Oxygen-regulated transferrin expression is mediated by hypoxia-inducible factor-1

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Transferrin (Tf) is a key iron transport protein synthesized in the liver.
• Plasma Tf concentrations rise under hypoxic conditions.
• Understanding the regulation of Tf gene expression during hypoxia is crucial.

Purpose of the Study:

• To develop a cell culture model for studying oxygen-dependent Tf mRNA expression.
• To identify the specific DNA elements responsible for hypoxia-induced Tf gene regulation.
• To elucidate the role of hypoxia-inducible factor-1 (HIF-1) in Tf gene expression.

Main Methods:

• Utilized a reporter gene assay with Tf promoter/enhancer fragments in hepatoma and HeLa cells.
• Identified and mutated a hypoxia-responsive element (HRE) containing HIF-1 binding sites (HBSs).
• Performed co-expression studies with HIF-1 subunits and dominant-negative mutants, alongside DNA binding assays.

Main Results:

• A 300-bp liver-specific Tf enhancer conferred hypoxia responsiveness.
• A 32-bp element within the enhancer contained two functional HBSs crucial for hypoxic activation.
• HIF-1 (composed of HIF-1alpha and ARNT/HIF-1beta) was confirmed to bind these HBSs.
• Mutation of both HBSs abolished hypoxic reporter gene activation.
• Hypoxic induction of endogenous Tf mRNA was dependent on HIF-1 binding in Hepa1C4 cells.

Conclusions:

• HIF-1 directly regulates transferrin gene expression in response to hypoxia.
• The identified HBSs within the Tf enhancer are critical oxygen-regulated elements.
• This study provides a model for investigating oxygen-dependent gene regulation by HIF-1.