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 (delta psim), 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 (BNIP3 delta TM) markedly reduced cell death. Immunohistochemical imaging showed that
cyanide stimulated translocation of BNIP3 from cytosol to mitochondria and displacement studies with BNIP3 delta TM 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 delta psim 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 delta psim. This was followed by mitochondrial release of
cytochrome c to execute a
caspase-dependent cell death.