Transcriptional regulation of the human cholesterol 7 alpha-hydroxylase gene (CYP7A) in HepG2 cells

163

Fibrates are widely used hypolipidemic drugs that regulate the expression of many genes involved in lipid metabolism by activating the peroxisome proliferator-activated receptor ␣ (PPAR ␣ ). The objective of this study was to investigate the mechanism of action of peroxisome proliferators and PPAR ␣ on the transcription of cholesterol 7 ␣hydroxylase, the rate-limiting enzyme in the conversion of cholesterol to bile acids in the liver. When cotransfected with the expression vectors for PPAR ␣ and RXR ␣ , Wy14,643 reduced human and rat cholesterol 7 ␣ -hydroxylase gene ( CYP7A1 )/luciferase reporter activities by 88% and 43%, respectively, in HepG2 cells, but not in CV-1 or CHO cells. We have mapped the peroxisome proliferator response element (PPRE) to a conserved sequence containing the canonical AGGTCA direct repeats separated by one nucleotide (DR1). This DR1 sequence was mapped previously as a binding site for the hepatocyte nuclear factor 4 (HNF-4) which stimulates CYP7A1 transcription. Electrophoretic mobility shift assay (EMSA) showed no direct binding of in vitro synthesized PPAR ␣ /RXR ␣ heterodimer to the DR1 sequence. PPAR ␣ and Wy14,643 did not affect HNF-4 binding to the DR1. However, Wy14,643 and PPAR ␣ /RXR ␣ significantly reduced HNF-4 expression in HepG2 cells. These results suggest that PPAR ␣ and agonist repress cholesterol 7 ␣ -hydroxylase activity by reducing the availability of HNF-4 for binding to the DR-1 sequence and therefore attenuates the transactivation of CYP7A1 by HNF-4. -Marrapodi, M., and J. Y. L. Chiang. Peroxisome proliferatoractivated receptor ␣ (PPAR ␣ ) and agonist inhibit cholesterol 7 ␣ -hydroxylase gene ( CYP7A1 ) transcription.

Section: Plasmidsmentioning

confidence: 99%

mentioning

confidence: 99%

Peroxisome proliferator-activated receptor α (PPARα) and agonist inhibit cholesterol 7α-hydroxylase gene (CYP7A1) transcription

Marrapodi

Chiang

2000

163

“…The first and rate-limiting reaction of this metabolic pathway is the 7 ␣ -hydroxylation of cholesterol, which is catalyzed by cholesterol 7 ␣ -hydroxylase, a product of a liver-specific cytochrome P450 gene ( CYP7A ) (3). Many lines of evidence suggest that cholesterol 7 ␣hydroxylase is regulated mainly at the transcriptional level by a wide array of stimuli including bile acids, hormones, and second messengers (4)(5)(6)(7)(8). The expression of cholesterol 7 ␣ -hydroxylase activity is developmentally regulated (9,10) and shows a strong diurnal rhythm (11,12).…”

mentioning

confidence: 99%

“…These include the diurnally regulated albumin D-site binding protein (DBP) (11,12), C/EBP and LAP (12), hepatocyte nuclear factor (HNF)-3 (8,13), and HNF-4 (14). In previous studies we identified regions important for the regulation of CYP7A gene transcription by bile acids and hormones (7,8,(15)(16)(17). Hydrophobic bile acids are potent feedback inhibitors of CYP7A transcription and mediate their effects through two regions in the rat CYP7A promoter which we named bile acid response element (BAREs) (15,17).…”

mentioning

confidence: 99%

Transcriptional activation of the cholesterol 7α-hydroxylase gene (CYP7A) by nuclear hormone receptors

Crestani

Sadeghpour

Stroup

et al. 1998

136

The gene encoding cholesterol 7 ␣ -hydroxylase ( CYP7A ), the rate-limiting enzyme in bile acid synthesis, is transcriptionally regulated by bile acids and hormones. Previously, we have identified two bile acid response elements (BARE) in the promoter of the CYP7A gene. The BARE II is located in nt ؊ 149/ ؊ 118 region and contains three hormone response element (HRE)-like sequences that form two overlapping nuclear receptor binding sites. One is a direct repeat separated by one nucleotide DR1 ( ؊ 146-TGGACTtAGTTCA-134) and the other is a direct repeat separated by five nucleotides DR5 ( ؊ 139-AGTTCAaggccGGG TAA-123). Mutagenesis of these HRE sequences resulted in lower transcriptional activity of the CYP7A promoter/reporter genes in transient transfection assay in HepG2 cells. The orphan nuclear receptor, hepatocyte nuclear factor 4 (HNF-4) 1 , binds to the DR1 sequence as assessed by electrophoretic mobility shift assay, and activates the CYP7A promoter/reporter activity by about 9-fold. Cotransfection of HNF-4 plasmid with another orphan nuclear receptor, chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), synergistically activated the CYP7A transcription by 80-fold. The DR5 binds the RXR/RAR heterodimer. A hepatocyte nuclear factor-3 (HNF-3) binding site ( ؊ 175-TGTTTGTTCT-166) was identified. HNF-3 was required for both basal transcriptional activity and stimulation of the rat CYP7A promoter activity by retinoic acid. Combinatorial interactions and binding of these transcription factors to BAREs may modulate the promoter activity and also mediate bile acid repression of CYP7A gene transcription.

“…Reporter gene and receptor expression plasmids. Human CYP7A1 / Luc reporter gene, ph-1887/Luc and ph-372/Luc, and rat p-376/ Luc were constructed as described previously (19,20). An SHP / Luc reporter plasmid containing 2,080 bp of 5 Ј upstream sequence of human SHP and a mouse IBABP /Luc reporter plasmid containing nucleotides Ϫ 496 to ϩ 40 of the gene encoding mouse ileum bile acid-binding protein (IBABP) were kindly provided by D. Moore (Baylor College of Medicine, Houston, TX).…”

Section: Methodsmentioning

confidence: 99%

Nuclear receptor-mediated repression of human cholesterol 7α-hydroxylase gene transcription by bile acids

Chen

Owsley

Yang

et al. 2001

Hydrophobic bile acids strongly repressed transcription of the human cholesterol 7 ␣ -hydroxylase gene ( CYP7A1 ) in the bile acid biosynthetic pathway in the liver. Farnesoid X receptor (FXR) repressed CYP7A1 /Luc reporter activity in a transfection assay in human liver-derived HepG2 cells, but not in human embryonic kidney (HEK) 293 cells. FXR-binding activity was required for bile acid repression of CYP7A1 transcription despite the fact that FXR did not bind to the CYP7A1 promoter. FXR-induced liverspecific factors must be required for mediating bile acid repression. Bile acids and FXR repressed endogenous CYP7A1 but stimulated ␣ -fetoprotein transcription factor (FTF) and small heterodimer partner (SHP) mRNA expression in HepG2 cells. Feeding of rats with chenodeoxycholic acid repressed CYP7A1, induced FTF, but had no effect on SHP mRNA expression in the liver. FTF strongly repressed CYP7A1 transcription in a dose-dependent manner, and SHP further inhibited CYP7A1 in HepG2 cells, but not in HEK 293 cells. FXR only moderately stimulated SHP transcription, whereas FTF strongly inhibited SHP transcription in HepG2 cells. Results revealed that FTF was a dominant negative factor that was induced by bile acid-activated FXR to inhibit both CYP7A1 and SHP transcription. Differential regulation of FTF and SHP expression by bile acids may explain the wide variation in CYP7A1 expression and the rate of bile acid synthesis and regulation in different species. -Chen, W., E. Owsley, Y. Yang, D. Stroup, and J. Y. L. Chiang. Nuclear receptor-mediated repression of human cholesterol 7 ␣ -hydroxylase gene transcription by bile acids.