No abstract

Cholesterol 7␣-hydroxylase gene (CYP7A1) transcription is repressed by bile acids. The goal of this study is to elucidate the mechanism of CYP7A1 transcription by bile acid-activated farnesoid X receptor (FXR) in its native promoter and cellular context and to identify FXR response elements in the gene. In Chinese hamster ovary cells transfected with retinoid X receptor ␣ (RXR␣)/FXR, only chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) were able to stimulate a heterologous promoter/reporter containing an ecdysone response element. In HepG2 cells, all bile acids (25 M) were able to repress CYP7A1/luciferase reporter activity, and only CDCA and DCA further repressed reporter activity when cotransfected with RXR␣/FXR. The concentration of CDCA required to inhibit 50% of reporter activity (IC 50 ) was determined to be approximately 25 M without FXR and 10 M with FXR. Deletion analysis revealed that the bile acid response element located between nucleotides ؊148 and ؊128 was the FXR response element, but RXR␣/FXR did not bind to this sequence. These results suggest that bile acid-activated FXR exerts its inhibitory effect on CYP7A1 transcription by an indirect mechanism, in contrast to the stimulation and binding of FXR to intestinal bile acid-binding protein gene promoter. Results also reveal that bile acid receptors other than FXR are present in HepG2 cells.

The transcriptional activity of the cholesterol 7 alpha-hydroxylase gene CYP7A is repressed by bile acids. Taurine conjugates of chenodeoxycholate and deoxycholate, but not cholate and ursodeoxycholate, inhibited the CYP7A promoter/luciferase reporter activity in transient transfection assays in Hep G2 cells. A region from nucleotide (nt) -74 to -55 was found to mediate bile acid response. However, deletion of this bile acid response element (BARE-I) enhanced reporter activity but did not eliminate the bile acid response. This is due to the presence of another BARE-II located in a conserved region between nt -149 and -128. Deletion or mutations of these sequences reduced promoter activity and abolished bile acid repression. This BARE-II shares an identical AGTTCAAG core sequence with BARE-I. Electrophoretic mobility shift assays of BARE-I and BARE-II probes using Hep G2 nuclear extract and the partially purified binding activity of nt -65/-54 DNA-affinity column revealed that the same or a similar nuclear protein might bind to both BAREs. BARE-II is the major BARE involved in the transcriptional repression of the CYP7A gene by hydrophobic bile acids.