Abstract
We determined if fatty acids can regulate the murine Cyp7a1 and human CYP7A1 gene promoters via peroxisome proliferator-activated receptor alpha (PPARalpha)/9-cis-retinoic acid receptor alpha (RXRalpha). In transfected cells, the murine Cyp7a1 gene promoter displayed markedly lower basal activity, but greater sensitivity to fatty acid- or WY 14,643-activated PPARalpha/RXRalpha when compared with the human CYP7A1 gene promoter. PPARalpha/RXRalpha can bind to a site (Site II) located within the region at nucleotides -158 to -132 of both promoters. Mutagenesis of the human CYP7A1 Site II element abolished the response to activated PPARalpha/RXRalpha. The murine Cyp7a1 gene promoter contains an additional PPARalpha/RXRalpha-binding site (Site I) located within nucleotides -72 to -57. Replacement of a single residue in human CYP7A1 Site I with that found in the murine Cyp7a1 Site I sequence enabled PPARalpha/RXRalpha binding, and this mutation resulted in reduced basal activity, but substantially improved the response to activated PPARalpha/RXRalpha in transfected cells. We conclude that fatty acids can regulate the cyp7a gene promoter via PPARalpha/RXRalpha. The differential response of the murine Cyp7a1 and human CYP7A1 gene promoters to PPARalpha activators is attributable to the additional PPARalpha/RXRalpha-binding site in the murine Cyp7a1 gene promoter.
Highlights
Peroxisome proliferator-activated receptors (PPARs)1 belong to the superfamily of ligand-activated nuclear hormone receptors and regulate a wide spectrum of genes involved in lipid homeostasis [1,2,3]
Fatty Acids Stimulate the Murine Cyp7a1 and Human CYP7A1 Gene Promoters via PPAR␣/RXR␣—We observed that mice consuming a semipurified high fat (20%) diet in the absence of exogenous bile acids had a significantly higher (2–3fold, p Ͻ 0.01) abundance of Cyp7a1 mRNA compared with mice consuming a chow diet (5% fat content)
To investigate whether fatty acids directly regulate cyp7a gene transcription through PPAR␣/RXR␣, gene chimeras containing the murine Cyp7a1 or human CYP7A1 proximal promoter regions linked to the chloramphenicol acetyltransferase (CAT) structural gene were transfected into rat hepatoma cells in the absence or presence of plasmids encoding PPAR␣ and RXR␣
Summary
Peroxisome proliferator-activated receptors (PPARs) belong to the superfamily of ligand-activated nuclear hormone receptors and regulate a wide spectrum of genes involved in lipid homeostasis [1,2,3]. The regulation of gene transcription by PPARs involves their heterodimerization with 9-cis-retinoic acid receptor ␣ (RXR␣) and binding to specific sequences known as peroxisome proliferator response elements (PPREs) in the proximal regions of target genes [2, 4, 5]. It was shown recently that induction of the rat Cyp7a1 gene in response to oxysterols involves the nuclear receptor LXR␣ [39], whereas repression of cyp7a gene transcription by bile acids appears to be mediated via the farnesoid X receptor [40]. Given the number of nuclear hormone receptor-binding sites in the cyp7a gene promoter, it would seem that this class of transcription factors plays a major role in regulating cyp7a gene expression. We evaluated the role of fatty acids in regulating the cyp7a gene expression and whether PPAR␣/RXR␣ is involved in the process
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