| Co-Investigator(Kenkyū-buntansha) |
KONDO Ayami Aichi-Gakuin University, School of Dentistry Department of Pharamcology Research, 歯学部, 助手 (70301629)
MOGI Makio Aichi-Gakuin University, School of Dentistry Department of Pharamcology Assistan, 歯学部, 講師 (00174334)
ARAI Michitsugu Aichi-Gakuin University, School of Dentistry Department of Pharamcology Assistan, 歯学部, 講師 (20097538)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
Proinflammatory cytokines, a combination of IL-1beta, TNF-alpha, and IFN-gamma, caused mRNA expression of GTP cyclohydrolase I (GTP-CH), the rate-limiting enzyme in tetrahydrobiopterin (BH_4) biosynthesis, as well as that of inducible nitric oxide synthase (iNOS) in a well-characterized osteoblastic clone MC3T3-E1 cell line. We found the expression of GTP-CH gene in osteoblasts for the first time. The expression of GTP-CH and iNOS mRNAs was found to be maximal at 3 and 9 hr, respectively. The expression of both genes elicited increases in BH_4 and NO levels. Pharmacological studies using 2, 4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP-CH activity showed that BH_4 is involved in the activity of iNOS, but not in the induction of iNOS mRNA.The results using an inhibitor of nuclear factor (NF)-kappaB and activating protein-1 (AP-1) activation suggested that coinduction of the two genes in response to cytokines occurred via activation of NF-kappaB and AP-1.
Cytokines as well as
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S-nitroso-N-acetyl-D, L-penicillamine (SNAP), an NO generator, decreased cell viability ; but sepiapterin, which was converted intracellularly to BH_4, increased it. The examination of cytotoxicity measured in terms of lactate dehydrogenase (LDH) release and apoptotic cell death assessed by flow cytometric analysis showed that cytokine-induced reduction of cell viability may be based upon cell death by apoptosis, but not lytic death as in necrosis. In the presence of sepiapterin, cytokine treatment resulted in a statistically pronounced reduction in the amount of DNA fragmentation. Furthermore, the DNA fragmentation could be blocked by 2-(4-carboxy-phenyl)-4, 4, 5, 5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO), a NO scavenger. These results suggest that cytokine-induced apoptotic cell death is attributed to NO and is protected by BH_4, and that osteoblastic cells in response to proinflammatory cytokines operate both a stimulatory process resulting in NO production and an inhibitory one resulting in BH_4 production for apoptotic cell death. Cytokine-induced apoptotic cell death may be a consequence of the predominance of the stimlatory process over the inhibitory process. Less
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