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.摘要我们有初步的数据显示，除了其促进成骨细胞（OB）的作用， 成纤维细胞生长因子2（FGF 2）是间充质干细胞生长的负调节因子， 细胞分化为成熟脂肪细胞（AD）。我们假设FGF 2表达的缺失导致了 间充质祖细胞从成骨细胞分化为脂肪细胞的过程。拟议的研究将 增加我们对FGF 2影响骨老化的分子机制的理解， FGF 2在骨中PTH的成骨和抗脂肪形成作用中的作用。具体目标1：确定如何 在Col3.6-GFP或aP 2-GFP遗传学中使用Fgf 2 +/+和Fgf 2-/-小鼠调节脂肪细胞表型 背景我们将检验这样的假设，即在缺乏FGF 2的情况下，骨髓祖细胞的增殖能力降低， 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 在6-8周龄的年轻成年小鼠中，并将它们与已经表现出 骨质减少。目的1A：i）确定原发性肝癌中GFP表达的时间和定量起始， 来自携带OB转基因报告基因的Fgf 2 +/+和Fgf 2-/-小鼠的骨髓基质培养物（BMSC） ii）表征GFP阳性的AD和OB潜能，和 通过FACS分析分离GFP阴性细胞，然后在不存在和存在外源性GFP的情况下培养。 FGF 2和PTH;和iii）检查基因和蛋白质表达的变化。目标1B：定义的功能 体内成骨与成脂分化过程中的FGF 2。使用在目标1A中开发的小鼠，我们将i） 评估骨密度和全身变化与骨脂肪含量是否存在相关性。 ii）检查来自整个骨和来自骨形成细胞的关键脂肪形成和成骨细胞信号传导分子的表达。 新鲜分离的骨髓;和iii）评估PTH单独或与 FGF 2对离体BMSC培养物中脂肪生成的影响。具体目标2：确定FGF 2是否是必需的 PTH介导的促成骨和抗脂肪生成因子对间充质祖细胞的影响。我们 假设FGF 2通过调节间充质中Wnt 10 b和PPARg抑制脂肪形成 祖先我们还假设，在缺乏FGF 2的情况下，PTH不能抑制间充质干细胞的生长。 祖细胞向脂肪形成分化，这是通过Runx 2调节PPARg介导的 和Wnt 10 b下游效应。目的2A：研究FGF 2缺乏调节细胞凋亡的机制。 OB或AD表型的发展。我们将确定FGF 2是否调节Wnt 10 b和PPARg 2， 活性和什么信号通路介导这在CFU-OB和CFU-AD从年轻和成年小鼠在体外。 目的2B：通过PTH和FGF 2信号转导来确定PPARg调控的转录机制。我们 将测试一种可能的机制，通过该机制，FGF 2和PTH串扰可以调节 脂肪形成是通过Runx 2和Lef-1/β-连环蛋白介导的PPARg 2启动子的控制。项目叙述。 Fgf 2基因敲除小鼠具有老年性骨质疏松症的几个特征。 骨量随年龄增长，骨形成减少和松质骨重塑， 骨髓中的成骨细胞生成和破骨细胞生成减少， 与野生型同窝仔交配。骨髓脂肪生成增加的新观察 成年和老年Fgf 2-/-与进行性骨质减少相关，表明Fgf 2-/-小鼠 代表了一个有价值的模型，以研究与年龄相关的骨丢失的机制， 成骨细胞/脂肪细胞谱系测定。血清FGF 2对PTH的反应增加 治疗骨质疏松患者和Fgf 2中响应PTH的骨形成受损， /-小鼠支持FGF 2在PTH的促成骨、抗成脂作用中的作用。 了解FGF 2在骨中的作用以及差异调节的基因， 刺激骨形成和抑制骨髓中脂肪积累，可能导致发育 用于管理与低骨量相关的疾病的有用的治疗靶点。