OXYGEN-REGULATED TRANSFERRIN EXPRESSION IS MEDIATED BY HYPOXIA-INDUCIBLE FACTOR-I

Transferrin (Tf) is a liver-derived iron transport protein whose plasm a concentration increases following exposure to hypoxia, Here, we pres ent a cell culture model capable of expressing Tf mRNA in an oxygen-de pendent manner, A C-kilobase pair Tf promoter/enhancer fragment as wel l as the 300-base pair liver-specific Tf enhancer alone conveyed hypox ia responsiveness to a heterologous reporter gene construct in hepatom a but not HeLa cells. Within this enhancer, a 32-base pair hypoxia-res ponsive element was identified, which contained two hypoxia-inducible factor-1 (HIF-1) binding sites (HBSs). Mutation analysis showed that b oth HBSs function as oxygen-regulated enhancers in Tf-expressing as we ll as in non-Tf-expressing cell lines, Mutation of both HBSs was neces sary to completely abolish hypoxic reporter gene activation, Transient co-expression of the two HIF-1 subunits HIF-1 alpha and aryl hydrocar bon receptor nuclear translocator (ARNT)/HIF-1 beta resulted in enhanc ed reporter gene expression even under normoxic conditions, Overexpres sion of a dominant-negative ARNT/HIF-1 beta mutant reduced hypoxic act ivation, DNA binding studies using nuclear extracts from the mouse hep atoma cell line Hepa1 and the ARNT/HIF-1 beta-deficient subline Hepa1C 4, as well as antibodies raised against HIF-1 alpha and ARNT/HIF-1 bet a confirmed that HIF-1 binds the Tf HBSs. Mutation analysis and compet ition experiments suggested that the 5' HBS was more efficient in bind ing HIF-1 than the 3' HBS. Finally, hypoxic induction of endogenous Tf mRNA was abrogated in Hepa1C4 cells, confirming that HIF-1 confers ox ygen regulation of Tf gene expression by binding to tile two HBSs pres ent in the Tf enhancer.