CIRCADIAN MECHANISMS REGULATING OSTEOGENESIS

描述（由申请人提供）：人体表现出反映24小时光/暗周期的基因表达、激素分泌和行为活动的循环模式。最近的研究表明，下丘脑视交叉上核表达的PAS结构域转录因子在人体中央生物钟的建立和调节中起着至关重要的作用。然而，新的证据表明，中枢神经系统周围的组织也有自己独立的生物钟。文献中的线索表明，存在一个外围的“成骨”生物钟；在人类和动物模型中，骨钙素、碱性磷酸酶和C-末端肽的血清水平表现出可重复的、振荡的昼夜模式。本R21提案旨在通过测试以下假设来证实骨组织中存在“外周”生物钟：（假设1）在成骨组织中可以发现外周生物钟，并且“时钟”PAS蛋白家族的成员是其定时的机械调节器。目的1将使用实时PCR记录昼夜节律基因在小鼠骨骼中的时间表达谱。年轻小鼠（4周）和老年小鼠（8周）的队列将被带入12小时的亮12小时暗周期。皮质骨和膜内骨将在24小时的不同时间从小鼠组中收集总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。目的2将研究骨钙素启动子在昼夜节律机制方面的表达谱。目标1：体外研究将用骨钙素启动子/荧光素酶报告基因构建体共转染昼夜节律基因表达载体。荧光素酶活性将在转染组合中测定，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：体内研究将使用转基因小鼠进行骨钙素启动子/荧光素酶报告基因构建。荧光素诱导的单个小鼠完整骨骼的发光将在一天的不同时间使用生物成像室进行可视化。这一创新建议的发现对骨病患者每日用药方案的最佳时机以及选择性矫形和正畸手术具有临床意义。

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.