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THE EFFECT OF DEXAMETHASONE ON IL-33-MEDIATED MAST CELL ACTIVATIONChernushevich, Oksana I 01 January 2015 (has links)
Dexamethasone has been shown to inhibit IgE-mediated mast cell activation, and the present research investigated its role in suppressing IL-33-mediated mast cell activation. We have found that micromolar concentrations of Dexamethasone are capable of suppressing IL-33-mediated mast cell cytokine production, on several genetic backgrounds, and in not only bone marrow derived mast cells, but also peritoneal mast cells. Intracellular staining demonstrated that Dexamethasone significantly reduces expression of the IL-33 receptor, T1/ST2, in mast cells; however, the cytokine suppression is independent of T1/ST2 downregulation. At the same time, Dexamethasone pretreatment significantly reduced ERK phosphorylation, but our data suggests that inhibition occurs even prior to ERK blockade. Finally, Dexamethasone treatment in vivo reduced IL-33-mediated cytokine production and neutrophil infiltration in the murine peritoneum. Thus, Dexamethasone, a well-established therapy for inflammatory disease, can suppress IL-33-mediated mast cell activation, and may therefore be effective for treating diseases now being attributed to IL-33 effects.
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Role of oxidative stress in the pathogenesis of triple A syndrome and familial glucocorticoid deficiencyPrasad, Rathi January 2014 (has links)
Maintaining redox homeostasis is crucial for normal cellular functions. Electron leak by the cytochrome P450 enzymes renders steroidogenic tissues acutely vulnerable to redox imbalance and oxidative stress is implicated in several potentially lethal adrenal disorders. This thesis aims to further delineate the role of oxidative stress in triple A syndrome and familial glucocorticoid deficiency (FGD). Triple A syndrome incorporates adrenal failure and progressive neurodegenerative disease. The AAAS gene product is the nuclear pore complex protein ALADIN, of unknown function. Patient dermal fibroblasts are sensitive to oxidative stress, with failure of nuclear import of DNA repair proteins and ferritin heavy chain protein. To provide an adrenal and neuronal-specific disease model, I established AAAS-knockdown in H295R human adrenocortical tumour cells and SH-SY5Y human neuroblastoma cells. This had effects on cell viability, exacerbated by hydrogen peroxide treatment. Redox homeostasis was impaired in AAAS-knockdown H295R cells, with depletion of key components of the steroidogenic pathway and a significant reduction in cortisol production, with partial reversal following treatment with N-acetylcysteine. Mutations in the mitochondrial antioxidant, nicotinamide nucleotide transhydrogenase (NNT), causing FGD, have recently highlighted the importance of redox regulation in steroidogenesis. I investigated seven individuals from a consanguineous Kashmiri kindred, mutation negative for known causes of FGD. A stop gain mutation, p.Y447* in TXNRD2, encoding the mitochondrial selenoprotein thioredoxin reductase 2 segregated with the disease trait; with complete absence of the 56 kDa TXNRD2 protein in patients homozygous for the mutation. TXNRD2-knockdown led to impaired redox homeostasis in H295R cells. This is the first report of a homozygous mutation in any component of the thioredoxin antioxidant system leading to inherited disease in humans.
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Investigating the non-genomic actions of the glucocorticoid receptorKershaw, Stephen January 2018 (has links)
Glucocorticoids (GCs) are a class of steroid hormone that play essential roles in development, glucose homeostasis, and reducing inflammation. Clinically, GCs are potent anti-inflammatory and immunosuppressive agents used to treat a variety of diseases. However, the therapeutic benefit of GCs is negatively impacted by the induction of severe side effects. In this thesis, I present two studies that have contributed to the understanding of the non-genomic actions of GCs. GCs inhibit cell migration by a non-transcriptional pathway involving HDAC6: A negative side effect of GC therapy is impaired wound healing which is ascribed to inhibited cell migration. Using live-cell microscopy, I show that GCs inhibit cell migration within 30 minutes of administration. GCs alter the dynamics of the microtubule network through rapid induction of tubulin acetylation (by inhibition of HDAC6) which increases microtubule stability and slows cell movement. The inhibitory effect of GCs on cell migration is reversed by overexpressing HDAC6. Using quantitative imaging, I identified a rapid ligand-dependent association of the GR and HDAC6 within the cytoplasm that is absent in the nucleus. However, a very small proportion of HDAC6 enters the nucleus post-GC treatment, suggesting that HDAC6 accompanies the GR during nuclear translocation. This study demonstrates that GCs rapidly inhibit cell migration by a non-transcriptional mechanism involving HDAC6. Investigating the rapid effects of GCs on the phosphoproteome: Non-steroidal GCs are useful tool compounds to dissect glucocorticoid receptor (GR) activity. Here, I investigated the early, rapid effect of GCs on the phosphoproteome of A549 cells using SILAC-based phosphoproteomics. A consistent spectrum of phosphoproteins was differentially regulated by GC within 10 minutes of administration, notably including regulators of RNA polymerase II, chromatin remodifying proteins, transcription factors, cytoskeletal modifiers, regulators of intracellular calcium signalling and endocytosis. These phosphoproteins were validated by western blotting. This study shows a clear early effect of GCs on the phosphoproteome with implications for non-specific, non-transcriptional activity of GCs.
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Deficiency of 11β-HSD1 modulates energy homeostasis in the brain following systemic inflammationVerma, Manu January 2017 (has links)
Chronically elevated brain glucocorticoid (GC) levels impair cognition. Age-related cognitive deficits or "sickness" behaviour is often associated with neuroinflammation. In rodents, raised GC levels prior to lipopolysaccharide (LPS) administration potentiate neuroinflammation although GC suppresses neuroinflammation if administered after LPS. 11β-hydroxysteroid dehydrogenase-1 (11β-HSD1) reductase activity can increase intracellular GC levels, including in the brain, without alteration in circulating levels. Deficiency/pharmacological inhibition of 11β-HSD1 is protective against age related cognitive impairment in both rodent and humans. However, the underlying mechanism remains unclear. 11β-HSD1 reductase activity is coupled to hexose-6-phosphate dehydrogenase activity, itself dependent on cellular energy status. Processes affected by deficiency/inhibition of 11β- HSD1 (e.g. acute inflammation, angiogenesis) are associated with increased glycolysis. Additionally, compared to C57BL/6J controls, adipose tissue of 11β-HSD1 deficient mice shows increased expression of glycolytic and oxidative metabolism genes in a rodent model of obesity, characterised by low-grade chronic inflammation. I hypothesised that 11β-HSD1 has a role in regulation of cellular energetics basally and following inflammation. 11β-HSD1 expression in the brain will be up-regulated during systemic inflammation. Following inflammation, 11β-HSD1 deficiency will attenuate the pro-inflammatory response and subsequently alter energy substrate uptake and/or utilisation in the key areas of brain (i.e. hypothalamus and the hippocampus) that sense and respond to inflammation and energy balance. To test my hypothesis, global 11β-HSD1 KO mice, primary macrophages in vitro and murine models of inflammations were utilised. 11β-HSD1 mRNA and protein expression were confirmed in the hypothalamus and the hippocampus of C57BL/6J mice. In the absence of inflammation, expression of inflammatory markers is low or negligible in the brains of Hsd11b1-/- mice similar to C57BL/6J controls. However, compared to C57BL/6J, Hsd11b1-/- mice show altered mRNA levels of metabolic transporters and enzymes in the hypothalamus and the hippocampus. Overall, the mRNA profiling suggests reduced dependence on glucose in the brains of Hsd11b1-/- mice, either through increased lactate availability (in the whole brain and hippocampus) or through increased glycolysis and mitochondrial number/function (in the hypothalamus). Primary macrophages were utilised to investigate the role of 11β-HSD1 in cellular energetics in vitro. In these cell based assays, glycolysis was found to be the predominant glucose metabolising pathway in C57BL/6J primary macrophages, consistent with the literature. Preliminary data suggested reduced glycolytic activity in Hsd11b1-/- compared to C57BL/6J primary macrophages. However, initial attempts to utilise these cell based assays on primary microglia were unsuccessful. Moreover, Hsd11b1 mRNAs in the brain (down-regulation with inflammation, discussed later) was found to be differentially regulated in comparison to Hsd11b1 mRNA levels in the macrophages (up-regulation with inflammation) hence further investigation was not pursued. To identify a model of peripheral inflammation where 11β-HSD1 is regulated in the brain in vivo, Staph. aureus induced acute lung inflammation and the K/BxN serum transfer induced model of arthritis were utilised. Increased expression of inflammatory markers in the brain was associated with reduced Hsd11b1 mRNA levels in the hippocampus of control mice in these models. Comparison of Hsd11b1-/- and C57BL/6J mice showed increased levels of mRNAs encoding metabolic transporters in the hypothalamus and the hippocampus of Hsd11b1-/- mice following inflammation in the K/BxN serum transfer model of arthritis suggesting increased energy substrate availability. Additionally, increased levels of mRNA encoding metabolic enzymes suggested increased glycolytic capacity and mitochondrial oxidative phosphorylation activity in the hippocampus but not the hypothalamus of Hsd11b1- /-, compared to C57BL/6J mice, following K/BxN serum induced arthritis. Overall, these data suggest that the reduction in expression of 11β-HSD1 could be a potential mechanism to increase energy substrate availability, glycolytic capacity and mitochondrial activity in the hippocampus to provide metabolic support for neuronal metabolism and function following peripheral inflammation. The role of 11β-HSD1 in the pro-inflammatory response and cellular energetics in the hippocampus was further investigated in a well characterised sterile peritonitis model of systemic inflammation in which a low to moderate dose of LPS was used. Mice were administered LPS or vehicle (0.9% saline) by a single i.p. injection and culled 3h, 6h or 9h post injection. Inflammation resulted in significant reduction in burrowing activity both in Hsd11b1-/- and C57BL/6J mice suggesting sickness behaviour.. The number of circulating immune cells, as a measure of peripheral inflammation, did not differ between genotypes. Similarly, plasma corticosterone levels were elevated following inflammation but no genotype difference was observed. However, levels of plasma 11-dehydrocorticosterone, the inert substrate for 11β- HSD1, were significantly elevated in the Hsd11b1-/-, compared to C57BL/6J mice, following inflammation. Levels of mRNA encoding inflammatory markers were lower in the hippocampus of Hsd11b1-/-, compared to C57BL/6J mice, following inflammation. Also, Hsd11b1 mRNA levels were reduced in the hippocampus of C57BL/6J mice following inflammation, consistent with the finding above. Principal component analysis on levels of mRNA encoding metabolite transporters and enzymes revealed a distinct metabolic response in the hippocampus of Hsd11b1-/-, compared to C57BL/6J mice, 6h post LPS. At the same time point in the hippocampus, levels of mRNAs encoding metabolite transporters and enzymes suggested an attenuated switch to aerobic glycolysis with maintenance of mitochondrial function/activity. Quantification of hippocampal energy metabolites using targeted metabolomics in the Hsd11b1-/- compared to C57BL/6J mice 6h post LPS showed correspondence with the mRNA results. Overall, these results suggest that reduced expression of 11β-HSD1 could be a potential mechanism to reduce the pro-inflammatory response and provide better metabolic support for neuronal function and metabolism in the hippocampus, following systemic inflammation. In summary, the current work provides evidence for neuroprotection with 11β-HSD1 deficiency, following systemic inflammation. The suggestive neuroprotection is at least in part mediated via an attenuated pro-inflammatory responses and increased energy substrate uptake and/or utilisation providing better metabolic support for neuronal function following inflammation. It argues for the development of tissue specific small molecule inhibitors of 11β-HSD1 that can cross the blood brain barrier as therapeutic agents against the adverse cognitive effects of systemic inflammation and/or inflammaging.
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Defining the DNA binding energetics of the glucocorticoid receptorZhang, Liyang 01 December 2017 (has links)
DNA-binding proteins bind to specific sequences to direct their activity to defined loci in the genome. Regulation of gene expression, for example, is dependent on the recognition of specific DNA sequences by transcription factors (TFs). These TFs receive input from cellular signals to control panels of genes to meet the needs of the cells. Critical to this function is the recognition and binding of TFs to the correct DNA sequence. The main focus of this thesis is to quantitatively determine how proteins, including TFs, distinguish DNA sequences, and to understand how DNA sequence affect their function. Primarily using the Glucocorticoid receptor (GR) as the model TF, I developed novel methods to measure the DNA binding specificity over long binding sites. These methods: 1) Distinguished the sequence specificity of GR and closely related androgen receptor (AR), which helped to both account for differential genomic localization between the two factors, and explained how GR can functionally substitute for AR in castration-resistant prostate cancer (Chapter II); 2) Explored the effect of DNA sequence on GR-regulated transcription through the specification of monomeric versus dimeric binding. Sequence motifs that bias GR binding toward the monomeric state were discovered (Chapter III); 3) Demonstrated a conserved role of intrinsic specificity in directing the degree of GR genomic occupancy in vivo in a fixed chromatin context (Chapter V); 4) Quantitatively modeled and decoupled the DNA binding and cleavage specificities of CRISPR-Cas9 system, providing a rapid pipeline to characterize the genome-editing reagents (Chapter IV). In summary, we showed here that DNA binding specificity is only the initial step in directing the activity of the bound protein. Beyond the affinity-based recruitment, DNA sequences can regulate the protein activity through alternative mechanisms, such as modulating the binding cooperativity, or directly serving as an allosteric ligand for protein function that is independent of DNA binding affinity.
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Sequence and Effects of Glucocorticoid Receptor Nuclear Retention: An Aid to Understanding Nuclear Retention in Other Proteins?Carrigan, Amanda 27 January 2011 (has links)
Corticosteroid ligands activate the glucocorticoid receptor (GR). GR plays a role in glucose homeostasis, adipogenesis, inflammation, and mood and cognitive functions. Understanding the interplay of diverse forms of receptor regulation (including post-translational modification, cofactor interactions, ligand binding, and receptor localization) and their effects is important for understanding and developing more effective treatment for a variety of conditions. Prior to ligand binding, the naïve GR is primarily cytoplasmic, residing in a chaperone complex containing heat-shock proteins and immunophilins. Upon ligand-binding, alterations to the complex allow the receptor to dimerize and import into the nucleus. Nuclear GR interacts with transcriptional regulatory sequences and recruits cofactors to regulate specific gene expression. Upon hormone withdrawal, the original chaperone complex is reassembled and the receptor is exported to the cytoplasm. Interestingly, while the import of GR into the nucleus occurs very rapidly (t ½ = 5 min), the re-export is significantly slower (t ½ = 12-24h). Previous work by our lab and others has indicated the existence of a nuclear retention signal (NRS) within the GR. The NRS sequence of the GR, its interaction partners, and the role it might play in the activity of the receptor have not yet been fully defined. Work in the Hache lab indicates that mutation of the GR nuclear localization signal 1 (NL1) increases the export rate of nuclear GR to the cytoplasm, as well as compromising receptor import, suggesting that the NL1 overlapped an NRS sequence. In this work, I made a series of GR mutants, based on sequence from the SV40 large T antigen NLS, which lacks nuclear retention activity. Using these mutants, I found that GR nuclear retention is influenced by both specific residues within the hinge region and the location of the sequence within the receptor, as reintroduction of the NLS sequence at the N-terminus of the receptor retention mutant failed to reconstitute the retention activity. Agonist liganded and hormone-withdrawn receptor mutants showed a similar decrease in retention. By contrast, antagonist-withdrawn GR mutants were retained in the nucleus, possibly due to altered receptor configuration and interactions. Assays of GR-responsive promoter activation by receptor retention mutants showed that while no difference in the ability of retention mutants to activate transcription was seen at a simple promoter, activation of a complex promoter was compromised. This impaired transactivation for the SV506-523 mutant correlated with decreased histone H4 acetylation and PolII recruitment, while GR DNA-binding at the target promoter appeared to be unaffected. These results suggested that promoter-specific cofactor interactions might be implicated in GR nuclear retention. Loss of GR hinge interaction with Oct cofactors produced an incomplete loss of retention, suggesting overlapping signals, but not supporting Oct as a primary factor in GR retention. The overlap between important residues in GR nuclear retention and localization signals and the lack of retention shown by the SV40 NLS suggested that retention might be intrinsic to the sequence of particular NLS. Preliminary results suggest that the KT511-512 residues of GR may be of general importance in protein nuclear retention, while the role of proline is likely more variable. My research has focused on increasing our understanding of glucocorticoid receptor nuclear retention and its possible implications. I have determined that the KT511-512 residues of GR play an important role in its retention, and possibly also figure in nuclear retention of other proteins. These residues are involved in interactions which affect promoter-specific histone acetylation and transcriptional activation in GR, suggesting a reason for the existence of nuclear retention.
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Exploring the Suitability of a Specifici Glucocorticoid Receptor Antagonist as a Tool in the Study of the Regulation of Rat Lung Alveolarization by GlucocorticoidsLopez, Ana Sofia 10 January 2011 (has links)
Background: Intracellular glucocorticoid receptors (GRs) mediate the regulation of lung development, including alveolarization, by glucocorticoids (GCs). One potential approach to determining the role of GC-GR signalling in alveolar formation would be by pharmacologic blockade.
Hypothesis: CP472555, a novel GR antagonist with negligible anti-PR activity, is a suitable tool for the study of GC-GR regulation of rat alveolarization.
Design/Methods: CP472555 doses needed to block GR were estimated in vitro in fetal rat lung primary cultures. Postnatally, a variety of doses were administered intraperitoneally over a range of days.
Results: During postnatal days (PN)0-PN10, when GC levels are low, CP472555 induced changes consistent with GR agonist activity. While GC levels increase after PN11, animals exposed to CP472555 from PN11-PN21 exhibit changes consistent with anti-GR antagonist activity.
Conclusion: CP472555 causes a degree of GR blockade sufficient to permit further pharmacological investigation of the role of endogenous GC-GR signalling at the end of alveolarization.
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Exploring the Suitability of a Specifici Glucocorticoid Receptor Antagonist as a Tool in the Study of the Regulation of Rat Lung Alveolarization by GlucocorticoidsLopez, Ana Sofia 10 January 2011 (has links)
Background: Intracellular glucocorticoid receptors (GRs) mediate the regulation of lung development, including alveolarization, by glucocorticoids (GCs). One potential approach to determining the role of GC-GR signalling in alveolar formation would be by pharmacologic blockade.
Hypothesis: CP472555, a novel GR antagonist with negligible anti-PR activity, is a suitable tool for the study of GC-GR regulation of rat alveolarization.
Design/Methods: CP472555 doses needed to block GR were estimated in vitro in fetal rat lung primary cultures. Postnatally, a variety of doses were administered intraperitoneally over a range of days.
Results: During postnatal days (PN)0-PN10, when GC levels are low, CP472555 induced changes consistent with GR agonist activity. While GC levels increase after PN11, animals exposed to CP472555 from PN11-PN21 exhibit changes consistent with anti-GR antagonist activity.
Conclusion: CP472555 causes a degree of GR blockade sufficient to permit further pharmacological investigation of the role of endogenous GC-GR signalling at the end of alveolarization.
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The Relationship of Dominance, Reproductive State and Stress in a Non-Cooperative Breeder, The Domestic Horse (<em>Equus caballus</em>)Sinderbrand, Carly Anne 01 August 2011 (has links)
Animals that live in a social group are often organized in a hierarchy with rank determining access to resources. Maintaining a dominant position requires a high rate of energetically expensive aggressive displays and physical exertion. Lab based winnerloser studies, often conducted with individuals from non-social species, have shown that subordinates have higher stress hormone levels than dominant individuals (subordinatestress hypothesis). However, in carnivorous animals that are cooperative breeders, displays of aggression are associated with elevated stress hormone levels (dominancestress hypothesis). The effect of reproductive state on dominance and stress is not addressed within either of these hypotheses. The purpose of this study was to examine stress level in relation to dominance rank and reproductive state in a non-cooperative breeder and herbivore, the domestic horse. As rank and reproductive state can affect behavior, I examined activity budgets, behavioral patterns, and social interactions, as well as the proximity and identity of neighbors in the social group. At two facilities in Kentucky, I recorded the social interactions and measured fecal glucocorticoids in pastured, female horses that were either lactating or non-lactating. While fecal glucocorticoid level did not differ between reproductive state and rank, activity behavior demonstrated significant differences between reproductive states. Higher energetic requirements of lactation were reflected in significantly longer bouts of eating and significantly less time spent alert and socializing. The non-cooperative social nature of horses does not limit their reproduction or resource acquisition based upon rank, and therefore does not fit with the dominance-stress hypothesis or subordinate-stress hypothesis and instead offers the alternative of an independent-stress hypothesi
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Sequence and Effects of Glucocorticoid Receptor Nuclear Retention: An Aid to Understanding Nuclear Retention in Other Proteins?Carrigan, Amanda 27 January 2011 (has links)
Corticosteroid ligands activate the glucocorticoid receptor (GR). GR plays a role in glucose homeostasis, adipogenesis, inflammation, and mood and cognitive functions. Understanding the interplay of diverse forms of receptor regulation (including post-translational modification, cofactor interactions, ligand binding, and receptor localization) and their effects is important for understanding and developing more effective treatment for a variety of conditions. Prior to ligand binding, the naïve GR is primarily cytoplasmic, residing in a chaperone complex containing heat-shock proteins and immunophilins. Upon ligand-binding, alterations to the complex allow the receptor to dimerize and import into the nucleus. Nuclear GR interacts with transcriptional regulatory sequences and recruits cofactors to regulate specific gene expression. Upon hormone withdrawal, the original chaperone complex is reassembled and the receptor is exported to the cytoplasm. Interestingly, while the import of GR into the nucleus occurs very rapidly (t ½ = 5 min), the re-export is significantly slower (t ½ = 12-24h). Previous work by our lab and others has indicated the existence of a nuclear retention signal (NRS) within the GR. The NRS sequence of the GR, its interaction partners, and the role it might play in the activity of the receptor have not yet been fully defined. Work in the Hache lab indicates that mutation of the GR nuclear localization signal 1 (NL1) increases the export rate of nuclear GR to the cytoplasm, as well as compromising receptor import, suggesting that the NL1 overlapped an NRS sequence. In this work, I made a series of GR mutants, based on sequence from the SV40 large T antigen NLS, which lacks nuclear retention activity. Using these mutants, I found that GR nuclear retention is influenced by both specific residues within the hinge region and the location of the sequence within the receptor, as reintroduction of the NLS sequence at the N-terminus of the receptor retention mutant failed to reconstitute the retention activity. Agonist liganded and hormone-withdrawn receptor mutants showed a similar decrease in retention. By contrast, antagonist-withdrawn GR mutants were retained in the nucleus, possibly due to altered receptor configuration and interactions. Assays of GR-responsive promoter activation by receptor retention mutants showed that while no difference in the ability of retention mutants to activate transcription was seen at a simple promoter, activation of a complex promoter was compromised. This impaired transactivation for the SV506-523 mutant correlated with decreased histone H4 acetylation and PolII recruitment, while GR DNA-binding at the target promoter appeared to be unaffected. These results suggested that promoter-specific cofactor interactions might be implicated in GR nuclear retention. Loss of GR hinge interaction with Oct cofactors produced an incomplete loss of retention, suggesting overlapping signals, but not supporting Oct as a primary factor in GR retention. The overlap between important residues in GR nuclear retention and localization signals and the lack of retention shown by the SV40 NLS suggested that retention might be intrinsic to the sequence of particular NLS. Preliminary results suggest that the KT511-512 residues of GR may be of general importance in protein nuclear retention, while the role of proline is likely more variable. My research has focused on increasing our understanding of glucocorticoid receptor nuclear retention and its possible implications. I have determined that the KT511-512 residues of GR play an important role in its retention, and possibly also figure in nuclear retention of other proteins. These residues are involved in interactions which affect promoter-specific histone acetylation and transcriptional activation in GR, suggesting a reason for the existence of nuclear retention.
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