WNT13 & WNT13-signaling in Endothelial Cell Survival

内皮细胞（EC）在涉及血管损伤和慢性炎症如动脉粥样硬化的血管疾病的发生和发展中起关键作用。识别控制EC行为的所有因素对于设计适当的治疗至关重要。 最近的证据表明，Wnt蛋白和Wnt信号组件可能代表一类新的因素控制EC的行为。 我们和其他人已经表明，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，与血管细胞凋亡相关。 我们已经表明，锂，Wnt信号通路的激动剂，诱导细胞周期停滞和衰老样表型EC和激活肿瘤抑制p53依赖性途径。 这些发现暗示锂在EC中的存活途径的激活中。 此外，我们最近发现，Wnt 13基因在EC中表达，并通过EC增殖抑制剂（TNF α，锂）上调。 我们的目标是研究自分泌Wnt 13通路在EC存活控制中的作用及其潜在的作用机制。 在目的1中，我们将研究Wnt 13蛋白在EC存活控制中的功能作用。 这将通过操纵EC中的Wnt 13表达水平、通过过度表达和表达不足、以及通过跟踪所得到的i）在不存在或存在凋亡刺激的情况下EC存活的变化和ii）通过cDNA阵列的EC基因表达谱的变化、以及iii）测试Wnt 13是否介导锂对EC存活的影响来解决。 在目标2中，我们将确定EC中Wnt 13信号通路的下游效应物。 将通过测量i）细胞内Ca 2+浓度的变化，ii）Ca 2+依赖性激酶PYK 2的活性的变化，和iii）在存在Ca 2+依赖性途径的效应物的已知抑制剂或显性阴性同种型的情况下响应于Wnt 13的Wnt 13靶基因表达的变化来测试新型Ca 2+依赖性Wnt信号传导途径的激活。 随后将鉴定和表征iv）参与Wnt 13信号传导途径的新型转录因子。 目的3：研究生存信号对Wnt 13基因表达的调控。 这将通过以下方式实现：i）定量EC中响应于锂和其他EC存活或凋亡诱导剂的Wnt 13 mRNA水平，ii）表征Wnt 13基因启动子/增强子序列，iii）鉴定介导Wnt 13基因表达的锂依赖性调节的响应序列和转录因子。

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.