Regulation of glut1 mRNA by hypoxia-inducible factor-1. Interaction between H-ras and hypoxia.
Oncogenic transformation and hypoxia both induce glut1 mRNA. We studied the interaction between the ras oncogene and hypoxia in up-regulating glut1 mRNA levels using Rat1 fibroblasts transformed with H-ras (Rat1-ras). Transformation with H-ras led to a substantial increase in glut1 mRNA levels under normoxic conditions and additively increased glut1 mRNA levels in concert with hypoxia. Using a luciferase reporter construct containing 6 kilobase pairs of the glut1 promoter, we showed that this effect was mediated at the transcriptional level. Promoter activity was much higher in Rat1-ras cells than in Rat1 cells and could be down-regulated by cotransfection with a dominant negative Ras construct (RasN17). A 480-base pair (bp) cobalt/hypoxia-responsive fragment of the promoter containing a HIF-1 binding site showed significantly higher activity in Rat1-ras cells than in Rat1 cells, suggesting that Ras might mediate its effect through HIF-1 even under normoxic conditions. Consistent with this, Rat1-ras cells displayed higher levels of HIF1-alpha protein under normoxic conditions. In addition, a promoter construct containing a 4-bp mutation in the HIF1 binding site showed lower activity in Rat1-ras cells than a construct with an intact HIF1 binding site. The activity of the latter construct but not the former could be down-regulated by RasN17, supporting the importance of the HIF1 binding site in regulation by Ras. The phosphatidylinositol 3-kinase inhibitor LY29004 down-regulated glut1 promoter activity and mRNA levels under normoxia and also decreased HIF1alpha protein levels in these cells. Collectively these results indicate that H-Ras up-regulates the glut1 promoter, at least in part, by increasing HIF-1alpha protein levels leading to transactivation of promoter through the HIF-1 binding site.
Chen
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C
Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 , USA.
Pore
N
N
Behrooz
A
A
Ismail-Beigi
F
F
Maity
A
A
D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D002867:Chromones / Q000494:pharmacology; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D015536:Down-Regulation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology*; D051272:Glucose Transporter Type 1; D000860:Hypoxia / Q000378:metabolism*; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D009025:Morpholines / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology*; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D014157:Transcription Factors
D002867:Chromones; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D051272:Glucose Transporter Type 1; C497444:Hif1a protein, rat; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009004:Monosaccharide Transport Proteins; D009025:Morpholines; D009687:Nuclear Proteins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger; C495679:Slc2a1 protein, rat; D014157:Transcription Factors; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004247:DNA; D020928:Mitogen-Activated Protein Kinases; D015689:Oncogene Protein p21(ras); C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one