WNT/BETA-CATENIN SIGNALING IN ENDOCHONDRAL OSSIFICATION

描述(由申请人提供)：WNT蛋白是一种分泌型信号因子，在各种类型的组织中起着关键作用。这些蛋白质与细胞表面受体结合，通过几种途径传递信号，包括β-连环蛋白途径。当受体激活时，细胞质中的β-连环蛋白转移到细胞核，并与常驻转录因子(LEF或TCFs)形成复合体，结合反应序列并调节靶基因的表达。在最近的研究中，我们发现Wnt/β-catenin通路在生长板软骨细胞的骨骼形成过程中起作用。我们发现，在增殖和肥大前期细胞中，β-连环蛋白是胞质的，但在肥大的软骨细胞中经历了戏剧性的核重新定位。一致认为，启动子驱动的结构性活性Lef/TCF蛋白的表达促进了软骨细胞的成熟、基质金属蛋白酶-13等基因的表达和基质矿化。显性-否定结构阻止了这些事件。这些发现和其他发现引导我们得出我们的中心假设：Wnt/β-catenin信号的激活是软骨细胞肥大和功能以及软骨内成骨进展和完成所必需的。为了验证这一假说，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复合体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；鸟类和哺乳动物胚胎的体内分析；RNA干扰；转基因小鼠的创造和分析；启动子报告测试。这一结果将为揭示一条以前未知的途径提供重要线索，该途径在肥大的软骨细胞中运作，并控制它们在软骨内成骨中的功能和作用。这些数据还将建议未来的研究目标，以确定Wnt/β-catenin信号可能参与软骨和骨骼的病理，这一推定基于WRIT信号在其他组织和器官的病理中已确立的作用。

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.