Aryl hydrocarbon nuclear translocator (ARNT) promotes oxygen-independent stabilization of hypoxia-inducible factor-1alpha by modulating an Hsp90-dependent regulatory pathway.
Hypoxia-inducible factor-1 (HIF-1) is a potent cellular survival factor contributing to tumorigenesis in a broad range of cancers. The functional transcription factor exists as a heterodimeric complex consisting of HIF-1alpha and the aryl hydrocarbon receptor nuclear translocator (ARNT). Association of HIF-1 with ARNT is required for its activity; however, no other role has been ascribed to this interaction. We demonstrated previously that pharmacologic inhibition of Hsp90 by geldanamycin (GA) impairs HIF transcription and promotes VHL (Von Hippel-Lindau)-independent degradation of the protein, thus implicating Hsp90 as an essential interacting partner for HIF. In this study, we further explore the physiological role for Hsp90 in HIF function. We establish that the PAS (Per-ARNT-Sim) domain of HIF is required both to promote association with Hsp90 and confer sensitivity to GA. Coincidentally, this domain also associates with ARNT. Overexpression of ARNT in a VHL-deficient background resulted in substantially increased HIF-1 protein concomitant with increased protein stability. Conversely, down-regulation of endogenous ARNT protein by RNA interference decreased the steady-state HIF protein. ARNT-mediated stabilization of HIF is specific for the Hsp90-dependent pathway, as ARNT was unable to protect HIF from VHL-mediated degradation. We establish that the ability of ARNT to up-regulate HIF and diminish HIF sensitivity to GA is due to its ability to compete for the Hsp90 binding site on HIF. These data elucidate novel functions for ARNT and Hsp90 in regulating HIF function and further illustrate that cofactor association may significantly impact upon the sensitivity of Hsp90 clients to chaperone inhibitors.
Isaacs
Jennifer S
JS
Urologic Oncology Branch, Center for Cancer Research, NCI, National Institutes of Health, Rockville, Maryland 20850, USA. isaacsj@mail.nih.gov
Jung
Yun-Jin
YJ
Neckers
Len
L
D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D016227:Benzoquinones; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047029:Lactams, Macrocyclic; D009687:Nuclear Proteins / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011809:Quinones / Q000494:pharmacology; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D015854:Up-Regulation
C497396:ARNT protein, human; D016227:Benzoquinones; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D018841:HSP90 Heat-Shock Proteins; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047029:Lactams, Macrocyclic; D009687:Nuclear Proteins; D011809:Quinones; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D010100:Oxygen; C001277:geldanamycin