Functional analysis of nitric oxide metabolites in hard tissues

一氧化氮(NO)是一种细胞内和细胞间的信号分子。除了依赖于cGMP的信号外，也有人认为信号是通过NO和NO代谢产物对生物分子的化学修饰来传递的。在这项研究中，我们研究了NO及其代谢产物在构成硬组织的细胞分化和功能中的作用。首先，我们建立了它们的细胞培养模型。即成牙本质细胞分化、炎症诱导破骨细胞分化和软骨细胞培养作为炎症性关节疾病的模型。利用这些模型，我们发现牙齿磨损后，牙髓中诱导型一氧化氮合酶(INOS)的表达增加。然后研究了NO对牙髓细胞生长和分化的影响。NO抑制牙髓细胞增殖，促进成牙本质细胞分化和矿化，提示NO参与了f-…。牙齿磨损后修复性牙本质形态增多。诱导型一氧化氮合酶基因敲除的颅骨可强烈抑制肿瘤坏死因子-α诱导的体外骨吸收。此外，在诱导型一氧化氮合酶基因敲除培养中，破骨细胞的分化受到抑制，野生型细胞在成骨细胞分化过程中观察到硝化核苷酸的非依赖性形成。最后，我们研究了IL-1作用后软骨细胞外基质的代谢，发现IL-1通过激活TRAF-6和NF-κB来诱导和激活软骨细胞中的吞噬细胞型NADPH-氧化酶。我们已经报道了IL-1诱导软骨细胞中诱导型一氧化氮合酶的表达和过亚硝酸根的形成，过氧亚硝酸盐是一氧化氮和超氧化物的反应产物。目前的结果表明，吞噬细胞型NADPH-氧化酶是超氧化物歧化的来源之一。有趣的是，胞外和胞内的pH以NADPH-氧化酶依赖的方式降低，这反过来又激活了透明质酸酶，加速了细胞外基质的损失。较少

Nitric oxide (NO) acts as an infra- and intercellular signaling molecule. In addition to the cGMP-dependent signaling, it has been suggested that signals are transmitted by chemical modifications of biological molecules by NO and NO metabolites. In this study, we investigated the roles of NO and its metabolites in the differentiation and function of the cells which constitute hard tissues. First, we established their cell culture models. Namely, odontoblast differentiation, inflammation-induced osteoclast differentiation, and chondrocyte culture as a model of inflammatory joint diseases. We obtained the following findings using these models Expression of inducible type of NO synthase (iNOS) was induced in the pulp after abrasion of teeth without exposure of pulp. Then we studied the effects of NO on the pulp cell growth and differentiation. NO suppressed the proliferation of pulp cells and promoted the odontoblastic differentiation and mineralization, suggesting NO is involved in the f … More ormation of reparative dentin after tooth abrasion. TNF-α-induced bone resorption in ex-vivo was strongly suppressed in the iNOS-knockout calvaria In addition, osteoclast differentiation in the presence of TNF-α was retarded in the iNOS-knockout cultures NO-dependent formation of nitrated nucleotides was observed in the course of osteockast differentiation in the wild type cells. Finally, we studied the metabolism of extracellular matrix of chondmcytes after IL-1 exposure and found that IL-1 induced and activated the phagocyte-type NADPH-oxidase in the chondrocytes through the activation of TRAF-6 and NF-κB. We had already reported that IL-1 induced the expression of iNOS and formation of peroxynitrite, a reaction product of NO and superoxide in chondrocytes. The present results indicate that one of the sources of superoxide is the phagocyte type NADPH-oxidase. Interestingly, extra- and intracellular pH was decreased in the NADPH-oxidase-dependent manner, which in turn activated hyaluronidase and accelerated the loss of extracellular matrix. Less