Multiple mechanisms regulate circadian expression of the gene, for cholesterol 7α-hydroxylase (Cyp7a), a key enzyme in hepatic bile acid biosynthesis

Cholesterol 7 alpha-hydroxylase (CYP7A) and sterol 12 alpha-hydroxylase (CYP8B) in bile acid biosynthesis and 3-hydroxyl-3-methylglutaryl CoA reductase (HMGCR) in cholesterol biosynthesis are the key enzymes in hepatic metabolic pathways, and their transcripts exhibit circadian expression profiles in rodent liver. The authors determined transcript levels of these enzymes and the regulatory factors for Cyp7a-including Dbp, Dec2, E4bp4, Hnf4 alpha, Ppar alpha, Lxr alpha, Rev-erb alpha, and Rev-erb beta-in the liver of wild-type and homozygous Clock mutant mice (Clock/Ciock) and examined the effects of these transcription factors on the transcription activities of Cyp7a. The expression profile of the Cyp7a transcript in wildtype mice showed a strong circadian rhythm in both the 12L:12D light-dark cycle and constant darkness, and that in Clock/Clock also exhibited a circadian rhythm at an enhanced level with a lower amplitude, although its protein level became arrhythmic at a high level. The expression profile of Cyp8b mRNA in wild-type mice showed a shifted circadian rhythm from that of Cyp7a, becoming arrhythmic in Clock/Clock at an expression level comparable to that of wild-type mice. The expression profile of Hingcr mRNA also lost its strong circadian rhythm in Clock/Clock, showing an expression level comparable to that of wild-type mice. The expressions of Dbp, Dec2, Rev-erba, and Rev-erbp-potent regulators for Cyp7a expression-were abolished or became arrhythmic in Clock/Clock, while other regulators for Cyp7a-Lxr alpha, Hnf4 alpha, Ppar alpha, and E4bp4-had either less affected or enhanced expression in Clock/Clock. In luciferase reporter assays, REV-ERB alpha/P, DBP, LXR alpha, and HNF4 alpha increased the promoter activity of Cyp7a, whereas DEC2 abolished the transcription from the Cyp7a promoter: E4BP4 and PPAR alpha were moderate negative regulators. Furthermore, knockdown of REV-ERB alpha/beta with siRNA suppressed Cyp7a transcript levels, and in the electrophoretic mobility shift assay, REV-ERB alpha/beta bound to the promoter of Cyp7a. These observations suggest that (1) active CLOCK is essential for the robust circadian expression of hepatic metabolic enzymes (Cyp7a, Cyp8b, and Hmgcr); (2) clock-controlled genes-DBP, DEC2, and REV-ERB alpha/beta - are direct regulators required for the robust circadian rhythm of Cyp7a; and (3) the circadian rhythm of Cyp7a is regulated by multiple transcription factors, Including DBP, REV-ERB alpha/beta, LXR alpha, HNF4 alpha DEC2, E4BP4, and PPAR alpha.