Conditional Knockout of Calcium Receptors in Bone Cells

骨细胞钙受体的条件敲除

• 批准号: 8255611
• 负责人: DOLORES M. SHOBACK
• 金额: $ 33.15万
• 依托单位: NORTHERN CALIFORNIA INSTITUTE/RES/EDU
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-10 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8255611
• 关键词: Acute; Address; Adenoviruses; Affect; Alkaline Phosphatase; Animal Model; Animals; Ankylosis; Apoptosis; Biochemical Markers; Birth; Bone Development; Bone Diseases; Bone Marrow; Bone Resorption; Bone remodeling; Bromodeoxyuridine; Calcium; Calcium-Sensing Receptors; Cartilage; Cell Culture Techniques; Cell Differentiation process; Cell Proliferation; Cell membrane; Cell physiology; Cells; Cessation of life; Chemistry; Chemotaxis; Collagen Type I; Coupled; Deterioration; Development; Differentiation and Growth; Disease; Environment; Event; Excretory function; Exons; Fourier Transform; Fourier transform infrared spectrometry; Galactosidase; Gene Expression; Genes; Goals; Growth; Growth Factor; Health; Hematopoietic stem cells; Homeostasis; Hormonal; Hormones; Hypercalcemia; Hyperparathyroidism; Immunoblotting; In Situ Nick-End Labeling; In Vitro; Kidney; Knock-out; Knockout Mice; Laboratories; Learning; Length; Ligands; Longevity; Mediating; Membrane; Metabolic; Metastatic Neoplasm to the Bone; Minerals; Mitogen-Activated Protein Kinases; Modeling; Mus; Nucleotide pyrophosphatase; Osteoblasts; Osteocalcin; Osteoclasts; Osteogenesis; Osteoporosis; Parathyroid gland; Pathway interactions; Phenotype; Phospholipase; Physiological; Physiology; Population; Production; Property; Proteins; RNA; RNA Splicing; Reporter; Rickets; Role; Safety; Serum; Signal Pathway; Signal Transduction; Signaling Molecule; Signaling Pathway Gene; Skin; Staging; Staining method; Stains; Stromal Cells; System; TRANCE protein; Testing; Time; Transgenic Mice; Tumor necrosis factor receptor 11b; Water; Work; bisphosphonate; bone; bone cell; bone mass; bone metabolism; bone turnover; cell growth; cell type; dentin matrix protein 1; design; effective therapy; extracellular; immunocytochemistry; in vivo; indexing; kidney cell; meetings; mineralization; mouse model; novel; osteopontin; overexpression; phosphoric diester hydrolase; prevent; promoter; recombinase; response; skeletal; therapeutic target; tomography; vector; vector control

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.

描述（由申请人提供）：骨重建是一系列事件，其中骨吸收与成骨细胞驱动的形成相结合。再吸收将Ca 2+、基质蛋白、生长因子和信号分子释放到微环境中。骨和骨髓中的细胞响应于[Ca 2 +]e中的流动，其启动信号级联。在成骨细胞中，[Ca 2 +]e的变化影响对基质产生和矿化、趋化性和增殖至关重要的基因的表达。大量证据支持[Ca 2 +]e的变化与汽车的激活相关联的观点。已知汽车控制PTH分泌、甲状旁腺细胞生长以及肾Ca 2+和水处理。这些方面的CaR生理学在Ho等人于1995年开发的原始（全球）CaR敲除小鼠中得到证实。这种敲除是通过一种靶向策略产生的，后来人们了解到该策略可以产生选择性剪接的汽车，这些CaR实际上在骨骼、骨髓、软骨、皮肤和其他细胞中表达。因此，为了了解汽车在体内骨细胞和骨骼稳态中的作用，需要一种不同的敲除模型-一种选择性靶向骨细胞群体（条件性敲除）并且不允许形成任何膜锚定的，具有信号传导能力的汽车的模型。我们的实验室最近开发了一种新的floxed CaR小鼠，符合这些规格。我们建议使用该模型和在特定骨启动子（1型胶原、骨钙素、osterix、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小鼠来开发靶向不同分化阶段的骨细胞群体的CaR的条件性敲除。我们将检验以下假设：汽车介导高[Ca 2 +] 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;汽车在骨发育中的特定时间点起作用; CaR激活至少部分通过Wnt/<$-连环蛋白途径介导其作用。我们的目的是（1）通过分析成骨细胞发育过程中不同时间点条件性缺失CaR的小鼠，确定CaR在成骨细胞系细胞生长和分化中的作用;（2）确定成骨细胞中敲除CaR的细胞和表型特征是否通过Wnt/<$-连环蛋白信号转导介导。将通过体内显微CT、组织形态计量学、转换的生化标志物以及血清化学和激素参数来表征体内Skeleton表型。将培养这些动物的细胞，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中汽车的治疗方法奠定基础，作为治疗低骨形成和过度重塑疾病（如骨质疏松症、甲状旁腺功能亢进和骨转移）的一种手段。公共卫生关系：钙敏感受体是细胞膜上的分子，它能感知环境中的钙水平，并将此信息传达给细胞内部。钙浓度的波动作为骨细胞的信号，导致它们改变它们的反应和活动。该提案的目标是通过在小鼠模型中选择性地从骨中不同类型的细胞中去除钙敏感受体来弄清楚钙在骨细胞中的作用，以更好地了解骨代谢和骨疾病，并找到更有效的治疗方法。