Glucocorticoids (Gc) are vital for development, maintenance of glucose homeostasis and the resolution of inflammation. As potent modulators of the immune response Gc are routinely prescribed in the management of a variety of inflammatory diseases including asthma and rheumatoid arthritis. However clinical use of Gc is limited by variation in patient sensitivity to Gc treatment and development of a wide range of side effects. In this thesis I present two studies that have advanced our understanding of Gc action in vivo. The first defines and characterises the cause of familial glucocorticoid resistance, and the second describes the action of two potent non-steroidal Gc in a cell line model. Familial Gc Resistance: Cases of primary generalised Gc resistance are very rare and typically present as mineralocorticoid and androgen excess leading to hypertension, hypokalemia and hirsutism. Gc resistance is attributed to loss of function mutations within the glucocorticoid receptor (GR). Here I identify a family with a novel mutation in GR exon 6 that gives rise to a very mild phenotype. Analysis of transformed patient peripheral blood lymphocytes revealed a 50% reduction in full length GR but no expression of a mutant form. As this did not rule out expression in vivo, the mutant receptor (Δ612GR) was characterised in a cell line. Investigation using reporter genes revealed that Δ612GR lacked any activity, but had dominant negative action when co expressed with full length GR. In response to Gc Δ612GR was not phosphorylated or targeted for degradation. Fluorophore tagged Δ612GR was unable to translocate to the nucleus in response to Gc, but delayed the translocation of full length GR when co-expressed. Together this indicates that Δ612GR is unable to bind ligand but has dominant negative action upon full length GR most likely due to heterodimerisation. Therefore I describe a novel GR mutation that results in Gc resistance but presents with a mild very phenotype. Novel Non-steroidal Gc: Non-steroidal Gc can be used as tools to determine how ligand structure directs GR function. Here I describe two highly potent non steroidal Gc ligands, GSK47867A and GSK47869A which alter the kinetics of receptor activity. Treatment with either ligand induces slow GR nuclear translocation, promotes GR nuclear retention and prolongs transcriptional activity following ligand withdrawal. Crystal structure analysis revealed that GSK47867A and GSK47869A specifically alter the surface charge of the GR at a site important for Hsp90 binding. GR bound to GSK47867A and GSK47869A shows prolonged activity in the presence of Hsp90 inhibitor geldanamycin. Therefore this work identifies a new chemical series that could prolong GR activity due to altered pharmacodynamics rather than altered pharmacokinetics.In summary this work uses a combination of genetic and chemical biology approaches to broaden our understanding of GR function. Characterisation of naturally occurring GR mutations gives insight into the complex function of the GR, and non-steroidal Gc act as useful tools that will aid in the design of improved therapeutics.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:588151 |
| Date | January 2013 |
| Creators | Trebble, Peter |
| Contributors | Ray, David; Matthews, Laura |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/glucocorticoid-receptor-function-new-insights-from-genetic-and-chemical-biology-approaches(b55c612b-eda1-4908-a0df-ec916f03ade2).html |
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