Functional Interference between Hypoxia and Dioxin Signal Transduction Pathways: Competition for Recruitment of the Arnt Transcription Factor

Abstract

Hypoxia-inducible factor 1α (HIF-1α) 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-1α 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-1α. Strikingly, HIF-1α 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-1α function in vivo or overexpression of HIF-1α inhibited ligand-dependent induction of DNA binding activity by the dioxin receptor and dioxin receptor function on minimal reporter gene constructs. However, HIF-1α- and dioxin receptor-mediated signalling pathways were not mutually exclusive, since activation of dioxin receptor function did not impair HIF-1α-dependent induction of target gene expression. Both HIF-1α 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-1α may be conditionally regulated by a mechanism that is distinct from induced expression levels, the prevalent model of activation of HIF-1α function. Interestingly, we observed that HIF-1α 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-1α is regulated by a similar mechanism, possibly by binding an as yet unknown class of ligands.