Molecular mechanism of estrogen action to inhibit obesity

众所周知，雌激素可以调节啮齿类动物的脂肪生成。去卵巢的啮齿动物给予雌激素可降低脂肪组织中脂蛋白脂酶(LPL)基因的表达。在本研究中，我们研究了雌激素对小鼠LPL基因转录调控的机制。将LPL基因5‘侧翼区(-1980个碱基)与CAT报告基因融合，用雌激素受体表达载体导入COS-1细胞，检测雌二醇对COS-1细胞CAT活性的影响。雌二醇抑制COS-1细胞pLPL-(-1980)-CAT活性20倍。接下来，我们通过系统地删除5‘-侧翼区域来确定LPL启动子上的雌激素抑制元件。雌激素依赖抑制作用最强的区域位于-1980~-1570bp之间。我们用-1980到-1570个碱基之间的50-410个核苷酸片段与最小的启动子-CAT基因融合，缩小了该区域。我们发现50bp-(-1620/-1570)元件在雌激素依赖性抑制LPL基因转录中起重要作用。当从pLPL(-1980)-CAT构建中删除这个50个碱基的元件时，雌激素依赖的抑制作用降低到5倍。通过凝胶迁移率改变分析，观察到COS-1细胞中的核蛋白与该50bp元件特异性结合。这些结果共同表明，50bp(-1620/-1570)的元件不包含传统的雌激素反应元件，它可能包含一个新的顺式作用元件，该元件对雌激素依赖抑制小鼠LPL基因转录至关重要。

Estrogen is known to regulate adipogenesis in rodents. Estrogen administration in ovariectomized rodents reduces lipoprotein lipase (LPL) gene expression in fat tissues. In this study we studied the mechanisms of transcriptional regulation of the murine LPL gene by estrogen. The 5'-flanking region of LPL gene (-1980 bp) was fused to CAT reporter gene, introduced into COS-1 cells with estrogen receptor expression vector and CAT activities were determined in the presence or the absence of estradiol. Estradiol at 10^<-6> M suppressed pLPL- (-1980) -CAT activity by 20-fold in COS-1 cells. We next determined the estrogen-suppressive element on the LPL promoter by systematically deleting the 5'-flanking region. The most potent region for the estrogen-dependent suppression was located between -1980 and -1570 bp. We narrowed down the region using 50 - 410 bp fragments between -1980 and -1570 bp fused to the minimal promoter-CAT gene. We found that the 50-bp- (-1620/-1570) element was important for estrogen-dependent suppression in LPL gene transcription. When this 50-bp element was deleted from the pLPL (-1980) -CAT construct, estrogen-dependent suppressiveness was decreased to 5-fold. Nuclear proteins from COS-1 cells specifically bound to this 50-bp element were observed by the electrophoretic mobility shift assay. These results collectively suggest that the 50-bp (-1620/-1570) element, which harbors no conventional estrogen-responsive element, may contain a novel cis-acting element which is crucial for estrogen-dependent suppression of the murine LPL gene transcription.