Reactive oxygen species enhances the induction of inducible nitric oxide synthase by sphingomyelinase in RAW264.7 cells.
The sphingomyelin pathway, activated by stimuli, such as inflammatory cytokines, results in the formation of ceramide, a second messenger molecule. The purpose of the present study was to examine the mechanism by which macrophage-type nitric oxide synthase (NOS II) is induced by stimulation of the sphingomyelin pathway. When RAW264.7 cells were incubated with sphingomyelinase (SMase), nitrite production, NOS II activity, and NOS II mRNA were increased in a dose-dependent manner. Sphingosine, dihydrosphingosine, N-acetylsphingosine (C2-ceramide), and N-acylsphingosine (natural ceramide) had no effect on nitrite production, suggesting that signal molecules other than these were concomitantly produced by SMase treatment and required for NOS II induction. We then investigated the possible involvement of intracellular reactive oxygen species (ROS) in gene induction. SMase treatment increased the level of intracellular ROS, as assessed by flow cytometric analysis using a ROS-sensitive dye, dichlorofluorescin diacetate. Antioxidants, such as N-acetyl-l-cysteine and alpha-tocopherol, inhibited gene induction as well as nitrite production by SMase. These results suggest that activation of the sphingomyelin pathway induces gene expression and that the elevated ROS were somehow involved in this process.
Hatanaka
Y
Y
Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Fujii
J
J
Fukutomi
T
T
Watanabe
T
T
Che
W
W
Sanada
Y
Y
Igarashi
Y
Y
Taniguchi
N
N
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004790:Enzyme Induction; D048031:JNK Mitogen-Activated Protein Kinases; D051379:Mice; D020928:Mitogen-Activated Protein Kinases; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology*; D013109:Sphingomyelins / Q000378:metabolism
D000975:Antioxidants; D009573:Nitrites; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013109:Sphingomyelins; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D013108:Sphingomyelin Phosphodiesterase; D000111:Acetylcysteine