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 mR NA levels using Rat1 fibroblasts transformed with H-ras (Rat1-ras), Transfo rmation 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 p airs 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 dom inant 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, suggest ing that Ras might mediate its effect through HIF-1 even under normoxic con ditions. Consistent with this, Rat1-ras cells displayed higher levels of HI F1-alpha protein under normoxic conditions. In addition, a promoter constru ct containing a 4-bp mutation in the HIF1 binding site showed lower activit y 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 regulatio n by Ras, The phosphatidylinositol 3-kinase inhibitor LY29004 down-regulate d glut1 promoter activity and mRNA levels under normoxia and also decreased HIF1 alpha protein levels in these cells. Collectively these results indic ate that H-Ras up-regulates the glut1 promoter, at least in part, by increa sing HIF-1 alpha protein levels leading to transactivation of promoter thro ugh the HIF-1 binding site.