Stimulation of GATA-2 as a mechanism of hydrogen peroxide suppression in hypoxia-induced erythropoietin gene expression.

Abstract

Hydrogen peroxide (H2O2) has previously been shown to inhibit the DNA binding activity of hypoxia inducible factor-1 (HIF-1), the accumulation of HIF-1alpha protein and erythropoietin (Epo) gene expression. Epo gene expression has been previously shown to be down-regulated through a GATA binding site at its promoter region. In this study, the effect of H2O2 on Epo gene expression under hypoxic conditions through a GATA transcription factor was investigated. Hypoxic induction was found to be inhibited upon the addition of H2O2, and this effect could be reversed through the addition of catalase. Hypoxic induction was found to be suppressed by co-transfection with a human GATA-2 cDNA expression plasmid. Transfection of Hep3B cells with a reporter gene bearing a mutation at the promoter GATA binding site was found to be only mildly affected by the addition of H2O2. Electrophoretic gel mobility shift assays (EMSAs), using the Epo promoter GATA site as a probe and the GATA-2 protein extracted from Hep3B cells, showed that addition of H2O2 enhanced the binding of GATA-2 while addition of catalase inhibited this binding. From these results, we conclude that H2O2 increases the binding activity of GATA-2 in a specific manner, thereby suppressing the activity of the Epo promoter and thus inhibiting Epo gene expression.