CIRCADIAN MECHANISMS REGULATING OSTEOGENESIS

描述(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，反映出24小时的光/暗周期。最近的研究表明，在下丘脑视交叉上核表达的PAS结构域转录因子在建立和调节身体的中心生物钟方面起着关键作用。然而，新的证据表明，中枢神经系统周围的组织含有自己独立的生物钟。文献中的线索表明，外周的“成骨”生物钟存在；在人类受试者和动物模型中，血清骨钙素、碱性磷酸酶和C-端肽的水平表现出可重复的、振荡的昼夜模式。这项R21提案试图通过检验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计时的机械调节器。目的1利用实时定量聚合酶链式反应(Real Time PCR)记录小鼠骨骼中昼夜节律基因的时间表达谱。一群年轻(4周)和老年(8周)的小鼠将被带入12小时光照12小时的黑暗周期。全天候在不同的时间从各组小鼠的皮质骨和膜内骨中获取总RNA。昼夜节律基因的表达谱将通过实时定量聚合酶链式反应来确定。(假设2)骨骼特异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体外研究将与骨钙素启动子/荧光素酶报告载体共转染昼夜节律基因的表达载体。荧光素酶的活性将在转染组合中确定，以确定昼夜节律蛋白是增强还是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构建骨钙素启动子/荧光素酶报告结构。利用生物成像室，荧光素诱导的光发射将在一天中的不同时间显示单个小鼠完整的骨骼。这一创新方案的发现对骨科疾病的日常用药方案以及选择性矫形和正畸程序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.