Gata4 and Sp1 regulate expression of the erythropoietin receptor in cardiomyocytes

Abstract

Experimental studies indicate significant cardioprotective effects of recombinant erythropoietin (Epo) by binding to the Epo receptor (EpoR) and by inducing various molecular mechanisms, including activation of Gata4, a transcription factor that induces anti-apoptotic genes. However, specific molecular mechanisms ofEpoRregulation in cardiomyocytes are unknown. We identified a 774 bp regulatory domain in theEpoR5' flanking region by reporter gene assays in murine HL-1 cardiomyocytes. The binding sites for Gata and Sp transcription factors both significantly contributed toEpoRpromoter activity. DNA-binding studies (EMSA and ChIP assays) identified Gata4 and Sp1 asEpoRpromoter-binding proteins in HL1 cardiomyocytes. Although Sp1 alone stimulatesEpoRonly slightly, forced expression ofGata4significantly inducedEpoRmRNA expression. In addition, knockdown of Gata4 (but also of Sp1) resulted in a significant decrease ofEpoRtranscript levels in HL-1 cardiomyocytes. Cumulativein vitrodata suggest that function of the Sp1 site is essential for the Gata4-mediated transcription. In vivo, analysis of transgenic mice expressing an inducible small-hairpin RNA againstGata4confirmed suppression ofEpoRexpression in the heart. Treating mice with high-dose doxorubicin not only resulted in Gata4 protein depletion, but also down-regulatedEpoR, followed by up-regulation ofEpoRtranscripts when Gata4 levels recovered. In conclusion, we identified Gata4 as novel regulator ofEpoRtranscription in cardiomyocytes. In models of cardiac injury, down-regulation of Gata4 or Sp1 may limit the accessibility of the EpoR for binding of erythropoiesis-stimulating agents (ESA). Thereby our data underline the essential role of Gata4 in mediating cardioprotective effects. © 2011 The Authors Journal compilation © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.