Non-steroidal progesterone receptor specific ligands.

The nuclear receptor for progesterone is a target for contraception and for several therapeutic indications. Progestin agonists and antagonists in clinical use mimic the steroidal backbone of the cognate ligand, progesterone. Thus, they have significant cross-reactivity with other steroid receptors. Recently, non-steroidal progesterone receptor ligands have begun to appear in the literature. This review will describe the current status of research into these promising new chemical entities.

Induction of the progesterone receptor gene in estrogen target cells monitored by branched DNA signal amplification.

Estrogens have multiple effects on the growth and development of cells in their target tissues, including the uterus, ovary, breast, bone marrow and brain. The hormone regulates the transcription of diverse genes in these tissues via the estrogen receptor, a nuclear transcription factor. Naturally occurring estrogens and estrogen analogs including selective estrogen receptor modulators (SERMs), constitute important therapies for breast cancer and osteoporosis, and are major components of oral contraceptives. The in vitro biologic activities of pharmaceutical estrogen agonists and antagonists have frequently been monitored by cotransfection assay, where exogenous estrogen receptor and reporter genes are transiently inserted into a heterologous, non receptor-containing cell line, such as those derived from kidney cells. Here we describe an alternative to this method, where induction of an endogenous estrogen-responsive gene, the progesterone receptor gene, is monitored by branched DNA signal amplification. Assays are performed with cultured cells derived from estrogen-responsive tissues; namely, breast, uterine endothelium and bone. Hormonal induction occurs via the endogenous estrogen receptor of these cells. Our data show that SERMs, which are estrogen agonists on bone in vivo, antagonize estrogen-dependent target gene induction in conditionally immortalized osteoblast-like cells.

Nonsteroidal progesterone receptor ligands with unprecedented receptor selectivity.

We have characterized a series of nonsteroidal progesterone receptor ligands, the tetrahydropyridazines. Compounds in this series, exemplified by RWJ 26819, demonstrate high affinity and unprecedented specificity for the progesterone receptor relative to other steroid hormone receptors. Like steroidal progestins, RWJ 26819 induces binding of the receptor to a progesterone response element in vitro, and stimulates gene expression in and proliferation of T47D human breast cancer cells. When administered to rabbits orally or subcutaneously, the compound induces histological changes in the uterine lining comparable to those induced by levonorgestrel. It also inhibits ovulation in monkeys. Though less potent in cells and in animal models than would be predicted from binding affinity alone, their enhanced selectivity suggests that they could be effectively used in a clinical setting. Most of the tetrahydropyridazines synthesized are progestin agonists or mixed agonists and antagonists in vitro; however, one compound with antagonist activity in the rabbit uterine transformation assay has been identified.

An ultrahigh-throughput screening assay for estrogen receptor ligands.

Members of the steroid and thyroid hormone receptor superfamily (nuclear receptors) play diverse roles in mammalian physiology, in both normal and pathological states. For this reason, and because nuclear receptors are natural receptors for lipophilic small molecules, they are important therapeutic targets for the pharmaceutical industry. Here we describe a method for screening for ligands for one of these receptors, the estrogen receptor. The method is rapid, robust, and reliable, and has been used in an ultrahigh-throughput robotic screen. The receptor is crosslinked to a scintillant-containing solid support (FlashPlate) via a receptor-specific antibody. Test compounds are assayed for competition with a radiolabeled estrogen for binding to the immobilized receptor. Receptor-ligand complexes are allowed to form and receptor-bound radioactivity is detected in a scintillation counter. The assay has been designed for both isoforms of the estrogen receptor, alpha and beta, using separate antibodies for each. Given a radioactive tracer and an appropriate antibody, many of which are now commercially available, the assay could be established for any nuclear receptor.

Induction of a novel conformation in the progesterone receptor by ZK299 involves a defined region of the carboxyl-terminal tail.

Progesterone receptor antagonists are a promising class of therapeutic drugs indicated for the treatment of a variety of reproductive conditions. Understanding their mechanism of action at the molecular level is an important prerequisite for the development of future generations of these drugs. Using limited proteolytic analysis to monitor conformational changes in the progesterone receptor, we can detect three distinct classes of progestin antagonist. The effect of the first, RU486, on the conformation of the carboxyl terminus of the receptor has been previously described. The second, exemplified by RWJ 47626, a nonsteroidal compound with in vitro antiprogestin activity, induces a proteolytic fragment pattern indistinguishable from that induced by the agonist R5020. Finally, ZK299 induces a fragment pattern intermediate between that induced by R5020 and RU486. Site-directed mutagenesis of the carboxyl-terminal tail of the progesterone receptor indicates that the region containing the putative activation function AF-2 is differentially exposed to proteolytic attack depending on the nature of the antagonist bound. The differentially exposed region is most accessible when the antagonist RU486 is bound, less accessible when the antagonist ZK299 is bound, and least accessible when the antagonist RWJ47626 or agonist R5020 is bound. The results suggest that multiple types of antiprogestin can be defined in terms of their effects on the conformation of the carboxyl-terminal activation function of the progesterone receptor.

Maternal protein reserves and their influence on lactational performance in rats. 4. Tissue protein synthesis and turnover associated with mobilization of maternal protein.

The present study was undertaken to investigate the changes in muscle protein turnover involved in the rapid mobilization of protein in rats subjected to severe protein restriction during lactation. Estimates of mammary gland and liver protein synthesis were also made during lactation. Multiparous female Sprague-Dawley rats, caged individually following mating, were offered a high-protein diet (H; 215 g crude protein (N x 6.25; CP)/kg dry matter (DM)) ad lib. until parturition. Following parturition, half the females continued to receive diet H, whilst the remainder were offered a diet low in protein (L; 90 g CP/kg DM) ad lib. On days 2, 4, 8 and 12 of lactation, groups of females were used in the estimation of tissue protein synthesis (flooding dose of [3H]phenylalanine) immediately after a milk sample had been obtained. Rates of muscle protein synthesis were unchanged during lactation in group H. The feeding of diet L during lactation reduced the muscle protein synthesis on day 12 to rates that were lower than group H and also the rate on diet L on day 2 (P < 0.01). However, this fall in muscle protein synthesis was not rapid and muscle fractional synthesis rate (FSR) was different from group H only from day 8 (P < 0.05). Estimated rates of mammary protein synthesis appeared to be generally unchanged by dietary treatment or stage of lactation. Liver FSR was also unchanged by dietary protein supply or stage of lactation. The effect of dietary protein restriction on liver size and protein content during lactation influenced liver absolute synthesis rate (ASR), and on days 8 and 12 of lactation liver ASR was lower in group L than in group H (P < 0.001). The loss of muscle protein in rats fed on diet L during lactation (133 mg) occurred mainly between days 2 and 8 of lactation and was primarily associated with a dramatic increase in degradation (13.0% per d), with the decline in synthesis having a much smaller role. A decline in muscle protein degradation during the latter half of lactation was part of the mechanism that prevented excessive muscle protein catabolism. It is thought that the estimation of mammary protein synthesis in the present study was impaired by the milk sampling procedure previously used.

Maternal protein reserves and their influence on lactational performance in rats. 2. Effects of dietary protein restriction during gestation and lactation on tissue protein metabolism and Na+, K(+)-ATPase (EC 3.6.1.3) activity.

Changes in tissue protein synthesis and an associated membrane transport system in rats were investigated during lactation and under conditions of dietary protein restriction. Following mating, female Sprague-Dawley rats (second parity) were caged individually and offered a high-protein diet (H; 215 g crude protein (N x 6.25; CP)/kg dry matter (DM)) ad lib. until day 12 of gestation. Subsequently half continued to receive diet H, whilst the remainder were offered a low-protein diet (L; 65 g CP/kg DM) until parturition. On day 1 of lactation females were then allocated to either diet H or another low-protein diet (L2; 90 g CP/kg DM) which were offered ad lib. until day 13 of lactation, giving four lactation groups HH, LH, HL2 and LL2. On days 1 and 13 of lactation groups of females were used in the estimation of tissue protein synthesis (flooding dose of [3H] phenylalanine) and Na+, K(+)-ATPase (EC 3.6.1.3) activity (polarographically) in skeletal muscle, mammary gland, liver and duodenal mucosa. By day 1 of lactation diet L had reduced fractional and absolute synthesis rates (FSR and ASR) of muscle protein (P < 0.05) and the O2 consumption associated with Na+, K(+)-ATPase, although not significantly (P < 0.10). Rates of protein synthesis in the other tissues studied were not affected on day 1 of lactation by the gestation dietary treatment. By day 13 of lactation the feeding of diet L2 had reduced muscle FSR and ASR of group HL2 to rates that were lower than those on day 1 (P < 0.05), comparable to those of group LL2 and lower than those of groups HH and LH (P < 0.05). Diet H had allowed group LH to increase their muscle protein synthesis compared with that on day 1 (P < 0.05). Muscle Na+, K(+)-ATPase activity on day 13 of lactation was also lower in groups offered diet L2 (P < 0.05). Mammary protein synthesis was increased during lactation with the feeding of diet H (P < 0.05), which was prevented by diet L2 such that rates of groups HL2 and LL2 were lower than those of the two high-protein groups on day 13 (P < 0.01). Mammary respiration and in particular Na+, K(+)-ATPase activity was increased during lactation by the feeding of diet H (P < 0.05). Rates of protein synthesis and respiration in liver and duodenal mucosa were not significantly affected by the gestational or lactational dietary treatments.(ABSTRACT TRUNCATED AT 400 WORDS)

Detection of potential ligands for nuclear receptors in cellular extracts.

In response to external stimuli, steroid receptors are directly influenced to transactivate gene expression. Assuming they exist, identification of ligands for orphan steroid receptors is a key to understanding their physiology. In the orphan subgroup of the steroid receptor superfamily, the putative carboxyl terminal ligand-binding domain (LBD) is well conserved among members of the superfamily, which suggests a role in ligand binding. A consequence of ligand binding is the induction of a significant conformational change within the LBD which is necessary for the transactivation function. This characteristic conformational change can be detected by partial proteolytic digestion and has been localized by mutational analysis and epitopic mapping of the LBD using monoclonal antibodies. Based on this finding, a sensitive in vitro assay was developed for the rapid screening and identification of potential ligands for orphan receptors. We examined the patterns of conformational changes in the androgen receptor, glucocorticoid receptor, and progesterone receptor induced by binding of their cognate agonists and antagonists. We demonstrated that the conformational changes induced by ligands can serve as characteristic and reliable markers to distinguish between the ligand-bound and apoprotein states of a receptor. The sensitivity and feasibility of employing this assay to detect new endogenous ligands using fractionated cellular extracts were also tested. The results strongly suggest that unknown compounds can be defined as potential ligands for orphan receptors using this approach.

Transcriptional activation by the estrogen receptor requires a conformational change in the ligand binding domain.

The estrogen receptor (ER) is a strong hormone-inducible transcription factor that regulates the expression of many genes. It was shown for the human progesterone receptor that the binding of hormone causes distinct conformational changes in the ligand binding domain (LBD) and that these changes in LBD conformation are crucial for events after DNA binding. We now show that conformational changes in the LBD of the human ER are a prerequisite for trans-activation. Under the appropriate conditions ER binds to its response element and activates transcription only in the presence of ligand. Binding of the ligand causes changes in the conformation of the LBD. Antihormones induce distinct conformational changes, the differences between the conformations lying in the carboxy-terminal end of the receptor. Changing the experimental conditions results in a receptor that can bind to DNA and activate transcription in a ligand-independent manner. Under these conditions the LBD has a transcriptionally active conformation in the absence of ligand. Taken together, our data indicate that the conformational change induced by ligand is required for converting a receptor to the transcriptionally active form.

Ligand-dependent conformational changes in the progesterone receptor are necessary for events that follow DNA binding.

Hormones and antihormones induce related, but distinct, conformational changes in the progesterone receptor [Allan, G. F., Leng, X., Tsai, S. Y., Weigel, N. L., Edwards, D. P., Tsai, M.-J. & O'Malley, B. W. (1992) J. Biol. Chem. 267, 19513-19520]. In both cases the conformational change precedes the dissociation of heat shock proteins and binding to DNA. We have now investigated the steps in hormone action which are dependent upon this conformational change. We show that in the absence of ligand, monoclonal antibodies directed against different regions of the progesterone receptor can induce high-affinity binding to its response element in vitro. This antibody-induced DNA binding is presumably facilitated by enhanced dimerization of receptor monomers. However, antibodies do not induce the hormone-specific conformational change in the progesterone receptor and do not induce in vitro transcription by the receptor. In contrast, the antiprogestin ZK98299, which inhibits receptor binding to DNA, fully induces the antihormone-specific conformational change. Thus, our data imply that steroids induce a conformational change in their receptors which is necessary for events subsequent to DNA binding, most likely for transactivation.

Hormone and antihormone induce distinct conformational changes which are central to steroid receptor activation.

Antihormones are potent antagonists of hormone action in vivo, but the mechanism underlying this antagonism is not understood. Several steroid hormones transform (activate) their receptors from a cytosolic, non-DNA binding 8 S sedimentation form to a nuclear, DNA binding 4 S form. Transformation is accompanied by the loss of associated heat shock proteins. We have previously demonstrated that an additional hormone-dependent step, separate from heat shock protein removal, is required for activation of the human progesterone receptor. We have devised an assay in which the human progesterone receptor translated in vitro binds to its specific response element in a hormone-dependent manner. As assessed by limited proteolytic digestion, hormone treatment of the nascent receptor induces a dramatic conformational change within the protein. The conformational change occurs in the absence of DNA and renders the entire ligand binding domain resistant to digestion by proteases. A number of antiprogestins, including RU486, induce an equally dramatic, but distinct, structural alteration of the ligand binding domain. The distinction centers upon the final 30 to 40 amino acids at the carboxyl terminus. The conformational change can be induced by ligand prior to dissociation of the 8 S complex and is not induced by heat shock protein removal in the absence of hormone. Remarkably, virtually identical hormone-induced conformational changes were detected following proteolytic analysis of in vitro translated retinoic acid receptors. Our data indicate that the sole necessary event in the activation of steroid receptors is conformational modification by the ligand. Furthermore, we conclude that transcriptional inactivation of steroid receptors by antihormones involves the induction of an inappropriate structural conformation at the extreme carboxyl terminus of the ligand binding domain.

The mechanism of RU486 antagonism is dependent on the conformation of the carboxy-terminal tail of the human progesterone receptor.

The human progesterone receptor form B (hPR-B) was expressed in Saccharomyces cerevisiae together with a specific reporter plasmid. To understand the mechanism underlying antagonist ligand activity, libraries of hormone binding domain (HBD)-mutated hPR-B molecules were prepared. A mutant receptor was identified that had lost the ability to bind either progesterone or R5020; it could still bind RU486 and, surprisingly, fully activated transcription in the presence of this "antagonist" and other antiprogestins. When this receptor mutant was assayed in mammalian cells, RU486 again demonstrated agonistic activity. Sequence analysis indicated that the mutant phenotype was due to truncation of the carboxy (C)-terminal 42 aa. We conclude that amino acids in the extreme C-terminal region are required for the receptor to bind progesterone, while antagonists bind to a site located more N-terminal of the HBD. Our results suggest that the extreme C-terminal region of the receptor contains an inhibitory function that silences receptor transactivation in the absence of agonist and in the presence of antagonist.

PIP: The function of the hormone-binding domain of the human progesterone receptor was examined in a yeast cell system and in mammalian HeLa cells using mutant receptors, progesterone, the progesterone agonist R502, and the antagonists RU-486, Org31806, and Org31376. The hormone-binding domain, located on the carboxy terminal of the peptide, is known to initiate a conformational change in the receptor upon binding an agonist or antagonist, then shedding of associated proteins including the heat shock protein, dimerization of the receptor, and finally, binding to DNA, leading to transcription. Binding of a progesterone antagonist such as RU-486 elicits all these events except transcription. First the progesterone receptor was inserted in yeast with a plasmid, and a set of mutants were generated, using beta galactosidase as an indicator. A mutant progesterone receptor, U-P1, was selected for mechanistic studies, that binds and was activated by antagonists, but was inactive with progesterone. This receptor had a deletion at base 2636, resulting in a shift of reading frame so that a stop codon 36 nucleotides downstream caused truncation of 54 amino acids at the C-terminus and addition of 12 novel amino acids. Western blot analysis confirmed the expected molecular weight. The mutant receptor was active with RU-486, suggesting that the C-terminus may be responsible for poor transcription with RU-486, suggesting in normal receptors. 2 other truncated mutants were inactive with progesterone. These data suggested that the terminal 42 amino acids of the progesterone receptor are needed to bind progesterone, and that the antagonist is contacting different amino acids than the native receptor, possibly inducing a different conformational change. The activity of the UP-1 mutant was also confirmed in HeLa cells, with the chloramphenicol acetyltransferase reporter system. The results were interpreted to mean that progesterone agonists and antagonists contact at least some different amino acids in the hormone binding domain of the receptor, and that the conformational changes resulting from binding these agents are different. It appears that the C-terminus of the receptor contains an inhibitory domain which, when removed, turns antagonists into agonists.

Synergism between steroid response and promoter elements during cell-free transcription.

We have analyzed quantitatively the influence of distal promoter elements on steroid-responsive gene expression in vitro. Functional synergism between enhancer and distal promoter elements was examined using two model promoters, one containing a natural promoter (mouse mammary tumor virus long terminal repeat) and one constructed artificially. Human glucocorticoid receptor (GR) expressed in baculovirus induces transcription from a mouse mammary tumor virus long terminal repeat-containing DNA template. Transcription is diminished by oligonucleotides containing a nuclear factor 1 (NF-1)-binding site or a glucocorticoid/progesterone response element. Quantitative analysis indicates that NF-1 and GR act synergistically during transcriptional activation. In contrast, efficient activation by GR or purified chick progesterone receptor of a glucocorticoid/progesterone response element-linked ovalbumin promoter does not require interaction with the chicken ovalbumin upstream promoter (COUP) element in the distal promoter. Lack of synergism is not related to enhancer strength, since the glucocorticoid/progesterone response elements can be moved further from the promoter or reduced to a single copy response element without increasing the dependence upon COUP. Strong synergism is restored following substitution of an NF-1 distal promoter element for the COUP element in this construct. Our results suggest that synergism between steroid response and distal promoter elements is dependent upon the identity of the promoter element rather than upon the inherent strength of the enhancer element.

Steroid hormone receptors and in vitro transcription.

Steroid hormone receptors are ligand-inducible transcription factors that exhibit potent effect on gene expression in living cells. Precise dissection of their mode of action at the molecular level can best be carried out in functional cell-free systems. This article will describe the benefits of such systems and review their development up to the recent establishment of steroid receptor-dependent in vitro transcription. Subsequent advances in our knowledge of receptor function arising from the exploitation of this powerful experimental tool will be described. Particular emphasis will be placed upon two key problems: the role of steroid hormone in receptor action and the mechanisms by which steroid receptors activate gene transcription.

Recombinant human glucocorticoid receptor induces transcription of hormone response genes in vitro.

A recombinant full length human glucocorticoid receptor stimulates transcription in vitro of test genes containing synthetic glucocorticoid and progesterone response elements or murine mammary tumor virus promoter. The receptor expressed in a baculoviral vector is highly active, enhancing transcription of hormone response genes greater than 30-fold even at a receptor concentration of 1.2 nM. The enhancement of transcription is glucocorticoid and progesterone response element-dependent, suggesting that it is a receptor mediated event. In vitro and in vivo treatment with the agonist dexamethasone or with the antagonist Ru486 did not alter significantly the functional activity of partially purified receptor. Kinetic studies suggest that both glucocorticoid receptor and HeLa cell extracts are required for formation of a stable committed transcriptional complex. Our results indicate that the action of glucocorticoid receptor on gene transcription is similar to that defined recently for the progesterone receptor and may be a general mechanism for all steroid receptors.

The progesterone receptor stimulates cell-free transcription by enhancing the formation of a stable preinitiation complex.

Highly purified chicken progesterone receptor (cPR) is shown to stimulate RNA synthesis directly in an in vitro transcription assay. Stimulation of transcription by cPR requires the presence of progesterone response elements (PREs) in the template and can be specifically inhibited by addition of competitor oligonucleotides containing PREs. Binding of receptor to two PREs is cooperative and leads to synergistic (27-fold) stimulation of transcription. A purified fusion protein containing the DNA binding domain of cPR linked to yeast ubiquitin was produced in E. coli and also functions in the transcription assay. Using this in vitro transcription system, we demonstrate that hormone-free cPR activated by salt treatment induces transcription of a test gene in a hormone-independent manner. Finally, we present evidence that the progesterone receptor acts by facilitating the formation of a stable preinitiation complex at the target gene promoter and thus augments the initiation of transcription by RNA polymerase II.