targeting of iNOS with antisense DNA plasmid prevents cytokine-induced inhibition of osteoblastic differentiation

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Purpose :Proinflammatry cytokines, tumor necrosis factor (TNF) -α combined with interleukin-1 (IL-1) -β, induce excessive production of nitric oxide (NO) and its cytotoxic metabolite, peroxynitrite (ONOO^-) via indueible nitric oxide synthase (iNOS) in mouse osteoblasts. In this investigation, to estimate the effects of antisense DNA of iNOS on osteoblastic activity, we produced transformed cell lines with antisense plasmid targeting the iNOS gene for long-lasting inhibition The identifications of the transformed antisense cell lines were 1) the detection of antisense transcripts, 2) the attenuated expression of iNOS protein, 3) the reduction of NOS activity and 4) the level of NO production. These cell lines targeting which showed decreased production of both NO and ONOO^-, prevented the inhibition iNOS, of osteoblastic differentiation as was assayed by the mRNA expression of alkaline phosphatase (ALPase) in the presence of proinflammatory cytokines. Present results indicate that the antisense DNA plasmid of iNOS is potent to prevent the cytokine-induced reduction of osteoblastic activity.