Glucocorticoid Receptor Transactivation Research Articles

Mitogen- and stress-activated protein kinase 1 MSK1 regulates glucocorticoid response element promoter activity in a glucocorticoid concentration-dependent manner

The glucocorticoid receptor is a nuclear receptor, and can be activated by glucocorticoid ligands. Mitogen- and stress-activated protein kinase (MSK1), when activated by p38 and ERK mitogen-activated protein kinases (MAPKs), plays a major role in chromatin relaxation via phosphorylation of histone H3 S10. The glucocorticoid receptor can target MSK1 as part of its anti-inflammatory mechanism. Here, we studied the converse mechanism, i.e. the impact of MSK1 on glucocorticoid receptor-mediated transactivation.Upstream MSK1-activating kinases concentration-dependently enhanced glucocorticoid response element (GRE)-regulated promoter activity. Correspondingly, MSK1 inhibition, via H89, or combined p38 and ERK MAPK inhibition, via SB203580 and U0126, diminished maximally stimulated GRE-regulated promoter activity using high concentrations of glucocorticoids. Concomitantly, the combination of these agents does not seem to alter site-specific phosphorylations of murine glucocorticoid receptor S212 or S220. Paradoxically, we reveal that a sub-maximally activated GRE-mediated promoter activity, by using lower concentrations of glucocorticoids, is consistently enhanced by H89 or a combination of SB203580 and U0126, irrespective of the GRE promoter context. Furthermore, we show that the glucocorticoid-induced nucleocytoplasmic translocation of MSK1 occurs in a glucocorticoid concentration-dependent manner. The observed glucocorticoid concentration-dependent effect of MSK1 or MAPK inhibition on glucocorticoid receptor transactivation warrants further research into the applicability of combined glucocorticoid and kinase inhibitor strategies for anti-inflammatory purposes.

Pro-Asthmatic Cytokines Regulate Unliganded and Ligand-Dependent Glucocorticoid Receptor Signaling in Airway Smooth Muscle

To elucidate the regulation of glucocorticoid receptor (GR) signaling under pro-asthmatic conditions, cultured human airway smooth muscle (HASM) cells were treated with proinflammatory cytokines or GR ligands alone and in combination, and then examined for induced changes in ligand-dependent and -independent GR activation and downstream signaling events. Ligand stimulation with either cortisone or dexamethsone (DEX) acutely elicited GR translocation to the nucleus and, comparably, ligand-independent stimulation either with the Th2 cytokine, IL-13, or the pleiotropic cytokine combination, IL-1β/TNFα, also acutely evoked GR translocation. The latter response was potentiated by combined exposure of cells to GR ligand and cytokine. Similarly, treatment with either DEX or IL-13 alone induced GR phosphorylation at its serine-211 residue (GRSer211), denoting its activated state, and combined treatment with DEX+IL-13 elicited heightened and sustained GRSer211 phosphorylation. Interestingly, the above ligand-independent GR responses to IL-13 alone were not associated with downstream GR binding to its consensus DNA sequence or GR transactivation, whereas both DEX-induced GR:DNA binding and transcriptional activity were significantly heightened in the presence of IL-13, coupled to increased recruitment of the transcriptional co-factor, MED14. The stimulated GR signaling responses to DEX were prevented in IL-13-exposed cells wherein GRSer211 phosphorylation was suppressed either by transfection with specific serine phosphorylation-deficient mutant GRs or treatment with inhibitors of the MAPKs, ERK1/2 and JNK. Collectively, these novel data highlight a heretofore-unidentified homeostatic mechanism in HASM cells that involves pro-asthmatic cytokine-driven, MAPK-mediated, non-ligand-dependent GR activation that confers heightened glucocorticoid ligand-stimulated GR signaling. These findings raise the consideration that perturbations in this homeostatic cytokine-driven GR signaling mechanism may be responsible, at least in part, for the insensirtivity to glucocorticoid therapy that is commonly seen in individuals with severe asthma.

Dexamethasone-Mediated Changes in Adipose Triacylglycerol Metabolism Are Exaggerated, Not Diminished, in the Absence of a Functional GR Dimerization Domain

The glucocorticoid (GC) receptor (GR) has multiple effector mechanisms, including dimerization-mediated transactivation of target genes via DNA binding and transcriptional repression mediated by protein-protein interactions. Much attention has been focused on developing selective GR modulators that would dissociate adverse effects from therapeutic anti-inflammatory effects. The GR(dim/dim) mouse has a mutation in the dimerization domain of GR and has been shown to have attenuated transactivation with intact repression. To understand the role of GR dimerization-dependent targets in multiple tissues, we measured metabolic fluxes through several disease-relevant GC target pathways using heavy water labeling and mass spectrometry in wild-type and GR(dim/dim) mice administered the potent GC dexamethasone (DEX). Absolute triglyceride synthesis was increased in both wild-type and GR(dim/dim) mice by DEX in the inguinal and epididymal fat depots. GR(dim/dim) mice showed an exaggerated response to DEX in both depots. De novo lipogenesis was also greatly increased in both depots in response to DEX in GR(dim/dim), but not wild-type mice. In contrast, the inhibitory effect of DEX on bone and skin collagen synthesis rates was greater in wild-type compared with GR(dim/dim) mice. Wild-type mice were more sensitive to DEX-dependent decreases in insulin sensitivity than GR(dim/dim) mice. Wild-type and GR(dim/dim) mice were equally sensitive to DEX-dependent decreases in muscle protein synthesis. Chronic elevation of GCs in GR(dim/dim) mice results in severe runting and lethality. In conclusion, some metabolic effects of GC treatment are exaggerated in adipose tissue of GR(dim/dim) mice, suggesting that selective GR modulators based on dissociating GR transactivation from repression should be evaluated carefully.

PA1 Protein, a New Competitive Decelerator Acting at More than One Step to Impede Glucocorticoid Receptor-mediated Transactivation

Numerous cofactors modulate the gene regulatory activity of glucocorticoid receptors (GRs) by affecting one or more of the following three major transcriptional properties: the maximal activity of agonists (A(max)), the potency of agonists (EC(50)), and the partial agonist activity of antisteroids (PAA). Here, we report that the recently described nuclear protein, Pax2 transactivation domain interaction protein (PTIP)-associated protein 1 (PA1), is a new inhibitor of GR transactivation. PA1 suppresses A(max), increases the EC(50), and reduces the PAA of an exogenous reporter gene in a manner that is independent of associated PTIP. PA1 is fully active with, and strongly binds to, the C-terminal half of GR. PA1 reverses the effects of the coactivator TIF2 on GR-mediated gene induction but is unable to augment the actions of the corepressor SMRT. Analysis of competition assays between PA1 and TIF2 with an exogenous reporter indicates that the kinetic definition of PA1 action is a competitive decelerator at two sites upstream from where TIF2 acts. With the endogenous genes IGFBP1 and IP6K3, PA1 also represses GR induction, increases the EC(50), and decreases the PAA. ChIP and re-ChIP experiments indicate that PA1 accomplishes this inhibition of the two genes via different mechanisms as follows: PA1 appears to increase GR dissociation from and reduce GR transactivation at the IGFBP1 promoter regions but blocks GR binding to the IP6K3 promoter. We conclude that PA1 is a new competitive decelerator of GR transactivation and can act at more than one molecularly defined step in a manner that depends upon the specific gene.

Respiratory syncytial virus (RSV) suppression of glucocorticoid receptor phosphorylation does not account for repression of transactivation

Respiratory syncytial virus (RSV)-induced bronchiolitis in infants, although inflammatory in nature, is not responsive to glucocorticoids. We have recently shown that RSV-infected lung epithelial cells have impaired glucocorticoid receptor (GR)-mediated transactivation. In this study, we show that the N-terminal region of GR is required for RSV repression of GR transactivation and that RSV infection of lung epithelial cells reduces ligand-dependent GR phosphorylation at serine 211 and serine 226. However, we also show that these changes in GR phosphorylation do not account for the RSV repression of GR transactivation suggesting other regions of the GR N-terminus must also be involved.

The Plant-Derived Glucocorticoid Receptor Agonist Endiandrin A Acts as Co-Stimulator of Colonic Epithelial Sodium Channels (ENaC) via SGK-1 and MAPKs

In a search for secondary plant compounds that bind to the glucocorticoid receptor (GR), the cyclobutane lignan endiandrin A was discovered from the rainforest tree Endiandra anthropophagorum Domin. Our present study aims to characterize the effect of endiandrin A on GR-dependent induction of colonic sodium transport. The effect of endiandrin A was analyzed in GR-expressing colonic HT-29/B6 cells (HT-29/B6-GR). GR transactivation and subcellular localization were investigated by reporter gene assay and immunofluorescence. Epithelial sodium channel (ENaC) was analyzed by qRT-PCR and by measuring amiloride-sensitive short-circuit current (Isc) in Ussing chambers. Endiandrin A (End A) has been identified as GR receptor binder. However, it did not cause significant GR transactivation as pGRE-luciferase activity was only 7% of that of the maximum effect of dexamethasone. Interestingly, endiandrin A had a significant impact on dexamethasone-dependent sodium absorption in cells co-exposed to tumor necrosis factor (TNF)-α. This was in part due to up-regulation of β- and γ-ENaC subunit expression. Endiandrin A potentiated GR-mediated transcription by increasing GR protein expression and phosphorylation. It inhibited c-Jun N-terminal kinase (JNK) activation induced by dexamethasone and/or TNF-α and increased levels of GR localized to the nucleus. Additionally, endiandrin A increased the serum- and glucocorticoid-induced kinase (sgk)-1 via activation of p38. Finally, the regulation of ENaC function by endiandrin A was confirmed in rat native colon. In conclusion, endiandrin A potentiates glucocorticoid-driven activation of colonic epithelial sodium channels via JNK inhibition and p38 activation due to transcriptional up-regulation of β- and γ-ENaC-subunits along with induction of sgk-1.

Modulation of transcriptional mineralocorticoid receptor activity by nitrosative stress

The mineralocorticoid receptor (MR) plays an important role in salt and water homeostasis and pathological tissue modifications, such as cardiovascular and renal fibrosis. Importantly, MR activation by aldosterone per se is not sufficient for the deleterious effects but requires the additional presence of a certain pathological milieu. Phenomenologically, this milieu could be generated by enhanced nitrosative stress. However, little is known regarding the modulation of MR transcriptional activity in a pathological milieu. The glucocorticoid receptor (GR), the closest relative of the MR, binds to the same hormone-response element but elicits protective effects on the cardiovascular system. To investigate the possible modulation of MR and GR by nitrosative stress under controlled conditions we used human embryonic kidney (HEK) cells and measured MR and GR transactivation after stimulation with the nitric oxide (NO)-donor SNAP and the peroxynitrite-donor Sin-1. In the presence of corticosteroids NO led to a general reduced corticosteroid receptor activity by repression of corticosteroid receptor–DNA interaction. The NO-induced diminished transcriptional MR activity was most pronounced during stimulation with physiological aldosterone concentrations, suggesting that NO treatment prevented its pathophysiological overactivation. In contrast, single peroxynitrite administration specifically induced the MR transactivation activity whereas genomic GR activity remained unchanged. Mechanistically, peroxynitrite permitted nuclear MR translocation whereas the cytosolic GR distribution was unaffected. Consequently, peroxynitrite represents a MR-specific aldosterone mimetic. In summary, our data indicate that the genomic function of corticosteroid receptors can be modulated by nitrosative stress which may induce the shift from physiological toward pathophysiological MR effects.

Abstract 549: Role of the glucocorticoid receptor in skin carcinogenesis and stem cell maintenance: Implication for the use of selective GR activators (SEGRA) as anti-cancer drugs

Abstract Glucocorticoids are important physiological and pharmacological regulators of epidermal proliferation, differentiation and skin tumorigenesis. The glucocorticoid effects are mediated via the glucocorticoid receptor (GR), a transcription factor that regulates genes expression by DNA-binding dependent transactivation, and DNA-binding independent transrepression through negative interaction with other transcription factors. Our previous studies demonstrated that keratin5.GR transgenic animals are resistant to skin carcinogenesis. We also found that GR diminishes the number of follicular epithelial stem cells (SC), reduces their proliferative and survival potential and affects the expression of follicular SC “signature” genes. To further explore the tumor suppressor role of GR in skin carcinogenesis we generated skin-specific GR knockout animals by breeding of GRflox/flox animals with Keratin5.Cre recombinase animals in C57Bl genetic background. Skin-specific GR KO (Cre/GRfl/fl) mice do not have gross skin or hair phenotype. However, the detailed analysis of keratinocyte markers revealed decreased expression of some intermediate/late markers of differentiation including Keratin 10 and loricrin. We also found that Cre/GRfl/fl mice are more sensitive to the effects of UV irradiation and tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The ongoing DMBA/TPA skin carcinogenesis experiment showed that Cre/GRfl/fl mice are more prone to skin carcinogenesis: they have higher tumor incidence and tumor multiplicity compared to GRfl/fl littermates. The analysis of global effect of the GR overexpression and GR silencing on gene expression in follicular epithelial SC, basal keratinocytes, and mouse skin tumors 1) revealed important GR targets in keratinocytes including STAT3 and REDD1 - the major regulator of mTOR; 2) provided an unexpected evidence that gene transrepression by GR plays critical role in the maintenance of SC and in inhibition of skin carcinogenesis. It is well understood that therapeutic effects of glucocorticoids are mediated by GR transreprssion, while many adverse metabolic effects are mediated by GR transactivation. Thus, our findings are important in light of drug discovery programs focused on the development of selective GR acivators (SEGRA) that preferentially induce GR transrepression. Some of SEGRA entered preclinical studies or clinical trials as anti-inflammatory GR ligands with reduced side effects. Our results suggest that SEGRA could be also used for skin cancer prevention/treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 549. doi:1538-7445.AM2012-549

Abstract 3814: Combination of selective modular of the glucocorticoid receptor (SEGRA) with proteasome inhibitors as a novel strategy for chemotherapy of hematologic malignancies

Abstract Glucocorticoids are widely used for the treatment of hematological malignancies; however chronic treatment with steroids results in numerous adverse effects. Glucocorticoid receptor (GR) regulates gene expression via transactivation that requires GR homodimer binding to gene promoters; and transrepression mediated via negative interaction between GR and other transcription factors. GR transactivation is linked to metabolic side effects, while GR transrepression underlies glucocorticoid therapeutic action. One of the novel GR modulators is 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium-chloride also called CpdA. CpdA is a synthetic analogue of a aziridine precursor isolated from the Namibian shrub Salsola tuberculatiformis Botschantzev. Using representative human T cell (CEM) and B cell (NCEB) lymphoma cell lines expressing functional GR, and their counterparts with blocked GR expression, we showed that CpdA indeed acted as dissociated GR ligand, and inhibited growth and survival of these cells via GR. Sensitivity to therapeutic effects of GCs directly depends on the amount of functional GR. The 26S protesome controls GR protein stability, and is responsible for desensitization to GCs via hormone-induced GR degradation and following development of glucocorticoid resistance in patients. Thus, the use of proteasome inhibitors represents a pharmacological approach to prevent GR down-regulation and to elevate the level of GR in cells. Proteasome inhibitor Bortezomib (BZ) was approved by FDA for the treatment of patients with multiple myeloma and mantle cell lymphoma. Since proteasome inhibitors stabilize GR, we hypothesized that BZ will augment CpdA effects as a selective GR modulator and enhance its chemotherapeutic activity. We showed that pretreatment of CEM and NCEB cells with proteasome inhibitor Bortezomib resulted in GR accumulation, and enhanced CpdA ligand properties further shifting GR activity towards transrepression evaluated by inhibiton of NF-kB and AP-1 factors. We also revealed remarkable GR-dependent synergy between CpdA and BZ in suppressing growth and survival of T and B lymphoma cells. Overall, our data provide the rationale for novel GR-based therapy for lymphoma and leukemia based on the combination of non-steroidal GR modulators with proteasome inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3814. doi:1538-7445.AM2012-3814

The possible separation of 12‐O‐tetradecanoylphorbol‐13‐acetate‐induced skin inflammation and hyperplasia by compound A

Activated glucocorticoid receptor (GR) acts via two different mechanisms: transcriptional regulation that requires DNA-binding, and protein-protein interaction between GR and other transcription factors, such as nuclear factor kappa B (NF-κB) or activator protein 1 (AP-1). It has been postulated that many important effects of glucocorticoids, including their anti-inflammatory properties, depend on GR's transrepressive effects on NF-κB and AP-1. In the present study, we have employed a TPA-induced model of skin inflammation and epidermal hyperplasia to determine whether partial activation of the glucocorticoid receptor by compound A (CpdA) is sufficient to reverse the effect of TPA treatment. CpdA is a nonsteroidal GR modulator with high binding affinity, is capable of partial activation of GR. Topical application of TPA twice per week for 2 wk results in inflammation and epidermal hyperplasia. TPA treatment also elevates levels of c-jun (AP-1 component), cyclooxygenase-2 (COX-2), p50 (NF-κB component), interleukin-6 (IL-6), and tumor necrosis factor (TNF) in the skin. Fluocinolone acetonide (FA) (a full GR agonist) was able to completely reverse the above effects of TPA. When applied alone, CpdA increased the epidermal thickness and keratinocyte proliferation as well as levels of c-jun, COX-2, IL-6, and IFN-γ. However, CpdA treatment resulted in a decrease in the number of p50 positive cells induced by TPA, suggesting its role in inhibition of NF-κB. The level of metallothionein-1 mRNA, regulated by GR was also significantly decreased in skin samples treated with CpdA. Our results suggest that CpdA is able to inhibit GR transactivation and activate only some transrepression properties of GR.

Defective glucocorticoid hormone receptor signaling leads to increased stress and anxiety in a mouse model of Angelman syndrome

Angelman syndrome (AS) is a neurodevelopmental disorder caused due to deletions or loss-of-function mutations in maternally inherited UBE3A. Ube3a functions as an ubiquitin ligase as well as a transcriptional coactivator of steroid hormone receptors. However, the mechanisms by which maternal Ube3a deficiency gives rise to phenotypic features of AS are not clear. We report here that Ube3a regulates glucocorticoid receptor (GR) transactivation and GR signaling pathway is disrupted in Ube3a-maternal-deficient mice brain. The expression of several GR-dependent genes is down-regulated in multiple brain regions of Ube3a-maternal-deficient mice. AS mice show significantly higher level of blood corticosterone, selective loss of GR and reduced number of parvalbumin-positive inhibitory interneurons in their hippocampus that could ultimately lead to increased stress. These mice also exhibit increased anxiety-like behavior, which could be due to chronic stress. Altogether, our findings suggest that chronic stress due to altered GR signaling might lead to anxiety-like behavior in a mouse of model of AS.

New models to investigate complex glucocorticoid receptor functions

New models to investigate complex glucocorticoid receptor functions

Antitumor effect of non-steroid glucocorticoid receptor ligand CpdA on leukemia cell lines CEM and K562

Glucocorticoids (GCs) are widely used in chemotherapy of hematological malignancies, particularly leukemia. Their effect is mediated by glucocorticoid receptor (GR), a well-known transcription factor. Besides their therapeutic impact, GCs may cause a number of side effects leading to various metabolic complications. The goal of immediate interest is testing glucocorticoid analogs capable of induction/enhancement of GR transrepression, but preventing GR dimerization and transactivation leading to side effects. In this work we have investigated effects of a promising new selective GR agonist, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride (CpdA), on CEM and K562 leukemia cells. Both cell lines express functional GR. CpdA compared with the glucocorticoid fluocinolone acetonide (FA) exerted more prominent cytostatic and apoptotic effects on the cells. Both cell lines exhibited sensitivity to CpdA, demonstrating a good correlation with the effects of FA on cell growth and viability. In contrast to FA, CpdA did not induce GR transactivation evaluated by no obvious increase in expression of GR target (and dependent) gene FKBP51. At the same time, luciferase assay showed that CpdA efficiently activated transrepression of NF-κB and AP-1 factors. We also evaluated the effect of combined action of CpdA and the proteasome inhibitor Bortezomib. The latter induced a caspase-dependent apoptosis in both T-cell leukemia cell lines. By treatment of CEM cells with different CpdA/GC and Bortezomib doses, we have designed a protocol where CpdA shows potentiating effect on Bortezomib cytotoxic activity. Generally, the present work characterizes a novel non-steroid GR ligand, CpdA, as a promising compound for possible application in leukemia chemotherapy.

Cell Cycle Phase Regulates Glucocorticoid Receptor Function

The glucocorticoid receptor (GR) is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors. In contrast to many other nuclear receptors, GR is thought to be exclusively cytoplasmic in quiescent cells, and only translocate to the nucleus on ligand binding. We now demonstrate significant nuclear GR in the absence of ligand, which requires nuclear localisation signal 1 (NLS1). Live cell imaging reveals dramatic GR import into the nucleus through interphase and rapid exclusion of the GR from the nucleus at the onset of mitosis, which persists into early G1. This suggests that the heterogeneity in GR distribution is reflective of cell cycle phase.The impact of cell cycle–driven GR trafficking on a panel of glucocorticoid actions was profiled. In G2/M-enriched cells there was marked prolongation of glucocorticoid-induced ERK activation. This was accompanied by DNA template-specific, ligand-independent GR transactivation. Using chimeric and domain-deleted receptors we demonstrate that this transactivation effect is mediated by the AF1 transactivation domain. AF-1 harbours multiple phosphorylation sites, which are consensus sequences for kinases including CDKs, whose activity changes during the cell cycle.In G2/M there was clear ligand independent induction of GR phosphorylation on residues 203 and 211, both of which are phosphorylated after ligand activation. Ligand-independent transactivation required induction of phospho-S211GR but not S203GR, thereby directly linking cell cycle driven GR modification with altered GR function. Cell cycle phase therefore regulates GR localisation and post-translational modification which selectively impacts GR activity. This suggests that cell cycle phase is an important determinant in the cellular response to Gc, and that mitotic index contributes to tissue Gc sensitivity.

Abstract 669: Anticancer effects of novel non steroidal glucocorticoid receptor ligand, Compound A, in T-cell leukemia lines

Abstract Glucocorticoids (GCs) are widely used in chemotherapy of hematologic malignancies, including T cell leukemia. The biological effects of GCs are mediated by the glucocorticoid receptor (GR), a well known transcription factor. Therapeutic effects of GCs are mainly mediated via GR transrepression that involves negative interaction between GR and other transcription factors. In contrast, side effects of glucocorticoids are mediated by GR transactivation that requires binding of GR homodimer to hormone-responsive elements in gene promoters. Selective GR activators (SEGRA) that shift GR functions towards transrepression have improved therapeutic index and hold a great potential for the GR-targeted chemotherapies. Recently we and others characterized a novel non-steroidal GR ligand, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride also called Compound A (CpdA). CpdA prevents GR dimerization and transactivation, but strongly enhances GR transrepression. We also revealed that CpdA possesses anti-cancer activity: it inhibited growth and viability of prostate cancer cells in GR-dependent fashion. Here we analyzed CpdA effects in K562 and CEM T-cell leukemia lines. We found that both cell lines express functional GR. CpdA at 10-6-10-5M concentration range exerted cytostatic and apoptotic effects in both cell lines. CEM cells were more sensitive to CpdA than K562 that correlated with the higher amount of GR protein in these cells. We found that in contrast to GCs, CpdA did not induce GR transactivation evaluated by RT-PCR of known GR-target genes such as FKBP51. At the same time, Luciferase assay showed that CpdA efficiently activated transrepression of NF-kB and AP-1 factors. Currently, GCs are used for the treatment of blood cancers in combined chemotherapy with different anti-cancer drugs including proteasome inhibitor Bortezomib (BZ). It is known that proteasome inhibitors increase GR stability. Thus, we expected that BZ will maximize ligand properties of CpdA as GR modulator and will increase its anti-cancer effects. We found that BZ induced caspase-dependent apoptosis in both T-cell leukemia cell lines. The effect of BZ on GR expression and function and on apoptosis induced by CpdA is under study. In addition, we have performed Ames test and Topoisomerase I assay as first steps towards the evaluation of toxicological profile of CpdA. We have not revealed mutagenic effect of CpdA either in frameshift or in base-substitution indicator strains at the wide range of concentrations (10-9-10-4M). CpdA also did not inhibit eukaryotic topoisomerase I activity. Overall, our data suggest that novel GR modulator CpdA has a high chemotherapeutic potential for the treatment of T-cell leukemia. The detailed mechanisms of its anti-cancer effects in transformed T-cells are under investigation. Work is supported by grants: RFBR 10-04-00979, RO1CA118890, ACS IL 160185, UICC ICRETT-09-137. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 669. doi:10.1158/1538-7445.AM2011-669

Antidepressants increase human hippocampal neurogenesis by activating the glucocorticoid receptor.

Antidepressants increase adult hippocampal neurogenesis in animal models, but the underlying molecular mechanisms are unknown. In this study, we used human hippocampal progenitor cells to investigate the molecular pathways involved in the antidepressant-induced modulation of neurogenesis. Because our previous studies have shown that antidepressants regulate glucocorticoid receptor (GR) function, we specifically tested whether the GR may be involved in the effects of these drugs on neurogenesis. We found that treatment (for 3–10 days) with the antidepressant, sertraline, increased neuronal differentiation via a GR-dependent mechanism. Specifically, sertraline increased both immature, doublecortin (Dcx)-positive neuroblasts (+16%) and mature, microtubulin-associated protein-2 (MAP2)-positive neurons (+26%). This effect was abolished by the GR-antagonist, RU486. Interestingly, progenitor cell proliferation, as investigated by 5′-bromodeoxyuridine (BrdU) incorporation, was only increased when cells were co-treated with sertraline and the GR-agonist, dexamethasone, (+14%) an effect which was also abolished by RU486. Furthermore, the phosphodiesterase type 4 (PDE4)-inhibitor, rolipram, enhanced the effects of sertraline, whereas the protein kinase A (PKA)-inhibitor, H89, suppressed the effects of sertraline. Indeed, sertraline increased GR transactivation, modified GR phosphorylation and increased expression of the GR-regulated cyclin-dependent kinase-2 (CDK2) inhibitors, p27Kip1 and p57Kip2. In conclusion, our data suggest that the antidepressant, sertraline, increases human hippocampal neurogenesis via a GR-dependent mechanism that requires PKA signaling, GR phosphorylation and activation of a specific set of genes. Our data point toward an important role for the GR in the antidepressant-induced modulation of neurogenesis in humans.

Glucocorticoid Insensitivity at the Hypoxic Blood–Brain Barrier Can Be Reversed by Inhibition of the Proteasome

Glucocorticoids potently stabilize the blood-brain barrier and ameliorate tissue edema in certain neoplastic and inflammatory disorders of the central nervous system, but they are largely ineffective in patients with acute ischemic stroke. The reasons for this discrepancy are unresolved. To address the molecular basis for the paradox unresponsiveness of the blood-brain barrier during hypoxia, we used murine brain microvascular endothelial cells exposed to O(2)/glucose deprivation as an in vitro model. In an in vivo approach, mice were subjected to transient middle cerebral artery occlusion to induce brain infarctions. Blood-brain barrier damage and edema formation were chosen as surrogate markers of glucocorticoid sensitivity in the presence or absence of proteasome inhibitors. O(2)/glucose deprivation reduced the expression of tight junction proteins and transendothelial resistance in murine brain microvascular endothelial cells in vitro. Dexamethasone treatment failed to reverse these effects during hypoxia. Proteasome-dependent degradation of the glucocorticoid receptor impaired glucocorticoid receptor transactivation thereby preventing physiological glucocorticoid activity. Inhibition of the proteasome, however, fully restored the blood-brain barrier stabilizing properties of glucocorticoid during O(2)/glucose deprivation. Importantly, mice treated with the proteasome inhibitor Bortezomib in combination with steroids several hours after stroke developed significantly less brain edema and functional deficits, whereas respective monotherapies were ineffective. We for the first time show that inhibition of the proteasome can overcome glucocorticoid resistance at the hypoxic blood-brain barrier. Hence, combined treatment strategies may help to combat stroke-induced brain edema formation in the future and prevent secondary clinical deterioration.

Characterization of activity and binding mode of glycyrrhetinic acid derivatives inhibiting 11β-hydroxysteroid dehydrogenase type 2

Modulation of intracellular glucocorticoid availability is considered as a promising strategy to treat glucocorticoid-dependent diseases. 18β-Glycyrrhetinic acid (GA), the biologically active triterpenoid metabolite of glycyrrhizin, which is contained in the roots and rhizomes of licorice ( Glycyrrhiza spp.), represents a well-known but non-selective inhibitor of 11β-hydroxysteroid dehydrogenases (11β-HSDs). However, to assess the physiological functions of the respective enzymes and for potential therapeutic applications selective inhibitors are needed. In the present study, we applied bioassays and 3D-structure modeling to characterize nine 11β-HSD1 and fifteen 11β-HSD2 inhibiting GA derivatives. Comparison of the GA derivatives in assays using cell lysates revealed that modifications at the 3-hydroxyl and/or the carboxyl led to highly selective and potent 11β-HSD2 inhibitors. The data generated significantly extends our knowledge on structure–activity relationship of GA derivatives as 11β-HSD inhibitors. Using recombinant enzymes we found also potent inhibition of mouse 11β-HSD2, despite significant species-specific differences. The selected GA derivatives potently inhibited 11β-HSD2 in intact SW-620 colon cancer cells, although the rank order of inhibitory potential differed from that obtained in cell lysates. The biological activity of compounds was further demonstrated in glucocorticoid receptor (GR) transactivation assays in cells coexpressing GR and 11β-HSD1 or 11β-HSD2. 3D-structure modeling provides an explanation for the differences in the selectivity and activity of the GA derivatives investigated. The most potent and selective 11β-HSD2 inhibitors should prove useful as mechanistic tools for further anti-inflammatory and anti-cancer in vitro and in vivo studies. Article from the Special issue on Targeted Inhibitors.

LXRβ is required for glucocorticoid-induced hyperglycemia and hepatosteatosis in mice

Although widely prescribed for their potent antiinflammatory actions, glucocorticoid drugs (e.g., dexamethasone) cause undesirable side effects that are features of the metabolic syndrome, including hyperglycemia, fatty liver, insulin resistance, and type II diabetes. Liver x receptors (LXRs) are nuclear receptors that respond to cholesterol metabolites and regulate the expression of a subset of glucocorticoid target genes. Here, we show LXRβ is required to mediate many of the negative side effects of glucocorticoids. Mice lacking LXRβ (but not LXRα) were resistant to dexamethasone-induced hyperglycemia, hyperinsulinemia, and hepatic steatosis, but remained sensitive to dexamethasone-dependent repression of the immune system. In vivo, LXRα/β knockout mice demonstrated reduced dexamethasone-induced expression of the key hepatic gluconeogenic gene, phosphoenolpyruvate carboxykinase (PEPCK). In perfused liver and primary mouse hepatocytes, LXRβ was required for glucocorticoid-induced recruitment of the glucocorticoid receptor to the PEPCK promoter. These findings suggest a new avenue for the design of safer glucocorticoid drugs through a mechanism of selective glucocorticoid receptor transactivation.

Increased Glucocorticoid Receptor Expression and Activity Mediate the LPS Resistance of SPRET/EI Mice

SPRET/Ei mice are extremely resistant to acute LPS-induced lethal inflammation when compared with C57BL/6. We found that in vivo SPRET/Ei mice exhibit strongly reduced expression levels of cytokines and chemokines. To investigate the role of the potent anti-inflammatory glucocorticoid receptor (GR) in the SPRET/Ei phenotype, mice were treated with the GR antagonist RU486 or bilateral adrenalectomy. Under such conditions, both C57BL/6 and SPRET/Ei mice were strongly sensitized to LPS, and the differences in LPS response between SPRET/Ei and C57BL/6 mice were completely gone. These results underscore the central role of GR in the LPS hyporesponsiveness of SPRET/Ei mice. Compared with C57BL/6, SPRET/Ei mice were found to express higher GR levels, which were reflected in increased GR transactivation. Using a backcross mapping strategy, we demonstrate that the high GR transcription levels are linked to the Nr3c1 (GR) locus on chromosome 18 itself. Unexpectedly, SPRET/Ei mice exhibit a basal overactivation of the hypothalamic-pituitary-adrenal axis, namely strongly increased corticosterone levels, ACTH levels, and adrenocortical size. As a consequence of the excess of circulating glucocorticoids (GCs), levels of hepatic gluconeogenic enzymes are increased, and insulin secretion from pancreatic β-cells is impaired, both of which result in hyperglycemia and glucose intolerance in SPRET/Ei mice. We conclude that SPRET/Ei mice are unique as they display an unusual combination of elevated GR expression and increased endogenous GC levels. Hence, these mice provide a new and powerful tool for the study of GR- and GC-mediated mechanisms, including immune repressive functions, neuroendocrine regulation, insulin secretion, and carbohydrate metabolism.