NITRIC OXIDE, OSTEOCLASTS AND METABOLIC BONE DISEASE

描述(来自申请者的逐字记录)：骨骼重塑是一种敏感的 多核骨吸收调节的动态过程 破骨细胞(OCs)通常紧随其后的是成骨细胞(OB)介导的骨 队形。多功能信号分子，一氧化氮(NO)，作为一种 正常情况下重要的细胞间自分泌和旁分泌信号调节器，如 以及在病理性的骨骼建模和重塑过程中，没有出现 对卵巢癌的增殖/存活、卵巢癌的发展和 功能。NO是由L-精氨酸在NO催化的氧化反应中生成的 合成酶(NOS)同工酶 钙激活(内皮型eNOS和神经型nNOS亚型)，或 炎症反应中的转录诱导(诱导型一氧化氮合酶亚型) 刺激物。升高的NO水平在体外和体外都能有效地抑制骨吸收 在体内，与其他细胞不同，对OCS的作用不涉及cGMP依赖 机制。NO对OCS可能的自分泌作用也开始 检查，因为现在已经很好地确定了OCs同时表达iNOS和eNOS。 虽然禽类OCs同时表达iNOS信使RNA(MRNA)和蛋白质， 炎性细胞因子或内毒素不能诱导iNOS mRNA或蛋白 成熟的OCS，而钙水平升高则会。与OCS相比，LPS和 炎性细胞因子在许多其他类型的细胞中诱导iNOS和NO，包括 禽类非吸收骨髓源性巨细胞(MAGC)。因此，我们 假设OCS分化为全功能再吸收 感受态细胞中，iNOS表达的调节被修饰，这种变化 诱导型一氧化氮合酶调节对正常和病理性卵巢癌组织功能的影响 功能。修订后的续期申请的目标是开始 了解这种独特的OC iNOS涉及的细胞和分子机制 监管。提出了以下具体目标：(1)确定是否 细胞因子和内毒素介导的OC iNOS调节的变化是 OC分化过程中信号通路的变化。AS 作为这一目标的一部分，我们将比较鸡、小鼠和人类OC的发育 模型；(2)确定启动子调控序列和转录因子 介导(A)钙、PMA和PMA对iNOS基因的差异诱导 MAGCs中的炎性物质与成熟的吸收OC的比较，以及(B) 黏附后抑制炎症介导的iNOS基因诱导 MAGCs向矿化骨基质的转化；(3)信号转导机制分析 通过它，没有改变OC的活动。这样的研究有望揭示出新的 正常的骨重建机制的方面，并有可能提供洞察力 骨骼病理，如种植体松动、骨关节炎等 炎症性骨骼疾病和骨质疏松。

DESCRIPTION (Verbatim from the Applicant): Bone remodeling is a sensitively regulated dynamic process in which bone resorption by multinucleated osteoclasts (OCs) is normally closely followed by osteoblast (OB)-mediated bone formation. The multifunctional signal molecule, nitric oxide (NO), serves as an important intercellular autocrine and paracrine signal modulator in normal, as well as in pathological bone modeling and remodeling processes, and NO appears to exert profound effects on OB proliferation/survival, OC development and function. NO is formed from L-arginine in an oxidative reaction catalyzed by NO synthase (NOS) isoenzymes that are either constitutively expressed and calcium-activated (endothelial eNOS and neuronal nNOS isoforms), or transcriptionally induced (inducible NOS isoform) in response to inflammatory stimuli. Elevated NO levels potently inhibit bone resorption, both in vitro and in vivo, and NO actions on OCs, like other cells, involves a cGMP-dependent mechanism. The possible autocrine effect of NO on OCs has also begun to be examined, since it is now well established that OCs express both iNOS and eNOS. Although avian OCs express both iNOS messenger RNA (mRNA) and protein, inflammatory cytokines or LPS do not induce iNOS mRNA or protein in authentic mature Ocs, whereas elevated levels of calcium do. In contrast to Ocs, LPS and inflammatory cytokines induce iNOS and NO in many other cell types, including avian non-resorptive marrow-derived giant cells (MAGC). Therefore, we hypothesize that as OCs differentiate into fully functional resorption competent cells, the regulation of INOS expression is modified and this change in iNOS regulation impacts on OC-function in normal, as well as pathological OC function. The goal of this revised renewal application is to begin to understand the cell and molecular mechanisms involved in this unique OC iNOS regulation. The following Specific Aims are proposed: (1) to determine if the change in cytokine- and LPS-mediated OC iNOS regulation is a consequence of alterations in signaling pathways during the process of OC differentiation. As part of this aim, we will compare chicken, mouse, and human OC developmental models; (2) to identify promoter regulatory sequences and transcription factors that mediate (a) the differential induction of iNOS gene by calcium, PMA and inflammatory agents in MAGCs compared to mature resorbing OC, and (b) the inhibition of inflammatory-mediated iNOS gene induction following attachment of MAGCs to mineralized bone matrices; and (3) to analyze the signaling mechanisms by which NO alters OC activity. Such studies are anticipated to reveal new aspects of normal bone remodeling mechanisms and have potential to lend insight into skeletal pathologies such as implant loosening, osteoarthritis, other inflammatory skeletal disorders, and osteoporosis.