Cell Cycle Mechanisms of PTH/PTHrP Action in Osteoblasts

描述（申请人提供）：骨重塑的生理过程受到局部和内分泌因素的严格调控，任何干扰都会导致颅面畸形、骨科疾病等各种骨骼疾病。甲状旁腺激素（PTH）已被证明在临床上可诱导骨形成。然而，甲状旁腺激素骨形成和再生的确切生物学机制尚不清楚。我们广泛的长期目标是确定PTH/PTHrP （PTH相关蛋白）作用和骨转换的分子机制。初步数据表明，细胞周期控制可能是PTHrP影响成骨细胞寿命和成骨活性的一种机制。cyclin D1的下调和JunB表达的升高表明AP-1转录因子参与了成骨细胞的细胞周期机制，并使我们假设PTHrP对cyclin D1的作用是发育阶段特异性的，并与特异性的信号转导介质相关。使用体外和体内模型，两个特定的目标将验证这一假设。在特定目标中，1周期蛋白D1启动子构建和荧光素酶测定将确定PTHrP对周期蛋白D1的影响是否依赖于成骨细胞增殖和分化阶段。我们将利用RNA干扰（RNAi）技术、显性负AP-1蛋白（TAM 67）和CDK1的过表达来确定JunB是否在调节cyclin D1表达中发挥作用。特异性目的2将利用一种新的异位小骨模型系统来验证cyclin D1是体内PTHrP信号传导的下游介质之一。最后，利用免疫组化技术，阐明PTHrP在体内成骨细胞分化的不同阶段对细胞周期蛋白D1的影响。这项研究的结果将促进我们对PTH/PTHrP在骨中的作用的认识，并有助于理解PTH的合成代谢作用。确定这些与甲状旁腺激素作用相关的机制将有可能作为治疗骨质疏松症和牙周病等对骨骼有不利影响的疾病的新治疗药物的潜在靶点。

DESCRIPTION (provided by applicant): The physiological process of bone remodeling is tightly regulated by local and endocrine factors, and any disturbance will lead to various skeletal diseases including craniofacial deformities and orthopedic disorders. Parathyroid hormone (PTH) has been shown clinically to induce bone formation. However the exact biological mechanisms of bone formation and regeneration by PTH remain elusive. Our broad, long-term goal is to identify molecular mechanisms of PTH/PTHrP (PTH related protein) action and bone turnover. Preliminary data suggest that cell cycle control may be a mechanism through which PTHrP impacts the life span and bone forming activity of osteoblasts. Down-regulation of cyclin D1 and elevated expression of JunB suggested involvement of AP-1 transcription factors to the cell cycle machinery of osteoblasts and led us to hypothesize that the action of PTHrP on cyclin D1 is developmental stage specific and associated with specific signal transduction mediators. Using in vitro and in vivo models, 2 specific aims will test this hypothesis. In specific aim 1 cyclin D1 promoter constructs and Luciferase assays will determine if PTHrP effects on cyclin D1 are dependent on osteoblast proliferation and differentiation stage. We will determine if JunB plays a role in modulating cyclin D1 expression utilizing RNA interference (RNAi) technology, over expression of dominant negative AP-1 protein (TAM 67) and CDK1. Specific Aim 2 will utilize a novel ectopic ossicle model system to verify cyclin D1 as 1 of the downstream mediators of PTHrP signaling in vivo. Finally, using immunohistochemistry, effects of PTHrP on cyclin D1 will be elucidated at various stages of osteoblast differentiation in vivo. The outcome of this investigation will advance our knowledge of PTH/PTHrP action in bone and contribute to understanding the anabolic actions of PTH. Identifying these mechanisms related to PTH action will have applicability as potential targets for new therapeutic agents for the treatment of diseases which adversely affect bone, including osteoporosis and periodontal diseases.