The role of 24(S),25-epoxycholesterol in cellular cholesterol homeostasis.

Cellular cholesterol homeostasis ensures that cells are able to acquire enough cholesterol for cellular needs including membrane renewal, but avoid excess accumulation. Cholesterol homeostasis is maintained by two transcription factors, the Sterol Regulatory Element-Binding Protein-2 (SREBP-2) which regulates the genes involved in cholesterol biosynthesis and uptake; and the Liver X Receptor (LXR), which regulates the genes involved in maintaining Reverse Cholesterol Transport (RCT). Both LXR and SREBP-2 are regulated by oxidized cholesterol derivatives, referred to as oxysterols. 24(S),25-epoxycholesterol (24,25EC) is unique amongst the physiologically produced oxysterols as it is synthesized de novo in the mevalonate pathway. The overall aim of this thesis was to investigate the role of 24,25EC in cellular cholesterol homeostasis. In Chapter 3, using the statin class of HMG-CoA reductase inhibitors, we showed that 24,25EC synthesis correlates with the expression of the LXR-target gene, the ATP-binding cassette transporter, A1 (ABCA1) in human macrophages. In Chapters 4 and 5, we investigated this further showing that the effect of statins on ABCA1 transcription can be modulated by cellular cholesterol status and the extent of macrophage differentiation. Our results also indicated a critical role of SREBP-2 as a positive regulator of ABCA1 transcription by enabling the generation of 24,25EC. In Chapter 6, we showed that 24,25EC synthesis paralleled cholesterol synthesis, suggesting that this oxysterol may protect against the accumulation of newly-synthesized cholesterol. In Chapter 7, we investigated this contention further using a novel strategy of overexpressing the enzyme 2,3-Oxidosqualene Cyclase (OSC) in Chinese Hamster Ovary cells to selectively inhibit 24,25EC synthesis. In cells lacking 24,25EC, fine-tuning of the acute regulation of cholesterol homeostasis was lost, supporting the hypothesis that 24,25EC functions to protect the cell against the accumulation of newly-synthesized cholesterol. In Chapter 8, we focused on 24,25EC in a neurological context since cholesterol is an essential component of the central nervous system. Both astrocytes and neurons had the capacity to synthesize 24,25EC. Furthermore, both added 24,25EC and stimulated cellular production of 24,25EC modulated expression of LXR and SREBP-2 target genes. The evidence present in this thesis demonstrates the importance of 24,25EC as an endogenously synthesized regulator of cholesterol homeostasis