Upregulation of BNIP3 and translocation to mitochondria mediates cyanide-induced apoptosis in cortical cells

Abstract

Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a Bcl-2 homology domain 3 (BH3) domain only protein, has been identified as a mitochondrial mediator of hypoxia-induced cell death. Since cyanide produces histotoxic anoxia (chemical hypoxia), the present study was undertaken in primary rat cortical cells to determine involvement of the BNIP3 signaling pathway in cyanide-induced death. Over a 20 h exposure KCN increased BNIP3 expression, followed by a concentration-related apoptotic death. To determine if BNIP3 plays a role in the cell death, expression was either increased with BNIP3 cDNA (BNIP3 +) or knocked down with small interfering RNA (RNAi). In BNIP3 + cells, cyanide-induced apoptotic death was markedly enhanced and preceded by reduction of mitochondrial membrane potential (Δ ψ m ), release of cytochrome c from mitochondria and elevated caspase 3 and 7 activity. Pretreatment with the pan-caspase inhibitor N-benzyloxycarbonyl-Ala-Asp-fluoromethyl ketone (zVAD-fmk) suppressed BNIP3 +-mediated cell death, thus confirming a caspase-dependent apoptosis. On the other hand, BNIP3 knockdown by RNAi or antagonism of BNIP3 by a transmembrane-deleted dominant-negative mutant (BNIP3ΔTM) markedly reduced cell death. Immunohistochemical imaging showed that cyanide stimulated translocation of BNIP3 from cytosol to mitochondria and displacement studies with BNIP3ΔTM showed that integration of BNIP3 into the mitochondrial outer membrane was necessary for the cell death. In BNIP3 + cells, cyclosporin-A, an inhibitor of mitochondrial pore transition, blocked the cyanide-induced reduction of Δ ψ m and decreased the apoptotic death. These results demonstrate in cortical cells that cyanide induces a rapid upregulation of BNIP3 expression, followed by translocation to the mitochondrial outer membrane to reduce Δ ψ m . This was followed by mitochondrial release of cytochrome c to execute a caspase-dependent cell death.