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The rat tyrosine aminotransferase (TAT) gene is a liver-specific and glucocorticoid-inducible gene. Previous studies have shown that the TAT promoter (TAT0.35; nt -350 to +1) is able to sustain liver-specific gene expression both in transient transfection and in a transcription assay in vitro [Schweizer-Groyer, Groyer, Cadepond, Grange, Baulieu and Pictet (1994) Nucleic Acids Res. 22, 1583-1592]. Here we report that the basal transcriptional activity generated from TAT0.35 in the presence of crude liver nuclear extracts is enhanced by added human glucocorticoid receptor (hGRalpha), provided that TAT0.35 sequences were flanked (5') with a glucocorticoid responsive unit (GREII of the TAT gene, including its 5'-CCAAT flanking sequence). Two sources of hGRalpha were used: nuclear extracts prepared from Sf9 insect (Sf9-NEs) cells over-expressing hGRalpha, and hGRalpha from pRShGRalpha-transfected COS-7 cells, enriched by high-performance ion-exchange chromatography. The enhancement of transcription in vitro (1.5-4.5-fold) was dependent on the amount of added hGRalpha and independent of the nature (agonist or antagonist) of the ligand. Moreover, the hGRalpha-mediated stimulation of transcription was (i) dependent on GRE/progesterone response element (PRE) (it was inhibited by a 25-fold excess of GRE/PRE but not by a 100-fold excess of oestrogen response element) and (ii) receptor-dependent (Sf9-NEs prepared from uninfected Sf9 cells or from Sf9 cells infected with wild-type baculoviral DNA did not enhance transcription). Taken together, these experiments support the conclusions that in vitro the glucocorticoid receptor is able to enhance transcription from genomic, liver-specific, promoter sequences (those of the TAT gene), and that this enhancement of transcription from the liver-specific TAT0.35 promoter is dependent both on the glucocorticoid receptor and on the latter's interaction with its cognate response elements.

The metabolism of 27-, 25- and 24-hydroxycholesterol in cultures of rat astrocytes, Schwann cells and neurons was studied. 27- and 25-Hydroxycholesterol, but not 24-hydroxycholesterol, underwent 7 alpha-hydroxylation with subsequent oxidation to 7 alpha-hydroxy-3-oxo-delta 4 steroids in all three cell types. When cells were incubated for 24 h with 0.28 nmol of 27-hydroxycholesterol in 10 ml of medium, the rates of conversion into 7 alpha-hydroxylated metabolites were 0.21, 0.12 and 0.02 nmol/24 h per 10(6) cells in the media of astrocytes, Schwann cells and neurons respectively. The corresponding values for 25-hydroxycholesterol were 0.26, 0.16 and 0.04. A minor fraction of 27-hydroxycholesterol and its 7 alpha-hydroxylated metabolites was oxidized to 3 beta-hydroxy-5-cholestenoic acid. 3 beta, 7 alpha-dihydroxy-5-cholestenoic acid and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid. In addition to the two hydroxycholesterols, other 3 beta-hydroxy-delta 4 steroids, dehydro-epiandrosterone, pregnenolone, 3 beta-hydroxy-5-cholestenoic acid and 3 beta-hydroxy-5-cholenoic acid underwent 7 alpha-hydroxylation. Competitive experiments did not distinguish between the presence of one or several 7 alpha-hydroxylases. In astrocyte incubations, 27-hydroxycholesterol also underwent 25-hydroxylation, and 12% of its metabolites carried a 25-hydroxy group. 25-Hydroxylation of added 24-hydroxycholesterol was also observed in the astrocyte incubations, as was the formation of 7 alpha, 25-dihydroxy-4-cholesten-3-one, 25-hydroxycholesterol and 7 alpha, 25-dihydroxycholesterol from endogenous precursor(s). Our study indicates that side-chain oxygenated cholesterol can undergo metabolic transformations that may be of importance for cholesterol homoeostasis in the brain.

The role of heat-shock protein 90 (hsp90) in the regulation of the oestrogen receptor (ER) function is less well understood than for other steroid-hormone receptors because hsp90 is not involved in the stabilization or induction of a high-affinity ligand-binding state of ER nor in the inhibition of receptor dimerization. Electrophoretic mobility-shift assays, using purified ER and hsp90, were employed to investigate directly the effect of hsp90 on the ability of ER to bind to the oestrogen-response element (ERE) from the vitellogenin A2 gene. Contrary to models in which hsp90 binds to and passively inactivates steroid-hormone receptors, our studies show that the binding of ER to ERE is inversely dependent on the relative concentration of hsp90. Exposure of purified ER-hsp90 complexes to ERE led to the dissociation of hsp90 and concomitant specific binding of ER to ERE. We demonstrate that the amount of ER-ERE complex decreased with increasing concentrations of hsp90. Furthermore hsp90 dissociated preformed high-affinity ER-ERE complexes. Kinetic dissociation experiments indicate the hsp90 acts in a dynamic and specific process rather than by simple trapping of ER owing to its inherent off-rate. The receptor released from the ERE-bound state by hsp90 was recovered associated with hsp90 and was able to rebind to ERE. These results indicate that hsp90 does not suppress ER function merely by steric hindrance. On the basis of these results and others, we propose that, in vivo, hsp90 may play a dual role in ER function: (i) at a physiological temperature, hsp90 stabilizes an active form of the receptor in accordance with its general molecular chaperone role; (ii) at elevated temperatures or under other environmental stress, the increased cellular concentration of hsp90 negatively interferes with ER-dependent transcription, in accordance with the inhibition of gene transcription attributed to hsp90 after heat shock.

Recent studies have confirmed that the 90 kDa heat-shock protein (hsp90) interacts both in vitro and in vivo with steroid receptors, encouraging further detailed physicochemical and functional analysis of its chaperone role. Thus, to explore the relationship between hsp90 and receptors, the baculovirus system was used to overexpress the chick hsp90 alpha (chsp90) along with the chick oestradiol receptor (cER) or the human mineralocorticosteroid receptor (hMR). These receptors were able to form 9 S complexes with chsp90, demonstrating the association of the co-expressed recombinant proteins. Three mutants of chsp90 (delta A, delta B and delta Z) have been created by deletion of the A (residues 221-290) and B (530-581) regions, rich in charged amino acids, and the Z (392-419) region, a putative leucine zipper. After co-expression, anti-receptor antibodies immunoprecipitated the cER or hMR complexed with the wild-type chsp90, the delta B or the delta Z mutant, but not with the delta A chsp90, indicating that deletion of the A region of chsp90 leads to a lack of interaction with these receptors. The hormone binding capacity of the cER was unaffected after its co-expression with each of the three mutants. In contrast, the hMR co-expressed with the delta B mutant failed to bind aldosterone, a finding confirmed in vivo by the absence of hormone-induced hMR nuclear translocation. Thus the B region is required for high-affinity ligand binding by the hMR. Our results suggest that the A region (but not the B or Z regions) is involved in binding of chsp90 to the cER and hMR, while the B region is essential for hormone binding by the hMR, consistent with a chaperone function for hsp90.

The effect of aldosterone and antimineralocorticoids on the intracellular localization of human mineralocorticosteroid receptor (hMR) was studied using a new monoclonal anti-peptide antibody FD4. This antibody was directed against the peptide hMR-(412-422). As demonstrated by ultracentrifugation analysis, immunoprecipitation assays and Western blot, FD4 recognized both the native and denatured form of the receptor overexpressed in the baculovirus expression system. In whole-cell assays, the amount of hMR recovered in high-salt extracts was significantly lower after exposure to the antimineralocorticoid ZK91587 than to aldosterone, suggesting a lack of nuclear MR translocation. FD4 was also used for immunohistochemical studies on hMR-expressing High Five cells. In the absence of hormone, immunoreactive hMR was detected almost exclusively in the cytoplasmic compartment of cells. After aldosterone exposure, intense nuclear immunostaining appeared in a time-dependent manner, consistent with stable nuclear localization of the receptor. Immunohistochemistry showed that antimineralocorticosteroids (ZK91587, SC9420, 18-vinylprogesterone) predominantly maintained a cytoplasmic distribution of hMR and inhibited its aldosterone-dependent nuclear localization. Thus, in our model, the nuclear/cytoplasmic partition of hMR is drastically different in the presence of antagonists from that in the presence of aldosterone. This phenomenon may contribute to their mechanism of action by preventing productive interaction of antagonist-receptor complex with specific DNA sequences in aldosterone target cells.

Although the mineralocorticosteroid receptor (MR) belongs to the superfamily of hormone-dependent transcription factors, the molecular mechanism by which it regulates gene expression is poorly understood. Binding of the MR to target gene promoters has never been characterized, and specific mineralocorticosteroid response elements (MREs) remain to be identified. The human MR (hMR) was overexpressed in Sf21 insect cells using the baculovirus system. The high degree of similarity between the glucocorticosteroid receptor (GR) and the MR prompted us to examine the DNA-binding properties of the recombinant MR with glucocorticosteroid-regulated genes. Gel shift mobility assays demonstrated that the recombinant receptor interacted with oligonucleotides containing perfect and imperfect palindromic sequences of GRE. A monoclonal anti-hMR antibody (FD4) induced a supershift of protein-DNA complexes and identified the MR in Western blot analysis. In vitro DNAse I protection assays with the hormone-regulated murine mammary tumour virus promoter showed that recombinant hMR generated four footprints whose limits encompassed the GRE motifs. By means of these two complementary approaches, no difference between the interaction of free, agonist- or antagonist-bound MR and DNA was detected. We provide evidence that hMR functions as a sequence-specific DNA-binding protein.

Two 'neurosteroids', dehydroepiandrosterone (DHEA) and pregnenolone (PREG), are converted by rat brain microsomes into polar metabolites, identified as the respective 7 alpha-hydroxylated (7 alpha-OH) derivatives by the 'twin ion' technique of g.l.c.-m.s. with deuterated substrates. The enzymic reaction requires NADPH and is stimulated 2-4-fold by EDTA. Under optimal conditions (pH 7.4, 0.5 mM-NADPH, 1 mM-EDTA), the Km values for DHEA and PREG are 13.8 and 4.4 microM respectively, and the Vmax. values are 322 and 38.8 pmol/min per mg of microsomal protein respectively. Trace amounts of putative 7 beta-OH derivatives of DHEA and PREG are detected. Oestradiol, at a pharmacological concentration of 5 microM, inhibits DHEA and PREG 7 alpha-hydroxylation. Formation of 7 alpha-hydroxylated metabolites is low in prepubertal rats and increases 5-fold in adults. Derivatives of PREG and DHEA, such as PREG sulphate, DHEA sulphate, progesterone and 3 alpha-hydroxy-5 alpha-pregnan-20-one, are known to be neuroactive. Therefore the quantitatively important metabolism to 7 alpha-OH compounds may contribute to the control of neurosteroid activity in brain.

To elucidate the mechanism of action of aldosterone antagonists, we studied the interaction of spironolactone with the chick mineralocorticosteroid receptor (MR). Intestinal cytosol contains specific spironolactone-binding sites (Kd approximately 3 nM; max. no. of binding sites approximately 100 fmol/mg of protein) that have been identified as MRs by competition experiments with steroid ligands and with the monoclonal anti-idiotypic antibody H10E that interacts with aldosterone-binding domain of the MR. Binding studies indicate that aldosterone and spironolactone bind to the MR through a common site that encompasses the epitope recognized by H10E. At 4 degrees C, spironolactone dissociates much more rapidly from the cytosol 8-9 S form of MR (t1/2 38 min) than does aldosterone (t1/2 3240 min). A high dissociation rate was also observed for progesterone, a natural aldosterone antagonist (t1/2 84 min). The covalent linkage of the 90 kDa heat shock protein (hsp90) to the ligand-binding subunit of MR with dimethyl pimelimidate did not notably modify the rate of dissociation of spironolactone from the receptor (t1/2 96 min), excluding the possibility that the rapid dissociation rate of the antagonist was related to hsp90 release. The effects of aldosterone and the two anti-mineralocorticosteroids on the 8-9 S heterooligomeric structure of the MR differed strikingly. Using low-salt density-gradient centrifugation analysis, aldosterone-labelled receptors were recovered as 8-9S complexes, whereas 4 S entities were detected after spironolactone and progesterone binding. This indicated that, under the experimental conditions used, aldosterone antagonists facilitate hsp90 release and thus do not stabilize the non-DNA-binding 8-9S form of MR. We propose that the combination of rapid dissociation of the ligand and a weakened hsp90-receptor interaction is involved in the anti-mineralococorticosteroid activity of aldosterone antagonists.

R 5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) is a synthetic analogue of progesterone, which is the physiological hormone that reinitiates germinal vesicle breakdown in Xenopus laevis oocytes. U.v.-driven photoaffinity labelling experiments were conducted with [3H]R 5020 in oocyte subcellular fractions, and covalently bound radioactivity was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In P-10000 (the pellet sedimenting between 1000 and 10000 g and which contains plasma membrane), a major radioactive band migrating as a 30kDa peptide was found. Non-radioactive progesterone competed with the [3H]R 5020 labelling of this fraction, but not with the labelling of minor [3H]R 5020-binding fractions. It displayed the required characteristics of a specific progesterone-binding membrane 'receptor', postulated from previous studies with intact oocytes and with cell-free P-10000 preparations of membrane-bound adenylate cyclase. The apparent Ki of approx. 4 microM for progesterone was compatible with the active concentration of the hormone. Binding specificity, as determined in competition studies, was highly correlated with the germinal vesicle breakdown activity of the steroids and analogues tested. The receptor was not found in the vitelline envelope, in vitelline platelets, in melanosome-enriched or microsomal fractions, in cytosol, nor in germinal vesicles of oocytes. The properties of this membrane steroid receptor are different from those of the already known soluble intracellular steroid receptors, in particular regarding ligand binding specificity and subcellular distribution.

Oestrogen-primed and withdrawn chicks were injected with oestradiol benzoate, progesterone, and/or the anti-oestrogens tamoxifen and 4-hydroxytamoxifen. Oestrogen receptors were studied in oviduct chromatin solubilized by mild digestion of purified nuclei with micrococcal nuclease. After a single injection of oestradiol benzoate, ultracentrifugation on sucrose gradients of chromatin extracts labelled with [3H]-oestradiol showed two peaks of oestradiol binding sites, sedimenting at 13--14 S and 7--8 S. After repeated injections of oestradiol benzoate, the 13--14 S peak increased more than the 7--8 S peak. After injection of anti-oestrogen alone or together with oestradiol benzoate, no [3H]oestradiol-binding or 4-hydroxy[3H]tamoxifen-binding peaks were detected in the chromatin. Injection of progesterone also produced an increase of the 13--14 S and 7--8 S chromatin oestradiol receptor. Progesterone receptor could only by detected in chromatin early after progesterone administration, and it sedimented in density gradients with the 12 S mononucleosome fraction. Tamoxifen injected together with progesterone gave higher levels of 13--14 S oestrogen binding sites than did progesterone alone. The presence of a 13--14 S peak of oestrogen binding sites in hormonal situations which promote a biological response in the chick oviduct, and the absence of this peak after administration of anti-oestrogens, suggest that this subfraction of chromatin contains elements involved in gene regulation.

Studies of the proton-transfer reaction by Pseudomonas testosteroni 3-oxo steroid Delta(4)-Delta(5)-isomerase with Delta(5(6))- and Delta(5(10))-steroid substrates demonstrate the importance of the position of the double bond for the efficiency of the isomerization process. Thus 3-oxo-Delta(5(6))-substrates have markedly high k(cat.) values, whereas those of 3-oxo-Delta(5(10))-substrates are very low and their apparent K(m) values approach equilibrium dissociation constants. The first step in the isomerization process is: [Formula: see text] which is governed by the k(-1)/k(+1) ratio and is shown to be very similar for the two classes of substrates (3-oxo-Delta(5(6))- and -Delta(5(10))-steroids). They therefore differ in the steps distal to the initial formation of the Michaelis-Menten complex. The use of the deuterated androst-5(6)-ene-3,17-dione substrate enabled us to calculate individual rate constants k(+1) and k(-1) as well as to determine the apparent rate-limiting step in the isomerization process. With the deuterated oestr-5(10)-ene-3,17-dione substrate, no significant isotope effect was observed suggesting that a different rate-limiting step may be operative in this isomerization process. Data are presented that indicate that under optimal concentrations of the efficient androst-5(6)-ene-3,17-dione substrate, the forward reaction for ES complex formation (as defined by k(+1)) is limited only by diffusion and the apparent K(m) does not approach the equilibrium constant, suggesting that the evolution of this enzyme has proceeded close to ;catalytic perfection'.

The existence of unoccupied nuclear oestradiol-receptor sites in normal human endometrium was investigated. Nuclei were prepared from endometrial samples obtained by curettage and exposed to [3H]oestradiol, which became maximmaly bound at 0 degrees C within 1 h. This result contrasted with the binding kinetics of oestradiol--receptor complexes, since the exchange of hormone took at least 3 h at 30 degrees C and no displacement occurred at 0 degrees C. Before concluding that the nuclear sites were unoccupied, the presence of endogenous low-affinity ligands was excluded, because the association rate of oestradiol was unchanged after nuclei were stripped from their putative ligands, and the displacement of oestrone bound to nuclear receptor by oestradiol was very slow at 0 degrees C. The available sites had high affinity for oestradiol (KD 1.3 nM) and binding-specificity characteristics of oestradiol receptors. Similar results were observed with crude and purified nuclear preparations. It was concluded that a significant proportion of nuclear oestradiol receptors in normal human endometrium is unoccupied by endogenous hormones.

1. The nuclear-myofibrilar (800g pellet) fraction of the uterus from immature (22-23 days old) rats not exposed to oestrogen exhibits saturable binding of oestradiol. The nuclear binding capacity represents approximately 10% of that of the cytosol fraction (approx. 3.5 fmol/mug of DNA). The predominant part (0.3.5 fmol/mug of DNA) of the nuclear binind sites are present in the residual pellet after extraction with 0.5 M-KC1. 2. By using an exchange technique in vitro, determinations of the nuclear binding sites have been carried out after administration of 1 mug of oestradiol in vivo. Within 0.5h after the hormone injection, the concentration of nuclear bindng sites increased to approx. 0.4 fmol/mug of DNA in the 0.5 M-KC1-extractable fraction, and to approx. 1.2 fmol/mug of DNA in the residual fraction. Meanwhile the cytosol oestrogen-receptor concentration decreased to approx. 10% of its initial value. In the following period from 0.5 h after the oestradiol injection onwards, the concentration of nuclear oestrogen receptors decreased with halflife values of approx. 140 and 200 min for the KC1(0.5 M)-extractable and residual form respectively. At the same time, the cytosol receptor concentration increased to reach approx. 50% of the initial value by the 6h. This increase could not be blocked by cycloheximide. The initial concentration of cytosol receptor was restored approx. 11h after the injection and the increase during the 6-11h period was sensitive to cycloheximide inhibition, suggesting protein-synthesis-dependence of the process. 3. With the (more) physiological dose of oestradiol (0.1 mug), the decrease the cytosol receptor was only 50% by 4h and this was followed by a period (up to 12h after injection) during which the initial concentration was restored. During this period the increase of the receptor can be blocked by cycloheximide.

Salt (NaCl)-extracted nuclear oestrogen receptor from hen oviduct was incubated with salt-depleted oviduct chromatin and dialysed to low salt. The oestrogen receptor (re)associated with chromatin to form a 13-14S-sedimenting fraction, as found in 'native' chromatin, and saturation of this interaction was obtained for very low receptor concentrations (approx. 0.04 nM). Similarly, purified progesterone receptor from chick oviduct cytosol associated with depleted chromatin to form an 11-12S-sedimenting fraction, as in 'native' chromatin; this interaction tended towards saturation for much higher concentrations of progesterone receptor (approx. 8 nM) than that observed for oestrogen receptor. When the two receptors were incubated with depleted chromatin from hen kidney or erythrocytes, their s values were as for oviduct chromatin. However, no saturation of these interactions was seen, even for high concentrations of receptor. Steroid-hormone receptors can therefore bind in vitro to particular subfractions of non-target-tissue chromatin, but with a much lower affinity than to target-tissue chromatin.

Activation (transformation) of the chick oviduct progesterone receptor was found to be induced at 0 degrees C by heparin free in solution as well as by chromatography on a column of heparin linked to acrylamide/agarose. The transformed molecule displayed properties of the activated form of [3H]progesterone-receptor complex obtained by heat treatment or by high ionic strength: smaller size (s20,w = 3.9 S, Stokes radius = 5.2 nm), lower rate of dissociation (t 1/2 approx. 50 h at 0 degrees C compared with approx. 20 h for the 'native' form) and increased binding to phosphocellulose. In all cases, molybdate was an effective inhibitor of transformation and stabilized a large 'native' form (s20,w = 7.9 S, Stokes radius = 7.6 nm). Transformation by neither KCl nor heparin depended on the presence of ligand bound to the receptor, and the properties of the receptor molecule produced by treatment of ligand-free receptor with high ionic strength or with heparin were identical with those of the activated progesterone-receptor complex, demonstrating that receptor activation can be obtained experimentally in the absence of hormone. Our data are compatible with a model in which activation implies separation of the 4 S units, which compose the approx. 8 S 'native' form.

We have examined the regulation of murine mammary-gland 90 kDa heat-shock protein (hsp-90) as a function of normal development and differentiation. We find that both hsp-90 and amounts of its mRNA are modulated during development and differentiation, with the highest concentrations of mRNA and protein being present in tissues from pregnant and lactating animals respectively. Metabolic labelling experiments with [35S]methionine reveal that the rate of synthesis of hsp-90 also varies among tissues from various developmental states and correlates with the relative hsp-90 mRNA content. These data also suggest that the highest concentration of hsp-90 found in lactating mammary tissues may be due to a greater stability of this protein in this developmental state. The possible significance of the developmental modulation of mammary hsp-90 to mammary steroid-receptor properties is discussed.

A 110kDa component of the chick oviduct progesterone receptor (PR) has been purified to homogeneity according to electrophoretic criteria and specific activity (assuming one progestagen-binding site/110kDa). The procedure involved affinity chromatography of 0.3 M-KCl-prepared cytosol, followed by DEAE-Sephacel chromatography (elution at 0.2 M-KCl). The final yield was about 12% in terms of binding activity. Properties of the 110kDa component indicate that it is identical with the 'B' subunit described previously [Stokes radius approximately 6.1 nm; sedimentation coefficient, (S20, w) approximately 4S; frictional ratio approximately 1.77]. It reacted with the IgG-G3 polyclonal antibody, but not with BF4 monoclonal antibody raised against the 8S molybdate-stabilized chick oviduct PR and reacting with its 90kDa component. Another progesterone-binding component, corresponding to the 'A' subunit, also previously described, was eluted from the DEAE-Sephacel column at approximately 0.08 M-KCl, and contained a peptide of molecular mass approx. 75-80kDa, which had S20, w approximately 4S in a sucrose gradient. This component was also recognized by IgG-G3, but not by BF4; it was very unstable in terms of hormone-binding activity.