Enhanced Human Decidual Cell-Expressed FKBP51 May Promote Labor-Related Functional Progesterone Withdrawal.

Sustained plasma progesterone (P4) levels suggest initiation of human term labor by functional P4 withdrawal, reflecting reduced progesterone receptor (PR) and/or glucocorticoid receptor (GR) expression or activity. The steroid-induced immunophilin cochaperone FKBP51 inhibits PR- and GR-mediated transcription, suggesting a labor-initiating role. Gestational age-matched decidual sections were immunostained for FKBP51 and decidual cell (DC) and interstitial trophoblast (IT) markers, vimentin and cytokeratin, respectively. Term DC cultures were incubated with vehicle (control), estradiol (E2) with or without medroxyprogesterone acetate, dexamethasone (Dex), or Organon 2058. FKBP51 histologic scoring was significantly higher in DC nuclei during labor versus prelabor decidua, whereas FKBP51 immunostaining was undetected in interstitial trophoblasts (P < 0.05). In term DC cultures, E2 + medroxyprogesterone acetate or E2 + Dex enhanced FKBP51 expression (P < 0.01), whereas E2 + Organon 2058 inhibited PR expression (P < 0.05), and E2 + Dex inhibited GR expression (P < 0.05). Unlike term DCs, FKBP51 was undetected in control or Dex-treated cultured third-trimester trophoblasts. Electrophoretic mobility shift assays revealed that FKPB51 overexpression or silencing in cultured DCs altered PR-DNA binding. Increased FKBP51 levels in term DCs during labor complement our prior in situ observations of significantly lower PR in labor versus prelabor DCs. In a milieu of sustained plasma P4 levels, these reciprocal changes will amplify functional P4 withdrawal in DCs via FKBP51-mediated PR resistance coupled with declining PR levels, whereas the lack of FKBP51 expression in interstitial trophoblasts suggests unopposed constitutive GR action.

Demonstration of progesterone receptors in rat Schwann cells.

We have recently shown that progesterone promotes myelin formation in peripheral nerves of rodents. In this study, we demonstrate the presence of progesterone receptors (PR) in primary cultures of rat Schwann cells, the glial cells of the PNS, prepared from sciatic nerves of 4-5 days old rats. After 3 weeks of culture, the presence of PR was measured by whole cell assay after incubating living cells for 1 h at 37 degrees C with [3H]-Organon 2058 as a ligand, and about 5000 specific binding sites per cell were found. In contrast to the PR of rat glial cells from the central nervous system (CNS), which is induced by estrogens, treatment of Schwann cells with estradiol did not increase the PR-binding, even after exposure of cells to high doses of estrogen under various culture conditions. Progesterone receptors were also visualized in Schwann cells by indirect immunofluorescence staining with a monoclonal anti-PR antibody. Again, treatment of the cells with estradiol did not increase the immunofluorescence staining of the PR. Specific PR binding was also measured in sciatic nerves of adult female rats. Cytosol was prepared and labeled with [3H]-Organon 2058 for 15 h at 2 degrees C. After treatment with dextran-coated charcoal, specific ligand binding was about 30 fmol/mg cytosolic protein. When castrated adult female rats were treated with estradiol (20 micrograms EB/day for 3 days), no PR-induction was observed in the cytosol of sciatic nerves. In contrast, PR-binding sites in cytosols prepared from pituitary gland and uteri of the same animals were significantly increased by the estrogen.

Kinetic analysis of the binding of nomegestrol acetate to the progesterone receptors in rat uterus by competition studies.

The characteristics of binding (Kinetic and equilibrium binding analysis) of nomegestrol acetate (NOM, 17 alpha-acetoxy-6 alpha-methyl-19-nor-pregna-4.6-diene-3.20-dione) to the progesterone receptor (PgR) in rat uterine cytosolic fraction were determined in comparison to progesterone (P), to fully appreciate the amplitude and specificity of the induced biological response. Since an appropriate radio-labelled form of this steroid molecule was not available, competition studies were performed against the synthetic progestin: [3H]-Organon 2058 [( 3H]-ORG). This allowed a direct comparison between the unlabelled forms of NOM and P, the kinetic constants of which were respectively: Inhibition constant (Ki): 22.8 and 34.3 nM; Association rate constant (k1): 0.39 X 10(3) and 0.21 X 10(3) M-1.s-1; Dissociation rate constant (k-1): 1.81 X 10(-5) and 2.16 X 10(-5) s-1. These results are much more informative than the mere determination of relative binding affinities which only reflect the specificity of the PgR. It was concluded that NOM behaves like the natural hormone in the cytosol of rat uterus.

In vitro evaluation of estrogenic, estrogen antagonistic and progestagenic effects of a steroidal drug (Org OD-14) and its metabolites on human endometrium.

The human endometrial model for in vitro evaluation of estrogenic, estrogen antagonistic, and progestagenic effects of endogenous steroids, natural products or synthetic drugs was applied to the study of Org OD-14, an analog of norethynodrel developed by Organon International, Oss, The Netherlands, and some of its metabolites. Estrogen antagonistic actions of Org OD-14 and its 4-ene isomer were evident from their ability to suppress the enhancement of PGF2 alpha output elicited by estradiol on fragments of secretory endometrium and to decrease the rate of output of the prostaglandin by proliferative tissue, already stimulated by endogenous estrogens. These inhibitory effects were similar to those obtained with progesterone and do not appear to involve competition for the estrogen receptor since the antiestrogen 4-hydroxyamoxifen was not active in parallel incubations of proliferative endometrium. The progestagenic effects of Org OD-14 and its 4-ene isomer were also evident from their capability to enhance estradiol 17 beta-dehydrogenase activity and glycogen accumulation in specimens of proliferative endometrium. Estrogenic effects of the 3 alpha- and 3 beta-hydroxy metabolites of Org OD-14 were demonstrated by their stimulatory actions on PGF2 alpha output during incubations of secretory endometrium. The estrogenic and progestagenic actions of these compounds are in general agreement with their relative affinity for binding to the estradiol and progesterone receptors, although their actions may be influenced by intracellular metabolism in the endometrial tissue. For instance, the similarity in progestagenic activity of Org OD-14 and the 4-ene isomer, contrasting with their different affinities for the progesterone receptor, may result from in situ isomerization of Org OD-14 to the 4-ene metabolite.

Generation of C127-derived cell lines expressing estrogen or progesterone receptors for studying gene regulation on bovine papilloma virus minichromosomes.

To study regulation of transcription by multiple steroid hormones we have stably introduced expression vectors for human estrogen and rabbit progesterone receptors into the genome of the murine fibroblast cell line C127. These cells express functional endogenous glucocorticoid receptor and support bovine papilloma virus minichromosomes, a useful system for studying the role of chromatin structure on gene expression. Three clones containing progesterone receptor integrates and six containing estrogen receptors integrates were selected and characterized. All three progesterone and four of the estrogen receptors containing cell lines expressed functional receptors that were able to transactivate transcription from a mouse mammary tumor virus and Xenopus vitellogenin promoter, respectively, in steroid-specific manner. Levels of steroid binding varied between 38 and 890 fmol/mg protein for progesterone receptor, and between 22 and 94 fmol/mg protein for estrogen receptor. The observed dissociation constants of 1.8-2.5 nM (Organon.2058) and 0.75-2.8 nM (17 beta-Estradiol) are consistent with previously reported values for wild type rabbit progesterone receptor and the estrogen receptor derivative employed. Finally, we demonstrate that transcription of a mouse mammary tumor virus construct in a novel nontransforming bovine papilloma virus vector is regulated by both progestin and glucocorticoid agonists in line C127PR9.