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: 8073004
• 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 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8073004
• 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

DESCRIPTION (provided by applicant): 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 PPAR( 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 PPAR( 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 PPAR(2 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 PPAR( 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 PPAR(2 promoter. PUBLIC HEALTH RELEVANCE: 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.

描述（申请人提供）：我们的初步数据显示，成纤维细胞生长因子2（FGF2）除了具有促进成骨细胞（OB）功能和骨形成的作用外，还是间充质干细胞分化为成熟脂肪细胞（AD）的负调节因子。 ）。我们假设 FGF2 表达缺失导致基质间充质祖细胞从 OB 分化转向脂肪形成。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的理解。具体目标 1：使用 Col3.6-GFP 或 aP2-GFP 遗传背景中的 Fgf2+/+ 和 Fgf2-/- 小鼠确定 FGF2 如何调节脂肪细胞表型。我们将检验以下假设：在缺乏 FGF2 的情况下，骨髓祖细胞选择成骨途径的能力会降低。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骨量减少的 4-5 个月大的成年小鼠进行比较。目标 1A：i) 确定来自携带 OB (Col3.6-GFP) 或 AD (aP2) 转基因报告基因的 Fgf2+/+ 和 Fgf2-/- 小鼠的原代骨髓基质培养物 (BMSC) 中 GFP 表达的时间和定量起始时间-GFP或-Cyan)谱系； ii) 表征通过 FACS 分析分离的 GFP 阳性和 GFP 阴性细胞的 AD 和 OB 潜力，然后在不存在和存在外源 FGF2 和 PTH 的情况下培养； iii) 检查基因和蛋白质表达的变化。目标 1B：定义 FGF2 在体内成骨与脂肪分化过程中的功能。使用目标 1A 中开发的小鼠，我们将 i) 评估骨矿物质密度与全身和骨脂肪含量的变化是否存在相关性。 ii) 检查来自整个骨骼和新鲜分离的骨髓的关键脂肪形成和成骨细胞信号分子的表达； iii)评估单独给予小鼠或与FGF2组合给予小鼠的PTH对离体BMSC培养物中脂肪形成的影响。具体目标 2：确定 FGF2 是否是 PTH 介导的间充质祖细胞促成骨和抗脂肪生成作用的必要因子。我们假设 FGF2 通过调节间充质祖细胞中的 Wnt 10b 和 PPAR() 来抑制脂肪生成。我们还假设，在 FGF2 缺失的情况下，PTH 无法抑制间充质祖细胞向脂肪生成分化，这是通过 Runx2 对 PPAR( 的调节) 介导的和 Wnt 10b 下游效应 目标 2A：检查 FGF2 缺乏调节的机制。我们将确定 FGF2 是否调节 Wnt 10b 和 PPAR(2 活性，以及​​在体外年轻和成年小鼠的 CFU-OB 和 CFU-AD 中介导这种作用的信号通路。目标 2B：定义转录机制潜在的 PPAR（受 PTH 和 FGF2 信号传导调节。我们将检验这一假设，即 FGF2 和 PTH 相互作用调节脂肪生成的一种可能机制是通过 Runx2 和Lef-1/(-连环蛋白介导的 PPAR(2 启动子) 控制。公共健康相关性：Fgf2 缺失小鼠具有老年骨质疏松症的几个特征。与野生型同窝动物相比，它们表现出随着年龄的增长，骨量减少、骨形成和松质骨重塑减少、骨髓中成骨细胞生成和破骨细胞生成减少。成人和老年 Fgf2-/- 骨髓中脂肪生成增加与进行性骨质减少相关的新观察表明，Fgf2-/- 小鼠是研究年龄相关骨质流失和成骨细胞/脂肪细胞谱系测定机制的一个有价值的模型。骨质疏松患者 PTH 治疗后血清 FGF2 增加，Fgf2-/- 小鼠中 PTH 导致骨形成受损，这支持了 FGF2 在 PTH 的促骨、抗脂肪作用中的作用。了解 FGF2 在骨骼中的作用以及被差异调节以刺激骨形成和抑制骨髓中脂肪积累的基因，可能会导致开发出有用的治疗靶点来管理与低骨量相关的疾病。