PDGF AND PI-3-KINASE IN OSTEOBLASTLIKE CELLS--PROLIFERATION AND DIFFERENTIATION

增殖分化 PDGF与成骨细胞的有丝分裂和趋化作用有关。 而PDGF与磷脂酰肌醇-3-激酶的激活有关 成纤维细胞、肝细胞和血小板中的信号转导途径 生长因子仅被证明能刺激磷脂酶C和 成骨细胞中典型的磷脂酰肌醇途径。这样做的目的是 项目是确定PI-3-Kinase是否参与了增殖 和成骨细胞在PDGF作用下的分化。小鼠 成骨样细胞MC3T3-E1将在不同的时间暴露于PDGF 浓度和时间间隔。然后这些细胞将被裂解并 免疫沉淀将与各种抗体一起进行 PI-3-Kinase途径的蛋白质。西方的印迹将被建造成 免疫沉淀法和PI-3-Kinase法分析蛋白质 以表明这些蛋白质与该信号有关 转导途径。由于蛋白质复合体在不同的 PI-3-Kinase途径的亚基，我们的目标是将这一途径映射到 成骨细胞从受体到细胞核与细胞活性。 到目前为止，我们已经发现PI-3-Kinase确实在MC3T3-E1中被激活 成骨细胞在PDGF浓度范围内 5-100 ng/m1。PDGF暴露1至10分钟可产生最大的PI-3-Kinase 活动。Wortmannin，p110亚单位的不可逆抑制物 PI-3-Kinase可抑制PDGF诱导的心肌细胞PI-3-Kinase活性。 体内和体外培养的MC3T3-E1细胞。为了确定 PI-3-Kinase在成骨细胞中的生理作用，我们正在初步研究 趋化性和碱性磷酸酶作为分化的指示物。PDGF 刺激MC3T3-E1细胞的趋化作用。当这些成骨细胞暴露在 对Wortmannin来说，趋化性显著降低，表明 PI-3-Kinase信号转导通路是启动心肌梗死的必要途径 移动。确定PI-3-Kinase是否发挥作用的实验 碱性评估对成骨细胞分化的调控作用 在Wortmanmin存在和不存在的情况下的磷酸酶目前正在 指挥。 EGF、胰岛素和钙也被发现能刺激PI-3-Kinase MC3T3-E1成骨细胞的信号转导途径。在未来，我们希望确定 这些生长控制着成骨细胞分化的各个方面。 通过PI-3-Kinase途径的因子/制剂。

Proliferation and Differentiation PDGF has been associated with mitogenesis and chemotaxis in osteoblasts. While PDGF has been linked to activation of phosphatidylinositol-3-kinase signal transduction pathway in fibroblasts, hepatocyte, and platelets, this growth factor has only been shown to stimulate phospholipase C and the canonical phosphatidylinositide pathway in osteoblasts. The purpose of this project is to determine whether PI-3-Kinase is involved in the proliferation and differentiation of osteoblasts when subjected to PDGF. Murine osteoblast-like cells, MC3T3-E1, will be exposed to PDGF at various concentrations and time intervals. These cells will then be lysed and immunoprecipitation will be performed with various antibodies against proteins of the PI-3-Kinase pathway. Western blots will be constructed to analyze the proteins of the immunoprecipitation and PI-3-Kinase assays will be performed to show that these proteins are associated with this signal transduction pathway. Since protein complexes form between the various subunits of the PI-3-Kinase pathway, our goal is to map this pathway in the osteoblasts from receptor to nucleus and cellular activity. Thus far, we have found the PI-3-Kinase is indeed activated in MC3T3-E1 osteoblasts when subjected to concentrations of PDGF ranging from 5-100ng/m1. One to ten minute PDGF exposures produce maximum PI-3-Kinase activity. Wortmannin, an irreversible inhibitor of the p110 subunit of PI-3-Kinase, was found to inhibit PDGF-induced PI-3-Kinase activity of MC3T3-E1 cells both in vivo and in vitro. In order to determine the physiological role of PI-3-Kinase in osteoblasts, we are initially examining chemotaxis and alkaline phosphatase as indicators of differentiation. PDGF stimulates chemotaxis in MC3T3-E1 cells. When these osteoblasts are exposed to Wortmannin, chemotaxis is significantly reduced, indicating that the PI-3-Kinase signal transduction pathway is necessary for initiation of locomotion. Experiments to determine whether PI-3-Kinase functions to regulate differentiation of osteoblasts through evaluation of alkaline phosphatasein the presence and absence of Wortmanmin are presently being conducted. EGF, insulin, and calcium have also been found to stimulate the PI-3-Kinase pathway in MC3T3-E1 osteoblasts. In the future, we hope to determine what aspects of osteoblast differentiation are controlled by these growth factors/agents via the PI-3-Kinase pathway.