Studies on the transcriptional regulation of sterol 12alpha-hydroxylase (CYP8B1)

Abstract

The conversion of cholesterol into bile acids is mainly via two pathways, which are initiated by CYP7A1 and CYP27A1 respectedly. CYP8B1, a microsomal P450 cytochrome, hydroxylates of the steroid nucleus at the C-12alpha position to form 7alpha, 12alpha-dihydroxy-4-cholesten-3-one, a facultative precursor of CA. The activity of the enzyme determines the ratio between CA and CDCA. The enzyme activity is affected by bile acids, cholesterol, hormones and fasting. In the present studies, we have focused on the structure-function relationship of the rat gene to explore the mechanisms of regulation.

The cDNA for rat CYP8B1 coded for a protein of 499 amino acid residues. The gene, like the human and mouse gene, also lacked introns. The promoter region contains several putative transcriptional response elements including DR1, c-jun, NF-1, Sp1 and repeated SRE-1 motifs. Thyroidectomy in rats caused a four-fold increase in CYP8B1 mRNA in the liver together with a 2,2-fold increase of enzyme activity. The treatment of intact animals with L-thyroxine caused a 60% reduction of the enzyme activity and a 50% reduction in CYP8B1 mRNA. No putative DR4 or thyroid hormone response elements could be recognized in the promoter fragment.

Previous studies in rats showed that CYP8B1 activity and mRNA levels were increased by fasting and clofibrate. In present studies, fasting for 24 hours or administration of WY-14,643 did not change the CYP8B1 activity and mRNA levels in PPAR-alpha null mice, whereas a significant increase was recorded in wild-type animals. An relative increase of CA was found in the bile. In vitro, PPAR-alpha was found to directly bind to a DR1 motif in the promoter. Co-transfection with PPAR-alpha expression plasmids induced a 2,5-fold increase of the activity of the CYP8B1 promoter in a ligand-dependent manner in Hep G2 cells.

Similar to CYP7A1, CYP8B1 is subjected to a negative feed-back regulation by bile acids. Feeding rats with chenodeoxycholic acid caused a 40% decrease of the liver CYP8B1 mRNA levels, while cholestyramine increased the mRNA 120%. It was associated with an increased mRNA levels for FTF and decreased for HNF4-alpha. A bile acid response element (BARE), DR1 motif overlapped with two M binding sites, was identified. FTF strongly repressed the CYP8B1 promoter activity probably by a competitive binding to BARE with HNF4-alpha.

In contrast to the up-regulation of CYP7A1, cholesterol feeding of rodents decreases the activity of CYP8B1. Treatment with cholesterol/25-hydroxycholesterol inhibited the activity of the rat CYP8B1 promoter in a dose-dependent manner. Overexpression of SREBPl a and l c stimulated the activity of promoter from rat, mouse and man, while SREBP2 did not have any effects. SREBP1 a and SREBP1 c directly bound to SRE and E box motifs in the rat promoter. Cholesterol feeding also decreased the mRNA levels for SREBP1, but not for SREBP2 in rat liver.

In summary, the HNF4-alpha, FTF and PPAR-alpha mediate the regulation of CYP8B1 by bile acids and fatty acids through BARE in the gene promoter. The coordinative effect between HNF4-alpha and FTF seems to be crucial for the expression level of CYP8B. PPAR-alpha mediates the up-regulation of CYP8B1 during starvation and fibrate administration. SREBP1 promotes the transcription of CYP8B1 gene by interaction with SRE and E box motifs, thereby constituting a new regulatory link between the cholesterol and bile acid metabolism. The CYP8B1 gene is also subjected to down-regulation by thyroid hormone, but responsive elements have not yet been identified in the promoter.