It is now recognized that there is an alternate pathway for endogenous synthesis of oxysterols which arises from the diversion of squalene 2, 3 monoxide to squalene 2, 3:22,23 diepoxide. This pathway can generate oxysterol molecules which are potential inhibitory regulators of cholesterol biosynthesis. We have found that U18666A ketoconazole and progesterone can inhibit the formation of regulatory oxysterols and allow accumulation of precursors of elements of the alternate pathway. When the effector molecules are removed the accumulated intermediates are converted to inhibitors of HMG CoA reductase (HMG-R). The same compounds which affect the alternate pathway also prevent the down regulation of HMG-R by LDL-cholesterol without affecting the processing of LDL. A common link among all of these substances is that they are cytochrome P-450 inhibitors. We propose to investigate the mechanism by which these substances act by first identifying the regulatory oxysterol products and to determine which cytochrome P-450 linked steps are involved in their formation. This will be followed by studies on the metabolic fate of these products. Since these same effectors are inhibitors of the cytochrome P-450 linked reactions of the arachidonic acid cascade we will also include studies on the role of intermediates of the cascade on the down regulation of HMG-R by LDL. We will also examine the role of Ca++ ions in the down regulation of reductase by oxysterols. In all of the above studies changes in the level of HMG-R activity will be monitored by determing whether synthesis or degradation of HMG-R protein is involved and whether transcriptional or translational controls on the expression of the HMG-R gene also play a role. These studies will provide a deeper knowledge of how cells regulate their metabolism of cholesterol in response to physiological and dietary stimuli, and the role that interruption of these processes play in the genesis of atherosclerosis.
