Oxygen-regulated Transferrin Expression Is Mediated by Hypoxia-Inducible factor-1

J Biol Chem. 1997 Aug 8;272(32):20055-62. doi: 10.1074/jbc.272.32.20055.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aryl Hydrocarbon Receptor Nuclear Translocator
  • Binding Sites
  • Carcinoma, Hepatocellular / metabolism
  • DNA / metabolism
  • DNA-Binding Proteins / metabolism*
  • Helix-Loop-Helix Motifs*
  • Humans
  • Hypoxia-Inducible Factor 1
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Liver Neoplasms / metabolism
  • Mice
  • Nuclear Proteins / metabolism*
  • Oxygen / metabolism*
  • RNA, Messenger / metabolism
  • Receptors, Aryl Hydrocarbon / metabolism
  • Repetitive Sequences, Nucleic Acid
  • Transcription Factors / metabolism*
  • Transferrin / genetics*
  • Transferrin / metabolism
  • Tumor Cells, Cultured

Substances

  • ARNT protein, human
  • Arnt protein, mouse
  • DNA-Binding Proteins
  • HIF1A protein, human
  • Hif1a protein, mouse
  • Hypoxia-Inducible Factor 1
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Nuclear Proteins
  • RNA, Messenger
  • Receptors, Aryl Hydrocarbon
  • Transcription Factors
  • Transferrin
  • Aryl Hydrocarbon Receptor Nuclear Translocator
  • DNA
  • Oxygen