Pharmacological study on NO and biopterin synthesis in osteoblastic cells

由IL-1β、肿瘤坏死因子-α和干扰素-γ组成的致炎细胞因子可诱导四氢生物蝶呤(BH_4)生物合成的限速酶GTP环水解酶I(GTP-CH)和诱导型一氧化氮合酶(INOS)的mRNA表达。首次在成骨细胞中发现了GTP-CH基因的表达。GTP-CH和iNOSmRNAs的表达分别在3h和9h达到高峰。两个基因的表达均引起BH_4和NO水平升高。GTP-CH活性抑制剂2，4-二氨基-6-羟基嘧啶的药理研究表明，BH_4参与诱导型一氧化氮合酶的活性，但不参与诱导型一氧化氮合酶…的诱导。使用核因子-kappaB的抑制剂和激活蛋白-1(AP-1)的结果表明，这两个基因对细胞因子的共同诱导是通过激活核因子-kappaB和AP-1以及细胞因子NO生成物S-亚硝基-N-乙酰基-D、L-青霉胺(SNAP)使细胞存活率降低，而细胞内转化为BH4的七叶蝶呤则使细胞存活率增加。细胞毒性检测以乳酸脱氢酶(LDH)释放和细胞凋亡性细胞死亡为指标，流式细胞仪分析表明，细胞因子诱导的细胞活性降低可能是基于细胞凋亡引起的细胞死亡，而不是像坏死那样的溶血性死亡。在Sepiapterin存在的情况下，细胞因子治疗导致DNA片段量在统计上显著减少。此外，NO清除剂2-(4-羧基苯基)-4，4，5，5-四甲基咪唑-1-氧基-3-氧化物(Carxy-PTIO)可阻断DNA断裂。这些结果表明，细胞因子诱导的细胞死亡归因于NO，并受BH_4的保护，成骨细胞对促炎细胞因子的反应既有促进NO产生的过程，又有抑制BH_4产生的过程，从而导致细胞死亡。细胞因子诱导的细胞凋亡可能是刺激过程优于抑制过程的结果。较少

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 … More 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