Abstract
The transcription of the cholesterol 7alpha-hydroxylase gene (CYP7A1) is greatly decreased in cholesterol-fed rabbits. To determine whether the molecular structure of the promoter is responsible for this downregulation, we cloned the rabbit CYP7A1 promoter, identified the binding sites for alpha-fetoprotein transcription factor (FTF) and liver X receptor (LXRalpha), and studied the effects of FTF, LXRalpha, and SHP on its transcription. Adding LXRalpha/retinoid X receptor together with their ligands (L/R) to the promoter/reporter construct transfected into HepG2 cells greatly increased its activity. FTF did not increase promoter activity, nor did it enhance the stimulatory effect of L/R. Mutating the FTF binding site abolished the promoter baseline activity. Increasing amounts of SHP abolished the effect of L/R, and FTF enhanced the ability of SHP to decrease promoter activity below baseline levels. Thus, downregulation of CYP7A1 in cholesterol-fed rabbits is attributable secondarily to the activation of farnesoid X receptor, which increases SHP expression to override the positive effects of LXRalpha. Although FTF is a competent factor for maintaining baseline activity, it does not further enhance and may suppress CYP7A1 transcription.
Highlights
The transcription of the cholesterol 7a-hydroxylase gene (CYP7A1) is greatly decreased in cholesterol-fed rabbits
In contrast to what is observed in the rat, we found that CYP7A1 activity was downregulated in cholesterolfed rabbits [8] and hypothesized that this specific response led to the accumulation of dietary cholesterol in plasma that, in the rat, would have been destined for bile acid synthesis and excretion
Clone the promoter and identify the binding sites 999 these sites are functional, we used electrophoretic mobility shift assays to determine whether LXRa and fetoprotein transcription factor (FTF) can bind to their respective sites (Fig. 2). 32P-labeled rabbit LXRa probe (LXRE) bound to LXRa/retinoid X receptor (RXR) (Fig. 2A, lane 3), whereas excess cold LXRa probe but not mutated LXR probe (LXRm; lane 5) competed with the labeled probe for binding
Summary
The transcription of the cholesterol 7a-hydroxylase gene (CYP7A1) is greatly decreased in cholesterol-fed rabbits. FTF did not increase promoter activity, nor did it enhance the stimulatory effect of L/R. Mutating the FTF binding site abolished the promoter baseline activity. Increasing amounts of SHP abolished the effect of L/R, and FTF enhanced the ability of SHP to decrease promoter activity below baseline levels. Downregulation of CYP7A1 in cholesterol-fed rabbits is attributable secondarily to the activation of farnesoid X receptor, which increases SHP expression to override the positive effects of LXRa. FTF is a competent factor for maintaining baseline activity, it does not further enhance and may suppress CYP7A1 transcription.—Shang, Q., L. Xu. The stimulatory effect of LXRa is blocked by SHP despite the presence of a LXRa binding site in the rabbit CYP7A1 promoter.
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