Action of Anabolic Factors on Bone Formation in Mice

描述(申请人提供)：我们有初步数据表明，除了促进成骨细胞(OB)功能和骨形成，成纤维细胞生长因子2(FGF2)是间充质干细胞分化为成熟脂肪细胞(AD)的负调控因子。我们假设FGF2的表达缺失会导致间质间充质祖细胞从OB分化向成脂转变。这些研究将加深我们对成纤维细胞生长因子2影响骨老化的分子机制的理解(S)，以及成纤维细胞生长因子2在甲状旁腺激素成骨和抗脂肪作用中的作用。具体目标1：确定在Col3.6-GFP或aP2-GFP遗传背景下，FGF2如何利用FGF2+/+和FGF2-/-小鼠调节脂肪细胞表型。我们将检验这一假设，即在缺乏FGF2的情况下，骨髓前体细胞选择成骨途径的能力降低。为了评估表型的年龄依赖性，我们将检查6-8周大的年轻成年小鼠，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。目的：1)检测携带OB(Col3.6-GFP)或AD(aP2-GFP或-Cyan)转基因报告的FGF2+/+和FGF2-/-小鼠原代骨髓基质细胞(BMSC)中GFP表达的时间和数量；ii)通过流式细胞仪分析分离的GFP阳性和GFP阴性细胞的AD和OB潜能，然后在没有和存在外源FGF2和PTH的情况下培养；以及iii)检测基因和蛋白表达的变化。目的1B：明确FGF2在体内成骨和成脂分化过程中的作用。使用在Aim 1A培育的小鼠，我们将评估骨骼密度的变化与全身和骨脂肪含量是否存在相关性。Ii)检测从整个骨骼和新鲜分离的骨髓中关键的成脂和成骨信号分子的表达；以及iii)评估PTH单独或与FGF2联合应用对体外培养的BMSC成脂的影响。特异性目的2：确定成纤维细胞生长因子2是否是甲状旁腺激素对间充质祖细胞的促成骨和抗脂肪生成作用的必需因子。我们假设FGF2通过调节WnT 10b和PPAR(在间充质祖细胞中)来抑制脂肪的形成。我们还假设，在缺乏FGF2的情况下，PTH不能抑制间充质祖细胞向成脂方向的分化，这是通过调节PPAR(通过Runx2和Wnt 10b下游效应)介导的。目的：研究FGF2缺乏调节OB或AD表型发育的机制。我们将确定FGF2是否调节WNT 10b和PPAR(2)的活性，以及哪些信号通路在体外调节幼年和成年小鼠的CFU-OB和CFU-AD。目的2B：确定PPAR(受PTH和FGF2信号调节)的转录机制。我们将检验这一假设，即FGF2和PTH串扰可能调节脂肪形成的一个可能机制是通过Runx2和Lef-1/(-catenin)介导的PPAR(2)启动子的控制。公共卫生相关性：FGF2缺失的小鼠具有老年性骨质疏松症的几个特征。与野生型相比，它们表现出随年龄增长的骨量减少，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-/-小鼠骨髓脂肪生成增加与进行性骨量减少相关的新观察表明，FGF2-/-小鼠是研究年龄相关性骨丢失机制和成骨/脂肪细胞谱系确定的一个有价值的模型。甲状旁腺激素治疗骨质疏松症患者的血清FGF2增加，甲状旁腺激素引起的骨形成障碍，在甲状旁腺激素的促成骨和抗脂肪作用中，FGF2的作用是支持的。了解FGF2在骨骼中的作用以及哪些基因被差异调控以刺激骨形成和抑制骨髓中的脂肪积聚，可能会导致开发有用的治疗靶点来管理与低骨量相关的疾病。

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.