Identification of epigenetic mechanisms regulating the isoform 1 of sodium calcium exchanger (NCX1) in in vitro and in vivo models of brain ischemia and brain ischemic preconditioning

The Na+-Ca2+ exchanger 1 (NCX1) is reduced in stroke by the RE1-silencing transcription factor (REST), whereas it is increased in Ischemic Brain Preconditioning (PC) by Hypoxia-Inducible Factor 1 (HIF-1). Since ncx1 brain promoter (ncx1-Br) has five putative consensus sequences for the Specificity protein (Sp) family of transcription factors (Sp1, Sp2, Sp3, Sp4), named Sp1 A-E, we investigated the role of this family in regulating ncx1 transcription in cortical neurons. Here we found that Sp1 is a transcriptional activator, whereas Sp3 is a transcriptional repressor of ncx1, and that both bind ncx1 brain promoter (ncx1-Br) in a sequence-specific manner, modulating ncx1 transcription through the Sp1 sites C-E. Furthermore, in brain ischemia (tMCAO) the transcriptional repressors Sp3 and REST colocalize with histone-deacetylases (HDACs) HDAC1-2 at the ncx1 promoter, with a consequent hypoacetylation. By contrast, in PC+tMCAO the transcriptional activators Sp1 and HIF-1 colocalize with Histone-Acetyltransferase p300 (p300) on ncx1 promoter sequence with a consequent hyperacetylation. Moreover, when brain ischemia-induced REST and Sp3 up-regulation was prevented by intracerebroventricular (icv) injection of siRNAs for REST and Sp3, NCX1 downregulation was reverted. Interestingly, also siRNAs for HDAC1-2 was able to completely revert NCX1 reduction. On the contrary, siRNA for Sp1 and HIF-1, during PC+tMCAO, blocked NCX1 increase, that was almost completely reverted by double siRNA for HIF-1 and Sp1 or by siRNA for p300. In addition, in neurons transfected for siNCX1, and subjected to Oxygen Glucose Deprivation (OGD) (3 hours) plus Reoxygenation (RX) (24 hours), the protective effect of class I HDAC inhibitor MS-275 was counteracted, whereas neurons overexpressing NCX1 and subjected to ischemic preconditioning (PC+OGD\Rx), the detrimental effect of p300 inhibitor C646 was prevented. Collectively, these results demonstrate that NCX1 expression is regulated by Sp3\REST\HDAC1-2 complex in tMCAO and by the Sp1\HIF-1\p300 complex in PC+tMCAO, and that epigenetic therapy modulating the acetylation of ncx1 gene promoter may be a new strategy to reduce the neurodetrimental effect of stroke