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 protein complex originally described in the transcriptional activation of the erythropoietin gene by hypoxia. This protein complex is composed of two subunits, HIF-1alpha and -1beta (aryl hydrocarbon receptor nuclear translocator, ARNT). In this study, we used ARNT-deficient cells, derived from the mouse hepatoma cell line Hepa1c1c7, to further characterize HIF-1 complex formation and its relationship with gene activation by hypoxia and desferrioxamine (Df). Gel shift assays revealed that ARNT is absolutely required for the formation of the HIF-1 DNA-binding complex. Results from RNase protection assays and Northern blots showed that the lack of functional HIF-1 complex completely abrogated the response to hypoxia of vascular endothelial growth factor (VEGF) and the glycolytic enzymes aldolase A (ALDA) and phosphoglycerate kinase 1 (PGK-1), genes known to be upregulated by low oxygen tension. Desferrioxamine induction of VEGF and PGK-1 genes was reduced in the ARNT-deficient cells, but at difference with hypoxia, it was not completely suppressed. These results suggest that Df is able to activate gene transcription through HIF-1-independent mechanisms. Exposure to hypoxia or Df did not induce any changes in HIF-1alpha and -1beta mRNA levels, suggesting that posttranscriptional mechanisms are involved in HIF-1 complex activation.
Salceda
S
S
Cardeza Foundation for Hematologic Research, Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Beck
I
I
Caro
J
J
D000199:Actins / Q000096:biosynthesis; D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D015152:Blotting, Northern; D015687:Cell Hypoxia; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003676:Deferoxamine / Q000494:pharmacology; D016228:Endothelial Growth Factors / Q000096:biosynthesis; D005634:Fructose-Bisphosphate Aldolase / Q000096:biosynthesis; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008114:Liver Neoplasms, Experimental; D008222:Lymphokines / Q000096:biosynthesis; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D010735:Phosphoglycerate Kinase / Q000096:biosynthesis; D018336:Receptors, Aryl Hydrocarbon / Q000096:biosynthesis*; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D000199:Actins; C497397:Arnt protein, mouse; D004268:DNA-Binding Proteins; D016228:Endothelial Growth Factors; C497443:Hif1a protein, mouse; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008222:Lymphokines; D009687:Nuclear Proteins; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D010735:Phosphoglycerate Kinase; D005634:Fructose-Bisphosphate Aldolase; D003676:Deferoxamine