CIRCADIAN MECHANISMS REGULATING OSTEOGENESIS

描述（由申请人提供）：人体显示基因表达、激素分泌和行为活动的周期模式，反映了24小时光照/黑暗周期。近年来的研究表明，下丘脑视交叉上核内表达的PAS结构域转录因子在建立和调节机体中枢生物钟中起着重要作用。然而，新的证据表明，中枢神经系统周围的组织包含自己独立的生物钟。文献中的线索表明存在外周“成骨”生物钟;骨钙素、碱性磷酸酶和C端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。这个R21建议试图通过测试以下假设来证实骨组织内存在“外周”生物钟：（假设1）外周生物钟可以在成骨组织内发现，并且“时钟”PAS蛋白家族的成员是其计时的机械调节器。目的1利用真实的时间PCR技术研究小鼠骨中昼夜节律基因的时间表达谱。将年轻（4周）和年长（8周）小鼠的群组带入12小时光照12小时黑暗周期。将在24小时的不同时间从各组小鼠中收获皮质骨和膜内骨的总RNA。昼夜节律基因的表达谱将通过定量真实的时间PCR来确定。（假设2）骨特异性基因可以表现出昼夜节律的表达模式。目的2将研究骨钙素启动子的表达谱与昼夜节律机制的关系。目标1体外研究将昼夜节律基因的表达载体与骨钙素启动子/荧光素酶报告基因构建体共转染。将在转染组合中测定荧光素酶活性，以确定昼夜节律蛋白是否增强或抑制骨钙蛋白启动子的表达。 目标2体内研究将使用骨钙素启动子/荧光素酶报告基因构建体的转基因小鼠。将使用生物成像室在一天中的不同时间观察来自个体小鼠的完整骨骼的荧光素诱导的光发射。这项创新建议的发现对骨疾病和择期矫形和正畸手术的每日药物治疗方案的最佳时机具有临床意义。

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.