Functional analysis of nitric oxide metabolites in hard tissues

一氧化氮（NO）是细胞内和细胞间的信号分子。除了cgmp依赖性信号外，有研究表明，信号还可以通过NO和NO代谢物对生物分子进行化学修饰来传递。在本研究中，我们研究了一氧化氮及其代谢物在构成硬组织的细胞的分化和功能中的作用。首先，我们建立了他们的细胞培养模型。即，成牙细胞分化，炎症诱导的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。通过这些模型，我们得到了以下结果：磨蚀牙后，牙髓中诱导型NO合成酶（iNOS）的表达在不暴露牙髓的情况下被诱导。研究了一氧化氮对牙髓细胞生长和分化的影响。NO抑制牙髓细胞增殖，促进成牙细胞分化和矿化，提示NO参与了牙磨蚀后修复性牙本质的形成。TNF-α-诱导的离体骨吸收在inos敲除的颅骨中被强烈抑制。此外，在inos敲除的培养物中，TNF-α的存在阻碍了破骨细胞的分化。最后，我们研究了IL-1暴露后软骨细胞细胞外基质的代谢情况，发现IL-1通过激活TRAF-6和NF-κB，诱导并激活了软骨细胞内吞噬细胞型nadph氧化酶。我们已经报道了IL-1诱导软骨细胞中iNOS的表达和过氧亚硝酸盐的形成，过氧亚硝酸盐是NO和超氧化物的反应产物。目前的研究结果表明，超氧化物的来源之一是吞噬细胞型nadph氧化酶。有趣的是，细胞外和细胞内的pH值以nadph氧化酶依赖的方式降低，这反过来激活透明质酸酶并加速细胞外基质的损失。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