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

Hydrogen peroxide (H₂O₂) has previously been shown to inhibit the DNA binding activity of hypoxia inducible factor-1 (HIF-1), the accumulation of HIF-1α 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.