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Conditional Knockout of Calcium Receptors in Bone Cells

骨细胞钙受体的条件敲除

基本信息

批准号：
8461650
负责人：
DOLORES M. SHOBACK
金额：
$ 31.49万
依托单位：
NORTHERN CALIFORNIA INSTITUTE/RES/EDU
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-10 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8461650
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

项目摘要

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.

描述（由申请人提供）：骨重塑是一系列事件，其中破骨细胞吸收与成骨细胞驱动的形成相结合。吸收将 Ca2+、基质蛋白、生长因子和信号分子释放到微环境中。骨骼和骨髓中的细胞对 [Ca2+]e 通量做出反应，从而启动信号级联反应。在成骨细胞中，[Ca2+]e 的变化会影响对基质生成和矿化、趋化性和增殖至关重要的基因表达。大量证据支持 [Ca2+]e 的变化与 CaR 的激活相关的观点。 CaR 已知可控制 PTH 分泌、甲状旁腺细胞生长以及肾脏 Ca2+ 和水处理。 CaR 生理学的这些方面在 Ho 等人于 1995 年开发的原始（全局）CaR 敲除小鼠中得到了证实。这种敲除是通过靶向策略产生的，后来了解到该策略允许产生可变剪接的 CaR，而这些 CaR 实际上在骨、骨髓、软骨、皮肤和其他细胞中表达。因此，为了了解 CaR 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（条件敲除）并且不允许形成任何膜锚定、具有信号传导功能的 CaR 的模型。我们的实验室最近开发了一种符合这些规格的新型 floxed CaR 小鼠。我们建议使用该模型和在特定骨促进剂（1型胶原、骨钙素、osterix、牙本质基质蛋白1）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的骨细胞群的CaR的条件敲除。我们将检验以下假设：CaR 介导高 [Ca2+]e 诱导的信号传导以及成骨细胞分化、基因表达和矿化的变化，CaR 在骨发育过程中暂时作用于特定点，并且 CaR 激活至少部分通过 Wnt/¿-连环蛋白途径介导其作用。我们的目标是（1）通过分析在成骨细胞发育的不同时间点条件性删除 CaR 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)确定在成骨细胞中敲除CaR的细胞和表型特征是否是通过Wnt/¿-连环蛋白信号传导介导的。体内骨骼表型将通过体内显微 CT、组织形态计量学、周转生化标志物以及血清化学和激素参数来表征。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 CaR 的疗法奠定基础，作为治疗骨质疏松症、甲状旁腺功能亢进和骨转移等骨形成低和过度重塑疾病的手段。