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PubMed1995

Progesterone receptor and the mechanism of action of progesterone antagonists.

Currently available progesterone antagonists have been suggested to fall into two categories based on differences in how they interact with and inactivate the progesterone receptor (PR). The anti-progestin ZK98299 (Type I) impairs PR association with DNA, while Type II compounds (RU486, ZK112993, ZK98734) promote PR binding to DNA. Type II agents, therefore, appear to inhibit receptor activity at a step downstream of DNA binding, presumably failing to induce conformational changes in PR structure requird for enhancement of transcription. This paper discusses both published and unpublished data supporting the concept of two types of progestin antagonists. Using PR-mediated induction of reporter genes in breast cancer cells as an assay for biological response, both types of anti-progestins, after correction for difference in steroid binding affinity, inhibit progestin induction substoichiometrically. However, Type II anti-progestins are more potent, inhibiting at lower ratios of antagonist to agonist than ZK98299. This suggests that in addition to behaving by classical competitive mechanisms these compounds (in particular Type II) may exhibit additional activity as transrepressors of PR in the same cell bound to hormone agonist. Transrepression may occur by the combined mechanisms of heterodimerization and competition for binding to DNA. In support of this, mixed ligand dimers form readily in solution between a PR subunit bound to agonist and another bound to either type of anti-progestin, whereas these mixed ligand dimers bind poorly, if at all, to specific progesterone response elements (PREs) in vitro. Additionally, when added as a single ligand, Type II agents increase PR dimerization in solution and PR affinity for PREs as compared with single ligand dimers formed by progestin agonist. This contrasts with ZK98299, when given as a single ligand, which reduces PR affinity for PREs without disrupting solution dimerization. Thus the higher affinity of PR for PREs may account for the greater biological potency of Type II compounds as compared with ZK98299. As a further distinction between types of antiprogestins, ZK98299 minimally stimulates phosphorylation of PR whereas RU486 increases site-specific phosphorylation of PR in a manner indistinguishable from that of hormone agonist. Additionally, ZK98299 is not susceptible in vivo to functional switching to a partial agonist by cross talk with cAMP signal transduction pathways, as occurs with Type II compounds. Thus, ZK98299 under certain conditions may be a more pure antagonist than Type II compounds.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2005

Effects of cryo-injury on progesterone receptor(s) of canine spermatozoa and its response to progesterone.

The integrity of sperm progesterone (P4) receptor(s) and its response to steroid stimulation might be crucial for the maintenance of sperm fertilizing ability after cryopreservation. The aim of the current investigation was to study the effect of cryo-procedures on canine sperm P4 receptor(s). In addition, alteration of P4 receptor(s) at the molecular level and their functional integrity following cryo-procedures was evaluated. Fresh and frozen-thawed semen samples (n=6 same dogs) after capacitation were treated with 10 microg/mL P4 to induce the acrosome reaction (AR, FITC-PNA staining). Parallel samples were treated with 50% canine seminal plasma (SP) prior to AR induction with P4. The percentages of AR in capacitated fresh and frozen-thawed semen samples after treatment with P4 were 31.0+/-6.7 and 21.6+/-4.1% (P<0.05), respectively. The percentage of AR in fresh and frozen-thawed semen samples pretreated with SP and incubated with P4 was; 11.5+/-4.8 and 16.5+/-2.0% (P<0.05), respectively. The incidence of the spontaneous AR (P>0.05) in fresh and frozen-thawed semen samples at the onset (5.5+/-2.2 and 6.1+/-1.8%; respectively) and after a 2h (9.6+/-5.1 and 10.4+/-2.7%; respectively) capacitation, avoiding P4 stimulation, were not different. The percentage of progesterone-BSA-FITC staining over the acrosomal region was 18.3+/-10.3% in fresh semen, 36.0+/-11.9% in capacitated (P<0.05) and less than 5% in SP treated spermatozoa. This staining was barely visible in frozen-thawed spermatozoa regardless of capacitation status. In western blot analysis, mAb C262 recognized two bands (54 and 65 kDa). Digitonin treated fresh and frozen-thawed spermatozoa, labeled with [3H]-progesterone, revealed that the P4 binding capacity decreased from 6.0+/-4.4 in fresh to 3.0+/-2.1 nM in frozen-thawed spermatozoa. In nearly all samples tested (except one) 65 kDa protein band decreased significantly after freeze-thaw procedures while the 54kDa protein was increased. These results indicate that the reduced incidence of AR in response to P4 in frozen spermatozoa is possibly due to the conformational changes of P4 receptor(s) and/or reduced P4 receptor density derived from freezing injury.

Acrosome↗
PubMedDate not supplied

Endocrine pharmacological characterization of progesterone antagonists and progesterone receptor modulators with respect to PR-agonistic and antagonistic activity.

Studies were conducted to assess progesterone antagonists (PAs) and progesterone receptor modulators (PRMs) with respect to PR agonistic and antagonistic activities in vivo. These properties are not always adequately reflected in transactivation in vitro models. Studies were performed in pregnant rats, estrogen-primed rabbits (McPhail -Test), and cycling and pregnant guinea pigs. Tested compounds included mifepristone (RU486), onapristone, J867, J956, J1042, and ZK137316. J-compounds induced sub-maximum endometrial transformation and, paradoxically, inhibited effects of progesterone in rabbits. Mifepristone, onapristone, and ZK137316 behaved as 'pure' antagonists in this species. Inhibition of uterine PGF(2alpha) secretion and inhibition of luteolysis in cycling guinea pigs were more sensitive parameters of PR-agonistic and antagonistic properties. 'Pure' PAs inhibited uterine PGF(2alpha) secretion and luteal regression completely. The PR agonist R5020 reversed both effects which demonstrates a PR mediation. Agonistic PRMs (J-substances and mifepristone) showed no or blunted antiluteolytic effects compared to the 'pure' PR antagonist onapristone. When tested in pregnant guinea pigs for their labor-inducing potential, PR agonistic PRMs had much reduced or abolished abortifacient activity compared to mifepristone (mifepristone > J956 > J867/J912 > J1042). However, in cycling animals, superior antiovulatory and antiproliferative properties of the J-substances were seen. Antiovulatory effects of 'pure' and agonistic PRMs are probably due to different mechanisms. The relevance of rodent studies for antiovulatory and uterine antiproliferative effects for the human is still uncertain. The non-abortifacient PRM J1042 induced stromal compaction and inhibition of endometrial proliferation in monkeys, but this effect was not stronger than that of the 'purer' PAs. 'Pure' PAs are important pharmacological tools analogous to PRKO models to study the role of PR in the menstrual cycle and in pregnancy.

Abortion, Induced↗
PubMedDate not supplied

The use of progesterone antagonists and progesterone receptor modulators in contraception.

Progesterone antagonists (PAs) and progesterone receptor modulators (PRMs) have contraceptive potential by suppressing follicular development, delaying the surge of luteinizing hormone (LH), retarding endometrial maturation, and promoting endometrial bleeding. Mifepristone, in daily doses of 2-10 mg, blocks the LH surge and ovulation. Many of the studies were conducted in women not at risk of pregnancy, and thus the contraceptive efficacy is not yet known. Nevertheless, there is evidence that daily doses of 2 or 5 mg of mifepristone have contraceptive potential. Because of anovulation, there may be an unopposed estrogen effect on the endometrium, although this risk may be mitigated by the noncompetitive anti-estrogenic activity exhibited by both PAs and PRMs. Low doses of PAs and PRMs, which do not affect ovulation, retard endometrial maturation, indicating that the endometrium is exquisitely sensitive to these compounds. This raises the prospect of endometrial contraception, i.e. prevention of endometrial maturation without disturbing ovulation or producing alterations in bleeding patterns. This approach works well in monkeys but was not found to be very promising when given to women not using contraception. On the other hand, 200 mg mifepristone administered 48 h after the LH surge, which has minimal or no effect on ovulation and bleeding patterns, is an effective contraceptive; yet, it is not a practical approach to contraception. Late luteal phase administration of mifepristone produces menstrual bleeding. However, when mifepristone was administered every month at the end of the cycle either alone or together with prostaglandins, it was not very effective in preventing pregnancy. In contrast, a mifepristone-prostaglandin combination has been shown to be a very effective treatment for occasional menstrual regulation, with vaginal bleeding induced in 98% of pregnant women, with menses delay of 11 days or less. Mifepristone is an excellent agent for emergency contraception when used within 120 h of unprotected intercourse. It is also possible that PAs and PRMs may be used to reduce the occurrence of bleeding irregularities induced by progestin-only contraceptive methods. Both classes of progesterone receptor ligands may also have contraceptive efficacy by having a pharmacological effect on the embryo or altering tubal transport or other aspects of tubal physiology.

Animals↗
PubMed1999

Investigation of the binding interactions of progesterone using muteins of the human progesterone receptor ligand binding domain designed on the basis of a three-dimensional protein model.

The aim of this study was to investigate the binding interactions of the human progesterone receptor (hPR) with its natural ligand. Therefore, a homology-derived model of the hPR ligand binding domain has been constructed and used to predict residues potentially involved in interactions with progesterone. These residues and the free cysteines have been mutated (in total 13 residues with 15 mutations). All exchanges have been designed to preserve the three-dimensional structure of the protein. With respect to the binding characteristics towards progesterone, the muteins fall into three groups displaying no, reduced, or wildtype-like binding activity.

Amino Acid Sequence↗
PubMedDate not supplied

Novel mechanisms of progesterone antagonists and progesterone receptor.

The progesterone receptor (PR), as a member of the nuclear receptor superfamily of ligand-dependent transcription factors, activates gene transcription through binding to specific palindromic progesterone response elements (PRE) in the promoter region of progestin-responsive genes. The progesterone antagonists ZK98299 (Onapristone) and RU 486 (Mifepristone) inhibit the transcriptional activity of PR by complex mechanisms at concentrations much lower than the progestins. Altered conformation is central to antagonist inhibition of the transcriptional activity of PR. Antagonists also promote inappropriate association of PR with corepressors. We speculate that the different PR conformations induced by agonist and antagonists results in an asymmetric agonist/antagonist heterodimer that binds inefficiently to palindromic PREs. PR, under the same cellular conditions but with different promotor contexts, can repress (beta-casein) or enhance (3 beta-HSD) signal transducer and activator of transcription (Stat5)-mediated gene activation. The beta-casein promoter appears to contain a composite DNA-binding element for PR and Stat5 and that occupancy by PR in response to progestins or antagonists suppresses Stat5 transactivation function.

Animals↗
PubMed1987

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)

Animals↗
PubMed1977

pH dependence of progesterone interaction with progesterone-binding globulin. Kinetic and equilibrium studies.

The kinetics of binding and dissociation for the progesterone-binding globulin (PBG)-progesterone complex have been measured as a function of pH. The association rate constant appears to be independent of pH from pH to 10 with an average value of kon = 8.5 X 10(7)M-1 S-1. The dissociation rate constant is strongly pH dependent with the dependency defined by: koff = k0 (1 + [H+]/K1 + K2/[H+])(1 + K3*/[H+])/(1 + K3/[H+]). The best values for the various parameters were k0 = 0.0785 s-1, pK1 = 5.30, pK2 = 10.54, pK3* = 7.41, and pK3 = 7.21. Simpler expressions were inadequate to fit the data, and it was concluded that at least three ionizing residues are responsible for the stability of the PBG-progesterone complex. The affinity constant was determined by equilibrium dialysis over the range of pH 3 to 12. The ratio of the association and dissociation rate constants is in agreement with the affinity constant from pH 6.5 to 10.5. The influence of pH on the conformation and binding activity of PBG was also investigated. Denaturation by acid, base, or guanidine hydrochloride leads to a reversible loss of binding activity. Regain of binding activity in all cases is slow with half-times of 0.5 to 2.7 h, depending on conditions. The rate of acid denaturation was found to be incompletely protonated at pH 1.4, suggesting a buried carboxylic acid residue. The slow renaturation of PBG might be due to the difficulty of burying a charged residue in the protein's interior coupled with steric hindrance by the large carbohydrate moiety of PBG.

Alpha-Globulins↗
PubMed1997

Characterization of ligand binding, DNA binding and phosphorylation of progesterone receptor by two novel progesterone receptor antagonist ligands.

In order to gain a better understanding of the distinctive mechanisms of the various types of antiprogestins, we have characterized in vitro ligand binding, specific DNA binding and phosphorylation of progesterone receptor (PR) from T47D cells after treatment of cells with progestins (progesterone, R5020) and antiprogestins (RU486, ZK98299, Org 31806 and Org 31710). Treatment of the cells with R5020 or PR antagonists, with the exception of ZK98299, resulted in a quantitative upshift of PR-A and PR-B indicative of ligand/DNA-induced phosphorylation of PR. Treatment of cells with RU486, Org 31710 or Org 31806, but not R5020 or ZK98299 resulted in detectable PR-progesterone response element complexes (PR-PREc) as assessed by gel mobility shift assay. Although treatment of cells with ZK98299, a type I PR antagonist, did not induce phosphorylation, the antiprogestins, Org 31806 and Org 31710, in a manner identical to RU486, did. Our data suggest that Org 31806 and Org 31710 affect properties of PR from T47D cells that are similar to RU486.

Binding, Competitive↗
PubMed2003

A decline in the levels of progesterone receptor coactivators in the pregnant uterus at term may antagonize progesterone receptor function and contribute to the initiation of parturition.

The molecular events that lead to the onset of labor in humans and in other mammalian species remain unclear. We propose that a decline in coactivators containing histone acetylase activity in myometrium may contribute to the onset of labor by impairing the function of the progesterone-progesterone receptor (PR) complex. As assessed by semiquantitative and real-time RT-PCR, immunohistochemistry, and immunoblotting, expression of the PR coactivators cAMP-response element-binding protein (CREB)-binding protein and steroid receptor coactivators 2 and 3 was decreased in fundal uterine tissue of women in labor. Using the mouse as an animal model, we also found decreased coactivator levels in uterine tissues at term. In both human and mouse, the levels of acetylated histone H3 were also decreased in uterine tissues at term. Administration of trichostatin A, a specific and potent histone deacetylase inhibitor, to pregnant mice late in gestation increased histone acetylation and delayed the initiation of parturition by 24-48 h, suggesting the functional importance of the decline in histone acetylation in the initiation of labor. These findings suggest that the decline in PR coactivator expression and in histone acetylation in the uterus near term may impair PR function by causing a functional progesterone withdrawal. The resulting decrease in expression of PR-responsive genes should increase sensitivity of the uterus to contractile stimuli.

Acetyltransferases↗
PubMed1992

Hormone-induced progesterone receptor phosphorylation consists of sequential DNA-independent and DNA-dependent stages: analysis with zinc finger mutants and the progesterone antagonist ZK98299.

Human progesterone receptors (hPRs) are phosphorylated at multiple serine residues, first in a basal step and then in a hormone-induced step. To determine whether hormone-induced phosphorylation precedes or follows the interaction of hPRs with DNA two strategies were used. (i) DNA binding was prevented or altered with site-specific mutants of the A form of hPR; (ii) DNA binding of wild-type hPR forms A and B was prevented with the progesterone antagonist ZK98299. Two hPRA mutants were constructed: DBDCys, which lacks a critical cysteine residue in the first zinc finger, and DBDsp, which is mutated at three discriminatory amino acids to change its DNA binding specificity from a progesterone response element to an estrogen response element. Receptors were transiently expressed in PR-negative cells and were intranuclear. DBDCys did not bind DNA in vitro and DBDsp bound only the estrogen response element. Transiently expressed hPRA and DBDsp showed the upward shift in electrophoretic mobility characteristic of hormone-induced phosphorylation; it was absent with DBDCys. Hormone-induced [32P] orthophosphate incorporation into transiently expressed DBDCys was reduced 60% compared to hPRA and DBDsp but was not eliminated. ZK98299 binds hPRs but prevents their interaction with DNA. Compared to R5020, the antagonist reduced phosphorylation of hPRB and hPRA in T47D breast cancer cells by 60% and totally prevented the mobility shift. We conclude that the hormone-induced phosphorylation of hPR includes DNA-independent and DNA-dependent stages and that only DNA-dependent sites contribute to the mobility shift.

Animals↗
PubMed2003

Selective interactions of Kruppel-like factor 9/basic transcription element-binding protein with progesterone receptor isoforms A and B determine transcriptional activity of progesterone-responsive genes in endometrial epithelial cells.

The Sp/KLF transcription factor basic transcription element-binding protein (BTEB1) regulates gene transcription by binding to GC-rich sequence motifs present in the promoters of numerous tissue-specific as well as housekeeping genes. Similar to other members of this family, BTEB1 can act as a transactivator or transrepressor depending on cell and promoter context, although the molecular mechanism underlying these distinct activities remains unclear. Here we report that BTEB1 can mediate signaling pathways involving the nuclear receptor for the steroid hormone progesterone in endometrial epithelial cells by its selective interaction with the progesterone receptor (PR) isoforms, PR-A and PR-B. Functional interaction with ligand-activated PR-B resulted in superactivation of PR-B transactivity, facilitated the recruitment of the transcriptional integrator CREB-binding protein within the PR-dimer, and was dependent on the structure of the ligand bound by PR-B. By contrast, BTEB1 did not influence agonist-bound PR-A transactivity, although it augmented PR-A inhibition of PR-B-mediated transactivation as well as potentiated ligand-independent PR-A transcriptional activity in the presence of CREB-binding protein. We also demonstrate similar positive modulatory actions of BTEB1-related family members Krüppel-like family (KLF) 13/FKLF2/BTEB3 and Sp1 on PR-B transactivity. Further, we provide support for the potential significance of the selective functional interactions of PR isoforms with BTEB1 in the peri-implantation uterus using mouse and pig models and in the breast cancer cell lines MCF-7 and T47D. Our results suggest a novel mechanism for the divergent physiological consequences of PR-A and PR-B on progesterone-dependent gene transcription in the uterus involving select KLF members.

Animals↗
PubMed1996

Non-competitive anti-oestrogenic actions of progesterone antagonists in primate endometrium: enhancement of oestrogen and progesterone receptors with blockade of post-receptor proliferative mechanisms.

Previous studies have shown that the progesterone antagonists (antiprogestins) inhibit oestrogen-dependent endometrial proliferation in ovariectomized monkeys, without having affinity to the oestrogen receptor (ER). This study was designed to investigate the effect of the antiprogestins mifepristone (RU 486) and onapristone (ZK 98,299), on the concentration of ER and progesterone receptor (PR) in the endometrium of long-term ovariectomized cynomolgus monkeys (Macaca fascicularis). In untreated monkeys, tissue preparations bound in total 228 +/- 68 pmol [3H]-oestradiol/g protein (ER), and 119 +/- 42 pmol [3H]-R5020/g protein (PR). These values were not significantly different from the total binding capacities of tissues from monkeys treated with RU 486 alone or primates treated with oestradiol plus progesterone. Treatment with oestradiol alone almost doubled the ER and PR concentrations. Combined treatment with oestradiol and RU 486 enhanced the ER and PR concentrations in a dose-dependent manner: 1 mg/kg body weight (bw) RU 486/kg increased both ER and PR contents about 3-fold. The dose of 5mg/kg bw RU 486 or onapristone increased the ER and PR concentrations almost 6- and 5-fold respectively, compared with the oestradiol-treated controls. Our results demonstrate that RU 486 and onapristone increased the endometrial ER and PR concentrations far beyond the physiological level in ovariectomized, oestradiol-treated monkeys. Whether the over-expression of ER and PR in the presence of antiprogestins is causally related to the antiproliferative impact of antiprogestins in the endometrium (non-competitive anti-oestrogenic effects) or is an independent action in unknown.

Animals↗
PubMed1984

High affinity binding of [3H]R5020 and [3H]progesterone by putative progesterone receptors in cytosol and nuclear extract of turtle oviduct.

The purpose of this investigation was to identify and characterize putative cytosolic and nuclear forms of progesterone receptor in the female reproductive tract of a turtle, Chrysemys picta. A dextran-coated charcoal adsorption assay and DNA-cellulose affinity chromatography were used as the primary methodologies with [3H]R5020 [3H-labeled 17 alpha-dimethyl-19-norpregna-4,9-diene-3,20-dione) and [3H]progesterone (P4) as the ligands. The receptor was of high affinity (Kd = 4.7 X 10(-10) M for [3H]P4; 2.2 X 10(-10) M for [3H]R5020) and limited capacity (500-6000 fmol/g tissue wet wt). Association was rapid (apparent equilibrium being reached in 30-40 min), as was dissociation (t1/2 = 45 min for [3H]P4 and 180 min for [3H] R5020). The putative receptor demonstrated strict steroid specificity, binding progestins but not estrogens, androgens, or glucocorticoids. Heterogeneity of the cytosolic receptor was demonstrated as two forms eluting off DNA-cellulose columns at 0.2- and 0.3-M salt concentrations. Binding of cytosolic receptor to DNA-cellulose was not increased by preexposure of cytosol to 25 C for 30 min. Some variations in cytosolic, but not nuclear, receptor were associated with different stages of the reproductive cycle and were positively correlated with body weight. Preliminary studies using an explant culture system suggest that the progesterone receptor in turtle oviduct may be maintained by estrogen and translocated from the cytosol to the nucleus by P4. In summary, we have partially characterized a putative P4 receptor in the oviduct of the turtle that is similar to mammalian and avian P4 receptors in specificity, affinity, and other physicochemical properties, supporting the idea that steroid receptor proteins have been highly conserved in vertebrate evolution. However, temperature sensitivity of activation and DNA affinity are different in the turtle and suggest modifications that may be related to physiological adaptation in such a poikilothermic species.

Animals↗
PubMed2004

Prostaglandins differentially modulate progesterone receptor-A and -B expression in human myometrial cells: evidence for prostaglandin-induced functional progesterone withdrawal.

We tested the hypothesis that prostaglandin (PGs), PGE2, and PGF2 alpha, stimulate labor and delivery in women, in part, by inducing functional progesterone withdrawal in myometrial cells by increasing the progesterone receptor (PR)-A/PR-B expression ration. PHM1-31 cells (an immortal pregnant human myometrial cell line) were exposed to PGE2, PGF2 alpha, cyclic-8-bromoadenosine monophosphate (8-Br-cAMP) and phorbol 12-myristate 13-acetate (PMA) at various concentrations for 24h. Effects on PR-A and PR-B expression were then assessed by quantitative RT-PCR. PGF2 alpha dose dependently increased PR-A mRNA and the PR-A/PR-B expression ration but did not effect PR-B mRNA. PGE2 dose-dependently increased mRNAs encoding PR-A and PR-B. The PGE2 dose-threshold for PR-A (0.01 nM) was lower than that for PR-B (0.1 nM), which resulted in an initial rise then a gradual fall in PR-A/PR-B expression ration to basal levels in response to PGE2. Activation of the protein kinase (PK)-A signaling pathway with 8-Br-cAMP coordinately increased expression of PR-A and PR-B and therefore did not alter the PR-A/PR-B expression ration. In contrast, activation of the PKC signaling pathway with PMA increased expression of PR-A without affecting PR-B and therefore significantly (P<0.05) increased the PR-A/PR-B expression ration. These data demonstrate differential control of myometrial PR-A and PR-B expression by PGE2 and PGF2 alpha and by specific intracellular signaling pathways. We conclude that PGs acting via the PKC pathway facilitate functional progesterone withdrawal by increasing the myometrial PR-A/PR-B expression ratio.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2004

Progesterone-independent effects of human progesterone receptors (PRs) in estrogen receptor-positive breast cancer: PR isoform-specific gene regulation and tumor biology.

Progesterone receptors (PRs) are prognostic markers in breast cancers irrespective of the patient's progestational status. However, there are two PR isoforms, PR-A and PR-B, that are equimolar in the normal breast but dysregulated in advanced disease. Postmenopausal, tamoxifen-treated patients with estrogen receptor (ER)-positive, PR-A-rich tumors have much faster disease recurrence than patients with PR-B-rich tumors. To study the mechanisms we engineered ER+ breast cancer cells that express each PR isoform under control of an inducible promoter. We identified 79 genes regulated by progesterone (P), mainly by PR-B, and 51 genes regulated without progesterone, mainly by PR-A. Only nine genes were regulated with and without ligand, leading to definition of three classes: I) genes regulated only by liganded PR; II) genes regulated only by unliganded PR; III) genes regulated by both. Unliganded PR-A and PR-B differentially regulate genes that coordinate extracellular signaling pathways and influence tumor cell biology. Indeed, in the absence of P, compared with ER+/PR-B+ or PR- cells, ER+, PR-A+ cells exhibit an aggressive phenotype, are more adhesive to an extracellular matrix, and are more migratory. Additionally, unliganded PR-A and PR-B both inhibit cell growth and provoke resistance to Taxol-induced apoptosis. We propose that PR-A:PR-B ratios, even in the absence of P, influence the biology and treatment response of ER+ tumors, that PR-A isoforms are functionally dominant in P-deficient states, and that PR-A rich tumors are especially aggressive.

Apoptosis↗
PubMed2001

New antiprogestins with partial agonist activity: potential selective progesterone receptor modulators (SPRMs) and probes for receptor- and coregulator-induced changes in progesterone receptor induction properties.

A pharmacologically relevant property of steroid hormone-regulated gene induction is the partial agonist activity of antisteroid complexes. We now report that dexamethasone-mesylate (Dex-Mes) and dexamethasone-oxetanone (Dex-Ox), each a derivative of the glucocorticoid-selective steroid dexamethasone (Dex), are two new antiprogestins with significant amounts of agonist activity with both the A and B isoforms of progesterone receptor (PR), for different progesterone-responsive elements, and in several cell lines. These compounds continue to display activity under conditions where another partial antiprogestin (RTI-020) is inactive. These new antiprogestins were used to determine whether the partial agonist activity of PR complexes can be modified by changing concentrations of receptor or coregulator, as we have recently demonstrated for glucocorticoid receptors (GRs). Because GR and coregulator concentrations simultaneously altered the position of the physiologically relevant dose-response curve, and associated EC(50), of GR-agonist complexes, we also examined this phenomenon with PR. We find that elevated PR or transcriptional intermediary factor 2 (TIF2) concentrations increase the partial agonist activity of Dex-Mes and Dex-Ox, and the EC(50) of agonists, independently of changes in total gene transactivation. Furthermore, the corepressors SMRT (silencing mediator for retinoid and thyroid receptors) and NCoR (nuclear receptor corepressor) each suppresses gene induction but NCoR acts opposite to SMRT and, like the coactivator TIF2, reduces the EC(50) and increases the partial agonist activity of antiprogestins. These comparable responses of GR and PR suggest that variations in receptor and coregulator concentrations may be a general mechanism for altering the induction properties of other steroid receptors. Finally, the magnitude of coregulator effects on PR induction properties are often not identical for agonists and the new antagonists, suggesting subtle mechanistic differences. These properties of Dex-Mes and Dex-Ox, plus the sensitivity of their activity to cellular differences in PR and coregulator concentrations, make these steroids potential new SPRMs (selective progesterone receptor modulators) that should prove useful as probes of PR induction properties.

Animals↗
PubMed1996

Effects of progesterone, estrogen, and androgen on progesterone receptor binding in hen oviduct uterus (shell gland).

The specific [3H] progesterone binding per milligram of protein of the cytosolic fraction of the hen uterus was found to have decreased at 1 and 4 h after the injection of progesterone (P4), but increased at 4 and 8 h after the injection of estradiol-17 beta (E2), and at 1 and 4 h after the injection of testosterone (T). The equilibrium dissociation constant obtained by Scatchard analysis was not significantly different between the hens receiving steroid injections [P4], E2, T, and dihydrotestosterone (DHT)] and controls. The maximum binding capacity was lower in hens injected with P4 and greater in hens injected with E2, T, or DHT than in uninjected hens. The results suggest that the cytosolic progesterone receptor binding in the hen uterus may be modulated by the sex steroid hormones.

Analysis of Variance↗