Overexpression of protein kinase C isoforms protects RAW 264.7 macrophagesfrom nitric oxide-induced apoptosis: Involvement of c-Jun N-terminal kinase stress-activated protein kinase, p38 kinase, and CPP-32 protease pathways

Nitric oxide (NO) induces apoptotic cell death in murine RAW 264.7 macropha ges. To elucidate the inhibitory effects of protein kinase C (PKC) on NO-in duced apoptosis, we generated clones of RAW 264.7 cells that overexpress on e of the PKC isoforms and explored the possible interactions between PKC an d three structurally related mitogen-activated protein (MAP) kinases in NO actions. Treatment of RAW 264.7 cells with sodium nitroprusside (SNP), a NO -generating agent, activated both c-dun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 kinase, but did not activate extracellula r signal-regulated kinase (ERK)-1 and ERK-2, In addition, SNP-induced apopt osis was slightly blocked by the selective p38 kinase inhibitor (SB203580) but not by the MAP/ERK1 kinase inhibitor (PD098059). PKC transfectants (PKC -beta II, -delta, and -eta) showed substantial protection from cell death i nduced by the exposure to NO donors such as SNP and S-nitrosoglutathione (G SNO), In contrast, in RAW 264.7 parent or in empty vector-transformed cells , these NO donors induced internucleosomal DNA cleavage. Moreover, overexpr ession of PKC isoforms significantly suppressed SNP-induced JNK/SAPK and p3 8 kinase activation, but did not affect ERK-1 and -2, We also explored the involvement of CPP32-like protease in the NO-induced apoptosis, Inhibition of CPP32-like protease prevented apoptosis in RAW 264.7 parent cells. In ad dition, SNP dramatically activated CPP32 in the parent or in empty vector-t ransformed cells, while slightly activated CPP32 in PKC transfectants, Ther efore, we conclude that PKC protects NO-induced apoptotic cell death, presu mably nullifying the NO-mediated activation of JNK/SAPK, p38 kinase, and CP P32-like protease in RAW 264.7 macrophages.