Metón Isidoro, Egea Miriam, Anemaet Ida G, Fernández Felipe, Baanante Isabel V
Departament de Bioquímica i Biologia Molecular, Facultat de Farmàcia, Universitat de Barcelona, Diagonal 643, 08028 Barcelona, Spain.
Endocrinology. 2006 Jul;147(7):3446-56. doi: 10.1210/en.2005-1506. Epub 2006 Apr 13.
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) catalyzes the synthesis and degradation of fructose-2,6-bisphosphate, a key modulator of glycolysis-gluconeogenesis. To gain insight into the molecular mechanism behind hormonal and nutritional regulation of PFKFB expression, we have cloned and characterized the proximal promoter region of the liver isoform of PFKFB (PFKFB1) from gilthead sea bream (Sparus aurata). Transient transfection of HepG2 cells with deleted gene promoter constructs and electrophoretic mobility shift assays allowed us to identify a sterol regulatory element (SRE) to which SRE binding protein-1a (SREBP-1a) binds and transactivates PFKFB1 gene transcription. Mutating the SRE box abolished SREBP-1a binding and transactivation. The in vivo binding of SREBP-1a to the SRE box in the S. aurata PFKFB1 promoter was confirmed by chromatin immunoprecipitation assays. There is a great deal of evidence for a postprandial rise of PFKB1 mRNA levels in fish and rats. Consistently, starved-to-fed transition and treatment with glucose or insulin increased SREBP-1 immunodetectable levels, SREBP-1 association to PFKFB1 promoter, and PFKFB1 mRNA levels in the piscine liver. Our findings demonstrate involvement of SREBP-1a in the transcriptional activation of PFKFB1, and we conclude that SREBP-1a may exert a key role mediating postprandial activation of PFKFB1 transcription.
6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶（PFKFB）催化果糖-2,6-二磷酸的合成与降解，果糖-2,6-二磷酸是糖酵解-糖异生的关键调节因子。为深入了解PFKFB表达的激素和营养调节背后的分子机制，我们克隆并鉴定了金头鲷（Sparus aurata）肝脏同工型PFKFB（PFKFB1）的近端启动子区域。用缺失基因启动子构建体对HepG2细胞进行瞬时转染以及电泳迁移率变动分析，使我们能够鉴定出一个固醇调节元件（SRE），固醇调节元件结合蛋白-1a（SREBP-1a）可与之结合并反式激活PFKFB1基因转录。突变SRE框可消除SREBP-1a的结合和反式激活。通过染色质免疫沉淀分析证实了SREBP-1a在体内与金头鲷PFKFB1启动子中SRE框的结合。有大量证据表明，鱼类和大鼠在餐后PFKB1 mRNA水平会升高。同样，饥饿到进食的转变以及用葡萄糖或胰岛素处理会增加SREBP-1的免疫检测水平、SREBP-1与PFKFB1启动子的结合以及鱼肝中PFKFB1 mRNA水平。我们的研究结果表明SREBP-1a参与了PFKFB1的转录激活，并且我们得出结论，SREBP-1a可能在介导餐后PFKFB1转录激活中发挥关键作用。
