Mitochondrial reactive oxygen species (ROS) produced during early reperfusion are important triggers for the development of ischemia/reperfusion injury (IRI). However, antioxidants yielded little clinical benefit in its prevention, most likely because of the failure to timely and efficiently target them to the site of ROS production and action. Cytosolic p66SHC, which translocates to the mitochondria under stress to oxidize cytochrome c, is a major contributor of ROS under stress conditions. Gene knockout in mice confirmed its important role in the development of IRI. Here we identified pathways essential for the activation of p66SHC, which may be targeted for therapeutic intervention.

Using MEFs and HL-1 cardiomyocytes we failed to detect the previously reported PKCbeta-dependent phosphorylation of p66SHC on S36 and instead identified S139 as critical target. In p66SHC-deficient MEFs reconstituted with the S139A mutant of p66SHC we observed significantly reduced ROS production. MAPK (ERK, JNK, p38) activity, p66SHC S36 phosphorylation and ROS production are frequently increased during ischemia/reperfusion (IR) as they are under prooxidant treatment and HR. Inhibition of signaling through MAPKs in vitro with specific inhibitors showed a pronounced decrease in p66SHC S36 phosphorylation only for JNK, which also directly interacted with p66SHC. Moreover, JNK inhibition resulted in decreased ROS production and reduced DNA damage. We further confirmed JNK-dependent regulation of p66SHC S36 phosphorylation, ROS production and cell death using JNK 1, 2 deficient MEFs. Finally, the low ROS phenotype of JNK 1, 2 knockout MEFs was reversed by introducing p66SHC mutated in S36E.

Interference with p66SHC activation by targeting upstream pathways (PKCbeta, JNK), that are commonly activated during IR and for which specific inhibitors are available provides a possible therapeutic approach for decreasing damage to cells and organs during ischemia/reperfusion.

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