Erythropoietin-receptor gene regulation in neuronal cells.

Because erythropoietin (Epo) is intensively studied as neuroprotective agent, Epo receptor (EpoR) regulation in neurons is of particular interest. Herein, we investigated molecular mechanisms of EpoR regulation in neuronal cells including the role of GATA transcription factors. First, developmental downregulation of EpoR expression in murine brain was observed. A differential expression pattern of the Gata factors was found in these specimens as well as in murine adult neural stem cells (NSC) and primary rat neurons, astrocytes, and microglia. Human SH-SY5Y cells served as a model to analyze EpoR regulation. In vitro binding of GATA-2, -3, and -4 to the 5'-flanking region was demonstrated. In reporter gene assays, the activity of a region containing two GATA binding sites was significantly induced when these GATA factors were overexpressed. However, GATA factors alone did not affect endogenous EpoR expression. Importantly, EpoR transcripts have doubled under hypoxia. Furthermore, we analyzed the methylation pattern close to the GATA motifs. Indeed, demethylation with 5-Aza-2'-deoxycytidine (Aza) resulted in upregulation of EpoR mRNA. Additionally, several CpGs were mostly nonmethylated in SH-SY5Y cells, but methylated in specific regions of the human adult brain. Thus, methylation may be involved in developmental EpoR downregulation.