Grosfeld Alexandra, Andre Jocelyne, Hauguel-De Mouzon Sylvie, Berra Edurne, Pouyssegur Jacques, Guerre-Millo Michele
INSERM U 465, Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, 15 Rue de l'Ecole de Médecine, 75006 Paris, France.
J Biol Chem. 2002 Nov 8;277(45):42953-7. doi: 10.1074/jbc.M206775200. Epub 2002 Sep 4.
Increased placental leptin has been demonstrated in preeclampsia, a pregnancy disorder associated with placental hypoxia. This suggests that leptin gene expression is enhanced in response to oxygen deficiency in this organ. In support of this hypothesis, we have previously shown that hypoxia activates the leptin promoter in trophoblast-derived BeWo cells. Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric HIF-1alpha/HIF-1beta complex that regulates the transcription of hypoxia-responsive genes. To test whether this factor is involved in hypoxia-induced leptin promoter activation, BeWo cells were transiently transfected with a HIF-1alpha expression vector. Exogenous HIF-1alpha markedly increased luciferase reporter activity driven by the leptin promoter when HIF-1beta was co-expressed in the same cells. This effect was similar to that elicited by CoCl2, an agent known to stabilize endogenous HIF-1alpha. These data suggest that HIF-1alpha/HIF-1beta dimers are involved in the effect of CoCl2 to activate the leptin promoter. To confirm the implication of HIF-1, the cells were transfected with a dominant negative form of HIF-1alpha producing transcriptionally inactive HIF-1beta/HIF-1alpha dimers. This mutant HIF-1alpha protein abolished CoCl2 activation of the leptin promoter, providing direct evidence that the effect of CoCl2 is mediated by endogenous HIF-1alpha. Deletion analysis and site-specific mutagenesis demonstrated that a HIF-1 consensus binding site (HRE) spanning -120 to -116 bp relative to the start site was required for CoCl2 and exogenous HIF-1alpha induction of leptin promoter activity. Electrophoretic mobility shift assays performed with in vitro-translated HIF-1alpha and HIF-1beta proteins demonstrated binding to this HRE and not to mutated sequences only when both subunits were used together. These data demonstrate that leptin is a new hypoxia-inducible gene, which is stimulated in a placental cell line through HIF-1 interaction with a consensus HRE site located at -116 in the proximal promoter.
子痫前期（一种与胎盘缺氧相关的妊娠疾病）患者的胎盘瘦素水平升高。这表明该器官中瘦素基因表达会因缺氧而增强。为支持这一假说，我们之前已表明缺氧可激活滋养层来源的BeWo细胞中的瘦素启动子。缺氧诱导因子1（HIF - 1）是一种异二聚体HIF - 1α/HIF - 1β复合物，可调节缺氧反应基因的转录。为检测该因子是否参与缺氧诱导的瘦素启动子激活，将BeWo细胞用HIF - 1α表达载体进行瞬时转染。当HIF - 1β在同一细胞中共同表达时，外源性HIF - 1α显著增加了由瘦素启动子驱动的荧光素酶报告基因活性。这种效应与已知可稳定内源性HIF - 1α的氯化钴所引发的效应相似。这些数据表明HIF - 1α/HIF - 1β二聚体参与了氯化钴激活瘦素启动子的效应。为证实HIF - 1的作用，将细胞用产生转录无活性的HIF - 1β/HIF - 1α二聚体的显性负性形式的HIF - 1α进行转染。这种突变的HIF - 1α蛋白消除了氯化钴对瘦素启动子的激活作用，提供了直接证据表明氯化钴的效应是由内源性HIF - 1α介导的。缺失分析和位点特异性诱变表明，相对于起始位点跨越 - 120至 - 116 bp的HIF - 1共有结合位点（HRE）是氯化钴和外源性HIF - 1α诱导瘦素启动子活性所必需的。用体外翻译的HIF - 1α和HIF - 1β蛋白进行的电泳迁移率变动分析表明，只有当两个亚基一起使用时才会与该HRE结合而不与突变序列结合。这些数据表明瘦素是一种新的缺氧诱导基因，其在胎盘细胞系中通过HIF - 1与位于近端启动子 - 116处的共有HRE位点相互作用而受到刺激。
