Mechanisms of actions and roles of 5α-reduced glucocorticoids during inflammation and wound repair
Abernethie, Amber Jean
Topical inflammatory diseases are most commonly treated with glucocorticoids, such as hydrocortisone, which have debilitating side effects including a range of systemic metabolic side effects as well as local effects such as to thin the skin and delay wound healing. Safer anti-inflammatory therapies are required and this thesis investigates a novel drug called 5α-tetrahydrocorticosterone (5α-THB) as a safer topical anti-inflammatory treatment. The main foci of this thesis are to assess the effects of 5αTHB on wound repair, as well as to characterise its mechanisms of action. Defective angiogenesis accounts for impaired wound healing brought about by steroids in many cases. 5αTHB suppressed vessel growth in a mouse ex vivo model of angiogenesis, but was less potent in this action than hydrocortisone, suggesting a safer therapeutic profile. To understand the underlying mechanisms, the effect of 5αTHB on gene expression in the mouse aorta during angiogenesis was compared with that of dexamethasone (a selective GR agonist) and hydrocortisone. Whereas dexamethasone and hydrocortisone caused differential expression of genes involved in inflammatory signalling and extracellular matrix remodelling, 5αTHB did not and instead selectively regulated Pecam1, involved in vasculature remodelling. This suggested that 5αTHB suppresses angiogenesis through different mechanisms of action in comparison to dexamethasone, and thus may not act through GR. Supporting this, dexamethasone increased the abundance of GR responsive transcripts (Per1, Hsd11b1, Fkbp51) whereas 5αTHB only increased the abundance of Per1. Furthermore, whereas the GR antagonist RU486 attenuated dexamethasone-regulation of genes, it had no effect on gene regulation by 5αTHB. To assess GR-mediation of 5αTHB effects, model systems were used to investigate whether 5αTHB is able to bind GR, stimulate its nuclear translocation, and initiate changes in its interaction with co regulator peptides. In a competitive binding assay, dexamethasone and hydrocortisone both decreased the fluorescence polarisation of a GR specific ligand, consistent with GR binding. In contrast, 5αTHB only displaced the specific GR ligand at very high concentrations. In terms of nuclear translocation, 5αTHB also did not have an effect on the ratio of GR in the nucleus and cytoplasm (N/C) of A549 cells, suggesting that GR remained predominantly in the cytoplasm after 5αTHB treatment and did not translocate into the nucleus, whereas dexamethasone increased the N/C ratio at three different time points. Likewise, whereas dexamethasone stimulated changes in the interaction between GR and many co regulator peptides, 5αTHB had no effect. Collectively these results from model systems suggest that 5αTHB does not work through the conventional GR mechanism of action. Finally, a hypothesis generating approach was taken in order to gain hints into how 5αTHB may be working. A microarray was performed to compare the effects of 5αTHB and dexamethasone on gene expression in human peripheral blood derived macrophages. Both dexamethasone and 5αTHB were able to cause differential expression of genes in these cells. However unexpectedly, out of the 350 genes regulated by dexamethasone, and the 165 genes regulated by 5αTHB, only 35 genes were commonly regulated by both steroids. This suggested that 5αTHB mainly acts through different mechanisms to dexamethasone also in macrophages. In an enrichment analysis of the differentially expressed genes, whereas the NFκB signalling pathway was the top enriched pathway in genes only regulated by dexamethasone, enriched pathways in genes only regulated by 5αTHB included those related to phagocytosis, the TGF-beta signalling pathway, and Th1-Th2 cell differentiation. This thesis therefore provides evidence to suggest that 5αTHB may provide a safer topical anti-inflammatory steroid, less harmful to wound repair processes. In addition, the mechanisms underlying the action of 5αTHB differ from those of classical GCs, consistent with its reduced side-effect profile. Other potential mechanisms, such as actions through the mineralocorticoid receptor, must now be explored.