Nuclear Localization Of Glucocorticoid Receptor Research Articles

Phosphodiesterase 5 inhibitor mirodenafil ameliorates Alzheimer-like pathology and symptoms by multimodal actions

BackgroundAlzheimer’s disease (AD) pathology is associated with complex interactions among multiple factors, involving an intertwined network of various signaling pathways. The polypharmacological approach is an emerging therapeutic strategy that has been proposed to overcome the multifactorial nature of AD by targeting multiple pathophysiological factors including amyloid-β (Aβ) and phosphorylated tau. We evaluated a blood-brain barrier penetrating phosphodiesterase 5 (PDE5) inhibitor, mirodenafil (5-ethyl-2-7-n-propyl-3,5-dihydrro-4H-pyrrolo[3,2-d]pyrimidin-4-one), for its therapeutic effects on AD with polypharmacological properties.MethodsTo evaluate the potential of mirodenafil as a disease-modifying AD agent, mirodenafil was administered to test its effects on the cognitive behaviors of the APP-C105 AD mouse model using the Morris water maze and passive avoidance tests. To investigate the mechanisms of action that underlie the beneficial disease-modifying effects of mirodenafil, human neuroblastoma SH-SY5Y cells and mouse hippocampal HT-22 cells were used to show mirodenafil-induced alterations associated with the cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG)/cAMP-responsive element-binding protein (CREB) pathway, apoptotic cell death, tau phosphorylation, amyloidogenesis, the autophagy-lysosome pathway, glucocorticoid receptor (GR) transcriptional activity, and the Wnt/β-catenin signaling.ResultsHere, mirodenafil is demonstrated to improve cognitive behavior in the APP-C105 mouse model. Mirodenafil not only reduced the Aβ and phosphorylated tau burdens in vivo, but also ameliorated AD pathology induced by Aβ through the modulation of the cGMP/PKG/CREB signaling pathway, glycogen synthase kinase 3β (GSK-3β) activity, GR transcriptional activity, and the Wnt/β-catenin signaling in neuronal cells. Interestingly, homodimerization and nuclear localization of GR were inhibited by mirodenafil, but not by other PDE5 inhibitors. In addition, only mirodenafil reduced the expression levels of the Wnt antagonist Dickkopf-1 (Dkk-1), thus activating the Wnt/β-catenin signaling.ConclusionsThese findings strongly suggest that the PDE5 inhibitor mirodenafil shows promise as a potential polypharmacological drug candidate for AD treatment, acting on multiple key signaling pathways involved in amyloid deposition, phosphorylated tau burden, the cGMP/PKG/CREB pathway, GSK-3β kinase activity, GR signaling, and the Wnt/β-catenin signaling. Mirodenafil administration to the APP-C105 AD mouse model also improved cognitive behavior, demonstrating the potential of mirodenafil as a polypharmacological AD therapeutic agent.

Machine Learning Based Real-Time Image-Guided Cell Sorting and Classification.

Cell classification based on phenotypical, spatial, and genetic information greatly advances our understanding of the physiology and pathology of biological systems. Technologies derived from next generation sequencing and fluorescent activated cell sorting are cornerstones for cell- and genomic-based assays supporting cell classification and mapping. However, there exists a deficiency in technology space to rapidly isolate cells based on high content image information. Fluorescence-activated cell sorting can only resolve cell-to-cell variation in fluorescence and optical scattering. Utilizing microfluidics, photonics, computation microscopy, real-time image processing and machine learning, we demonstrate an image-guided cell sorting and classification system possessing the high throughput of flow cytometer and high information content of microscopy. We demonstrate the utility of this technology in cell sorting based on (1) nuclear localization of glucocorticoid receptors, (2) particle binding to the cell membrane, and (3) DNA damage induced γ-H2AX foci. © 2019 International Society for Advancement of Cytometry.

Theophylline suppresses interleukin-6 expression by inhibiting glucocorticoid receptor signaling in pre-adipocytes

Adipose tissues in obese individuals are characterized by a state of chronic low-grade inflammation. Pre-adipocytes and adipocytes in this state secrete pro-inflammatory adipokines, such as interleukin 6 (IL-6), which induce insulin resistance and hyperglycemia. Theophylline (1,3-dimethylxanthine) exerts anti-inflammatory effects, but its effects on pro-inflammatory adipokine secretion by pre-adipocytes and adipocytes have not been examined. In this study, we found that theophylline decreased IL-6 secretion by 3T3-L1 pre-adipocytes and mouse-derived primary pre-adipocytes. The synthetic glucocorticoid dexamethasone (DEX) induced IL-6 expression in 3T3-L1 pre-adipocytes, and this effect was suppressed by theophylline at the mRNA level. Knockdown of CCAAT/enhancer binding protein (C/EBP) δ inhibited DEX-induced IL-6 expression, and theophylline suppressed C/EBPδ expression. Furthermore, theophylline suppressed transcriptional activity of the glucocorticoid receptor (GR) through suppression of nuclear localization of GR. In vivo, glucocorticoid corticosterone treatment (100 μg/mL) increased fasting blood glucose and plasma IL-6 levels in C57BL/6 N mice. Theophylline administration (0.1% diet) reduced corticosterone-increased fasting blood glucose, plasma IL-6 levels, and Il6 gene expression in adipose tissues. These results show that theophylline administration attenuated glucocorticoid-induced hyperglycemia and IL-6 production by inhibiting GR activity. The present findings indicate the potential of theophylline as a candidate therapeutic agent to treat insulin resistance and hyperglycemia.

The Calreticulin control of human stress erythropoiesis is impaired by JAK2V617F in polycythemia vera.

Calreticulin (CALR) is a Ca2+-binding protein that shuttles among cellular compartments with proteins bound to its N/P domains. The knowledge that activation of the human erythropoietin receptor induces Ca2+ fluxes prompted us to investigate the role of CALR in human erythropoiesis. As shown by Western blot analysis, erythroblasts generated invitro from normal sources and JAK2V617F polycythemia vera (PV) patients expressed robust levels of CALR. However, Ca2+ regulated CALR conformation only in normal cells. Normal erythroblasts expressed mostly the N-terminal domain of CALR (N-CALR) on their cell surface (as shown by flow cytometry) and C-terminal domain (C-CALR) in their cytoplasm (as shown by confocal microscopy) and expression of both epitopes decreased with maturation. In the proerythroblast (proEry) cytoplasm, C-CALR was associated with the glucocorticoid receptor (GR), which initiated the stress response. In these cells, Ca2+ deprivation and inhibition of nuclear export increased GR nuclear localization while decreasing cytoplasmic detection of C-CALR and C-CALR/GR association and proliferation in response to the GR agonist dexamethasone (Dex). C-CALR/GR association and Dex responsiveness were instead increased by Ca2+ and erythropoietin. In contrast, JAK2V617F proErys expressed normal cell-surface levels of N-CALR but barely detectable cytoplasmic levels of C-CALR. These cells contained GR mainly in the nucleus and were Dex unresponsive. Ruxolitinib rescued cytoplasmic detection of C-CALR, C-CALR/GR association, and Dex responsiveness in JAK2V617F proErys and its effects were antagonized by nuclear export and Ca2+ flux inhibitors. These results indicates that Ca2+-induced conformational changes of CALR regulate nuclear export of GR in normal erythroblasts and that JAK2V617F deregulates this function in PV.

The Carboxy-Terminal Domain of Calreticulin (CALR) Exports the Glucocorticoid Receptor (GR) from the Nucleus to the Cytoplasm of Human Erythroid Cells Resetting Their Stress Response

Steady state erythropoiesis is regulated by interaction of EPO with its receptor, EPO-R which activates Ca2+signaling and terminal maturation [Miller, Blood 1989;73:1188]. Under stress, GR switches EPO-R signaling to a proliferation mode allowing erythroblast (Ery) amplification [Zhang, Nature 2013;499:92]. However, for recovery from anemia to occur, a mechanism, still to be identified, must counteract GR and switch EPO-R signaling back to a maturation mode.CALR chaperones other proteins to their active sites[Michalak, Biochem J 1999;344: 281]. By importing GR from the nucleus, CALR resets the stress response of murine fibroblasts. This function is regulated by the exposure of its C-terminal domain (C-CALR) sustained by Ca2+ [Holaska, Mol Cell Biol 2002;22:6286]. Whether CALR regulates nuclear import of GR in human Ery and how this regulation is modulated by Ca2+and EPO is unknown. Studies were performed to fill this gap.By WB, human Ery expanded in vitro from adult blood expressed robust levels of CALR, detectable with N-CALR- and C-CALR antibodies, and of GR. By flow cytometry, N-CALR and C-CALR were detectable on the surface of cells from the proerythroblast (proEry) to polychromatic stage. Expression of N-CALR, the epitope most exposed on the surface, was up-regulated by EPO. By confocal microscopy, both N-CALR and C-CALR were detected in non-permeabilized Ery but only C-CALR was detectable in cells permeabilized to reveal their cytoplasmic content. Intra-cytoplasmic C-CALR staining was robust in proEry, barely detectable in orthochromatic cells and enhanced by adding Ca2+ to the permeabilization buffer. Double staining of C-CALR with GR or LaminB1 (an inner nuclear membrane protein) indicated that C-CALR/GR were associated in the perinuclear area of the cytoplasm but that C-CALR remained distinct from Lamin B1, suggesting that C-CALR/GR co-localize at the cytoplasm/nuclear boundary. These results suggest that C-CALR mediates nuclear export of GR and that this function is regulated by Ca2+.This hypothesis was tested first by determining content and localization of GRα and C-CALR in nuclear and cytoplasmic fractions of Ery treated with Dex, EPO and SCF by WB. Although none of these stimulations altered the total content of these proteins, some of them affected their compartmentalization: Dex rapidly (15’) induced GR S211 phosphorylation and increased the levels of GRα in nuclear fractions. These effects were antagonized by addition of the GR inhibitor RU486. The nuclear levels of GRα were modestly increased also by SCF but strongly decreased by EPO. By contrast, regardless of the stimuli, C-CALR was detected mostly in cytoplasmic fractions.The effects of these stimulations were also assessed by confocal microscopy. Exposure to Dex and EPO increased by 15' but decreased by 4h the nuclear localization of GR and significantly increased the levels of C-CALR and of C-CALR/GR co-localization detected in the cytoplasm by 4h, suggesting that C-CALR drives nuclear export of GR. In agreement with this hypothesis, multi-regression analyses comparing single C-CALR and total and cytoplasmic GR signals as independent parameters against merged signals as dependent parameter in 410 individual proEry indicated that the levels of C-CALR/GR co-localization directly correlated with the cytoplasmic levels of both C-CALR and GR but that the greatest p-values were observed for C-CALR (p=0.0000 vs 0.045). Moreover, treatment with the nuclear export inhibitor Leptomycin greatly decreased C-CALR/GR co-localization, increased nuclear localization of GR in proEry, both in untreated and EPO-treated cells, and reduced Ery proliferation in response to Dex down to levels sustained by Dex with RU486.To assess the role of Ca2+ in the nuclear export activity of C-CALR confocal microscopy and proliferation studies on Ery exposed or not to the Ca2+ chelator BAPTA in combination with Ionomycin (Io, a raiser of intracellular Ca2+) or Thapsigargin (TG, a reducer of Ca2+in endoplasmic reticulum) were performed. These treatments greatly decreased C-CALR and C-CALR/GR co-localization in the cytoplasm, increased GR signals in nuclei and reduced the proliferation of Ery in response to Dex.These results suggest that conformation changes of C-CALR induced by Ca2+signaling downstream to EPO-R sustain nuclear export of GR, resetting the stress-response of normal proEry allowing them to undergo terminal maturation. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 4995: HO1 induction interrupts the glucocorticoid receptor signaling in prostate cancer cells

Abstract Although initially treatable, prostate cancer can recur in a hormone refractory form that is not responsive to current available therapies. The mortality rate associated with hormone refractory prostate cancer is high, and there is an urgent need for new therapeutic agents to treat prostate cancer. In order to overcome the toxicity of chemotherapy, glucocorticoids have long been used in the treatment of hormone-resistant prostate cancer. Nevertheless, many authors reported that the glucocorticoid receptor (GR) can substitute the androgen receptor and activate a similar but distinguishable set of genes. A common feature of prostate cancer is the dependence on activated signal transducer and activator of transcription 3 (STAT3), a transcription factor for survival and a coactivator of GR signaling. We have previously demonstrated that the induction of HO1, an antioxidant and anti-inflammatory protein, increases STAT3 cytoplasmic retention, interfering with its signaling and promotes HO1 nuclear localization. Our aim is to study the effect of HO1 induction on GR signaling in prostate cancer. PC3 cells were treated with Dexamethasone (Dex) at different concentrations (10−7, 10−8 and 10−9 M) during 24 h in 10% charcoaled-serum media and were exposed to the pharmacological inducer of HO1 (hemin, 80 uM, 24 h). Control cells were treated with vehicles. No significant differences in cell viability were detected by MTT assay. In order to investigate whether HO1 interrupts the GR transcriptional activity, PC3 cells were transfected with a luciferase reporter plamid containing three glucocorticoid response elements. Transfected cells were treated either with hemin (80 uM, 24 h), Dex (10−8 M, 6 h) or the combination of both drugs. The GR transcriptional activation was confirmed when cells were treated with the agonist Dex (p<0.01). HO1 induction significantly reduced either the GR basal transcriptional activity (p<0.01) as well as the Dex induced (p<0.01). The expression of Ikb, a GR regulated gene, was screened by RT-qPCR and was confirmed its inhibition in hemin exposed cells treated or not with dexamethasone. The repressive activity of HO1 was also demonstrated using an NFkB luciferase reporter assay. Using confocal fluorescence microscopy, GR nuclear translocation upon Dex treatment was confirmed, and a decrease of GR nuclear localization was demonstrated under hemin+Dex treatment (p<0.01). These results correlate with the diminished luciferase activity of the MMTV-luc plasmid in the presence of hemin+Dex vs Dex alone. Strikingly this is the first report showing the immunoprecipitation of GR and HO1, showing physical interaction between these proteins, both in the nucleus and in the cytoplasm. In conclusion, these results provide a novel function for HO1 downmodulating GR transcriptional activity, repressing the expression of its target genes and interfering with the receptor's nuclear localization in prostate cancer cells. Citation Format: Daiana B. Leonardi, Alejandra V. Paez, Federico Schuster, Nicolas Anselmino, Javier N. Brandani, Mercedes Abbate, Geraldine Gueron, Javier H. Cotignola, Elba S. Vazquez. HO1 induction interrupts the glucocorticoid receptor signaling in prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4995. doi:10.1158/1538-7445.AM2015-4995

Abstract 2074: Selective inhibitor of nuclear export (SINE) compounds show synergistic anti-tumor activity in combination with dexamethasone in multiple myeloma

Abstract Background: The primary nuclear export protein, Exportin 1 (XPO1/CRM1), is overexpressed in most cancers, which is frequently correlated with poor prognosis. SINE compounds are small-molecule bioavailable drugs that bind covalently to XPO1, which leads to nuclear retention of major tumor suppressor proteins, such as p53, FOXO and IκB, resulting in selective cancer cell death. Selinexor is the most advanced SINE with >500 hematological and solid tumor cancer patients treated to date in Phase I/II clinical trials. Glucocorticoid Receptor (GR) is an XPO1 cargo and dexamethasone (Dex) acts through GR agonism and inhibition of NF-κB activity. The combination of selinexor with Dex seems likely to have enhanced anti-tumor potency and this hypothesis was tested in multiple myeloma models. Methods: Total RNA and whole protein cell lysates from multiple myeloma cell line (MM1S) treated with selinexor with or without dexamethasone were analyzed by quantitative PCR and by immunoblots. Localization of GR was evaluated by immunofluorescence. GR and NF-κB transcriptional activity was analyzed using ELISA assays. MM1S xenograft model in NOD-SCID mice were treated with selinexor with or without dexamethasone to evaluate the effect on tumor growth. Results: In MM.1S cells, we found Dex, but not selinexor, induced phosphorylation of GR resulting in GR nuclear localization. Selinexor prevented nuclear export of phosphorylated GR, leading to the synergistic induction of GR-dependent transcriptional activity. RNA levels of GR regulated genes such as MNK2 were induced by this combination treatment. Interestingly, between the two MNK2 isomers, the combination treatment increased the expression of MNK2α, which is a tumor suppressor protein but not the MNK2β isoform. NF-κB transcriptional activity was inhibited additively by this treatment in MM1S cells. The combination treatment of suboptimal doses of selinexor (5 mg/kg; 60% TGI) and Dex (1 mg/kg; 47% TGI) led to nearly complete growth suppression (91% TGI) in the MM1.S multiple myeloma xenografts. Conclusions: We confirmed the hypothesized synergistic anti-tumor activity for the combination of selinexor with dexamethasone and provided evidence that this synergy is mediated through enhanced GR transcriptional activity, inhibition NF-κB and upregulation of the tumor suppressor MNK2α. This work provides a rationale basis for conducting clinical trials of combinations of selinexor with dexamethasone against cancers known to be sensitive to dexamethasone, such as multiple myeloma. Promising anti tumor activity for the combination of selinexor and dexamethasone in patients with relapsed/refractory MM was reported in the ongoing Phase 1 study (NCT01607892). Citation Format: Yosef Landesman, Trinayan Kashyap, Boris Klebanov, Sivan Elloul, Marsha Crochiere, Sharon Friedlander, William Senapedis, Robert Carlson, Michael Kauffman, Sharon Shacham. Selective inhibitor of nuclear export (SINE) compounds show synergistic anti-tumor activity in combination with dexamethasone in multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2074. doi:10.1158/1538-7445.AM2015-2074

The impact of prenatal alcohol exposure on hippocampal-dependent outcome measures is influenced by prenatal and early-life rearing conditions.

The clinical course of individuals exposed to alcohol in utero is influenced by multiple factors, including the social environments of the gravid female and offspring. In the present studies we focused on the effects of prenatal alcohol exposure (PAE) and the prenatal and early-life social environments on the hippocampal formation, as impaired development and functioning of this brain region have been implicated in several of the adverse cognitive outcomes associated with PAE. We combined our PAE mouse model with 2 conditions of housing pregnant dams and their preweanling offspring: the standard nest (SN), in which a dam is individually housed prior to parturition and then remains isolated with her offspring, and the communal nest (CN), in which multiple dams are housed together prior to parturition and then following delivery the moms and their litters share a nest. Mouse dams consumed either 10% (w/v) ethanol in 0.066% (w/v) saccharin (SAC) or 0.066% (w/v) SAC alone using a limited (4-hour) access, drinking-in-the-dark paradigm. Immunoblotting techniques were used to measure levels of the glucocorticoid receptor (GR), 11-β-hydroxysteroid dehydrogenase 1, the FK506-binding proteins 51 and 52, and corticotropin-releasing hormone receptor type 1 in the hippocampal formation isolated from male adolescent offspring. We also determined the effect of PAE and rearing conditions on context discrimination, a hippocampal-dependent learning/memory task. SN PAE offspring displayed impaired context discrimination and neurochemical changes in the hippocampal formation consistent with increased GR nuclear localization. These effects of PAE were, in general, ameliorated in mice reared in a CN. The CN also altered neurochemical measures and improved learning/memory in SAC control animals. These studies demonstrate a complex interplay between the effects of PAE and social environments. The findings have important translational implications, as well as highlight the importance of considering rearing conditions in the interpretation of research findings on PAE.

Selinexor Demonstrates Marked Synergy with Dexamethasone (Sel-Dex) in Preclinical Models and in Patients with Heavily Pretreated Refractory Multiple Myeloma (MM)

Introduction: The nuclear export protein exportin 1, (XPO1) is overexpressed in a wide variety of cancers including MM and often correlate with poor prognosis. Selinexor (KPT-330) is an oral Selective Inhibitor of Nuclear Export (SINE) XPO1 antagonist in Phase 1 and 2 clinical studies. Selinexor forces nuclear retention and reactivation of tumor suppressor proteins (TSPs) and reduction of many proto-oncogenes, including MDM2, MYC and Cyclin D. In addition, selinexor potently deactivates NF-κB, through forced nuclear retention of IκBα. Together these effects induce selective apoptosis in MM cells and inhibition of NF-κB dependent osteoclast activation. XPO1 is also responsible for nuclear export of the glucocorticoid receptor (GR). We hypothesized that selinexor will enhance the activity of dexamethasone (DEX)-bound GR, resulting in synergistic tumor cell killing. Methods: In vitro tumor cell viability measurements were based on MTT (CellTiter 96¨/Promega) and combination indices were calculated using CalcuSyn software. For xenograft studies, utilized NOD-SCID mice with subcutaneous inoculation of MM.1s cells. GR nuclear localization was measured with immunofluorescent anti-GR (phosphor-S211) antibody and quantitative imaging. To assess GR transcriptional activation, GR binding to a GCR consensus sequence was measured in nuclear extracts using an ELISA method (GR ELISA kit/Affymetrix). Patients (pts) with heavily pretreated refractory MM were dosed with oral selinexor at doses of up to 60 mg/m2 (8-10 doses/4 wk cycle) as part of a Phase 1 program in advanced hematological malignancies. Response we defined based on the IMWG criteria. The effect of combining DEX with selinexor was analyzed in all pts who received selinexor at moderate to high doses (30-60 mg/m2). Safety and efficacy were analyzed separately in three groups: no DEX, Disclosures Chen:Celgene: Honoraria; Janssen: Honoraria. Off Label Use: Lenalidomide maintenance therapy after ASCT. Gutierrez:Senesco: PI Other. Siegel:Celgene, Millennium, Onyx: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau. Baz:Celgene: Research Funding; Millennium: Research Funding; Bristol Myers Squibb: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding. Kukreti:Celgene: Honoraria. Azmi:Karyopharm Therpeutics: Research Funding. Kashyap:Karyopharm Therapeutics: Employment. Landesman:Karyopharm Therapeutics: Employment. Marshall:Karyopharm Therpeutics: Employment. McCartney:Karyopharm Therpeutics: Employment. Saint-Martin:Karyopharm Therpeutics: Employment. Norori:Karyopharm Therpeutics: Consultancy. Savona:Karyopharm Therpeutics: Membership on an entity9s Board of Directors or advisory committees. Rashal:Karyopharm Therapeutics: Employment. Carlson:Karyopharm Therapeutics: Employment. Mirza:Karyopharm Therpeutics: Consultancy, Membership on an entity9s Board of Directors or advisory committees. Shacham:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics: Employment, Equity Ownership. Reece:Millennium: Honoraria, Research Funding; Millennium: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Merck: Research Funding; Merck: Research Funding; BMS: Research Funding; BMS: Research Funding; Novartis: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen : Honoraria; Amgen : Honoraria.

Prenatal alcohol exposure modifies glucocorticoid receptor subcellular distribution in the medial prefrontal cortex and impairs frontal cortex-dependent learning.

Prenatal alcohol exposure (PAE) has been shown to impair learning, memory and executive functioning in children. Perseveration, or the failure to respond adaptively to changing contingencies, is a hallmark on neurobehavioral assessment tasks for human fetal alcohol spectrum disorder (FASD). Adaptive responding is predominantly a product of the medial prefrontal cortex (mPFC) and is regulated by corticosteroids. In our mouse model of PAE we recently reported deficits in hippocampal formation-dependent learning and memory and a dysregulation of hippocampal formation glucocorticoid receptor (GR) subcellular distribution. Here, we examined the effect of PAE on frontal cortical-dependent behavior, as well as mPFC GR subcellular distribution and the levels of regulators of intracellular GR transport. PAE mice displayed significantly reduced response flexibility in a Y-maze reversal learning task. While the levels of total nuclear GR were reduced in PAE mPFC, levels of GR phosphorylated at serines 203, 211 and 226 were not significantly changed. Cytosolic, but not nuclear, MR levels were elevated in the PAE mPFC. The levels of critical GR trafficking proteins, FKBP51, Hsp90, cyclophilin 40, dynamitin and dynein intermediate chain, were altered in PAE mice, in favor of the exclusion of GR from the nucleus, indicating dysregulation of GR trafficking. Our findings suggest that there may be a link between a deficit in GR nuclear localization and frontal cortical learning deficits in prenatal alcohol-exposed mice.

Prenatal Alcohol Exposure Is Associated with Altered Subcellular Distribution of Glucocorticoid and Mineralocorticoid Receptors in the Adolescent Mouse Hippocampal Formation

BackgroundAccumulating evidence indicates that several of the long-term consequences of prenatal alcohol exposure (PAE) are the result of changes in the development and function of cortico-limbic structures, including the hippocampal formation. The glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) are key regulators of hippocampal formation development, structure, and functioning and, thus, are potential mediators of PAE’s effects on this brain region. In the present studies, we assessed the impact of PAE on components of corticosteroid signaling pathways in the mouse hippocampal formation.MethodsThroughout pregnancy, mouse dams were offered either 10% (w/v) ethanol sweetened with 0.066% (w/v) saccharin (SAC) or 0.066% (w/v) SAC alone using a limited (4-hour) access, drinking-in-the-dark paradigm. The hippocampal formation was isolated from naïve postnatal day 40 to 50 offspring, and subcellular fractions were prepared. Using immunoblotting techniques, we measured the levels of GR, MR, 11-β-hydroxysteroid dehydrogenase 1 (11β-HSD1), and the FK506-binding proteins 51 (FKBP51, FKBP5) and 52 (FKBP52, FKBP4). Finally, we determined the effect of PAE on context discrimination, a hippocampal-dependent learning/memory task.ResultsPAE was associated with reduced MR and elevated GR nuclear localization in the hippocampal formation, whereas cytosolic levels of both receptors were not significantly altered. FKBP51 levels were reduced, while FKBP52 levels were unaltered, and 11β-HSD1 levels were increased in postnuclear fractions isolated from PAE mouse hippocampal formation. These neurochemical alterations were associated with reduced context discrimination.ConclusionsThe data support a model in which PAE leads to increased nuclear localization of GRs secondary to reductions in FKBP51 and increases in 11β-HSD1 levels in the adolescent mouse hippocampal formation. Persistent dysregulation of GR subcellular distribution is predicted to damage the hippocampal formation and may underlie many of the effects of PAE on hippocampal-dependent functioning.

Importin‐7 mediates glucocorticoid receptor nuclear import and is impaired by oxidative stress, leading to glucocorticoid insensitivity

Some patients with severe inflammatory disease fail to respond to glucocorticoids, and oxidative stress contributes to this insensitivity. Importin receptors are associated with nuclear translocation of the glucocorticoid receptor (GR), which is essential for glucocorticoid function. We hypothesized that importin-7 is central to GR nuclear translocation and glucocorticoid sensitivity. We investigated the effects of importin-7 siRNA on fluticasone propionate (FP)-induced GR nuclear localization and suppression of IL-1β-induced CXCL8 and the effects of hydrogen peroxide (H2O2) plus IL-1β costimulation on importin-7 expression, function, and glucocorticoid responsiveness in a human macrophagecell line (U937). H2O2 significantly reduced FP-induced GR nuclear localization (3.4±0.51- vs. 5.7±0.85-fold increase, P<0.05) and suppression of IL-1β-induced CXCL8 (62.3±2.3 vs. 85.1±7.0%, P<0.05). Knockdown of importin-7 by 38.4 ± 11.5% (compared with control siRNA) significantly reduced FP-mediated GR nuclear localization (3.5±0.5- vs. 5.7±0.85-fold increase, P<0.05) and suppression of IL-1β-induced CXCL8 expression (40.2±16.1 vs. 68.4±3.0%, P<0.05). H2O2 plus IL-1β had no direct effect on importin-7 but caused a significant loss (61.2±12.6% compared with baseline) of nuclear RanGTP, an essential cofactor for importin-7-mediated nuclear import of cargo proteins. The importin-7 complex is essential for glucocorticoid function, and the expression of its cofactor RanGTP is reduced during oxidative stress-induced glucocorticoid insensitivity.

Glucocorticoid receptor concentration and the ability to dimerize influence nuclear translocation and distribution

Glucocorticoid receptor (GR) concentrations and the ability of the GR to dimerize are factors which influence sensitivity to glucocorticoids. Upon glucocorticoid binding, the GR is actively transported into the nucleus, a crucial step in determining GR function. We examined the effects of GR concentration and the ability to dimerize on GR nuclear import, export and nuclear distribution using both live cell microscopy of GFP-tagged GR and immunofluorescence of untagged GR, with both wild type GR (GRwt) and dimerization deficient GR (GRdim). We found that the observed rate of GR nuclear import increases significantly at higher GR concentrations, at saturating concentrations of dexamethasone (10−6M) using GFP-tagged GR, while with untagged GR it is only discernable at sub-saturating ligand concentrations (10−10–10−9M). Loss of dimerization results in a slower observed rate of nuclear import (2.5- to 3.3-fold decrease for GFP-GRdim) as well as a decreased extent of GR nuclear localization (18–27% decrease for untagged GRdim). These results were linked to an increased rate of GR export at low GR concentrations (1.4- to 1.6-fold increase for untagged GR) and where GR dimerization is abrogated (1.5- to 1.7-fold increase for GFP-GRdim). Furthermore, GR dimerization was shown to be required for the appearance of discrete GC-dependent GR nuclear foci, the loss of which may explain the increased rate of GR export for the GRdim. The reduction in the observed rate of nuclear import and increased rate of nuclear export displayed at low GR concentrations and by the GRdim could explain the lowered glucocorticoid response under these conditions.

Differential nuclear localisation and promoter occupancy play a role in glucocorticoid receptor ligand-specific transcriptional responses

The glucocorticoid receptor (GR) is a ligand-activated transcription factor for which a number of endogenous and synthetic ligands exist. A key question in steroid receptor biology is how different ligands elicit different maximal transcriptional responses via the same receptor and on the same promoter. This question was addressed quantitatively for the GR, using a panel of agonists, partial agonists and antagonists, on the endogenous GILZ gene in two different human cell lines. It was found that the extent of GR nuclear localization correlated with the efficacy for GILZ transactivation by the GR in U2OS cells. However, in A549 cells there was no significant correlation, with all ligands resulting in similar levels of GR nuclear localization, despite different levels of transcriptional activation of the GILZ gene. Chromatin immunoprecipitation analysis on the other hand, revealed ligand-specific differences in GILZ promoter occupancy in the A549 cells, which correlated with the transcriptional efficacy of the subset of ligands investigated. This suggests that ligand-specific differences in promoter occupancy by activated GR play a major role in discrimination between agonist, partial agonist and antagonist responses on the endogenous GILZ gene in A549 cells, while differences in nuclear localisation of liganded GR play a role in determining the transcriptional outcome in U2OS cells. These cell line-specific differences were not dependent on the amount of GR present, since transient overexpression of GR in U2OS did not alter the relative ligand-selective nuclear localisation. Our results show that there is a relationship between ligand-specific transactivation efficacy, extent of nuclear translocation and recruitment of GR to the promoter. However, the relative contribution of nuclear translocation and GR promoter recruitment to ligand-specific transactivation efficacy is cell-specific.

Microglial glucocorticoid receptors play a pivotal role in regulating dopaminergic neurodegeneration in parkinsonism

Among the pathogenic processes contributing to dopaminergic neuron (DN) death in Parkinson disease (PD), evidence points to non-cell-autonomous mechanisms, particularly chronic inflammation mounted by activated microglia. Yet little is known about endogenous regulatory processes that determine microglial actions in pathological states. We examined the role of glucocorticoid receptors (GRs), activated by glucocorticoids released in response to stress and known to regulate inflammation, in DN survival. Overall GR level was decreased in substantia nigra of PD patients and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. GR changes, specifically in the microglia after MPTP treatment, revealed a rapid augmentation in the number of microglia displaying nuclear localization of GR. Mice with selective inactivation of the GR gene in macrophages/microglia (GR(LysMCre)) but not in DNs (GR(DATCre)) showed increased loss of DNs after MPTP intoxication. This DN loss in GR(LysMCre) mice was not prevented by corticosterone treatment, in contrast to the protection observed in control littermates. Moreover, absence of microglial GRs augmented microglial reactivity and led to their persistent activation. Analysis of inflammatory genes revealed an up-regulation of Toll-like receptors (TLRs) by MPTP treatment, particularly TLR9, the level of which was high in postmortem parkinsonian brains. The regulatory control of GR was reflected by higher expression of proinflammatory genes (e.g., TNF-α) with a concomitant decrease in anti-inflammatory genes (e.g., IL-1R2) in GR(LysMCre) mice. Indeed, in GR(LysMCre) mice, alterations in phosphorylated NF-κB levels indicated its protracted activation. Together, our data indicate that GR is important in curtailing microglial reactivity, and its deregulation in PD could lead to sustained inflammation-mediated DN injury.

Modulation of glucocorticoid receptor nuclear translocation in neurons by immunophilins FKBP51 and FKBP52: Implications for major depressive disorder

Mood disorders associated with dysfunction of the hypothalamic–pituitary–adrenal (HPA) axis are common psychiatric conditions. The glucocorticoid receptor (GR) is a steroid-activated nuclear receptor that, upon binding to cortisol, translocates to the nucleus where it targets genes related to neuronal metabolism and plasticity. In patients suffering from major depressive disorder (MDD), hypercortisolemia is a common finding. In the current study we investigated the molecular events associated with the FK506 binding proteins (FKBP) -52 and -51 response to cortisol exposure in neuronal cell cultures and their effect on GR translocation. We noted that FK506 altered nuclear localization of the GR and inhibited expression of GR-responsive genes. Furthermore, siRNA knockdown of FKBP4 gene, coding for the immunophilin FKBP52, inhibited cortisol-activated GR nuclear translocation, while knockdown of FKBP5, coding for immunophilin FKBP51, was associated with increased baseline GR nuclear localization. We propose that immunophilins are modulators of the cortisol–HPA axis response to stress and related chronic brain disorders.

Selective Cancer Targeting via Aberrant Behavior of Cancer Cell-associated Glucocorticoid Receptor

Glucocorticoid receptors (GRs) are ubiquitous, nuclear hormone receptors residing in cell types of both cancer and noncancerous origin. It is not known whether cancer cell-associated GR alone can be selectively manipulated for delivery of exogenous genes to its nucleus for eliciting anticancer effect. We find that GR ligand, dexamethasone (Dex) in association with cationic lipoplex (termed as targeted lipoplex) could selectively manipulate GR in cancer cells alone for the delivery of transgenes in the nucleus, a phenomenon that remained unobserved in normal cells. The targeted lipoplex (i) showed GR-targeted transfections in all cancer cells experimented (P < 0.01), (ii) significantly diminished transfection in cancer cells when GR is downregulated (P < 0.01), and (iii) elicited specific nuclear translocation of targeted lipoplex in cancer cells, followed by upregulated transactivation of glucocorticoid response element (GRE)- promoted gene. Using anticancer gene, targeted lipoplex induced significant tumor growth retardation in mice in comparison to different control groups (P < 0.05). Interestingly, cell surface-associated Hsp90 in cancer cells assisted the intracellular uptake of GR-targeted lipoplex. Moreover, selective inhibition of Hsp90 in noncancer cells resulted in cancer cell-like, aberrant, GR activation. The current study discovers a therapeutically important, unique property of cancer cell associated-GR that may be linked to a compromised role of Hsp90.Molecular Therapy (2009) 17 4, 623-631 doi:10.1038/mt.2009.4.

Selective increases in regional brain glucocorticoid: A novel effect of chronic alcohol

The hypothalamo-pituitary–adrenal axis shows functional changes in alcoholics, with raised glucocorticoid release during alcohol intake and during the initial phase of alcohol withdrawal. Raised glucocorticoid concentrations are known to cause neuronal damage after withdrawal from chronic alcohol consumption and in other conditions. The hypothesis for these studies was that chronic alcohol treatment would have differential effects on corticosterone concentrations in plasma and in brain regions. Effects of chronic alcohol and withdrawal on regional brain corticosterone concentrations were examined using a range of standard chronic alcohol treatments in two strains of mice and in rats. Corticosterone was measured by radioimmunoassay and the identity of the corticosterone extracted from brain was verified by high performance liquid chromatography and mass spectrometry. Withdrawal from long term (3 weeks to 8 months) alcohol consumption induced prolonged increases in glucocorticoid concentrations in specific regions of rodent brain, while plasma concentrations remained unchanged. This effect was seen after alcohol administration via drinking fluid or by liquid diet, in both mice and rats and in both genders. Shorter alcohol treatments did not show the selective effect on brain glucocorticoid levels. During the alcohol consumption the regional brain corticosterone concentrations paralleled the plasma concentrations. Type II glucocorticoid receptor availability in prefrontal cortex was decreased after withdrawal from chronic alcohol consumption and nuclear localization of glucocorticoid receptors was increased, a pattern that would be predicted from enhanced glucocorticoid type II receptor activation. This novel observation of prolonged selective increases in brain glucocorticoid activity could explain important consequences of long term alcohol consumption, including memory loss, dependence and lack of hypothalamo-pituitary responsiveness. Local changes in brain glucocorticoid levels may also need to be considered in the genesis of other mental disorders and could form a potential new therapeutic target.

T1843 Fructose-Induced Translocation of the Glucocorticoid Receptor in Developing Rat Small Intestine

Despite dramatic increases in per capita consumption of fructose (F) and its potential links to the obesity epidemic, particularly in young children, little is known about neonatal intestinal F absorptionmediated by the brushborder F transporter GLUT5. GLUT5 is normally expressed at low baseline levels throughout the suckling (0 ~ 13 d of age) and weaning (14 ~ 28 d) stages in neonatal rats. GLUT5 expression and activity, however, are dramatically enhanced by precocious perfusion of its substrate F for 4 h into the intestinal lumen, but only after ~14 d of age. Priming the gut with dexamethasone (D, a glucocorticoid analog) allowed F to stimulate GLUT5 even in suckling pups < 14 d old. Since an earlier study showed that the D effect is mediated by the glucocorticoid receptor (GR), we tested the hypothesis that GR must translocate to the nucleus to allow F to stimulate GLUT5 transcription. Four groups of 10 d old pups were injected with D or saline (S) before intestinal perfusion for 4h with F or glucose (G) solutions (10FD, 10FS, 10GD and 10GS). Using primary antibodies specific for GLUT5, GR or GR-P (phosphorylated GR), and propidium iodide (PI) as a nuclear marker we compared the (co)localization of GR and/or GLUT5 in the jejunum. In 10GS (control) or 10FS intestines double stained with anti GR and PI, we saw only cytoplasmic GR. GR remained in the cytoplasm of the 10GD intestines while in the 10FD pups, GR translocated into the nucleus. This GR translocation was blocked if the pups were treated with RU (specific antagonist of GR) before D injection and F perfusion. We then double stained the intestine with anti-rat GLUT5 and GR. The 10FD pups had the highest intensity of GLUT5 staining on the apical membrane, in correlation with a nuclear localization of GR. In the other groups, GR remained in the cytoplasm and the intensity of the GLUT5 staining was low or undetectable, suggesting that GR translocation may be necessary for D-allowed, F-mediated upregulation of GLUT5. We also probed the tissue with a primary antibody specific for GR phosphorylated at Ser-211 (GR-P), a known phosphorylation site that activates GR. However, GR-P was not detectable in the nucleus of any group of pups, indicating that GR phosphorylation is not necessary in the D-allowed, F-induced precocious increases in GLUT5 transcription. We conclude that the developmental regulation of GLUT5 expression in response to D and F requires translocation of GR to the nucleus but does not involve GR phosphorylation.

Glucocorticoid Receptor Nuclear Translocation in Airway Cells after Inhaled Combination Therapy

Clinical evidence is accumulating for the efficacy of adding inhaled long-acting beta(2)-agonists (LABAs) to corticosteroids in asthma. Corticosteroids bind to cytoplasmic glucocorticoid receptors (GRs), which then translocate to the nucleus where they regulate gene expression. This article reports the first evidence in vivo of an interaction between inhaled LABA and corticosteroid on GR nuclear translocation in human airway cells using immunocytochemistry. We initially demonstrated significant GR activation 60 minutes after inhalation of 800 microg beclomethasone dipropionate in six healthy subjects. Subsequently, we determined the effects of salmeterol and fluticasone propionate (FP) in seven steroid-naive patients with asthma. We observed dose-dependent GR activation with 100- and 500-microg doses of FP, and to a lesser extent with 50 microg salmeterol alone. However, combination therapy with 100 microg FP and salmeterol augmented the action of FP on GR nuclear localization. In vitro, salmeterol enhanced FP effects on GR nuclear translocation in epithelial and macrophage-like airway cell lines. In addition, salmeterol in combination with FP enhanced glucocorticoid response element (GRE)-luciferase reporter gene activity and mitogen-activated protein kinase phosphatase 1 (MKP-1) and secretory leuko-proteinase inhibitor (SLPI) gene induction. Together, our data confirm that GR nuclear translocation may underlie the complementary interactions between LABAs and corticosteroids, although the precise signal transduction mechanisms remain to be determined.