Schoonjans K, Gelman L, Haby C, Briggs M, Auwerx J
Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, 67404, France.
J Mol Biol. 2000 Dec 1;304(3):323-34. doi: 10.1006/jmbi.2000.4218.
Overexpression of the adipocyte differentiation and determination factor-1 (ADD-1) or sterol regulatory element binding protein-1 (SREBP-1) induces the expression of numerous genes involved in lipid metabolism, including lipoprotein lipase (LPL). Therefore, we investigated whether LPL gene expression is controlled by changes in cellular cholesterol concentration and determined the molecular pathways involved. Cholesterol depletion of culture medium resulted in a significant induction of LPL mRNA in the 3T3-L1 preadipocyte cell line, whereas addition of cholesterol reduced LPL mRNA expression to basal levels. Similar to the expression of the endogenous LPL gene, the activity of the human LPL gene promoter was enhanced by cholesterol depletion in transient transfection assays, whereas addition of cholesterol caused a reversal of its induction. The effect of cholesterol depletion upon the human LPL gene promoter was mimicked by cotransfection of expression constructs encoding the nuclear form of SREBP-1a, -1c (also called ADD-1) and SREBP-2. Bioinformatic analysis demonstrated the presence of 3 potential sterol regulatory elements (SRE) and 3 ADD-1 binding sequences (ABS), also known as E-box motifs. Using a combination of in vitro protein-DNA binding assays and transient transfection assays of reporter constructs containing mutations in each individual site, a sequence element, termed LPL-SRE2 (SRE2), was shown to be the principal site conferring sterol responsiveness upon the LPL promoter. These data furthermore underscore the importance of SRE sites relative to E-boxes in the regulation of LPL gene expression by sterols and demonstrate that sterols contribute to the control of triglyceride metabolism via binding of SREBP to the LPL regulatory sequences.
脂肪细胞分化与决定因子-1（ADD-1）或固醇调节元件结合蛋白-1（SREBP-1）的过表达可诱导许多参与脂质代谢的基因表达，包括脂蛋白脂肪酶（LPL）。因此，我们研究了LPL基因表达是否受细胞胆固醇浓度变化的调控，并确定了其中涉及的分子途径。培养基中的胆固醇耗竭导致3T3-L1前脂肪细胞系中LPL mRNA显著诱导，而添加胆固醇则使LPL mRNA表达降至基础水平。与内源性LPL基因的表达相似，在瞬时转染实验中，人LPL基因启动子的活性因胆固醇耗竭而增强，而添加胆固醇则导致其诱导作用逆转。编码SREBP-1a、-1c（也称为ADD-1）和SREBP-2核形式的表达构建体共转染可模拟胆固醇耗竭对人LPL基因启动子的影响。生物信息学分析表明存在3个潜在的固醇调节元件（SRE）和3个ADD-1结合序列（ABS），也称为E-box基序。通过体外蛋白质-DNA结合实验和对每个位点含有突变的报告构建体进行瞬时转染实验相结合，一个称为LPL-SRE2（SRE2）的序列元件被证明是赋予LPL启动子固醇反应性的主要位点。这些数据进一步强调了SRE位点相对于E-boxes在固醇对LPL基因表达调控中的重要性，并证明固醇通过SREBP与LPL调控序列的结合对甘油三酯代谢的控制起作用。
