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.
