NADPH-cytochrome P450 reductase (CPR) is a flavoprotein containing both FAD and FMN and functions as the electron donor protein for several oxygenase enzymes found on the endoplasmic reticulum of eukaryotic cells, including cytochrome P450s involved in drug metabolism and cholesterol biosynthesis. As many as three enzymes in the cholesterol biosynthetic pathway have been demonstrated, or proposed, to use CPR as a redox partner: squalene monooxygenase, which converts squalene to 2,3-oxidosqualene; lanosterol demethylase, a cytochrome P450 (CYP51); and 7-dehydrocholesterol reductase, the final step in cholesterol synthesis. In yeast CPR can be replaced by the NADH-cytochrome b5 pathway, but this has not been demonstrated in animals or plants. My studies with hepatic cytochrome P450 reductase-null mice have revealed a second microsomal reductase for squalene monooxygenase that was not previously detected. Studies carried out with hepatocytes from CPR-null mice demonstrate that this second reductase is active in whole cells and leads to the accumulation of 24-dihydrolanosterol, indicating that lanosterol demethylation, catalyzed by CYP51, is blocked. These results demonstrate that this second reductase plays a significant role in supporting squalene monooxygenase but not cytochrome P450-mediated reactions. 7-Dehydrocholesterol reductase (E.C. 1.3.1.21) catalyzes the reduction of the 7-8 double bond of 7-dehydrocholesterol to yield cholesterol. It has been suggested that cytochrome-P450 reductase is required for this reaction. My studies show that 7-dehydrocholesterol reductase is enzymatically active in CPR-null microsomes, with activity equal to or greater than that found in preparations from wild-type mice. Mammalian cytochrome b5, which can accept electrons from either cytochrome P450 reductase or NADH-cytochrome b5 reductase, is known to be involved in augmenting some P450-dependent monooxygenase reactions. Cytochrome P450 2E1 has been found to exhibit reasonable rates of turnover via an NADHcytochrome b5 pathway in reconstituted enzyme systems and in heterologous hosts. Using microsomes from hepatic CPR-null mice, I have determined that NADH-dependent CYP2E1 activity in the absence of NADPH-dependent activity constituted approximately 10% of CYP2E1 activity observed in microsomal preparations with NADPH from wild-type mice. However, little or no CYP2E1 activity could be detected in primary hepatocytes isolated from CPR-null mice.

7-Dehydrocholesterol (7-DHC) is the substrate of 7-dehydrocholesterol reductase (DHCR7) in the cholesterol synthesis pathway. Keratinocytes in human skin possess the enzymes necessary for cholesterol synthesis but are also responsible for vitamin D3 synthesis from 7-DHC by exposure to UVB irradiation. It has been well established that DHCR7 is regulated by the SREBP pathway in the regulation of cholesterol synthesis, but little is known about the regulation of DHCR7 by the vitamin D pathway. In this study, the regulation of DHCR7 activity by vitamin D was explored. Treatment of adult human epidermal keratinocyte (HEKa) cells with vitamin D3 resulted in a rapid decrease in DHCR7 activity which was not due to changes in the amount of enzyme present. This suppression of activity was observed only in HEKa cells, a primary cell line cultured from normal human skin, and not in an immortalized skin cell line (HaCaT cells) nor in two liver-derived hepatoma cell lines. Because vitamin D3 treatment of HEKa cells did not change the content of lanosterol nor 7-DHC, these results suggest that vitamin D3 rapidly down-regulates the entire cholesterolgenesis pathway, presumably at a very early step in the pathway. 25-Hydroxyvitamin D3, the first metabolite and circulating form of vitamin D3, had a lesser effect on DHCR7 activity, while 1,25-dihydroxyvitamin D3, the activated form of the vitamin, had no effect on DHCR7, indicating that the vitamin D receptor is not involved. The decrease in DHCR7 activity was due neither to the dephosphorylation of the enzyme, an established mechanism of inactivation, nor to direct inhibition by vitamin D3. Vitamin D3 markedly inhibited proliferation and induced differentiation of HEKa cells, suggesting a possible role for hedgehog signaling in the decrease in DHCR7 activity.