KROX 20 AND BONE MASS

描述(申请人提供)：Krox20和骨量。我们建议描述转录因子Krox20在成骨细胞功能调节和骨量控制中的作用。在这个项目的前一个资金周期中，我们发现Krox20的表达和转录活性被糖皮质激素(GCs)强烈抑制，这有助于抑制培养的成骨细胞中的骨钙素转录。我们建议将该项目扩展到GC诱导的骨质疏松症(GIO)问题之外，并研究Krox20在体内和体外基础成骨细胞生物学中的作用。这种作用被认为是：(I)Krox20-/-小鼠的骨小梁形成发育停止；(Ii)成熟Krox20-/-小鼠的骨小梁质量降低和骨形成率下降；(Iii)Krox20和Krox20-/-成骨细胞培养的矿化受损；(Iv)通过Wnt信号通路刺激Krox20的表达和活性；以及(V)Krox20在体内和体外的表达模式与Krox20在成骨细胞功能中的作用一致。我们将分析Krox20不足和/或成骨细胞特异性过量的小鼠的骨骼表型随年龄的变化。体内方法将包括微型计算机断层扫描(MU CT)以及组织学和血清学分析。Krox20的细胞和分子靶点将通过分析成骨细胞培养来确定，在成骨细胞培养中，Krox20的表达已因遗传操作或病毒感染而改变。体外分析将在成骨细胞表型发育的短时间间隔内进行，以确定Krox20敏感的分化阶段。Krox20的具体作用将从细胞周期进程、细胞凋亡、生化标记物的发展和促进成骨细胞表型的基因表达方面确定，包括由Krox20直接和间接通过Wnt信号通路调节的基因。最后，我们将回到对新闻局的调查。我们将在对小鼠GIO发育的详细描述(包括MU CT和细胞凋亡)的背景下，评估GCs对体内Krox20表达的影响；我们将确定Krox20病毒或重组Wnt3a治疗是否拮抗GCs在成骨细胞中的不利影响。这些研究将阐明在健康和疾病中控制成骨细胞功能和骨形成的新的调节机制，并为追求骨合成代谢开辟新的途径。

DESCRIPTION (provided by applicant): Krox20 and Bone Mass. We propose to delineate the role of the transcription factor Krox20 in the regulation of osteoblast function and bone mass control. During the previous funding cycle of this project, we discovered that Krox20 expression and transcriptional activity were strongly inhibited by glucocorticoids (GCs), contributing to the repression of osteocalcin transcription in cultured osteoblasts. We propose to expand the project beyond the problem of GC-induced osteoporosis (GIO) and study the role of Krox20 in basic osteoblast biology in vivo and in vitro. Such a role is suggested by: (i) developmental arrest of trabecular bone formation in Krox20-/- mice; (ii) low trabecular bone mass and decreased bone formation rate in mature Krox20 mice; (iii) impaired mineralization in Krox20 and Krox20-/- osteoblast cultures; (iv) stimulation of Krox20 expression and activity by the Wnt signaling pathway; and (v) expression pattern in vivo and in vitro consistent with a role for Krox20 in osteoblast function. We will analyze as a function of age the skeletal phenotype of mice with insufficient and/or osteoblast-specific excess of Krox20. In vivo approaches will include microcomputed tomography (mu CT) as well as histological and serological analyses. The cellular and molecular targets of Krox20 will be determined analyzing osteoblast cultures in which Krox20 expression has been altered by either genetic manipulations or viral infection. The in vitro analyses will be performed at short intervals during the development of the osteoblast phenotype to identify the Krox20-sensitive differentiation stage. The specific roles of Krox20 will be determined with respect to cell cycle progression, apoptosis, development of biochemical markers and expression of genes that promote the osteoblast phenotype, including genes potentially regulated directly by Krox20 and indirectly by the Wnt signaling pathway. Finally, we will return to the investigation of GIO. We will assess the effect of GCs on Krox20 expression in vivo in the context of a detailed description (including mu CT and apoptosis) of GIO development in the mouse; and, we will determine whether treatment with a Krox20 virus or recombinant Wnt3A antagonize adverse effects of GCs in osteoblasts. These studies will elucidate novel regulatory mechanisms that control osteoblast function and bone formation in health and disease, and open new avenues in the pursuit of bone anabolics.