Purification and characterization of the activated mineralocorticoid receptor from rat myocardium.

Cardiac cytosol from adrenalectomized rats was radiolabelled with 10 nM tritiated RU 26752, R 5020 or aldosterone, to saturate the mineralocorticoid receptor (MCR) in the presence of 1 microM RU 38486 to block the glucocorticoid and progestin receptors. Free steroids were removed by charcoal treatment and the radiolabelled cytosol was passed through a phosphocellulose column. The MCR peak in the phosphocellulose eluate was activated at 25 degrees C for 45 min, adsorbed onto the DNA-cellulose and finally extracted once each with buffers containing 1 M potassium chloride or 25 mM magnesium chloride. The pooled DNA-cellulose extracts, reequilibrated with 10 nM [3H]RU 26752, were resolved as a single, homogeneous band of 78 kDa upon polyacrylamide gel electrophoresis. Ion-exchange analysis of the purified MCR on DEAE-cellulose-52 revealed a single peak in the 0.017 M sodium phosphate region with both RU 26752 and R 5020, but aldosterone dissociated during this procedure. Molecular filtration on Ultrogel AcA-44 columns revealed a major 145 kDa peak, with some smaller components of 40 and 80 kDa. These hydrodynamic properties of the purified MCR are at variance with those of the native receptor in crude myocardial cytosol, and suggest that some post-translational modifications in vivo may be required for the expression of MCR-mediated responses.

The distribution of progesterone receptor in the 20-day-old fetal mouse: an autoradiographic study with [125I]progestin.

The distribution of progestin target sites in 20-day-old fetuses of estrogen-primed pregnant mice was investigated by thaw-mount autoradiography. Pregnant mice received a Silastic estradiol implant on day 17 and were ovariectomized on day 19 of pregnancy. Twenty-four hours after ovariectomy 10 prematurely delivered fetuses were each injected with 0.33 microgram/100 g BW [125I]progestin (SA, 2200 Ci/mM). To show specificity of progestin localization two additional fetuses were each injected sc with 20 micrograms R5020, a synthetic progestin, 1 h before the injection of [125I]progestin. The fetuses were frozen 2 h after injection of [125I]progestin, sectioned, and processed for thaw-mount autoradiography. Cells with nuclear uptake and retention of radioactivity were observed in numerous tissues, including certain regions of the oral mucosa and developing teeth, esophagus, larynx, skin, mammary gland, skeletal muscle, kidney, and reproductive glands and ducts. Injection of unlabeled R5020 1 h before [125I]progestin prevented nuclear concentration of radioactivity in all target tissues. The results indicate that progesterone receptors are expressed with a regional, cellular, and subcellular distribution in term fetal mouse tissues and suggest that progesterone is important to the growth and development of certain fetal tissues.

The leucine zippers of c-fos and c-jun for progesterone receptor dimerization: A-dominance in the A/B heterodimer.

Human progesterone receptors (hPR) exist as two isoforms: 120 kDa B-receptors (hPRB) and N-terminally truncated 94 kDa A-receptors (hPRA). When transfected separately, each isoform exhibits different transcriptional properties that are ligand- and promoter-specific. In human target tissues, both receptor isoforms are present, so that a mixture of three dimeric species, A/A, A/B, and B/B, bind to DNA at progesterone response elements (PRE), and regulate transcription. To study the transcriptional phenotype of pure A/B heterodimers uncontaminated by A/A or B/B homodimers, we exploited the property of the leucine zipper (zip) domains of fos and jun, to form pure heterodimers. Chimeric constructs were made linking the zip of either c-fos or c-jun to the C-terminus of hPRB or hPRA (hPR-zip) to produce A-fos, B-fos, A-jun or B-jun. To determine whether the A- or B-isoform is functionally dominant in the A/B heterodimer, cells expressing hPR-zip chimeras were treated with the progestin antagonist RU486, which produces opposite transcriptional effects with the two isoforms. Gel mobility shift and immune co-precipitation assays show that in the presence of RU486 only pure heterodimers form between A-fos/B-jun or A-jun/B-fos, and bind DNA at PREs. Thus, in these pairs, interactions between the extrinsic fos/jun zipper domains override interactions between the intrinsic hPR dimerization domains. We find that under these conditions, antagonist-occupied B-zip homodimers stimulate transcription, while antagonist-occupied A-zip homodimers are inhibitory, and that pure A/B zip heterodimers have the inhibitory transcriptional phenotype of the A-zip homodimers. We conclude that, in pure heterodimers, A-receptors are dominant negative inhibitors of B-receptors. Additionally, the pure PR-zip heterodimers, unlike wild-type receptors, bind a PRE in the absence of hormone but do not activate transcription. Thus, PR dimerization and PRE binding are necessary but, without hormone, not sufficient to activate transcription.

Myometrial estrogen and progesterone receptor binding in pregnancy: inhibition by the detergent action of phospholipids.

We characterized the phospholipid inhibition of estradiol and progesterone binding to guinea-pig and human myometrial receptors. Of twelve compounds studied, phosphatidylinositol (PI), lysophosphatidic acid and lysophosphatidylcholine (lyso-PC) were the most active inhibitors (50% inhibition at 10(-5) M). Lyso-PC with fatty acid chain length C14:0 inhibited ligand binding both to estrogen receptor (ER) and progesterone receptor (PR), C16:0 only to PR and C18:0 neither to ER nor to PR. The lyso-derivates were more inhibitory than the parent compounds. The ionic detergent (sodium taurocholate) inhibited both ER and PR binding, but the non-ionic detergent (Triton X-100) only PR. Triton X-100 enhanced the PI-induced inhibition of ER binding by a factor of 10. PR was more sensitive to inhibition than ER in all cases. The type of inhibition was non-competitive. At term pregnancy, ligand binding to myometrial ER or PR was low or absent in humans, but moderate in the guinea-pig. Phospholipid extracts of human decidua and fetal membranes contained PI and phosphatidylserine rather than lyso-PC. The extract was a potent inhibitor of ligand binding to PR (50% inhibition at 10(-6) M phospholipid phosphorus), but not to ER. The physicochemical environment, modulated by phospholipids acting as detergents, may regulate sex steroid function also in vivo. This might have special significance for pregnancy maintenance.

Sex steroid receptors in transformed human cervical tissue.

Investigation of the binding affinity, the concentration and the DNA binding ability of the sex steroid receptors in transformed human cervical tissue has shown considerable changes in comparison to normal specimens. Lower level of hormone receptors and higher dissociation constants are the essential characteristics of the majority of analysed transformed tissue specimens. The DNA binding of steroid-receptor complexes is in agreement with mentionated data. These preliminary results are just one more evidence on the existence of relationship between modified properties of sex steroid receptors and different histological alterations of their target tissues. Further investigations are necessary to make some conclusion about the possible involvement of estrogen and progesterone receptors in pathological transformation of the portio vaginalis uteri.

Characterization of 7-8S progestin binding protein in human prostate using vertical tube rotor.

The specific binding of the synthetic progestin, 17 alpha-methyl[3H]promegestone (R5020), to the cytosol of human benign hyperplastic prostate has been studied in sucrose density gradients using a vertical tube rotor. The cytosol of human prostate was shown to contain substantial amounts of a 7-8S macromolecule with a high affinity (Kd = 0.5-1 nM) for R5020 which is saturated at low concentrations (10 nM). The conventional technique of sucrose density gradient analysis in a swinging bucket rotor was not suitable for reproducible optimal analysis of a 7-8S high affinity complex. The use of the salt, Na2MoO4, had a stabilizing effect on the complex. Comparison of saturation analysis assays using dextran charcoal assay and vertical tube rotor assay showed that the charcoal assay can give an over-estimation of the 7-8S saturable binding. Progestational steroids competed with R5020 for binding to 7-8S, whereas androgenic steroids, with the exception of 19-nor-testosterone, did not compete. Incubation of cytosol at elevated temperatures in the presence of DNA-cellulose resulted in the binding of the hormone-protein complex to DNA-cellulose. High ionic strength buffer was required to extract the complex which sedimented at 4.5S in sucrose gradients prepared in 0.4 M KCl. Based on the data presented, progestin binding in human prostate is clearly similar in physical chemical properties to progesterone receptors in "classical" target tissues. However, rapid sucrose gradient analysis with a vertical tube rotor is preferred over conventional techniques to evaluate progestin receptor binding in human prostate.

Characterization and stabilization of progesterone receptors in human benign prostatic hypertrophy.

Experimental conditions for the optimum detection and measurement of the cytosolic progestogen receptor in human prostatic hyperplasia and neoplasia are described. In presence of 20 mM molybdate ion and a reaction time of 0.5-2 h at 0-2 degrees C, we are able to detect the appearance of a [6.7-3H]-17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione ([3H]-R5020) and [3H]-progesterone binding moiety in human prostatic cytosol. The [3H]-R5020 binding protein sediments at approximately 8-11S in a glycerol density gradient centrifuged in a Sorvall TV865 rotor (vertical rotor). Maximal binding of [3H]-R5020 occurs at 30 min at 25 degrees C and 1 h at 0 degree C. Considerable overall improvement in receptor detection and measurement was made when gradient centrifugation was carried out in a vertical rotor instead of swing bucket rotor. The specifically bound [3H]-R5020 is displaced by progesterone, triamcinolone acetonide and R5020, but not by cortisol, dihydrotestosterone, 17 beta-estradiol, or diethylstilbestrol. High affinity was demonstrated by Scatchard analysis on binding of [3H]-5020 to receptors from cytosol of benign prostatic hypertrophy (BPH) giving a KD of 2.4 X 10(-10)M and a binding site concentration of 50 fmol/mg protein. Molybdate ion maintains the hormone receptor complex in 8-11S form which is totally converted to the 4-5S form in the presence of 0.6 M K Cl. The optimum concentration of fluoride ion for enhancing the specific binding of [3H]-R5020 was determined to be between 20 and 50 mM. Cytosol from human BPH in the presence of 25 mM sodium fluoride contains [3H]-R5020 receptors which sedimented mostly in the 4-5S region of glycerol density gradient. Sodium-molybdate and sodium fluoride stabilize and stimulate the specific [3H]-R5020 receptor complex at 25 and 0 degree C. The loss of specific receptor hormone binding capacity can be reversed by fluoride and, to a lesser extent, by molybdate.

RU486, a progestin antagonist, binds to progesterone receptors in a human endometrial cancer cell line and reverses the growth inhibition by progestins.

The human endometrial cancer cell line, IK-90 cells, contains estrogen-independent progesterone receptors (PR) and is progestin sensitive. Accumulation of glycogen in the cytoplasm of IK-90 cells as well as growth inhibition of the cells in response to progestins are observed. In the present study, the effects of RU486, a progestin antagonist, on IK-90 cells were investigated in a serum-supplemented medium. Scatchard plot analysis of cytoplasmic binding data in the cells revealed a high affinity binding site for RU486 (Kd, 2.6 nM) with maximum binding sites of 169 fmol/mg protein. However, the binding ability to DNA-cellulose of heat activated [3H]RU486-PR complexes was lower when compared with that of the progestin agonist [3H]R5020-PR complexes, suggesting a decrease in progestin activity of RU486 in IK-90 cells. The addition of 1 microM RU486 to culture medium produced periodic acid-Schiff-positive granules in the cytoplasm of the cells. On the other hand, RU486 (1 nM-1 microM) did not significantly inhibit the growth of cells. However, RU486 (0.1-1 microM) totally prevented the growth-inhibitory effect of R5020 (0.1-1 microM) on IK-90 cells. In conclusion, RU486, an antiprogestin, had a dual activity both a progestin antagonist and weak agonist in human endometrial cancer cells, which was not mediated through the estrogen receptor system.

Effects of the steroid antagonist RU486 on dimerization of the human progesterone receptor.

We previously reported, using a coimmunoprecipitation assay, that the B form (PR-B) of the human progesterone receptor from T47D human breast cancer cells dimerizes in solution with the A receptor (PR-A) and that the extent of dimerization correlates with receptor binding activity for specific DNA sequences [DeMarzo, A.M., Beck, C.A., Oñate, S.A., & Edwards, D.P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 72-76]. This suggested that solution dimerization is an intermediate step in the receptor activation process. The present study has tested the effects of the progesterone antagonist RU486 on solution dimerization of progesterone receptors (PR). As determined by the coimmunoprecipitation assay, RU486 binding did not impair dimerization of receptors; rather, the antagonist promoted more efficient solution dimerization than the progestin agonist R5020. This enhanced receptor dimerization correlated with a higher DNA binding activity for transformed receptors bound with RU486. RU486 has been shown previously to produce two other alterations in the human PR when compared with R5020. PR-RU486 complexes in solution exhibit a faster sedimentation rate (6 S) on salt-containing sucrose density gradients than PR-R5020 complexes (4 S), and PR-DNA complexes have a faster electrophoretic mobility on gel-shift assays in the presence of RU486. We presently show that the 6 S PR-RU486 complex is a receptor monomer, not a dimer. The increased sedimentation rate and increased mobility on gel-shift assays promoted by RU486 were also observed with recombinant PR-A and PR-B separately expressed in insect cells from baculovirus vectors. These results suggest that RU486 induces a distinct conformational change both in PR monomers in solution and in dimers bound to DNA. We also examined whether conformational changes in PR induced by RU486 would prevent a PR polypeptide bound to RU486 from heterodimerization with another PR polypeptide bound to R5020. To evaluate this, PR-A and PR-B that were separately bound to R5020 or RU486 in whole cells were mixed in vitro. PR-A-RU486 was capable of dimerization with PR-B-R5020, and this was demonstrated for heterodimers both formed in solution and bound to specific DNA. The capability to form heterodimers in vitro raises the possibility that the antagonist action of RU486 in vivo could in part be imposed in a dominant negative fashion through heterodimerization between one receptor subunit bound to an agonist and another bound to RU486.