WNT/BETA-CATENIN SIGNALING IN ENDOCHONDRAL OSSIFICATION

描述（由申请人提供）：Wnt蛋白是分泌的信号传导因子，在各种类型的组织组织中具有关键作用。这些蛋白质与细胞表面受体结合，并通过几种途径传递信号，包括β-连环蛋白途径。在受体活化后，细胞质β-连环蛋白转运到细胞核并与结合应答序列并调节靶基因表达的驻留转录因子（LEF或TCF）形成复合物。在最近的研究中，我们已经表明，Wnt/β-连环蛋白途径在骨骼形成过程中的生长板软骨细胞中起作用。我们发现，β-连环蛋白是细胞质中的增殖和prehypertrophic细胞，但经历了显着的核重新定位在肥大的软骨细胞。在良好的协议，启动子驱动的组成型活性LEF/TCF蛋白的表达促进软骨细胞成熟，基因的表达，如MMP-13，和基质矿化。显性负性结构阻断了这些事件。这些和其他研究结果使我们得出了我们的中心假设：Wnt/β-连环蛋白信号的激活是软骨细胞肥大和功能以及软骨内骨化的进展和完成所必需的。为了验证这一假设，我们将鉴定生长板软骨细胞表达的Wnt和LEF/TCF分子，并确定它们在软骨细胞成熟和软骨内骨化过程中的活性和功能。我们还将描述β-连环蛋白-LEF/TCF复合物调节肥大细胞基因表达的机制。将使用的方法包括：细胞培养;禽类和哺乳动物胚胎的体内分析; RNA干扰;转基因小鼠的创建和分析;启动子报告基因测试。这些结果将为以前未知的在肥大软骨细胞中起作用并控制其功能和在软骨内骨化中的作用的途径提供重要的线索。这些数据还将建议未来的研究目标，以确定Wnt/β-连环蛋白信号传导在软骨和骨病理学中的可能参与，这是基于Writ信号传导在其他组织和器官病理学中的既定作用的假设。

DESCRIPTION (provided by applicant): Wnt proteins are secreted signaling factors with critical roles in various types of tissue organization. The proteins bind to cell surface receptors and transmit signals by several pathways, including the beta-catenin pathway. Upon receptor activation, cytoplasmic beta-catenin transits to the nucleus and forms complexes with resident transcription factors (LEF or TCFs) that bind response sequences and modulate target gene expression. In recent studies, we have shown that the Wnt/beta-catenin pathway operates in growth plate chondrocytes during skeletal formation. We found that beta-catenin is cytoplasmic in proliferating and prehypertrophic cells, but undergoes a dramatic nuclear re-localization in hypertrophic chondrocytes. In good agreement, promoter-driven expression of constitutive-active LEF/TCF proteins boosted chondrocyte maturation, expression of genes such as MMP-13, and matrix mineralization. Dominant-negative constructs blocked these events. These and other findings lead us to our central hypothesis: activation of Wnt/beta-catenin signaling is required for chondrocyte hypertrophy and function and for progression and completion of endochondral ossification. To test this hypothesis, we will identify Wnt and LEF/TCF molecules expressed by growth plate chondrocytes and determine their activity and function during chondrocyte maturation and endochondral ossification. We will also characterize mechanisms by which beta-catenin-LEF/TCF complexes regulates gene expression in hypertrophic cells. Approaches to be used will include: cell cultures; in vivo analyses of avian and mammalian embryos; RNA interference; creation and analysis of transgenic mice; promoter reporter tests. The results will shed important light into a previously unsuspected pathway that operates in hypertrophic chondrocytes and controls their function and roles in endochondral ossification. The data will also suggest future targets of investigation to determine the possible involvement of Wnt/beta-catenin signaling in pathologies of cartilage and bone, a presumption based on well-established roles of Writ signaling in the pathology of other tissues and organs.