WNT13 & WNT13-signaling in Endothelial Cell Survival

血管内皮细胞(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。确定所有控制EC行为的因素对于设计合适的治疗方法至关重要。最近的证据表明，Wnt蛋白和Wnt信号组件可能代表了一类新的控制EC行为的因素。我们和其他人已经证明，Wnt信号通路拮抗剂是FRP家族的成员，在血管损伤过程中表达增加，与血管细胞凋亡有关。我们已经证明，Wnt信号通路的激动剂锂诱导EC细胞周期停滞和衰老样表型，并激活肿瘤抑制因子P53依赖的途径。这些发现表明，锂在EC中激活了一条生存途径。此外，我们最近发现Wnt13基因在EC中表达，并被EC增殖抑制剂(TNFpha、锂)上调。我们的目标是研究Wnt13自分泌途径在控制EC存活中的作用及其潜在的作用机制。在目标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Wnt13在EC中的表达水平，通过过度和低表达，并通过以下方法来解决：i)在没有或存在凋亡刺激的情况下EC存活率的变化；ii)通过基因芯片检测EC基因表达谱的变化；以及iii)测试Wnt13是否介导了锂对EC存活率的影响。在目标2中，我们将确定Wnt13信号通路在EC中的下游效应。一条新的钙依赖的Wnt信号通路的激活将通过测量：1)细胞内钙离子浓度的变化，2)钙依赖的激酶PYK2的活性的变化，以及3)在已知的抑制剂或钙依赖的通路的显性-负效应亚型存在的情况下，Wnt13靶基因表达的变化。随后将对参与WNT13信号通路的新的转录因子进行鉴定和鉴定。在目标3中，我们将研究生存信号对Wnt13基因表达的调控。这将通过量化WNT13基因在EC中对锂和其他EC存活或凋亡诱导剂的反应水平，II)通过鉴定WNT13基因的启动子/增强子序列，以及III)通过鉴定介导锂依赖的WNT13基因表达的反应序列(S)和转录因子(S)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.