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. 2011 Aug;52(8):1509-16.
doi: 10.1194/jlr.M014084. Epub 2011 May 16.

Cholesterol 25-hydroxylation Activity of CYP3A

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Free PMC article

Cholesterol 25-hydroxylation Activity of CYP3A

Akira Honda et al. J Lipid Res. .
Free PMC article

Abstract

To date, many studies have been conducted using 25-hydroxycholesterol, which is a potent regulator of lipid metabolism. However, the origins of this oxysterol have not been entirely elucidated. Cholesterol 25-hydroxylase is one of the enzymes responsible for the metabolism of 25-hydroxycholesterol, but the expression of this enzyme is very low in humans. This oxysterol is also synthesized by sterol 27-hydroxylase (CYP27A1) and cholesterol 24-hydroxylase(CYP46A1), but it is only a minor product of these enzymes. We now report that CYP3A synthesizes a significant amount of 25-hydroxycholesterol and may participate in the regulation of lipid metabolism. Induction of CYP3A by pregnenolone-16α-carbonitrile caused the accumulation of 25-hydroxycholesterol in a cell line derived from mouse liver. Furthermore, treatment of the cells with troleandomycin, a specific inhibitor of CYP3A, significantly reduced cellular 25-hydroxycholesterol concentrations. In cells that overexpressed human recombinant CYP3A4, the activity of cholesterol 25-hydroxylation was found to be higher than that of cholesterol 4β-hydroxylation, a known marker activity of CYP3A4. In addition, 25-hydroxycholesterol concentrations in normal human sera correlated positively with the levels of 4β-hydroxycholesterol (r = 0.650, P < 0.0001, n = 78), but did not significantly correlate with the levels of 27-hydroxycholesterol or 24S-hydroxycholesterol. These results demonstrate the significance of CYP3A on the production of 25-hydroxycholesterol.

Figures

Fig. 1.
Fig. 1.
Effects of PCN, troleandomycin (TAM), and desmosterol (DES) on sterol concentrations in AML12 cells. Cells were incubated with PCN (10 μM), TAM (100 μM), DES (30 μM), or DES (30 μM) plus PCN (10 μM) for 7 days. A Hypersil GOLD column was used for HPLC separation of oxysterols. This column cannot distinguish between 26- and 27-hydroxycholesterol and between 24R- and 24S-hydroxycholesterol. Each column and error bar represents the mean and SD obtained in triplicate assay. ***P < 0.001, **P < 0.01, *P < 0.05.
Fig. 2.
Fig. 2.
Effects of PCN, troleandomycin (TAM), and desmosterol (DES) on relative mRNA expression of Cyp3a11, Ch25h, Cyp46a1, and Cyp27a1 in AML12 cells. Cells were incubated with PCN (10 μM), TAM (100 μM), DES (30 μM), or DES (30 μM) plus PCN (10 μM) for 72 h. Each column and error bar represents the mean and SD obtained in triplicate assay. **P < 0.01, *P < 0.05. NS, not significant.
Fig.
Fig.
Effects of PCN, troleandomycin (TAM), and desmosterol (DES) on CYP3A and CH25H protein in AML12 cells. Cells were incubated with PCN (10 μM), TAM (100 μM), DES (30 μM), or DES (30 μM) plus PCN (10 μM) for 72 h. Cell homogenates (10 μg protein per lane) were subjected to SDS-PAGE analysis.
Fig. 4.
Fig. 4.
SRM chromatograms obtained during HPLC-ESI-MS/MS analysis of the oxysterol fraction from an incubation mixture of overexpressed recombinant human CYP3A4 (A, B) or boiled CYP3A4 (C, D) with 200 μM cholesterol. The oxysterol fraction was derivatized to picolinyl esters by two different methods, 80°C for 60 min (A, C) and room temperature for 30 min (B, D). The former produces di-picolinyl esters of 25-hydroxycholesterol (25HC) and 4β-hydroxycholesterol (4βHC), whereas the latter produces the mono-picolinyl ester of 25HC. 25HC-d3 (1 ng) and 4βHC-d7 (5 ng) were added to each incubated mixture as internal standards. The same Hypersil GOLD column and the same mobile phase were used for HPLC separation of both di- and mono-picolinyl esters of 25HC. The numbers on the right upper side of each chromatogram represent the full scale of the chromatogram.
Fig. 5.
Fig. 5.
Effects of cholesterol (substrate) concentrations on 25-, 4β-, 22R-, 24R-, 24S-, 26-, and 27-hydroxylase activities in overexpressed recombinant human CYP3A4. A Hypersil GOLD aQ column was used for HPLC separation of oxysterols. Data points represent the mean of duplicate determinations.
Fig. 6.
Fig. 6.
Correlations between serum concentrations of 25-hydroxycholesterol and 4β-hydroxycholesterol (A), 24S-hydroxycholesterol (B), or 27-hydroxycholesterol (C) in 78 normal subjects. NS, not significant.

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