Oxygen-regulated transferrin expression is mediated by hypoxia-inducible factor-1.
Transferrin (Tf) is a liver-derived iron transport protein whose plasma concentration increases following exposure to hypoxia. Here, we present a cell culture model capable of expressing Tf mRNA in an oxygen-dependent manner. A 4-kilobase pair Tf promoter/enhancer fragment as well as the 300-base pair liver-specific Tf enhancer alone conveyed hypoxia responsiveness to a heterologous reporter gene construct in hepatoma but not HeLa cells. Within this enhancer, a 32-base pair hypoxia-responsive element was identified, which contained two hypoxia-inducible factor-1 (HIF-1) binding sites (HBSs). Mutation analysis showed that both HBSs function as oxygen-regulated enhancers in Tf-expressing as well as in non-Tf-expressing cell lines. Mutation of both HBSs was necessary to completely abolish hypoxic reporter gene activation. Transient co-expression of the two HIF-1 subunits HIF-1alpha and aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF-1beta resulted in enhanced reporter gene expression even under normoxic conditions. Overexpression of a dominant-negative ARNT/HIF-1beta mutant reduced hypoxic activation. DNA binding studies using nuclear extracts from the mouse hepatoma cell line Hepa1 and the ARNT/HIF-1beta-deficient subline Hepa1C4, as well as antibodies raised against HIF-1alpha and ARNT/HIF-1beta confirmed that HIF-1 binds the Tf HBSs. Mutation analysis and competition experiments suggested that the 5' HBS was more efficient in binding HIF-1 than the 3' HBS. Finally, hypoxic induction of endogenous Tf mRNA was abrogated in Hepa1C4 cells, confirming that HIF-1 confers oxygen regulation of Tf gene expression by binding to the two HBSs present in the Tf enhancer.
Rolfs
A
A
Institute of Physiology, University of Zürich-Irchel, CH-8057 Zürich, Switzerland.
Kvietikova
I
I
Gassmann
M
M
Wenger
R H
RH
D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008113:Liver Neoplasms / Q000378:metabolism; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism*; D014168:Transferrin / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured
C497396:ARNT protein, human; C497397:Arnt protein, mouse; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; C497443:Hif1a protein, mouse; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D014168:Transferrin; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D004247:DNA; D010100:Oxygen