NITRIC OXIDE, OSTEOCLASTS AND METABOLIC BONE DISEASE

一氧化氮、破骨细胞和代谢性骨疾病

• 批准号: 6534426
• 负责人: Philip A Osdoby
• 金额: $ 21.71万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-30 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6534426
• 关键词: CD14 molecule; RNase protection assay; bone metabolism disorder; cell differentiation; chickens; confocal scanning microscopy; gel mobility shift assay; glucocorticoids; in situ hybridization; interleukin 1; isozymes; laboratory mouse; molecular cloning; nitric oxide; nitric oxide synthase; northern blottings; nucleic acid sequence; osteoclasts; pathologic bone resorption; polymerase chain reaction

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.

描述（来自申请人的逐字描述）：骨重建是一种敏感的 调节多核细胞骨吸收的动态过程， 破骨细胞（OCs）通常紧随其后的是成骨细胞（OB）介导的骨 阵多功能信号分子一氧化氮（NO）充当 在正常细胞中重要细胞间自分泌和旁分泌信号调节剂，如 以及病理性骨建模和重建过程中， 对OB增殖/存活、OC发育和 功能NO是由L-精氨酸在NO催化的氧化反应中形成的 合成酶（NOS）同工酶，其组成性表达， 钙激活（内皮eNOS和神经元nNOS亚型），或 转录诱导（诱导型NOS同工型）对炎症反应的反应 刺激。在体外和体外，升高的NO水平有效地抑制骨吸收。 在体内，和一氧化氮对OC的作用，像其他细胞一样，涉及cGMP依赖性 机制NO对OC可能的自分泌作用也开始受到关注。 因为现在已经确定OC表达iNOS和eNOS两者。 虽然鸟类OC表达iNOS信使RNA（mRNA）和蛋白质， 炎性细胞因子或LPS不诱导iNOS mRNA或蛋白在真实的 成熟的OCs，而钙水平升高。与OC相比，LPS和 炎性细胞因子在许多其他细胞类型中诱导iNOS和NO，包括 禽类非吸收性骨髓源性巨细胞（MAGC）。所以我们 假设随着OC分化为完全功能性吸收 在感受态细胞中，INOS表达的调节被修改，并且这种变化 iNOS调节对正常和病理性OC中OC功能的影响 功能修订后的更新申请的目标是开始 了解这种独特的OC iNOS所涉及的细胞和分子机制 调控提出了以下具体目标：（1）确定 细胞因子和LPS介导的OC iNOS调节的变化是 OC分化过程中信号通路的改变。作为 作为这一目标的一部分，我们将比较鸡、小鼠和人的OC发育 （2）鉴定启动子调控序列和转录因子 其介导（a）钙、PMA和 与成熟的再吸收OC相比，MAGC中的炎性因子，和（B）MAGC中的炎性因子， 粘附后炎症介导iNOS基因诱导的抑制 MACs对矿化骨基质的作用;（3）分析其信号转导机制 NO通过这种方式改变OC活性。这些研究预计将揭示新的 正常骨重建机制的方面，并有可能提供洞察力 例如植入物松动、骨关节炎、其他 炎性骨骼疾病和骨质疏松症。