Presence Of RU486 Research Articles

Cortisol/glucocorticoid receptor: a critical mediator of the ovulatory process and luteinization in human periovulatory follicles.

Do cortisol/glucocorticoid receptors play an active role in the human ovary during ovulation and early luteinization? The ovulatory hCG stimulation-induced glucocorticoid receptor signaling plays a crucial role in regulating steroidogenesis and ovulatory cascade in human periovulatory follicles. Previous studies reported an increase in cortisol levels in the human follicular fluid after the LH surge or ovulatory hCG administration. However, little is known about the role of cortisol/glucocorticoid receptors in the ovulatory process and luteinization in humans. This study was an experimental prospective clinical and laboratory-based study. An in vivo experimental study was accomplished utilizing the dominant ovarian follicles from 38 premenopausal women undergoing laparoscopic sterilization. An in vitro experimental study was completed using the primary human granulosa/lutein cells (hGLC) from 26 premenopausal women undergoing IVF. This study was conducted in a private fertility clinic and academic medical centers. Dominant ovarian follicles were collected before the LH surge and at defined times after hCG administration from women undergoing laparoscopic sterilization. Primary hGLC were collected from women undergoing IVF. hGLC were treated without or with hCG in the absence or presence of RU486 (20 µM; dual antagonist for progesterone receptor and glucocorticoid receptor) or CORT125281 (50 µM; selective glucocorticoid receptor antagonist) for 12 or 36 h. The expression of genes involved in glucocorticoid receptor signaling, steroidogenesis, and ovulatory cascade was studied with RT-quantitative PCR and western blotting. The production of cortisol, corticosterone, and progesterone was assessed by hormone assay kits. hCG administration upregulated the expression of hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1), nuclear receptor subfamily 3 group C member 1 (NR3C1), FKBP prolyl isomerase 5 (FKBP5), and FKBP prolyl isomerase 4 (FKBP4) in human ovulatory follicles and in hGLC (P < 0.05). RU486 and CORT125281 reduced hCG-induced increases in progesterone and cortisol production in hGLC. The expression of genes involved in glucocorticoid receptor signaling, steroidogenesis, and the key ovulatory process was reduced by RU486 and/or CORT125281 in hGLC. N/A. The role of cortisol/glucocorticoid receptors demonstrated using the hGLC model may not fully reflect their physiological roles in vivo. Successful ovulation and luteinization are essential for female fertility. Women with dysregulated cortisol levels often suffer from anovulatory infertility. Deciphering the functional role of glucocorticoid receptor signaling in human periovulatory follicles enhances our knowledge of basic ovarian physiology and may provide therapeutic insights into treating infertility in women. This study was supported by P01HD71875 (to M.J., T.E.C., and M.B.) and R01HD096077 (to M.J.) from the Foundation for the National Institutes of Health and the BTPSRF of the University of Kentucky Markey Cancer Center (P30CA177558). The authors report no competing interests. N/A.

Prunetin Protects Against Dexamethasone-Induced Pancreatic Β-Cell Apoptosis via Modulation of p53 Signaling Pathway

Long-term administration of dexamethasone results in insulin resistance and pancreatic β-cell apoptosis. Prunetin (an O-methylated isoflavone, a type of flavonoid) is demonstrated to protect diabetes, but the molecular mechanism of this protection is still unclear. This study thus aims to investigate how prunetin protects against dexamethasone-induced pancreatic β-cell apoptosis. Rat insulinoma (INS-1) cells were cultured in medium with or without dexamethasone in the presence or absence of prunetin or pifithrin-α, a p53 inhibitor. Cell apoptosis was measured by Annexin V/propidium iodide staining. Dexamethasone significantly induced INS-1 apoptosis but dexamethasone plus prunetin significantly reduced INS-1 apoptosis. Dexamethasone-treated INS-1 upregulated p53 protein expression; the induction of p53 was also reduced in the presence of RU486, a glucocorticoid receptor (GR) inhibitor. This suggested that dexamethasone induced P53 via GR. Dexamethasone-treated INS-1 significantly increased p53, Bax, and Rb protein expressions, whereas treatments of dexamethasone plus prunetin or pifithrin-α significantly decreased these protein expressions. In addition, dexamethasone significantly decreased B-cell lymphoma 2 (Bcl2), while dexamethasone plus prunetin or pifithrin-α significantly increased Bcl2. Dexamethasone significantly increased caspase-3 activity while co-treatment of dexamethasone plus prunetin or pifithrin-α significantly decreased caspase-3 activity to the control level. Taken together, our results revealed that prunetin protected against dexamethasone-induced pancreatic β-cells apoptosis via modulation of the p53 signaling pathway.

Probing Dominant Negative Behavior of Glucocorticoid Receptor β through a Hybrid Structural and Biochemical Approach.

Glucocorticoid receptor β (GRβ) is associated with glucocorticoid resistance via dominant negative regulation of GRα. To better understand how GRβ functions as a dominant negative inhibitor of GRα at a molecular level, we determined the crystal structure of the ligand binding domain of GRβ complexed with the antagonist RU-486. The structure reveals that GRβ binds RU-486 in the same ligand binding pocket as GRα, and the unique C-terminal amino acids of GRβ are mostly disordered. Binding energy analysis suggests that these C-terminal residues of GRβ do not contribute to RU-486 binding. Intriguingly, the GRβ/RU-486 complex binds corepressor peptide with affinity similar to that of a GRα/RU-486 complex, despite the lack of helix 12. Our biophysical and biochemical analyses reveal that in the presence of RU-486, GRβ is found in a conformation that favors corepressor binding, potentially antagonizing GRα function. This study thus presents an unexpected molecular mechanism by which GRβ could repress transcription.

Dexamethasone induced miR-155 up-regulation in differentiating 3T3-L1 preadipocytes does not affect adipogenesis

Dexamethasone is a synthetic glucocorticoid that is widely used as an adipogenic inducer in both murine and human in vitro models. Glucocorticoids have been shown to regulate early transcriptional events in adipogenesis. MicroRNAs (miRNAs) have been also implicated in the regulation of preadipocyte differentiation; however, the effects of glucocorticoids on miRNA expression levels during this process have not been studied. In this study we investigated the effects of glucocorticoids on the expression levels of miR-155 in differentiating 3T3-L1 preadipocytes. We found that miR-155 levels were up-regulated (2.4-fold) by glucocorticoids in differentiating 3T3-L1 preadipocytes, and this enhancement was abolished in the presence of RU486, a glucocorticoid receptor antagonist. In contrast, treatment with rosiglitazone, another adipogenic inducer decreased the expression levels of miR-155 in these cells. Further, our data show that endogenous miR-155 is unlikely to be involved in adipogenesis as we show that both dexamethasone and rosiglitazone induced adipogenesis to similar levels. Furthermore, using miR-155 inhibitor, we showed that the dexamethasone mediated miR-155 enhancement did not alter adipogenesis. Our data show that dexamethasone but not rosiglitazone increases miR-155 expression and that the increased expression of miR-155 is not involved in the dexamethasone-mediated adipogenesis in the 3T3-L1 model.

HCG activates Epac-Erk1/2 signaling regulating Progesterone Receptor expression and function in human endometrial stromal cells.

How does hCG signal in human endometrial stromal cells (ESCs) and what is its role in regulating ESC function? hCG signaling in ESCs activates the extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) pathway through exchange protein activated by cyclic AMP (cAMP) (Epac) and transiently increases progesterone receptor (PR) transcript and protein expression and its transcriptional function. hCG is one of the earliest embryo-derived secreted signals in the endometrium, which abundantly expresses LH/hCG receptors. hCG signals through cAMP/protein kinase A (PKA) in gonadal cells, but in endometrial epithelial cells, hCG induces Erk1/2 activation independent of the cAMP/PKA pathway. Few data exist concerning the signal transduction pathways triggered by hCG in ESCs and their role in regulation of ESC function. This is an in vitro study comprising patients undergoing benign gynecological surgery (n = 46). Endometrial samples were collected from normal cycling women during the mid-secretory phase for ESCs isolation. The study conducted in an academic research laboratory within a tertiary-care hospital. The activation of the Erk1/2 signal transduction pathway elicited by hCG was evaluated in ESC. Signaling pathway inhibitors were used to examine the roles of PKA, PI3K, PKC, adenylyl cyclase and Epac on the hCG-stimulated up-regulation of phospho-Erk1/2 (pErk1/2). Erk1/2 phosphorylation was determined by immunoblot. siRNA targeting Epac was used to investigate the molecular mechanisms. To assess the role of Erk1/2 signaling induced by hCG on ESC function, gene expression regulation was examined by immunofluorescence and real-time quantitative PCR. The role of PR on the regulation of transcript levels was studied using progesterone and the PR antagonist RU486. All experiments were conducted using at least three different cell culture preparations in triplicate. Addition of hCG to ESCs in vitro induced the phosphorylation of Erk1/2 through cAMP accumulation. Such induction could not be blocked by inhibitors for PKA, PKC and PI3K. Epac inhibition and knockdown with siRNA prevented pErk1/2 induction by hCG. ESCs stimulated with hCG for up to 72 h showed a significant increase in PR mRNA and immunofluorescent label at 48 h only; an effect that was abrogated with the mitogen-activated protein kinase kinase inhibitor UO126. In addition, the hCG-activated Erk1/2 pathway significantly decreased the mRNA levels for secreted frizzled-related protein 4 (SFRP4) at 24 h, whereas it increased those for homeobox A10 (HOXA10) at 48 h (P = 0.041 and P = 0.022 versus control, respectively). Prolactin mRNA levels were not significantly modified. HOXA10 mRNA up-regulation by hCG was not enhanced by co-stimulation with progesterone; however, it was completely abolished in the presence of RU486 (P = 0.036 hCG versus hCG + RU486). N/A. This is an in vitro study utilizing stromal cell cultures from human endometrial tissues. Furthermore, results obtained should also be confirmed in vivo in the context of the whole human endometrial tissue and hormonal milieu. The in vitro experiments using hCG have been conducted without other hormones/factors that may also modulate the ESCs response to hCG. We have determined that hCG induces the PR through the Erk1/2 pathway in ESCs which may render them more sensitive to progesterone, increasing our understanding about the effects of hCG at the embryo-maternal interface. The activation of such a pathway in the context of the hormonal milieu during the window of implantation might contribute to a successful dialog between the embryo and the uterus, leading to appropriate endometrial function. Defective hCG signaling in the endometrial stromal tissue may lead to an incomplete uterine response, compromising embryo implantation and early pregnancy. This work was supported by the National Fund for Scientific and Technological Development, Government of Chile (FONDECYT) grants 11100443 and 1140614 (A.T.-P.). The authors have no conflicts of interest to declare.

A novel dissociative steroid VBP15 reduces MUC5AC gene expression in airway epithelial cells but lacks the GRE mediated transcriptional properties of dexamethasone

Overproduction of secretory mucins contributes to morbidity/mortality in inflammatory lung diseases. Inflammatory mediators directly increase expression of mucin genes, but few drugs have been shown to directly repress mucin gene expression. IL-1β upregulates the MUC5AC mucin gene in part via the transcription factors NFκB while the glucocorticoid Dexamethasone (Dex) transcriptionally represses MUC5AC expression by Dex-activated GR binding to two GRE cis-sites in the MUC5AC promoter in lung epithelial cells. VBP compounds (ReveraGen BioPharma) maintain anti-inflammatory activity through inhibition of NFκB but exhibit reduced GRE-mediated transcriptional properties associated with adverse side-effects and thus have potential to minimize harmful side effects of long-term steroid therapy in inflammatory lung diseases. We investigated VBP15 efficacy as an anti-mucin agent in two types of airway epithelial cells and analyzed the transcription factor activity and promoter binding associated with VBP15-induced MUC5AC repression. VBP15 reduced MUC5AC mRNA abundance in a dose- and time-dependent manner similar to Dex in the presence or absence of IL-1β in A549 and differentiated human bronchial epithelial cells. Repression was abrogated in the presence of RU486, demonstrating a requirement for GR in the VBP15-induced repression of MUC5AC. Inhibition of NFκB activity resulted in reduced baseline expression of MUC5AC indicating that constitutive activity maintains MUC5AC production. Chromatin immunoprecipitation analysis demonstrated lack of GR and of p65 (NFκB) binding to composite GRE domains in the MUC5AC promoter following VBP15 exposure of cells, in contrast to Dex. These data demonstrate that VBP15 is a novel anti-mucin agent that mediates the reduction of MUC5AC gene expression differently than the classical glucocorticoid, Dex.

Downregulation of sphingosine 1-phosphate (S1P) receptor 1 by dexamethasone inhibits S1P-induced mesangial cell migration.

Sphingosine 1-phosphate (S1P) is generated by sphingosine kinase (SK)-1 and -2 and acts mainly as an extracellular ligand at five specific receptors, denoted S1P1-5. After activation, S1P receptors regulate important processes in the progression of renal diseases, such as mesangial cell migration and survival. Previously, we showed that dexamethasone enhances SK-1 activity and S1P formation, which protected mesangial cells from stress-induced apoptosis. Here we demonstrate that dexamethasone treatment lowered S1P1 mRNA and protein expression levels in rat mesangial cells. This effect was abolished in the presence of the glucocorticoid receptor antagonist RU-486. In addition, in vivo studies showed that dexamethasone downregulated S1P1 expression in glomeruli isolated from mice treated with dexamethasone (10 mg/kg body weight). Functionally, we identified S1P1 as a key player mediating S1P-induced mesangial cell migration. We show that dexamethasone treatment significantly lowered S1P-induced migration of mesangial cells, which was again reversed in the presence of RU-486. In summary, we suggest that dexamethasone inhibits S1P-induced mesangial cell migration via downregulation of S1P1. Overall, these results demonstrate that dexamethasone has functional important effects on sphingolipid metabolism and action in renal mesangial cells.

Effects of RU486 and indomethacin on meiotic maturation, formation of extracellular matrix, and progesterone production by porcine oocyte-cumulus complexes

This study was designed to determine whether inhibition of either cyclooxygenase-2 (COX-2) by indomethacin or progesterone receptor (PR) by PR antagonist, RU486, affects oocyte maturation, progesterone production, and covalent binding between hyaluronan (HA) and heavy chains of inter-α trypsin inhibitor, as well as expression of cumulus expansion-associated proteins (HA-binding protein, tumor necrosis factor α-induced protein 6, pentraxin 3) in oocyte-cumulus complexes (OCCs). The experiments were based on freshly isolated porcine OCC cultures in which the consequences of PR and COX-2 inhibition on the final processes of oocyte maturation were determined. Granulosa cells (GCs) and OCCs were cultured in medium supplemented with FSH/LH (both 100 ng/mL) in the presence/absence of RU486 or indomethacin. Western blot analysis, 3H-glucosamine hydrochloride assay, immunofluorescence, and radioimmunoassay were performed. Only treatment with RU486 (25 μM) caused a decrease in the number of oocytes that reached germinal vesicle breakdown and metaphase II stage compared with indomethacin (100 μM) or FSH/LH treatment alone after 44 h. All treated OCCs synthesized an almost equal amount of HA. Heavy chains (of inter-α trypsin inhibitor)-HA covalent complexes were formed during in vitro FSH/LH-stimulated expansion in RU486- or indomethacin-treated OCCs. Follicle-stimulating hormone/LH-induced progesterone production by OCCs was increased in the presence of RU486 after 44 h. In contrast, a decrease of FSH/LH-stimulated progesterone production by GCs was detected in the presence of either RU486 or indomethacin after 72 h. We suggest that the PR-dependent pathway may be involved in the regulation of oocyte maturation. Both PR and COX-2 regulate FSH/LH-stimulated progesterone production by OCCs and GCs.

Glucocorticoid dexamethasone regulates the differentiation of mouse conducting airway epithelial progenitor cells

Inhaled glucocorticoid dexamethasone is the most effective treatment of asthma currently available. Epithelial damage and shedding represents a clear manifestation of asthmatic pathologies. However it remains unknown if dexamethasone regulates functions of airway progenitor cells that are responsible for epithelial repair. In present study Secretoglobin1a1 (Scgb1a1) lineage tracing mice were injected intraperitoneally with tamoxifen to induce the expression of green fluorescence protein (GFP) in Scgb1a1-expressing conducting airway progenitor cells. Scgb1a1-expressing progenitor cells were isolated from lungs of Scgb1a1 lineage tracing mice via flow activated cell sorting. In vitro three-dimensional matrigel culture of these progenitor cells revealed that dexamethasone has little effect on the colony forming ability of airway epithelial progenitor cells, but exhibits significant effects on the differentiation of the progenitor cells. Compared to the untreated group, dexamethasone treatment inhibited the expression of forkhead box J1 (FoxJ1) and mucin subtype A & C (Muc5Ac), but promoted the expression of calcium activated chloride channel 3 (Clca3) and cystic fibrosis transmembrane conductance regulator (Cftr). Dexamethasone-induced effects on the expression of FoxJ1, Muc5Ac and Clca3 were abolished or even reversed in the presence of RU486, an antagonist of glucocorticoid receptor, indicating that glucocorticoid receptor plays a role in the regulation of airway epithelial progenitor cells by dexamethasone. These data suggested that, though effective to reduce airway inflammation, dexamethasone treatment alone fails to fully restore the mucociliary clearance function in the treatment of asthma patients.

Essential roles of endogenous glucocorticoids and TNF/TNFR1 in promoting bone-marrow eosinopoiesis in ovalbumin-sensitized, airway-challenged mice

AimsStress mechanisms paradoxically contribute to allergic episodes in humans and mice. Glucocorticoids (GC) and interleukin (IL)-5 synergically upregulate murine bone-marrow eosinophil production. Here we explored the role of endogenous GC in allergen-stimulated bone-marrow eosinophil production in ovalbumin-sensitized/challenged mice. Main methodsIn BALB/c or C57BL/6 mice, sensitized and intranasally challenged with ovalbumin, we monitored eosinophil numbers in freshly harvested or cultured bone-marrow, and plasma corticosterone levels. Metyrapone (MET) was used to inhibit GC synthesis, and RU486 to block GC actions. In sensitized mice challenged intraperitoneally, we examined the relationship between eosinophilia of bone-marrow and peritoneal cavity, in the absence or presence of RU486. In experiments involving in vivo neutralization of tumor necrosis factor-α (TNF) by specific antibodies, or using mice which lack functional type I TNF receptors (TNFRI), we evaluated the relationship between TNF blockade, corticosterone levels, RU486 or MET treatment and challenge-induced bone-marrow eosinophilia. Key findingsRU486 or MET pretreatments abolished challenge-induced increases in eosinophil numbers in bone-marrow (in vivo and ex vivo), and in the peritoneal cavity. MET, but not RU486, prevented the challenge-induced increase in corticosterone levels. Challenge-induced bone-marrow eosinophilia and corticosterone surge were abolished in TNFRI-deficient mice. Anti-TNF-treatment very effectively prevented challenge-induced bone-marrow eosinophilia, in the absence of RU486 or MET, but had no independent effect in the presence of either drug. SignificanceEndogenous GC was essential for allergen challenge-induced increases in eosinophil numbers inside bone-marrow. This effect required TNF and TNFRI, which suggests an immunoendocrine mechanism.

58 STAGE–SPECIFIC EFFECTS OF THE PROGESTERONE RECEPTOR ANTAGONIST, RU486, ON EARLY BOVINE EMBRYO DEVELOPMENT

Progesterone plays a key role in the reproductive events associated with pregnancy establishment and maintenance. High concentrations of circulating progesterone in the immediate post-conception period are associated with an advancement of conceptus elongation, an associated increase in interferon-tau production and higher pregnancy rates in cattle. Progesterone-induced changes in the uterine environment are thought to be responsible for the reported advancement in conceptus elongation; however, the function of the progesterone receptor in embryos is not known. Therefore, the aim of the current study was to examine the effect of adding a progesterone receptor antagonist (mifepristone, RU486) at various stages of early embryonic development and at varying concentrations to examine the effects on subsequent embryo development in vitro. Bovine zygotes (n = 2902), 2-cell (n = 1991) and 8-cell (n = 1244) embryos, derived by in vitro maturation and fertilization, were cultured in synthetic oviduct fluid medium in the absence or presence of RU486 at concentrations ranging from 0.0004 to 20 μg mL–1. Cleavage rate (of zygotes), 8-cell development rate (of 2-cell embryos) and development to the blastocyst stage (for all cell stages) were recorded at Day 2, 3 and 8 post-insemination (day of IVF = Day 0), respectively. Cultures of zygotes in the presence of RU486 at concentrations of 0.004 and 0.04 μg mL–1 resulted in a decline in cleavage rate (62.5 ± 2.55% and 48.8 ± 5.07% for respective treatments vs controls without RU486 81.9 ± 5.97%; P ≤ 0.05). These same concentrations resulted in a significant decline in blastocyst development on Day 8 (18.8 ± 1.82% and 17.4 ± 4.85% for respective treatments compared to controls 35.1 ± 4.89%; P ≤ 0.05). Cultures at concentrations of 0.4 μg mL–1 resulted in a 10-fold decrease in blastocyst development (3.3 ± 1.3%; P ≤ 0.05) and concentrations in excess of 10 μg mL–1 completely ablated blastocyst development (P ≤ 0.05). Cultures of 2-cell embryos with RU486 at concentrations below 8 μg mL–1 had no effect on 8-cell rate or blastocyst development. However, cultures with RU486 at 10 μg mL–1 resulted in a significant decline in the proportion reaching the 8-cell stage (59.1 ± 4.59% vs 38.1 ± 2.13% for control and treated, respectively) and developing to the blastocyst stage (32.8 ± 4.68% vs 17.8 ± 3.77% for control and treated, respectively; P ≤ 0.05). Cultures with RU486 at a concentration of 20 μg mL–1 resulted in a dramatic effect in 8-cell rate (16.3 ± 2.55%; P ≤ 0.05) and prevented blastocyst development. Similarly, cultures of 8-cell embryos with RU486 at concentrations at or below 10 μg mL–1 had no effect on blastocyst development. However, cultures at concentrations of 15 or 20 μg mL–1 resulted in no blastocyst development. In conclusion, addition of the progesterone and glucocorticoid receptor antagonist RU486 to culture media has a clear stage-specific and concentration-dependent effect on bovine embryo development, which is more pronounced at earlier developmental stages. Supported by Science Foundation Ireland (07/SRC/B1156).

In vitro effects of RU486 on proliferation and differentiation capabilities of human bone marrow mesenchymal stromal cells

Although exogenous glucocorticoids (GC) play a role in the regulation of bone marrow mesenchymal stem/stromal cells (MSCs) proliferation and differentiation, the function of endogenous GC is not well understood. The purpose of this study was to investigate the effect of the blockage of endogenous GC using RU486, an antagonist of the glucocorticoid receptor, on the in vitro proliferation and differentiation capabilities of human MSCs. We quantitatively measured cell proliferation of human MSCs after treatment with increasing concentrations of RU486. We also evaluated multiple MSC differentiation capabilities, as well as the expression of stemness and senescence genes after proliferation of these human cells in vitro in the presence of RU486 at 10 −8 M. It was observed that RU486 treatment significantly increases the proliferation of human MSCs, although the optimal dose of RU486 for this increase in proliferation differs depending on the gender of the MSC donor. This improvement in MSC proliferation with RU486 treatment was higher in MSCs from male donors than that from females. No effect of RU486 on MSC proliferation was observed in a steroid-free medium. RU486 pretreatment significantly increased the expression of mRNA for alkaline phosphatase in human MSCs and the mRNA expression of osteocalcin of these cells up-regulated earlier after their exposure to osteogenic differentiation medium. Although no statistical significance in terms of chondrogenic differentiation markers was detected, mRNA expression for aggrecan and collagen type 2 were higher in a majority of the RU486-pretreated donor MSCs than their untreated controls. No significant difference in terms of MSC adipogenic differentiation capabilities were observed after RU486 treatment. RU486 treatment up-regulated the expressions of FGF-2 and Sox-11 in human MSCs. These results indicate that blockage of endogenous GCs may be developed as a novel approach to effectively improve the proliferation and osteochondral differentiation capabilities of human MSCs for potential clinical applications. Additional studies will be required to determine the potential long-term effects of RU486 treatment on these bone marrow cells.

Effect of the steroid receptor antagonist RU486 (mifepristone) on an IFNγ-induced persistent Chlamydophila pneumoniae infection model in epithelial HEp-2 cells

We have previously demonstrated that the steroid receptor antagonist mifepristone (RU486) causes growth inhibition of Chlamydophila pneumoniae by binding to and subsequently destroying the bacteria during their normal developmental cycle in epithelial HEp-2 cells. In the present study, we assessed the efficacy of treatment with RU486 against persistent C. pneumoniae infection in interferon (IFN)γ-treated HEp-2 cells. Assessment of bacterial growth modification, the number of infectious progenies, the formation of inclusions, and the expressions of the C. pneumoniae genes 16S rRNA and hsp60 were investigated in cells with or without IFNγ stimulation in the presence of RU486, using an inclusion-forming unit (IFU) assay, fluorescence microscopic analysis, and reverse transcription polymerase chain reaction (RT-PCR), respectively. Our results indicated that RU486 treatment produced growth inhibition and an absence of C. pneumoniae gene expression in normal HEp-2 cells and that this treatment failed to inhibit C. pneumoniae growth in HEp-2 cells stimulated with IFNγ. These results indicate that treatment with RU486 had a limited effect on C. pneumoniae growth only during the active developmental stage of the bacteria, suggesting that the bacterial target molecule of RU486 is not expressed sufficiently during persistent infection in which there is an aberrant developmental cycle. Thus, our findings provide valuable insight into the complicated chlamydial biological processes involved in the recurrent cycling between normal and persistent infections.

Dexamethasone Suppresses Parathyroid Hormone-related Protein Expression by Human Oral Squamous Carcinoma Cells (HSC-3)

We examined the effect of dexamethasone on parathyroid hormone-related protein (PTHrP) mRNA expression and PTHrP production in HSC-3 cells, a human oral squamous carcinoma cell line. Dexamethasone suppressed PTHrP mRNA expression and PTHrP production in a dose-dependent manner. A time course study demonstrated a significant decrease in PTHrP mRNA from as early as 6 h after the initiation of exposure to 10−1 M dexamethasone, and the suppressive effect was stronger at 24 h. Dexamethasone-induced suppression of PTHrP mRNA expression and PTHrP production was abolished in the presence of RU486, a glucocorticoid receptor antagonist. Dexamethasone decreased the transcriptional activity of the PTHrP promoter. These findings show that dexamethasone acts through binding to functional glucocorticoid receptors and down-regulates PTHrP gene expression via a transcriptional mechanism in HSC-3 cells.

Identification of the functional domain of glucocorticoid receptor involved in RU486 antagonism

Mifepristone, also known as RU486, is a potent glucocorticoid receptor (GR) antagonist that inhibits GR-mediated transactivation. As an alternative to existing antidepressants, RU486 has been shown to rapidly reverse psychotic depression, most likely by blocking GR. Although a number of studies have demonstrated RU486-induced GR antagonism, the precise mechanism of action still remains unclear. To identify the GR domain involved in RU486-induced suppression, GR transactivation and nuclear translocation were examined using cells transfected with human GR (hGR), Guyanese squirrel monkey GR (gsmGR), and GR chimeras into COS-1 cells. RU486 showed a much more potent suppressive effect in gsmGR-expressing cells versus hGR-expressing cells, without significant cortisol- or RU486-induced changes in nuclear translocation. A GR chimera containing the gsmGR AF1 domain (amino acids 132–428) showed a marked decrease in luciferase activity, suggesting that this domain plays an important role in RU486-induced GR antagonism. Furthermore, fluorescence recovery after photobleaching (FRAP) analysis indicated that, in the presence of RU486, gsmGR AF1 domain contributes to GR mobility in living COS-1 cells. Taken together, these results demonstrate, for the first time, that the antagonistic effects of RU486 on GR transactivation involve a specific GR domain.

The Paradoxical Increase in Cortisol Secretion Induced by Dexamethasone in Primary Pigmented Nodular Adrenocortical Disease Involves a Glucocorticoid Receptor-Mediated Effect of Dexamethasone on Protein Kinase A Catalytic Subunits

Context: Primary pigmented nodular adrenocortical disease (PPNAD) results in most cases from mutations of the protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene. Patients with PPNAD exhibit a paradoxical increase in cortisol secretion in response to dexamethasone. Objective: The aim was to investigate the mechanism of the action of dexamethasone on adrenocortical cells removed from patients with PPNAD and a transgenic model of PPNAD [Tg(tTA/X2AS) mice]. Design and Setting: We performed an in vitro study in an academic research laboratory. Patients: Eleven patients with histologically proven PPNAD were included in the study. Intervention: Cultured PPNAD cells were incubated with dexamethasone in the presence of various modulators of the cAMP/PKA pathway and the glucocorticoid receptor antagonist RU486. Main Outcome Measure: Cortisol and corticosterone were measured by radioimmunological assays in cell culture supernatants. Results: Dexamethasone stimulated in vitro cortisol secretion from PPNAD tissues in six patients. The stimulatory effect of dexamethasone on cortisol release was not reduced by the adenylyl cyclase inhibitor SQ22536 or potentiated by the phosphodiesterase inhibitor IMBX and the cAMP analog 8Br-cAMP. Conversely, the PKA inhibitor H89 and RU486 inhibited the cortisol response to dexamethasone. Dexamethasone had no effect on cortisol production from normal human adrenocortical cells but stimulated corticosteroidogenesis in the presence of RU486. Similarly, dexamethasone failed to influence corticosterone release by adrenocortical cells removed from Tg(tTA/X2AS) mice but stimulated corticosteroidogenesis in the presence of RU 486. Conclusions: These results indicate that, in human PPNAD tissues, dexamethasone paradoxically stimulates cortisol release through a glucocorticoid receptor-mediated effect on PKA catalytic subunits. Mol Endocrinol, June 2009, 23(6):943–945 mend.endojournals.org 945

The Paradoxical Increase in Cortisol Secretion Induced by Dexamethasone in Primary Pigmented Nodular Adrenocortical Disease Involves a Glucocorticoid Receptor-Mediated Effect of Dexamethasone on Protein Kinase A Catalytic Subunits

Primary pigmented nodular adrenocortical disease (PPNAD) results in most cases from mutations of the protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene. Patients with PPNAD exhibit a paradoxical increase in cortisol secretion in response to dexamethasone. The aim was to investigate the mechanism of the action of dexamethasone on adrenocortical cells removed from patients with PPNAD and a transgenic model of PPNAD [Tg(tTA/X2AS) mice]. We performed an in vitro study in an academic research laboratory. Eleven patients with histologically proven PPNAD were included in the study. Cultured PPNAD cells were incubated with dexamethasone in the presence of various modulators of the cAMP/PKA pathway and the glucocorticoid receptor antagonist RU486. Cortisol and corticosterone were measured by radioimmunological assays in cell culture supernatants. Dexamethasone stimulated in vitro cortisol secretion from PPNAD tissues in six patients. The stimulatory effect of dexamethasone on cortisol release was not reduced by the adenylyl cyclase inhibitor SQ22536 or potentiated by the phosphodiesterase inhibitor IMBX and the cAMP analog 8Br-cAMP. Conversely, the PKA inhibitor H89 and RU486 inhibited the cortisol response to dexamethasone. Dexamethasone had no effect on cortisol production from normal human adrenocortical cells but stimulated corticosteroidogenesis in the presence of RU486. Similarly, dexamethasone failed to influence corticosterone release by adrenocortical cells removed from Tg(tTA/X2AS) mice but stimulated corticosteroidogenesis in the presence of RU 486. These results indicate that, in human PPNAD tissues, dexamethasone paradoxically stimulates cortisol release through a glucocorticoid receptor-mediated effect on PKA catalytic subunits.

The Paradoxical Increase in Cortisol Secretion Induced by Dexamethasone in Primary Pigmented Nodular Adrenocortical Disease Involves a Glucocorticoid Receptor-Mediated Effect of Dexamethasone on Protein Kinase A Catalytic Subunits

Primary pigmented nodular adrenocortical disease (PPNAD) results in most cases from mutations of the protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene. Patients with PPNAD exhibit a paradoxical increase in cortisol secretion in response to dexamethasone. The aim was to investigate the mechanism of the action of dexamethasone on adrenocortical cells removed from patients with PPNAD and a transgenic model of PPNAD [Tg(tTA/X2AS) mice]. We performed an in vitro study in an academic research laboratory. Eleven patients with histologically proven PPNAD were included in the study. Cultured PPNAD cells were incubated with dexamethasone in the presence of various modulators of the cAMP/PKA pathway and the glucocorticoid receptor antagonist RU486. Cortisol and corticosterone were measured by radioimmunological assays in cell culture supernatants. Dexamethasone stimulated in vitro cortisol secretion from PPNAD tissues in six patients. The stimulatory effect of dexamethasone on cortisol release was not reduced by the adenylyl cyclase inhibitor SQ22536 or potentiated by the phosphodiesterase inhibitor IMBX and the cAMP analog 8Br-cAMP. Conversely, the PKA inhibitor H89 and RU486 inhibited the cortisol response to dexamethasone. Dexamethasone had no effect on cortisol production from normal human adrenocortical cells but stimulated corticosteroidogenesis in the presence of RU486. Similarly, dexamethasone failed to influence corticosterone release by adrenocortical cells removed from Tg(tTA/X2AS) mice but stimulated corticosteroidogenesis in the presence of RU 486. These results indicate that, in human PPNAD tissues, dexamethasone paradoxically stimulates cortisol release through a glucocorticoid receptor-mediated effect on PKA catalytic subunits.

Hepatic transcriptome response to glucocorticoid receptor activation in rainbow trout

Cortisol, the principal corticosteroid in teleosts, is thought to play a key role in the metabolic adjustments critical for regaining homeostasis. However, the target tissue molecular mechanisms involved in this adaptive response to corticosteroid stimulation are still unclear. Cortisol signaling is mediated predominantly by the glucocorticoid receptor (GR), and previous studies have shown that RU486 (a GR antagonist) offsets corticosteroid signaling in teleosts. To elucidate the molecular basis of GR-mediated metabolic readjustments, we exposed primary culture of trout hepatocytes in vitro to cortisol (to mimic stressed levels seen in fish), RU486, or a combination of both for 24 h. The gene expression was analyzed using a low-density custom-made rainbow trout cDNA array enriched with endocrine-, metabolic-, and stress-related genes. The microarray results for select genes were further validated using quantitative real-time PCR. Cortisol treatment significantly increased glucose production in hepatocytes, and this response was blocked by RU486, confirming GR-mediated corticosteroid signaling. Cortisol also elevated GR transcript levels, and this response was abolished by RU486, whereas both cortisol and RU486, either alone or in combination, reduced GR protein content in trout hepatocytes. Cortisol treatment significantly modulated the expression of several genes known to be involved in intermediary metabolism, cellular stress response, reproduction, and xenobiotic metabolism. Most of these cortisol-mediated transcript changes were abolished in the presence of RU486, suggesting a key role for GR-specific signaling in this adaptive response. Taken together, our results suggest a key role for genomic cortisol signaling in the liver molecular reprogramming that is critical for coping with stress in fish.

Progesterone-dependent and -independent expression of the multidrug resistance type I gene in porcine granulosa cells

A primary role of plasma membrane P-glycoprotein (P-gp), encoded by multidrug resistance type I (MDR1), is to protect against naturally occurring xenotoxics. Progesterone (P(4)) profoundly influences MDR1 expression in granulosa cells and luteal cells. Here, P(4) regulation of MDR1 expression was investigated in porcine granulosa cells using the P(4)-mediated promoter activity assay and a P4 receptor (PR) antagonist (RU-486). The promoter activity was measured chronologically for 48 h in cells transfected with the PR response element-containing pGL3. LH could stimulate the promoter activity through endogenous P4, with a maximum activity at 5 h. MDR1 mRNA level was highly maintained at 24-36 h. Conversely, exogenous P4 prolonged the promoter activity to further 10 h, and the high level of MDR1 mRNA was maintained even at 48 h. RU-486 completely inhibited the promoter activity, but the level of MDR1 mRNA rapidly increased in the presence of RU-486. The granulosa cells may become susceptible to RU-486 as a xenotoxic to rapidly express MDR1 for protection against it. These results indicate that MDR1 is expressed in porcine granulosa cells through P4-dependent and -independent regulations.