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.
Gotoh
T
T
Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Honjo, Kumamoto, Japan. tomomi@gpo.kumamoto-u.ac.jp
Terada
K
K
Oyadomari
S
S
Mori
M
M
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D022762:CCAAT-Enhancer-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000378:metabolism; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000502:physiology*; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism / Q000494:pharmacology*; D009569:Nitric Oxide / Q000037:antagonists & inhibitors*; D021381:Protein Transport / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014162:Transfection; D051028:bcl-2-Associated X Protein
C494827:Bax protein, mouse; D022762:CCAAT-Enhancer-Binding Proteins; C497250:Ddit3 protein, mouse; C494639:Dnaja1 protein, mouse; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014157:Transcription Factors; D051028:bcl-2-Associated X Protein; D051742:Transcription Factor CHOP; D009569:Nitric Oxide; D000251:Adenosine Triphosphatases