Mechanisms of Altered Glucocorticoid Sensitivity

Abstract

Glucocorticoids (GCs) are the most potent, and widely used anti-inflammatory agents known. Despite their wide therapeutic use over many years much remains unclear about how GCs mediate their broad spectrum of cellular effects. The reasons why some patients fail to respond to GCs are also poorly understood. GCs mediate their cellular effects through the glucocorticoid receptor (GR) which is a ligand activated transcription factor. After binding GC, GR translocates to the nucleus to modulate transcription either by direct DNA binding (transactivation), or by tethering to other DNA bound transcription factors such as AP1 and NF-kB (transrepression). My findings show that raised cellular cholesterol selectively attenuates GC sensitivity. GR transactivation of MT1X and FKBP5 were impaired, but not GR transrepression of IL-6 or IL-8. The impaired MT1X induction was rapidly reversed by washout into regular medium for 4 hours. The underlying mechanism was not a direct effect on GR expression or localisation, but was due to increased AP1 activity as a consequence of JNK pathway activation. Hypoxic culture also selectively reduced cellular GC sensitivity. My results implicated this was not due to altered activity or localisation of GR, but was through altered DNA binding. To explore genome wide changes in GR binding, I developed ChIP-sequencing protocols and identified 595 highly-specific GR binding sites. Motif analysis demonstrated a template specific switch with cooperative binding with GR and KLF4 in normoxia and GR and FOXC1 in hypoxia.Results present in this thesis identify two novel mechanisms operating within the local tissue microenvironment to modify GC sensitivity, which may be operational in inflammatory disease.

Date of Award	31 Dec 2013
Original language	English
Awarding Institution	The University of Manchester
Supervisor	David Ray (Supervisor) & Laura Matthews (Supervisor)