AbstractDavid Jamey Morgan; The University of Manchester; PhD; September 2013Application of Systems Biology to Dissect Glucocorticoid Receptor FunctionGlucocorticoids (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, GRalpha 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 GRalpha 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 alpha-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.
|Date of Award||31 Dec 2013|
|Supervisor||Andrew Brass (Supervisor), David Ray (Supervisor) & Laura Matthews (Supervisor)|
- Glucocorticoid Receptor
- Systems Biology