Redox Signaling in Osteoblast Differentiation

描述(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建的状态。成骨因子骨形态发生蛋白-2(BMP-2)在骨重建的微环境中高表达。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在成骨前细胞中表达的机制以及成骨细胞分化的潜在信号转导途径正在被活跃地描述。我们的初步数据首次证明BMP-2增加了调节成骨细胞分化的磷脂酰肌醇3激酶(PI 3K)/Akt激酶信号。此外，我们还证明了BMP-2通过激活两个转录因子NFKappaB和MEF-2A来调节自己的表达。此外，BMP-2诱导原代成骨细胞中的活性氧(ROS)，伴随着PI 3 K依赖的NADPH氧化酶活性的增加。他汀类药物最近被证明通过诱导BMP-2的表达来诱导新骨形成。我们的初步数据显示，他汀类药物能刺激前成骨细胞的PI-3K/Akt信号传导。在这项研究中，我们将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信号协同作用通过NFKappaB和MEF-2A调节BMP-2表达以诱导成骨细胞分化的假设。在第一个特定目标中，我们计划研究PI3K/Akt信号通路在NFKappaB和MEF-2A转录因子调控中的作用。在第二个特定目标中，我们将研究PI3K的下游中介ROS在BMP-2反应的成骨细胞分化中的作用。在具体目标3中，我们将研究PI3K/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的，将使用免疫沉淀、免疫印迹、免疫复合体激酶分析、电泳迁移率改变分析、报告基因转染法、腺病毒介导的突变酶基因转移和蛋白质的条件表达等技术。了解成骨细胞分化的信号转导途径可能有助于开发治疗以缺乏骨形成为病理基础的人类疾病的方法。

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.