Induction and nuclear translocation of hypoxia-inducible factor-1 (HIF-1):heterodimerization with ARNT is not necessary for nuclear accumulation of HIF-1 alpha

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of mammalian oxyge n homeostasis. HIF-1 consists of two subunits, HIF-1 alpha and the aryl hyd rocarbon receptor nuclear translocator (ARNT), Whereas hypoxia prevents pro teasomal degradation of HIF-1 alpha,ARNT expression is thought to be oxygen -independent. We and others previously showed that ARNT is indispensable fo r HIF-1 DNA-binding and transactivation function. Here, we have used ARNT-m utant mouse hepatoma and embryonic stem cells to examine the requirement of ARNT for accumulation and nuclear translocation of HIF-1 alpha in hypoxia, As shown by immunofluorescence, HIF-1 alpha accumulation in the nucleus of hypoxic cells was independent of the presence of ARNT, suggesting that nuc lear translocation is intrinsic to HIF-1 alpha. Coimmunoprecipitation of HI F-1 alpha together with ARNT could be performed in nuclear extracts but not in cytosolic fractions, implying that formation of the HIF-1 complex occur s in the nucleus. A proteasome inhibitor and a thiol-reducing agent could m imic hypoxia by inducing HIF-1 alpha in the nucleus, indicating that escape from proteolytic degradation is sufficient for accumulation and nuclear tr anslocation of HIF-1 alpha, During biochemical separation, both HIF-1 alpha and ARNT tend to leak from the nuclei in the absence of either subunit, su ggesting that heterodimerization is required for stable association within the nuclear compartment, Nuclear stabilization of the heterodimer might als o explain the hypoxically increased total cellular ARNT levels observed in some of the cell lines examined.