Redox Signaling in Osteoblast Differentiation

描述（由申请人提供）：许多正常和异常的身体状况，如更年期、衰老、骨折等疾病会改变骨重塑的状态。成骨因子骨形态发生蛋白-2 （BMP-2）在骨重塑的微环境中高度表达。BMP-2是成骨细胞生长、分化和存活形成成熟骨所必需的。BMP-2在成骨前细胞中的表达机制和成骨细胞分化的潜在信号转导途径正在被积极地表征。我们的初步数据提供了BMP-2增加磷脂酰肌醇3激酶(PI 3k)/Akt激酶信号通路的第一个证据，该信号通路调节成骨细胞分化。此外，我们证明BMP-2通过激活两个转录因子NFKappaB和MEF-2A来调节自身的表达。此外，BMP-2在原代成骨细胞中诱导活性氧（ROS），同时增加PI 3k依赖性NADPH氧化酶活性。他汀类药物最近被证明通过诱导BMP-2表达来诱导新骨形成。我们的初步数据显示，他汀类药物刺激成骨前细胞的PI 3k /Akt信号。本研究利用成骨前细胞系和原代胎鼠颅骨细胞，验证氧化还原和PI 3k /Akt信号通过NFKappaB和MEF-2A调控BMP-2表达诱导成骨细胞分化的假说。在第一个具体目标中，我们计划研究PI 3k /Akt信号级联在调控NFKappaB和MEF-2A转录因子中的作用。在第二个具体目标中，我们将研究ROS （pi3k的下游介质）在BMP-2诱导成骨细胞分化中的作用。在具体目标3中，我们将研究PI 3k /Akt信号通路作为他汀类药物诱导BMP-2表达和成骨细胞分化的机制。为了解决这些特定的目标，将使用包括免疫沉淀、免疫印迹、免疫复合物激酶测定、电泳迁移转移测定、报告基因转染测定、腺病毒介导的突变酶基因转移和蛋白质的条件表达在内的技术。了解成骨细胞分化的信号转导途径可能会导致以骨形成不足为病理的人类疾病的治疗模式的发展。

DESCRIPTION (provided by applicant): Many normal and abnormal physical conditions, such as, menopause, aging, fractures and other diseases alter the state of bone remodeling. The osteogenic factor bone morphogenetic protein-2 (BMP-2) is highly expressed in the microenvironment of bone remodeling. BMP-2 is necessary for osteoblast growth, differentiation and survival to form mature bone. The mechanism by which BMP-2 is expressed in preosteoblasts and the underlying signal transduction pathways of osteoblast differentiation are being actively characterized. Our preliminary data provide the first evidence that BMP-2 increases phosphatidylinositol 3 kinase (PI 3 K)/Akt kinase signaling, which regulates osteoblast differentiation. Furthermore, we demonstrate that BMP-2 regulates its own expression by activating two transcription factors, NFKappaB and MEF-2A. Moreover, BMP-2 induces reactive oxygen species (ROS) in primary osteoblasts with concomitant increase in PI 3 K-dependent NADPH oxidase activity. Statins have recently been shown to induce new bone formation by inducing BMP-2 expression. Our preliminary data show that statin stimulates PI 3 K/Akt signaling in preosteoblasts. In this proposal, using preosteoblast cell line and primary fetal rat calvarial cells, we will test the hypothesis that concerted action of redox and PI 3 K/Akt signaling regulates BMP-2 expression via NFKappaB and MEF-2A to induce osteoblast differentiation. In the first specific aim, we plan to investigate the role of PI 3 K/Akt signaling cascade in the regulation of NFKappaB and MEF-2A transcription factors. In the second specific aim, we will examine the role of ROS, a downstream mediator of PI 3 K, in osteoblast differentiation in response to BMP-2. In the specific aim 3, we will study the PI 3 K/Akt signaling pathway as mechanism for statin-induced BMP-2 expression and osteoblast differentiation. To address these specific aims, techniques including immunoprecipitation, immunoblotting, immunecomplex kinase assays, electrophoretic mobility shift assay, reporter transfection assays, adenovirus-mediated gene transfer of mutant enzymes and conditional expression of proteins will be used. Understanding the signal transduction pathways of osteoblast differentiation may result in the development of therapeutic modalities for the human diseases where lack of bone formation is the pathology.