Conditional Knockout of Calcium Receptors in Bone Cells

描述(申请人提供)：骨重建是破骨细胞吸收与成骨细胞驱动的形成相结合的一系列事件。再吸收作用会向微环境中释放钙离子、基质蛋白、生长因子和信号分子。骨骼和骨髓中的细胞对[Ca~(2+)]e中的流量做出反应，后者启动信号级联反应。在成骨细胞中，[Ca~(2+)]e的变化会影响基质生成、矿化、趋化和增殖的关键基因的表达。相当多的证据支持这样一种观点，即[Ca~(2+)]e的变化与汽车的激活有关。众所周知，CARS可以控制甲状旁腺激素的分泌、甲状旁腺细胞的生长、肾脏的钙离子和水的处理。汽车生理学的这些方面在Ho等人于1995年开发的原始(全球)汽车基因敲除小鼠中得到了证实。这种敲除是由一种靶向策略产生的，后来人们学会了这种策略，以允许生产实际上在骨骼、骨髓、软骨、皮肤和其他细胞中表达的选择性剪接CARS。因此，为了了解CARS在体内骨细胞和骨骼内稳态中的作用，需要一种不同的基因敲除模型--一种选择性地针对骨细胞群体(条件性基因敲除)的模型，并且不允许形成任何膜锚定的、具有信号功能的CARS。我们实验室最近开发了一种符合这些规格的新型车载带牙线鼠标。我们建议使用该模型和表达Cre重组酶的成骨细胞特异性TG小鼠，在特定的骨促进剂(I型胶原、骨钙素、Osterix、牙本质基质蛋白1)的控制下，开发针对不同分化阶段的骨细胞群体的CAR的条件性敲除。我们将检验这样一种假设，即CARS介导高[Ca~(2+)]e诱导的信号传递和成骨细胞分化、基因表达和矿化的变化，CARS在骨发育的特定时间点起作用，CAR激活至少部分通过Wnt/β-catenin途径介导其影响。我们的目标是(1)通过分析成骨细胞发育过程中不同时间点CAR条件性缺失的小鼠，确定CAR在成骨细胞谱系中的生长和分化中的作用；(2)确定成骨细胞中CAR基因敲除的细胞和表型特征是否通过Wnt/β-catenin信号介导。活体骨骼的表型将通过体内显微CT、组织形态计量学、周转生化标记物以及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针对不同骨细胞群的CARS的治疗药物奠定基础，作为治疗低骨形成和过度重塑的疾病的手段，如骨质疏松症、甲状旁腺功能亢进症和骨转移。与公共健康相关：钙感应受体是细胞膜上的分子，它感知环境中的钙水平，并将这一信息传达给细胞内部。钙浓度的波动对骨细胞来说是一个信号，导致它们改变自己的反应和活动。这项建议的目的是通过在小鼠模型中选择性地从不同类型的骨骼细胞中移除钙感受器，来弄清楚钙在骨骼细胞中的作用，以更好地了解骨骼代谢和骨骼疾病，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.