title: Ubiquitin-like protein MNSFβ covalently binds to Bcl-G and enhances lipopolysaccharide/interferon γ-induced apoptosis in macrophages.
abstract: Monoclonal non-specific suppressor factor β (MNSFβ) is a ubiquitously expressed member of the ubiquitin-like family that is involved in various biological functions. Previous studies have demonstrated that MNSFβ covalently binds to intracellular pro-apoptotic protein Bcl-G and regulates the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade in the mouse macrophage cell line Raw264.7. In this study, we demonstrate that MNSFβ promotes lipopolysaccharide (LPS)/interferon γ (IFNγ)-induced apoptosis of Raw264.7 macrophages. In Raw264.7 cells treated with MNSFβ small interfering RNA (siRNA), LPS/IFNγ- or NO donor S-nitrosoglutathione-induced apoptosis was inhibited. siRNA-mediated knockdown of MNSFβ did not affect inducible nitric-oxide synthase (iNOS) expression in LPS/IFNγ-stimulated Raw264.7 cells. Conversely, co-transfection with MNSFβ and Bcl-G greatly enhanced LPS/IFNγ- induced apoptosis in Raw264.7 cells, accompanied by increased expression of p53 and decreased Cox-2 activity. Unlike co-transfection with wild-type MNSFβ, co-transfection of a mutant MNSFβ (G74A) and Bcl-G did not result in enhancement of LPS/IFNγ-induced apoptosis. Co-over-expression of MNSFβ and Bcl-G reduced S-nitrosoglutathione-induced ERK1/2 phosphorylation. Furthermore, electrophoretic mobility shift assay experiments revealed that MNSFβ down-regulates the ERK/activator protein 1 (AP-1) signaling cascade which leads to Cox-2 activation. We also observed that MNSFβ-Bcl-G promotes LPS/IFNγ-induced apoptosis of mouse peritoneal macrophages, together with a decrease in Cox-2 expression. Taken together, our data indicate an apoptosis-enhancing effect of MNSFβ-Bcl-G is due in part to down-regulation of Cox-2 activation in macrophages.
authors.affiliation: Department of Cooperative Medical Research, Collaboration Center, Shimane University, Izumo, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Watanabe
authors.forename: Mai
authors.initials: M
authors.lastname: Nakagawa
authors.forename: Natsuko
authors.initials: N
authors.lastname: Watanabe
authors.forename: Morihiko
authors.initials: M
authors.lastname: Nakamura
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation; D007371:Interferon-gamma / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013491:Suppressor Factors, Immunologic / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: C576156:BCL2L14 protein, mouse; D008070:Lipopolysaccharides; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D013491:Suppressor Factors, Immunologic; C072169:monoclonal nonspecific suppressor factor; D009569:Nitric Oxide; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2