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

增殖和分化 PDGF与成骨细胞的有丝分裂和趋化性有关。 虽然PDGF与磷脂酰肌醇-3-激酶的激活有关， 成纤维细胞、肝细胞和血小板中信号转导途径， 生长因子仅显示刺激磷脂酶C， 成骨细胞中典型的磷脂酰肌醇途径。 这样做的目的 本项目旨在确定PI-3-激酶是否参与了细胞增殖 和成骨细胞的分化。 鼠 成骨细胞样细胞MC 3 T3-E1将暴露于不同浓度的PDGF， 浓度和时间间隔。 然后这些细胞将被裂解， 免疫沉淀将用各种抗 PI-3-激酶途径的蛋白质。 将构建蛋白质印迹， 分析免疫沉淀和PI-3-激酶测定的蛋白质将 来显示这些蛋白质与该信号相关 转导途径 由于蛋白质复合物的形成之间的各种 PI-3-激酶通路的亚基，我们的目标是在PI-3-激酶通路中定位该通路。 成骨细胞从受体到细胞核和细胞活性。 到目前为止，我们已经发现PI-3-激酶确实在MC 3 T3-E1中被激活， 成骨细胞，当受到浓度范围为 5-100ng/m1。 1 - 10分钟PDGF暴露产生最大PI-3-激酶 活动 Wortmannin是一种不可逆的p110亚基抑制剂， PI-3-激酶，发现抑制PDGF诱导的PI-3-激酶活性， MC 3 T3-E1细胞在体内和体外。 为了确定 PI-3-激酶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标。 PDGF 刺激MC 3 T3-E1细胞的趋化性。 当这些成骨细胞暴露在 对渥曼青霉素，趋化性显著降低，表明 PI-3-激酶信号转导通路是启动细胞凋亡所必需的。 运动 确定PI-3-激酶是否起作用以 通过评估碱性成骨细胞的分化调节 磷酸酶在Wortmanmin存在和不存在的情况下，目前正在 进行。 EGF，胰岛素和钙也被发现刺激PI-3-激酶 MC 3 T3-E1成骨细胞中的信号通路。 在未来，我们希望确定 成骨细胞分化的各个方面受这些生长的控制， 通过PI-3-激酶途径调节因子/试剂。

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.