Abstract 1498: Agonist-modulated Activation of AMP-activated Protein Kinase: Evidence for a Novel AMPK-Rac1-Akt-eNOS Pathway in Endothelial Cells
| Autor: | Yehoshua Levine, Thomas Michel |
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| Rok vydání: | 2007 |
| Předmět: | |
| Zdroj: | Circulation. 116 |
| ISSN: | 1524-4539 0009-7322 |
| DOI: | 10.1161/circ.116.suppl_16.ii_309-a |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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