites. From this, non-specific and specific moieties could be estimated. Binding capacity of specific sites was of the order of 50-80 pmol uterus. Half saturation was attained between 300 and 600 nM E2(3)H, which approximated the Kdiss of these sites, at variance with the Kdiss of 15-30 nM originally reported for type II binding sites. In conclusion, these results show that secondary binding sites were present in uterine nuclei of Ad and OxPS rats. Binding capacity was about 30-fold higher than that of type I sites. Affinity was however very low, and casts some doubt on the role of these sites as active estradiol binders in physiological situations. Their increase under the influence of estrogen may however be related to some as yet undetermined role.     

Binding of glucocorticoid receptors to mammary chromatin acceptor sites

We have recently characterized the interaction of mouse mammary estrogen receptors (ER) with mammary chromatin acceptor sites and demonstrated that ER from estrogen resistant lactating mammary glands do not bind to chromatin. In this study we have characterized the chromatin binding of the glucocorticoid receptor from mouse mammary glands isolated from nulliparous and lactating mice in order to better understand the relationship between receptor binding to chromatin and steroidogenic sensitivity of the tissue. Mammary chromatin was linked covalently to cellulose and deproteinized sequentially by 0-8 M Gdn-HCl. Binding to intact chromatin as well as to chromatin deproteinized by Gdn-HCl was determined using partially purified [3H]dexamethasone labelled glucocorticoid-receptor complexes (GR) obtained by fractionation on DEAE-cellulose columns. The binding of [3H]GR from mammary glands of nulliparous mice to chromatin fractions from the same tissue revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 5-6 M Gdn-HCl. Binding of [3H]GR was of high affinity (Kd = 0.2 nM) and saturable. A simultaneous comparison of the chromatin binding patterns for [3H]ER and [3H]GR isolated from mammary glands of nulliparous mice revealed that the chromatin subfractions obtained with 4-6 M Gdn-HCl extraction contained acceptor sites for both [3H]ER and [3H]GR; however, while the [3H]ER bound to a 4.5 M and a 5.5 M site, the [3]GR bound a 5 M and a 6 M site. Competition experiments supported the steroid receptor specificity of the chromatin acceptor sites. Thus, the 4-6 M chromatin fractions contain distinct acceptor sites for the glucocorticoid receptor and for the estrogen receptor. In addition our studies reveal that the binding patterns of [3H]GR isolated from mammary glands of nulliparous and lactating mice to their homologous chromatin is essentially similar. Thus, in contrast to estrogen receptors, glucocorticoid receptors from lactating mammary glands are able to effectively bind to chromatin acceptor sites which supports our previous suggestion that the estrogenic insensitivity of lactating mouse mammary glands may at least be in part due to the impeded interaction of ER with chromatin acceptor sites.     

Studies on the molecular mechanism of the translocation of estrogen receptor from the cytoplasm into the nucleus

A detailed study of the molecular mechanism of the translocation of estrogen receptor (ER) from the cytoplasm into the nucleus was undertaken in an in vitro system of porcine uterus. The capabilities of vero-ER . E (basic ER molecular bound with estradiol) (sedimentation coefficient 4.5S; Stokes radius 44 A) and the complexes ["5S" ER . E, (vero-ER . E) . (component A); "6S" ER . E, (vero-ER . E) . (component B)6; "8S" ER . E, (vero-ER . E) . (component B)6 . (component A)] with ER-binding factors (ERBFs) to translocate into the isolated nuclei were estimated by subtracting the amounts of ER adsorbed by the nuclear envelopes from those of ER bound to the whole nuclei. The results strongly supported our previous assumption that vero-ER . E translocates into the nuclei, and the complexes with ERBFs do not. The results suggested also that the binding site of vero-ER to ERBFs is required to be unoccupied in the process of the translocation of ER from the cytoplasm into the nucleus. The presence of a cytoplasmic factor (component C) which binds specifically with "5S" ER . E under low salt conditions was indicated. The complex, ("5S" ER . E) . (component C), was shown to possess relatively high affinity towards nuclear envelopes, but not to translocate into the nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of cyproterone acetate with rat prostatic androgen receptors

We have investigated the binding of cyproterone acetate (CA) to cytosolic androgen receptors (RC) and translocation of the RCCA complex into the nucleus. In a cell-free system (3H)CA binds to cytosolic androgen receptors with high affinity (KD = 11.6 nM) and limited capacity (180-200 femtomoles/mg protein). (3H)CA, however, dissociates very rapidly from the cytosolic and nuclear androgen receptors (Rn) at 0 degree C. Incubation of RC (3H)CA at 20 degrees C increased its ability to bind to nuclei. Translocation of RC (3H)CA to nuclei of intact cells was demonstrated after incubation of prostatic tissue with (3H)CA in tissue culture medium at 37 degrees C. In vivo administration of CA to castrated rats promoted RCCA translocation but did not induce androgen receptor replenishment. These data demonstrate that CA binds to and translocates androgen receptors to nuclei without concomitant receptor replenishment.     

Androgen receptor in nuclei of rat testis.

Testis nuclei of hypophysectomized rats selectively accumulate labeled testosterone and 5alpha-dihydrotestosterone following the injection of tritiated testosterone in vivo. Testosterone and 5alpha-dihydrotestosterone are bound to macromolecules in nuclei and can be extracted with 0.5 M KCl. Accumulation of protein bound radioactive androgens in nuclei of isolated seminiferous tubules is similar to that of whole testis. The relative amounts of testosterone and dihydrotestosterone in purified nuclei were similar to the relative amounts bound to cytoplasmic receptors, suggesting that cytoplasmic androgen-receptor complexes may be transported into the nuclei. Binding of labeled androgen is saturable and inhibited by prior injection of unlabeled testosterone or cyproterone acetate. Nuclear binding sites are destroyed by the proteolytic enzyme pronase, but not by DNase. Like the cytoplasmic androgen-receptor complexes in rat testis, nuclear androgen-protein complexes are heat labile and dissociate slowly at 0 degrees C. androgens fail to accumulate in testis nuclei of the Stanley-Gumbreck androgen insensitive rat, a species lacking cytoplasmic androgen receptors in testis and other androgen target tissues.     

Manipulation of the amount of RNA in post-mitotic nuclei

Because all (or almost all) nuclear RNAs are liberated to the cytoplasm during mitosis and then return to the post-mitotic nuclei, we expected that if cytoplasm were amputated from mitotic cells the post-division nuclei would possess less than normal amounts of RNA. Experiments performed with amebae (A. proteus) show that this is in fact what happens. Furthermore, since the enucleate fragment cut from a mitotic cell possesses an “excess” of returnable nuclear RNAs, a normal interphase nucleus implanted into such mitotic cytoplasm might be expected to acquire above-normal amounts of RNA. Experiments reported here show that this expectation also is realized. Thus, the regulation of the normal nuclear concentration of these RNAs involves mechanisms other than a limited number of intranuclear “binding” sites and most likely is restricted by the rate of synthesis of these RNAs.The demonstration that nuclei can be depleted or enriched for RNAs, many of which are unique to nuclei, makes it possible to determine the consequences for cell metabolism of altered amounts of nuclear RNA. Hopefully, such studies will reveal the function(s) of these RNAs.     

Mechanisms of biphasic action of glucocorticoids on alpha-lactalbumin production by rat mammary gland explants

The highly selective Type II glucocorticoid ligand RU28362 showed a clear biphasic effect on alpha-lactalbumin (alpha-LA) production in rat mammary gland explants, with a peak at 1 nM and a return to basal levels at 30-300 nM; dexamethasone showed a similar profile. Corticosterone, which has a higher affinity for Type I than Type II receptors, produced a variable response. In six out of eleven studies this was biphasic, with a maximum at 300 nM; in five no increase above baseline was seen. Classical Type I receptor ligands--aldosterone and deoxycorticosterone--showed responses parallel to their Type II agonist activity. We interpret these data as follows occupancy of Type I receptors does not increase alpha-LA production the response to selective Type II receptor ligands is truly biphasic and one explanation of this pattern may be the existence of both "turn-on" and "turn-off" acceptor sites in the nucleus.     

Effect of adrenalectomy and aldosterone on the modulation of mineralocorticoid receptors in rat kidney

Adrenalectomized rat kidney is commonly used for the study of mineralocorticoid mechanism of action in mammals. In this model, aldosterone is known to bind to two classes of binding sites: type I (mineralocorticoid) and type II (glucocorticoid). The study of the aldosterone binding in normal rat kidney requires the elimination of endogenous hormones bound to each type of receptor. Thus, a suitable technique was developed using in situ perfusion of the kidneys. The efficacy of this method was of about 85 to 90% at the level of both cytoplasm and nucleus. Aldosterone binding capacity was checked in normal rat kidney after in situ perfusion and was found to be 300 to 500% lower than in adrenalectomized rat kidney, both in cytoplasm and nuclei. Computer analysis of aldosterone binding parameters in the cytoplasm (30,000 X g supernatant) of rat kidney suggested that adrenalectomy might induce an important rise in the number of mineralocorticoid receptors (congruent to 260%). An increase in the number of glucocorticoid receptors was also observed but appeared to be lower. Aldosterone, when perfused during 24 h in adrenalectomized rats, lowered the number of type I sites to the same level as observed in normal rat kidney. This effect was fully reversible after interruption of aldosterone perfusion. These results suggested an aldosterone-induced down regulation of mineralocorticoid receptors.