Abstract
Cholesterol is required for normal cellular and physiological function, yet dysregulation of cholesterol metabolism is associated with diseases such as atherosclerosis. Cholesterol biosynthesis is regulated by end product negative feedback inhibition where the levels of sterols and oxysterols regulate the expression of cholesterologenic enzymes. Sterol regulatory element-binding protein-2 is responsive to both sterols and oxysterols and has been shown to mediate the transcriptional response of the cholesterologenic enzymes to these lipids. Here, we show that the nuclear hormone receptor for oxysterols, the liver X receptor alpha (LXRalpha), regulates cholesterol biosynthesis by directly silencing the expression of two key cholesterologenic enzymes (lanosterol 14alpha-demethylase (CYP51A1), and squalene synthase (farnesyl diphosphate farnesyl transferase 1)) via novel negative LXR DNA response elements (nLXREs) located in each of these genes. Examination of the CYP51A1 gene revealed that both the SRE and nLXRE are required for normal oxysterol-dependent repression of this gene. Thus, these data suggest that LXRalpha plays an important role in the regulation of cholesterol biosynthesis.
Highlights
Tion of the expression of several cholesterol biosynthetic genes by the sterol sensing sterol regulatory element-binding protein-2 (SREBP-2) [2]
These data clearly indicate that both the negative LXR DNA response elements (nLXREs) and SRE play a role in mediating the oxysterol responsiveness of transcription of this gene and indicate that the nLXRE has a basal repressor effect on transcription of this gene. These findings indicate that, beyond the role of LXR␣ in the regulation of cholesterol transport and elimination, this nuclear receptor plays an important role in end product inhibition of cholesterol biosynthesis (Fig. 9)
It is interesting to note that the two enzymes whose expression is directly repressed by LXR␣ are in steps of cholesterologenesis where the pathway has committed to sterol synthesis
Summary
Plasmid Construction—The CYP51A1 promoter (Ϫ2217 to ϩ155) was amplified from genomic DNA isolated from HepG2 cells and cloned into pTAL-Luc luciferase report vector (Clontech, CA) by introducing KpnI and BglII into the primers. The proximal portion (Ϫ2217 to Ϫ1749) containing LXRE and the distal portion (Ϫ1749 to ϩ155) containing the SRE of the promoter were amplified from the CYP51A1 promoter vector and cloned into pTAL-Luc vector using the same restriction sites. LXR␣ and LXR were cloned into pcDNA3.1 vector for overexpression analysis. Cell Culture and Cotransfections—HepG2 cells were maintained and routinely propagated in minimum essential medium supplemented with 10% fetal bovine serum at 37 °C under 5% CO2. HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 °C under 5% CO2. In experiments where lipids and sterols were depleted, cells were maintained on charcoal treated serum (10% fetal bovine serum) and treated with 7.5 M lovastatin and VOLUME 283 NUMBER 39 SEPTEMBER 26, 2008
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