Regulation of glucocorticoid receptor expression and downstream gene targets in murine lymphocytes

Glucocorticoids (GCs) are homeostatic steroid hormones with essential roles in the regulation of development, integrated metabolism, immune and neurological responses. GCs act via the widely expressed Glucocorticoid Receptor (GR), which is expressed from multiple untranslated exon 1s to yield 11 alternatively spliced transcripts in humans (1A-1H) and five in mice (1A-1E). These transcript isoforms are under the control of their own promoters which confers tissue specificity and a higher level of regulation to this transcription factor. In thymocytes activity from the GR1A promoter is implicated with increasing sensitivity to Glucocorticoid Induced Cell Death (GICD). CD4+CD8+ Double Positive (DP) cells and NKT cells in particular are hypersensitive to GICD. The main objectives of this study were to investigate further the molecular mechanisms involved in GICD, to examine more closely the role of GR in the development of T-lymphocytes, and to characterise the expression and regulation of the GR1A promoter in mouse tissues previously not examined. To explore the molecular pathway driving GICD in thymocytes we performed whole genome microarray analysis in mouse GR null thymocytes. Interesting direct GR targets included P21, Bim and Nfil3. Regulation of these targets by GCs was validated using qRT-PCR in WT thymocytes. Nfil3 in particular has been studied further. Previous studies demonstrated that GC-mediated up-regulation of Nfil3 is dependent on intracellular calcium levels, and correlates with GICD of GC-sensitive leukemic CEM cells. In silico promoter analysis revealed a putative Glucocorticoid Response Element in the Nfil3 5’UTR which was confirmed to interact with the GR by ChIP. Immunohistochemical staining of Nfil3 in whole thymus has localised NFIL3 protein primarily to the medullary region. Double labelling has co-localised NFIL3 to apoptotic cells and macrophages. Using siRNA technology we have shown that NFIL3 does in fact confer greater sensitivity to GICD in Ctll-2 cells. Previous to our studies transcripts initiating from the GR1A promoter had only been localised to the cortex of the brain and to T-lymphocytes. Using qRTPCR and in situ hybridisation we have detected transcripts initiating from the GR1A promoter in the anterior lobe of the pituitary. However, the role of the GR1A promoter activity in the brain is unknown. In the brain, particularly in the cortex, and also in the hypothalamus and pituitary (components of the Hypothalamic- Pituitary-Adrenal axis) GCs and their receptors have a key role in the response to stress. A 2.5 fold increase in the level of GR1A promoter usage in the pituitary was observed in response to treatment with the synthetic GC Dexamethasone. It is possible that a tissue/cell specific increase in activity of the GR1A promoter during periods of elevated levels of circulating GCs may help to make those cells more sensitive to these rising levels of GCs and serve as a fine tuning mechanism to aid in a rapid return to the normal state after stress. The significance of increased sensitivity of some T-cell lineages to GICD is controversial, with a proposed function being that it is involved in T-cell development. The direct role of the GR in development of T-cell populations and some specific lymphocyte lineages in T-cell specific GR-null mice (TGRKO) was examined using FACS. Major differences in CD8 CD4 cellularity was observed in spleen and liver and to a lesser extent in thymus. In Spleen NKT cellularity was reduced and TReg cell CD25 populations were altered. This study has shed light on some of the regulatory mechanisms and molecular interactions associated with GR function in the pathways of stress, T-cell development and GICD. We have identified rapidly induced GR target genes in GR null thymocytes and characterized the regulation of the transcriptional repressor Nfil3 by GR in normal mouse thymocytes. We have shown that NFIL3 is necessary for GICD in Ctll2-T cells. Additionally we have shown that the regulation of GR expression in the pituitary may occur via auto-regulation and maintenance of the GR1A promoter, an action which likely increases sensitivity to elevated GC levels and may account for the rapid return to a normal state following stress. Finally we have demonstrated T-cell development is subtly affected by loss of GR in T-cells.