Submitted on June 17, 2002
Accepted on March 14, 2003
Targeting of iNOS with antisense DNA plasmid reduces cytokine-induced inhibition of osteoblastic activity
Takahiro Abe1, Hisako Hikiji2*, Wee Soo Shin3, Noboru Koshikiya2, Sei-ichi Shima2, Jumi Nakata2, Takafumi Susami1, Tsuyoshi Takato1, and Teruhiko Toyo-oka3
1 Department of Oral and Maxillofacial Surgery, University of Tokyo, Tokyo, Japan
2 Department of Oral and Maxillofacial Surgery, University of Tokyo, Tokyo, Japan; Health Service Centre, University of Tokyo, Tokyo, Japan
3 Department of Organ Pathophysiology and Internal Medicine, University of Tokyo, Tokyo, Japan; Health Service Centre, University of Tokyo, Tokyo, Japan
* To whom correspondence should be addressed. E-mail: hikiji-ora{at}h.u-tokyo.ac.jp.
Proinflammatry cytokines, tumor necrosis factor (TNF) -
combined with interleukin-1 (IL-1) -
, 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 which 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 NOS 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 (COL I), alkaline phosphatase (ALPase), osteocalcin (OSC) and Core binding factor (Cbfa1) 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.
