ABSOLUTE REQUIREMENT OF ARYL-HYDROCARBON RECEPTOR NUCLEAR TRANSLOCATOR PROTEIN FOR GENE ACTIVATION BY HYPOXIA

Hypoxia-inducible factor 1 (HIF-1) is a DNA-binding heterodimeric prot ein complex originally described in the transcriptional activation of the erythropoietin gene by hypoxia. This protein complex is composed o f two subunits, HIF-1 alpha and -1 beta (aryl hydrocarbon receptor nuc lear translocator, ARNT). In this study, we used ARNT-deficient cells, derived from the mouse hepatoma cell. line Hepa1c1c7, to further char acterize HIF-1 complex formation and its relationship with gene activa tion by hypoxia and desferrioxamine (Df). Gel shift assays revealed th at ARNT is absolutely required for the formation of the HIF-1 DNA-bind ing complex. Results from RNase protection assays and Northern blots s howed that the lack of functional HIF-1 complex completely abrogated t he response to hypoxia of vascular endothelial growth factor (VEGF) an d the glycolytic enzymes aldolase A (ALDA) and phosphoglycerate kinase 1 (PGK-1), genes known to be upregulated by low oxygen tension. Desfe rrioxamine induction of VEGF and PGK-1 genes was reduced in the ARNT-d eficient cells, but at difference with hypoxia, it was not completely suppressed. These results suggest that Df is able to activate gene tra nscription through HIF-1-independent mechanisms. Exposure to hypoxia o r Df did not induce any changes in HIF-1 alpha and -1 beta mRNA levels , suggesting that posttranscriptional mechanisms are involved in HIF-1 complex activation. (C) 1996 Academic Press, Inc.