Bag-1 Protects Nucleus Pulposus Cells from Oxidative Stress by Interacting with HSP70

Bcl-2-associated athanogene 1 (Bag-1) is a multifunctional prosurvival protein that binds to several intracellular targets and promotes cell survival. HSP70 and Raf-1 are important targets of Bag-1; however, the protective function of Bag-1 in nucleus pulposus (NP) cells remains unclear. In this study, we determined the effects of Bag-1 on NP cells under oxidative stress induced by treatment with hydrogen peroxide (H(2)O(2)). We found that Bag-1 was bound to HSP70, but Bag-1–Raf1 binding did not occur in NP cells. Bag-1 overexpression in NP cells enhanced cell viability and mitochondrial function and significantly suppressed p38/MAPKs phosphorylation during oxidative stress, although NP cells treated with a Bag-1 C-terminal inhibitor, which is the binding site of HSP70 and Raf-1, decreased cell viability and mitochondrial function during oxidative stress. Furthermore, the phosphorylation of the ERK/MAPKs was significantly increased in Bag-1 C-terminal inhibitor-treated NP cells without H(2)O(2) treatment but did not change with H(2)O(2) exposure. The phosphorylation of Raf-1 was not influenced by Bag-1 overexpression or Bag-1 C-terminal binding site inhibition. Overall, the results suggest that Bag-1 preferentially interacts with HSP70, rather than Raf-1, to protect NP cells against oxidative stress.