Action of Anabolic Factors on Bone Formation in Mice

A.总结。我们有初步数据表明，除了它在促进成骨细胞(OB)方面的作用外， 成纤维细胞生长因子2(FGF2)是间充质干细胞的负调控因子 细胞分化为成熟脂肪细胞(AD)。我们假设FGF2表达的缺失会导致移位 间质间充质祖细胞从成骨分化到成脂。拟议的研究将 加深对成纤维细胞生长因子2影响骨骼老化的分子机制的理解(S) 成纤维细胞生长因子2在甲状旁腺素成骨和抗成脂作用中的作用具体目标1：确定如何 在Col3.6-GFP或aP2-GFP基因中，FGF2通过FGF2+/+和FGF2-/-小鼠调节脂肪细胞表型 背景。我们将检验这一假设，即在缺乏FGF2的情况下，骨髓祖细胞的数量减少 选择成骨途径的能力。为了评估表型的年龄相关性，我们将检查 6-8周的年轻成年小鼠，并与4-5个月大的成年小鼠进行比较，这些小鼠已经表现出 骨量减少。目的1A：i)确定绿色荧光蛋白在原发肿瘤中表达的时间和数量 携带OB转基因报告的FGF2+/+和FGF2-/-小鼠骨髓基质细胞培养 (Col3.6-GFP)或AD(aP2-GFP或-Cyan)谱系；ii)表征GFP阳性和 用流式细胞仪分离GFP阴性细胞，然后在无外源和有外源的情况下培养 FGF2和PTH；以及iii)检测基因和蛋白质表达的变化。目标1B：确定的功能 在体内成骨和成脂分化过程中的FGF2。使用在目标1A中培育的小鼠，我们将I) 评估骨密度变化与全身和骨脂含量之间是否存在相关性。 二)研究关键的成脂和成骨信号分子在整个骨骼和 新鲜分离的骨髓；以及iii)评估甲状旁腺素单独或联合应用于小鼠的效果 成纤维细胞生长因子2对体外培养骨髓间充质干细胞成脂的影响。特定目标2：确定FGF2是否是必需的 甲状旁腺素对间充质祖细胞的促成骨和抗脂肪生成作用的因子。我们 FGF2通过调节WnT 10b和PPARg抑制间充质脂肪形成的假说 祖先。我们还假设，在缺少FGF2的情况下，PTH不能抑制间充质 Runx2调控PPARg介导的前体细胞向成脂分化 和WNT 10b的下游效应。目标2a：研究FGF2缺乏调节 OB或AD表型的发展。我们将确定FGF2是否调制WNT 10b和PPARg2 在体外幼年和成年小鼠的CFU-OB和CFU-AD中，活性以及什么信号通路介导了这一点。 目的2B：明确PTH和FGF2信号调节PPARg的转录机制。我们 将检验这样一种假设，即FGF2和PTH串扰可能通过一种可能的机制调节 脂肪的形成是通过Runx2和Lef-1/-catenin介导的PPARg2启动子调控来实现的。

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.