Addition Of RU486 Research Articles

Studies on the contribution of PPAR Gamma to tuberculosis physiopathology.

Tuberculosis (TB) is a major health problem characterized by an immuno-endocrine imbalance: elevated plasma levels of cortisol and pro- and anti-inflammatory mediators, as well as reduced levels of dehydroepiandrosterone. The etiological agent, Mycobacterium tuberculosis (Mtb), is captured by pulmonary macrophages (Mf), whose activation is necessary to cope with the control of Mtb, however, excessive activation of the inflammatory response also leads to tissue damage. Glucocorticoids (GC) are critical elements to counteract the immunoinflammatory reaction, and peroxisome proliferator-activated receptors (PPARs) are also involved in this regard. The primary forms of these receptors are PPARϒ, PPARα, and PPARβ/δ, the former being the most involved in anti-inflammatory responses. In this work, we seek to gain some insight into the contribution of PPARϒ in immuno-endocrine-metabolic interactions by focusing on clinical studies in pulmonary TB patients and in vitro experiments on a Mf cell line. We found that TB patients, at the time of diagnosis, showed increased expression of the PPARϒ transcript in their peripheral blood mononuclear cells, positively associated with circulating cortisol and related to disease severity. Given this background, we investigated the expression of PPARϒ (RT-qPCR) in radiation-killed Mtb-stimulated human Mf. The Mtb stimulation of Mf derived from the human line THP1 significantly increased the expression of PPARϒ, while the activation of this receptor by a specific agonist decreased the expression of pro- and anti-inflammatory cytokines (IL-1β and IL-10). As expected, the addition of GC to stimulated cultures reduced IL-1β production, while cortisol treatment together with the PPARϒ agonist lowered the levels of this proinflammatory cytokine in stimulated cultures. The addition of RU486, a glucocorticoid receptor antagonist, only reversed the inhibition produced by the addition of GC. The current results provide a stimulating background for further analysis of the interconnection between PPARs and steroid hormones in the context of Mtb infection.

Glycyrrhizic acid and 18β-glycyrrhetinic acid recover glucocorticoid resistance via PI3K-induced AP1, CRE and NFAT activation

Glucocorticoids are widely used in the clinical setting as remedies for inflammatory diseases, such as asthma and chronic obstructive pulmonary disease. However, the constant increase in the number of patients suffering from glucocorticoid resistance could present a serious problem for clinicians. In these cases, it may be reasonable to use additional treatments to restore the therapeutic effect of glucocorticoids. Glycyrrhizic acid (GA) and 18β-glycyrrhetinic acid (18βGA) are bioactive compounds in licorice that have been used for thousands of years in traditional Chinese medicine to treat coughs. We showed that GA and 18βGA exhibit potential anti-inflammatory and antioxidant properties. GA and 18βGA induced dual specificity protein phosphatase 1 (DUSP1) expression, and this effect was unchanged by the addition of RU486, a glucocorticoid receptor antagonist. The stimulation of DUSP1 expression by GA and 18βGA occurred via both glucocorticoid receptor (GR) and PI3K signaling, and the simultaneous activation of transcription elements, such as AP1 (activator protein 1), CRE (cAMP response element), GRE (glucocorticoid receptor element) and NFAT (nuclear factor of activated T-cells), was confirmed. Furthermore, we designed an in vitro glucocorticoid resistance model to verify the effects of GA and 18βGA on glucocorticoid resistance that was induced by ROS. The data showed that these two phytochemicals restored glucocorticoid sensitivity by depleting ROS through HO-1 expression. p38 and NO, which are factors that are induced by reactive oxygen species and caused depletion of GR signaling, were inhibited by GA and 18βGA treatment. This phenomenon was considered to be related to the coordinated modulation of GR and PI3K signaling by GA and 18βGA, in conjugation with AP1, CRE, GRE and NFAT activation. This study provides a possible strategy for enhancing the efficacy of glucocorticoids and may improve the prognosis of patients with serious inflammatory diseases.

The neurosteroids, allopregnanolone and progesterone, induce autophagy in cultured astrocytes

Recent studies have suggested that neurosteroids such as pregnenolone, progesterone (PG) and their derivatives, have a role in activating autophagy in addition to diverse other functions. In our previous studies, we demonstrated that cellular free Zn2+ is involved in oxidative stress-induced autophagy and autophagic cell death in astrocytes. In the present study, we examined the possibility that neurosteroids, allopregnanolone (Allo) and PG, also activate autophagy in cultured mouse astrocytes through modulation of intracellular Zn2+.Exposure of astrocytes to 250nM Allo or 500nM PG caused cytosolic vacuoles to appear within a few hours of treatment onset. Live-cell confocal microscopy of astrocytes transfected with red fluorescent protein-conjugated LC3 (RFP-LC3), a marker for autophagic vacuoles (AVs), as well as transmission electron microscopy, revealed that these vacuoles were AVs. In addition, Western blots showed increases in LC3-II levels. Interestingly, mTOR and Akt were concurrently activated, and their blockade further increased LC3-II levels and caused some cell death. These results indicate that co-activation of mTOR and Akt may act to limit neurosteroid-induced autophagy and thus inhibit autophagic cell death. As in other cases of autophagy, cellular Zn2+ levels increased after treatment with neurosteroids. The neurosteroid-induced increase in LC3-II levels was inhibited by addition of the Zn2+ chelator TPEN. Both the increase in LC3-II levels and activation of Akt and mTOR by neurosteroids were all mediated by PG receptors, as the effects were blocked by the addition of RU-486, a PG receptor antagonist. Moreover, mutant huntingtin (mHtt) aggregates in GFP-mHttQ74-transfected astrocytes were substantially reduced by neurosteroid treatment, indicating that neurosteroid-induced autophagy may be functional.Present results demonstrate that Allo and PG activate autophagy in astrocytes. Notably, unlike several other autophagy inducers that, in excess, may cause autophagic cell death, Allo and PG are relatively non-toxic, possibly because of concurrent Akt and mTOR activation. Thus, as natural endogenous brain substances, Allo and PG may have a potential as therapeutic agents in neurodegenerative conditions in which abnormal protein aggregates are involved.

ORIGINAL ARTICLE: Effect of Progesterone on HLA‐E Gene Expression in JEG‐3 Choriocarcinoma Cell Line

Problem Among class Ib human leukocyte antigen (HLA) molecules, HLA‐E is known to be a major ligand of CD94/NKG2 receptor on natural killer (NK) cells, and to play a pivotal role in recognition of extravillous trophoblasts (EVTs) by maternal immune cells. However, it is scarcely known how HLA‐E expression is regulated in EVTs.Method of study In this study, we investigated whether progesterone, an essential hormone in maintaining pregnancy, regulated HLA‐E expression in EVT‐like cell line, JEG‐3. HLA‐E mRNA amount in cultured JEG‐3 cells was assessed by real‐time PCR and cell‐surface HLA‐E protein was analyzed by flowcytometry.Results Real‐time PCR showed 3.5‐fold increase 1 hour after the addition of 1000 ng/ml progesterone. This response was dimished by the addition of RU486, an antagonist for progesterone receptor. Flowcytometry indicated that 1000 ng/ml progesterone slightly enhanced HLA‐E expression on the surface of JEG‐3.Conclusion These results suggest that progesterone up‐regulates HLA‐E expression in JEG‐3 cells through the pathway mediated by progesterone receptor. Our findings might give a new insight into immunomodulatory function of progesterone at fetomaternal interface.

Response of the ABCG2 promoter in T47D cells and BeWo cells to sex hormone treatment

The aim of this study was to elucidate the effects of sex hormones on activity of the ABCG2 promoter in different cell lines. T47D cells and BeWo cells were used as models for ABCG2-expressing cell lines, and luciferase assays using ABCG2 promoter-luciferase constructs were performed. It was shown that progesterone increased the response of the ABCG2 promoter in T47D cells but not in BeWo cells. On the other hand, estradiol had no effect on response of the ABCG2 promoter in either cell line. However, response of the ABCG2 promoter was enhanced by overexpression of ERalpha in both T47D cells and BeWo cells. T47D cells had higher sensitivity to ERalpha than did BeWo cells. Furthermore, it was shown that the inductive effect of progesterone on the ABCG2 promoter was inhibited by addition of RU486 or mithramycin A. Therefore, it was thought that the ABCG2 promoter responded to stimulation of the progesterone receptor (PR)-Sp1 pathway in T47D cells. Furthermore, progesterone suppressed the response of the ABCG2 promoter by changing the expression levels of PR-A and PR-B in BeWo cells. These findings suggested that there are differences between cell lines in the regulation mechanism of ABCG2 expression by sex hormone treatment.

Isolated Autosomal Dominant Growth Hormone Deficiency: Stimulating MutantGH-1Gene Expression DrivesGH-1Splice-Site Selection, Cell Proliferation, and Apoptosis

The majority of mutations that cause isolated GH deficiency type II (IGHD II) affect splicing of GH-1 transcripts and produce a dominant-negative GH isoform lacking exon 3 resulting in a 17.5-kDa isoform, which further leads to disruption of the GH secretory pathway. A clinical variability in the severity of the IGHD II phenotype depending on the GH-1 gene alteration has been reported, and in vitro and transgenic animal data suggest that the onset and severity of the phenotype relates to the proportion of 17.5-kDa produced. The removal of GH in IGHD creates a positive feedback loop driving more GH expression, which may itself increase 17.5-kDa isoform productions from alternate splice sites in the mutated GH-1 allele. In this study, we aimed to test this idea by comparing the impact of stimulated expression by glucocorticoids on the production of different GH isoforms from wild-type (wt) and mutant GH-1 genes, relying on the glucocorticoid regulatory element within intron 1 in the GH-1 gene. AtT-20 cells were transfected with wt-GH or mutated GH-1 variants (5'IVS-3 + 2-bp T->C; 5'IVS-3 + 6 bp T->C; ISEm1: IVS-3 + 28 G->A) known to cause clinical IGHD II of varying severity. Cells were stimulated with 1 and 10 mum dexamethasone (DEX) for 24 h, after which the relative amounts of GH-1 splice variants were determined by semiquantitative and quantitative (TaqMan) RT-PCR. In the absence of DEX, only around 1% wt-GH-1 transcripts were the 17.5-kDa isoform, whereas the three mutant GH-1 variants produced 29, 39, and 78% of the 17.5-kDa isoform. DEX stimulated total GH-1 gene transcription from all constructs. Notably, however, DEX increased the amount of 17.5-kDa GH isoform relative to the 22- and 20-kDa isoforms produced from the mutated GH-1 variants, but not from wt-GH-1. This DEX-induced enhancement of 17.5-kDa GH isoform production, up to 100% in the most severe case, was completely blocked by the addition of RU486. In other studies, we measured cell proliferation rates, annexin V staining, and DNA fragmentation in cells transfected with the same GH-1 constructs. The results showed that that the 5'IVS-3 + 2-bp GH-1 gene mutation had a more severe impact on those measures than the splice site mutations within 5'IVS-3 + 6 bp or ISE +28, in line with the clinical severity observed with these mutations. Our findings that the proportion of 17.5-kDa produced from mutant GH-1 alleles increases with increased drive for gene expression may help to explain the variable onset progression, and severity observed in IGHD II.

Dickkopf-1, an Inhibitor of Wnt Signaling, Is Regulated by Progesterone in Human Endometrial Stromal Cells

Some members of the Wnt family, including ligands, receptors, inhibitors, and signaling components, are expressed in human endometrium. Dickkopf-1 (Dkk-1), a potent inhibitor of the Wnt signaling pathway, was recently found to be up-regulated in decidualizing endometrial stromal cells during the secretory phase of the menstrual cycle, suggesting regulation by progesterone. To test the hypothesis that progesterone regulates Dkk-1 expression in human endometrial stromal cells, we investigated the following effects on stromal cell expression of Dkk-1 mRNA and protein: decidualizing stimuli (progesterone or cAMP), RU486 (an inhibitor of progesterone action), and withdrawal of progesterone. Short-term treatment (up to 72 h, which corresponds to the full decidualized phenotype in response to cAMP and an early response to progesterone) did not reveal regulation of Dkk-1 mRNA or protein by cAMP but did show induction of Dkk-1 expression when the cells were treated with progesterone, an effect that was blocked by RU486. In long-term cultures (from 14 to 23 d, which corresponds to the full decidualized phenotype in response to progesterone), a significant increase in Dkk-1 mRNA and protein production was observed. Addition of RU486 or withdrawal of progesterone after long-term decidualization resulted in a decrease of Dkk-1 mRNA and protein to control levels. Estradiol alone had no effect on stromal Dkk-1 expression. These data strongly support regulation by progesterone of Dkk-1 mRNA synthesis and protein expression in human endometrial stromal cells and that the response is specific for progesterone and independent of cAMP and estradiol.

22. Generation of Circular Sleeping Beauty DNA Transposition Substrates by RU486-Induced Flp Recombination In Vitro – Steps towards a Single Integrating Adeno-Transposon Vector

DNA transposons, incorporated in nonviral or viral gene delivery systems, represent promising new tools for chromosomal transgene insertion and establishment of persistent gene expression in vivo. In a nonviral gene therapy approach based on a single plasmid vector containing (i) a transgene-tagged Sleeping Beauty (SB) transposon and (ii) a SB-derived transposase we have previously demonstrated efficient transposition and therapeutic gene expression in mouse liver (Mikkelsen et al., Mol. Ther. 2003, 8:654). Although this vector technology holds great promise as a nonviral therapeutic tool, incorporation of the transposon integration machinery into viral vector systems may be required for some applications. We have previously shown that circular DNA serves as an efficient substrate for SB transposition, whereas linear DNA only supports limited transposition. Therefore, to create a gutless 'all-in-one' adenoviral vector containing all components required for transposition we have generated adenoviral vector constructs from which plasmid-like circular DNA carrying a transgene-marked transposon and a transposase expression cassette can be released by Flp-recombinase-based recombination. To reduce possible premature DNA circle formation during vector preparation, we fused Flpe with a modified human progesterone receptor (hPR) domain that facilitates specific binding to and activation by the synthetic steroid RU486. By co-transfections with a |[beta]|-gal reporter plasmid, the Flpe-hPR protein was found to retain 20 to 50% of the wildtype Flpe activity (depending on cell line) in the presence of RU486 and was induced 6- to 44-fold by addition of RU486. Next, by using plasmid vectors containing both (i) a neo-tagged transposon and (ii) a Flpe-activated transposase expression cassette we found that the level of transposition in Flpe-hPR-transfected HeLa cells was 5-fold higher than the background level in the presence of RU486 and only slightly reduced (1.4-fold) compared to cells transfected with wildtype Flpe plasmid. We have successfully produced 'all-in-one' adenoviral vectors carrying the transposase coding sequence, the Flpe-hPR recombinase gene, and a human factor IX-marked transposon and are currently studying RU486-induced generation of circular substrates for SB-mediated transposition in in vitro-transduced cells. In summary, this study demonstrates a new approach for drug-inducible SB transposition and its use in a novel integrating adenoviral vector system with potential applications in gene therapy approaches which require long-term transgene expression.

Role of Apoptosis Controlled by Cytochrome c Released from Mitochondria for Luteal Function in Human Granulosa Cells

The aim of this study was to investigate the relationship between apoptosis by the mitochondrial pathway and luteal function in human granulosa cells. Granulosa cells were obtained by ultrasound-guided follicular aspiration from patients undergoing in vitro fertilization and embryo transfer. After the addition of RU486, cells were stained with a mitochondria-specific fluorescent dye, MitoTracker Red CM x Ros. Using flow cytometry and National Institute of Health image, the mitochondrial fluorescent area was measured. After staining with Hoechst 33258 dye, the number of apoptotic bodies per 1000 cells were counted at random on photomicrographs. Homogenates were used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis using antibodies against cytochrome c or caspase-3. The incidence of apoptotic bodies increased and the mitochondrial membrane potential decreased time dependently. The opposite effect was observed dose dependently with RU486 treatment. Western blot analysis showed increased cytochrome c expression, after treatment with 1-2 microg/mL of RU486 which then decreased with 5-10 microg/mL of RU486. Caspase-3 expression increased dose dependently with RU486. These results suggest that the activation of caspase-3 caused by cytochrome c released from mitochondria plays an important role in apoptosis-related luteal function in human granulosa cells.

Expression of the functional glucocorticoid receptor in mouse and human lens epithelial cells.

Studies have questioned the reported presence of a classic glucocorticoid receptor (GR) in the mammalian lens. The purpose of this study is to determine whether the functional GR is expressed in human and mouse lens epithelial cells. GR mRNA was determined by RT-PCR in freshly isolated human lens epithelia, mouse lens, immortalized human (HLE B-3) and mouse (alphaTN4) lens epithelial cells and in mouse lung, NIH-3T3 cells, and HeLa cells, which served as positive controls. Western blot analysis with the GR-specific antibody H-300 was performed on protein extracts from human lens epithelia, HLE B-3 cells, and alphaTN4 cells and from HeLa cells, NIH-3T3 cells, and partially purified GR, which served as positive controls. pGRE.Luc drives the expression of firefly luciferase. HLE B-3 and alphaTN4 cells were transfected with pGRE.Luc and cotreated with dexamethasone, with and without the competitive inhibitor RU-486. PCR products of the expected size were detected in all samples, sequenced in both directions, and found to have 97% to 100% homology with the GR. A band in the appropriate molecular weight range was identified by Western blot analysis in the lens extracts. Active GR binding to the GRE was demonstrated by an increase in firefly luciferase expression in transfected cells treated with dexamethasone. The dexamethasone-induced increase in luciferase activity was inhibited with the addition of RU-486. These results demonstrate expression of the functional glucocorticoid receptor in mouse and human lens epithelial cells. This finding suggests that glucocorticoids may act on the mouse and human lens directly during normal lens development and/or cataractogenesis.

Dexamethasone inhibits the antigen presentation of dendritic cells in MHC class II pathway

Glucocorticoids (GC) are physiological inhibitors of inflammatory responses and are widely used as anti-inflammatory and immunosuppressive agents in treatment of many autoimmune and allergic diseases. In the present study, we demonstrated that one of the mechanisms by which GC can suppress the immune responses is to inhibit the differentiation and antigen presentation of dendritic cells (DC). DC were differentiated from murine bone marrow hematopoietic progenitor cells by culture with GM-CSF and IL-4 with or without dexamethasone (Dex). Our data showed that Dex, in a dose dependent manner, down-regulated surface expression of CD86, CD40, CD54 and MHC class II molecules by DC, but the expression of MHC class I, CD80, CD95 and CD95L were not affected. In addition, Dex-treated DC showed an impaired function to activate alloreactive T cells and to secrete IL-Iβ and IL-12p70. Moreover, Dex inhibited DC to present antigen by MHC class II pathway. However, the endocytotic activity of DC was not affected. The inhibitory effect of Dex on the expression of costimulatory molecules and the antigen-presenting capacity of DC could be blocked by the addition of RU486, a potent steroid hormone antagonist, suggesting the requirement of binding to cytosolic receptors in the above-described action of Dex. Since DC have the unique property to present antigen to responding naive T cells and are required in the induction of a primary response, the functional suppression of DC by Dex may be one of the mechanisms by which GC regulate immune responses in vivo.

Dexamethasone-induced suppression of apoptosis in human neutrophils requires continuous stimulation of new protein synthesis.

We examined the mechanisms of corticosteroid inhibition of cell death by apoptosis in human neutrophils. Suppression of apoptosis by dexamethasone was abolished by co-treatment with the protein synthesis inhibitor cycloheximide. At doses of 1 microg/mL cycloheximide did not reduce basal survival of neutrophils but effectively inhibited dexamethasone-induced increases in 24-h survival (24.4 +/- 8.7 vs. 49.6 +/- 10%, P < 0.01). Similar results were obtained with actinomycin D, an inhibitor of mRNA synthesis. The factor(s) responsible for mediating increased survival following dexamethasone treatment is not active extracellularly because dexamethasone-treated neutrophil-conditioned medium (CM) had no effect on the survival of naive neutrophils when the direct effects of dexamethasone were neutralized with the steroid antagonist RU-486. In contrast, LPS-treated neutrophil CM significantly increased neutrophil survival even after addition of polymyxin b. The survival effect of dexamethasone required the continuous presence of the agonist because addition of RU-486 caused prompt development of apoptosis in dexamethasone-treated cells. When naive and dexamethasone-treated cells were examined by mRNA differential display, a limited number of cDNA bands were consistently and reproducibly detected that were increased in intensity, indicating up-regulation by dexamethasone. Thus, corticosteroid regulation of neutrophil apoptosis is a specific effect that depends on continuous stimulation of synthesis of a (protein) survival factor.

Circadian rhythm of in vitro bone-resorbing activity in human serum.

Calciotropic hormones as well as biochemical parameters of bone formation and resorption show circadian rhythms. In a previous study in the rat, we observed a circadian rhythm in serum bone-resorbing activity (SBRA). In the present study, we investigated whether there was a circadian rhythm of SBRA in human serum. For this purpose, we studied 10 healthy premenopausal women and 5 healthy men. Blood was collected every 2 h, and urine samples were collected during 4-h periods for 24 h. For the determination of SBRA, media were prepared by reconstituting serum samples with Dulbecco's Modified Eagle's Medium at a ratio of 20% serum and 80% Dulbecco's Modified Eagle's Medium. Limb bones were dissected from 19-day old fetal rats prelabelled with 45Ca and were cultured for 72 h in the presence of the sera. Bone resorption was assessed from the 45Ca released into the culture medium and from that retained in the bone and was expressed as percentage 45Ca release. Serum calcium, phosphorus, PTH, cortisol, and urinary pyridinium cross-links were also determined. SBRA in human serum followed a circadian rhythm with a peak at about 0300 h and a nadir at 0700 h. There was no significant difference between the rhythm of SBRA of women and men. At concurrent time points, SBRA and serum PTH were positively correlated (r = 0.629; P < 0.01), and SBRA and serum cortisol were negatively correlated (r = 0.797; P < 0.01). To further investigate the possible contribution of these hormones to SBRA, either neutralizing anti-PTH antibody or RU-486 (mifepristone), a glucocorticoid receptor antagonist, was added to the serum samples of 6 subjects. Neutralizing the effect of PTH did not change the pattern of SBRA rhythm. The addition of RU-486 had a significant effect on the rhythm of SBRA, reducing the peak and nadir amplitudes. Thus we conclude that cortisol plays a major role in the rhythm of SBRA present in human serum; however, the influence of other factors cannot be excluded. Cortisol may be an important determinant of the circadian rhythm of bone resorption in vivo.

Hepatic apolipoprotein production in liver cirrhosis

The liver represents the major site of apolipoprotein metabolism, and therefore determines concentrations of lipoprotein particles. So far there are no data about arterial and hepatic venous apolipoprotein concentrations in patients with documented liver cirrhosis. We have studied 20 patients (Child A=4, Child B=9, Child C=7) of different etiology (cholestatic liver disease= 7, non-cholestatic=13). All studied patients received hepatic venous catherization. Liver blood flow was assessed by ICG steady state infusion method. Arterial and hepatic venous blood samples were obtained and assayed for the concentrations of apolipoprotein (apo) A-I, apoB and apoE using a standardized, nephelome~c assay. The mean concentrations of apoA-I were 96 mg/dl in the artery and 93 mg/dl in the hepatic vein. The liver extraction rate was 16.7 rag/rain (range -51,9-97.0 mg/min). The mean concentrations of apnB were 75 mg/dl in the artery and 76 mg/dl in the hepatic vein. The mean synthesis rate was 8.9 mg/min (range -21.8-43.7). The mean concentrations of apoE Were 9.1 mg/dl in the artery and 10.4 mg/dl in the hepatic vein. ApoA-I and apoB concentrations are significantly higher in Child A patients compared to Child B and C (p=0.009 and p=0.03, respectively). However apoA-I concentration is not correlated with ICG clearance and ICG half-life time. These data confirm the data of the lipoprotein subfractions determined in cirrhotic patients. Interestingly, patients with cholestatic disease have decreased apoB levels as well as decreased production rates (p<0.05) in contrast to apoE, which is significantly increased (p<0.05). In summary, although apoA-I is a good predictor for liver synthesis, it does not correlate with liver function tests in our studied patients and the degree of portal hypertension. ApoE seems to be inverse correlated in cholestatic liver disease compared to apoA-I and apoB. Further studies are required to get more insights into the liver dependent apolipoprotein metabolism. Q ENDOGENOUS GLUCOCORTICOIDS INHIBIT LEUKOCYTE RECRUITMENT TO INFLAMMATORY SITES IN CHOLESTATIC RATS. K. Tiandra, M. Marie, P. Kubcs, M.G. Swain, GIRG, Calgary, Canada. Cholestatic patients have a high incidence of septic complications. Glucocorticoids inhibit leukocyte recruitment to inflammatory sites and we have recently documented elevated plasma glucocorticoid levels (corticosterone; C) in cholestatic rats (Gastroenterology 107:1469,1994). Therefore, we investigated the role of endogenous C in leukocyte recruitment during carrageenan-induced inflammation in cholestatic rats. Rats were bile duct (BDR) or sham (sham) reseeted and injected 5 days later with a 2% carragecnan solution into a preformed air pouch on their hacks. 5 hrs later rats were sacrificed, plasma C levels determined, and the inflammatory response quantitated by measuring exudate cell count (cell#/ml) and MPO activity (U/ml). BDR rats exhibited an impaired inflammatory response reflected by 52 % and 42 % decreases in exudatc cell count and MPO activity respectively compared to sham rats (all p<0.05). Exudate cell differentials by cytospot were similar in BDR and sham rats. Total C levels were similar in BDR and sham rats; however, free C levels (bioactive form) were 5-fold higher in BDR than sham rats (p<0.001). To investigate whether the elevated C levels in BDR rats were suppressing inflammation, we prctreated BDR and sham rats with RU486 (20mg/kg), a C receptor antagonist, before earrageenan injection. Treatment with RU486 significantly increased the inflammatory response in BDR (to sham levels) but not in sham rats; suggesting endogenous C was suppressing the inflammatory response in BDR rats. We have previously shown that BDR plasma is anti-adhesive to lcukocytcs in vitro (Gastroenterology 106; A992,1993). To investigate whether endogenous C in BDR plasma was inhibiting the inflammatory resEonsc by interfering with leukocyte adhesion, we examined PMA-stimulated 51C-labelled rat leukocyte adhesion to a rat biological substratum in vitro. Leukocyte adhesion was inhibited to a similar extent by plasma derived from either BDR or adrenalectomized BDR (devoid of C) rats. Furthermore, the addition of RU486 with BDR plasma did not alter leukocyte adhesion from that observed in the presence of BDR plasma alone. In conclusion, endogenous C contributes to an inhibition of the inflammatory response in BDR rats. This C effect is not due to an inhibition of leukocyte adhesion. Glucocorticoids may inhibit leukocyte recruitment to inflammatory sites in cholestasis by suppressing inflammatory mediator generation or by preventing the upregulation of endothelial adhesion molecules (eg.ICAM-1).

Dexamethasone negatively regulates the activity of a chimeric dihydrofolate reductase/glucocorticoid receptor protein.

A chimeric gene was constructed encoding the entire murine dihydrofolate reductase (DHFR) protein with a carboxyl-terminal extension encompassing amino acids 494-795 of the rat glucocorticoid receptor (GR). The chimeric DHFR/GR gene encoded a functional DHFR protein, as measured by the ability to transform DHFR-deficient Chinese hamster ovary (CHO) cells to a DHFR-positive phenotype. The DHFR/GR protein bound [3H]dexamethasone with a similar affinity as wild-type GR. Selection of stable CHO transformants in increasing concentrations of methotrexate resulted in increased expression of DHFR/GR. Addition of dexamethasone, a synthetic glucocorticoid agonist, decreased the activity of the chimeric protein, as measured by colony formation in selective medium, binding of fluoresceinated methotrexate, and direct enzymatic assay for DHFR. Addition of RU486, a glucocorticoid antagonist, antagonized the effect of dexamethasone. In the absence of dexamethasone, the chimeric protein was primarily localized to the cytoplasm. In the presence of dexamethasone or RU486, DHFR/GR translocated into the nucleus. However, RU486 did not decrease DHFR activity, distinguishing subcellular location from functional activity. These results demonstrate that glucocorticoids negatively affect the function of DHFR/GR.

Direct effects of progesterone and antiprogesterone on human sperm hyperactivated motility and acrosome reaction

To study the direct effects of progesterone (P) and its antagonist RU486 (mifepristone) on sperm hyperactivation (HA) and acrosome reaction. Prospective evaluation of semen samples incubated in capacitation media with P and/or RU486. University-affiliated tertiary care center. Normal healthy volunteers. Semen samples were incubated in media with P or RU486 alone or in combination, and aliquots were taken at 10 minutes, 1, 5, and 24 hours for HA analyses by computer-aided sperm analysis system, and at 0, 5, and 24 hours for assessment of acrosome reaction by fluorescein-labeled Pisum sativum (pea) agglutinin. HA and acrosome reaction. Sperm HA was significantly increased at 10 minutes by P both at 10−7 M (9.27 ± 1.59%; mean ± SEM) and 10− 6 M (9.39 ± 1.94%) when compared with untreated spermatozoa (5.62 ± 1.59%). The stimulatory effect of P on sperm HA was transient because this was not observed after 1, 5, and 24 hours of incubation. The antiprogesterone RU486 (10−6 M) alone had no effect and did not abolish the stimulatory effect of P on HA. The %HA was further enhanced by the addition of RU486 at 10−6 M to P at 10−7 M (12.43 ± 3.31%) or P at 10−6 M (13.52 ± 4.10%); however, this effect was not significantly different from P alone. Coincubation of P or RU486 with spermatozoa during capacitation did not stimulate the acrosome reaction in the concentrations tested. Progesterone directly stimulates human sperm HA transiently. Progesterone has no significant effect on acrosome reaction in capacitating spermatozoa. The effects of P are rapid and not counteracted by RU486, suggesting that the mechanism of action of P may not be mediated by specific P nuclear receptors.

Are human luteinizing granulosa cells a site of action for progesterone and relaxin?

Specific nuclear staining for progesterone receptor (PR) was detected by immunocyto-chemistry in human granulosa cells (GCs) obtained from in vitro fertilization protocols. The percent of PR-positive cells (60% to 80%) remained unchanged during 7 days of culture in media containing fetal calf serum, in the absence or presence of human chorionic gonadotropin (hCG) or the progesterone antagonist RU486. Progesterone (P) production by GCs cultured on extracellular matrix from bovine corneal endothelial cells was stimulated by hCG and prostaglandin E 2 (PGE 2 ). However, addition of RU486 or human relaxin had no effect on control, hCG-, or PGE 2 -stimulated P production. Thus, the receptor data are consistent with an autocrine action of P in luteinizing GCs, but initial experiments in cell culture did not define a role for P or relaxin in modulating luteal steroidogenesis.

Stimulatory action of progesterone on the synthesis of glycogen in primary cell culture of human endometrium.

AbstractThe effects of progesterone and its antagonist, RU486, on the synthesis of glycogen were investigated using a primary cell culture of human endometrium. Progesterone stimulated the glycogen synthesis in a dose‐dependent manner. The minimal effective dose of progesterone is 10‐9 M with maximal effect occurring at 10‐7 M. Stimulation of glycogen synthesis was apparent as early as after 6 hours of incubation. Progesterone induced the activity of glycogen synthetase and reduced the activity of glycogen phosphorylase. The addition of RU486 with progesterone completely eliminated the action of progesterone in both the synthesis of glycogen and the activity of glycogen synthetase. These results clearly demonstrate the stimulatory action of progesterone in glycogen synthesis in cultured human endometrial cells. Moreover, the antiprogesterone, RU486, is shown to counteract the progesterone action in glycogen synthesis in vitro.