Demonstration of steroid hormone receptors and steroid action in primary cultures of rat glial cells

Primary cultures of rat glial cells were established from newborn rat forebrains. A mixed population of oligodendrocytes and astrocytes was obtained, as confirmed by indirect immunofluorescence staining with specific markers for each cell type. Receptors were measured 3 weeks after primary culture in glial cells cultured in the presence or not of 50 nM estradiol and we have identified progesterone, glucocorticoid, estrogen, and androgen receptors (PR, GR, ER and AR), but only PR was inducible by the estrogen treatment. This estrogen-induction of PR was more dramatic in glial cells derived from female offsprings than from males, as measured by binding studies and by immunohistochemical techniques with the KC 146 anti-PR monoclonal antibody. The antiestrogen tamoxifen inhibited the estrogen induction, but had no effect by itself on PR concentration. Specific binding sites for PR, GR, ER and AR were measured by whole cell assays after labeling cells with, respectively, [³H]R5020, [³H]dexamethasone, [³H]OH-tamoxifen or [³H]R1881. PR and GR were also analyzed by ultracentrifugation and after exposure of cells to agonists, both receptors were recovered from cytosol as a 9S form, and from the nuclear high-salt, tungstate ions-containing fraction as a 4–6S form. In contrast, when the antiprogestin- and antiglucocorticosteroid RU486 was used as a ligand, a non-activated 8.5S receptor complex was found for both receptors in this nuclear fraction. The 8.5S complex of the GR was further analyzed in the presense of specific antibodies and, in addition to GR, the presence of the heat shock protein hsp90 and of a 59 kDa protein was found.

During primary culture, the effects of progesterone (P) and estradiol (E₂) were tested on glial cell multiplication, morphology and differentiation. Cell growth was inhibited by P and stimulated by E₂. Both hormones induced dramatic morphologic changes in oligodendrocytes and astrocytes and increased synthesis of the myelin basic protein in oligodendrocytes and of the glial fibrillary acidic protein in astrocytes.     

ZK98299, a novel antiprogesterone that does not interact with chicken oviduct progesterone receptor

Steroid antagonists, at receptor level, are valuable tools for elucidating the mechanism of steroid hormone action. We have examined and compared the interaction of avian and mammalian progesterone receptors with progestins; progesterone and R5020, and a newly synthesized antiprogesterone ZK98299. In the chicken oviduct cytosol, [3H]R5020 binding to macromolecule(s) could be eliminated with prior incubation of cytosol with excess radioinert steroids progesterone or R5020 but not ZK98299. Alternatively, [3H]ZK98299 binding in the chicken oviduct was not abolished in the presence of excess progesterone, R5020, or ZK98299. In the calf uterine cytosol, [3H]R5020 or [3H]ZK98299 binding was competeable with progesterone, R5020 and ZK98299 but not estradiol, DHT or cortisol. Furthermore, immunoprecipitation and protein A-Sepharose adsorption analysis revealed that in the calf uterine cytosol, the [3H]R5020-receptor complexes were recognized by anti-progesterone receptor monoclonal antibody PR6. This antibody, however, did not recognize [3H]ZK98299-receptor complexes. When phosphorylation of progesterone receptor was attempted in the chicken oviduct mince, presence of progesterone resulted in an increased phosphorylation of the known components A (79 kDa) and B (110 kDa) receptor proteins. Presence of ZK98299 neither enhanced the extent of phosphorylation of A and B proteins nor did it reverse the progesterone-dependent increase in the phosphorylation. The avian progesterone receptor, therefore, has unique steroid binding site(s) that exclude(s) interaction with ZK98299. The lack of immunorecognition of calf uterine [3H]ZK98299-receptor complexes, suggests that ZK98299 is either interacting with macromolecule(s) other than the progesterone receptor or with another site on the same protein. Alternatively, the antisteroid binds to the R5020 binding site but the complex adopts a conformation that is not recognized by the PRG antibodies.     

Novel antiprogestins Org 31806 and 31710: interaction with mammalian progesterone receptor and DNA binding of antisteroid receptor complexes.

We have examined steroid binding parameters and transformation of calf uterine progesterone receptor (PR) liganded with progestins (progesterone and R5020) and the newly synthesized antiprogestins (Org 31806 and 31710). Species specificity analysis indicated that [3H]R5020 binding in the chicken oviduct cytosol could be eliminated in the presence of 100-fold excess radioinert progesterone and R5020 but not Org 31806 and 31710. In the calf uterine cytosol, the progestins and the antiprogestins appeared to interact with the same PR as revealed by the displacement of [3H]R5020 by all of the above steroids. When the extent of [3H]R5020 binding was examined in the presence of different concentrations of radioinert steroids, the relative affinity with which these compounds interacted with the uterine PR was found to be comparable. A 23 degrees C incubation of cytosol transformed the progestin-bound PR complexes increasing their binding to DNA-cellulose from 5 (0 degrees C, nontransformed) to 35%. Under these conditions, 20% Org 31710- and RU486-occupied PR complexes bound to DNA-cellulose whereas only 10% Org 31806-receptor complexes were retained by the resin. Transformation (23 degrees C) of cytosol receptor caused a loss of the larger 8 S form and an increase in the smaller 4 S form. In its unliganded state or when it was complexed with R5020 or the antiprogestins, incubation of PR at 23 degrees C led to dissociation of the receptor-associated 90 kDa heat-shock protein (hsp90). The PR-hsp90 association was stabilized in the presence of 10 mM iodoacetamide when the ligand binding site was occupied by Org 31806 and 31710. The R5020-receptor complexes, however, allowed release of hsp90 under the above transforming conditions. Our results indicate that although Org 31806 and 31710 show no affinity for the avian PR, these steroids interact with the mammalian PR. We propose that the reported antiprogestational effects of Org 31806 and 31710 are mediated via their interaction with PR which appears similar to one that exists between PR and RU486.     

Characterization of R5020 and RU486 binding to progesterone receptor from calf uterus

We have examined and compared the binding characteristics of the progesterone agonist R5020 [promegestone, 17,21-dimethylpregna-4,9(10)-diene-3,20-dione] and the progesterone antagonist RU486 [mifepristone, 17 beta-hydroxy-11 beta-[4-(dimethylamino) phenyl]-17 alpha-(prop-1-ynyl)-estra-4,9-dien-3-one] in calf uterine cytosol. Both steroids bound cytosol macromolecule(s) with high affinity, exhibiting Kd values of 5.6 and 3.6 nM for R5020 and RU486 binding, respectively. The binding of the steroids to the macromolecule(s) was rapid at 4 degrees C, showing saturation of binding sites at 1-2 h for [3H]progesterone and 2-4 h for both [3H]R5020 and [3H]RU486. Addition of molybdate and glycerol to cytosol increased the extent of [3H]R5020 binding. The extent of [3H]RU486 binding remained unchanged in the presence of molybdate, whereas glycerol had an inhibitory effect. Molybdate alone or in combination with glycerol stabilized the [3H]R5020- and [3H]RU486-receptor complexes at 37 degrees C. Although the rate of association of [3H]RU486 with the cytosolic macromolecule was slower than that of [3H]R5020, its dissociation from the ligand-macromolecule complex was significantly slower than [3H]R5020. Competitive steroid binding analysis revealed that [3H]progesterone, [3H]R5020, and [3H]RU486 compete for the same site(s) in the uterine cytosol, suggesting that all three bind to the progesterone receptor (PR). Sedimentation rate analysis showed that both steroids were bound to a molecule that sediments in the 8S region. The 8S [3H]R5020 and [3H]RU486 peaks were abolished by excess radioinert progesterone, RU486, or R5020.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mammalian progesterone receptor shows differential sensitivity to sulfhydryl group modifying agents when bound to agonist and antagonist ligands

Modulation of calf uterine progesterone receptor (PR), in relation to its binding to synthetic steroids with known agonist (R5020) and antagonist (RU486) properties, was studied in the presence of iodoacetamide (IA), N-ethylmaleimide (NEM), beta-mercaptoethanol (MER), and dithiothreitol (DTT). Pretreatment of uterine cytosol at 4 degrees C with NEM (4-10 mM) reduced the binding of [3H]RU486 to PR by 40%, but [3H] R5020 binding was completely abolished. Whereas IA (2-10 mM) treatment did not affect [3H]RU486 binding, [3H]R5020 binding was totally eliminated. DTT or MER increased the binding of both steroids slightly (15%). [3H]R5020- or [3H]RU486-receptor complexes (Rc) migrated in the 8 S region and were eliminated upon pretreatment with NEM. At 23 degrees C, DTT increased the amount of 4 S [3H]R5020-Rc, but had no effect on the [3H]RU486-Rc. In the control, [3H]RU486 binding to the 8 S PR could be competed with radioinert R5020 or RU486, but R5020 failed to compete in the presence of IA. The heat-treated [3H]R5020- and [3H]RU486-Rc showed reduced binding to DNA-cellulose in the presence of NEM and IA. The results of our study suggest that SH group modifications differentially influence the properties of mammalian PR complexed with either R5020 or RU486. In the presence of IA, the [3H]RU486-Rc remained in the 8 S form when incubated at 23 degrees C, indicating that RU486 binding causes conformational changes in PR which are distinct from those that result upon R5020 binding.     

Transformation of calf uterine progesterone receptor: analysis of the process when receptor is bound to progesterone and RU38486

Effects of different transforming agents were examined on the sedimentation characteristics of calf uterine progesterone receptor (PR) bound to the synthetic progestin [3H]R5020 or the known progesterone antagonist [3H]RU38486 (RU486). [3H]R5020-receptor complexes [progesterone-receptor complexes (PRc)] sedimented as fast migrating 8S moieties in 8-30% linear glycerol gradients containing 0.15 M KCl and 20 mM Na2MoO4. Incubation of cytosol containing [3H]PRc at 23 degrees C for 10-60 min, or at 0 degrees C with 0.15-0.3 M KCl or 1-10 mM ATP, caused a gradual transformation of PRc to a slow sedimenting 4S form. This 8S to 4S transformation was molybdate sensitive. In contrast, the [3H]RU486-receptor complex exhibited only the 8S form. Treatment with all three activation agents caused a decrease in the 8S form but no concomitant transformation of the [3H]RU486-receptor complex into the 4S form. PR in the calf uterine cytosol incubated at 23 or at 0 degrees C with 0.3 M KCl or 10 mM ATP could be subsequently complexed with [3H]R5020 to yield the 4S form of PR. However, the cytosol PR transformed in the absence of any added ligand failed to bind [3H]RU486. Heat treatment of both [3H]R5020- and [3H]RU486-receptor complexes caused an increase in DNA-cellulose binding, although the extent of this binding was lower when RU486 was bound to receptors. An aqueous two-phase partitioning analysis revealed a significant change in the surface properties of PR following both binding to ligand and subsequent transformation. The partition coefficient (Kobsd) of the heat-transformed [3H]R5020-receptor complex increased about 5-fold over that observed with PR at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

ZK98299--a new antiprogesterone: biochemical characterization of steroid binding parameters in the calf uterine cytosol.

We have examined steroid binding characteristics of a newly synthesized antisteroid, ZK98299 [onapristone, 11 beta-(4-dimethylaminophenyl)-17 alpha-hydroxy-17 beta-(3-hydroxypropyl)- 13 alpha-methyl-4,9-gonadien-3-one], in the calf uterus cytosol and compared the nature of this interaction with the binding of progesterone receptor (PR) agonist R5020 [promegestone, 17,21-dimethylpregna-4,9-diene-3,20-dione]. In the freshly prepared cytosol, [3H]ZK98299 interacted specifically with a macromolecule: the binding was abolished in the presence of excess progestins (R5020 and progesterone) and the antiprogesterone ZK98299. The high affinity (Kd = 2.5 nM) interaction between [3H]ZK98299 and PR was temperature- and time-dependent, reaching an optimum by 2-3 h at 0 degrees C, and was facilitated by 20 mM Na2MoO4. Under nontransforming conditions, [3H]ZK98299-receptor complexes sedimented as 8 S species in 8-30% linear glycerol gradients. Upon salt or thermal transformation, there was a loss of the 8 S form, with only a small fraction of total complexes (5-7%) binding to DNA-cellulose. In contrast, transformed [3H]R5020-receptor complexes exhibited a greater extent of binding (25-55%) to DNA-cellulose. [3H]ZK98299-receptor complexes could be resolved into two ionic species over DEAE-Sephacel following incubation of the complexes at 0 or 23 degrees C. [3H]ZK98299 binding was sensitive to sulfhydryl group modification as beta-mercaptoethanol increased the extent of steroid binding. Although treatment with iodoacetamide (IA) abolished [3H]R5020 binding, there was a significant (nearly twofold) increase in the [3H]ZK98299 binding. The results of this study point to similarities and differences between the steroid binding properties of the uterine PR occupied by R5020 and ZK98299: both steroids appear to bind the same 8 S receptor but exhibit differential DNA binding and sensitivity to IA. The reported antagonist properties of ZK98299 may, therefore, be explained on the basis of a distinct receptor conformation induced by the antisteroid.     

Evidence for a Leydig cell progesterone receptor in the rat

Tritiated promegestone [3H] R 5020 is bound with high affinity by charcoal-treated cytosol prepared from purified Leydig cells. The binding is characterized by high affinity (Kd = 2 x 10(-9) M) and specificity (R 5020 = progesterone greater than testosterone = dehydrotestosterone greater than hydroxyprogesterone greater than cortisol = dexamethasone greater than estradiol) appropriate for progesterone receptors. In vitro, progestin-bound cytosol was quantitatively translocated to nuclei fractions, only if cytosol samples were previously labeled at 25 degrees C. However no translocation of binding activity was observed when previous cytosol labeling was done in the presence of sodium molybdate. Effects of glucocorticoids, androgens and estrogens on the Leydig cell are well documented, the demonstration of a putative progesterone receptor raises the possibility of direct effect of progesterone on the Leydig cell.     

Immunologically distinct binding molecules for progesterone and RU38486 in the chick oviduct cytosol

[3H]Progesterone and [3H]RU38486 binding in the chick oviduct cytosol is associated with macromolecules which sediment as 8 S and 4 S moieties, respectively, in molybdate-containing 5-20% sucrose gradients. The [3H]progesterone binding could be displaced by excess progesterone, but not by RU38486. Conversely, the [3H]RU38486 binding was able to compete with RU38486 but not by excess progesterone. A preparation containing antibodies against chick oviduct progesterone receptor recognized only the [3H]progesterone-receptor complex but not the 4 S, [3H]RU38486 binding component of the chick cytosol. In the calf uterus cytosol, [3H]R5020 (a synthetic progestin) and [3H]RU38486 were associated with 8 S molecules and the peaks of radioactivity were displaceable upon preincubation with radionert steroids. In addition, the complexes were recognized by antibodies to chick oviduct progesterone receptor. Our data suggest that in the chick oviduct cytosol, RU38486 does not bind to progesterone receptor, but interacts with an immunologically distinct macromolecule.     

Specific progesterone receptors in dimethylbenzanthracene (DMBA)-induced mammary tumors.

Properties of a progesterone receptor present in the cytosol (105,000 xg supernatant) of dimethylbenzanthracene (DMBA)-induced mammary tumors were studied using the highly potent progestin [3H]R 5020 (17, 21-dimethyl-19-nor-pregna-4, 9-diene-3,20-dione). As shown by sucrose gradient analysis, specific binding of [3H] R 5020 is associated with components migrating at 7-8S and 4S. Low affinity binding of the synthetic progestin is eliminated by treatment with dextran-coated charcoal. [3H] R 5020 binding is highly progestin-specific since it is easily displaced by unlabelled norgestrel, R 5020 and progesterone while estradiol-17beta, dihydrotestosterone, testosterone, testosterone and diethylstilbestrol have much lower activity. Dexamethasone and cortisol have little, if any, effect on [3H] R 5020 binding.     

Rat uterine progesterone receptor: Stabilization of hormone-binding components for biochemical analyses

A method was developed for quantitative recovery of the labile rat uterine progesterone receptor hormone-binding components. Initial conditions were established by the sucrose gradient procedure. Upon centrifugation through low-salt 5–20% sucrose gradients prepared in 10% glycerol, the well-known 6–8 S progesterone receptor components were observed either when cytosol was prelabeled with [³H]17,21-dimethyl-19-nor-4,9-pregnadiene-3, 20-dione ([³H]R5020) or when prelabeled with [³H]progesterone followed by postlabeling the fractions collected after centrifugation with either [³H]progesterone or [³H]R5020. Recovery of progesterone receptor binding was improved by prelabeling with [³H]R5020, by adding 1.5 mm ethylene glycol bis(β-aminoethylether)N,N′-tetraacetic acid (EGTA) to all buffers, and at high tissue concentrations. Under these conditions quantitative conversion of the receptor to specific [³H]R5020-binding 4S components was achieved with 150 or 400 mm KCl. Similar conditions proved unsuitable for receptor analysis by gel filtration (Bio-Rad agarose A0.5M or A1.5M), apparently due to [³H]R5020 dissociation from the receptor in the large volume of elution buffer. However, excellent receptor recovery (97.2 ± 6.7%) was achieved by including 10 nm unlabeled progesterone in all preparation and elution buffers. Receptors were then detected by the addition of 5 nm [³H]R5020 to the column fractions, exchange incubation for 3–6 h at 4 °C, and subsequent separation of bound and free steroid by the hydroxyapatite assay. This method resulted in a consistent elution pattern suggestive of receptor heterogeneity. Identity of the peak(s) as progesterone receptor components(s) was confirmed by the lack of competition by 2 μm cortisol when added either to cytosol or during the post-labeling-exchange process. Neither the qualitative nor quantitative results of the column profiles were changed substantially in the presence of 20 mm molybdate. Although the receptor structure has yet to be established, both statistical analysis of the column profiles by computer curve-fitting procedures and rechromatography of peak fractions suggested that the rat uterine progesterone receptor may be composed of multiple components. This ligand-stabilization/postlabeling-exchange procedure provides a method for further studies of progesterone receptor biochemistry in mammalian systems. Additionally, similar procedures may stabilize other labile ligand-binding proteins for biochemical analyses and/or purification.     

Induction and inhibition of estradiol hydroxylase activities in MCF-7 human breast cancer cells in culture

The effects of estradiol, progesterone, and tamoxifen on the activity of estradiol 2- and 16 alpha-hydroxylases were studied in human breast cancer cell cultures using a radiometric assay. After 5 days' exposure to these compounds, incubations in the presence of either [2-3H]estradiol or [16 alpha-3H]estradiol as substrate were carried out. In MCF-7 cells, estradiol (10(-8) M), progesterone (10(-6) M) and tamoxifen (10(-6) M) significantly increased 16 alpha-hydroxylase activity (estradiol; 21% progesterone 10% to 32%; tamoxifen 21% to 31%; P less than 0.01). Synergistic effects were observed when the cells were successively exposed to tamoxifen and progesterone. Simultaneous treatment with tamoxifen plus estradiol or estradiol plus progesterone showed no change from estradiol alone. On the other hand, although estradiol had no direct effects on 2-hydroxylase activity, tamoxifen decreased this enzymatic activity significantly at 10(-6) M (23% to 37%). Progesterone acted synergistically to further decrease this reaction. Treatment with only progesterone caused an increase in 2-hydroxylation. In contrast, a subline of MCF-7 cells with low estrogen receptor levels showed only minimal enzyme-hormone responses. Likewise, treatment of the estrogen receptor-negative MDA-MB-231 human breast cancer cell line with these compounds showed no effects on either 2- or 16 alpha-hydroxylase activity. In the progesterone receptor-rich T47D cell line, estradiol decreased both activities while progesterone increased both.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a single steroid-binding protein in the rabbit progesterone receptor

The rabbit uterine progesterone receptor copurifies as two molecular weight (Mr) forms of about 105,000 and 78,000. To investigate whether these are different proteins, we have used protease digestion, reversible denaturation, and photoaffinity labeling in studies on the steroid-binding domain of the receptor. Digestion of the Mr 105,000 and 78,000 forms, photoaffinity labeled with [3H]R5020, with Staphylococcus aureus V8 protease revealed identical peptide fragments of Mr 43,000, 39,000, and 27,000-30,000. When receptor in cytosol was denatured, separated by electrophoresis, and then reconstituted, [3H]progesterone bound specifically to a single form at about Mr 105,000. After partial purification, the reversible denaturation procedure revealed both the larger and the smaller progesterone-binding species similar to the photoaffinity-labeled species in this preparation. Receptor in uterine cytosol prepared under mild conditions appeared as a predominant large molecular weight form on photoaffinity labeling with [17 alpha-methyl-3H]R5020, [6,7-3H]R5020, or [3H]RU27987. Further purification of this cytosol showed the generation of a smaller labeled species. These results from three different approaches reinforce the view that the rabbit progesterone receptor contains a single steroid-binding protein.     

Antiprogestin-receptor complexes: differences in the interaction of the antiprogestin RU38,486 and the progestin R5020 with the progesterone receptor of human breast cancer cells

In order to understand the molecular basis for antiprogestin action, we have compared the interaction of the antiprogestin [3H]RU38, 486 (RU486) and the progestin [3H]R5020 with the progesterone receptor (PR). In both MCF-7 and T47D human breast cancer cells, we have observed marked differences in the sedimentation properties of the PR on high salt sucrose gradients: while the R5020-receptor complexes sediment at approximately 4 S (4.4 +/- 0.1 S), the RU486-receptor sediments as a prominent 6 S species as well as a 4 S species. This binding is abolished by excess unlabelled R5020, RU486 or progesterone, but is unaffected by excess unlabelled hydrocortisone or dexamethasone, indicating that both the 4 S and 6 S species represent the PR and not glucocorticoid receptor. Although the relative distribution of 4 S and 6 S forms is not altered by treatment with DNAse or RNAse, exposure to 10 mM thioglycerol or to 3 M urea results in conversion of the 6 S to the 4 S form, suggesting that disulfide bonds and hydrophobic interactions are important in maintaining the integrity of the 6 S form. These findings suggest that the 6 S antiprogestin complex is formed as a result of the interaction of PR units with each other or with a different protein. This change in receptor association state may be an important aspect of the antiprogestin activity of RU486.     

Progesterone receptor quantification with radiolabeled promegestone (R 5020) in frozen sections of endometrium and breast cancer tissue

A technique for the determination of the progesterone receptor content at sections was developed. Series of coverglass-mounted unfixed frozen sections were incubated with [3H]R5020 only, to determine total binding, or with excess unlabeled R5020, to determine non-specific binding. Ligand binding in the tissue sections was measured by liquid scintillation counting after repeated washing of the coverslips. Elution of ligand binding proteins into the incubation buffer was quantitated with the dextran-coated charcoal method. Specific ligand binding was related to the total tissue protein content which was determined on parallel, unmounted sections. Scatchard analysis showed specific saturable and high affinity (Kd = 0.01-2 nM) section-bound and soluble binding sites in cryostat sections of calf uterus, human endometrium and breast cancer samples. Ligand specificity was studied by competition of [3H]R5020 with a 100-fold excess of various steroid receptor ligands. The competition was excellent for R5020 and progesterone, negligible for estrogens and slight for androgens and corticosteroids. These binding characteristics provide evidence that with this assay progesterone receptors are determined. Exchange experiments showed that with this method total, free as well as occupied, progesterone receptors can be measured. A highly significant linear correlation, and agreement in PR status classification between assay on cytosol and sections was obtained for a series of 21 breast cancer samples. Finally, progesterone receptor analysis using cryostat sections results in the recovery of 2-3 times more PR from the same amount of tissue as compared to the use of cytosol. These results indicate that progesterone receptors can be reliably assayed with Scatchard analysis using cryostat sections, which requires less tissue than the cytosol assay. This method, which is simple and easy to perform could be of practical importance, particularly when only small tissue samples (which also have to be analyzed morphologically or histochemically) are available and when quantitative radiochemical progesterone receptor data are required for direct comparison with (immuno-) histochemical information.     

Progestins stimulate the differentiation of 3T3-L1 preadipocytes

The effect of progesterone on the differentiation of the 3T3-L1 preadipocytes was investigated and compared with other sex steroids (estradiol and testosterone), with cortisol, with the synthetic progestin R5020 and with the progestin/glucocorticoid antagonist RU38486. At 10⁻⁸ M, progesterone stimulated the activity of glycerol-3-phosphate dehydrogenase and triglyceride deposition. Progesterone, R5020, cortisol, and RU38486 increased triglycerides about 2-fold at 10⁻⁷ M. Only minimal effects were observed with testosterone and estradiol even at 10⁻⁶ M. When the cells were cultured in presence of 10⁻⁵ M metyrapone the effect of progesterone was unchanged, suggesting that the progesterone was not metabolized to a glucocorticoid. Progesterone, R5020 and RU38486 competed efficiently with [³H]dexamethasone for the glucocorticoid receptor in 3T3-L1 cytosol. These results indicate a significant, reproducible dose-dependent effect of progestins on differentiation of the preadipocytes, which appears to be mediated via the glucocorticoid receptor.     

Effects of antiestrogen versus antiprogestin on transformed and nontransformed steroid receptors

In order to determine if different physicochemical properties exist among antihormone-receptor complexes, we have compared the interaction of the antiprogestin RU486 with progesterone receptor (PR) versus the triphenylethylene antiestrogen H1285 (4-(N,N-diethyl-aminoethoxy)-4'-methoxy-alpha-(p-hydroxyphenyl-alp ha'- ethylstilbene] with estrogen receptor (ER) from rabbit uterine tissue. Contrary to other reports, we observed no difference in the sedimentation properties of transformed PR (4S) when bound by the antagonist RU486 versus the progesterone agonist R5020 in either cytosol or DEAE partially-purified receptor preparations analyzed on sucrose gradients containing 0.3 M KCl. In addition, we found no difference in the sedimentation properties of these receptor preparations in the presence of 10 mM sodium molybdate: the nontransformed RU486-PR and nontransformed R5020-PR both sedimented as a 6S species. These same results were obtained when the receptor preparation and gradient analysis were performed in the absence of monothioglycerol. Likewise, there was no change in the sedimentation properties of the transformed PR when the receptor, partially purified in the absence of molybdate, was analyzed on sucrose gradients containing 10 mM sodium molybdate to prevent receptor alteration during centrifugation. From DNA-cellulose assays performed with partially purified PR in the absence of molybdate we determined that the 4S form of R5020-PR and RU486-PR is transformed receptor; whereas in the presence of molybdate, the 6S species is nontransformed. In contrast, we found a different pattern of sedimentation when comparing transformed antiestrogen-receptor complexes with transformed estrogen-receptor complexes. In this case, transformed H1285-ER sedimented as 6S and estradiol-ER sedimented as 4S. We conclude from these experiments that these two antihormones, RU486 and H1285, may have different mechanisms of action in their antagonism of steroid hormone action. Antiestrogen stabilizes the salt-transformed ER as a dimer while antiprogestin appears to permit dissociation of the oligomeric form of the receptor to the monomeric form.     

Evidence for two structurally related progesterone receptors in chick oviduct cytosol

The existence of two progesterone receptor forms present in crude cytosol of chick oviduct has been demonstrated by photoaffinity labelling using [3H]R5020. On SDS-polyacrylamide gels these two forms exhibit app. Mr-values of 79000 and 109000 corresponding to the progesterone receptor forms A and B. Peptide maps of photoaffinity-labelled steroid receptors have been established by limited proteolysis with alpha-chymotrypsin. The peptide map obtained for chick oviduct cytosol progesterone receptor crosslinked with [3H]R5020 proved to be the sum of peptides obtained from partially purified preparations of forms A and B. The peptide maps of both progesterone receptor forms were identical for peptides below the Mr-value of form A, indicating extensive homology of the two forms. A significantly different peptide pattern was observed for the rat liver glucocorticoid receptor crosslinked with [3H]triamcinolone acetonide. Prolonged proteolysis with chymotrypsin gave rise to peptides with Mr-values of 6000 and 10000 from the hormone-binding domain of progesterone and glucocorticoid receptors, respectively.     

Effect of photoaffinity labeling on rabbit uterine progesterone receptor

Photoaffinity labeling with [17 alpha-methyl-3H]promegestone ([ 3H]R5020) is an effective technique for the covalent labeling of the progesterone receptor (PR), which allows monitoring of the steroid receptor complex under denaturing conditions. The present study was initiated to evaluate whether photolabeled PR could be used also as a marker for PR under nondenaturing conditions. Accordingly, the effect of irradiation on each component of the reaction was evaluated separately. When [3H]R5020 alone was irradiated, there was a rapid (less than 5 min), light dependent destruction of [3H]R5020, as evident from increased formation of a more polar tritiated product on TLC and a concomitant decrease in the ability of the irradiated preparation to bind to PR. When rabbit uterine PR was irradiated in the absence of steroid, a gradual decrease in the binding capacity was observed, reaching 70% of the nonirradiated control in 10 min. The optimal irradiation time for covalent [3H]R5020-PR complex formation was determined by irradiation for up to 5 min, and separation of the products by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. Specific labeling of proteins of Mr 116,000 and 85,000 was observed, with the rate of labeling of the two being similar, and reaching a plateau by 4 min of irradiation. The photolabeling efficiency ranged from 2 to 12%. Sucrose gradient ultracentrifugation of photolabeled PR revealed that both the irradiated sample and the nonirradiated control sedimented to the same position. Subsequent SDS-polyacrylamide gel electrophoresis of the sucrose gradient peak from the photolabeled sample showed the presence of both labeled proteins of Mr 116,000 and 85,000. In addition, photolabeled rabbit uterine PR (Mr 116,000 and 85,000) could be immunoprecipitated with a guinea pig antiserum raised against rabbit uterine PR. Analysis of the photoaffinity labeling procedure in our system revealed that the photodestruction of [3H]R5020 was very rapid. However, maximal labeling with [3H]R5020 was obtainable with minimal photodestruction of PR which suggests that photolabeled receptor can be used as a marker for PR under nondenaturing conditions.     

The nonactivated progesterone receptor is a nuclear heterooligomer

The discovery of the nuclear localization of estradiol and progesterone receptors in the absence of the steroid hormone has led to reconsideration of the model of cytoplasmic to nuclear translocation of these receptors upon exposure to hormone. Unoccupied nonactivated receptors are thought to be weakly bound to nuclei of target cells from which they are leaking during tissue fractionation and thus found in the cytosol fraction of homogenates in a nontransformed heterooligomeric "8-9 S" form, which includes hsp90. However, no direct biochemical evidence has yet been obtained for the presence of such heterooligomers in the target cell nucleus, possibly because it dissociates in high ionic strength medium used for extraction of the nuclear receptor. We took advantage of the combined stabilizing effects of tungstate ions and antiprogestin RU486 to extract a nuclear non-DNA binding nontransformed 8.5 S-RU486-progesterone receptor complex from estradiol-treated immature rabbit uterine explants incubated with the antagonist. As demonstrated by immunological criteria and by irreversible cross-linking with dimethylpimelimidate, the complex contained, in addition to the hormone binding unit, hsp90, and p59, another nonhormone binding protein. Control experiments carried out with the progestin R5020 yielded the expected nuclear transformed DNA binding 4.5 S-R5020-progesterone receptor complex. These results offer evidence for two distinct forms of steroid receptor in target cell nuclei. Besides the classical "4 S" agonist-receptor complex, tightly bound to the DNA-chromatin structure and in all probability able to trigger the hormonal response, we have observed in the RU486-bound state a non-DNA binding nontransformed 8.5 S form, presumably already present in the nucleus in the absence of hormone and representing the native nonactive form of the receptor.