Action of Anabolic Factors on Bone Formation in Mice

描述（由申请人提供）：我们的初步数据显示，除了促进成骨细胞（OB）功能和骨形成的作用外，成纤维细胞生长因子2（FGF 2）是间充质干细胞分化为成熟脂肪细胞（AD）的负调节因子。我们推测FGF 2表达的缺失导致间质间充质祖细胞从OB分化向脂肪形成的转变。拟议的研究将增加我们对FGF 2影响骨老化的分子机制以及FGF 2在骨中PTH的成骨和抗脂肪形成作用中的作用的理解。具体目的1：使用Col 3.6-GFP或aP 2-GFP遗传背景中的Fgf 2 +/+和Fgf 2-/-小鼠确定FGF 2如何调节脂肪细胞表型。我们将测试的假设，在没有FGF 2，骨髓祖细胞有一个降低的能力，选择成骨途径。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的转基因报告基因的Fgf 2 +/+和Fgf 2-/-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，（Col3.6-GFP）或AD（aP 2-GFP或-Cyan）谱系; ii）第二阶段表征通过FACS分析分离的GFP阳性和GFP阴性细胞的AD和OB潜能，然后在不存在和存在外源FGF 2的情况下培养和PTH;和iii）检测基因和蛋白质表达的变化。目的1B：明确FGF 2在体内成骨与成脂分化过程中的功能。使用目标1A中开发的小鼠，我们将i）评估骨矿物质密度和全身变化与骨脂肪含量之间是否存在相关性。ii）检查来自全骨和来自新鲜分离的骨髓的关键脂肪形成和成骨细胞信号传导分子的表达;和iii）评估单独或与FGF 2组合施用于小鼠的PTH对离体BMSC培养物中脂肪形成的影响。具体目标2：确定FGF 2是否是PTH介导的对间充质祖细胞的促成骨和抗成脂作用的必要因子。我们假设FGF 2通过调节Wnt 10 b和PPAR（在间充质祖细胞中）抑制脂肪形成。我们还假设，在缺乏FGF 2的情况下，PTH不能抑制间充质祖细胞向脂肪形成的分化，并且这是通过调节PPAR（通过Runx 2和Wnt 10 b下游效应）介导的。目的2A：研究FGF 2缺乏调节OB或AD表型发生的机制。我们将确定FGF 2是否调节Wnt 10 b和PPAR β 2活性，以及在体外，什么样的信号通路介导来自年轻和成年小鼠的CFU-OB和CFU-AD中的Wnt 10 b和PPAR β 2活性。目的2B：定义PPAR的转录机制（通过PTH和FGF 2信号调节）。我们将检验这样的假设，即FGF 2和PTH相互作用可能调节脂肪形成的一种可能机制是通过Runx 2和Lef-1/β-连环蛋白介导的对PPAR β 2启动子的控制。公共卫生相关性：Fgf 2缺失小鼠具有老年性骨质疏松症的几个特征。与野生型同窝仔相比，它们表现出骨量随年龄增加而减少、骨形成减少和松质骨重塑、骨髓中成骨细胞生成减少以及破骨细胞生成减少。新的观察结果表明，成年和老年Fgf 2-/-小鼠骨髓中脂肪生成增加与进行性骨量减少相关，这表明Fgf 2-/-小鼠代表了一个值得研究的模型，年龄相关的骨丢失和成骨细胞/脂肪细胞谱系确定的机制。在Fgf 2- /-小鼠中，响应于PTH治疗而增加的血清FGF 2和响应于PTH而受损的骨形成支持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以刺激骨形成并抑制骨髓中脂肪积累的基因，可能会导致开发用于管理与低骨量相关的疾病的有用治疗靶点。

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.