CIRCADIAN MECHANISMS REGULATING OSTEOGENESIS

描述（由申请人提供）：人体表现出基因表达、激素分泌和行为活动的循环模式，反映了 24 小时的光/暗周期。最近的研究表明，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人体中央生物钟方面发挥着关键作用。然而，新的证据表明，中枢神经系统周围的组织拥有自己独立的生物钟。文献中的线索表明存在外周的“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重复的振荡昼夜模式。该 R21 提案旨在通过测试以下假设来确认骨组织内存在“外周”生物钟：（假设 1）在成骨组织内可以发现外周生物钟，并且“时钟”PAS 蛋白家族的成员是其计时的机械调节器。目标 1 将使用实时 PCR 来记录小鼠骨骼中昼夜节律基因的时间表达谱。年轻小鼠（4 周）和老年小鼠（8 周）组将接受 12 小时光照12 小时黑暗周期。全天候不同时间从小鼠组中采集皮质骨和膜内骨以获取总 RNA。昼夜节律基因的表达谱将通过实时定量PCR确定。 （假设 2）骨骼特异性基因可以表现出昼夜节律的表达模式。目标 2 将检查骨钙素启动子在昼夜节律机制方面的表达谱。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素启动子的表达。 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。荧光素诱导的单个小鼠完整骨骼的光发射将使用生物成像室在一天的不同时间进行可视化。这项创新提案的研究结果对于骨病每日用药方案以及选择性骨科和正畸手术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.