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: 7528585
• 负责人: Marja Marie Hurley
• 金额: $ 30.34万
• 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/7528585
• 关键词: Adipocytes; Adult; Affect; Age; Age-Related Bone Loss; Aging; Biological Assay; Bone Density; Bone Marrow; 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 Proteins; Genes; Genetic; Genotype; Green Fluorescent Proteins; Immunohistochemistry; In Vitro; Knockout Mice; Lead; Maintenance; Marrow; Mediating; Mesenchymal; Mesenchymal Stem Cells; Modeling; Molecular; Mus; Osteoblasts; Osteogenesis; Pathway interactions; Patients; Peroxisome Proliferator-Activated Receptors; Phenotype; Public Health; RNA; Regulation; Reporter; Reverse Transcriptase Polymerase Chain Reaction; Role; Senile Osteoporosis; Serum; Signal Pathway; Signal Transduction; Signaling Molecule; Site-Directed Mutagenesis; Standards of Weights and Measures; Stromal Cells; Testing; Transfection; Transgenes; Visual; Week; Western Blotting; Work; aged; base; bone; in vivo; lipid biosynthesis; novel; osteoclastogenesis; progenitor; promoter; protein expression; response; therapeutic target; young adult; Adipocytes; Adult; Affect; Age; Age-Related Bone Loss; Aging; Biological Assay; Bone Density; Bone Marrow; 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 Proteins; Genes; Genetic; Genotype; Green Fluorescent Proteins; Immunohistochemistry; In Vitro; Knockout Mice; Lead; Maintenance; Marrow; Mediating; Mesenchymal; Mesenchymal Stem Cells; Modeling; Molecular; Mus; Osteoblasts; Osteogenesis; Pathway interactions; Patients; Peroxisome Proliferator-Activated Receptors; Phenotype; Public Health; RNA; Regulation; Reporter; Reverse Transcriptase Polymerase Chain Reaction; Role; Senile Osteoporosis; Serum; Signal Pathway; Signal Transduction; Signaling Molecule; Site-Directed Mutagenesis; Standards of Weights and Measures; Stromal Cells; Testing; Transfection; Transgenes; Visual; Week; Western Blotting; Work; aged; base; bone; in vivo; lipid biosynthesis; novel; osteoclastogenesis; progenitor; promoter; protein expression; response; 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（由申请人提供）：我们有初步数据表明，除了其在促进成骨细胞功能和骨形成中的作用外，成纤维细胞生长因子2（FGF2）还是间质细胞细胞分化为成熟脂肪细胞（AD）的间质干细胞的负调节剂。我们假设FGF2表达的丧失导致基质间充质祖细胞从OB分化向脂肪形成转移。拟议的研究将增加我们对FGF2影响骨骼衰老的分子机制的理解，以及FGF2在PTH在骨骼中的成骨和抗氧化作用中的作用。具体目标1：确定FGF2如何使用FGF2+/+和FGF2 - / - 小鼠在Col3.6-GFP或AP2-GFP遗传背景中调节脂肪细胞表型。我们将检验以下假设：在没有FGF2的情况下，骨髓祖细胞的选择能力降低了。为了评估表型的年龄依赖性，我们将在6-8周龄检查年轻的成年小鼠，并将其与已经表现出降低骨骼质量的4-5个月大的成年小鼠进行比较。 AIM 1A：i）确定来自FGF2+/+和FGF2 - / - 携带OB（COL3.6-GFP）或AD（AP2-GFP或-CYAN）lineage的FGF2+/+和FGF2 - / - 小鼠的原代骨髓基质培养物（BMSC）的时间和定量发作； 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的情况下进行培养； iii）检查基因和蛋白质表达的变化。 AIM 1B：定义在体内成骨与成脂分化过程中FGF2的功能。使用在AIM 1A中开发的小鼠，我们将评估骨矿物质密度与全身和骨骼脂肪含量的变化是否存在相关性。 ii）检查整个骨骼以及新鲜分离的骨髓的关键成脂和成骨细胞信号分子的表达； iii）评估PTH的作用，单独或与FGF2结合给小鼠对过体BMSC培养物中的脂肪形成。具体目标2：确定FGF2是否是PTH介导的促骨和抗磷酸对间充质祖细胞的必要因素。我们假设FGF2通过调节Wnt 10b和PPAR（在间充质祖细胞中的调节中抑制脂肪生成。我们还假设，在没有FGF2的情况下，PTH无法抑制间质祖细胞的分化促成脂肪生成，并通过PPAR的pPAR介导了2a和Wnt的原理。 FGF2缺乏调节OB或AD表型的发展。 FGF2和PTH串扰可能调节脂肪形成是通过RUNX2和LEF-1/（ - Catenin介导的PPAR的控制（2个启动子。公共卫生相关性：FGF2无效的小鼠具有老年骨质疏松症的几种特征。与野生型同窝仔相比，它们表现出随着年龄的增长，骨形成减少，骨形成减少，骨骼的重塑，骨髓中的成骨细胞生成以及骨髓中的骨质碎屑。与进行性骨质骨相关的成人和老年FGF2 - / - 肥大中脂肪形成增加的新观察结果表明，FGF2 - / - 小鼠代表了一个有价值的模型，用于研究年龄相关的骨质流失和成骨细胞/成骨细胞/脂肪细胞谱系的机制。血清FGF2响应于PTH治疗骨质疏松患者的PTH治疗，并在FGF2- / - 小鼠中响应PTH的骨骼形成受损，这支持FGF2在PTH的亲稳态，抗促性作用中的作用。了解FGF2在骨骼中的作用和受差异调节以刺激骨骼形成和抑制骨髓中脂肪积累的基因，可能会导致发展有用的治疗靶标，以治疗与低骨骼相关的疾病。