Evidence that cyclophilin-A protects cells against oxidative stress

Cyclophilin-A is the cytosolic isoform of a family of peptidyl-proline cis- rr uns-isomerases that bind cyclosporin A. This study investigates the role of cyclophilin-A in necrotic cell death, induced by chemical ischaemia and by t-butylhydroperoxide. An 18-mer antisense phosphorothioate oligodeoxynu cleotide was used to target a translated region of cyclophilin-a mRNA in ra t neonatal cardiomyocytes. After a 24 h exposure to the oligonucleotide, th e amount of cyclophilin-A in the cells was decreased by at least 93% as jud ged bq immunological and enzymic criteria. For the enzyme assays, peptidyl proline cis-trans-isomerase activity was measured fluorimetrically in small (10 mu l) volumes of cell extract. Immunoblots were developed with a polyc lonal anti-cyclophilin-A antibody after sample isoelectric focusing and SDS /PAGE. Cyclophilin-A suppression had no effect on cyanide-plus-3-deoxygluco se-induced cell death. However, cyclophilin-A-suppressed cells were markedl y more sensitive to t-butylhydroperoxide. Cyclosporin A conferred some resi stance to the peroxide in both types of cell, bur protection was greater in cyclophilin-A-suppressed cells, where cyclosporin A increased the survival time 2-fold. It is concluded that two cyclophilin isoforms are involved, i n quite different ways, in peroxide-induced cell death. Cyclophilin-A has a protective role. Another isoform, possibly mitochondrial cyclophilin-D, ha s a deleterious cole, such that blockade by cyclosporin A leads to protecti on.