title: Nitric oxide-induced apoptosis in RAW 264.7 macrophages is mediated by endoplasmic reticulum stress pathway involving ATF6 and CHOP.
abstract: Excess nitric oxide (NO) induces apoptosis in some cell types including macrophages; however, the cascade of NO-mediated apoptosis is not fully understood. We investigated the initial steps of NO-mediated apoptosis in mouse macrophage-like RAW 264.7 cells. When cells were treated with bacterial lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma), NO-mediated apoptosis occurred. Under these conditions, p53 accumulation was not observed, indicating that DNA damage is not the main trigger of NO-mediated apoptosis. On the other hand, mRNA and protein for CHOP, a transcription factor known to be induced by endoplasmic reticulum (ER) stress, were induced. The CHOP induction by LPS/IFN-gamma treatment preceded cytochrome c release from mitochondria. In addition, p90ATF6, an ER membrane-bound transcription factor involved in ER stress response, was cleaved to its active soluble form p50ATF6, which was transported to nucleus and bound to the ER stress response element of the CHOP gene. In the luciferase reporter assay, both the CHOP-binding element of the Rous sarcoma virus long terminal repeat and ER stress response element of the CHOP gene were activated by LPS/IFN-gamma treatment. When RAW 264.7 cells or COS-7 cells were transfected with expression plasmids for CHOP, p90ATF6, or p50ATF6, cell death was observed. In addition, apoptosis induced by p50ATF6 was prevented by a CHOP dominant negative form as well as by an ATF6 dominant negative form, and LPS/IFN-gamma-induced apoptosis was prevented by the CHOP dominant negative form. Peritoneal macrophages from CHOP knockout mice showed resistance to NO-induced apoptosis. These results indicate that the ER stress pathway involving ATF6 and CHOP plays a key role in NO-mediated apoptosis in macrophages.
authors.affiliation: Department of Molecular Genetics, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan. tomomi@gpo.kumamoto-u.ac.jp
authors.forename: Tomomi
authors.initials: T
authors.lastname: Gotoh
authors.forename: Seiichi
authors.initials: S
authors.lastname: Oyadomari
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Mori
authors.forename: Masataka
authors.initials: M
authors.lastname: Mori
mesh_terms: D051702:Activating Transcription Factor 6; D000818:Animals; D017209:Apoptosis; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005799:Genes, Dominant; D015151:Immunoblotting; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D051702:Activating Transcription Factor 6; C497112:Atf6 protein, mouse; D022762:CCAAT-Enhancer-Binding Proteins; D003574:Cytochrome c Group; D004268:DNA-Binding Proteins; C497250:Ddit3 protein, mouse; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D051742:Transcription Factor CHOP; D009569:Nitric Oxide; D007371:Interferon-gamma; D008156:Luciferases