Descripción: Cytokine-induced glucocorticoid secretion and glucocorticoid inhibition of cytokine synthesis and pleiotropic actions act as important safeguards in preventing cytokine overreaction. We found that TNF-α increased glucocorticoid-induced transcriptional activity of the glucocorticoid receptor (GR) via the glucocorticoid response elements (GRE) in L-929 mouse fibroblasts transfected with a glucocorticoid-inducible reporter plasmid. In addition, TNF-α also enhanced GR number. The TNF-α effect on transcriptional activity was absent in other cell lines that express TNF-α receptors but not GRs, and became manifest when a GR expression vector was cotransfected, indicating that TNF-α, independent of any effect it may have on GR number, has a stimulatory effect on the glucocorticoid-induced transcriptional activity of the GR. Moreover, TNF-α increased GR binding to GRE. As a functional biological correlate of this mechanism, priming of L- 929 cells with a low (noncytotoxic) dose of TNF-α significantly increased the sensitivity to glucocorticoid inhibition of TNF-α-induced cytotoxicity/apoptosis. TNF-α and IL-1β had the same stimulatory action on glucocorticoid-induced transcriptional activity of the GR via the GRE, in different types of cytokine/glucocorticoid target cells (glioma, pituitary, epithelioid). The phenomenon may therefore reflect a general molecular mechanism whereby cytokines modulate the transcriptional activity of the GR, thus potentiating the counterregulation by glucocorticoids at the level of their target cells.

Descripción: The cellular resistance to tumor necrosis factor (TNF) of most cell types has been attributed to both a protective pathway induced by this cytokine and the preexistence of protective factors in the target cell. NF-κB has been postulated as one of the principal factors involved in antiapoptotic gene expression control on TNF-resistant cells. We have previously shown that glucocorticoids protect the naturally TNF-sensitive L-929 cells from apoptosis. Here we analyze the role of NF-κB and glucocorticoids on TNF-induced apoptosis in L-929 cells. We found that inhibition of NF-κB enhanced the sensitivity to TNF-induced apoptosis. Glucocorticoids inhibited NF-κB transactivation via IκB induction. Moreover, glucocorticoids protected from TNF-induced apoptosis even when NF-κB activity was inhibited by stable or transient expression of the superrepressor IκB. These results demonstrate that although glucocorticoids inhibit NF-κB transactivation in these cells, this is not required for their protection from TNF-induced apoptosis. (C) 2000 Elsevier Science B.V.

Descripción: The bcl-X gene plays a critical role in apoptosis. Six different isoforms generated by tissue-specific promoter usage and alternative splicing were described. Some of them exert opposite effects on cell death. In mammary epithelial cells glucocorticoids induce bcl-X expression and increase the ratio bcl-XL (antiapoptotic)/bcl-XS (apoptotic) by activating P4 promoter, which contains two hormone response elements. Here we show that, on mouse thymocytes and T lymphocyte derivative S49 cells, glucocorticoids inhibited transcription from P4 and decreased the ratio bcl-X L/bcl-XS favoring apoptosis. Upon hormonal treatment, glucocorticoid receptor (GR), steroid receptor coactivator-1, and RNA polymerase II were transiently recruited to P4 promoter, whereas STAT5B was also recruited but remained bound. Concomitant with the release of GR, silencing mediator for retinoic acid receptor and thyroid hormone receptor and histone deacetylase 3 were recruited, histone H3 was deacetylated, and RNA polymerase II left the promoter. Inhibition of STAT5 activity reverted glucocorticoid repression to activation of transcription and was accompanied by stable recruitment of GR and RNA polymerase II to P4. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: It has been shown that the molecular mechanism by which cytokines and glucocorticoids mutually antagonize their functions involves a mutual glucocorticoid receptor (GR)/nuclear factor-κB (NF-κB) transrepression. Here we report a role for the nuclear receptor coactivator RAC3, in modulating NF-κB transactivation. We found that RAC3 functions as a coactivator by binding to the active form of NF-κB and that overexpression of RAC3 restores GR-dependent transcription neglecting GR/NF-κB transrepression. The competition between GR and NF-κB for binding to RAC3 may represent a general mechanism by which both transcription factors mutually antagonize their activity. (C) 2000 Federation of European Biochemical Societies.

Descripción: Glucocorticoids are potent regulators of the innate immune response, and alteration in this inhibitory feedback has detrimental consequences for the neural tissue. This study profiled and investigated functionally candidate genes mediating this switch between cell survival and death during an acute inflammatory reaction subsequent to the absence of glucocorticoid signaling. Oligonucleotide microarray analysis revealed that following lipopolysaccharide (LPS) intracerebral administration at striatum level, more modulated genes presented transcription impairment than exacerbation upon glucocorticoid receptor blockage. Among impaired genes we identified ceruloplasmin (Cp), which plays a key role in iron metabolism and is implicated in a neurodegenative disease. Microglial and endothelial induction of Cp is a natural neuroprotective mechanism during inflammation, because Cp-deficient mice exhibited increased iron accumulation and demyelination when exposed to LPS and neurovascular reactivity to pneumococcal meningitis. This study has identified genes that can play a critical role in programming the innate immune response, helping to clarify the mechanisms leading to protection or damage during inflammatory conditions in the CNS. © 2007 Glezer et al.

Descripción: Background: The glucocorticoid receptor (GR) is a transcription factor that regulates gene expression in a ligand-dependent fashion. This modular protein is one of the major pharmacological targets due to its involvement in both cause and treatment of many human diseases. Intense efforts have been made to get information about the molecular basis of GR activity. Methodology/Principal Findings: Here, the behavior of four GR-ligand complexes with different glucocorticoid and antiglucocorticoid properties were evaluated. The ability of GR-ligand complexes to oligomerize in vivo was analyzed by performing the novel Number and Brightness assay. Results showed that most of GR molecules form homodimers inside the nucleus upon ligand binding. Additionally, in vitro GR-DNA binding analyses suggest that ligand structure modulates GRDNA interaction dynamics rather than the receptor's ability to bind DNA. On the other hand, by coimmunoprecipitation studies we evaluated the in vivo interaction between the transcriptional intermediary factor 2 (TIF2) coactivator and different GR-ligand complexes. No correlation was found between GR intranuclear distribution, cofactor recruitment and the homodimerization process. Finally, Molecular determinants that support the observed experimental GR LBD-ligand/TIF2 interaction were found by Molecular Dynamics simulation. Conclusions/Significance: The data presented here sustain the idea that in vivo GR homodimerization inside the nucleus can be achieved in a DNA-independent fashion, without ruling out a dependent pathway as well. Moreover, since at least one GR-ligand complex is able to induce homodimer formation while preventing TIF2 coactivator interaction, results suggest that these two events might be independent from each other. Finally, 21-hydroxy-6,19-epoxyprogesterone arises as a selective glucocorticoid with potential pharmacological interest. Taking into account that GR homodimerization and cofactor recruitment are considered essential steps in the receptor activation pathway, results presented here contribute to understand how specific ligands influence GR behavior. © 2010 Presman et al.

Descripción: The antiapoptotic effect of melatonin (MEL) has been described in several systems. In particular, MEL inhibits glucocorticoid-mediated apoptosis. Our group previously demonstrated that in the thymus, MEL inhibits the release of Cytochrome C from mitochondria and the dexamethasone-dependent increase of bax mRNA levels. In this study we analyzed the ability of MEL to regulate the activation of the glucocorticoid receptor (GR) in mouse thymocytes. We found that even though the methoxyindole does not affect the ligand binding capacity of the receptor, it impairs the steroid-dependent nuclear translocation of the GR and also prevents transformation by blocking the dissociation of the 90-kDa heat shock protein. Coincubation of the methoxyindole with dexamethasone did not affect the expression of a reporter gene in GR-transfected Cos-7 cells or HC11 and L929 mouse cell lines that express Mel-1a and retinoid-related orphan receptor-α (RORα) receptors. Therefore, the antagonistic effect of MEL seems to be specific for thymocytes, in a Mel 1a- and RORα-independent manner. In summary, the present results suggest a novel mechanism for the antagonistic action of MEL on GR-mediated effects, which involves the inhibition of 90-kDa heat shock protein dissociation and the cytoplasmic retention of the GR. Copyright © 2006 by The Endocrine Society.

Descripción: Rapid, ligand-dependent movement of glucocorticoid receptors (GR) from cytoplasm to the nucleus is hsp90-dependent, and much of the movement system has been defined. GR-hsp90 heterocomplexes isolated from cells contain one of several hsp90-binding immunophilins that link the complex to cytoplasmic dynein, a molecular motor that processes along microtubular tracks to the nucleus. The immunophilins link to dynein indirectly via the dynamitin component of the dynein-associated dynactin complex (Galigniana, M. D., Harrell, J. M., O'Hagen, H. M., Ljungman, M., and Pratt, W. B. (2004) J. Biol. Chem. 279, 22483-22489). Although it is known that rapid, hsp90-dependent GR movement requires intact microtubules, it has not been shown that the movement is dynein-dependent. Here, we show that overexpression of dynamitin, which blocks movement by dissociating the dynein motor from its cargo, inhibits ligand-dependent movement of the GR to the nucleus. We show that native GR·hsp90·immnunophilin complexes contain dynamitin as well as dynein and that GR heterocomplexes isolated from cytosol containing paclitaxel and GTP to stabilize microtubules also contain tubulin. The complete movement system, including the dynein motor complex and tubulin, can be assembled under cell-free conditions by incubating GR immune pellets with paclitaxel/GTP-stabilized cytosol prepared from GR - L cells. This is the first evidence that the movement of a steroid receptor is dynein-dependent, and it is the first isolation of a steroid receptor bound to the entire system that determines its retrograde movement.

Descripción: Glucocorticoid receptor (GR) is cytoplasmic in the absence of ligand and localizes to the nucleus after steroid binding. Previous evidence demonstrated that the hsp90-based heterocomplex bound to GR is required for the efficient retrotransport of the receptor to the nuclear compartment. We examined the putative association of GR and its associated chaperone heterocomplex with structures of the nuclear pore. We found that importin β and the integral nuclear pore glycoprotein Nup62 interact with hsp90, hsp70, p23, and the TPR domain proteins FKBP52 and PP5. Nup62 and GR were able to interact in a more efficient manner when chaperoned by the hsp90-based heterocomplex. Interestingly, the binding of hsp70 and p23 to Nup62 does not require the presence of hsp90, whereas the association of FKBP52 and PP5 is hsp90 dependent, as indicated by the results of experiments where the hsp90 function was disrupted with radicicol. The ability of both FKBP52 and PP5 to interact with Nup62 was abrogated in cells overexpressing the TPR peptide. Importantly, GR cross-linked to the hsp90 heterocomplex was able to translocate to the nucleus in digitonin-permeabilized cells treated with steroid, suggesting that GR could pass through the pore in its untransformed state. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Descripción: Confocal microscopy images revealed that the tetratricopeptide repeat motif (TPR) domain immunophilin FKBP51 shows colocalization with the specific mitochondrial marker Mito-Tracker. Signal specificity was tested with different antibodies and by FKBP51 knockdown. This unexpected subcellular localization of FKBP51 was confirmed by colocalization studies with other mitochondrial proteins, biochemical fractionation, and electron microscopy imaging. Interestingly, FKBP51 forms complexes in mitochondria with the glucocorticoid receptor and the Hsp90/Hsp70-based chaperone heterocomplex. Although Hsp90 inhibitors favor FKBP51 translocation from mitochondria to the nucleus in a reversible manner, TPR domain-deficient mutants of FKBP51 are constitutively nuclear and fully excluded from mitochondria, suggesting that a functional TPR domain is required for its mitochondrial localization. FKBP51 overexpression protects cells against oxidative stress, whereas FKBP51 knockdown makes them more sensitive to injury. In summary, this is the first demonstration that FKBP51 is a major mitochondrial factor that undergoes nuclear-mitochondrial shuttling, an observation that may be related to antiapoptotic mechanisms triggered during the stress response. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: The antiapoptotic effect of melatonin has been described in several systems. In this study, the antagonistic effect of the methoxyindole on dexamethasone-induced apoptosis in mouse thymocytes was examined. Melatonin decreased both DNA fragmentation, and the number of annexin V-positive cells incubated in the presence of dexamethasone. Analysis of the expression of the members of the Bcl-2 family indicated that the synthetic glucocorticoid increased Bax protein levels without affecting the levels of Bcl-2, Bcl-X L, Bcl-X S, or Bak. This effect correlated with an increase in thymocytes bax mRNA levels. Dexamethasone also increased the release of cytochrome C from mitochondria. All of these effects were reduced in the presence of melatonin, which was ineffective per se on these parameters. In addition, the involvement of cAMP on glucocorticoid/melatonin antagonism was examined. Both melatonin and dexamethasone decreased the levels of this nucleotide in mouse thymocytes, indicating that the antagonistic action between both hormones involves a cAMP-independent pathway. In summary, the present results suggest that the antiapoptotic effect of melatonin on glucocorticoid-treated thymocytes would be a consequence of an inhibition of the mitochondrial pathway, presumably through the regulation of Bax protein levels.

Descripción: Background: Compound A (CpdA) is a dissociating non-steroidal glucocorticoid receptor (GR) ligand which has anti-inflammatory properties exerted by down-modulating proinflammatory gene expression. By favouring GR monomer formation, CpdA does not enhance glucocorticoid (GC) response element-driven gene expression, resulting in a reduced side effect profile as compared to GCs. Considering the importance of Th1/Th2 balance in the final outcome of immune and inflammatory responses, we analyzed how selective GR modulation differentially regulates the activity of T-bet and GATA-3, master drivers of Th1 and Th2 differentiation, respectively. Results: Using Western analysis and reporter gene assays, we show in murine T cells that, similar to GCs, CpdA inhibits T-bet activity via a transrepressive mechanism. Different from GCs, CpdA induces GATA-3 activity by p38 MAPK-induction of GATA-3 phosphorylation and nuclear translocation. CpdA effects are reversed by the GR antagonist RU38486, proving the involvement of GR in these actions. ELISA assays demonstrate that modulation of T-bet and GATA-3 impacts on cytokine production shown by a decrease in IFN-γ and an increase in IL-5 production, respectively. Conclusions: Taken together, through their effect favoring Th2 over Th1 responses, particular dissociated GR ligands, for which CpdA represents a paradigm, hold potential for the application in Th1-mediated immune disorders. © 2012 Liberman et al.

Descripción: Specific receptors for the different gp130 cytokines, as well as the cytokines themselves, are expressed in anterior pituitary cells, providing the basis for the regulation of hormone secretion and cell growth (Figure 2). During an inflammatory response, both IL-6 and LIF increase (15, 17). LPS stimulates intrapituitary IL-6 production in FS cells via specific Toll receptors using the p38 MAPK-NF-κB pathway (20). Anti-IL-6 antibodies block the ACTH response of rat anterior pituitary cell cultures to LPS, showing the involvement of locally produced IL-6 (U. Renner et al., unpublished observations). Thus, during acute or chronic inflammation or infection, systemic, hypothalamic, or hypophyseal gp 130 cytokines may act on anterior pituitary cells, integrating the neuroendocrine response. The action of gp130 cytokines through the STAT3 transcription factor represents a powerful mechanism for regulation of pituitary corticotroph function. In response to different stressful stimuli, CRH stimulates the corticotrophs through cAMP/protein kinase A-mediated and calcium-mediated pathways and AP-1, CREB, and Nurr transcription factors. Cytokines may act on corticotrophs through different mechanisms; whereas IL-1 acts through Nur77, gp130 employs STAT3 for transcriptional activation. Cooperation between STAT3 and other transcription factors, such as NF-κB, AP-1, or the glucocorticoid receptor, has been described in other tissues (6), but it remains to be established whether this occurs in the pituitary. Future research clarifying the molecular mechanisms of gp130 action on pituitary cells will provide new clues regarding their involvement in neuro-endocrine responses to immune stimulation and will be of great importance for understanding pituitary pathophysiology.

Descripción: The mammary epithelium undergoes cyclical periods of cellular proliferation, differentiation, and regression. During lactation, the signal transducer and activator of transcription factor (STAT)-5A and the glucocorticoid receptor (GR) synergize to induce milk protein expression and also act as survival factors. During involution, STAT3 activation mediates epithelial cell apoptosis and mammary gland remodeling. It has been shown that the administration of glucocorticoids at weaning prevents epithelial cell death, probably by extracellular matrix breakdown prevention. Our results show that the synthetic glucocorticoid dexamethasone (DEX) modulates STAT5A and STAT3 signaling and inhibits apoptosis induction in postlactating mouse mammary glands, only when administered within the first 48 h upon cessation of suckling. DEX administration right after weaning delayed STAT5A inactivation and degradation, preserving gene expression of target genes as β-casein (bcas) and prolactin induced protein (pip). Weaning-triggered GR down-regulation is also delayed by the hormone treatment. Moreover, DEX administration delayed STAT3 activation and translocation into epithelial cells nuclei. In particular, DEX treatment impaired the increment in gene expression of signal transducer subunit gp130, normally up-regulated from lactation to involution and responsible for STAT3 activation. Therefore, the data shown herein indicate that glucocorticoids are able to modulate early involution by controlling the strong cross talk that GR, STAT5, and STAT3 pathways maintains in the mammary epithelium. Copyright © 2010 by The Endocrine Society.

Descripción: Prior to this work, we found that adrenal as well as extra-adrenal factors activate the response of renal l 11β-hydroxysteroid dehydrogenase 2 to stressful situations. These results -showing ways through which the organism hinders the pathological occupation of mineralocorticoid receptors by glucocorticoids leading to sodium retention and hypertension- prompted the present study on the nature of the above-mentioned extra-adrenal factors. Serotonin was chosen because of its properties as a widely distributed neurohormone, known to interact with glucocorticoids at many sites, also exhibiting increased levels and effects under stressful situations. We studied serotonin effects on 11β-hydroxysteroid dehydrogenase 2 activity in a cell line derived from distal nephron polarized-epithelium, employing 3H-corticosterone as substrate. The end-product, 3H-11- dehydrocorticosterone was separated from the substrate by HPLC and quantified. Serotonin stimulated 11β-hydroxysteroid dehydrogenase 2 activity only at 2nM and 25pM, the magnitude of the response depending also on substrate concentration. The stimulation was blocked by the specific inhibitors methiothepin and ketanserin. We postulate that the organism partially prevents renal mineralocorticoid receptor occupancy by glucocorticoids, circulating at enhanced levels under stressful situations, through serotonin-mediated catabolic regulation of the 11β-hydroxysteroid dehydrogenase 2 activity. Given many, mostly positive, interactions between both hormones, this might eventually pave the way to studies on a new regulatory axis.