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Abstract
p66Shc-dependent ROS production contributes to many pathologies including ischemia/reperfusion injury (IRI) during solid organ transplantation. Inhibiting p66Shc activation may provide a novel therapeutic approach to prevent damage, which is poorly managed by antioxidants in vivo. Previous work suggested that pro-oxidant and a pro-apoptotic function of p66Shc required mitochondrial import, which depended on serine 36 phosphorylation. PKCß has been proposed as S36 kinase but cJun N-terminal kinases (JNKs) may also phosphorylate this residue. To simulate the early stages of ischemia/reperfusion (IR) we either used H2O2 treatment or hypoxia/reoxygenation (HR). As during reperfusion in vivo, we observed increased JNK and p38 activity in mouse embryonic fibroblasts (MEFs) and HL-1 cardiomyocytes along with significantly increased p66ShcS36 phosphorylation, ROS production and cell damage. Application of specific inhibitors caused a pronounced decrease in p66ShcS36 phosphorylation only in the case of JNK1/2. Moreover, S36 phosphorylation of recombinant p66Shc by JNK1 but not PKCß was demonstrated. We further confirmed JNK1/2-dependent regulation of p66ShcS36 phosphorylation, ROS production and cell death using JNK1/2 deficient MEFs. Finally, the low ROS phenotype of JNK1/2 knockout MEFs was reversed by the phosphomimetic p66ShcS36E mutant. Inhibiting JNK1/2-regulated p66Shc activation may thus provide a therapeutic approach for the prevention of oxidative damage.
Highlights
Physiological levels of reactive oxygen species (ROS) are important for the maintenance of cellular homeostasis while excessive production causes aberrant signaling, inflammasome activation, cell death and organ damage, which leads to many pathological conditions ranging from diabetes, cancer, atherosclerosis, neurodegenerative diseases, rheumatoid arthritis to ischemia/reperfusion injury (IRI) during solid organ transplantation[1,2,3]
ROS p66Shc may be a promising candidate for therapeutic intervention: its activation in the cytosol is controlled by signaling proteins, which respond to cellular stress, p66Shc directly causes mitochondrial ROS production and cell death, and presence of survival signals and normoxic conditions precludes p66Shc activation
Mouse embryonic fibroblasts (MEFs) were exposed to two different treatments commonly used to mimic ischemia/reperfusion (IR), prooxidant treatment with H2O2 and HR
Summary
Physiological levels of reactive oxygen species (ROS) are important for the maintenance of cellular homeostasis while excessive production causes aberrant signaling, inflammasome activation, cell death and organ damage, which leads to many pathological conditions ranging from diabetes, cancer, atherosclerosis, neurodegenerative diseases, rheumatoid arthritis to ischemia/reperfusion injury (IRI) during solid organ transplantation[1,2,3]. In the work presented here we systematically addressed a possible role of JNK in controlling the activation of p66Shc, ROS production and cell death in a setting close to ischemia and early reperfusion. Our experiments demonstrate that JNK1/2 regulate p66Shc S36 phosphorylation and mitochondrial ROS production under the conditions studied here and blocking this molecular route may provide a therapeutic mean to prevent cellular damage and death under oxidative stress
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