Regulation of glut1 mRNA by hypoxia-inducible factor-1. Interaction between H-ras and hypoxia

• Published in: The Journal of biological chemistry Vol. 276; no. 12; pp. 9519 - 9525
• Main Authors: Chen, C, Pore, N, Behrooz, A, Ismail-Beigi, F, Maity, A
• Format: Journal Article
• Language: English
• Published: United States 23.03.2001
• Subjects: Animals; Base Sequence; Cell Line, Transformed; Chromones - pharmacology; DNA; DNA-Binding Proteins - metabolism; DNA-Binding Proteins - physiology; Down-Regulation - drug effects; Enzyme Inhibitors - pharmacology; Flavonoids - pharmacology; Gene Expression Regulation - physiology; Glucose Transporter Type 1; glut1 gene; H-ras gene; Hypoxia - metabolism; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; LY29004; Mitogen-Activated Protein Kinases - antagonists & inhibitors; Molecular Sequence Data; Monosaccharide Transport Proteins - genetics; Morpholines - pharmacology; Nuclear Proteins - metabolism; Nuclear Proteins - physiology; Oncogene Protein p21(ras) - metabolism; Phosphoinositide-3 Kinase Inhibitors; Promoter Regions, Genetic; Protein Binding; Rats; RNA, Messenger - genetics; Transcription Factors
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M010144200

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.