Transcriptional Regulation of Cholesterol Biosynthetic Gene by Nuclear Receptors

利用缺失的大鼠和人类角鲨烯环氧酶(SE)启动子进行的荧光素酶活性分析表明，SE基因受SREBP和NF-Y的调控，LXR的过度表达导致SE基因的表达减少。接下来，我们利用LxRβ、RxRα和LxRα突变体检测了LxRE和SE启动子表达的变化。LXRα、LXRβ和RXRα的过表达以及LXR和RXRα的共同过表达导致了LXRE的诱导，但LXRα突变体1和突变体2没有表现出任何LXRE的诱导，并且在RXR共转染组中诱导非常轻微的诱导。这些结果有力地表明，LxRα的突变体1和2对LxRE的表达起显性负突变作用。LxRE的诱导与SE启动子报告质粒的抑制相互关联，表明LxR参与了SE基因的表达。为了确定可能的LxRα配体，我们测试了激活LxRE表达的能力。如本幻灯片所示，在24(S)、25-环氧胆固醇和22(R)-羟基胆固醇处理的HeLa细胞中，LXRE嵌合体被显著激活，而20α-羟基胆固醇和22(S)-羟基胆固醇的作用较弱。这表明24(S)、25-环氧胆固醇和22(R)-羟基胆固醇是LXR的候选配体。抑制SE启动子的表达也再次与LXRE报告基因的诱导相关，为LXR参与SE启动子的抑制提供了另一种支持。

Luciferase assay using deleted rat and human squalene epoxidase (SE) promoter, reveals that SE gene is regulated by SREBP and NF-Y.Over-expression of LXR cause reduction of SE gene expression. Next we tested the changes of LXRE and SE promoter expression, using LXRβ, RXRα and LXRα mutants. Over-expression of LXRα, LXRβ, RXRα and co-overexpression of LXRs and RXRα cause the induction of LXRE.But LXRα mutant 1 and mutant-2 did not show any induction of LXRE as well as very slight induction with RXR cotransfection. These results strongly suggest that mutant 1 and 2 of LXRα functioned as a dominant negative mutants to LXRE expression. LXRE induction reciprocally correlated with the suppression of SE promoter reporter plasmid, indicating the LXR involvement of SE gene expression.To identify possible LXRα ligands, we tested for ability to activate LXRE expression. As shown in this slide, significant activation of LXRE chimeras was obtained in transfected HeLa cells treated with 24(S), 25-epoxycholesterol and 22(R)-hydroxycholesterol, but 20α-hydroxycholesterol and 22(S)-hydroxycholesterol was less effective. This suggests 24(S), 25-epoxycholesterol and 22(R)-hydroxycholesterol are candidates of LXR ligands. Suppression of SE promoter expression also correlated again with LXRE reporter plasmid induction, provided another support for LXR participation in SE promoter suppression.