KROX 20 AND BONE MASS

描述（由申请人提供）：Krox20和Bone Mass。我们建议描述转录因子Krox20在成骨细胞功能调控和骨量控制中的作用。在本项目的上一个资助周期中，我们发现糖皮质激素（GCs）强烈抑制Krox20的表达和转录活性，从而抑制骨钙素在培养成骨细胞中的转录。我们建议将项目扩展到gc诱导的骨质疏松症（GIO）问题之外，研究Krox20在体内和体外基础成骨细胞生物学中的作用。Krox20-/-小鼠的骨小梁形成发育阻滞表明了这种作用；（ii）成熟Krox20小鼠骨小梁骨量低，骨形成率降低；（iii） Krox20和Krox20-/-成骨细胞培养中矿化受损；（iv）通过Wnt信号通路刺激Krox20的表达和活性；(v)体内和体外表达模式与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小鼠的骨骼表型随年龄的变化。体内方法将包括微计算机断层扫描（mu CT）以及组织学和血清学分析。Krox20的细胞和分子靶标将通过分析通过基因操作或病毒感染改变Krox20表达的成骨细胞培养物来确定。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敏感分化阶段。Krox20的具体作用将在细胞周期进程、细胞凋亡、生化标志物的发展和促进成骨细胞表型的基因表达方面确定，包括可能由Krox20直接调节和间接由Wnt信号通路调节的基因。最后，我们将回到GIO的调查。我们将在小鼠GIO发育的详细描述（包括mu CT和凋亡）的背景下评估GCs对体内Krox20表达的影响；并且，我们将确定用Krox20病毒或重组Wnt3A治疗是否能拮抗成骨细胞中GCs的不良反应。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调控机制，并为骨合成代谢的研究开辟新的途径。

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.