Functional interference between hypoxia and dioxin signal transduction pathways: competition for recruitment of the Arnt transcription factor.
Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and the intracellular dioxin receptor mediate hypoxia and dioxin signalling, respectively. Both proteins are conditionally regulated basic helix-loop-helix (bHLH) transcription factors that, in addition to the bHLH motif, share a Per-Arnt-Sim (PAS) region of homology and form heterodimeric complexes with the common bHLH/PAS partner factor Arnt. Here we demonstrate that HIF-1 alpha required Arnt for DNA binding in vitro and functional activity in vivo. Both the bHLH and PAS motifs of Arnt were critical for dimerization with HIF-1 alpha. Strikingly, HIF-1 alpha exhibited very high affinity for Arnt in coimmunoprecipitation assays in vitro, resulting in competition with the ligand-activated dioxin receptor for recruitment of Arnt. Consistent with these observations, activation of HIF-1 alpha function in vivo or overexpression of HIF-1 alpha inhibited ligand-dependent induction of DNA binding activity by the dioxin receptor and dioxin receptor function on minimal reporter gene constructs. However, HIF-1 alpha- and dioxin receptor-mediated signalling pathways were not mutually exclusive, since activation of dioxin receptor function did not impair HIF-1 alpha-dependent induction of target gene expression. Both HIF-1 alpha and Arnt mRNAs were expressed constitutively in a large number of human tissues and cell lines, and these steady-state expression levels were not affected by exposure to hypoxia. Thus, HIF-1 alpha may be conditionally regulated by a mechanism that is distinct from induced expression levels, the prevalent model of activation of HIF-1 alpha function. Interestingly, we observed that HIF-1 alpha was associated with the molecular chaperone hsp90. Given the critical role of hsp90 for ligand binding activity and activation of the dioxin receptor, it is therefore possible that HIF-1 alpha is regulated by a similar mechanism, possibly by binding an as yet unknown class of ligands.
Gradin
K
K
Department of Medical Nutrition, Karolinksa Institute, Huddinge, Sweden.
McGuire
J
J
Wenger
R H
RH
Kvietikova
I
I
fhitelaw
M L
ML
Toftgård
R
R
Tora
L
L
Gassmann
M
M
Poellinger
L
L
D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D006528:Carcinoma, Hepatocellular; D015687:Cell Hypoxia; D003035:Cobalt / Q000494:pharmacology; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000502:physiology*; D004147:Dioxins / Q000494:pharmacology*; D005260:Female; D017930:Genes, Reporter; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006367:HeLa Cells; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008113:Liver Neoplasms; D008156:Luciferases / Q000096:biosynthesis; D008297:Male; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000502:physiology*; D009928:Organ Specificity; D012333:RNA, Messenger / Q000032:analysis; D018336:Receptors, Aryl Hydrocarbon / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
C497396:ARNT protein, human; D004268:DNA-Binding Proteins; D004147:Dioxins; C497442:HIF1A protein, human; D018841:HSP90 Heat-Shock Proteins; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D003035:Cobalt; D008156:Luciferases; D019363:Cytochrome P-450 CYP1A1; C018021:cobaltous chloride