A special estrogen-binding protein in the rat liver: the effect of nuclear accumulation of estradiol-receptor complexes in vitro

The accumulation of [3H]estradiol-receptor complexes by liver nuclei after preliminary incubation of the hormone with rat liver cytosol was studied. It was demonstrated that addition to female rat liver cytosol of a purified preparation of the unusual estrogen-binding protein (UEBP) from male rat liver causes a dose-dependent inhibition of subsequent accumulation of specifically bound [3H]estradiol in the nuclei. Addition to male rat liver cytosol of 1.5 microM 2 alpha-hydroxytestosterone, testosterone, 1-dehydrotestosterone, 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-dihydrotestosterone, i. e. compounds possessing marked affinity for UEBP, resulted in a 2-5-fold increase of the subsequent nuclear accumulation of estrogen-receptor complexes. The use of UEBP-deficient female rat liver cytosol revealed that the afore-mentioned steroids are ineffective with respect to estrogen reception. It is concluded that UEBP of male rat liver is capable of modulating estrogen reception.     

Purification and comparative study of the properties of glucocorticoid- and estrogen-receptor complexes in the rat liver

Parallel purification of glucocorticoid- and estrogen-receptor complexes from rat liver cytosol has been accomplished. Some properties of purified steroid-receptor complexes (SRC) were determined. The procedure developed earlier when the two-step treatment of cytosol with DNA-cellulose alternated with SRC ammonium sulphate precipitation, was shown to be universally applicable for purification of various SRC. Certain modifications have been devised allowing some increase in the degree of receptor purification. The amount of estrogen-receptor complexes (ERC) isolated from male liver cytosol was 20-40 times less than that of glucocorticoid-receptor complexes (GRC) isolated simultaneously from the equal volume of the same cytosol. Both SRC types bind intensively to homologous DNA but not to poly(A). The elution of GRC from DNA cellulose was mainly achieved at 0.4 M NaCl. With this, GRC and ERC showed small, but reliable differences in the salt resistance of their associates with DNA: the ERC-DNA link was stable toward NaCl up to 0.1 M, whereas an appreciable amount of GRC was eluted from DNA-cellulose at 0.1 M NaCl. The stability of purified ERC exceeded that of purified GRC, which apparently reflects the differences in the hormone-receptor binding constants. The receptor stability under various environmental conditions is discussed and some recommendations on the improvement of the SRC stability and its control are given.     

Nuclear translocation of estrogen-receptor complex and stimulation of RNA synthesis by estrogens of different biological potencies in the female rat pituitary

Stimulation of RNA synthesis and of nuclear translocation of estrogen-receptor complexes was investigated in isolated nuclei of anterior pituitaries of castrated female rats after injection with estrogens of different biological potencies. The assay system for the estimation of total RNA synthesis was validated and data suggest that incorporation of [3H]UMP into acid-precipitable material is consistent with RNA synthesis. An increase in RNA synthesis was seen 30 min after application of either 17 beta-estradiol, estriol or 1,3-diacetyl-17 alpha-ethinyl-7 alpha-methyl-1,3,5,(10)estratriene-17,3-ol (DMEE). RNA synthesis was maximal 90 min after estrogen application. Thereafter, RNA synthesis decreased slowly and reached pretreatment levels 3, 8 and 30 h after application of estriol, 17 beta-estradiol and the diacetyl derivative of ethinyl-estradiol, respectively. All estrogens were found to stimulate rapidly nuclear translocation of estrogen-receptor complexes. Peak levels of nuclear receptor contents were reached 30 min after administration of estrogens. A concomitant depletion of cytosol receptor levels was noted. Nuclear retention of estrogen-receptor complexes paralelled duration of enhanced RNA synthesis and correlated with biological potencies of the steroids. Data of present experiments combine to suggest that long-term nuclear retention is a requisite for expression of biological activity of estrogens at the anterior pituitary. Furthermore, the degree of biological activity seems to be associated with duration of stimulation of RNA synthesis, amount of estrogen-receptor complexes translocated to the nucleus, and duration of nuclear retention.     

Activation of [3H]estradiol--and [3H]H1285-receptor complexes: effect of salt versus ATP on molybdate-stabilized estrogen receptors

The activation by salt or ATP of [3H]estradiol- and [3H]H1285-receptor complexes from rabbit uterus and their binding capacity to DNA-cellulose, phosphocellulose and ATP-Sepharose has been studied. The estrogen-receptor was prepared in 1 mM molybdate which stabilized the receptor; but both salt- and ATP-transformation of estrogen receptors occurred. The binding of molybdate-stabilized cytosol [3H]estradiol-receptor complexes to the various resins revealed that salt-activation by 0.3 M KCl caused the greatest binding (5-6-fold) to DNA-cellulose as compared to other resins. However, 5 mM ATP-dependent activation of receptor-complexes resulted in preferential binding to ATP-Sepharose. Activated cytosol [3H]H1285-receptor complexes bound all the resins to a lesser degree when compared to [3H]estradiol-receptor complexes. Partially purified receptor complexes also showed different resin-binding patterns for salt- and ATP-mediated activation. These findings suggest that salt-activation is different than ATP-activation. Further, the differential magnitude of [3H]estradiol- and [3H]H1285-receptor activation suggests that estrogen-receptor complexes are "fully" activated as compared to "partially" activated antiestrogen-receptor complexes.     

Assessment of “activation” of estrogen receptors in lactating mammary gland using DNA-cellulose binding

To circumvent the need for isolated nuclei in studies on activation of estrogen-receptor complexes in mammary gland of the rat, a DNA-cellulose binding assay was employed using a cell-free system. Incubation at 28°C for 30 min of receptors previously charged with [³H]estradiol markedly enhanced their association with DNA-cellulose. Once activated, estrogen-receptor complexes bound maximally to DNA-cellulose within 20–30 min. The temperature optimum for activation was 28 ± 2°C using cytosol preparations. The temperature-induced activation required the presence of both steroid and cytosolic receptors simultaneously. Density gradient centrifugation revealed that, unlike those of uterus, both activated and charged estrogen-receptor complexes of lactating mammary tissue sedimented as a 4 S species in sucrose gradients containing 0.4 M KCl.     

Interaction of estrogen receptor complexes with the promoter region of genes that are negatively regulated by estrogens: the alpha 2u-globulins.

Since estrogens strongly suppress the expression of alpha 2u-globulin genes in the rat liver, we studied the binding of estrogen-receptor complexes to fragments derived from alpha 2u-globulin gene RAO 01 using a DNA-cellulose competition assay. Rat uterus cytosol labelled with [3H]estradiol was used as a source of the estrogen receptor. As a positive control in these experiments we used an oligonucleotide containing the estrogen response element (ERE) cloned into pUC18. Our experiments indicate that estrogen-receptor complexes bind specifically to the ERE and to a fragment of RAO 01 located in the 5'-upstream region (bp -606 up to -575). This fragment is conserved among other members of this gene family. This is the first time that in vitro estrogen receptor binding is observed to gene fragments derived from a gene that is repressed by this steroid in vivo.     

Effects of Charcoal on Dissociation Kinetics of Nuclear and Cytosolic Steroid-Receptor Complexes from Hen Oviduct

Abstract

The observed rate of dissociation of radioactive estradiol from nuclear estrogen-receptor complexes from hen oviduct has been shown to depend to a large extent on the method used to initiate dissociation. The present study indicates that nuclear progesterone receptors display the same pattern of behavior. Dissociation kinetics of nuclear progesterone receptor extracted from hen oviducts were affected by the method of initiation of dissociation; the rate of dissociation at 25°C when dissociation was initiated with unlabeled steroid was three times that observed when dissociation was initiated by addition of a charcoal/dextran suspension. In contrast to nuclear receptors, both estrogen and progesterone receptors prepared from cytosol displayed only a single rate of dissociation, no matter what the method of initiation of dissociation. These results strengthen the idea that nuclear receptors contain a factor or subunit which may be removed by charcoal, which alters the rate of dissociation of steroid from the complex.     

Nanomolar concentrations of untransformed glucocorticoid receptor in nuclei of intact cells

The subcellular distribution of untransformed glucocorticoid-receptor complex in vivo has been studied by chemical crosslinking of intact cells, and using a procedure adequate for correction of experimental errors due to redistribution of components between cytosolic and nuclear fractions. We found that in HeLa S₃ cells 85.4% of total glucocorticoid-receptor complexes are located in nuclei, and 14.6% are cytosolic. If measurements were performed with MCF-7 cells, we determined that the nuclear pool of glucocorticoid-receptor complexes accounts for 75.2% of the total cellular content, whereas the remaining 24.8% are cytosolic. When the subcellular distribution of estrogen-receptor complexes was determined, instead, we found that they are almost exclusively located in nuclei of MCF-7 cells, which contain 88.9% of the total. In order to estimate the molar concentration of receptors in cytosol and nuclei of intact cells, we determined the free water content of the two compartments. The volume of solvent was found to vary in the three cell lines we have studied, and our data showed that these variations are due to the cytosolic fractions, as the free water content of nuclei is essentially the same in those cells. When the free water content and the levels of glucocorticoid-receptor complexes we have measured were used to estimate the molar ncentrations of receptors, we found that these range between 0.4 and 18.9 nM in cytosols, and between 3.9 and 6.3 nM in nuclei of the three cell lines we have studied. We then concluded that the relative distribution of untransformed glucocorticoid-receptor complexes between cytosol and nuclei is cell-specific but their molar concentration in the nuclear compartment does not greatly vary among different cells.     

Receptor binding in the rat liver nuclear matrix

3H-Dexamethasone (Dex)-receptor complexes prepared from the rat liver cytosol efficiently bound to the nuclear matrix from the same tissue. The binding was increased with the concentration of the 3H-Dex-receptor complex added and reached a maximum plateau. However, when the partially purified 3H-Dex-receptor complex was used, saturation of the binding sites in the nuclear matrix was not observed in the range of concentration of 3H-Dex-receptor complex used. Therefore, it was considered that the apparent saturability observed in the binding of the unpurified receptor complexes is caused by the translocation inhibitor(s) in the cytosol. When the binding capacity was expressed on the basis of unit weight of DNA, the nuclear matrix exhibited 20 times more of that of the unfractionated nuclei. However, no line of evidence of enrichment of the binding sites in the DNA isolated from the nuclear matrix was observed. These observations show that the role of the nuclear matrix in the action of glucocorticoid is quite uncertain.     

Duration of antagonizing effect of RU486 on the agonist induction of tyrosine aminotransferase via glucocorticoid receptor

The duration of the antagonizing activity of RU486 on tyrosine aminotransferase (TAT) induction and the glucocorticoid receptor in rat liver was studied. A single dose of RU486 (10 mg/kg) caused occupation of the cytosol glucocorticoid receptor in rat liver at 1h. During this time no nuclear binding of [³H]dexamethasone ([³H]Dex) receptor complex was recorded, and TAT induction was completely blocked. TAT inducibility recovery parallelled receptor binding in both the cytosol and the nuclei, reaching maximum at 12 h. In contrast, nuclear binding recovered in 24 h, and [³H]Dex receptor binding in cytosol 48 h after RU486 application. It is concluded that the inhibitory effect of a single dose of RU486 on TAT induction is of rather short duration. At concomitant presence of agonist and antagonist in vivo, no direct correlation between agonist receptor occupancy and TAT induction could be observed.     

Microsomal receptor for steroid hormones: functional implications for nuclear activity

Target tissues for steroid hormones are responsive by virtue of and to the extent of their content of functional intracellular receptors. Recent years have seen a shift in considerations of the cellular dynamics and distribution of these receptors, with current views favoring predominant intranuclear localization in the intact cell. This paper summarizes our analyses of the microsomal estrogen and androgen binding capability of rat uterine and ventral prostate tissue, respectively; these studies have revealed a set of high affinity sites that may act as a conduit for estrogen traversing the cell en route to the nucleus. These sites have many properties in common with cytosolic receptors, with the salient difference of a failure to activate to a more avid DNA-binding form under conditions which permit such activation of cytosolic receptors. The microsomal estrogen-binding proteins also have appreciable affinity for progesterone, another distinction from other known cellular estrogen receptor species. Various experimental approaches were employed to demonstrate that the microsomal receptors were not simply cytosol contaminants; the most convincing evidence is the recent successful separation of the cytosolic and microsomal forms by differential ammonium sulfate precipitation. Discrete subfractionation of subcellular components on successive sucrose gradients, with simultaneous assessments of binding capability and marker enzyme concentrations, indicates that the major portion of the binding is localized within the vesicles of the endoplasmic reticulum free of significant plasma membrane contamination. The microsomal receptors are readily solubilized by extraction with high- or low-salt-containing buffers or with steroid. The residual microsomes following such extraction have the characteristics of saturable acceptor sites for cytosolic estrogen-receptor complexes. The extent to which these sites will accept the cytosolic complexes is equal to the concentration of microsomal binding sites extracted. These observations suggest three possible roles for the microsomal receptor-like proteins: (a) modulation of estrogen access to nuclear binding sites; (b) formation of functional complexes which diffuse to other extranuclear sites to alter non-genomic cellular processes; (c) regulation of nuclear concentration of estrogen-receptor complexes by virtue of producing microsomal acceptor sites for uptake of free or loosely associated nuclear complexes, previously thought to exist in the cytoplasm.     

Effect of estradiol and other endocrine factors on the level and dynamics of estrogen receptors in the liver cells of rats

The authors studied the effects of different doses of estradiol (E2) on the level of estrogen receptors (ER) in female rat hepatocytes and the dynamics of ER distribution between the cytosol and nuclear cell fractions and compared the changes in the ER level in the liver in different endocrine states of the body. It was shown that the ER level in hepatocytes was far lower than in uterine cells and drastically increased during puberation and after ovariectomy. It was also found that the ER level in hepatocytes was dependent on pituitary functions. After a single injection of E2, the ER level in cytosol descended and that in the nucleus rose. The degree of ER reduction in cytosol and increase in the nucleus correlated with the dose of E2. The dynamics of intercombined changes in the levels of cytosol and nuclear ER was studied at different time intervals following the injection of different doses of the hormone. Some essential organ-specific features of E2 reception in the liver were revealed. Different mechanisms of the control of ER content in the liver and uterus are postulated.     

Interaction of glucocorticoid · receptor complexes with rat liver nuclei

Some previous reports on acellular binding of glucocorticoid · receptor complexes to rat liver nuclei have pointed to the conclusion that there exists a small number of high affinity nuclear “receptor” sites. Various investigations lead us to the opposite conclusion and suggest that these results were actually due to the presence, in the cytosol, of one or several macromolecules which inhibited the binding to nuclei of steroid · receptor complexes. The mechanism of this inhibition was examined. It appeared to be due not to a competition between both molecules for the same nuclear acceptor site but to an interaction in the cytosol between teh inhibitor and the steroid · receptor complex which prevented the binding of the latter to the nuclei. The search for high affinity specific acceptor sites was also negative for physiological saline conditions and for the non-salt-extractable fraction of the nuclear receptor. When 940-fold purified receptor · steroid complexes were used, very high concentrations of complexes could be achieved and saturation of nuclei was then observed, but only under physiological ionic strength conditions. However, the interaction was of relatively low affinity (K_A = 3.8 · 10⁷ M^?1) and to a great number of acceptor sites (N = 26.2 pmol/mg DNA), largely exceeding the cellular concentration of receptor (5.8 pmol/mg DNA).These results suggested that saturation of nuclei by steroid · receptor complexes should not occur in the intact liver cell. They were confirmed by studies on the distribution of steroid · receptor complexes in liver slices incubated with various concentrations of [³H]dexamethasone. For all hormone concentrations a constant proportion (90%) of the complexes was found in the nuclei, thus showing no saturation of the nuclear acceptor sites.     

Studies on the nuclear binding of steroid hormone-receptor complex; binding of liver and thymus dexamethasone-receptor complex and prostate dihydrotestosterone-receptor complex to nuclei from various tissues.

To examine the binding specificity of steroid hormone-cytoplasmic receptor complexes to nuclei, binding of 3H-dexamethasone (Dex)-liver, 3H-Dex-thymus and 3H-dihydrotestosterone (DHT)-prostate receptor complexes to nuclei from liver, prostate, thymus, spleen and kidney was studied. It was observed that a significant amount of steroid-receptor complexes was bound to any nuclei used in the present study and the extent of the binding of receptor complexes to nuclei from homologous tissues was not always greater than that to nuclei from heterogenous tissues. However, a significant portion of the 3H-Dex-liver and 3H-DHT-prostate receptor complexes was not absorbed by nuclei from kidney, spleem, and thymus, and the unabsorbed complexes were efficiently bound to liver and prostate nuclei. The results obtained indicate that two types of receptor complex with regard to nuclear binding were present in cytosols of liver and prostate; one binds to nuclei from kidney, spleen, thymus, liver and prostate and the other does not bind to nuclei from kidney, spleen and thymus but does bind to nuclei of liver and prostate. The latter type of receptor complex was not observed in the cytosol from the thymus.     

Exploring the priming mechanism of liver regeneration: proteins and protein complexes

The liver has the ability to restore its functional capacity following injury or resection and the priming of liver regeneration is a complex process that has not been completely elucidated. In the current research, to further reveal the priming mechanism of liver regeneration, hepatocyte total protein and hepatocyte cytosol of the rats at 4 h after 2/3 partial hepatectomy (PHx) were studied, respectively, by 2‐DE and 2‐D blue native gel electrophoresis. Seventeen unique differential proteins were identified in hepatocyte total protein samples. Nine differential protein complexes containing 41 protein components were identified in hepatocyte cytosol samples. For the first time, at the priming stage of liver regeneration, the variations of serine protease inhibitor 2c, sulfite oxidase and valosin‐containing protein (VCP) were presented and validated by Western blotting, and the VCP complex was further validated by antibody super‐shift experiments. The current results suggested that at 4 h after PHx, VCP complex was down‐regulated in hepatocyte cytosol, apoptosis pathways were inhibited, nuclear factor‐κB and interleukin 6 pathways worked together and triggered the liver regeneration.     

Comparison of cytosolic and nuclear poly(A) polymerases from rat liver and a hepatoma: structural and immunological properties and response to NI-type protein kinases

Poly(A) polymerases were purified from the cytosol fraction of rat liver and Morris hepatoma 3924A and compared to previously purified nuclear poly(A) polymerases. Chromatographic fractionation of the hepatoma cytosol on a DEAE-Sephadex column yielded approximately 5 times as much poly(A) polymerase as was obtained from fractionation of the liver cytosol. Hepatoma cytosol contained a single poly(A) polymerase species [48 kilodaltons (kDa)] which was indistinguishable from the hepatoma nuclear enzyme (48 kDa) on the basis of CNBr cleavage maps. Liver cytosol contained two poly(A) polymerase species (40 and 48 kDa). The CNBr cleavage patterns of these two enzymes were distinct from each other. However, the cleavage pattern of the 40-kDa enzyme was similar to that of the major liver nuclear poly(A) polymerase (36 kDa), and approximately three-fourths of the peptide fragments derived from the 48-kDa species were identical with those from the hepatoma enzymes (48 kDa). NI-type protein kinases from liver or hepatoma stimulated hepatoma nuclear and cytosolic poly(A) polymerases 4-6-fold. In contrast, the liver cytosolic 40- and 48-kDa poly(A) polymerases were stimulated only slightly or inhibited by similar units of the protein kinases. Antibodies produced in rabbits against purified hepatoma nuclear poly(A) polymerase reacted equally well with hepatoma nuclear and cytosolic enzyme but only 80% as well with the liver cytosolic 48-kDa poly(A) polymerase and not at all with liver cytosolic 40-kDa or nuclear 36-kDa enzymes. Anti-poly(A) polymerase antibodies present in the serum of a hepatoma-bearing rat reacted with hepatoma nuclear and cytosolic poly(A) polymerases to the same extent but only 40% as well with the liver cytosolic 48-kDa enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro

During entry, herpes simplex virus type 1 (HSV-1) releases its capsid and the tegument proteins into the cytosol of a host cell by fusing with the plasma membrane. The capsid is then transported to the nucleus, where it docks at the nuclear pore complexes (NPCs), and the viral genome is rapidly released into the nucleoplasm. In this study, capsid association with NPCs and uncoating of the viral DNA were reconstituted in vitro. Isolated capsids prepared from virus were incubated with cytosol and purified nuclei. They were found to bind to the nuclear pores. Binding could be inhibited by pretreating the nuclei with wheat germ agglutinin, anti-NPC antibodies, or antibodies against importin beta. Furthermore, in the absence of cytosol, purified importin beta was both sufficient and necessary to support efficient capsid binding to nuclei. Up to 60 to 70% of capsids interacting with rat liver nuclei in vitro released their DNA if cytosol and metabolic energy were supplied. Interaction of the capsid with the nuclear pore thus seemed to trigger the release of the viral genome, implying that components of the NPC play an active role in the nuclear events during HSV-1 entry into host cells.     

A low molecular weight inhibitor of steroid receptor activation.

Dilution at 0 degrees of rat liver cytosol incubated with [3H]triamcinolone acetonide provoked an enhanced binding of steroid-receptor complexes to nuclei. The explanation of this phenomenon was found to be an "activation" of the complexes. Dilution acted by decreasing the concentration of a cytosol inhibitor. This reaction was irreversible at 0 degrees: once activated the complexes could not be reversed to the nonactivated state by the addition of inhibitor. The presence of hormone was necessary, since hormone-free receptor molecules could not be activated by dilution. Removal of the inhibitor did not lead to activation of all complexes: after 24 h a "plateau" was attained where 55 to 70% of the complexes were activated. The inhibitor was shown to be a low molecular weight molecule by dialysis, Sephadex G-25 chromatography, ammonium sulfate precipitation, and ultrafiltration. Thus [3H]triamcinolone acetonide-receptor complexes present in a cytosol from which the inhibitor had been removed by Sephadex G-25 chromatography became spontaneously activated at low ionic strength and at 0 degrees. The inhibitor is not a steroid (at least of usual polarity) since it cannot be extracted by methylene chloride or adsorbed by activated charcoal. It is thermostable (resists to 30 min at 100 degrees). Its removal by incubation with a cation exchange resin suggests that it may be positively charged, however it is not complexed by EDTA. This inhibitor must be distinguished from a previously described inhibitor of steroid-receptor complexes binding to nuclei. The latter compound has been shown in various systems to be responsible for an artifactual saturation of nuclear acceptor by steroid-receptor complexes. It inhibits the binding to nuclear acceptors of already activated complexes and is probably a macromolecule. It is thus different from the low molecular weight activation inhibitor described in the present paper.     

Regulation of albumin synthesis in rat liver

The present report reviews our findings on the subcellular distribution of albumin mRNA in rat liver under normal and abnormal physiologic conditions, the identification of albumin mRNA in specific mRNP complexes in liver cytosol of starved rats, and evidence for albumin mRNA sequences in a higher molecular weight nuclear precursor to cytoplasmic albumin mRNA.