Electrophilic compounds exert cellular toxicity by compromising the redox balance of cells and causing oxidative stress. Relevant examples of such compounds are environmental pollutants such as AS3+ and Cd2+, products of the peroxidation of fatty acids, such as 4-hydroxy-2,3-nonenal, and chemotherapeutic agents, such as doxorubicin. The phase II detoxifying enzymes (a group of conjugating enzymes that includes, among others, glutathione S-transferases and quinone reductases) are the major cellular defense system against toxic environmental and metabolic electrophiles. Investigating the anti-oxidant response of mouse embryonic fibroblasts deficient in the genes encoding the stress-activated protein kinases JNK1 and JNK2, we have found that, like phase II detoxifying enzymes, JNKs are critical for the cytoprotective responses to electrophiles and oxidants. JNK-deficient cells are severely susceptible to oxidative stress and this increased susceptibility correlates with elevated production of reactive oxygen species by JNK-deficient cells and their inability to maintain sufficient levels of several phase II detoxification enzymes (e.g. the glutathione S-transferases of the A, M, and P functional classes) when challenged with electrophiles and oxidants. The research proposed in this grant proposal will elucidate the molecular mechanisms that mediate the regulation of phase II enzyme expression by JNK and the involvement of these JNK-regulated phase II enzymes in the resistance to environmental oxidants and electrophiles. To this end, we will utilize fibroblast cells from mice with genetic inactivation (""""""""knockout"""""""") of JNKs and various upstream regulators of JNK activity and we will employ molecular, cellular, and biochemical approaches (such as transfection, inducible expression of transgenic alleles, site-directed mutagenesis, analysis of gene expression at mRNA and protein levels, analysis of post-translational protein modifications, subcellular fractionation, and pharmacological interference with enzyme functions). The proposed research may lead to the development (on the basis of pharmacological effectors of the JNK signaling pathway) of novel strategies for counteracting the harmful pro-oxidant impact of important environmental pollutants.
