Regulation of the KLF2 Transcription Factor Gene in Endothelial Cells by Fluid Shear Stress

摘要

KLF2 is a mammalian zinc-finger transcription factor that is required for a number of normal embryonic processes, including lung development, adipocyte formation, T cell maturation and vasculogenesis. It is also expressed in a limited number of adult tissues, including vascular endothelial cells, where the KLF2 gene is specifically induced by fluid shear stress. This is important in light of the fact that shear stress promotes vascular health and protects endothelial cells from the inflammatory processes of atherosclerosis. These studies describe the promoter element/architecture, trans-activating factors, signal transduction pathway and sensor that transduce the physical force of fluid shear stress into upregulation of KLF2 expression. Luciferase reporter deletion analyses demonstrated that the shear stress response region of the promoter is an AT-rich tripartite palindrome motif. Electrophoretic mobility shift assays show that this region undergoes flow-induced increases in nuclear binding. Through combinations of DNA affinity chromatography/mass spectroscopy, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays, three factors were identified that bind the promoter in a shear stress-specific manner: P300/CBP-associated factor, heterogeneous nuclear ribonuclear protein D, and nucleolin. Through the use of a pharmacological inhibitor of the phosphatidylinositol 3-kinase (PI3K) pathway, LY294002, we have shown that these factors bind the promoter as components of a PI3K-dependent chromatin remodeling pathway the results in histone acetylation and opening of the chromatin to the general transcription machinery. Interestingly, constitutively active and dominant-negative mutants of the Akt kinase indicated that this process is independent of Akt. Extensive studies of nucleolin demonstrated that it interacts directly with the PI3K kinase and is absolutely required for the induction of KLF2 expression by fluid flow, as determined by RNAi technology. In addition, we identified the vascular endothelial growth factor receptor-2 as a mechanosensor that is required for full activation of the KLF2 promoter in response to flow. Functionally, we have also verified that KLF2 upregulates expression of the endothelial nitric oxide synthase gene, an important mediator of endothelial and vascular homeostasis. These studies describe a full mechanotransduction pathway, from plasma membrane to promoter element, by which fluid shear stress activates gene expression to promote vaso-protective endothelial phenotypes.