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., O?ate, 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.     

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.     

R5020 and RU486 act as progesterone receptor agonists to enhance Sp1/Sp4-dependent gene transcription by an indirect mechanism

It has been suggested that ligand-dependent gene activation by the progesterone receptor (PR) can result from recruitment of PR by the promoter bound Sp1. A detailed investigation of the Sp1-dependent agonistic activity of RU486 and R5020 on the folate receptor (FR) type alpha, p27, thymidine kinase 1 and p21 genes reveals a different mechanism. The FR-alpha P4 promoter and the endogenous FR-alpha gene were up-regulated by the PR agonist R5020 through either PR-A or PR-B. The classical antagonist RU486 also activated the promoter but only through PR-B. The most proximal (essential) G/C-rich (Sp1 binding) element and the initiator region constituted the minimal promoter responsive to PR regulation; substitution with a stronger cluster of G/C-rich elements enhanced the magnitude of the PR response. In contrast, substitution of the G/C-rich element with a TATA box resulted in the loss of regulation by PR. Overexpression of Sp1 and Sp4 but not Sp3 enhanced activation of the FR-alpha promoter by PR, knocking down Sp1 decreased the activation in a manner that was reversed by ectopic Sp1 or Sp4. The ligand-dependent action of PR on the promoter was delayed compared with its activation of a classical glucocorticoid response element-driven promoter and activation of both the promoter and the endogenous FR-alpha gene by PR required new protein synthesis. Activation by PR paralleled RNA polymerase II recruitment but was not accompanied by either association of PR or a change in the association of Sp1 with the endogenous FR-alpha P4 promoter. Similar observations were made for PR regulation of the genes encoding p27, thymidine kinase 1, and p21. The results contradict the current view of Sp1-dependent gene regulation by PR and point to the existence of one or more PR target genes whose promoter and cell context(s) must thus be key determinants of the agonistic activity of RU486 on a large group of important Sp1-dependent downstream target genes.     

Ligands induce conformational changes in the carboxyl-terminus of progesterone receptors which are detected by a site-directed antipeptide monoclonal antibody.

We have prepared a monoclonal antibody, C-262, to a synthetic peptide that contains the carboxy-terminal 14 amino acids from progesterone receptors (PR). This sequence is 100% conserved in all species of PRs that have been cloned to date, suggesting that this antibody will recognize all mammalian and avian PR. The C-262 antibody recognizes both native and denatured forms of the receptor. However, it does not recognize PR when they are bound to the hormone agonists progesterone or R5020. Surprisingly the antibody does recognize PR when they are bound to the steroid antagonist RU486. This suggests that progestin agonists induce a conformational change in the receptor that occludes the C-262 epitope in the carboxyl-terminus, whereas unliganded receptors and receptors bound with RU486 assume distinct conformations that leaves the C-terminal tail accessible to the C-262 antibody.     

A structural and in vitro characterization of asoprisnil: a selective progesterone receptor modulator

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.     

Progesterone receptors and the sexual differentiation of the medial preoptic nucleus

The central component of the medial preoptic nucleus (MPNc) of the rat has served as an excellent model of sexual differentiation. The MPNc is larger in adult males than in females, and its development is regulated by perinatal gonadal hormones. Although testosterone (T) and its metabolite estradiol (E) sexually differentiate this region, the exact mechanism by which they act during development is not known. There is a dramatic sex difference in the expression of progesterone receptors (PR) in the MPN during development; perinatal males express higher levels of PR than females. Additionally, PR expression during this time is dependent on exposure to T. Thus, PR induction may be one mechanism by which T sexually differentiates the MPN. The present study investigated the potential role of PR in the sexual differentiation of the MPNc. Anatomical examination of PR distribution within the MPN of neonatal males revealed the presence of PR immunoreactive cells within the MPNc, suggesting a direct route of action for PR in the development of the MPNc. Additionally, we measured the effects of neonatal RU486 treatment, a progesterone and glucocorticoid receptor antagonist, on subsequent MPNc volume in neonatally T-treated females and neonatally castrated males, given T. RU486 treatment reduced the MPNc volume of T-treated females while it increased the volume in T-treated, neonatally castrated males. These results, taken together with the expression of PR in the MPNc, suggest that PR may influence the sexual differentiation of the MPNc volume.     

A genomic approach to identify novel progesterone receptor regulated pathways in the uterus during implantation

The cellular actions of steroid hormone progesterone (P) are mediated via its nuclear receptors, which regulate the expression of specific target genes. The identity of gene networks that are regulated by the P receptors (PRs) in the uterus at various stages of the reproductive cycle and pregnancy, however, remain largely unknown. In this study, we have used oligonucleotide microarrays to identify mRNAs whose expression in the pregnant mouse uterus is modulated by RU486, a well-characterized PR antagonist, which is also an effective inhibitor of implantation. We found that, in response to RU486, expression of mRNAs corresponding to 78 known genes was down-regulated at least 2-fold in the preimplantation mouse uterus. The PR regulation of several of these genes was ascertained by administering P to ovariectomized wild-type and PR knockout (PRKO) mice. Detailed spatio-temporal analysis of these genes in the pregnant uterus indicated that their expression in the epithelium and stroma could be correlated with the expression of PR in those cell types. Furthermore, time-course studies suggested that many of these genes are likely primary targets of PR regulation. We also identified 70 known genes that were up-regulated at least 2-fold in the pregnant uterus in response to RU486. Interestingly, initial examination of a number of RU486-inducible genes reveals that their uterine expression is also regulated by estrogen. The identification of several novel PR-regulated gene pathways in the reproductive tract is an important step toward understanding how P regulates the physiological events leading to implantation.     

Differences in the binding mechanism of RU486 and progesterone to the progesterone receptor

The binding mechanism of the antagonist RU486 to the progesterone receptor was compared with that of the agonists progesterone and R5020. Both progesterone and RU486 bound to the receptor with a Hill coefficient of 1.2, indicating the binding of each ligand is positive cooperative. However, when each ligand was used to compete with [3H]progesterone for binding to the receptor at receptor concentrations near 8 nM, at which the receptor is likely a dimer, the competition curve for RU486 was significantly steeper than the curves for progesterone and R5020 (p less than 0.001). This indicated that a difference in the binding mechanism of RU486 and progesterone can be detected when both ligands are present. In contrast, at receptor concentrations near 1 nM, at which the receptor is likely a monomer, the competition curves for all three ligands were indistinguishable (p = 0.915). These results indicate that RU486 and agonists have different binding mechanisms for the receptor and further suggest that this difference may be related to site-site interactions within the receptor.     

Facilitation of Sexual Receptivity by Ventromedial Hypothalamic Implants of the Antiprogestin RU 486

RU 486 is known primarily as an antagonist to progestins and glucocorticoids. However, RU 486 has also been shown to have agonistic progestational properties in biochemical and behavioral studies. In the current study, RU 486 was implanted directly into tim ventromedial hypothalamus (VMH) to test for facilitative action on the receptive behavior of female ovariectomized Long-Evans rats primed with 5 μg of estradiol benzoate. Cannulae containing RU 486, progesterone (P), or empty cannulae were implanted 48 hr after estrogen priming. The lordosis quotient and the lordosis score were assessed 4 hr after the cannulae were lowered by a standardized test consisting of 10 mounts by a stimulus male. P and RU 486 significantly facilitated receptivity compared to blank implants in terms of lordosis quotient and lordosis score, with no significant difference between the hormone treatments. While only a single dose of each treatment was given in the current study, RU 486 facilitated lordosis when implanted to the VMH as well as progesterone in contrast to our previous results where the steroids were administered systemically.     

The antiprogesterone RU486 stabilizes the heterooligomeric, non-DNA-binding, 8S-form of the rabbit uterus cytosol progesterone receptor

The salt-induced (0.3 M KCl) transformation of the non-transformed, heterooligomeric 8S-form of the rabbit uterus cytosol progesterone receptor (PR) was analyzed by density gradient ultracentrifugation (8S----4S conversion) and DNA-cellulose chromatography (non-binding----binding forms). After 1 h treatment at 2 C, greater than 90% of agonist (R5020 or Org2058)-PR complexes were transformed, contrary to antagonist (RU486)-PR complexes, which did not undergo any transformation. Thus, there is stabilization of the non-transformed receptor form by RU486 as compared to the effect of agonist binding. The hydrodynamic parameters of both agonist- and antagonist-bound non-transformed receptors were similar and the calculated Mr were approximately 283,000 and approximately 293,000, respectively. In both cases, purification indicated the presence of a 90-kD non-hormone-binding protein associated with the hormone binding unit(s). Transformation of RU486-PR complexes occurred after exposure to high salt at increased temperature and was correlated to the dissociation of the 90-kD protein from the receptor. Both agonist- and antagonist-bound transformed forms of PR had apparent similar affinities for DNA-cellulose. Molybdate-stabilized and KCl-treated RU486-PR complexes were more stable, as assessed by steroid binding, than the corresponding R5020-PR complexes, arguing in favor of a stabilizing effect of both the 90-kD protein and RU486 against inactivation. These cell-free experiments support the concept that RU486 in the rabbit uterus system stabilizes the 8S non-DNA binding, non-transformed form of the receptor at low temperature. The possibility that impaired dissociation of the heterooligomeric receptor form is involved in the antiprogesterone activity of RU486 is discussed.