For more than fifty years glucocorticoids (GCs) has been used to treat a wide range of inflammatory diseases, such as allergies, asthma, rheumatoid arthritis, and autoimmune diseases, due to its potentiality on the antiinflammatory and immunomodulatory effects. The anti-inflammation actions of glucocorticoid were mediated by an intracellular receptor, glucocorticoid receptor (GR), a member of the nuclear receptor family of ligand-dependent transcription factor. Upon activation by their ligand, GRs translocated to the nuclear and then bound to glucocorticoid responsive element (GRE) or negative glucocorticoid responsive elemen (nGRE). The administration of GCs depended on the acuity of disease and on the responses of patient clinically. Although some Systemic Lupus Erythematosus (SLE) patients given the maximal steroid doses, the response to the therapy remained poorly, and thus called ¡§glucocorticoid resistance¡¨. Despite the fact that the side effects and complications in SLE patients may result from the restrictions of physic; it has been documented that there were some relationships between the glucocorticoid resistance with the polymorphisms of GR, and the levels of glucocorticoid receptor beta. However, no significant differences in the GR polymorphisns (TthIII, ER22/23EK, N363S, BclI and I559N) between controls and SLE patients were found and there were no significant differences found on the levels of SUMO-2 antibody between patients with active and inactive SLE in this study. Neverthless, a significant association on the the allelic polymorphism of BclI was observed in patients with glucocorticoid resistance. Additionally, the expression of GR£] in patients with SLE was higher than that of controls and the TthIII CT genotype was associated with GR£\ expression.

Glucocorticoid receptor (GR) is known to associate with KATs and KDACs to regulate transcription. The current model of GR-mediated transcription focuses on agonist-dependent recruitment of KATs to acetylate histones and casts KDACs as corepressors in the presence of antagonist. Recent studies have shown KDACs to function as coactivators in the GR-mediated activation of the MMTV promoter and inhibition of KDACs impairs this activation. Nevertheless, the effect of KDAC inhibition on the GR-regulated transcriptome is unknown. Our expression profiling studies in a glucocorticoid (GC) responsive hepatoma-derived cell line, show that the class I-selective KDACi, VPA, has a profound impact on the GR-regulated hepatic transcriptome. VPA treatment alone mimics GC signaling at some GR-target genes and cooperates with GC to activate a small number of genes. However, the predominant effect of VPA, seen in more than 50% of the GR-target genes, is impairment of normal GR-mediated activation. This suggests that KDACs play a significant role in facilitating GR signaling. We have shown that VPA does not impair GR processing and that the inhibitory effects of VPA are due to impaired transcription. We have also determined that apicidin, a structurally distinct class I-selective KDACi, impairs GR-transactivation similar to VPA, while valpromide, a structural analog of VPA without KDACi activity, does not. In addition, siRNA-mediated depletion of KDAC1 fully or partially mimics the effects of VPA at most of the VPA impaired GR-target genes and co-depletion of KDACs 1 and 2 caused full or partial impairment of Dex-activation at a few other genes. Collectively, our results show that class-I KDACs facilitate GR-mediated transcription at most of the GR-target genes and that KDAC1 alone or in co-operation with KDAC2 is required for efficient GR-mediated transactivation. Furthermore, ChIP assays have shown that active KDACs are constitutively present at the gene promoters and that KDAC inhibition does not affect GR binding to the DNA. Thus KDACs could potentially deacetylate the coregulators necessary for transcriptional activation. Finally, KDACs are known targets of a group of drugs either being used or evaluated in the treatment of cancer and other diseases. These results also pose ramifications for the clinical use of these drugs.

Epidemiological studies have identified psychological stress as a significant risk factor in breast cancer. The stress response is regulated by the HPA axis in the brain and is mediated by glucocorticoid receptor (GR) signalling. It has been found that early life events can affect epigenetic programming of GR, and methylation of the GR promoter has been reported in colorectal tumourigenesis. Decreased GR expression has also been observed in breast cancer. In addition, it has been previously demonstrated that unliganded GR can serve as a direct activator of the BRCA1 promoter in mammary epithelial cells. We propose a model whereby methylation of the GR promoter in the breast significantly lowers GR expression, resulting in insufficient BRCA1 promoter activation and an increased risk of developing cancer. Antibody-based methylated DNA enrichment was followed by qPCR analysis (MeDIP-qPCR) in a novel assay developed to detect locus-specific methylation levels. It was found that 13% of primary breast tumours were hypermethylated at the GR proximal promoter whereas no methylation was detected in normal tissue. RT-PCR and 5’ RACE analysis identified exon 1B as the predominant alternative first exon in the breast. Tumours methylated near exon 1B had decreased GR expression compared to unmethylated samples, suggesting that this region is important for transcriptional regulation of GR. It was also determined that GR and BRCA1 expression was decreased in breast tumour compared to normal tissue. Furthermore, the relative expression of GR and BRCA1 measured by qRT-PCR was correlated in normal tissue but this association was not found in tumour tissue. From this, it appears that lower GR levels with associated decreased BRCA1 expression in tissues may be a predisposing factor for breast cancer. Based on these results we propose a role for GR as a potential tumour suppressor gene in the breast due to its association with BRCA1, also a tumour suppressor gene, as well as its consistently decreased expression in breast tumours and methylation of its proximal promoter in a subset of cancer patients. / Thesis (Master, Biochemistry) -- Queen's University, 2012-09-26 18:19:11.006

Septic shock is one of the most common causes of mortality in intensive care, in spite of antibiotic treatment. Glucocorticoid treatment can be used to blunt an overwhelming immune response in severe inflammation. The varying effects of glucocorticoid treatment in sepsis are poorly understood, with consequences for the clinical guidelines for treatment. Glucocorticoids are potent anti-inflammatory mediators which exert their effects through the glucocorticoid receptor (GR). Deeper understanding about the mechanisms of GR signalling may help to guide and improve glucocorticoid treatment. The aim of this thesis was to analyse GR expression and binding capacity in experimental and human septic shock and severe inflammation with cellular specificity using flow cytometry. In the late phase of a murine sepsis model, we observed decreased GR expression in leukocytes. In a murine model of early endotoxic shock, we observed decreased GR binding capacity in spite of an increased expression, in neutrophils. Glucocorticoid treatment was beneficial only when administered early in both models. Compared to healthy subjects, GR expression was increased in leukocytes from patients during the initial sepsis phase, while GR binding capacity was only increased in lymphocytes and monocytes. In contrast, neutrophils and other leukocyte subsets displayed decreased GR binding capacity. Neutrophil numbers were increased in all patients with sepsis compared to healthy subjects. We also studied patients with burn injury after admission before any infectious event had likely occurred, and on day 7 post admission, when several of the patients had been diagnosed with sepsis. GR expression and binding capacity was increased in leukocytes on admission as compared to healthy subjects, and patients diagnosed with sepsis on day 7 had a further increased GR expression in T lymphocytes. GR binding capacity was decreased in proportion to the extent of the burn injury on day 14 post admission. In conclusion, sepsis and severe inflammation have significant impact on the expression and function of GR, likely to influence the efficiency of glucocorticoid treatment. In addition, glucocorticoid treatment is beneficial only when given early in these models of experimental sepsis.

Glucocorticoids (Gc) are essential for life. Clinically, Gcs are potent anti-inflammatory agents, prescribed as first line treatment for a range of inflammatory disorders, including rheumatoid arthritis and asthma. In this thesis I present two studies that advance our understanding of the diverse actions of Gcs by using bioinformatics approaches. I have identified the functional network of two glucocorticoid receptor (GR) isoforms and characterised the effects of Gcs on cell function. Study 1 Systems analysis of GR function: Gcs regulate a diverse range of biological processes through a single receptor. How this is achieved is unclear, but it is thought at least in part to be due to the tissue specific expression of GR isoforms. The constitutive splice variant, GRγ is conserved through mammalian evolution, suggesting a gain of function, but currently no clear biological role has been identified. GRγ differs from the most abundant GR isoform, GRα by a single arginine inserted in the DNA binding domain (DBD) of GRγ, and may therefore affect the transcriptome profile and protein interactions of the receptor. Indeed, marked differences between the GRα and GRγ interactomes were revealed by proteomic analysis, identifying a potential association of GRγ with the mitochondria. These differences in the protein interactomes were accompanied by altered intracellular distributions. A clear tangible result of these differences is an observed delay in both the translocation kinetics and transactivation potential of GRγ. Analysis of the GRγ regulated transcriptome revealed a clear distinction in the regulation of a subset of target genes. Gene ontology and gene enrichment analysis identified oxidative phosphorylation protein degradation and cell morphology as potential GRγ specific functions. These findings suggest a distinct biological role is conferred by the additional arginine in the level arm of the DBD.Study 2 Mathematical modelling of GR function: Cell migration is a fundamental biological process. Clinically, Gcs inhibit wound healing, yet the mechanisms by which Gcs regulate migration remain unclear. The impact of Gcs on cellular motility was initially characterised through traditional migration assays. However, as cell populations are heterogeneous, live cell microscopy and mathematical modelling were employed to monitor the response of single cells. Dynamic tracking of migration in individual cells revealed that the movement of A549 cells is modelled by an alpha stable distribution. Gcs changes the parameters of the distribution, without altering the nature of the walk statistics. Gcs reduce the overall displacement of a cell, by causing a significant shift in step length selection, resulting in a reduction of the large steps, and replacement with short steps. Changes in migration following treatment with Gcs were seen within hours, which is much faster than previously reported. To identify a potential mechanism a panel of actin cytoskeleton regulators were screened, but prolonged exposure of Gcs were required to see a response, implying that these markers are modified secondary to the shift in migration. As a more dynamic readout, I tracked microtubule reorganisation and found stabilisation of the microtubule network rapidly following Gc exposure. I identified acetylation of α-tubulin (within 10 minutes) as the earliest change following Gc treatment, which implicated the inactivation of HDAC6 as a candidate mechanism. Indeed, overexpression of HDAC6 was able to restore cell motility characteristics to the Gc-free state. I have used a combination of systems level, chemical biology and mathematical approaches to better understand how Gc work in vivo. Research of this kind will aid new drug development for more specific targeting of Gc action.

No description available.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The synthetic progestins, medroxyprogesterone acetate (MPA) and norethisterone (Net) and its derivatives (norethisterone enanthate (Net-EN) and norethisterone acetate (Net-A)), are widely used as contraceptives and in hormone replacement therapy (HRT). Several studies have indicated that synthetic progestins modulate immune function and increase the risk of sexually transmitted infections. However, little is known about the molecular mechanism of action of MPA and Net, in particular their regulation of gene expression in the female genital tract, as compared to progesterone (P4). In the first part of this thesis, the effect of P4, MPA and Net-A on the expression of the endogenous chemokine genes, macrophage inflammatory protein (MIP)-1α and MIP-1β, was investigated in a human vaginal epithelial cell line (Vk2/E6E7). Quantitative realtime PCR (QPCR) showed that both P4 and MPA upregulated the TNF-α-induced expression of MIP-1α and MIP-1β mRNA, while Net-A had no effect. Using siRNA technology, it was found that the responses to P4 and MPA on the MIP-1α gene, but not the MIP-1β gene, are mediated via the glucocorticoid receptor (GR). In the second part of the thesis, it was investigated whether the HIV-1 accessory protein, viral protein R (Vpr), could modulate the action of ligands on MIP-1α and MIP-1β gene expression. QPCR showed that Vpr abrogates the effects of P4 and MPA on the TNF-α induced expression of MIP-1α and MIP-1β. Silencing the GR with siRNA technology showed that the GR plays a role in the effect of Vpr on the P4 and MPA-induced expression of MIP-1α. Taken together, these results show that MPA and Net-A display differential effects on chemokine gene expression in a human vaginal epithelial cell line. Furthermore, this study shows that Vpr modulates the effects of MPA bound to the GR. Thus, the results of this thesis provide insight into the effect of synthetic progestins on the immune response in the vagina, and possibly how HIV-infection may alter these responses. / AFRIKAANSE OPSOMMING: Die sintetiese progestiene medroksieprogesteroon asetaat (MPA) en noretisteroon (Net) en derivate daarvan (noretisteroon enantaat (Net-EN) en noretisteroon asetaat (Net-A)), word op grootskaal gebruik as voorbehoedmiddels en in hormoonvervangingsterapie (HVT). Verskeie studies het al aangedui dat sintetiese progestiene immuunfunksie moduleer en die risiko vir seksuel oordraagbare infeksies verhoog. Daar is egter min bekend oor die molekulêre meganisme van aksie van MPA en Net, in die besonder die regulering van geenuitdrukking in die vroulike geslagskanaal in vergelyking met progesteroon (P4). In die eerste deel van hierdie tesis is die effek van P4, MPA en Net-A op die uitdrukking van endogene chemokiene gene, makrofaag inflammatoriese proteïen (MIP)-1α en MIP-1β, in 'n menslike vaginale epiteel sellyn (Vk2/E6E7) bestudeer. Kwantitatiewe intydse PKR (KPKR) het getoon dat beide P4 en MPA die TNF-α-geïnduseerde uitdrukking van beide die MIP-1α en MIP-1β mRNA uitdrukking op reguleer, terwyl Net-A geen effek getoon het nie. Met die gebruik van siRNA-tegnologie is daar bevind dat die effekte van P4 en MPA, bemiddel word deur die glukokortikoïd-reseptor (GR) op MIP-1α geen uitdrukking, maar nie op MIP-1β nie. In die tweede deel van die tesis, is ondersoek of die MIV-1-bykomstigheidsproteïen, virale proteïen R (Vpr), die aksie van die ligande op MIP 1α en MIP-1β geenuitdrukking kan moduleer. KPKR toon dat Vpr die uitwerking van P4 en MPA op die TNF-α-geïnduseerde uitdrukking van MIP 1α en MIP-1β kanselleer. Die verwydering van die GR met siRNA-tegnologie toon dat die GR 'n rol in die uitwerking van Vpr op die P4 en MPA-geïnduseerde uitdrukking van MIP-1α speel. Ter samevatting: hierdie resultate toon dat MPA en Net-A differensiële uitwerkings vertoon op chemokiene geenuitdrukking in 'n menslike vaginale epiteel sellyn, en dat Vpr hierdie uitwerkings moduleer van MPA gobonde aan die GR. Die resultate van hierdie tesis werp dus lig tot die uitwerking van sintetiese progestiene op die immuunreaksie in die vagina, sowel as hoe MIVinfeksie hierdie reaksies kan verander.

During stress conditions, glucocorticoids are secreted and exert most of their physiological responses by binding to and modulating the transcriptional activity of the glucocorticoid receptor (GR). Once activated, GR can regulate numerous cellular processes including inflammation, development, growth, metabolism and apoptosis. Although glucocorticoids have been used in the treatment of leukaemia for over 50 years, with the molecular mechanisms by which steroids exert their pro-apoptotic effect, the pathways responsible for the development of resistance to glucocorticoid treatment, as well as their role in the programmed cell death in other tissue types have not been precisely defined. Research has demonstrated that glucocorticoid-induced apoptosis requires a transcriptionally active form of GR and is executed by the induction of the intrinsic pathway of apoptosis. In addition, GR is regulated by diverse types of cytotoxic stress; including UV irradiation and hypoxia, which alter the receptor’s transcriptional activity through multiple mechanisms. These include post-translational modifications, subcellular localisation and interaction of the receptor with co-regulator proteins. The aims of this study are to identify novel members of the Bcl-2 family that are regulated at the transcriptional level by GR in both leukaemia and other tissue types where glucocorticoids promote cell survival. In addition, the molecular crosstalk between signalling pathways activated by cytotoxic stress conditions and the mechanisms by which they differentially modulate the apoptotic response will be investigated. Results obtained in this study have identified putative glucocorticoid response elements in the promoters of the BH3-only pro-apoptotic gene NOXA and the anti-apoptotic gene Mcl-1 and confirmed that both NOXA and Mcl-1 are direct GR transcriptional targets. The glucocorticoid-mediated expression of NOXA and Mcl-1 alters their protein-protein interaction pattern, leading to the subsequent destabilisation of Mcl-1 in cell lines that undergo glucocorticoid-induced apoptosis. Investigation into the effects that other cytotoxic stress pathways have on GR function have revealed that serine 226 phosphorylation of GR by JNK occurs in a rapid and transient manner. Phosphorylation has inhibitory effects on the transcription of GR targets in a gene-specific manner, including the differential regulation of NOXA gene expression. During hypoxia, glucocorticoids differentially regulate the GR and HIF-1 target genes, NOXA and Mcl-1, altering the apoptotic response. This study has provided additional insight into the molecular mechanisms that govern glucocorticoid-induced programmed cell death and revealed mechanisms by which glucocorticoids and cytotoxic stress pathways crosstalk, regulating apoptosis.

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.

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.