WNT/BETA-CATENIN SIGNALING IN ENDOCHONDRAL OSSIFICATION

描述（由申请人提供）：Wnt蛋白是分泌的信号因子，在各种类型的组织组织中起关键作用。这些蛋白与细胞表面受体结合，并通过几种途径传递信号，包括-连环蛋白途径。受体激活后，细胞质β -连环蛋白转运到细胞核，与结合应答序列并调节靶基因表达的常驻转录因子（LEF或tcf）形成复合物。在最近的研究中，我们已经证明Wnt/ β -连环蛋白途径在骨骼形成过程中在生长板软骨细胞中起作用。我们发现β -连环蛋白在增殖细胞和增生性细胞中是细胞质的，但在增生性软骨细胞中经历了戏剧性的核再定位。与此一致的是，启动子驱动的组成活性LEF/TCF蛋白的表达促进了软骨细胞成熟、MMP-13等基因的表达和基质矿化。显性负性构念阻止了这些事件。这些和其他的发现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成骨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成骨过程中的活性和功能。我们还将描述β -连环蛋白- lef /TCF复合物调节肥厚细胞基因表达的机制。将采用的方法包括：细胞培养；鸟类和哺乳动物胚胎的体内分析；RNA干扰;转基因小鼠的建立与分析；启动子报告程序测试。这一结果将为研究肥大软骨细胞和控制其在软骨内成骨中的功能和作用的先前未被怀疑的途径提供重要线索。这些数据还将提示未来的研究目标，以确定Wnt/ β -catenin信号在软骨和骨骼病理中的可能参与，这一假设基于Writ信号在其他组织和器官病理中的既定作用。

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.