Targeting of iNOS with antisense DNA plasmid reduces cytokine-induced inhibition of osteoblastic activity.
Proinflammatry cytokines, tumor necrosis factor-alpha combined with interleukin-1beta, induce excessive production of nitric oxide (NO) and its cytotoxic metabolite peroxynitrite (ONOO-) via inducible nitric oxide synthase (iNOS) in murine osteoblasts. In this study, to properly estimate the effects of antisense DNA of iNOS on osteoblastic activity, we produced transformed cell lines with antisense plasmid that specifically targets the iNOS gene for potential long-lasting inhibition. Transformed antisense cell lines were identified by 1) the detection of antisense transcripts, 2) the attenuated expression of iNOS protein, 3) the reduction of NO synthase activity, and 4) the level of NO production. These cell lines targeting iNOS, which showed decreased production of both NO and ONOO-, prevented the inhibition of osteoblastic differentiation as was assayed by the mRNA expression of type I collagen, alkaline phosphatase, osteocalcin, and Core binding factor in the presence of proinflammatory cytokines. Present results indicate that the antisense DNA plasmid of iNOS is potent to reduce the cytokine-induced inhibition of osteoblastic activity.
Abe
Takahiro
T
Department of Oral and Maxillofacial Surgery, University of Tokyo, Tokyo, Japan.
Hikiji
Hisako
H
Shin
Wee Soo
WS
Koshikiya
Noboru
N
Shima
Sei-ichi
S
Nakata
Jumi
J
Susami
Takafumi
T
Takato
Tsuyoshi
T
Toyo-oka
Teruhiko
T
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002461:Cell Line, Transformed; D024042:Collagen Type I / Q000235:genetics; D016373:DNA, Antisense / Q000494:pharmacology; D015870:Gene Expression; D005819:Genetic Markers; D007375:Interleukin-1 / Q000494:pharmacology*; D051379:Mice; D009252:NADPH Dehydrogenase / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D030421:Peroxynitrous Acid / Q000276:immunology / Q000378:metabolism; D010957:Plasmids / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000906:Antibodies; D000970:Antineoplastic Agents; D024042:Collagen Type I; D016373:DNA, Antisense; D005819:Genetic Markers; D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D030421:Peroxynitrous Acid; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D009252:NADPH Dehydrogenase; D000469:Alkaline Phosphatase