Glucocorticoid Receptor Phosphorylation Research Articles

The Endocrine Disrupting Chemical Tolylfluanid Alters Adipocyte Metabolism via Glucocorticoid Receptor Activation

Glucocorticoid signaling plays a critical role in regulating energy metabolism. Emerging data implicate environmental endocrine-disrupting chemicals as contributors to the obesity and diabetes epidemics. Previous studies have shown that the phenylsulfamide fungicide tolylfluanid (TF) augments glucocorticoid receptor (GR)-dependent luciferase expression in 3T3-L1 preadipocytes while modulating insulin action in primary murine and human adipocytes. Studies were performed to interrogate glucocorticoid signaling in primary adipocytes exposed to TF. TF mimicked the gene transcription profile of the murine glucocorticoid corticosterone (Cort). Cellular fractionation assays demonstrated that TF treatment promoted the activating serine phosphorylation of GR, augmenting its cytoplasmic-to-nuclear translocation as well as its enrichment at glucocorticoid response elements on the glucocorticoid-induced leucine zipper gene promoter. After acute treatment, Cort or TF promoted insulin receptor substrate-1 (IRS-1) gene and protein expression. Either treatment also enriched GR binding at an identified glucocorticoid response element in the IRS-1 gene. TF or Cort each increased insulin-stimulated lipogenesis, an effect resulting from increased lipogenic gene expression and enhanced insulin-stimulated dephosphorylation of acetyl-coenzyme A carboxylase. The augmentation of insulin-stimulated lipogenesis was mediated through a specific enhancement of Akt phosphorylation at T308. These findings support modulation of IRS-1 levels as a mechanism for glucocorticoid-mediated changes in insulin action in primary adipocytes. Albeit with less affinity than Cort, in silico analysis suggests that TF can interact with the ligand binding pocket of GR. Collectively, these studies identify TF as a structurally unique environmental glucocorticoid. Glucocorticoid signaling may thus represent a novel pathway by which environmental toxicants promote the development of metabolic diseases.

1381 – Phosphorylation of peripheral glucocorticoid receptor in the continuum from current depressive episode to vulnerability to depression

The impaired glucocorticoid receptor (GR) signaling has long been considered one of cornerstones in understanding the pathophysiology of depression. Phosphorylation of GR is very important for GR function, therefore we investigated: a) whether GR phosphorylation at serine 211 (pGR-S211) and serine 226 (pGR-S226) is altered in patients with episode of major depressive disorder (MDD); b) if neuroticism and negative affective states may be associated with altered GR phosphoisophorms of healthy adults. Phosphorylation at these sites is generally considered to be indicative of receptor activity. We studied leucocytes of : a) 30 MDD patients and 35 controls, and b) 37 healthy subjects, to assess the levels of nuclear total GR (tGR), pGR-S211 and pGR-S226 using Western blot technique. Our results demonstrated increased phosphorylation of GR at S226 and, to a less extent, at S211 in MDD compared to controls. Also, pGR-S226 positively correlated with neuroticism and self report of stress in healthy. In MDD, the pGRS211/ pGR-S226 ratio was decreased (p We examined whether easily accessible peripheral markers of GR activity and disease status in depression exist. Although we examined leucocytes, we propose that reduced GR transcriptional activity could be one of mechanisms of impaired GR function in the brain, resulting in dysfunction of HPA axis observed as a risk for depression.

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.

Dynamic Nucleotide-dependent Interactions of Cysteine- and Histidine-rich Domain (CHORD)-containing Hsp90 Cochaperones Chp-1 and Melusin with Cochaperones PP5 and Sgt1

Mammals have two cysteine- and histidine-rich domain (CHORD)-containing Hsp90 cochaperones, Chp-1 and melusin, which are homologs of plant Rar1. It has been shown previously that Rar1 CHORD directly interacts with ADP bound to the nucleotide pocket of Hsp90. Here, we report that ADP and ATP can bind to Hsp90 cochaperones Chp-1 and PP5, inducing their conformational changes. Furthermore, we demonstrate that Chp-1 and melusin can interact with cochaperones PP5 and Sgt1 and with each other in an ATP-dependent manner. Based on the known structure of the Rar1-Hsp90 complex, His-186 has been identified as an important residue of Chp-1 for ADP/ATP binding. His-186 is necessary for the nucleotide-dependent interaction of Chp-1 not only with Hsp90 but also with Sgt1. In addition, Ca(2+), which is known to bind to melusin, enhances the interactions of melusin with Hsp90 and Sgt1. Furthermore, melusin acquires the ADP preference for Hsp90 binding in the presence of Ca(2+). Our newly discovered nucleotide-dependent interactions between cochaperones might provide additional complexity to the dynamics of the Hsp90 chaperone system, also suggesting potential Hsp90-independent roles for these cochaperones.

Glucocorticoid receptor and FKBP5 expression is altered following exposure to chronic stress: modulation by antidepressant treatment.

Major depression is thought to originate from the interaction between susceptibility genes and adverse environmental events, in particular stress. The hypothalamus-pituitary-adrenal (HPA) axis is the major system involved in stress response and its dysregulation is an important element in the pathogenesis of depression. The stress response is therefore finely tuned through a series of mechanisms that control the trafficking of glucocorticoid receptors (GRs) to the nucleus, including binding to the chaperone protein FKBP5 and receptor phosphorylation, suggesting that these elements may also be affected under pathologic conditions. On these bases, we investigated FKBP5 and GR expression and phosphorylation in the hippocampus (ventral and dorsal) and in the prefrontal cortex of rats exposed to chronic mild stress (CMS) and we analyzed the effect of a concomitant antidepressant treatment. We found that animals exposed to CMS show increased expression of FKBP5 as well as enhanced cytoplasmic levels of GR, primarily in ventral hippocampus and prefrontal cortex. Chronic treatment with the antidepressant duloxetine is able to normalize such alterations, mainly in the prefrontal cortex. Moreover, we demonstrate that CMS-induced alterations of GR trafficking and transcription may be sustained by changes in receptor phosphorylation, which are also modulated by pharmacological intervention. In summary, while GR-related changes after CMS might be relevant for the depressive phenotype, the ability of antidepressant treatment to correct some of these alterations may contribute to the normalization of HPA axis dysfunctions associated with stress-related disorders.

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.

Phosphorylation of leukocyte glucocorticoid receptor in patients with current episode of major depressive disorder

The impaired glucocorticoid receptor (GR) signaling has long been considered one of the cornerstones in understanding the pathophysiology of depression. Since the phosphorylation of GR is very important for GR function, in this study we investigated whether GR phosphorylation at serine 211 (pGR-S211) and serine 226 (pGR-S226) is altered in patients with current episode of major depressive disorder (MDD). Particularly, in 30 MDD patients and 35 controls we assessed the levels of nuclear total GR (tGR), pGR-S211 and pGR-S226 in peripheral blood mononuclear cells (PBMC) using Western blot technique, along with plasma cortisol concentrations from the same blood samples. Our results demonstrated increased phosphorylation of GR at S226 (p<0.001) and, to a less extent, at S211 (p<0.05) in MDD patients compared to controls. Consequently, the pGR-S211/pGR-S226 ratio was decreased (p<0.05) implying reduced transcriptional activity of GR in MDD patients. MDD subjects had higher cortisol levels than controls and cortisol concentrations were positively correlated with PBMC pGR-S226 levels from the same blood samples. There was no difference in the levels of tGR between MDD and control subjects. The study showed that altered phosphorylation of GR could contribute to impaired GR function related to the pathophysiology of depression.

Antidepressant-like effects of Cortex Mori Radicis extract via bidirectional phosphorylation of glucocorticoid receptors in the hippocampus

Excessive and prolonged secretion of adrenal glucocorticoids leads to a wide range of pathophysiological processes, including depression. Glucocorticoids, which act at glucocorticoid receptors (GR), are key regulators of the limbic hypothalamic–pituitary–adrenocortical (HPA) axis. In the present study, the antidepressant-like effects of the alcohol extract Cortex Mori Radicis (CMR) and its role in GR signalling were investigated. Male Wistar rats were administered CMR extract (50, 100, 200mg/kg, p.o.) daily for 5 days and then exposed to the forced swim test (FST). Behavioural analyses showed that CMR extract dose-dependently decreased immobility time during forced swimming. CMR extract also decreased the limbic HPA axis response to the FST, as indicated by an attenuated corticosterone response and decreased c-fos immunoreactivity in the dentate gyrus. Reduced hippocampal GR expression following exposure to the FST was reversed by CMR treatment. Moreover, a prominent increase in GR phosphorylation at S232 and a decrease at S246 were noted following treatment with CMR. This resulted in a high pGR(S232)/(S246) ratio. CMR treatment also produced a downregulation of serine/threonine protein phosphatase 5 levels, producing a strong negative relationship with pGR(S232). Taken together, our findings suggest that the alcohol extract CMR promotes antidepressant-like effects through bidirectional phosphorylation of GR at S232 and S246.

Restoration of corticosteroid sensitivity by p38 mitogen activated protein kinase inhibition in peripheral blood mononuclear cells from severe asthma.

BackgroundSevere asthma accounts for a small number of asthmatics but represents a disproportionate cost to health care systems. The underlying mechanism in severe asthma remains unknown but several mechanisms are likely to be involved because of a very heterogeneous profile. We investigated the effects of a p38MAPK inhibitor in corticosteroid sensitivity in peripheral blood mononuclear cells (PBMCs) from severe asthmatics and the profile of its responders.Methodology/Principal FindingsCorticosteroid sensitivity was determined by measuring dexamethasone inhibition of CD3/28 and TNF-α induced IL-8 production in PBMCs by using ELISA. PBMCs from severe asthmatics were relatively less sensitive to dexamethasone (Dex) as compared to those of non-severe asthmatics and healthy volunteers. The IC50 values of Dex negatively correlated with decreased glucocorticoid receptor (GR) nuclear translocation assessed using immunocytochemistry (r = −0.65; p<0.0005) and with decreased FEV1 (% predicted) (r = 0.6; p<0.0005). A p38α/β inhibitor (SB203580) restored Dex-sensitivity in a subpopulation of severe asthma that was characterized by a defective GR nuclear translocation, clinically by lower FEV1 and higher use of oral prednisolone. We also found that SB203580 partially inhibited GR phosphorylation at serine 226, resulting in increased GR nuclear translocation in IL-2/IL-4 treated corticosteroid insensitive U937s.Conclusions/Significancep38MAPKα/β is involved in defective GR nuclear translocation due to phosphorylation at Ser226 and this will be a useful biomarker to identify responders to p38MAPKα/β inhibitor in the future.

Identification of Nuclear Protein Targets for Six Leukemogenic Tyrosine Kinases Governed by Post-Translational Regulation

Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases.

Plasticity of Button-Like Junctions in the Endothelium of Airway Lymphatics in Development and Inflammation

Endothelial cells of initial lymphatics have discontinuous button-like junctions (buttons), unlike continuous zipper-like junctions (zippers) of collecting lymphatics and blood vessels. Buttons are thought to act as primary valves for fluid and cell entry into lymphatics. To learn when and how buttons form during development and whether they change in disease, we examined the appearance of buttons in mouse embryos and their plasticity in sustained inflammation. We found that endothelial cells of lymph sacs at embryonic day (E)12.5 and tracheal lymphatics at E16.5 were joined by zippers, not buttons. However, zippers in initial lymphatics decreased rapidly just before birth, as buttons appeared. The proportion of buttons increased from only 6% at E17.5 and 12% at E18.5 to 35% at birth, 50% at postnatal day (P)7, 90% at P28, and 100% at P70. In inflammation, zippers replaced buttons in airway lymphatics at 14 and 28 days after Mycoplasma pulmonis infection of the respiratory tract. The change in lymphatic junctions was reversed by dexamethasone but not by inhibition of vascular endothelial growth factor receptor-3 signaling by antibody mF4-31C1. Dexamethasone also promoted button formation during early postnatal development through a direct effect involving glucocorticoid receptor phosphorylation in lymphatic endothelial cells. These findings demonstrate the plasticity of intercellular junctions in lymphatics during development and inflammation and show that button formation can be promoted by glucocorticoid receptor signaling in lymphatic endothelial cells.

The nuclear receptor cochaperone FKBP51 is required for diet‐induced visceral adiposity

Nuclear receptors are hormone‐regulated transcription factors that form heterocomplexes with Hsp90 and several cochaperones, including FK506‐binding protein‐51 (FKBP51), FKBP52, PP5, and Cyp40. We have evidence that loss of FKBP51 increases glucocorticoid receptor (GR) and decreases peroxisome proliferator‐activated receptor‐γ (PPARγ) transcriptional activity. In this study, we investigate the hypothesis that loss of FKBP51 leads to reciprocal dysregulation of GR and PPARγ, thereby increasing the expression of genes regulating lipid oxidation and/or energy expenditure. FKBP51‐KO mice were subjected to a high fat diet, compared to wild‐type mice, FKBP51‐KO animals were resistant to diet‐induced obesity, with reduced visceral adiposity, serum triglyceride, and free fatty acids. Food consumption, insulin secretion and clearance, and hepatic lipid accumulation were unchanged. Gene profiling of adipose showed elevated expression of thermogenic genes in the KO mice. In vitro adipogenesis studies showed FKBP51‐KO cells to be highly resistant to differentiation and lipid accumulation. The phosphorylation states of GR were tested using phospho‐specific antibodies for serines 212, 220 and 234 – the major sites of hormone‐induced phosphorylation. Results show FKBP51 controls GR phosphorylation at serines 220 and 234. These investigations have uncovered FKBP51 as a key regulator of adipogenesis and thermogenesis and may serve as a new drug target in the treatment of obesity and related disorders.Supported by NIH grant DK70127 to E.R.S.

Increased corticosteroid sensitivity by a long acting β2 agonist formoterol via β2 adrenoceptor independent protein phosphatase 2A activation

Long-acting β2-adrenoceptor agonists (LABAs) are reported to enhance anti-inflammatory effects of corticosteroids in vitro and in vivo, although the molecular mechanisms have not yet been elucidated. We investigated the role of serine/threonine protein phosphatase 2A (PP2A) on regulation of corticosteroid sensitivity via inhibition of glucocorticoid receptor (GR) phosphorylation as the target of formoterol, an LABA. Corticosteroid sensitivity was determined as IC50 to dexamethasone (Dex) on TNFα-induced IL-8 release in a U937 monocytic cell line (Dex-IC50). Phosphorylation levels of GR-Ser226 and c-Jun N-terminal kinase (JNK) were determined by western-blotting. Phosphatase activity of immunopurified PP2A was measured by fluorescence-based assay. Exposure to IL-2/IL-4 for 48 h decreased Dex sensitivity with a concomitant increase of GR phosphorylation at Ser226 with JNK1 activation. Formoterol restored Dex sensitivity by inhibiting phosphorylation of GR-Ser226 and JNK1. PP2A inhibition by okadaic acid, a phosphatase inhibitor, abrogated formoterol-mediated effects. In addition, formoterol enhanced PP2A activity in intact or IL-2/IL-4 treated U937 cells and human peripheral blood mononuclear cells. In addition, PP2A activation by formoterol was not antagonized by ICI-118551, and formoterol could activate PP2A directly in cell free system. Taken together, formoterol increases corticosteroid sensitivity via activation of PP2A in receptor independent manner, explaining its benefits as add-on therapy for the treatment of corticosteroid-insensitive diseases, such as severe asthma.

P.4.017 Cortisol decreases hippocampal neurogenesis by regulating the enzyme serum- and glucocorticoid-regulated kinase 1

Chronic stress increases circulating glucocorticoid hormones, which impair hippocampal neurogenesis by activating the glucocorticoid receptor (GR) [1], and thereby potentially contribute to the development of depressive symptoms [2]. However, the molecular mechanisms underlying this effect are unknown. The enzyme, serum-and glucocorticoid-regulated kinase 1 (SGK1) has been shown to mediate some glucocorticoid effects on the brain [3]. We therefore hypothesized that SGK1 may also mediate the effects of glucocorticoids on hippocampal neurogenesis in humans. We treated human hippocampal progenitor cells (HPC03A/07, ReNeuron, UK) with high concentrations of the glucocorticoid, cortisol (100mM), and with the SGK1-inhibitor, GSK650394 (100nM) (Tocris Biosciences). We analysed cell proliferation by 5′-bromodeoxyuridine (BrdU, 10mM) incorporation, and neuronal differentiation by immunocytochemistry for the neuronal marker, microtubule-associated protein-2 (MAP2). GR activation was examined by Western blot using specific antibodies for the GR phosphorylation-sites, S203, S211 and S226. Cortisol decreased BrdU-positive, proliferating cells (by −13±2%; p = 0.03, n = 3), and neuronal differentiation into MAP2positive neurons (by −16±2%; p = 0.04, n = 3). The SGK1inhibitor, GSK650394 (100nM), counteracted the effects of cortisol on proliferation (p = 0.0027, n = 4) and on neuronal differentiation (p = 0.03, n = 3). Moreover, cortisol induced GR phosphorylation at S203 (1.5±0.1 fold; p = 0.01, n = 3), S211 (3.0±0.5 fold; p = 0.004, n = 3) and S226 (1.6±0.2 fold; p = 0.04, n = 3). Cotreatment with GSK650394 (100nM) counteracted the cortisolinduced GR phosphorylation at S203 (p = 0.01, n = 3) and S211 (p = 0.03, n = 3), but not at the S226 phospho-site (p = 0.8, n = 3). In summary, our data identify the kinase, SGK1, as a novel mechanism mediating the effects of cortisol on human hippocampal neurogenesis and GR function.

Glucocorticoids reduce inflammation in cystic fibrosis bronchial epithelial cells

Reduction of lung inflammation is one of the goals of cystic fibrosis (CF) therapy. Among anti-inflammatory molecules, glucocorticoids (GC) are one of the most prescribed. However, CF patients seem to be resistant to glucocorticoid treatment. Several molecular mechanisms that contribute to decrease anti-inflammatory effects of glucocorticoids have been identified in pulmonary diseases, but the molecular actions of glucocorticoids have never been studied in CF. In the cytoplasm, glucocorticoids bind to glucocorticoid receptor (GR) and then, control NF-κB and MAPK pathways through direct interaction with AP-1 and NF-κB in the nucleus. Conversely, MAPK can regulate glucocorticoid activation by targeting GR phosphorylation. Together these pathways regulate IL-8 release in the lung. Using bronchial epithelial cell lines derived from non CF and CF patients, we analyzed GR-based effects of glucocorticoids on NF-κB and MAPK pathways, after stimulation with TNF-α. We demonstrate that the synthetic glucocorticoid dexamethasone (Dex) significantly decreases IL-8 secretion, AP-1 and NF-κB activity in CF cells in a pro-inflammatory context. Moreover, we show that p38 MAPK controls IL-8 release by determining GR activation through specific phosphorylation on serine 211. Finally, we demonstrate a synergistic effect of dexamethasone treatment and inhibition of p38 MAPK inducing more than 90% inhibition of IL-8 production in CF cells. All together, these results demonstrate the good responsiveness to glucocorticoids of CF bronchial epithelial cells and the reciprocal link between glucocorticoids and p38 MAPK in the control of CF lung inflammation.

O-34 - Phosphorylation of leukocyte glucocorticoid receptor as a measure of stress vulnerability in healthy women and men

The core mechanism of maladaptive stress response includs aberrant phosphorylation of the glucocorticoid receptor (GR) at serine 221 (pGR211) or GR serine 226 (pGR226), which enhance or attenuate GR transcriptional activity, respectively. In the current study we tested a hypothesis that alterations in the nuclear levels and/or ratios of GR, pGR211 and pGR226 in peripheral blood mononuclear cells (PBMC) may reflect individual level of distress and/or psychological vulnerability. In 36 healthy adults we evaluated the relation of total PMBC nuclear level of GR (tGR), pGR221, pGR226 and activated JNK1 (pJNK1) and its relation to environmental stress exposure, self reported distress (Depression Anxiety Stress Scale) and personality traits (Eysenck Personality Questionnaire). Molecular and psychometric data were cross-correlated by SPSS MannWhitney statistics. In women, self reported distress (DASS score) correlated more with neuroticism, while in men it correlated with environmental stressor. Also, in women, PBMC nuclear tGR level correlated with pGR211, while in men it correlated with pGR226. Cross-analysis of psychometric and molecular data indicated that in women both neuroticism and self reported distress correlated positively with nuclear pGR226, while in men the only correlation was between environmental stressor and pJNK1. This findings provide preliminary support that the nuclear level of pGR226 and pGR211/pGR226 ratio in PBMC may serve as a measure of affective vulnerability in healthy women, while nuclear level of pJNK1 may reflect environmentaly imposed distress in healthy men. The relevance of these molecular parameters as biomarkers requires broader verification in healthy and in clinical settings.

Post-Translational Modifications of Nuclear Receptors and Human Disease

Nuclear receptors (NR) impact a myriad of physiological processes including homeostasis, reproduction, development, and metabolism. NRs are regulated by post-translational modifications (PTM) that markedly impact receptor function. Recent studies have identified NR PTMs that are involved in the onset and progression of human diseases, including cancer. The majority of evidence linking NR PTMs with disease has been demonstrated for phosphorylation, acetylation and sumoylation of androgen receptor (AR), estrogen receptor α (ERα), glucocorticoid receptor (GR) and peroxisome proliferator activated receptor γ (PPARγ). Phosphorylation of AR has been associated with hormone refractory prostate cancer and decreased disease-specific survival. AR acetylation and sumoylation increased growth of prostate cancer tumor models. AR phosphorylation reduced the toxicity of the expanded polyglutamine AR in Kennedy’s Disease as a consequence of reduced ligand binding. A comprehensive evaluation of ERα phosphorylation in breast cancer revealed several sites associated with better clinical outcome to tamoxifen therapy, whereas other phosphorylation sites were associated with poorer clinical outcome. ERα acetylation and sumoylation may also have predictive value for breast cancer. GR phosphorylation and acetylation impact GR responsiveness to glucocorticoids that are used as anti-inflammatory drugs. PPARγ phosphorylation can regulate the balance between growth and differentiation in adipose tissue that is linked to obesity and insulin resistance. Sumoylation of PPARγ is linked to repression of inflammatory genes important in patients with inflammatory diseases. NR PTMs provide an additional measure of NR function that can be used as both biomarkers of disease progression, and predictive markers for patient response to NR-directed treatments.

Defects of Protein Phosphatase 2A Causes Corticosteroid Insensitivity in Severe Asthma

BackgroundCorticosteroid insensitivity is a major barrier of treatment for some chronic inflammatory diseases, such as severe asthma, but the molecular mechanism of the insensitivity has not been fully elucidated. The object of this study is to investigate the role of protein phosphate 2A (PP2A), a serine/threonine phosphatase, on corticosteroid sensitivity in severe asthma.Methodology/Principal FindingsCorticosteroid sensitivity was determined by the dexamethasone ability to inhibit TNFα-induced IL-8 or LPS-induced TNFα production. PP2A expression, glucocorticoid receptor (GR) nuclear translocation defined as the nuclear/cytoplasmic GR ratio and phosphorylation of GR-Ser226, c-Jun N-terminal kinase 1 (JNK1) and PP2A were analysed by Western-blotting. Phosphatase activity was measured by fluorescence-based assay. Okadaic acid (OA), a PP2A inhibitor, reduced corticosteroid sensitivity with reduced GR nuclear translocation and increased GR phosphorylation in U937 monocytic cells. PP2A knockdown by RNA interference showed similar effects. IL-2/IL-4 treatment to U937 reduced corticosteroid sensitivity, and PP2A expression/activity. In peripheral blood mononuclear cells (PBMCs) from severe asthma, the PP2A expression and activity were significantly reduced with concomitant enhancement of PP2AC-Tyr307 phosphorylation compared with those in healthy volunteers. As the results, GR-Ser226 and JNK1 phosphorylation were increased. The expression and activity of PP2A were negatively correlated with phosphorylation levels of GR-Ser226. Furthermore, co-immunoprecipitation assay in U937 cells revealed that PP2A associated with GR and JNK1 and IL-2/IL-4 exposure caused dissociation of each molecule. Lastly, PP2A overexpression increased corticosteroid sensitivity in U937 cells.Conclusions/SignificancePP2A regulates GR nuclear translocation and corticosteroid sensitivity possibly by dephosphorylation of GR-Ser226 via dephosphorylation of upstream JNK1. This novel mechanism will provide new insight for the development of new therapy for severe asthma.

P38 Mitogen-Activated Protein Kinase-γ Inhibition by Long-Acting β2Adrenergic Agonists Reversed Steroid Insensitivity in Severe Asthma

Corticosteroid insensitivity (CI) is a major barrier to treating severe asthma. Despite intensive research, the molecular mechanism of CI remains uncertain. The aim of this study was to determine abnormality in corticosteroid action in severe asthma and to identify the molecular mechanism of the long-acting β(2)-adrenergic agonists (LABAs) formoterol and salmeterol on restoration of corticosteroid sensitivity in severe asthma in vitro. Peripheral blood mononuclear cells (PBMCs) were obtained from 16 subjects with severe corticosteroid-insensitive asthma, 6 subjects with mild corticosteroid-sensitive asthma, and 11 healthy volunteers. Corticosteroid (dexamethasone) sensitivity was determined on tumor necrosis factor-α (TNF-α)-induced interleukin (IL)-8 production. Glucocorticoid receptor (GR) phosphorylation and kinase phosphorylation were evaluated by immunoprecipitation-Western blotting analysis and kinase phosphorylation array in IL-2/IL-4-treated corticosteroid insensitive model in PBMCs. In vitro corticosteroid sensitivity on TNF-α-induced IL-8 production was significantly lower in patients with severe asthma than in healthy volunteers and patients with mild asthma. This CI seen in severe asthma was associated with reduced GR nuclear translocation and with hyperphosphorylation of GR, which were reversed by LABAs. In IL-2/IL-4-treated PBMCs, LABAs inhibited phosphorylation of Jun-NH(2)-terminal kinase and p38 mitogen-activated protein kinase-γ (p38MAPK-γ) as well as GR. In addition, cells with p38MAPK-γ knockdown by RNA interference did not develop CI in the presence of IL-2/IL-4. Furthermore, p38MAPK-γ protein expression was up-regulated in PBMCs from some patients with severe asthma. In conclusion, p38 MAPK-γ activation impairs corticosteroid action and p38 MAPK-γ inhibition by LABAs has potential for the treatment of severe asthma.

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.