Exposure of human lung epithelial (A549) cells to asbestos fibers causes apoptosis, which is largely attributed to release of iron and generation of reactive oxygen species (ROS) within the cells. To mimic the highly oxidative environment generated by asbestos exposure in the absence of the actual fibers, we used two chemicals; buthione sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis and ferric ammonium citrate (FAC), a source of iron. Here, we report that exposure of A549 cells to crocidolite asbestos led to a significant time-dependent inactivation of signaling proteins, i.e. Akt and all mitogen-activated protein kinases (MAPKs) (p38, ERK1/2 and SAPK/JNK), and subsequently to apoptosis. Unlike crocidolite treatment, the use of BSO and FAC, independently or combined, did not change the phosphorylation status of proteins, nor did it induce apoptosis. Taken together, our results presented herein point to the possibility that crocidolite-induced apoptosis of human lung epithelial cells is not a mere consequence of generation of oxidants but also requires inactivation of major cell growth and differentiation pathways.
Sequestosome1 (p62) is a multifunctional signaling molecule and an autophagy adaptor protein. Previous work demonstrated that mice with whole-body p62 knockout recapitulated many detrimental features of aging. Of note, these mice developed late onset obesity and systemic abnormalities that could have contributed to their aging phenotype. Multiple studies have also shown that cardiac dysfunction can be linked to an increase in oxidative stress. The Nrf2-Keap1 pathway is critical for protection against oxidative stress and p62 has been shown to interact with Keap1, thus allowing Nrf2 activation to induce anti-oxidant responses. However, the role of p62 in the heart is not well known. We tested the hypothesis that p62 plays an important homeostatic role in the heart through the regulation of redox homeostasis via the Nrf2-Keap1 pathway. Wild-type and cardiomyocytes-specific p62 knockout (cp62 KO) mice at 8 weeks and 60 weeks of age were used. At 8 weeks, cp62KO mice exhibited mild but significant contractile dysfunction compared to the wild-type controls. By 60 weeks, the KO mice developed cardiac hypertrophy, fibrosis and increased oxidative stress. cp62 KO hearts had decreased Nrf2 nuclear translocation and activation as evidenced by a 50% (p<0.005) reduction in the expression of the Nrf2 target glutathione S-transferase A4 (
Gsta2
) gene. These findings were further validated by transcriptomic analysis followed by KEGG pathway analysis, which indicated that redox pathways were altered in the 60-week p62 null hearts. To examine the mechanisms involved in p62 regulation of Nrf2-Keap signaling, we utilized rat cardiac H9c2 myoblasts. Loss of p62 using p62 siRNA in H9c2 cells resulted in decreased Nrf2 levels and increased oxidative stress. These pathological consequences of suppressing p62 could be attributed to increased Nrf2 degradation via the proteasome. Together, these results reveal a previously uncharacterized role for p62 in the maintenance of cardiac redox signaling in the mouse heart.
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