Covalent attachment of a progestational steroid to chick oviduct progesterone receptor by photoaffinity labelling.
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Affinity Labels↗
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Sex difference in response to alphaxalone anaesthesia may be oestrogen dependent.
The steroid anaesthetic Althesin (Glaxo), which is a mixture of two C21 steroids, alphaxalone (3 alpha-hydroxy-5 alpha-pregnane-11, 20-dione--the active compound) and alphadolone acetate (21-acetoxy-3 alpha-hydroxy-5 alpha-pregnane-11, 20-dione), has been especially useful for the study of forebrain-autonomic and neuroendocrine functions. As determined by the loss of the righting reflex, Child et al. found no sex difference in the anaesthetic dose of Althesin administered intravenously (i.v.). However, in our neuroendocrine studies in which the anaesthetic was administered intraperitoneally (i.p.) and at dosage sufficient to produce surgical anaesthesia and analgesia, we observed a sex difference in the efficacy of Althesin. This may explain the difficulties that have been encountered in obtaining adequate anaesthesia (blockade of the somatomotor response to pain) with Althesin. Here we report, using cortical electroencephalography, that Althesin is a more potent anaesthetic than either sodium pentobarbitone or urethane, and that anaesthesia in the male rat requires about four times more Althesin (administered i.p.) than in the female. This sex difference is age dependent, can be abolished by administering oestrogen to the male, does not depend on sexual differentiation of the brain, and cannot be attributed to a sex difference in the metabolic clearance rate of alphaxolone. These results, taken together with those of Richards and Hesketh, suggest that the effect of alphaxalone may be mediated by interactions with synaptic membranes that are more specific than simply a generalized change in membrane structure, and that these interactions are affected by sex steroids.
Age Factors↗
Glucocorticoid- and progesterone-specific effects are determined by differential expression of the respective hormone receptors.
Although glucocorticoids and progestins control vastly different physiological processes, the receptors mediating the effects of these hormones interact with the same DNA sequences. Transfer experiments involving synthetic genes and in vitro binding studies have shown that progesterone and glucocorticoid receptors both recognize the same 15-base pair DNA element (TGTACAGGATGTTCT), raising the question of how the two steroids affect gene expression selectively. We considered the possibility that their selectivity arises from either the differential expression of the receptors in target cells or the differential dependence of receptor function on additional transcription factors. To test these alternatives we introduced a progesterone-receptor expression plasmid into the rat hepatoma cell line Fto2B-3 which contains glucocorticoid receptor but is devoid of progesterone receptor. We report that expression of the progesterone receptor in Fto2B-3 cells renders endogenous glucocorticoid-regulated genes inducible by progestins. Our data show that the responsiveness of a cell to external stimuli can be reprogrammed by the expression of a single transcription factor and that differential expression of hormone receptors is at least one mechanism by which steroid-specific gene activation is achieved.
Animals↗
Progesterone receptor stimulates transcription of mouse mammary tumour virus in a cell-free system.
In higher eukaryotes, steroid receptors are general modulators of gene activity which bind to DNA hormone response elements (HREs) in the vicinity of regulated promoters. Analysis of mutant and chimaeric receptor proteins in gene transfer experiments has identified the domains responsible for hormone binding, interaction with the HREs and transactivation, but the transactivating function of hormone receptors has proved difficult to reproduce in cell-free assays. Here we describe a crude in vitro system in which transcription from the mouse mammary tumour virus (MMTV) promoter is increased up to 10-fold by native progesterone receptor from rabbit uterus. The stimulatory effect must depend on receptor binding to the MMTV-HRE as it is abolished by deletion or mutation of the HRE and by HRE-oligonucleotide competition. The nuclear factor-I binding site immediately downstream of the HRE, however, does not appear to be essential to progesterone receptor-mediated in vitro stimulation of MMTV transcription. The transactivation activity of the progesterone receptor depends on binding of a functional ligand and so this assay should be useful for dissection of the mechanism of transcription activation by steroid hormones.
Animals↗
Variation of receptor status in cancer of the breast.
One hundred and nineteen patients with breast cancer had 2 or more lesions removed for oestrogen (REc) or progesterone receptor (RPc) assay, either synchronously (on 38 occasions) or after an interval (on 91 occasions). In all but 7 both receptors were assayed for each lesion. The assays did not agree on the presence or absence of REc alone, RPc alone or the combination of both receptors in 11, 13 and 16% respectively of the synchronous samples, compared with 23, 30 and 43% of the asynchronous samples. The differences between the synchronous and asynchronous samples were significant for the combined receptors (P = 0.007) but not for REc (P = 0.176) or RPc alone (P = 0.077). Variation between asynchronous biopsies was greater when the earlier lesion contained RPc (18/37 disagreed) than when it did not (8/50) disagreed, P = 0.0023). This was not true for oestrogen receptor. In those remaining receptor positive there was only a weak correlation between the first and second values (Spearman rank correlation coefficient, rho = 0.39 for REc, P less than 0.02, and 0.45 for RPc, 0.05 less than P less than 0.1). Receptor levels and receptor status may change with time. Biopsy is most appropriate at the time when systemic treatment is proposed.
Biopsy↗
Anti-oestrogens induce the secretion of active transforming growth factor beta from human fetal fibroblasts.
The clinical use of anti-oestrogens in breast cancer therapy has traditionally been restricted to tumours that contain measurable oestrogen receptor protein. However, it is now widely recognised that the clinical response to adjuvant anti-oestrogen therapy appears to be independent of the oestrogen receptor content of the primary tumour. The study reported here was designed to investigate the possibility that human stromal cells can respond to anti-oestrogens by an increased synthesis of the inhibitory growth factor, transforming growth factor beta (TGF-beta). Two established human fetal fibroblast strains were used as models for the breast cancer stromal fibroblasts. These cells were found to respond to the addition of anti-oestrogens by a large increase in their synthesis of biologically active TGF-beta. Despite the application of ligand binding, immunoassay and Northern analysis, no oestrogen receptor or oestrogen receptor mRNA was detected in either of the human fetal fibroblasts strains. These observations may provide a mechanism of action of anti-oestrogens that is independent of the presence of oestrogen receptor in the tumour epithelial cells, and thus provide an explantation for the counter-intuitive results of adjuvant anti-oestrogen action.
Blotting, Northern↗
Progesterone crosstalks with insulin-like growth factor signaling in breast cancer cells via induction of insulin receptor substrate-2.
Both progesterone and the insulin-like growth factors (IGFs) are critically involved in mammary gland development and also in breast cancer progression. However, how the progesterone and IGF signaling pathways interact with each other to regulate breast cancer cell growth remains unresolved. In this study, we investigated progesterone regulation of IGF signaling components in breast cancer cells. We found that insulin receptor substrate-2 (IRS-2) levels were markedly induced by progesterone and the synthetic progestin R5020 in MCF-7 and other progesterone receptor (PR) positive breast cancer cell lines, whereas IRS-1 and the IGF-I receptor were not induced. The antiprogestin RU486 blocked the R5020 effect on IRS-2 expression. Ectopic expression of either PR-A or PR-B in C4-12 breast cancer cells (estrogen receptor and PR negative) showed that progestin upregulation of IRS-2 was mediated specifically by PR-B. The IRS-2 induction by R5020 occurred via an increase of IRS-2 mRNA levels. Furthermore, progestin treatment prior to IGF-I stimulation resulted in higher tyrosine-phosphorylated IRS-2 levels, increased binding of IRS-2 to Grb-2 and the PI3K regulatory subunit p85, and correspondingly enhanced ERK and Akt activation, as compared with IGF-I-only conditions. Taken together, our data suggest that IRS-2 may play an important role in crosstalk between progesterone and the IGFs in breast cancer cells.
Breast Neoplasms↗
Distinction between progestin- and glucocorticoid-binding sites in mammary glands. Apparent lack of cytoplasmic progesterone receptors in lactating mammary glands.
The cytosol fraction of the lactating mammary glands of mice does not appear to contain detectable amounts of progesterone receptors. Mixing experiments indicate that the absence of receptors is not due to interference by other factors in the cytosol. However, in the cytosol of mammary glands, there is specific binding or progestins to certain low-affinity sites which have characteristics of specific glucocorticoid-binding sites.
20-alpha-Dihydroprogesterone↗
Progesterone receptor characterized by photoaffinity labelling in the plasma membrane of Xenopus laevis oocytes.
R 5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) is a synthetic analogue of progesterone, which is the physiological hormone that reinitiates germinal vesicle breakdown in Xenopus laevis oocytes. U.v.-driven photoaffinity labelling experiments were conducted with [3H]R 5020 in oocyte subcellular fractions, and covalently bound radioactivity was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In P-10000 (the pellet sedimenting between 1000 and 10000 g and which contains plasma membrane), a major radioactive band migrating as a 30kDa peptide was found. Non-radioactive progesterone competed with the [3H]R 5020 labelling of this fraction, but not with the labelling of minor [3H]R 5020-binding fractions. It displayed the required characteristics of a specific progesterone-binding membrane 'receptor', postulated from previous studies with intact oocytes and with cell-free P-10000 preparations of membrane-bound adenylate cyclase. The apparent Ki of approx. 4 microM for progesterone was compatible with the active concentration of the hormone. Binding specificity, as determined in competition studies, was highly correlated with the germinal vesicle breakdown activity of the steroids and analogues tested. The receptor was not found in the vitelline envelope, in vitelline platelets, in melanosome-enriched or microsomal fractions, in cytosol, nor in germinal vesicles of oocytes. The properties of this membrane steroid receptor are different from those of the already known soluble intracellular steroid receptors, in particular regarding ligand binding specificity and subcellular distribution.
Affinity Labels↗
Increased resistance to activated protein C after short-term oral hormone replacement therapy in healthy post-menopausal women.
As hormone replacement therapy is associated with an early excess risk of venous thrombosis, we investigated the effect of different oral hormone replacement therapies on resistance to activated protein C, and on levels of factor VIII antigen (FVIII:Ag) and factor XI antigen (FXI:Ag). In a prospective, randomized, placebo-controlled 12-week study, 60 healthy post-menopausal women daily received either placebo (n = 16) or 2 mg of micronized 17beta-oestradiol, either alone (E2, n = 16) or sequentially combined with dydrogesterone 10 mg (E2 + D, n = 14) or trimegestone 0.5 mg (E2 + T, n = 14). Medication was given orally. Normalized activated protein C sensitivity ratios (nAPCsr) were determined by quantifying the effect of activated protein C on the endogenous thrombin potential. FVIII:Ag and FXI:Ag were determined by enzyme-linked immunosorbent assay. Compared with baseline and placebo, the nAPCsr increased (92% to 142%; all P < 0.001) in all active treatment groups after both 4 and 12 weeks. Compared with placebo, hormone replacement therapy was not associated with significant changes in FVIII:Ag. After 4 and 12 weeks, FXI:Ag levels were significantly decreased in the E2 group (mean percentage changes from baseline versus placebo: -15.0%, P = 0.001 at 4 weeks and -16.6%, P = 0.003 at 12 weeks) and in the E2 + D group (-10.4%, P = 0.02 and -10.4%, P = 0.02). In conclusion, all hormone replacement regimens were associated with a large increase in resistance to activated protein C. In contrast, hormone replacement therapy had no effect on FVIII:Ag. Oral E2 and E2 + D had a small, favourable effect on FXI:Ag.
Activated Protein C Resistance↗
Short-term hormone replacement therapy: reduced plasma levels of soluble adhesion molecules.
BACKGROUND: Epidemiological data have suggested that the use of hormone replacement therapy (HRT) is associated with a decreased risk of cardiovascular disease. Vascular endothelium and adhesion molecules play an important role in the initiation and progression of atherosclerosis. MATERIAL AND METHODS: Prospective, randomized, placebo-controlled 12-week study. Sixty healthy, normotensive postmenopausal women received either micronised oestradiol 2 mg alone (n = 16, E2 group), or sequentially combined with a progestagen; E2 + P groups trimegestone 0.5 mg (E2 + T, n = 14) or dydrogesterone 10 mg (E2 + D group, n = 14) or placebo (n = 16). Data were collected at baseline and at 4 and 12 weeks. RESULTS: Twelve weeks of treatment with E2 or E2 + P was associated with a significant decrease in the plasma concentrations of soluble intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), and thrombomodulin (sTM). The average decrease in these markers was about 9%. In women treated with trimegestone the decreases were larger than in those treated with dydrogesterone; for sICAM-1 (-15% vs. -2%; P < 0.0001), sVCAM-1 (-15% vs. +3%; P = 0. 003) and sTM (-9% vs. -4%; P = 0.11). Plasma levels of endothelin-1 (ET-1) decreased (by 13%) only in women treated with E2 + P. In the E2 group, flow-mediated, endothelium-dependent vasodilatation increased by 6 percentage points after 12 weeks (P = 0.07 vs. baseline, P = 0.02 vs. E2 + P, and P = 0.17 vs. placebo). CONCLUSION: Short-term treatment with E2 or E2 + trimegestone reduces plasma levels of sICAM-1, sVCAM-1 and sTM. ET-1 decreased only in the E2 + P groups. Different types of progestagens may differentially affect sICAM-1, sVCAM-1 and sTM levels, which may be relevant for the choice of type HRT.
Cholesterol↗
Effects of hormone replacement therapy on blood platelets.
BACKGROUND: Hormone replacement therapy (HRT) increases the risk of cardiovascular morbidity in postmenopausal women under certain circumstances. Part of this effect may be the result of the influence of HRT on blood platelets. We studied the effect of short-term oral hormone replacement therapy (unopposed oestradiol or sequentially combined oestradiol and trimegestone or dydrogesterone) on platelet activation parameters in healthy postmenopausal women. DESIGN: We designed a prospective, randomised, placebo-controlled 12-week study. Sixty healthy, normotensive, nonhysterectomised, postmenopausal women received daily micronised oestradiol (E2) 2 mg (n = 16), or 2 mg E2 daily sequentially combined with either trimegestone 0.5 mg daily (n = 14) or dydrogesterone 10 mg daily (n = 14), or placebo (n = 16). Data on platelet activation were collected at baseline and after 12 weeks of treatment using flow cytometry. RESULTS: Twelve weeks of treatment with combined HRT was associated with an increase in platelet activation parameters P-selectin and glycoprotein 53 (by 17% and 14%, respectively, P = 0.04 vs. the placebo group for both comparisons), suggesting alpha granule and lysosome degranulation. E2 replacement therapy was associated with an increase in P-selectin labelling by 22% (P = 0.04 vs. the placebo group). CONCLUSION: Short-term treatment with oestradiol or combined HRT increases the amount of circulating activated platelets as measured by flow cytometry. This could be a mechanism by which short-term HRT might increase the risk of thrombosis.
Antigens, CD↗
Oral oestradiol/trimegestone replacement reduces procarboxypeptidase U (TAFI): a randomized, placebo- controlled, 12-week study in early postmenopausal women.
OBJECTIVE: To investigate the effects of short-term postmenopausal oral hormone administration on plasma levels of procarboxypeptidase U (proCPU, thrombin-activatable fibrinolysis inhibitor, EC 3.4.17.20), an inhibitor of fibrinolysis, in healthy early postmenopausal women. DESIGN: A prospective, randomized, placebo-controlled study. SETTING: Outpatient clinic of the Department of Obstetrics and Gynaecology. SUBJECTS: Seventy-seven healthy early postmenopausal women were screened of whom 65 were randomized. Analyses were based on 60 participants. INTERVENTIONS: The women received oral micronized oestradiol 2 mg either alone (E2 group, n=16), or sequentially combined with dydrogesterone 10 mg (E2 + D group, n=14) or trimegestone 0.5 mg (E2 + T, n=14), or placebo (n=16) for 12 weeks. MAIN OUTCOME MEASURE: ProCPU concentrations at baseline, and at 4 and 12 weeks of treatment. RESULTS: Four weeks of E2 + T was associated with a significant decrease in the fasting proCPU concentration, which was sustained after 12 weeks [t=0: 636 +/- 57 U L(-1) (mean +/- SD); t=4: 583 +/- 63UL-1; t=12: 589 +/- 48 U L(-1); ANCOVA versus placebo: P=0.011]. The percentage change from baseline versus placebo in this group was -8.4% [95% confidence interval (CI) -15.7 to -1.1] after 4 weeks and -5.9% (95% CI -11.7 to -0.1) after 12 weeks. There were no significant changes versus placebo in the E2 group nor in the E2 + D group. CONCLUSION: Short-term treatment with E2 + T, but not E2 alone or E2 + D, lowers proCPU concentration. These findings add to accumulating evidence suggesting that different progestagens added to oestrogen replacement may differentially affect the risk of arterial and venous disease.
Carboxypeptidase B2↗
Inhibition of brain oestrogen receptors by RU 58668.
The purpose of this study was to determine if injections of the 11 beta-substituted steroidal antioestrogen, RU 58668, would block two measures of oestrogen receptor action in ovariectomized adult female rats. Using an in vitro nuclear exchange assay, it was found that RU 58668 reduced cell nuclear [3H]-oestradiol binding in brain tissue 24 h after it was injected. However, pituitary cell nuclear [3H]-oestradiol binding was significantly reduced just 2 h after the antioestrogen was injected. Our results demonstrate that RU 58668 can reach the brain following a subcutaneous injection, but that it needs more time to reach the brain than it does to reach the pituitary. Since the levels of hypothalamic and pituitary progestin receptors are known to be regulated by oestradiol, cytosolic [3H]-R5020 binding was used as an in vitro assay of endogenous oestrogen receptor action. RU 58668 blocked induction by oestradiol of cytosolic [3H]-R5020 binding in both the brain and pituitary 48 h after it was injected. In the absence of oestradiol, RU 58668 did not stimulate cell nuclear [3H]-oestradiol binding or cytosolic [3H]-R5020 binding in either brain or pituitary. In conclusion, injections of RU 58668 blocked two measures of oestrogen receptor action in the brain and pituitary without showing oestrogenic activity itself.
Animals↗
Human papillomavirus type 16 cooperates with activated ras and fos oncogenes in the hormone-dependent transformation of primary mouse cells.
The effect of human papillomavirus (HPV) and activated oncogenes on the growth and morphology of primary baby mouse kidney (BMK) cells has been studied. Early region DNA from HPV types 16, 18, 31, and 33, but not type 6, under the transcriptional control of a heterologous, retroviral promoter cooperated with EJ-ras to produce cell lines that gave rise to carcinomas in syngeneic immunocompetent animals. The same HPV constructs, when cotransfected with a plasmid containing sequences from the Finkel-Biskis-Jinkins murine sarcoma virus provirus (v-fos), produced cell lines that were tumorigenic in nude mice. None of the other activated oncogenes tested, including activated c-myc, displayed any activity with HPV in this cotransfection assay. When the heterologous promoter was replaced by the homologous HPV16 promoter, the transforming effect of HPV16 with either EJ-ras or v-fos required the presence of either glucocorticoid or progestogen. Cell lines derived from transfection of HPV16 with either EJ-ras or v-fos required the continued presence of hormones for proliferation.
Animals↗
Antiprogestins prevent progesterone receptor binding to hormone responsive elements in vivo.
Antiprogestins inhibit progesterone action by competing for binding to the progesterone receptor and are potentially important pharmaceuticals in fertility control and cancer therapy. Why the complex of antiprogestins and progesterone receptor is functionally inactive is unclear. Present models are based on indirect evidence, such as transfection competition assays and in vitro DNA binding studies, partly because of difficulties in visualizing the receptor bound to DNA in vivo. Here we used genomic footprinting analysis to show ligand-dependent binding of endogenous progesterone receptor to the hormone responsive elements (HREs) of a chromosomally integrated mouse mammary tumor virus long terminal repeat in a human mammary carcinoma cell line. The antiprogestins RU 486 and ZK 98299 do not promote binding of the progesterone receptor to this natural HRE in vivo, even at concentrations that completely inhibit the agonistic effects of potent synthetic progestins. Moreover, antiprogestins cause a rapid disappearance of the agonist-induced progesterone receptor footprint. We conclude that antiprogestins interfere with receptor function by preventing its specific DNA binding.
Base Sequence↗
Disruption of estrogen signaling does not prevent progesterone action in the estrogen receptor alpha knockout mouse uterus.
Estrogen is known to increase progesterone receptor (PR) levels in the wild-type mouse uterus, and this estrogen induction was thought to be important for progesterone action through the PR. The estrogen receptor alpha knockout (ERKO) mouse uterus was observed to express PR mRNA that cannot be induced by estrogen. Progesterone action was characterized to determine whether it was diminished in ERKO mice. The PR protein is present in the ERKO uterus at 60% of the level measured in a wild-type uterus. The PR-A and PR-B isoforms are both detected on Western blot, and the ratio of isoforms is the same in both genotypes. Although the level of PR is reduced in the ERKO uterus, the receptor level is sufficient to induce genomic responses, since both calcitonin and amphiregulin mRNAs were increased after progesterone treatment. Finally, the ERKO uterus can be induced to undergo a progesterone-dependent decidual response. Surprisingly, the decidual response is estrogen independent in the ERKO, although it remains estrogen dependent in a wild type. These results indicate that estrogen receptor alpha modulation of PR levels is not necessary for expression of the PR or genomic and physiologic responses to progesterone in the ERKO uterus.
Amphiregulin↗
Phosphorylation of human progesterone receptors at serine-294 by mitogen-activated protein kinase signals their degradation by the 26S proteasome.
Ligand-dependent down-regulation that leads to rapid and extensive loss of protein is characteristic of several nuclear steroid receptors, including human progesterone receptors (PRs). In breast cancer cells, >95% of PRs are degraded 6 h after the start of progestin treatment. The mechanism for down-regulation is unknown. We examined the role of PR phosphorylation by mitogen-activated protein kinases (MAPKs) in this process. Lactacystin and calpain inhibitor I, specific inhibitors of the 26S proteasome, blocked progestin-induced down-regulation, and ubiquitinated conjugates of PR accumulated in cells. Ligand-dependent PR degradation was also blocked by specific inhibition of p42 and p44 MAPKs. To define the targets of phosphorylation by this kinase, two serine/proline MAPK consensus sites on PR were mutated. We demonstrate that mutation of PR serine-294 to alanine (S294A) specifically and completely prevents ligand-dependent receptor down-regulation. We also find that rapid, ligand-independent degradation of immature PR intermediates occurs by a proteasome-mediated pathway. These results demonstrate that PR destruction, by either of two alternate routes, is mediated by the 26S proteasome. Specifically, down-regulation of mature PRs occurs by a mechanism in which ligand binding activates PR phosphorylation by MAPKs at a unique serine residue, which then targets the receptors for degradation.
Acetylcysteine↗