Abstract 1498: Agonist-modulated Activation of AMP-activated Protein Kinase: Evidence for a Novel AMPK-Rac1-Akt-eNOS Pathway in Endothelial Cells
The endothelial isoform of nitric oxide synthase (eNOS) is regulated by diverse cell surface receptors. Activation of eNOS by vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) involves multiple protein kinases and other signaling proteins, yet the relationships between these pathways are incompletely understood. We designed and validated a series of potent and specific duplex siRNA constructs to knockdown expression of key signaling proteins in bovine aortic endothelial cells, and have identified a novel AMPK-Rac1-Akt hierarchical pathway that is critical for VEGF- and S1P-modulated eNOS activation and angiogenesis. We found that VEGF (10 ng/ml) and S1P (100 nM) promote a rapid ~4-fold increase in AMPK phosphorylation. siRNA constructs targeting AMPKα1 specifically suppress AMPKα1 expression by >90%, and markedly impair agonist-mediated eNOS activation (49 ± 14% decrease in VEGF-induced eNOS activity; 43 ± 19% decrease for S1P; n = 4, p < 0.05 for both by ANOVA) as well as Akt phosphorylation (38 ± 6% reduction for VEGF; 42 ± 7% reduction for S1P; n = 4, p < 0.01). In contrast, siRNA-mediated Akt1 knockdown attenuates eNOS activation but does not affect AMPK activation. These results establish that AMPK lies upstream of Akt in the pathway leading from receptor to eNOS. siRNA-mediated AMPKα1 knockdown attenuates agonist-mediated activation of the small GTPase Rac1 (51 ± 19% decrease; n = 3, p < 0.05). Conversely, Rac1 siRNA decreases the phosphorylation of AMPK substrates without affecting phosphorylation of AMPK, implicating Rac1 as an effector of AMPK in eNOS activation. siRNA-mediated knockdown of caveolin-1 significantly enhances AMPK phosphorylation, suggesting that AMPK is negatively regulated by caveolin-1. Finally, we found that cells transfected with AMPK, Rac1, or eNOS siRNA demonstrate severely impaired agonist-modulated endothelial migration and tube formation on Matrigel. These results suggest that VEGF and S1P regulate AMPK as a very key early step in an agonist-modulated AMPK → Rac1 → Akt hierarchy that controls eNOS activation as well as cell migration and endothelial tube formation. These findings may have important implications for the AMPK-dependent control of NO signaling pathways in the vascular wall.