Abstract
We have previously characterized the regulation of the KLF2 transcription factor gene by describing an induction complex that binds to and regulates its promoter. In the present study, by using DNA affinity chromatography and mass spectrometry, we have identified nucleolin as an additional protein that binds to a palindromic response region in the KLF2 promoter. The presence of nucleolin on the KLF2 promoter in macrophages was verified by electrophoretic mobility shift assays. Interestingly, in mouse and human endothelial cell lines, electrophoretic mobility shift assays and chromatin immunoprecipitation analyses indicated that nucleolin binds the KLF2 promoter only upon application of fluid shear stress. Pretreatment of the endothelial cells with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), blocked the shear stress-induced binding of nucleolin to the promoter, demonstrating its PI3K-dependent regulation. Additionally, nucleolin exhibited dynamic flow-specific, PI3K-dependent alterations in size. Anti-nucleolin antibodies interacted with a 110-kDa form in static endothelial cells and with several catalytic forms that changed in abundance after the application of shear stress. Immunoprecipitation experiments demonstrated that fluid flow induced the interaction of nucleolin with the p85 regulatory subunit of PI3K. Finally, introduction of small interfering RNAs targeting the nucleolin genetic sequence selectively reduced nucleolin expression and was sufficient to block the induction of KLF2 by shear stress. These data support a general role for nucleolin in gene regulation and identify it as a novel factor involved in regulation of KLF2 expression.
Highlights
Antiapoptotic and antiproliferative factor capable of maintaining T cell quiescence [5, 6]
We identify nucleolin as an additional factor that binds to the Ϫ138/Ϫ111 region of the KLF2 promoter
We have found that the phosphatidylinositol 3-kinase (PI3K) pathway is required for nucleolin binding in endothelial cells and that nucleolin is required for induction of the KLF2 gene by fluid shear stress
Summary
Antiapoptotic and antiproliferative factor capable of maintaining T cell quiescence [5, 6]. This region, which is evolutionarily conserved between mouse and human promoters, is critical for KLF2 expression in every cell type tested far In endothelial cells, it mediates the induction of KLF2 expression by fluid shear stress through the binding of flow-influenced nuclear factors [24]. We have recently demonstrated that p300/CREB-binding protein-associated factor (PCAF) and heterogeneous nuclear ribonucleoprotein D (hnRNP-D) bind this region upon the application of fluid shear stress to a mouse endothelial cell line [18] The binding of these factors is dependent upon the activity of the phosphatidylinositol 3-kinase (PI3K) pathway and is correlated with histone H3 and H4 acetylation to facilitate chromatin remodeling. We have found that the PI3K pathway is required for nucleolin binding in endothelial cells and that nucleolin is required for induction of the KLF2 gene by fluid shear stress
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