WNT13 & WNT13-signaling in Endothelial Cell Survival

内皮细胞（EC）在血管损伤和慢性炎症（如动脉粥样硬化）等血管疾病的发生和发展中起着关键作用。识别控制EC行为的所有因素对于设计适当的治疗方法至关重要。最近的证据表明，Wnt蛋白和Wnt信号成分可能代表了一类控制EC行为的新因素。我们和其他人已经证明，Wnt-信号通路拮抗剂（FRP家族成员）的表达在血管损伤期间增加，与血管细胞凋亡有关。我们已经证明，锂是Wnt信号通路的激动剂，在EC中诱导细胞周期阻滞和衰老样表型，并激活肿瘤抑制因子p53依赖通路。这些发现暗示锂在EC中激活了一种存活途径。此外，我们最近发现Wnt13基因在EC中表达，并被EC增殖抑制剂上调（TNFalpha, lithium）。我们的目标是研究自分泌Wnt13通路在控制EC存活中的作用及其潜在的作用机制。在目的1中，我们将研究Wnt13蛋白在控制EC存活中的功能作用。这将通过控制EC中Wnt13的表达水平，通过过度表达和低表达，以及通过跟踪结果来解决：i)在没有或存在凋亡刺激的情况下EC存活的变化，ii)通过cDNA阵列改变EC基因表达谱，以及iii)测试Wnt13是否介导锂对EC存活的影响。在目标2中，我们将确定EC中Wnt13信号通路的下游效应物。新的Ca2+依赖性wnt信号通路的激活将通过测量变化来测试i)细胞内Ca2+浓度，ii) Ca2+依赖性激酶PYK2活性和iii)在已知抑制剂或Ca2+依赖性通路效应物的显性阴性亚型存在下响应Wnt13的Wnt13靶基因表达。接下来将鉴定和表征参与wnt13信号通路的新型转录因子。在目标3中，我们将研究生存信号对Wnt13基因表达的调控。这将通过i)量化EC中响应锂和其他EC存活或凋亡诱导剂的Wnt13 mRNA水平，ii)通过表征Wnt13基因启动子/增强子序列，iii)通过鉴定介导Wnt13基因表达依赖锂调控的响应序列和转录因子来实现。

Endothelial cells (EC) play a key role in the development and progression of vascular diseases involving vascular injury and chronic inflammation such as atherosclerosis. The identification of all the factors controlling EC behavior is crucial to design appropriate therapy. Recent evidence suggest that Wnt proteins and Wnt-signaling components may represent a novel class of factors controlling EC behavior. We and others have shown that the expression of antagonists of the Wnt- signaling pathway, members of the FRP family, is increased during vascular injury, in association with vascular cell apoptosis. We have shown that lithium, an agonist of Wnt signaling pathway, induces a cell cycle arrest and a senescent-like phenotype in EC and activates the tumor suppressor p53-dependent pathway. These findings implicate lithium in the activation of a survival pathway in EC. Moreover, we recently showed that the Wnt13 gene is expressed in EC and up-regulated by inhibitors of EC proliferation (TNFalpha, lithium). Our goal is to investigate the role of an autocrine Wnt13 pathway in the control of EC survival and its underlying mechanisms of action. In aim 1, we will investigate the functional role of Wnt13 protein in the control of EC survival. This will be addressed by manipulating Wnt13 expression levels in EC, by over- and under-expression, and by following the resulting i) changes in EC survival in the absence or presence of apoptotic stimuli and ii) changes in EC gene expression profile by cDNA array, and by iii) testing whether Wnt13 mediates the effects of lithium on EC survival. In aim 2, we will identify downstream effectors of the Wnt13 signaling pathway in EC. The activation of a novel Ca2+- dependent Wnt-signaling pathway will be tested by measuring changes i) in intracellular Ca2+ concentration, ii) in the activity of the Ca2+-dependent kinase PYK2 and iii) in Wnt13- target gene expression in response to Wnt13 in the presence of known inhibitors or dominant-negative isoform of effectors of Ca2+-dependent pathways. This will be followed by the identification and characterization iv) of novel transcription factors involved in the Wnt13-signaling pathway. In aim 3, we will investigate the regulation of Wnt13 gene expression by survival signals. This will be achieved i) by quantification of Wnt13 mRNA levels in EC in response to lithium and other EC survival or apoptosis inducers, ii) by characterizing Wnt13 gene promoter/enhancer sequences and iii) by identifying the responsive sequence(s) and transcription factor(s) mediating lithium-dependent regulation of Wnt13 gene expression.