Involvement of nitric oxide and biopterin in proinflammatory cytokine-induced apoptotic cell death in mouse osteoblastic cell line MC3T3-E1

We previously demonstrated that the addition of proinflammatory cytokines ( tumor necrosis factor-alpha, interleukin-1 beta, and interferon-gamma) caus ed induction of mRNAs for inducible nitric oxide (NO) synthase and GTP cycl ohydrolase I, a rate-limiting enzyme for 5,6,7,8-tetrahydrobiopterin (BH4) biosynthesis, and produced their end products, NO and BH,, in osteoblastic cells. In the present study, we examined whether NO and BH,, biologically a ctive substances produced in response to proinflammatory cytokines, are inv olved in the effect of these cytokines on cell viability and apoptotic cell death involving DNA fragmentation. Cytokines as well as S-nitroso-N-acetyl -d,l-penicillamine, an NO generator, decreased cell viability, whereas sepi apterin, which was converted intracellularly to BH,, increased it. The exam ination of cytotoxicity measured in terms of lactate dehydrogenase release and apoptotic cell death assessed by flow cytometric analysis showed that c ytokine induced reduction of cell viability may be based upon cell death by apoptosis, but not lyric death as in necrosis. In the presence of sepiapte rin, cytokine treatment resulted in a statistically pronounced reduction in the amount of DNA fragmentation. Furthermore, this fragmentation could be blocked by 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide , an NO scavenger. These results suggest that cytokine-induced apoptotic ce ll death is attributed to NO and is protected by BH4, and that osteoblastic cells in response to proinflammatory cytokines operate both a stimulatory process resulting in NO production and an inhibitory one resulting in BH4 p roduction for apoptotic cell death. Cytokine-induced apoptotic cell death m ay be a consequence of the predominance of the stimulatory process over the inhibitory process. (C) 1999 Elsevier Science Inc.