Glucocorticoid Binding Research Articles

ENDOGENOUS RETROVIRUSES IN SYSTEMIC RESPONSE TO STRESS SIGNALS

Infection of germline cells with retroviruses initiates permanent proviral colonization of the germline genome. The germline-integrated proviruses, called endogenous retroviruses (ERVs), are inherited to offspring in a Mendelian order and belong to the transposable element family. Endogenous retroviruses and other long terminal repeat retroelements constitute ~8% and ~10% of the human and mouse genomes, respectively. It is likely that each individual has a distinct genomic ERV profile. Recent studies have revealed that a substantial fraction of ERVs retains the coding potentials necessary for virion assembly and replication. There are several layers of potential mechanisms controlling ERV expression: intracellular transcription environment (e.g., transcription factor pool, splicing machinery, hormones), epigenetic status of the genome (e.g., proviral methylation, histone acetylation), profile of transcription regulatory elements on each ERV's promoter, and a range of stress signals (e.g., injury, infection, environment). Endogenous retroviruses may exert pathophysiologic effects by infection followed by random reintegration into the genome, by their gene products (e.g., envelope, superantigen), and by altering the expression of neighboring genes. Several studies have provided evidence that ERVs are associated with a range of pathogenic processes involving multiple sclerosis, systemic lupus erythematosus, breast cancer, and the response to burn injury. For instance, the proinflammatory properties of the human ERV-W envelope protein play a central role in demyelination of oligodendrocytes. As reviewed in this article, recent advances in ERV biology and mammalian genomics suggest that ERVs may have a profound influence on various pathogenic processes including the response to injury and infection. Understanding the roles of ERVs in the pathogenesis of injury and infection will broaden insights into the underlying mechanisms of systemic immune disorder and organ failure in these patients.

Molecular Mechanisms of Corticosteroid Resistance

Most patients with asthma are successfully treated with conventional therapy. Nevertheless, there is a small proportion of asthmatic patients, including present cigarette smokers and former cigarette smokers, who fail to respond well to therapy with high-dose glucocorticoids (GCs) or with supplementary therapy. In addition, high doses of steroids have a minimal effect on the inexorable decline in lung function in COPD patients and only a small effect on reducing exacerbations. GC insensitivity, therefore, presents a profound management problem in these patients. GCs act by binding to a cytosolic GC receptor (GR), which is subsequently activated and is able to translocate to the nucleus. Once in the nucleus, the GR either binds to DNA and switches on the expression of antiinflammatory genes or acts indirectly to repress the activity of a number of distinct signaling pathways such as nuclear factor (NF)-kappaB and activator protein (AP)-1. This latter step requires the recruitment of corepressor molecules. Importantly, this latter interaction is mutually repressive in that high levels of NF-kappaB and AP-1 attenuate GR function. A failure to respond may therefore result from reduced GC binding to GR, reduced GR expression, enhanced activation of inflammatory pathways, or lack of corepressor activity. These events can be modulated by oxidative stress, T-helper type 2 cytokines, or high levels of inflammatory mediators, all of which may lead to a reduced clinical outcome. Understanding the molecular mechanisms of GR action, and inaction, may lead to the development of new antiinflammatory drugs or may reverse the relative steroid insensitivity that is characteristic of patients with these diseases.

Glucocorticoid binding and cytolethal responsiveness of hairy-cell and chronic lymphocytic leukaemia.

The glucocorticoid binding properties and cytolethal responsiveness of leukaemic cells were studied in vitro in seven patients with hairy-cell leukaemia (HCL) and five with chronic lymphocytic leukaemia (CLL). Substantial levels of glucocorticoid binding were detected both in whole cell and cytosol preparations from all patients although the level of binding by HCL cells always exceeded that of CLL cells (P less than 0.05). In both leukaemic cell types the uptake and binding of prednisolone in vitro was significantly greater than that of dexamethasone (P less than 0.05). CLL cells showed a variable dose-related cytolethal response to methylprednisolone sodium succinate (MPSS) treatment in vitro although cytolytic effects were not marked in the usual pharmacological dose range (10(-5)-10(-6)M). Treatment of CLL patients with conventional doses of prednisone for extended periods or high intravenous infusions of MPSS over shorter periods had no consistent effect on the in-vitro level of steroid binding or the cytolethal responsiveness of CLL cells to glucocorticoid treatment. Although HCL cells proved highly resistant to the cytolethal effects of MPSS in vitro, the substantial binding of glucocorticoids by leukaemic cells from all HCL patients indicates the potential value of steroid therapy in this disease should be explored further.

Mineralocorticoid Receptor and 11β-Hydroxysteroid Dehydrogenase Type II Expression in Renal Cell Neoplasms

The kidney is an important target for mineralocorticoids. Aldosterone, the major endogenously secreted mineralocorticoid, acts by binding to mineralocorticoid receptor (MR) in the distal renal tubule. The enzyme 11beta-hydroxysteroid dehydrogenase type II (11beta-HSD2) prevents the binding of glucocorticoids to the MR by inactivating cortisol to cortisone. Our goal was to determine whether MR and 11beta-HSD2 expression could be used to characterize the major types of renal cell neoplasms. Using immunohistochemistry we analyzed tissue microarray specimens from 132 patients with renal cell neoplasms, stratified into 84 clear cell renal cell carcinomas (CRCC), including 9 cases clear cell carcinoma with predominantly granular cytoplasm; 14 papillary RCC (PRCC); 20 chromophobe RCC (CHRCC); and 14 oncocytomas (OCs). MR and 11beta-HSD2 expression were also quantitated by real-time reverse transcription-polymerase chain reaction. Expression of both MR and 11-betaHSD2 was detected in the distal nephrons of normal kidneys. The CHRCC group stained for 11-betaHSD2 in a membranous and cytoplasmic pattern whereas diffuse cytoplasmic reactivity was seen in OCs. MR and 11beta-HSD2 were coexpressed in most of CHRCC (90% and 95%) and oncocytomas (93% and 100%). No MR staining was detected in CRCC, including clear cell carcinoma with predominantly granular cytoplasm, or in PRCC. Only 2 cases of CRCC (2.6%) showed focal positivity for 11beta-HSD2, whereas all PRCCs were negative. CHRCC and OC demonstrated significantly higher levels of MR and 11beta-HSD2 expression than CRCC and PRCC by real-time polymerase chain reaction. Moreover, CHRCC showed higher expression of MR and 11beta-HSD2, as compared with OC. Our study indicates MR and 11beta-HSD2 are both sensitive and specific markers of the distal nephron and its related neoplasms (CHRCC and OC). Additionally, the staining pattern and the level of MR and 11beta-HSD2 expression seems to be useful in the distinction of CHRCC from OC. MR and 11beta-HSD2 should be considered in the immunohistochemical panel to more accurately subtype renal cell tumors.

Glucocorticoid Gene Variant Blunts the Effects of Child Abuse on PTSD

In an adult exposed to trauma, the risk for post-traumatic stress disorder increases if the person had been abused as a child. Both child abuse and PTSD have been associated with dysregulation of cortisol and of the hypothalamic-pituitary-adrenal (HPA) axis. Single nucleotide polymorphisms (SNPs) in the FKBP5 gene (which modulates binding of glucocorticoid to its receptor) have been associated with peritraumatic dissociation in …

Mitochondrial effects of dexamethasone imply both membrane and cytosolic-initiated pathways in HepG2 cells

Glucocorticoid treatment is often linked to increased whole-body energy expenditure and hypermetabolism. Glucocorticoids affect mitochondrial energy production, notably in the liver, where they lead to mitochondrial uncoupling reducing the efficacy of oxidative phosphorylation. However, the signaling pathways involved in these phenomena are poorly understood. Here we treated HepG2 cells with dexamethasone for different times and, by using different combinations of inhibitors, we showed that dexamethasone treatment leads to recruitment of two main signaling pathways. The first one involves a G-protein coupled membrane glucocorticoid binding site and rapidly decreases complexes I and II activities while complex III activity is upregulated in a p38MAPK dependent mechanism. The second one implies the classical cytosolic glucocorticoid receptor and triggers long-term transcriptional increases of respiration rates and of complex IV activity and quantity. We concluded that mitochondria are the target of multiple dexamethasone-induced regulatory pathways that are set up gradually after the beginning of hormone exposure and that durably influence mitochondrial oxidative phosphorylation.

Signal Sequences for Targeting of Gene Therapy Products to Subcellular Compartments: The Role Of CRM1 in Nucleocytoplasmic Shuttling of the Protein Switch

The purpose of this study was to understand the mechanism of nuclear export of the protein switch, used for controlled intracellular delivery of gene products, by studying the involvement of classical export receptor CRM1. Transient transfections of protein switch constructs, isolated nuclear export and import signals were carried out. Effect of leptomycin B (inhibitor of export receptor) and geldanamycin (inhibitor of Hsp90) on localization of these constructs was studied using fluorescence microscopy. Putative nuclear export signals in the glucocorticoid and progesterone receptor ligand binding domains were identified and studied. It was observed that treatment with leptomycin B caused nuclear accumulation of the protein switch constructs. However, geldanamycin did not have any pronounced effect on the localization. The isolated nuclear export signal from glucocorticoid receptor localized mostly in the cytoplasm, while its mutated version was present everywhere. The localization controlled protein switch constructs are exported out of the nucleus by the classical CRM1 receptors. The ligand binding domain of these protein switch constructs plays an important role in maintaining these constructs in the cytoplasm in the absence of ligand, as well the re-export back to the cytoplasm from the nucleus after ligand washout.

Glucocorticoid receptor mediated repression of human insulin gene expression is regulated by PGC-1α

Transcriptional regulation of the insulin gene plays a critical role in maintenance of pancreatic beta cell function in response to various stimuli. Here, we used INS-1 cells to test the hypothesis that PGC-1α regulates human insulin gene transcription by modulating glucocorticoid (GR) binding to the insulin gene promoter. Analysis of the human insulin promoter region revealed that the suppressive region regulated by GR and PGC-1α is localized from −362 to −257 bp. To locate the GR binding site in the human insulin promoter region, EMSAs were performed with candidate GR binding sequences and confirmed that a palindromic region (Palin, −284 to −274 bp) specifically interacts with GR. We also found that the Palin-binding activity of GR is increased in the presence of PGC-1α. These findings suggest that PGC-1α elevates the binding of GR to Palin and thereby enhances the GR-mediated inhibition of human insulin transcription.

Phosphorylation of recombinant human low affinity glucocorticoid binding site recombinant protein in vitro reconstitutes its progesterone binding function

Phosphorylation of recombinant human low affinity glucocorticoid binding site recombinant protein in vitro reconstitutes its progesterone binding function

The glucocorticoid RU24858 does not distinguish between transrepression and transactivation in primary human eosinophils

BackgroundGlucocorticoids are used to treat chronic inflammatory diseases such as asthma. Induction of eosinophil apoptosis is considered to be one of the main mechanisms behind the anti-asthmatic effect of glucocorticoids. Glucocorticoid binding to its receptor (GR) can have a dual effect on gene transcription. Activated GR can activate transcription (transactivation), or by interacting with other transcription factors such as NF-κB suppress transcription (transrepression). RU24858 has been reported to transrepress but to have little or no transactivation capability in other cell types. The dissociated properties of RU24858 have not been previously studied in non-malignant human cells. As the eosinophils have a very short lifetime and many of the modern molecular biological methods cannot be used, a "dissociated steroid" would be a valuable tool to evaluate the mechanism of action of glucocorticoids in human eosinophils. The aim of this study was to elucidate the ability of RU24858 to activate and repress gene expression in human eosinophils in order to see whether it is a dissociated steroid in human eosinophils.MethodsHuman peripheral blood eosinophils were isolated under sterile conditions and cultured in the presence and/or absence RU24858. For comparison, dexamethasone and mometasone were used. We measured chemokine receptor-4 (CXCR4) and Annexin 1 expression by flow cytometry and cytokine production by ELISA. Apoptosis was measured by DNA fragmentation and confirmed by morphological analysis.ResultsRU24858 (1 μM) increased CXCR4 and Annexin 1 expression on eosinophils to a similar extent as mometasone (1 μM) and dexamethasone (1 μM). Like dexamethasone and mometasone, RU24858 did suppress IL-8 and MCP-1 production in eosinophils. RU24858 also increased spontaneous eosinophil apoptosis to a similar degree as dexamethasone and mometasone, but unlike dexamethasone and mometasone it did not reverse IL-5- or GM-CSF-induced eosinophil survival.ConclusionOur results suggest that in human eosinophils RU24858 acts as transactivator and transrepressor like classical glucocorticoids. Thus, RU24858 seems not to be a "dissociated steroid" in primary human eosinophils in contrast to that reported in animal cells. In addition, functionally RU24858 seems to be a less potent glucocorticoid as it did not reverse IL-5- and GM-CSF-afforded eosinophil survival similarly to dexamethasone and mometasone.

The native anti-glucocorticoid paradigm

Circulating 3β-hydroxysteroids including dehydroepiandrosterone (DHEA) are 7α-hydroxylated by the cytochrome P450-7B1 in the liver, skin and brain, which are the target organs of glucocorticoids. Anti-glucocorticoid effects with 7α-hydroxy-DHEA were observed in vivo without an interference with glucocorticoid binding to its receptor. In the organs mentioned above, the circulating inactive cortisone was reduced into active cortisol by the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). We demonstrated that 7α-hydroxy-DHEA was also a substrate for this enzyme. Studies of the 11β-HSD1 action on 7α-hydroxy-DHEA showed the reversible production of 7β-hydroxy-DHEA through an intermediary 7-oxo-DHEA, and the kinetic parameters favored this production over that of active glucocorticoids. Both the production of 7α-hydroxysteroids and their interference with the activation of cortisone into cortisol are basic to the concept of native anti-glucocorticoids efficient at their production site. This opens a promising new area for research.

Regulation of the Dynamics of hsp90 Action on the Glucocorticoid Receptor by Acetylation/Deacetylation of the Chaperone

It is known that inhibition of histone deacetylases (HDACs) leads to acetylation of the abundant protein chaperone hsp90. In a recent study, we have shown that knockdown of HDAC6 by a specific small interfering RNA leads to hyperacetylation of hsp90 and that the glucocorticoid receptor (GR), an established hsp90 "client" protein, is defective in ligand binding, nuclear translocation, and gene activation in HDAC6-deficient cells (Kovacs, J. J., Murphy, P. J. M., Gaillard, S., Zhao, X., Wu, J-T., Nicchitta, C. V., Yoshida, M., Toft, D. O., Pratt, W. B., and Yao, T-P. (2005) Mol. Cell 18, 601-607). Using human embryonic kidney wild-type and HDAC6 (small interfering RNA) knockdown cells transiently expressing the mouse GR, we show here that the intrinsic properties of the receptor protein itself are not affected by HDAC6 knockdown, but the knockdown cytosol has a markedly decreased ability to assemble stable GR.hsp90 heterocomplexes and generate stable steroid binding activity under cell-free conditions. HDAC6 knockdown cytosol has the same ability to carry out dynamic GR.hsp90 heterocomplex assembly as wild-type cytosol. Addition of purified hsp90 to HDAC6 knockdown cytosol restores stable GR.hsp90 heterocomplex assembly to the level of wild-type cytosol. hsp90 from HDAC6 knockdown cytosol has decreased ATP-binding affinity, and it does not assemble stable GR.hsp90 heterocomplexes when it is a component of a purified five-protein assembly system. Incubation of knockdown cell hsp90 with purified HDAC6 converts the hsp90 to wild-type behavior. Thus, acetylation of hsp90 results in dynamic GR.hsp90 heterocomplex assembly/disassembly, and this is manifest in the cell as a approximately 100-fold shift to the right in the steroid dose response for gene activation.

Glucocorticoid receptor antagonism in the basolateral amygdala and ventral hippocampus interferes with long-term memory of contextual fear

The binding of glucocorticoids to the type II or glucocorticoid receptor (GR) is known to play a role in memory consolidation and long-term memory. The present series of studies investigated the locus for GR effects on learning and memory of contextual fear conditioning. The GR antagonist RU 38486 was administered peripherally (10, 20, or 30 mg/kg/ml), as well as centrally into the lateral ventricle (75 or 150 ng/2 μL), basolateral amygdala (BLA; 0.3, 3, or 30 ng/0.2 μL), dorsal hippocampus (DH; 30 ng/μL) or ventral hippocampus (VH; 30 ng/μL) prior to contextual fear conditioning. Peripheral administration of RU 38486 did not affect fear-related levels of freezing immediately following a footshock or in a long-term memory test 24 h later. However, administration into the lateral ventricle, BLA, or VH decreased freezing in a 24 h retention test, while leaving post-shock levels of freezing intact. Both post-shock and retention levels of freezing were unaffected in rats that received RU 38486 in the DH compared to vehicle controls; however, vehicle rats also displayed low levels of freezing during retention. The data indicate that GR activation within the BLA and VH is important for the establishment of long-term memory for contextual fear conditioning.

The membrane glucocorticoid receptor on the pituitary is G protein coupled and interacts with the cytoskeleton

Background: While nongenomic glucocorticoid actions like fast feedback inhibition of ACTH release are well documented, only very little is known about possible receptors mediating them. We recently identified specific, positive-cooperative glucocorticoid binding sites on the cell membrane of the ACTH-producing pituitary cell line AtT-20. The bound dexamethasone molecules showed a high degree of mobility restriction suggesting tighter association e.g. with the cytoskeleton. Additionally, two fractions of fast (freely diffusing ligand) and intermediate mobility (diffusion on/inside the cell) were found. The aim of the present study was to further characterize these binding sites on living cells in relation to the intracellular glucocorticoid receptor (iGR), the actin cytoskeleton (which is involved in ACTH release) and the possible involvement of a G protein.

MRNA expression levels of (co)chaperone molecules of the glucocorticoid receptor are not involved in glucocorticoid resistance in pediatric ALL

Resistance to glucocorticoids (GC) is an important adverse risk factor in the treatment of acute lymphoblastic leukemia (ALL). To induce apoptosis, GC bind to the GC receptor (GR), which is regulated by various (co)chaperone proteins such as heat-shock protein 70 (HSP-70), HSP-40, HIP (HSP-70-interacting protein), BAG-1 (BCL-2-associated gene product-1), HOP (HSP-70/HSP-90-Organizing protein), HSP-90, P-23, FKBP-51, FKBP-52 and CYP-40. In this study, we tested the hypothesis that mRNA expression levels of these molecules are determinants of GC resistance in childhood ALL. In all, 20 children with ALL cells in vitro sensitive to prednisolone (LC(50) < 0.1 microg/ml) were compared each with a resistant patient (LC(50) >150 mug/ml), matched for immunophenotype, age and white blood cell count. mRNA expression levels of the (co)chaperone molecules were measured by quantitative real-time RT-PCR and normalized to GAPDH and RNaseP levels. In vitro resistance to prednisolone was measured by MTT assay. HSP-90 mRNA expression levels were 2000-fold higher as compared to HSP-70. Using matched pair analysis, mRNA expression levels of the various (co)chaperone molecules were not significantly different between in vitro-sensitive and -resistant patients. GC resistance in childhood ALL cannot be attributed to different mRNA expression levels of the investigated (co)chaperone molecules involved in GC binding and transport to the nucleus.

Dehydroepiandrosterone and its 7-hydroxylated metabolites do not interfere with the transactivation and cellular trafficking of the glucocorticoid receptor

The human brain is a target tissue for glucocorticoids (GC). Dehydroepiandrosterone (DHEA) is a neurosteroid produced in the brain where it is transformed into 7α-hydroxy-DHEA and 7β-hydroxy-DHEA. The antiglucocorticoid effects of both 7-hydroxylated metabolites have been investigated with evidence in mice that neither form of DHEA interfered with the binding of GC to its glucocorticoid receptor (GR), but contributed to a decreased nuclear uptake of the activated GR. Our objective was to use COS-7 cell culture to research DHEA, 7α-hydroxy-DHEA and 7β-hydroxy-DHEA interferences with GR trafficking. These cells did not carry out the 7α-hydroxylation of DHEA and the oxidation of cortisol into cortisone. The cDNA of the human GR was inserted into pcDNA3 for a transient transfection of COS-7 cells. Human GR transactivation activity was measured from a luciferase-MMTV reporter gene. The transfected COS-7 cells were cultured using 10 −12 to 10 −5 M dexamethasone (DEX) or cortisol, which triggered the reporter expression. Treatment with 10 −12 to 10 −5 M DHEA, 7α-hydroxy-DHEA and 7β-hydroxy-DHEA caused no change in the GC-induced GR transactivation. A reconstruction of the process associated EGFP to the human GR cDNA. Confocal microscopic examination of COS-7 cells transiently expressing the fusion protein EGFP-GR showed nuclear fluorescence 60 min after incubation with 10 −8 M DEX or cortisol. The addition of 10 −5 M DHEA, 7α-hydroxy-DHEA or 7β-hydroxy-DHEA did not change its kinesis and intensity. These results contribute to the knowledge of DHEA, 7α-hydroxy-DHEA and 7β-hydroxy-DHEA, in relation to antiglucocorticoid activity. We conclude that direct interference with GR trafficking can be discounted in the case of these hormones, therefore proposing new possibilities of investigation.

RNA Expression Levels of (Co)Chaperone Molecules of the Glucocorticoid Receptor Are Not Related to In Vitro Prednisolone Resistance in Childhood ALL.

In vitro resistance to Glucocorticoids (GC) is an important adverse risk factor in the treatment of ALL. To induce apoptosis in ALL cells, GC have to bind to the GC receptor (hGR), which is tightly regulated by various (co)chaperone molecules. HSP70, ST13 and HSP40 facilitate hGR binding to HSP90 and HOP in an energy dependent fashion. This complex is stabilised by immunophillins FKBP 51, FKBP59, CYP40 and P23 and is necessary for the hGR to be able to bind GC. The ATPase BAG1 can function as negative regulator of HSP70, and may downregulate hGR activity. After the GC-hGR complex is formed, it is transported to the nucleus to regulate GC responsive genes. In this study, we tested the hypothesis that RNA expression levels of these (co) chaperone molecules are important determinants for in vitro prednisolone sensitivity in childhood ALL.Methods: 20 children with leukemic cells in vitro sensitive to prednisolone (LC50<0.1μg/ml) were matched (according to age, immunophenotype and white blood cell count at diagnosis) each with an in vitro resistant patient (LC50 > 150 μg/ml). RNA expression levels of the different (co)chaperone molecules were measured by a quantitative real-time RT-PCR (Taqman) assay and standardised to endogenous GAPDH and RNAseP mRNA levels. In vitro resistance to prednisolone was measured by the MTT assay.Results. The highest median expression levels, indicated as percentage of GAPDH levels were found for HSP90 (29%) and p23 (10%), whereas FKBP51 and ST13 were expressed at 2% and 1.6% respectively. HSP70 (0.1%), HSP40 (0.44%), HOP (0.2%), FKBP59 (0.4%), PPID (0.3%) and BAG-1 (0.4%) were expressed at relatively low levels. Using matched pair analysis, no significant differences were found for the expression levels of the various (co)chaperone molecules between in vitro sensitive and resistant patients. The ratio of different (co)chaperone molecules with opposing effect (HIP versus BAG-1, FKBP-51 versus FKBP-52, HSP-90 versus P23, and HSP-90 versus the functional GR-alpha receptor, positive and negative effect respectively) was not related to GC resistance as well.Conclusion: Glucocorticoid resistance in childhood ALL can not be attributed to basal expression levels of the diverse (co)chaperone molecules involved in GC binding and transport of the GC-GR complex to the nucleus of the GR.

GLUCOCORTICOID RECEPTOR AND IN VITRO STEROID SENSITIVITY IN PRE-TRANSPLANT PATIENTS UNDERGOING LONG-TERM HEMODIALYSIS

P208 Aims: Glucocorticoid receptor (GR) number and binding affinity alterations have been observed in peripheral blood mononuclear cells (PBMC) from patients with inflammatory diseases. Chronic renal failure has been recently described as a chronic inflammatory state. Therefore, we evaluated glucocorticoid receptors and dexamethasone (Dex) mediated inhibition of concanavalin-A (Con-A) stimulated PBMC proliferation in pre kidney transplant patients undergoing long-term hemodialysis. Methods: PBMC from 61 healthy controls and 24 pre kidney transplant patients undergoing long-term hemodialysis were isolated for binding assay and cultured for the in vitro glucocorticoid sensitivity assay. Basal and Con-A-stimulated PBMC proliferation levels and the inhibitory effect of different doses of Dex(10-8, 10-6, 10-4M) were evaluated. The IC50 was defined as the Dex concentration that caused a 50% inhibition of cell proliferation and subject with an IC50>10-6M was considered corticosteroid resistant (CR). Results: There was lymphocyte proliferation after Con-A stimulation in both groups, which was inhibited by Dex in a dose dependent manner. As a group, controls and patients were steroid-sensitive by Dex mediated inhibition of Con-A proliferation test, but controls presented significant suppression of PBMC proliferation with lower Dex dose (10-8M) than patients (Dex 10-6M). Seven out of 61 controls (11.4%) and 5 out 15 patients (33.3%) were CR (p=0.05). The mean (±SEM) GR binding affinity (Kd) of the normal group was higher (7.1± 1.0nmol/L) than patients (12.8±2.0 nmol/L) (p=0.007). Two out of 38 controls (5.3%) and 6 out of 24 patients (25%) had Kd above the 95th percentile of normal group (p=0.04). There was concordance in both in vitro methods. There was no association between glucocorticoid resistance and acute rejection in a short term follow up. The possible impact of steroid sensitivity in the incidence of chronic allograft nephropathy and therefore long term graft outcome remains to be established. Conclusions: There is an alteration of GR sensitivity in pre kidney transplant patients undergoing long-term hemodialysis.

15-deoxy-Delta12,14-prostaglandin J2 inhibits glucocorticoid binding and signaling in macrophages through a peroxisome proliferator-activated receptor gamma-independent process.

15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) is involved in the control of inflammatory reaction. We tested the hypothesis that 15d-PGJ(2) would exert this control in part by modulating the sensitivity of inflammatory cells to glucocorticoids. Human U937cells and mouse RAW 264.7 cells were exposed to 15d-PGJ(2), and binding experiments were performed with [(3)H]dexamethasone as a glucocorticoid receptor (GR) ligand. 15d-PGJ(2) caused a transient and concentration-dependent decrease in [(3)H]dexamethasone-specific binding to either cells through a decrease in the number of GR per cell without significant modification of the K(d) value. These changes were related to functional alteration of the GR rather than to a decrease in GR protein. They did not require the engagement of peroxisome proliferator-activated receptor gamma (PPARgamma), because the response to 15d-PGJ(2) was neither mimicked by the PPARgamma agonist ciglitazone nor prevented by the PPARgamma antagonist bisphenol A diglycidyl ether. 15d-PGJ(2) altered GR possibly through the interaction of its cyclopentenone ring with GR cysteine residues because the cyclopentenone ring per se could mimic the effect of 15d-PGJ(2), and modification of GR cysteine residues with methyl methanethiosulfonate suppressed the response to 15d-PGJ(2). Finally, 15d-PGJ(2)-induced decreases in glucocorticoid binding to GR resulted in parallel decreases in the ability of GR to activate the transcription of a glucocorticoid-inducible reporter gene and to reduce the expression of monocyte chemoattractant protein-1. Together these data suggest that 15d-PGJ(2) limits glucocorticoid binding and signaling in monocytes/macrophages through a PPARgamma-independent and cyclopentenone-dependent mechanism. It provides a way in which 15d-PGJ(2) would exert proinflammatory activities in addition to its known anti-inflammatory activities.

Specific high-affinity glucocorticoid binding sites on the membrane of the pituitary cell line AtT-20 demonstrated by Fluorescence Correlation Spectroscopy

The fast feedback regulation of ACTH release from the pituitary by glucocorticoids is a physiological rapid steroid effect that has been well documented in vivo and in vitro. The mouse pituitary cell line AtT-20 is a model system for ACTH release and its fast (within 10 minutes) suppression by glucocorticoids. The existence of membrane Glucocorticoid Receptors on this cell line has been suggested by studies on isolated cell membranes. The aim of this study was to identify and characterize such binding sites on the membrane of single living cells using the single molecule technique Fluorescence Correlation Spectroscopy (FCS), a method that allows to monitor the diffusion behaviour of fluorescently labeled molecules with single-molecule sensitivity in a confocal volume measuring 0,35µm in the xy (lateral) and 2,4µm in the z (axial) dimension defined by a microscope objective epi-illuminated by a laser beam. Binding of a ligand to the cell membrane of single living cells can be studied in solution without the need of membrane isolation or other manipulations. We found that fluorescein-labeled dexamethasone binds indeed to the membrane of AtT-20 cells. The binding is specific, since it is washed out by unlabeled dexamethasone or the natural ligand corticosterone, but not estradiol or progesterone in 1000-fold excess. Incubation with different concentrations of dexamethasone resulted in a binding curve with a binding constant of 6 nM. Membrane binding is observed only in a subpopulation of the cells. The fraction of binding-positive cells is significantly reduced upon feeding of the cells. In summary we have shown the existence of specific glucocorticoid binding sites with high affinity on the membrane of living AtT-20 cells. Whether or not these binding sites are related to the rapid glucocorticoid effects on the pituitary remains to be elucidated, but the fact that they are downregulated upon feeding clearly points in the direction of their functionality.