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.

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FK506 binding protein 5 (FKBP5) is a peptidyl propyl cis-trans isomerase that has been shown to interact with cellular immune pathways such as calcineurin and NF-κB. During an influenza infection, FKBP5 is up-regulated at the lung in an in vivo ferret infection model, yet the effect of FKBP5 on influenza replication and immune response is not understood. An in vitro model of human alveolar epithelial cell line A549 was established to study the cause and the function of FKBP5 up-regulation during an influenza infection. In this in vitro model, FKBP5 was not up-regulated by influenza replication, but instead it was up-regulated when A549 cells were treated with glucocorticoid. FKBP5 up-regulation did not have any effect on rate of influenza replication. However, FKBP5 up-regulation mediated the suppressive effect of glucocorticoid on pro-inflammatory cytokine production, since FKBP5 knock-down by siRNA increased cytokine production in the presence of glucocorticoid. Overall, the results suggested that the up-regulation of FKBP5 is a physiological response of lung cells to the increase of glucocorticoid during influenza infections, which facilitates the suppressive effect of glucocorticoid on pro-inflammatory cytokine production.

FK506 binding protein 5 (FKBP5) is a peptidyl propyl cis-trans isomerase that has been shown to interact with cellular immune pathways such as calcineurin and NF-κB. During an influenza infection, FKBP5 is up-regulated at the lung in an in vivo ferret infection model, yet the effect of FKBP5 on influenza replication and immune response is not understood. An in vitro model of human alveolar epithelial cell line A549 was established to study the cause and the function of FKBP5 up-regulation during an influenza infection. In this in vitro model, FKBP5 was not up-regulated by influenza replication, but instead it was up-regulated when A549 cells were treated with glucocorticoid. FKBP5 up-regulation did not have any effect on rate of influenza replication. However, FKBP5 up-regulation mediated the suppressive effect of glucocorticoid on pro-inflammatory cytokine production, since FKBP5 knock-down by siRNA increased cytokine production in the presence of glucocorticoid. Overall, the results suggested that the up-regulation of FKBP5 is a physiological response of lung cells to the increase of glucocorticoid during influenza infections, which facilitates the suppressive effect of glucocorticoid on pro-inflammatory cytokine production.

Recent epidemiological data have implicated prenatal events in the development of cardiovascular disorders. Thus low birth weight strongly predicts the later occurrence of hypertension, type II diabetes mellitus, syndrome X and deaths from ischaemic heart disease. The mechanism linking prenatal events and later disease is not clear, although maternal malnutrition has been advocated. We have advanced the hypothesis that glucocorticoids might be important as they retard foetal growth and programme offspring hypertension in rats. The foetus has been thought to be protected from the 2-10 times higher maternal glucocorticoid levels by the placental enzyme 11B-hydroxysteroid dehydrogenase (11B-HSD), which is present in many tissues and in humans catalyses the conversion of the active glucocorticoid cortisol to inert cortisone (corticosterone to 11-dehydrocorticosterone in rats). The precise role of 11B-HSD as a barrier to maternal glucocorticoids during prenatal life has not been fully characterised. The role of 11B-HSD in controlling prenatal glucocorticoid exposure in humans and animals has thus been examined. Two isoforms of 11B-HSD exist, type 1, a widespread NADP dependent reversible enzyme and type 2, a high affinity NAD dependent dehydrogenase found mainly in placenta and kidney. 11B-HSD was found in abundance in the ovary and placenta. The main site of immunohistochemical staining and expression of mRNA (11B-HSD-1) in the rat ovary was in the oocyte. 11B-HSD was oxidative, inactivating corticosterone. In both rat placenta in-vitro (11B-HSD-2), and human placenta in-vitro and ex-vivo (11B-HSD-2) the bioactivity was also predominantly oxidative. The lowest placental enzyme activity at term (and hence the greatest foetal glucocorticoid exposure) was found in the smallest rats with the largest placentas, i.e. those in human studies who would be predicted to develop the highest adult blood pressures (birth weight vs. placental 11B-HSD activity: n = 56; r = 0.46; p < 0.0005). A method to examine 11B-HSD function in fresh intact human placentas was developed (ex-vivo dual circuit cotyledon perfusion) which allows close approximation to the in-vivo situation. The majority of cortisol, from low to high nanomolar concentrations, infused through the maternal circulation was metabolised to inert cortisone by the time it reached the foetal circulation, although considerable individual variation was observed. 118-HSD was the only significant contributor to placental cortisol metabolism at physiological maternal concentrations and inhibition of 118-HSD with either the liquorice constituent glycyrrhetinic acid or its hemi-succinate, carbenoxolone, resulted in abolition of the glucocorticoid barrier, allowing maternally administered cortisol to pass unmetabolised through the placenta. In a prospective study, on 16 normal primiparous women whose placentas were studied with this technique, a positive and significant correlation was found between the effectiveness of 118-HSD and offspring birth weight (r = 0. 67; p < 0. 005). The relationship between placental 118-HSD effectiveness in-vivo and term cord blood osteocalcin (a sensitive marker of glucocorticoid exposure) was prospectively examined in 19 women. Cord blood levels of the bone specific protein osteocalcin were determined with radioimmunoassay. The lowest cord blood osteocalcin levels were found in the foetuses whose placental 118-HSD barrier function was poorest (r = 0.58; p < 0.02), (and had presumably had the greatest glucocorticoid exposure), suggesting that term cord blood osteocalcin levels might be a useful predictor of hypertension, ischaemic heart disease and possibly metabolic bone disease. The findings presented in this thesis represent direct evidence that 118-HSD is the barrier to maternal glucocorticoids, its effectiveness correlating with foetal growth in rats (in-vitro), in humans (ex-vivo), and in-vivo with human cord blood osteocalcin levels (osteocalcin may be a marker of glucocorticoid exposure). In the light of studies on pregnant rats in which administration of exogenous glucocorticoids or 118-HSD inhibitors reduces birth weight and programmes hypertension in the offspring, it is reasonable to propose that increased foetal glucocorticoid exposure consequent upon attenuated placental 118-HSD function may play a role in intrauterine programming of later hypertension.

Mutations in the melanocortin 2 receptor (MC2R) and its accessory protein (MRAP), in the ACTH signalling pathway, and the antioxidant genes nicotinamide nucleotide transhydrogenase (NNT) and thioredoxin reductase 2 (TXNRD2) have been associated with familial glucocorticoid deficiency (FGD). Using a tandem affinity purification and mass spectrometry approach to identify interacting partners of MC2R and MRAP failed to identify putative candidate genes for further FGD cases. However in a male patient a homozygous mutation in another antioxidant gene, glutathione peroxidase 1 (GPX1), was identified. In vitro studies showed H295R cells with knockdown of GPX1 had 50% less basal GPX activity and were less viable than wild-type when exposed to oxidative stress. Adrenals from Gpx1-/- mice showed no gross morphological changes and corticosterone levels were not significantly different from their wild-type counterparts (in contrast to the Nnt mutants). Sequencing of >100 FGD patients did not reveal any other GPX1 mutations. This equivocal data lead to the hypothesis that there could be a second gene defect present in this proband contributing to his disease. Whole exome sequencing revealed a homozygous loss-of-function mutation in peroxiredoxin 3 PRDX3 (p.Q67X) in this patient, that was also present in his unaffected brother. In vitro studies revealed both single and double knockdown of the two genes in H295R cells reduced cell viability, but redox homeostasis and cortisol production were unaffected. GPXs and PRDXs work simultaneously to reduce H2O2, preventing cellular damage. My data suggest that loss of PRDX3 alone is insufficient to cause adrenal failure and further that mutation in GPX1, either alone or in combination with PRDX3 mutation, may tip the redox balance to cause FGD.