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 bindi ng activity of hypoxia inducible factor-1 (HIF-1), the accumulation of HIF- 1 alpha protein and erythropoietin (Epo) gene expression. Epo gene expressi on has been previously shown to be down-regulated through a GATA binding si te at its promoter region. In this study, the effect of H2O2 on Epo gene ex pression under hypoxic conditions through a GATA transcription factor was i nvestigated. 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 hu man GATA-2 cDNA expression plasmid. Transfection of Hep3B cells with a repo rter gene bearing a mutation at the promoter GATA binding site was found to be only mildly affected by the addition of H2O2. Electrophoretic gel mobil ity shift assays (EMSAs), using the Epo promoter GATA site as a probe and t he GATA-2 protein extracted from Hep3B cells, showed that addition of H2O2 enhanced the binding of GATA-2 while addition of catalase inhibited this bi nding. From these results, we conclude that H2O2 increases the binding acti vity of GATA-2 in a specific manner, thereby suppressing the activity of th e Epo promoter and thus inhibiting Epo gene expression. J. Cell. Physiol. 1 86:260-267, 2001. (C) 2001 Wiley-Liss, Inc.