Modulation of glucokinase expression by hypoxia-inducible factor 1 and upstream stimulatory factor 2 in primary rat hepatocytes

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.