Peroxynitrite production by TNF-alpha and IL-1beta : implication for suppression of osteoblastic differentiation

1 Department of Oral and Maxillofacial Surgery, 2 Second Department of Internal Medicine, Faculty of Medicine, and 3 Health Service Center, University of Tokyo, Bunkyo-ku, Tokyo 113-8865, Japan To determine the roles of nitric oxide (NO) and its metabolite, peroxynitrite (ONOO ), on osteoblastic activation, we investigated the effects of a NO donor [ethanamine, 2,2'-(hydroxynitrosohydrazono)bis- (dNO)], an O 2 donor (pyrogallol), and an ONOO scavenger (urate) on alkaline phosphatase (ALPase) activity and osteocalcin gene expression, which are indexes of osteoblastic differentiation. dNO elevated ALPase activity in the osteogenic MC3T3-E1 cell line. The combination of dNO and pyrogallol reduced both ALPase activity and osteocalcin gene expression. Because both indexes were recovered by urate, ONOO , unlike NO itself, inhibited the osteoblastic differentiation. Furthermore, treatment with a combination of the proinflammatory cytokines tumor necrosis factor- (TNF- ) and interleukin-1 (IL-1 ) was found to yield ONOO as well as NO and O 2 . The reductions in ALPase activity and osteocalcin gene expression were also restored by urate. We conclude that ONOO produced by TNF- and IL-1 , but not NO per se, would overcome the stimulatory effect of NO on osteoblastic activity and inhibit osteoblastic differentiation. nitric oxide; osteoblasts; proinflammatory cytokines