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.
Roth
Ulrike
U
Institut für Biochemie und Molekulare Zellbiologie, Georg-August-Universität, Humboldtallee 23, D-37073 Göttingen, Germany.
Jungermann
Kurt
K
Kietzmann
Thomas
T
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D004268:DNA-Binding Proteins / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic; D005941:Glucokinase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000201:enzymology*; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008297:Male; D009687:Nuclear Proteins / Q000502:physiology*; D010100:Oxygen / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020218:Response Elements / Q000235:genetics; D014157:Transcription Factors / Q000502:physiology*; D014162:Transfection; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; C497444:Hif1a protein, rat; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat; C497187:Usf2 protein, rat; D005941:Glucokinase; D010100:Oxygen