Conditional Knockout of Calcium Receptors in Bone Cells

描述（由申请人提供）：骨重塑是破骨细胞吸收与成骨细胞驱动形成相耦合的一系列事件。再吸收释放Ca2+，基质蛋白，生长因子和信号分子进入微环境。骨髓细胞响应[Ca2+]e的通量，从而启动信号级联反应。在成骨细胞中，[Ca2+]e的变化影响对基质产生和矿化、趋化和增殖至关重要的基因的表达。大量证据支持这一观点，即[Ca2+]e偶联的变化与CaRs的激活有关。已知car控制甲状旁腺激素分泌、甲状旁腺细胞生长和肾Ca2+和水处理。这些CaR生理学的方面在Ho等人1995年开发的原始（全局）CaR敲除小鼠中得到了证实。这种基因敲除是由一种靶向策略产生的，后来人们学会了这种策略，允许产生可选择拼接的car，这些car实际上在骨、骨髓、软骨、皮肤和其他细胞中表达。因此，为了理解car在骨细胞和体内骨骼稳态中的作用，需要一种不同的敲除模型——一种选择性地靶向骨细胞群（条件敲除），并且不允许形成任何膜锚定的、具有信号能力的car。我们的实验室最近开发了一种符合这些规格的新型粘接CaR鼠标。我们建议使用该模型和在特定骨启动子（1型胶原、骨钙素、骨基质、牙本质基质蛋白1）的控制下表达Cre重组酶的成骨细胞特异性Tg小鼠来开发针对不同分化阶段骨细胞群体的CaR的条件敲除。我们将验证以下假设：CaRs介导高[Ca2+]e诱导的信号传导和成骨细胞分化、基因表达和矿化的变化，CaRs在骨发育的特定时间点起作用，并且CaR激活至少部分通过Wnt/¿-catenin途径介导其作用。我们的目标是：(1)通过分析在成骨细胞发育的时间点上CaR条件缺失的小鼠，确定CaR在成骨细胞谱系中细胞生长和分化中的作用；(2)确定敲除成骨细胞中CaR的细胞和表型特征是否通过Wnt/¿-catenin信号传导介导。体内骨骼表型将通过体内显微ct、组织形态测定、代谢生化标志物、血清化学和激素参数来表征。这些动物的细胞将被培养以确定受影响的关键基因和信号通路。这些研究应该为开发靶向不同骨细胞群car的治疗方法奠定基础，作为治疗骨质疏松症、甲状旁腺功能亢进和骨转移等低骨形成和过度重塑疾病的手段。公共卫生相关性：钙感应受体是细胞膜上的分子，可感应环境中的钙水平并将此信息传递给细胞内部。钙浓度的波动作为骨细胞的信号，导致它们改变反应和活动。这项提议的目标是通过在小鼠模型中选择性地从不同类型的骨细胞中去除钙感应受体来弄清楚钙在骨细胞中的作用，从而更好地了解骨代谢和骨疾病，并找到更有效的治疗方法。

DESCRIPTION (provided by applicant): Bone remodeling is a sequence of events in which osteoclastic resorption is coupled to osteoblast-driven formation. Resorption releases Ca2+, matrix proteins, growth factors, and signaling molecules into the microenvironment. Cells in bone and marrow respond to fluxes in the [Ca2+]e which initiate signaling cascades. In osteoblasts, changes in the [Ca2+]e affect expression of genes critical for matrix production and mineralization, chemotaxis, and proliferation. Considerable evidence supports the idea that changes in the [Ca2+]e couple to the activation of CaRs. CaRs are known to control PTH secretion, parathyroid cell growth, and renal Ca2+ and water handling. These aspects of CaR physiology were confirmed in the original (global) CaR knockout mouse developed by Ho et al in 1995. This knockout was generated by a targeting strategy that was later learned to allow for the production of alternatively spliced CaRs that are in fact expressed in bone, marrow, cartilage, skin and other cells. Thus, to understand the role of CaRs in bone cells and skeletal homeostasis in vivo, a different knockout model is needed -- one that selectively targets bone cell populations (conditional knockout) and that does not allow for the formation of any membrane-anchored, signaling-competent CaRs. Our laboratory recently developed a novel floxed CaR mouse that meets these specifications. We propose to use this model and osteoblast- specific Tg mice expressing Cre recombinase under the control of specific bone promoters (type 1 collagen, osteocalcin, osterix, dentin matrix protein 1) to develop conditional knockouts of the CaR targeted to bone cell populations at different stages of differentiation. We will test the hypothesis that CaRs mediate high [Ca2+]e-induced signaling and changes in differentiation, gene expression, and mineralization in osteoblasts, that CaRs act at specific points temporally in bone development, and that CaR activation mediates its effects at least partly via the Wnt/¿-catenin pathway. Our aims are (1) to determine the role of the CaR in the growth and differentiation of cells in the osteoblastic lineage by analyzing mice with conditional deletion of the CaR at temporally different points in osteoblastic development; and (2) to determine whether cellular and phenotypic features of knocking-out the CaR in osteoblasts are mediated via Wnt/¿-catenin signaling. Skeletal phenotypes in vivo will be characterized by in vivo micro-CT, histomorphometry, biochemical markers of turnover, and serum chemistry and hormonal parameters. Cells from these animals will be cultured to determine the key genes and signaling pathways affected. These studies should lay the groundwork for developing therapeutics targeted to CaRs in different bone cell populations as a means to treat disorders of low bone formation and excessive remodeling such as osteoporosis, hyperparathyroidism, and bone metastases. PUBLIC HEALTH RELEVANCE: Calcium-sensing receptors are molecules on the membranes of cells that sense the level of calcium in the environment and communicate this information to the inside of the cell. Fluctuations in the concentration of calcium act as a signal to bone cells that cause them to change their responses and their activities. The goal of this proposal is to figure out what calcium is doing in bone cells by removing calcium-sensing receptors selectively from the different types of cells in bone in a mouse model, to better understand bone metabolism and bone diseases, and to find more effective treatments.