hsp70-DnaJ chaperone pair prevents nitric oxide- and CHOP-induced apoptosis by inhibiting translocation of Bax to mitochondria

We reported that the endoplasmic reticulum (ER) stress pathway involving CHOP, a member of the C/EBP transcription factor family, plays a key role in nitric oxide (NO)-mediated Apoptosis of macrophages and pancreatic beta cells. We also showed that the cytosolic chaperone pair of HSP70 and dj1 (HSP40/hdj-1) or dj2 (HSDJ/hdj-2) prevents NO-mediated Apoptosis upstream of cytochrome c release from mitochondria. To analyze roles of the chaperone pair in preventing Apoptosis, RAW 264.7 macrophages stably expressing HSP70 and dj1 or dj2 were established. The chaperone pair prevented LPS/IFN-gamma-induced and NO-mediated Apoptosis downstream of CHOP induction. HSP70 mutant protein lacking the ATPase domain or the C-terminal EEVD sequence were not effective in preventing CHOP-induced Apoptosis. A mutant dj2 lacking the C-terminal prenylation CaaX motif, was also not effective. When wild-type RAW 264.7 cells were treated with LPS/IFN-gamma, NO-mediated Apoptosis was induced, and proapoptotic Bcl-2 Family protein Bax was translocated from cytosol to mitochondria. This translocation was prevented in cells stably expressing HSP70/dj2, and in CHOP knockout cells. Overexpression of CHOP in wild-type cells also induced translocation of Bax and this translocation was prevented in cells expressing HSP70/dj2. CHOP-induced Apoptosis was prevented by Bax knock-down. Coimmunoprecipitation experiments showed that Bax interacts with both HSP70 and dj1/dj2. ATPase domain of HSP70 was necessary for the binding with Bax. These findings indicate that CHOP-induced Apoptosis is mediated by translocation of Bax from the cytosol to the mitochondria, and HSP70/dj1 or dj2 chaperone pair prevents Apoptosis by interacting with Bax and preventing translocation to the mitochondria.