Fluid shear stress maintains vascular homeostasis by influencing endothelial gene expression. One mechanism by which shear stress achieves this is through the induction of transcription factors including Krü ppel-like factor 2 (KLF2). We have previously reported that a 62-bp region of the KLF2 promoter is responsible for its shear stressinduced expression via the binding of nuclear factors. In this study, we find that the 62-bp shear stress response region contains a 30-bp tripartite palindrome motif. Electrophoretic mobility supershift and chromatin immunoprecipitation assays demonstrate that PCAF (P-300/ cAMP-response element-binding protein-binding protein-associated factor)) and heterogeneous nuclear ribonucleoprotein D bind this region as components of the shear stress regulatory complex. We have also characterized a PI3K-dependent/Akt-independent pathway responsible for shear stress-induced KLF2 nuclear binding, promoter activation, and mRNA expression. Furthermore, the shear stress response region of the KLF2 promoter was specifically immunoprecipitated by antibodies against acetylated histones H3 and H4 in shear-stressed but not static hemangioendothelioma cells. The acetylation of these histones was blocked by PI3K inhibition. Finally, we have found that KLF2 increases endothelial nitric-oxide synthase expression in murine endothelial cultures, an effect that is also blocked by PI3K inhibition. These results define the DNA regulatory element, signal transduction pathway, and molecular mechanism activating the flow-dependent expression of a vital endothelial transcription factor.Mechanical forces influence endothelial phenotypes through signal transduction and gene activation. Hemodynamic shear stress, the frictional force that results from viscous blood flow (1), is of primary importance to the endothelium. It stimulates an adaptive response to generate antioxidant, anti-proliferative, anti-apoptotic, and anti-atherosclerotic patterns of gene expression (2, 3).One of the most important factors in this response is the level of nitric oxide (NO) 1 production. In addition to being a potent vasodilator (4), NO inhibits inflammation (5), smooth muscle cell proliferation (6), platelet aggregation (7), and endothelial cell apoptosis (8). NO levels are increased, in part, through the activation of endothelial nitric-oxide synthase (eNOS) by phosphorylation on Ser 1177 via the pro-survival phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which plays an integral role in the response to fluid shear stress (9 -11). It is also responsible for the induction of eNOS mRNA by shear stress (12). Although Akt is the canonical immediate target of PI3K activation, PI3K-dependent/Akt-independent shear stress signal transduction pathways have been reported (13,14). However, little is known regarding the effectors of the PI3K mechanotransduction pathway or the molecular mechanisms underlying the signaling events from shear stress to gene expression in endothelial cells.The application of shear stress up-regulates mRNA levels ...

Fluid shear stress is crucial for maintenance of a properly functioning endothelium. In this study we demonstrate that the KLF2 transcription factor is greatly induced by pulsatile shear stress in murine microvascular endothelial cells. The promoter elements responsible for the induction were studied by transfection with luciferase-reporter plasmids including the 5' flanking region of the murine KLF2 gene. Deletion analysis reveals that the responses are regulated by a region from -157 to -95 bp from the start site of transcription. Furthermore, shear stress induces specific nuclear binding within this region. These results define a novel shear stress response region that is highly conserved between mouse and human homologs.

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. Antinucleolin 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.

The Na,K-ATPase transports three sodium ions out of the cell and two potassium ions into the cell using ATP hydrolysis for energy. The ion gradient formed by the Na,K-ATPase contributes to the resting membrane potential, maintains cellular excitability and is important for glucose and amino acid uptake in the cell. The α1 catalytic isoform is expressed in virtually all cell types. We have previously examined cardiac physiology of mice lacking one copy of the α1 isoform gene of the Na,K-ATPase. The observation of reduced cardiac contractility in the α1 heterozygous mice was unexpected since mice heterozygous for the α2 isoform displayed enhanced cardiac contractility similar to what would be observed with cardiac glycoside treatment. We further examined hearts from α1 heterozygous mice to identify genomic responses to reduced Na,K-ATPase capacity. Using microarray analyses, we identified groups of genes whose expressions were perturbed in the α1 heterozygous hearts compared to wild-type. Known functional relationships of these genes suggest that multiple biological pathways are altered by α1 hemizygosity including activation of the renin-angiotensin system, changes in genes of energy metabolism and transport and elevated brain natriuretic peptide. This suggests that Na,K-ATPase α1 isoform activity may be required in numerous cellular processes.