Abstract 303: The Shear Stress-Induced Activation of the AMP-Activated Protein Kinase Regulates FoxO1a and Angiopoietin-2 Expression in Endothelial Cells
Fluid shear stress, the frictional force generated on the luminal endothelial surface by the streaming blood, modulates endothelial cell signaling by activating protein kinases such as Akt and the AMPK. Here we assessed the influence of shear stress on the phosphorylation and downstream consequences of Akt and AMPK activation in cultured human umbilical vein endothelial cells. The application of fluid shear stress (12 dynes cm−2) to confluent human endothelial cells elicited the phosphorylation of Akt which was maintained as long as the stimulus was applied (up to 72 hours). This response was paralleled by the phosphorylation of the transcription factor FoxO1a (a reported Akt substrate), and by a decrease in FoxO1a protein levels. Indeed, while FoxO1a was clearly present in the nucleus of endothelial cells maintained under static conditions, shear stress elicited a rapid (within 30 minutes) nuclear exclusion of the transcription factor. FoxO1a remained excluded from the nucleus as long as shear stress was applied. However, neither Gab1 siRNA (which prevents the phosphorylation of Akt by fluid shear stress) nor a dominant negative (DN)-Akt mutant affected the shear stress-induced phosphorylation and degradation of FoxO1a indicating that another kinase is responsible for the shear stress-induced phosphorylation of FoxO1a. The shear stress-induced phosphorylation and nuclear exclusion of FoxO1a were attenuated by the AMPK inhibitor compound C (10 μ mol/L) as well as by a DN-AMPK. As shear stress regulates the expression of a number of genes in endothelial cells including that of angiopoietin-2 (Ang-2), we assessed the role of the AMPK-FoxO pathways in its expression. DN-AMPK significantly increased Ang-2 protein while the CA-AMPK mutant markedly decreased Ang-2 protein expression. Moreover, while Ang-2 expression was clearly evident in endothelial cells maintained under static conditions, the application of fluid shear stress elicited a down-regulation of the protein which was abrogated by the DN-AMPK. Given that Ang-2 plays a critical role in vessel remodeling, our data indicate that shear stress-induced angiogenesis, involves the AMPK-mediated regulation of the transcription factor FoxO1a and its target gene Ang-2.