Abstract 466: CD36 Mediates Mitochondrial Reactive Oxygen Species Production Through PKM2 In Macrophages

Introduction: Excessive mitochondria-derived reactive oxygen species (mtROS) are associated with foam cell formation and atherosclerosis progression in both animal models and human patients. We previously showed that oxLDL/CD36 signaling stimulated mtROS production and facilitated diet-induced atherosclerosis in mice. However, underlying mechanisms by which oxLDL/CD36 pathway mediates mtROS production during atherosclerosis remain elusive. Pyruvate kinase M2 (PKM2) is a glycolytic enzyme recently discovered to facilitate foam cell and atherosclerosis development. We hypothesize that oxLDL/CD36 signaling mediates mtROS production by stimulating PKM2 mitochondrial translocation. Methods and Results: We separated mitochondria and cytosol fractions in murine peritoneal macrophages. OxLDL treatment upregulated PKM2 protein level in mitochondria fractions in a time-dependent manner (peak at 3h, ~2.5 fold), but not in cytosol. Confocal imaging further confirmed a translocation of PKM2 to mitochondria induced by oxLDL. This phenomenon was not observed in CD36-deficient macrophages. To investigate the mechanism, we immunoprecipitated PKM2 followed by mass spectrometry analysis of proteins associated with PKM2. GRP75, a known chaperone protein that imports cytosol proteins to the mitochondria was identified. Co-IP assay confirmed the interaction between GRP75 and PKM2, which was augmented by oxLDL (~3 fold). Moreover, UQCRC1, a subunit of mitochondrial electron transport chain (ETC) complex III, a major site of mtROS production, was also identified from mass spectrometry. Increased interaction between PKM2 and UQCRC1 induced by oxLDL (~3 fold) was further validated by coIP and in situ proximity ligation assay, which was dependent on CD36.In addition, PKM2 inhibitor Shikonin and PKM2 siRNA both inhibited the oxLDL-induced mtROS generation (by 50% and 100% respectively) in murine macrophages and human monocytes-derived macrophages. Conclusions: OxLDL/CD36 axis promotes PKM2 translocation to mitochondrial which facilitates mtROS production in macrophages. It highlights the novel role of PKM2 in mtROS production during atherosclerosis and implicates GRP75-PKM2-UQCRC1 axis as a potential therapeutic target.