Action of Anabolic Factors on Bone Formation in Mice

合成代谢因子对小鼠骨形成的作用

• 批准号: 8278563
• 负责人: Marja Marie Hurley
• 金额: $ 28.87万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-15 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8278563
• 关键词: Adipocytes; Adult; Affect; Age; Age-Related Bone Loss; Aging; Biological Assay; Bone Density; Bone Marrow; Bone remodeling; Cell Differentiation process; Cells; Characteristics; DEXA; Data; Dependence; Development; Disease Management; Elderly; Electrophoretic Mobility Shift Assay; Exhibits; Extramedullary; Fatty acid glycerol esters; Fibroblast Growth Factor 2; Fibroblast Growth Factor Receptors; Funding; Gene Expression; Genes; Genetic; Genotype; Health; Immunohistochemistry; In Vitro; Knockout Mice; Lead; Maintenance; Marrow; Mediating; Mesenchymal; Mesenchymal Stem Cells; Modeling; Molecular; Mus; Osteoblasts; Osteogenesis; Osteopenia; Pathway interactions; Patients; Peroxisome Proliferator-Activated Receptors; Phenotype; RNA; Regulation; Reporter; Reverse Transcriptase Polymerase Chain Reaction; Role; Senile Osteoporosis; Serum; Signal Pathway; Signal Transduction; Signaling Molecule; Site-Directed Mutagenesis; Stromal Cells; Testing; Transfection; Transgenes; Visual; Western Blotting; Work; aged; base; bone; bone mass; in vivo; lipid biosynthesis; novel; osteoblast differentiation; osteoclastogenesis; osteogenic; progenitor; promoter; protein expression; response; stem cell differentiation; therapeutic target; young adult

A. Summary. We have preliminary data showing that in addition to its role in promoting osteoblast (OB) function and bone formation, fibroblast growth factor 2 (FGF2) is a negative regulator of mesenchymal stem cell differentiation into mature adipocytes (AD). We hypothesize that loss of FGF2 expression results in a shift of stromal mesenchymal progenitors from OB differentiation towards adipogenesis. The proposed studies will increase our understanding of the molecular mechanism (s) by which FGF2 affects aging bone as well as the role of FGF2 in the osteogenic and antiadipogenic effects of PTH in bone. Specific Aim 1: Determine how FGF2 modulates the adipocyte phenotype using Fgf2+/+ and Fgf2-/- mice in Col3.6-GFP or aP2-GFP genetic backgrounds. We will test the hypothesis that in the absence of FGF2, marrow progenitors have a reduced ability to choose the osteogenic pathway. To assess the age-dependence of the phenotype, we will examine young adult mice at 6-8 weeks of age and compare them to 4-5 month old adult mice that already exhibit reduced bone mass. Aim 1A: i) determine the temporal and quantitative onset of GFP expression in primary bone marrow stromal cultures (BMSC) from Fgf2+/+ and Fgf2-/- mice harboring transgene reporters for the OB (Col3.6-GFP) or AD (aP2-GFP or -Cyan) lineage; ii) characterize the AD and OB potential of GFP positive and GFP negative cells isolated via FACS analysis and then cultured in the absence and presence of exogenous FGF2 and PTH; and iii) examine changes in gene and protein expression. Aim 1B: Define the function of FGF2 during osteogenic versus adipogenic differentiation in vivo. Using mice developed in Aim 1A, we will i) assess whether there is a correlation of changes in bone mineral density and whole body and bone fat content. ii) examine the expression of key adipogenic and osteoblast signaling molecules from whole bones and from freshly isolated marrow; and iii) assess the effects of PTH, administered to mice alone or in combination with FGF2 on adipogenesis in ex vivo BMSC cultures. Specific AIM 2: Determine whether FGF2 is a necessary factor for PTH-mediated pro-osteogenic and anti-adipogeneic effect on mesenchymal progenitor cells. We hypothesize that FGF2 inhibits adipogenesis through modulation of Wnt 10b and PPARg in mesenchymal progenitors. We also hypothesize that in the absence of FGF2, PTH is unable to inhibit mesenchymal progenitor cell differentiation towards adipogenesis and this is mediated through regulation of PPARg by Runx2 and Wnt 10b downstream effects. Aim 2A: Examine the mechanisms by which FGF2 deficiency modulates the development of the OB or AD phenotype. We will determine whether FGF2 modulates Wnt 10b and PPARg2 activity and what signaling pathways mediate this in CFU-OB and CFU-AD from young and adult mice in vitro. Aim 2B: Define the transcriptional mechanisms underlying PPARg regulation by PTH and FGF2 signaling. We will test the hypothesis that one possible mechanism through which FGF2 and PTH crosstalk may regulate adipogenesis is through Runx2 and Lef-1/-catenin mediated control of the PPARg 2 promoter. Project Narrative. The Fgf2 null mice have several characteristics of senile osteoporosis.They exhibit decreased bone mass with age, diminished bone formation and remodeling of cancellous bones, decreased osteoblastogenesis as well as osteoclastogenesis in the bone marrow compared with wild type littermates. The novel observation of increased adipogenesis in bone marrow of adult and aged Fgf2-/- associated with progressive osteopenia suggests that the Fgf2-/- mice represents a worthwhile model to study the mechanism of age related bone loss and osteoblast/adipocyte lineage determination. Increased serum FGF2 in response to PTH treatment of osteoporotic patients and impaired bone formation in response to PTH in the Fgf2- /- mice support a role for FGF2 in the pro-osteogenic, anti-adipogenic effects of PTH. Understanding the role of FGF2 in bone and the genes that are differentially regulated to stimulate bone formation and inhibit fat accumulation in bone marrow, may lead to development of useful therapeutic targets for the management of disorders associated with low bone mass.

A、总结。我们有初步数据表明，除了促进成骨细胞（OB）的作用外， 功能和骨形成，成纤维细胞生长因子 2 (FGF2) 是间充质干的负调节因子 细胞分化为成熟脂肪细胞（AD）。我们假设 FGF2 表达的丧失会导致转变 基质间充质祖细胞从 OB 分化到脂肪形成的过程。拟议的研究将 增加我们对 FGF2 影响骨骼老化的分子机制以及 FGF2 在骨中 PTH 的成骨和抗脂肪作用中的作用。具体目标 1：确定如何 FGF2 使用 Col3.6-GFP 或 aP2-GFP 遗传中的 Fgf2+/+ 和 Fgf2-/- 小鼠调节脂肪细胞表型 背景。我们将检验以下假设：在缺乏 FGF2 的情况下，骨髓祖细胞的数量减少 选择成骨途径的能力。为了评估表型的年龄依赖性，我们将检查 6-8周龄的年轻成年小鼠，并将它们与已经表现出的4-5个月大的成年小鼠进行比较 骨量减少。目标 1A：i) 确定原代细胞中 GFP 表达的时间和定量起始时间 来自携带 OB 转基因报告基因的 Fgf2+/+ 和 Fgf2-/- 小鼠的骨髓基质培养物 (BMSC) (Col3.6-GFP) 或 AD (aP2-GFP 或 -Cyan) 谱系； ii) 表征 GFP 阳性的 AD 和 OB 潜力 通过 FACS 分析分离 GFP 阴性细胞，然后在不存在和存在外源性的情况下进行培养 FGF2 和 PTH； iii) 检查基因和蛋白质表达的变化。目标 1B：定义函数 FGF2 在体内成骨与脂肪形成分化过程中。使用 Aim 1A 中开发的小鼠，我们将 i) 评估骨矿物质密度的变化与全身和骨脂肪含量是否存在相关性。 ii) 检查来自整个骨骼和来自骨骼的关键脂肪形成和成骨细胞信号分子的表达 新鲜分离的骨髓； iii) 评估单独给予小鼠或联合给予小鼠 PTH 的效果 FGF2 对离体 BMSC 培养物中脂肪生成的影响。具体目标2：确定FGF2是否是必需的 PTH 介导的间充质祖细胞促成骨和抗脂肪生成作用的因子。我们 假设 FGF2 通过调节间充质中的 Wnt 10b 和 PPARg 抑制脂肪生成 祖先。我们还假设，在缺乏 FGF2 的情况下，PTH 无法抑制间充质细胞 祖细胞向脂肪生成分化，这是通过 Runx2 调节 PPARg 介导的 和 Wnt 10b 下游效应。目标 2A：检查 FGF2 缺乏调节的机制 OB或AD表型的发展。我们将确定 FGF2 是否调节 Wnt 10b 和 PPARg2 体外年轻和成年小鼠 CFU-OB 和 CFU-AD 中的活性以及哪些信号通路介导这种活性。 目标 2B：定义 PTH 和 FGF2 信号传导调节 PPARg 的转录机制。我们 将检验 FGF2 和 PTH 串扰可能调节的一种可能机制的假设 脂肪生成是通过 Runx2 和 Lef-1/-catenin 介导的 PPARg 2 启动子的控制。项目叙述。 Fgf2缺失小鼠具有老年骨质疏松症的几个特征。它们表现出骨质疏松症的减少 随着年龄的增长，骨量减少，骨形成和松质骨重塑减少， 相比之下，骨髓中的成骨细胞生成和破骨细胞生成减少 与野生型同窝动物。骨髓中脂肪生成增加的新观察 成年和老年 Fgf2-/- 与进行性骨质减少相关，表明 Fgf2-/- 小鼠 代表了一个有价值的模型来研究与年龄相关的骨质流失的机制 成骨细胞/脂肪细胞谱系测定。 PTH 导致血清 FGF2 升高 治疗骨质疏松症患者和 Fgf2- 中 PTH 引起的骨形成受损 /-小鼠支持 FGF2 在 PTH 的促骨、抗脂肪作用中发挥作用。 了解 FGF2 在骨骼中的作用以及差异调节的基因 刺激骨骼形成并抑制骨髓中的脂肪积累，可能导致发育 治疗低骨量相关疾病的有用治疗靶点。