Roth Ulrike, Jungermann Kurt, Kietzmann Thomas
Institut für Biochemie und Molekulare Zellbiologie, Georg-August-Universität, Humboldtallee 23, D-37073 Göttingen, Germany.
Biol Chem. 2004 Mar-Apr;385(3-4):239-47. doi: 10.1515/BC.2004.018.
Glucokinase (GK) is the key enzyme of glucose utilization in liver and is localized in the less aerobic perivenous area. Until now, the O2-responsive elements in the liver-specific GK promoter are unknown, and therefore the aim of this study was to identify the O2-responsive element in this promoter. We found that the GK promoter sequence -87/-80 matched the binding site for hypoxia inducible factor 1 (HIF-1) and upstream stimulatory factor (USF). In primary rat hepatocytes we could show that venous pO2 enhanced HIF-1alpha and USF-2a levels, both of which activated GK expression. Furthermore, transfection experiments revealed that the GK sequence -87/-80 mediated the HIF-1alpha- or USF-2-dependent activation of the GK promoter. The binding of HIF-1 and USF to the GK-HRE was corroborated by electrophoretic mobility shift assay (EMSA). However, the maximal response to HIF-1alpha or USF was only achieved when constructs with the -87/-80 sequence in context with a 3'-36 bp native GK promoter sequence containing a hepatocyte nuclear factor 4 (HNF-4) binding site were used. HIF-1alpha and HNF-4 additively activated the GK promoter, while USF-2 and HNF-4 together did not show this additive activation. Thus, HIF-1 and USF may play differential roles in the modulation of GK expression in response to O2.
葡萄糖激酶（GK）是肝脏中葡萄糖利用的关键酶，定位于需氧较少的肝静脉周围区域。迄今为止，肝脏特异性GK启动子中的氧反应元件尚不清楚，因此本研究的目的是鉴定该启动子中的氧反应元件。我们发现GK启动子序列-87/-80与缺氧诱导因子1（HIF-1）和上游刺激因子（USF）的结合位点匹配。在原代大鼠肝细胞中，我们可以证明静脉血氧分压可提高HIF-1α和USF-2α水平，二者均可激活GK表达。此外，转染实验表明，GK序列-87/-80介导了HIF-1α或USF-2依赖性的GK启动子激活。电泳迁移率变动分析（EMSA）证实了HIF-1和USF与GK缺氧反应元件（HRE）的结合。然而，只有当使用包含-87/-80序列且带有3'-36 bp天然GK启动子序列（含有肝细胞核因子4（HNF-4）结合位点）的构建体时，才能实现对HIF-1α或USF的最大反应。HIF-1α和HNF-4可协同激活GK启动子，而USF-2和HNF-4共同作用时则未表现出这种协同激活作用。因此，HIF-1和USF在响应氧气时对GK表达的调节中可能发挥不同作用。
