CIRCADIAN MECHANISMS REGULATING OSTEOGENESIS

调节成骨的昼夜节律机制

• 批准号: 6954223
• 负责人: Jeffrey Martin Gimble
• 金额: $ 18.93万
• 依托单位: LSU PENNINGTON BIOMEDICAL RESEARCH CTR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-28 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6954223
• 关键词: age difference; biological models; bone; bone metabolism; circadian rhythms; gene environment interaction; gene expression; gene expression profiling; genetic promoter element; genetically modified animals; juvenile animal; laboratory mouse; luciferin monooxygenase; mature animal; osteocalcin; osteogenesis; polymerase chain reaction; protein structure function; transcription factor; transfection

DESCRIPTION (provided by applicant): The human body displays cyclic patterns of gene expression, hormone secretion, and behavioral activity reflecting the 24-hour light/dark cycle. Recent studies reveal that the PAS domain transcription factors expressed within the suprachiasmic nucleus of the hypothalamus play a critical role in establishing and regulating the body's central circadian clock. However, new evidence indicates that tissues peripheral to the central nervous system contain their own, independent circadian clocks. Clues in the literature suggest that a peripheral, "osteogenic" circadian clock exists; serum levels of osteocalcin, alkaline phosphatase, and C- telopeptide exhibit a reproducible, oscillatory diurnal pattern in human subjects and animal models. This R21 proposal seeks to confirm the existence of a "peripheral" circadian clock within bone tissue by testing the following hypotheses: (Hypothesis 1) That a peripheral clock can be found within osteogenic tissues and that members of the "clock" PAS protein family are the mechanical regulators of its timing. Aim 1 will use real time PCR to document the temporal expression profile of circadian rhythm genes in murine bone. Cohorts of young (4 wk) and older (8 wk) mice will be entrained to a 12 hr light12 hr dark cycle. Cortical and intramembranous bones will be harvested for total RNA from groups of mice at different times around the clock. The expression profile of circadian rhythm genes will be determined by quantitative real time PCR. (Hypothesis 2) That bone-specific genes can demonstrate a circadian pattern of expression. Aim 2 will examine the expression profile of the osteocalcin promoter with respect to circadian mechanisms. Goal 1 in vitro studies will co-transfect expression vectors for the circadian rhythm genes with an osteocalcin promoter/luciferase reporter construct. Luciferase activity will be determined in transfection combinations to determine if the circadian rhythm proteins enhance or inhibit expression from the osteocalcin promoter. Goal 2 in vivo studies will use mice transgenic for the osteocalcin promoter/luciferase reporter construct. Luciferin induced light emission from the intact skeleton of individual mice will be visualized at different times of day using a bioimaging chamber. Findings from this innovative proposal have clinical implications regarding the optimal timing for daily medication regimens in bone diseases and for elective orthopedic and orthodontic procedures.

描述（由申请人提供）：人体显示基因表达，激素分泌和行为活性的环状模式，反映了24小时的光/暗周期。最近的研究表明，下丘脑上核表达的PAS结构域转录因子在建立和调节人体的中央昼夜节律方面起着至关重要的作用。但是，新的证据表明，中枢神经系统的组织包含其独立的昼夜节律。文献中的线索表明，存在外围的“成骨”昼夜节律。骨钙素，碱性磷酸酶和c-替肽的血清水平在人类受试者和动物模型中表现出可再现的振荡昼夜模式。该R21提案旨在通过测试以下假设来确认骨组织中“外围”昼夜节律的存在：（假设1）在骨组织中可以发现外围时钟，并且“时钟” PAS蛋白质家族的成员是其时间的机械调节剂。 AIM 1将使用实时PCR记录鼠骨中昼夜节律基因的时间表达谱。年轻（4周）及以上（8周）小鼠的队列将被夹在12小时的12小时12小时黑暗周期中。在全天候的不同时间，将收集从小鼠组的总RNA的皮质和膜内骨骼。昼夜节律基因的表达谱将通过定量实时PCR确定。 （假设2）骨特异性基因可以证明昼夜节律的表达方式。 AIM 2将检查骨钙素启动子相对于昼夜节律机制的表达谱。目标1体外研究将与骨钙蛋白启动子/荧光素酶报道构建体共同传递昼夜节律基因的表达载体。荧光素酶活性将在转染组合中确定，以确定昼夜节律蛋白是否增强或抑制骨钙素启动子的表达。 目标2体内研究将使用小鼠转基因进行骨钙蛋白启动子/荧光素酶报告基因构建体。荧光素诱导的单个小鼠完整骨骼的光发射将在一天的不同时间使用生物成像室进行可视化。这项创新提案的发现对骨骼疾病的每日药物治疗方案以及选举性骨科和正畸手术的最佳时机具有临床意义。