Ethyl pyruvate modulates acute inflammatory reactions in human endothelial cells in relation to the NF-kappaB pathway.

BACKGROUND AND PURPOSE: Endothelial cell activation plays a critical role in regulating leukocyte recruitment during inflammation and infection. Ethanol (EtOH) reduces host defence systems, including cell adhesion. However, well-known side effects of EtOH limit its clinical use as an anti-inflammatory drug. Instead, ethyl pyruvate (EtP) may represent a better alternative. Here, we compared effects of EtP and EtOH on neutrophil recruitment and activation of human umbilical vein endothelial cells (HUVECs). EXPERIMENTAL APPROACH: Adhesion of neutrophils to HUVEC monolayers, surface expression of intercellular cell adhesion molecule, E-selectin, vascular cell adhesion molecule, release of interleukin (IL)-8 and granulocyte colony-stimulating factor (G-CSF) from HUVECs were assessed as well as translocation of interleukin-1 receptor-associated kinase (IRAK-1), the nuclear factor-kappa B (NF-kappaB) subunits p50, p65 and IkappaB-alpha. NF-kappaB activation was analysed with a luciferase reporter plasmid. Cells were stimulated with IL-1beta, lipopolysaccharide (LPS) or tumour necrosis factor-alpha. KEY RESULTS: EtP was several-fold more potent than EtOH in reducing adhesion of neutrophils to activated HUVECs, generation of IL-8 or G-CSF and surface expression of the adhesion molecules. This last reaction was decreased by EtP even when added after cytokines or LPS. Translocation of IRAK-1, IkappaBalpha and the NF-kappaB p65 subunit to the HUVEC nucleus was inhibited by EtP for all stimuli, whereas the diminished p50 translocation was stimulus specific. When p65 was constitutively expressed in Cos7 cells, stimulation of an NF-kappaB-dependent reporter gene was not affected by EtP, suggesting that EtP acted upstream of gene activation. CONCLUSIONS AND IMPLICATIONS: EtP impedes adhesive, secretory and signalling events typical of the early inflammatory response in endothelial cells, suggesting EtP as a possible treatment for acute inflammatory conditions.

Pancreatic beta cells are important targets for the diabetogenic effects of glucocorticoids.

Abnormalities contributing to the pathogenesis of non-insulin-dependent diabetes mellitus include impaired beta cell function, peripheral insulin resistance, and increased hepatic glucose production. Glucocorticoids are diabetogenic hormones because they decrease glucose uptake and increase hepatic glucose production. In addition, they may directly inhibit insulin release. To evaluate that possible role of glucocorticoids in beta cell function independent of their other effects, transgenic mice with an increased glucocorticoid sensitivity restricted to their beta cells were generated by overexpressing the glucocorticoid receptor (GR) under the control of the insulin promoter. Intravenous glucose tolerance tests showed that the GR transgenic mice had normal fasting and postabsorptive blood glucose levels but exhibited a reduced glucose tolerance compared with their control littermates. Measurement of plasma insulin levels 5 min after intravenous glucose load demonstrated a dramatic decrease in acute insulin response in the GR transgenic mice. These results show that glucocorticoids directly inhibit insulin release in vivo and identify the pancreatic beta cell as an important target for the diabetogenic action of glucocorticoids.

Involvement of a C/EBP-like protein in the acquisition of responsiveness to glucocorticoid hormones during chick neural retina development.

The glucocorticoid receptor in chicken embryonic neural retina is expressed early in ontogeny, yet the tissue's response to the glucocorticoid hormone, i.e., induction of glutamine synthetase (GS), develops later, only during week 2 of ontogeny. Transient transfection of embryonic day 7 (E7) retinal cells, which are nonresponsive to glucocorticoids, with chimeric plasmids containing the chloramphenicol acetyltransferase reporter gene under the control of glucocorticoid-responsive promoters demonstrated that GR in E7 cells is a functional transactivating factor. We show that the limiting transcription factor that controls the developmental acquisition of responsiveness to glucocorticoids is similar to a CCAAT enhancer-binding protein (C/EBP). This protein recognizes a sequence in the promoter of the chick GS gene, which is required for eliciting the glucocorticoid response. Retinal C/EBP-like protein was not detected in the glucocorticoid-nonresponsive (E7) proliferating glioblasts but was found to be present in the glucocorticoid-responsive (E12) postmitotic cells. Premature expression of C/EBP in the nonresponsive E7 cells by transfection was shown to enhance the developmental acquisition of responsiveness to the glucocorticoid hormone, as deduced from the level of GS inducibility.

Expression level-dependent contribution of glucocorticoid receptor domains for functional interaction with STAT5.

The action of the glucocorticoid receptor (GR) on beta-casein gene transcription serves as a well-studied example of a case where the action of the GR is dependent on the activity of another transcription factor, STAT5. We have investigated the domain-requirement of the GR for this synergistic response in transfection experiments employing GR mutants and CV-1 or COS-7 cells. The results were influenced by the expression levels of the GR constructs. At low expression, STAT5-dependent transactivation by mutants of the GR DNA binding domain or N-terminal transactivation domain was impaired and the antiglucocorticoid RU486 exhibited a weak agonistic activity. When the N-terminal region of the GR was exchanged with the respective domain of the progesterone receptor, STAT5-dependent transactivation was reduced at low and high expression levels. Only at high expression levels did the GR exhibit the properties of a coactivator and enhanced STAT5 activity in the absence of a functional DNA binding domain and of GR binding sites in the proximal region of the beta-casein gene promoter. Furthermore, at high GR expression levels RU486 was nearly as efficient as dexamethasone in activating transcription via the STAT5 dependent beta-casein gene promoter. The results reconcile the controversial issue regarding the DNA binding-independent action of the GR together with STAT5 and provide evidence that the mode of action of the GR depends not only on the type of the particular promoter at which it acts but also on the concentration of the GR. GR DNA binding function appears to be mandatory for beta-casein gene expression in mammary epithelial cells, since the promoter function is completely dependent on the integrity of GR binding sites in the promoter.

Estradiol receptor analysis in human breast cancer tissue by isoelectric focusing in polyacrylamide gel.

Isoelectric focusing in polyacrylamide gel combined with limited proteolysis is a simple and specific method for quantitation of estradiol receptors in breast cancer tissue. At least eight different samples can be analyzed simultaneously on one gel, and the whole procedure, including sample preparation, takes less than 7 hr. In comparison with sucrose gradient centrifugation, isoelectric focusing is more sensitive, possibly due to the short time (1.5 to 2 hr) needed for the analysis. Furthermore, only one incubation with tritium-labeled estradiol is needed for an analysis, which means that a smaller amount of tumor tissue is needed than for most other methods. This fact allows analysis of the estrogen receptor content in tumor material obtained from fine-needle biopsy.

Absence in glucocorticoid-resistant mouse lymphoma P1798 of a glucocorticoid receptor domain responsible for biological effects.

Glucocorticoid-resistant (CR), in contrast to glucocorticoid-sensitive (CS), mouse lymphoma P1798 was shown to lack antiglucocorticoid receptor immunoactivity. Antibodies raised against the purified rat liver glucocorticoid receptor (GR) cross-reacted with the GR from CS, but not with the GR from CR, P1798 lymphoma. Using highly specific antisera against the GR in an indirect competitive enzyme-linked immunosorbent assay, it was demonstrated that alpha-chymotrypsin digestion of the GR from CS P1798 lymphoma caused a separation of a "resistant-like" nonimmunogenic steroid and DNA-binding domain (Stokes' radius, 3.3 nm) from an immunoactive domain (Stokes' radius, 2.6 nm). In contrast to CS P1798 lymphoma, neither before nor after alpha-chymotrypsin digestion, immunoactivity could be found in the cytosol from CR P1798 lymphoma. This was assayed after chromatography on DNA-cellulose or gel filtration on Agarose A (0.5 m). These results suggest that the domain of the CS GR containing the immunoactive determinant(s), normally removed by limited proteolysis by alpha-chymotrypsin, appears to be missing in CR P1798 lymphoma cytosol. It seems that this domain plays an important role in the mechanism of action of glucocorticoids. This might suggest that a mutation has occurred affecting the genome resulting in defective transcription of the receptor gene(s) in CR P1798 lymphoma.

Characterization of an antiserum against the glucocorticoid receptor.

An immunoglobulin (IgG) fraction from serum of a rabbit immunized with a highly purified preparation of glucocorticoid receptor from rat liver cytosol contained specific antibodies to glucocorticoid receptor. This was shown following incubation of the [3H]triamcinolone acetonide-glucocorticoid receptor (TA-GR) complex with the IgG fraction by (I) adsorption of the [3H]triamcinolone acetonide-glucocorticoid receptor (TA-GR) complex with the IgG fraction by (I) adsorption of the [3H]TA-GR-antibody complex to protein A linked to Sepharose, (II) an increased sedimentation rate of the [3H]TA-GR-antibody complex compared to that of the [3H]TA-GR complex, and (III) an increased molecular size of the [3H]TA-GR-antibody complex when compared to that of the [3H]TA-GR complex as judged from gel filtration. The antibody fraction was characterized with regard to titer, cross-reactivity and specificity. The antibodies cross-reacted with the glucocorticoid receptor from various rat tissues (liver, thymus and hippocampus), as well as with the glucocorticoid receptor from human normal lymphocytes, chronic lymphatic leukemia cells and human hippocampus. In the rat liver, the antibody bound to both the nuclear and the cytosolic glucocorticoid receptor (stokes radius 6.1 nm). It did not cross-react with the proteolytic fragments of the glucocorticoid receptors, the 3.6 nm complex or the 1.9 nm complex. Binding of the antibodies was not seen to the androgen, estrogen or progestin receptors in rat or to rat serum transcortin. With an indirect competitive ELISA (enzyme-linked immunosorbent assay) combined with various separation techniques, based on different physicochemical principles, it was shown that the glucocorticoid receptor was the only detectable antibody binding protein from rat liver cytosol using this assay system. These findings also indicate an immunochemical similarity between glucocorticoid receptors in different tissues as well as in different species, but not between glucocorticoid receptors and other steroid hormone receptor proteins. The cytosolic and nuclear glucocorticoid receptors in rat liver were shown to be immunochemically similar.

Mitogen stimulation of T-cells increases c-Fos and c-Jun protein levels, AP-1 binding and AP-1 transcriptional activity.

We have analysed the effect of mitogenic lectins on c-Fos and c-Jun protein levels as well as on activator protein-1 (AP-1) binding and enhancer activity in Jurkat T-cells. Both c-Fos and c-Jun protein levels were increased after Con A and PHA stimulation. Since T-cell stimulation increases both intracellular Ca2+ and cAMP levels and activates protein kinase C (PKC), the possible involvement of these intracellular messengers in c-Fos and c-Jun induction was tested. PMA, which directly activates PKC, mimicked the effect of the lectins on c-Fos and c-Jun, but elevation of either intracellular Ca2+ or cAMP levels had little or no effect. The mitogen-induced increase of c-Fos and c-Jun immunoreactivity was inhibited by H-7, a kinase inhibitor with relatively high specificity for PKC, and less efficiently by H-8, a structurally related kinase inhibitor less active on PKC, but more active on cyclic nucleotide-dependent kinases. Con A stimulation was found to increase both binding of AP-1 to the AP-1 consensus sequence, TRE, and AP-1 enhancer activity, in Jurkat cells. PMA was also found to increase the AP-1 enhancer activity, whereas elevation of Ca2+ or cAMP had only minor effects. We conclude that stimulation with mitogenic lectins is sufficient to increase both c-Fos and c-Jun protein levels, AP-1 binding and AP-1 enhancer activity in Jurkat cells and that they act via mechanisms that could involve the activation of PKC.

Nuclear factor-kappa B repression in antiinflammation and immunosuppression by glucocorticoids.

The transcription factor nuclear factor-kappaB (NF-kappaB) directs transcription of a large number of key molecules in immunological and inflammatory responses. The recently discovered inhibition of transcriptional activity of this factor by the activated glucocorticoid receptor (GR) provides a molecular basis for the potent antiinflammatory and immunosuppressive properties of glucocorticoids. This repressive activity of the GR can function independently of transcriptional activity. Because it has been shown that certain steroid receptor ligands can differentially address transactivation and transrepression functions, it may be possible to develop ligands that specifically suppress NF-kappaB activity and, as a result, are more efficient in treatment of a variety of important chronic inflammatory diseases with less severe side effects than those of currently available drugs.

Regulation of glucocorticoid receptor expression: evidence for transcriptional and posttranslational mechanisms.

The mechanism of ligand-induced (homologous) down-regulation of the glucocorticoid receptor (GR) has been studied. Dexamethasone caused a down-regulation of the levels of GR mRNA and protein both in hepatoma tissue culture cells and rat liver in vivo. The decrease in the level of rat liver GR mRNA was due to a reduced transcription rate of the GR gene, as assessed by nuclear run-on transcription experiments. The half-life of GR mRNA in hepatoma tissue culture cells was determined to be approximately 4.5 h and was unaffected by dexamethasone. In addition to the transcriptional regulation of GR gene expression, a dexamethasone-dependent posttranslational modification in the rate of GR protein turnover was observed. In the absence of dexamethasone, GR protein half life was approximately 25 h whereas it decreased to approximately 11 h in the presence of hormone. Down-regulation of GR protein occurred with a 6- to 24-h delay as compared to the decline in GR mRNA. This is most likely due to the differences in half-lives of GR mRNA and protein, respectively. Our results suggest that auto-regulation of GR by its cognate ligand is complex and occurs at both transcriptional and posttranslational levels.

p57Kip2, a glucocorticoid-induced inhibitor of cell cycle progression in HeLa cells.

Glucocorticoids exert antiproliferative effects on a number of cell types, including the HeLa cervical carcinoma cell line. However, the mechanism responsible for the antiproliferative effect is poorly understood. In this report we have investigated the role of the recently identified cyclin-dependent kinase inhibitor (CDI) p57Kip2 in the antiproliferative effect conferred by glucocorticoids. When HeLa cells were treated with the synthetic glucocorticoid dexamethasone (DEX), the doubling time of exponentially growing cells increased 2-fold. Within 11 h of DEX treatment, this was accompanied by an accumulation of cells in the G1 phase of the cell cycle with a corresponding decreased proportion of cells in the S phase and decreased CDK2 activity. DEX treatment of the HeLa cells dramatically induced the protein and mRNA expression of the CDI p57Kip2. This induction was seen within 4 h of DEX treatment, preceding a major DEX-induced accumulation of cells in the G1 phase. DEX-induced mRNA expression of p57Kip2 did not require de novo protein synthesis, and the transcription of the p57Kip2 gene was increased as determined by a run-on transcription assay. Furthermore, DEX induction of p57Kip2 was not a consequence of the cell cycle arrest, since other growth inhibition signals did not result in strong p57Kip2 induction. Overexpression of p57Kip2 using HeLa cells stably transfected with a tetracycline-inducible vector showed that p57Kip2 is sufficient to reconstitute an antiproliferative effect similar to that seen in DEX-treated cells. Selective p57Kip2 expression by the tetracycline analog doxycycline to levels comparable to those observed on DEX induction resulted in a 1.7-fold increase in the doubling time and a shift of HeLa cells to the G1 phase as well as a decrease in CDK2 activity. Taken together, these results suggest that glucocorticoid treatment directly induces transcription of the p57Kip2 gene and that the p57Kip2 protein is involved in the glucocorticoid-induced antiproliferative effect.

Negative cross-talk between RelA and the glucocorticoid receptor: a possible mechanism for the antiinflammatory action of glucocorticoids.

Glucocorticoids are efficient antiinflammatory agents, and their effects include transcriptional repression of several cytokines and adhesion molecules. Whereas glucocorticoids down-regulate the expression of genes relevant during inflammation, nuclear factor (NF)-kappa B/Rel proteins function as important positive regulators of these genes. The expression of intercellular adhesion molecule-1 (ICAM-1), which plays an essential role in recruitment and migration of leukocytes to sites of inflammation, is also down-regulated by glucocorticoids. We found that a functional NF-kappa B site in the ICAM-1 promoter, which can be activated by either 12-O-tetradecanoylphorbol-13-acetate or tumor necrosis factor-alpha (TNF alpha), is also the target for glucocorticoids. In this report we present evidence that the ligand-activated glucocorticoid receptor (GR) is able to repress RelA-mediated activation of the ICAM-1 NF-kappa B site. Conversely, transcriptional activation by GR via a glucocorticoid response element is specifically repressed by RelA, but not by other NF-kappa B/Rel family members. Mutational analysis of GR demonstrates that the DNA binding domain and the ligand binding domain are required for the functional repression of NF-kappa B activation. Despite the importance of the DNA binding domain, we found that the transcriptional repression of NF-kappa B, mediated by GR, is not caused by binding of GR to the ICAM-1 NF-kappa B element, but by a physical interaction between the GR and RelA protein. The repressive effect of GR on NF-kappa B-mediated activation was not shared by other steroid/thyroid receptors. Only the progesterone receptor, which belongs to the same subfamily as GR and which possesses high homology with GR, was able to repress NF-kappa B-mediated transcription. These studies highlight a possible molecular mechanism that can explain the antiinflammatory effects of glucocorticoid treatment during inflammation.

Intracellular localization of the glucocorticoid receptor: evidence for cytoplasmic and nuclear localization.

Using a monospecific, monoclonal antibody against the glucocorticoid receptor (GR), an immunocytochemical study was performed to investigate the intracellular localization of GR both in the presence or absence of ligand. With all fixation methods tested (paraformaldehyde, acetic acid in ethanol, Bouin's fixative, and bensochinone in PBS), it was possible to obtain specific GR staining. Fixation with paraformaldehyde was chosen for further studies on the effect of permeabilization, using several concentrations of Triton X-100 or saponin. A rat Rueber hepatoma (H-4-II-E) and a human uterus carcinoma (NHIK 3025) cell line were used as well as cultured hepatocytes from normal rat. The accessibility of the different cell compartments after fixation and permeabilization was tested for by using antibodies against cellular constituents with known locations (i.e. core-nucleosome proteins and tubulin), in combination with the anti-GR antibody in double immunofluorescence staining experiments. The specific GR stain obtained with the indirect peroxidase antiperoxidase technique or with fluorescein isothiocyanate-labeled second antibodies was shown to be present both in the cytoplasm and in the nucleus. Staining of all cellular compartments was abolished (peroxidase antiperoxidase) or diminished (fluorescein isothiocyanate) if the monoclonal antibody was preincubated with a 90% pure GR preparation. These findings are in contrast to recently reported immunocytochemical studies, where a strict nuclear existence of the estrogen and progestin receptors has been reported. Consequently, generalizations with regard to steroid receptor localization cannot be made. Furthermore, an in vitro model is described, where the effect of dexamethasone administration upon the localization of receptor staining in H-4-II-E cells can be studied.

Mapping of glucocorticoid receptor immunoreactive neurons in the rat tel- and diencephalon using a monoclonal antibody against rat liver glucocorticoid receptor.

By means of a monoclonal antibody against the rat liver glucocorticoid receptor (GR) in combination with the indirect immunoperoxidase technique it has been possible to demonstrate GR-immunoreactive nerve and glial cell nuclei all over the tel- and diencephalon of the male rat. Strongly GR-immunoreactive nerve cell nuclei were only present in the parvocellular part of the paraventricular hypothalamic nucleus, in the anterior periventricular hypothalamic nucleus, in the ventral part of the mediobasal hypothalamus, and in the CA1 and CA2 subregion of the hippocampal formation. Within the paraventricular hypothalamic nucleus a substantial overlap exists between the GR-immunoreactive area and the CRF-immunoreactive area. Medium to high densities of moderately GR-immunoreactive nerve cell nuclei were present all over the cortical hemispheres. Medium densities of moderately GR-immunoreactive nerve cells were demonstrated in many thalamic nuclei and in the central amygdaloid nucleus. After adrenalectomy the GR immunoreactivity was predominantly located in the pericaryon. Upon acute corticosterone treatment of adrenalectomized male rats, the GR immunoreactivity was again mainly demonstrated in the nerve cell nuclei indicating that corticosterone can translocate GR from the cytoplasm to the cell nuclei. It is suggested that the hypothalamic GR may be involved in the regulation of especially CRF secretion but also in the secretion of other anterior pituitary hormones such as TRH and somatostatin.

Evidence that rodent epididymal sperm contain the Mr approximately 94,000 glucocorticoid receptor but lack the Mr approximately 90,000 heat shock protein.

Monoclonal antibodies directed against four different polypeptide epitopes on the Mr approximately 94,000 steroid-binding subunit of the rat liver cytosolic glucocorticoid receptor (GcR) were used to probe Western blots of epididymal spermatozoa from rats and mice. Two sperm polypeptides with apparent molecular weights of 94,000 (indistinguishable in size from the liver GcR subunit) and 150,000 reacted with these antibodies. Other polypeptides that are present in a wide variety of somatic cells [lamin-A, -B, and -C; topoisomerase-I; poly(ADP-ribose) polymerase; the 62-kilodalton internal nuclear matrix protein; the nucleolar protein B23; and histone H1] could not be detected in these preparations of spermatozoa, thus appearing to rule out contamination by somatic cells. Rat and mouse pachytene spermatocytes and round spermatids contained much lower amounts of the Mr approximately 94,000 and 150,000 polypeptides. These results suggested that the steroid-binding subunit of the GcR might be accumulated late in spermatogenesis. Consistent with this view, a 6-kilobase mRNA (identical in size to a mRNA detected in mouse somatic cell lines) was detected when Northern blots of mouse round spermatid RNA were probed with a cDNA to the steroid-binding GcR subunit. Although the results described above suggest the presence of GcR in rodent sperm, high affinity binding of glucocorticoids to epididymal sperm could not be detected in a whole cell binding assay. Further analysis revealed that the Mr approximately 90,000 heat shock protein (hsp90), a component reportedly required for high affinity ligand binding to the GcR, was present in early germ cells, but absent from rodent epididymal sperm. These results suggest that the Mr approximately 94,000 steroid-binding subunit of the GcR and an immunologically related Mr approximately 150,000 polypeptide are specifically accumulated during the later stages of rodent spermatogenesis, but are not assembled into receptor complexes capable of binding steroid. In addition, these results support the view that hsp90 is required for high affinity binding of glucocorticoids to the Mr approximately 94,000 GcR subunit in intact cells.

A hormone response element upstream from the human alcohol dehydrogenase gene ADH2 consists of three tandem glucocorticoid receptor binding sites.

The 5'-flanking region of the human gene encoding beta-alcohol dehydrogenase (ADH2) was shown by DNase I footprinting to contain three tandem binding sites for purified glucocorticoid receptor. The three binding sites lie very close together between nucleotide (nt) positions -245 and -171 with respect to the transcription start point. DNase I footprinting using a rat liver nuclear extract indicated a lack of protection of the glucocorticoid receptor binding sites, but protection of a sequence between nt -209 and -191 which partially overlaps the glucocorticoid receptor binding sites I and II. This site has homology with the known binding site for hepatocyte nuclear factor 1 (HNF1). ADH2 promoter DNA fragments containing various lengths of 5'-flanking sequences were fused upstream from the gene encoding chloramphenicol acetyltransferase (cat) and transfected into the HepG2 human hepatoma cell line. The resulting cat expression was subject to induction by dexamethasone in constructions containing ADH2 DNA between nt -272 and -171. This indicates that the glucocorticoid receptor binding sites identified by footprint analysis function as a glucocorticoid response element (GRE) in a liver cell line. Heterologous ADH-cat fusions, in which the ADH2-GRE was fused to the adenovirus major late promoter, exhibited glucocorticoid induction of cat expression in CV-1B cells when cotransfected with a glucocorticoid receptor expression vector. Glucocorticoid regulation in CV-1B was observed when either all three glucocorticoid receptor binding sites (sites 0, I, II) or the two distal sites (sites 0, I) were present. Overall, these results indicate that the ADH2 gene possesses a functional GRE which can potentially regulate expression transcriptionally.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of glucocorticoid receptor expression.

A limiting factor determining the sensitivity of a cell to glucocorticoid hormones is the intracellular concentration of the glucocorticoid receptor (GR) protein. By regulating the expression of GR the cell is able to adapt to both changes in its hormone environment and to the varying requirements for biological response. Studies on the regulation of GR expression have shown this to be a complex process which involves both transcriptional and posttranscriptional mechanisms. Although GR is more or less constitutively expressed in most tissues its concentration varies under different physiological conditions. GR expression is regulated by a number of different agents including factors which act through a second messenger pathway. This allows the cell to control glucocorticoid regulated gene expression through a complex but integrated hormonal network. Here we summarize our studies on GR regulation with emphasis on: i), GR autoregulation; ii), the effect of cAMP on GR expression, and iii), GR expression during fetal rat lung development.

Identification of a functional glucocorticoid response element in the promoter of the cyclin-dependent kinase inhibitor p57Kip2.

Glucocorticoids are known regulators of the cell cycle, normally exerting an anti-proliferative effect. We have previously shown that glucocorticoids stimulate expression of p57(Kip2), a member of the Cip/Kip family of cyclin-dependent kinase inhibitors which, in some cell types, may account for the anti-proliferative responses seen after glucocorticoid treatment. The induction of p57(Kip2) involves primary transcriptional effects where no de novo protein synthesis is necessary, suggesting a direct interaction of the glucocorticoid receptor with the p57(Kip2) gene. In this study we have identified a functional glucocorticoid response element (GRE), located 5 kilo bases (kb) upstream of the transcription start site in the human p57(Kip2) promoter. This GRE was functional also when isolated, suggesting a direct transcriptional effect of the glucocorticoid receptor. Furthermore, mutation of this GRE abolished glucocorticoid induction of the reporter gene, whereas mutation of a nearby Sp1 site did not. Using electrophoretic mobility shift assays, we have shown that the -5 kb p57(Kip2) promoter GRE was able to compete with a well-known GRE for glucocorticoid receptor binding. Sequence comparisons with the mouse genome showed that this GRE is highly conserved, further strengthening the biological importance of this site. All these data emphasize the involvement of this GRE in the glucocorticoid-mediated induction of p57(Kip2) expression.

Mapping and computer assisted morphometry and microdensitometry of glucocorticoid receptor immunoreactive neurons and glial cells in the rat central nervous system.

By means of a monoclonal mouse immunoglobulin G2a antibody against the rat liver glucocorticoid receptor and the indirect immunoperoxidase technique, the distribution of glucocorticoid receptors in neuronal and glial cell populations was mapped in the central nervous system of the male rat. The mapping was complemented by computer-assisted morphometric and microdensitometric evaluation of glucocorticoid receptor immunoreactivity in many brain regions. The quantitative analysis allowed us to achieve for the first time an objective characterization of glucocorticoid receptor distribution in the CNS, thus avoiding the ambiguities of previous mapping studies based on subjective evaluations. In addition, a taxonomic analysis of central nervous system regions containing glucocorticoid receptor immunoreactivity was carried out utilizing the quantitative parameters obtained in the morphometric evaluation. Nuclei of neuronal and glial cells containing glucocorticoid receptor immunoreactivity were detected in a widespread, but still highly heterogeneous, fashion in the central nervous system, underlining the view that glucocorticoids can control a large number of central nervous system target cells via effects on gene expression. Many nerve cell populations have been shown to contain substantial amounts of nuclear glucocorticoid receptor immunoreactivity, whereas only a low density of glial cells, in both gray and white matter, show nuclear glucocorticoid receptor immunoreactivity. Thus, in most brain areas, the major target for glucocorticoids appears to be the nerve cells. Interestingly, an inverse correlation was found in the regional density of glucocorticoid receptor-immunoreactive nerve and glial cells, suggesting that glucocorticoids may influence a brain area either via glial cells or, more frequently, via nerve cells. The results on mapping highlight the impact of glucocorticoids in areas both traditionally and not traditionally involved in stress responses. The distribution of glucocorticoid receptor immunoreactivity also emphasizes a role of glucocorticoids in the regulation of the afferent regions of the basal ganglia and the cerebellar cortex, and of both afferent and efferent layers of the cerebral cortex. Glucocorticoid receptor immunoreactivity is widely distributed over the thalamus, probably leading to modulation of activity in the various thalamocortical pathways transmitting inter alia specific sensory information to the cerebral cortex. Many unspecific afferents to the cerebral cortex are potentially regulated by glucocorticoid receptors such as the noradrenaline and 5-hydroxytryptamine afferents, since their nerve cells of origin contain strong glucocorticoid receptor immunoreactivity. Eight brain regions involving sensory, motor and limbic areas were shown to have a similarity with regard to glucocorticoid receptor-immunoreactive parameters at the level of 95%. The density of glucocorticoid receptor-immunoreactive nerve cells appeared to be the main factor in determining such a very high level of similarity. Overall, our results emphasize that glucocorticoids may appropriately tune networks of different areas to obtain optimal integration and in this way improve survival of the animal under challenging conditions.

Colocalization of fos- and glucocorticoid receptor-like immunoreactivities in the rat amygdaloid complex after immobilization stress.

In the present paper we demonstrate the effect of immobilization stress on c-fos-like immunoreactivity (Fos-LI) in the rat amygdaloid complex. Furthermore, since the subnuclei of the amygdaloid complex contain numerous glucocorticoid receptor-immunoreactive (GR-IR) neurons, we also studied the possible colocalization of GR- and Fos-LI ie. Fos-Lls and the action of a synthetic glucocorticoid, dexamethasone, and an anti-glucocorticoid, RU 38486, on Fos-LI. Immobilization stress caused a remarkable increase in the number of the Fos-IR neurons in all the subnuclei of the amygdaloid complex except in the lateral nucleus. The majority of Fos-IR neurons also contained GR-LI, with the highest colocalization in the central amygdaloid nucleus. A similar induction of Fos-LI after immobilization was seen in the hypothalamic paraventricular nucleus and almost all the Fos-IR neurons in this nucleus also exhibited GR-LI. Treatments with dexamethasone or RU 38486 prior to stress did not have any marked effect on Fos-LI when compared to stress alone. The present findings suggest that Fos may function as a transcriptional regulator in the amygdaloid complex after stress and affect the synthesis of neurotransmitters and receptors in the amygdaloid neurons. Since we did not observe any effect of dexamethasone or RU 38486 on Fos-LI, it is likely that glucocorticoids do not directly regulate the expression of the c-fos gene or the formation of Fos protein. In view of the fact that Fos is capable of forming a stabile complex with GR and repress the transactivational capacity of GR, Fos may inhibit the negative feedback effect of circulating glucocorticoids and thus maintain elevated plasma glucocorticoid levels in stress.