Role of GSK3beta in Osteoblast Cell Cycle, Differentiat*

描述(申请人提供)：骨质疏松症是糖皮质激素(GC)治疗的一个主要副作用，用于改善自身免疫性和炎症性疾病的症状。GC治疗患者骨丢失最重要的单一机制是抑制成骨细胞骨形成。我们最近发现在培养的成骨细胞分化的承诺阶段(鹅卵石阶段)，GC发挥其抑制作用。在这一阶段，增殖持续存在，但与融合前对细胞周期的控制相比，它受到独特的调控。在这个承诺阶段，GC抑制细胞周期进程，这种抑制是通过减弱Ser9抑制的糖原合成酶3β的磷酸化(GSKbeta)来实现的。GSK3beta3在细胞增殖和分化中起关键作用。它参与了Wntβ-连环蛋白通路和生长因子激活的PI3K/Akt通路。大量研究表明，生长因子如IGF-I和FGF-2在成骨细胞分化中的作用，但GSK3β的参与还没有得到充分的研究。最近的一些报告表明Wnt信号在成骨细胞分化中起作用，但GSK3β的参与还没有得到测试。因此，我们建议研究GSK3β在成骨细胞分化相关细胞周期、成骨细胞表型发育以及GC介导的抑制这些过程中的作用。在具体目标1中，我们将研究GSK3β在PI3Kinase和Akt下游所起的作用。在具体目标2中，我们将研究GSK3β在Wnt下游所起的作用。在这两个特定的目标中，我们将测量GC处理的成骨细胞培养和未处理的成骨细胞培养中每个途径的各种成分的水平和活性。此外，为了定位直接受GC影响的步骤，我们将增强或抑制每个途径中的信号分子，并检测GC处理和未处理的培养物中GSK3β、细胞周期以及分化标志物的影响。这些研究将阐明成骨细胞生长和分化的新方面，以及糖皮质激素如何对这些过程产生不利影响。

DESCRIPTION (provided by applicant): Osteoporosis is a major adverse effect of glucocorticoid (GC) treatment for the amelioration of symptoms of autoimmune and inflammatory diseases. The most important single mechanism contributing to bone loss in GC-treated patients is inhibition of osteoblastic bone formation. We recently identified a commitment stage during osteoblast differentiation in culture (the "cobblestone" stage) in which GC exert their inhibitory effect. During this stage, proliferation persists, but is regulated uniquely compared to control of the cell cycle prior to confluence. At this commitment stage, but not earlier, GC inhibit cell cycle progression and this inhibition is mediated by attenuation of the Ser9 inhibitory phosphorylation of Glycogen Synthase Kinase 3beta(GSKbeta). GSK3beta3 plays pivotal roles in cell proliferation and differentiation. It has been implicated in both the Wnt beta - catenin pathway and the growth factor-activated PI3 kinase/Akt pathway. Numerous studies demonstrated roles for growth factors such as IGF-I and FGF-2 in osteoblast differentiation, but the involvement of GSK3beta has not been sufficiently studied. Several recent reports suggest a role for Wnt signaling in osteoblast differentiation, but again the involvement of GSK3beta has not been tested. Thus, we propose to examine the role of GSK3beta in the osteoblast differentiation-related cell cycle, in the development of the osteoblast phenotype and in GC-mediated inhibition of these processes. In Specific Aim 1, we will examine the role that GSK3beta plays down stream of PI3 Kinase and Akt. In Specific Aim 2, we will examine the role that GSK3beta plays downstream of Wnt. In both Specific Aims, we will measure levels and activities of various components of each pathway in GC-treated versus non-treated osteoblast cultures. In addition, to map the step that is directly affected by GC, we will either augmnent or suppress signaling molecules in each pathway, and examine the effects on GSK3beta, the cell cycle as well as differentiation markers in GC-treated and non-treated cultures. These studies will shed light on novel aspects of osteoblast growth and differentiation, and how these processes are adversely affected by glucocorticoids.