Globular adiponectin-induced RAW 264 apoptosis is regulated by a reactive oxygen species-dependent pathway involving Bcl-2.
Globular adiponectin (gAd), a truncated form of adipocyte-derived cytokine, stimulates RAW 264 cells to produce reactive oxygen species (ROS), which trigger an apoptotic cascade. In this study, we investigated the generation of intracellular and mitochondrial ROS in gAd-stimulated RAW 264 cells. Treatment with gAd efficiently induced the generation of intracellular and mitochondrial ROS, as detected by dichlorodihydrofluorescein diacetate and MitoSOX fluorescence, respectively. Furthermore, gAd treatment significantly increased 8-oxoguanine, a specific indicator of oxidative DNA damage. The transfection of RAW 264 cells with iNOS- and gp91(phox)-specific small interfering RNA reduced markedly the generation of intracellular, but not mitochondrial, ROS. Quantitative PCR revealed that the expression ratio of Bcl-2 to Bax was reduced in a time-dependent manner in gAd-treated RAW 264 cells. The overexpression of Bcl-2 markedly inhibited gAd-induced apoptosis in RAW 264 cells and also reduced both the intracellular and the mitochondrial ROS generation induced by gAd treatment. Moreover, the overexpression of Bcl-2 significantly suppressed gAd-induced NO secretion and NOS activity. In addition, the inhibition of NOS activity partially reduced the oxidative DNA damage induced by gAd. Taken together, these results demonstrate that the gAd-induced apoptotic pathway acting via ROS/RNS generation involves Bcl-2.
Akifusa
Sumio
S
Department of Preventive Dentistry, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan. akifusa@dent.kyushu-u.ac.jp
Kamio
Noriaki
N
Shimazaki
Yoshihiro
Y
Yamaguchi
Noboru
N
Nishihara
Tatsuji
T
Yamashita
Yoshihisa
Y
D052242:Adiponectin / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D005452:Fluoresceins; D005786:Gene Expression Regulation; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism* / Q000473:pathology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D010617:Phenanthridines; D015202:Protein Engineering; D017510:Protein Folding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction
C521281:5-(6'-triphenylphosphoniumhexyl)-5,6-dihydro-6-phenyl-3,8-phenanthridinediammine; D052242:Adiponectin; D005452:Fluoresceins; D008562:Membrane Glycoproteins; D010617:Phenanthridines; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C110423:dihydrofluorescein; C024829:8-hydroxyguanine; D006147:Guanine; D052247:Nitric Oxide Synthase Type II; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases