Action of Anabolic Factors on Bone Formation in Mice

描述（由申请人提供）：我们有初步数据显示，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 （FGF2）是间充质干细胞向成熟脂肪细胞（AD）分化的负调节因子。我们假设FGF2表达的缺失导致基质间充质祖细胞从OB分化向脂肪形成转变。所提出的研究将增加我们对FGF2影响骨骼老化的分子机制的理解，以及FGF2在骨骼中PTH的成骨和抗脂肪作用中的作用。特异性目的1：使用Col3.6-GFP或aP2-GFP遗传背景下的FGF2 +/+和FGF2 -/-小鼠，确定FGF2如何调节脂肪细胞表型。我们将验证在缺乏FGF2的情况下，骨髓祖细胞选择成骨途径的能力降低的假设。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，并将其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。目的1A: i)确定原代骨髓基质培养物（BMSC）中含有OB （Col3.6-GFP）或AD （aP2-GFP或- cyan）转基因报告基因的Fgf2+/+和Fgf2-/-小鼠中GFP表达的时间和定量开始；ii)通过FACS分析分离的GFP阳性和GFP阴性细胞，然后在缺乏和存在外源FGF2和PTH的情况下培养，表征其AD和OB电位；iii)检查基因和蛋白质表达的变化。目的1B：明确FGF2在体内成骨分化和成脂分化过程中的功能。使用Aim 1A中培养的小鼠，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)检测全骨和新鲜分离骨髓中关键的成脂和成骨信号分子的表达；iii)评估PTH单独给药或与FGF2联合给药对体外BMSC培养中脂肪生成的影响。特异性AIM 2：确定FGF2是否是甲状旁腺激素介导的间充质祖细胞促成骨和抗脂肪作用的必要因子。我们假设FGF2通过调节间质祖细胞中的Wnt 10b和PPAR(来抑制脂肪形成。我们还假设，在缺乏FGF2的情况下，PTH无法抑制间充质祖细胞向脂肪形成的分化，这是通过调控PPAR（通过Runx2和Wnt 10b的下游作用）来介导的。目的2A：研究FGF2缺乏调节OB或AD表型发展的机制。我们将在体外实验中确定FGF2是否调节Wnt 10b和PPAR(2活性，以及哪些信号通路介导CFU-OB和CFU-AD小鼠的Wnt 10b和PPAR(2活性。目的2B：明确PTH和FGF2信号调控PPAR的转录机制。我们将验证FGF2和PTH串扰调节脂肪形成的一种可能机制是通过Runx2和left -1/(-catenin)介导的PPAR(2启动子的控制。公共卫生相关性：Fgf2缺失小鼠具有老年性骨质疏松症的几个特征。与野生型幼崽相比，它们的骨量随着年龄的增长而减少，骨形成和松质骨重塑减少，骨髓中成骨细胞和破骨细胞的发生减少。成人和老年Fgf2-/-小鼠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机制和成骨细胞/脂肪细胞谱系测定的有价值的模型。骨质疏松症患者接受PTH治疗后血清FGF2升高，而FGF2 - /-小鼠接受PTH治疗后骨形成受损，这支持了FGF2在促成骨、抗PTH成脂作用中的作用。了解FGF2在骨骼中的作用，以及刺激骨骼形成和抑制骨髓脂肪积累的差异调控基因，可能会导致开发出与低骨量相关疾病管理的有用治疗靶点。

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.