Abstract 1914: Atorvastatin Activates CaMKK-beta as an Upstream Kinase of AMPK in Endothelium
Background: We reported previously that statins, at clinical dose, can activate AMP-activated protein kinase (AMPK) in mouse aorta and myocardium. As a consequence, the statin-activated AMPK phosphorylates endothelial NO synthase (eNOS) to enhance NO bioavailability, thereby exerting the pleiotropic effects in cardiovascular system. Here, we study the molecular mechanism by which AMPK is activated by statin.
Methods and Results: In vivo, mice receiving atorvastatin at a dose of 50 mg/kg body weight showed the activation of calmodulin kinase kinase (CaMKK)/AMPK/eNOS axis in aortas of C57BL/6 wild-type mice. However, such an atorvastatin-activated aortic AMPK/eNOS was abolished in CaMKK-beta KO mice. In vitro, atorvastatin treatment caused the activation of CaMKK in cultured bovine aortic endothelial cells (BAECs), revealed by an increase in CaMKI phosphorylation. The time- and dose-dependent CaMKI phosphorylation increase was consistent with the AMPK activation. CaMKK-beta immunoprecipitated from statin-treated BAECs was able to phosphorylate GST-AMPK fusion protein, indicating that CaMKK-beta is the upstream AMPK kinase responding to statin. Both STO-609, a CaMKK specific inhibitor, and siRNA against CaMKK-beta inhibited the activation of CaMKK/AMPK in BAECs. STO-609 or CaMKK-beta siRNA also blocked statin-induced NO production and endothelial cell migration. Thus, CaMKK-beta is the functional upstream of the AMPK/eNOS. Interestingly, calcium image study showed that the intracellular calcium concentration in BAECs was not affected by atorvastatin stimulation. Further, the statin-induced AMPK phosphorylation was not blocked by extracellular or intracellular calcium chelation.
Conclusions: Statin activates CaMKK-beta in a calcium independent manner, which in turn augments the AMPK-eNOS-NO pathway for enhanced endothelial biology.