Thrombin Activates AMP-Activated Protein Kinase in Endothelial Cells via a Pathway Involving Ca²⁺/Calmodulin-Dependent Protein Kinase Kinase β

Abstract

AMP-activated protein kinase (AMPK) is a sensor of cellular energy state in response to metabolic stress and other regulatory signals. AMPK is controlled by upstream kinases which have recently been identified as LKB1 or Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ). Our study of human endothelial cells shows that AMPK is activated by thrombin through a Ca²⁺-dependent mechanism involving the thrombin receptor protease-activated receptor 1 and G_q-protein-mediated phospholipase C activation. Inhibition of CaMKK with STO-609 or downregulation of CaMKKβ using RNA interference decreased thrombin-induced AMPK activation significantly, indicating that CaMKKβ was the responsible AMPK kinase. In contrast, downregulation of LKB1 did not affect thrombin-induced AMPK activation but abolished phosphorylation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside. Thrombin stimulation led to phosphorylation of acetyl coenzyme A carboxylase (ACC) and endothelial nitric oxide synthase (eNOS), two downstream targets of AMPK. Inhibition or downregulation of CaMKKβ or AMPK abolished phosphorylation of ACC in response to thrombin but had no effect on eNOS phosphorylation, indicating that thrombin-stimulated phosphorylation of eNOS is not mediated by AMPK. Our results underline the role of Ca²⁺ as a regulator of AMPK activation in response to a physiologic stimulation. We also demonstrate that endothelial cells possess two pathways to activate AMPK, one Ca²⁺/CaMKKβ dependent and one AMP/LKB1 dependent.

This work was supported by grants from Deutsche Forschungsgemeinschaft (Graduiertenkolleg 768) and Interdisziplinäres Zentrum für Klinische Forschung, Klinikum der Friedrich-Schiller-Universität Jena, to R.H. as well as by an Integrated Project (LSHM-CT-2004-005272) from the European Commission and the Medical Research Council (UK) to D.C.

We thank Gunda Guhr and Elke Teuscher for their excellent technical assistance.