Thrombin activates AMP-activated protein kinase in endothelial cells via a pathway involving Ca2+/calmodulin-dependent protein kinase kinase beta

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 beta (CaMKKbeta). 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 Gq-protein-mediated phospholipase C activation. Inhibition of CaMKK with STO-609 or downregulation of CaMKKbeta using RNA interference decreased thrombin-induced AMPK activation significantly, indicating that CaMKKbeta 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 CaMKKbeta 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+/CaMKKbeta dependent and one AMP/LKB1 dependent.

FIG. 1.

Effect of thrombin on AMPK activation in endothelial cells. HUVEC were stimulated with different thrombin concentrations for 1 min (A) or with 1 U/ml thrombin for the indicated times (B and C). A and B. Cell lysates were subjected to Western blot analysis using an antibody against AMPKα phosphorylated at threonine 172 or an anti-AMPKα antibody for counterstaining. Representative blots and densitometric analyses are shown (mean ± SEM, n = 3). C. AMPK was immunoprecipitated from 100 μg total protein using an anti-AMPKβ antibody, and the activity of the immune complexes was measured using the SAMS peptide assay. AMPK activity is shown as U/mg lysate protein (mean ± SEM, n = 3), where 1 unit equals 1 nmol ³²PO₄ incorporated into SAMS peptide per min. , P < 0.05 versus untreated controls.

FIG. 2.

Thrombin-induced AMPK activation is mediated via PAR-1, G_q coupling, and phospholipase C activation. A. HUVEC were stimulated with the PAR-1 agonist peptide TFLLR (10 μM) for the indicated times. B. HUVEC were pretreated with pertussis toxin (PTX) (500 ng/ml, 3 h) or with G_q-protein antagonist 2A (GPA-2A) (5 μM, 10-min preincubation in transiently permeabilized cells) and subsequently stimulated with thrombin (1 U/ml, 1 min). C. HUVEC were pretreated with U-73122 (10 μM, 30 min) and stimulated with thrombin (1 U/ml, 1 min) or alternatively treated with the phospholipase C activator m-3M3FBS (100 μM, 2 min). For panels A to C, cells were lysed and subjected to immunoblotting using antibodies against phosphorylated AMPKα (threonine 172) and total AMPKα for counterstaining. Representative figures and densitometry data (means ± SEMs) of three independent experiments for each treatment are shown. Phosphospecific signals from TFLLR-stimulated and nonstimulated cells (A) or from cells stimulated with thrombin in the absence or presence of inhibitors (B and C) were compared. , P < 0.05.

FIG. 3.

Role of Ca²⁺ in thrombin-induced AMPK activation. A. HUVEC were stimulated with ionomycin (2 μM) for the indicated times. B. HUVEC were pretreated with BAPTA-AM (20 μM, 30 min) and subsequently stimulated with thrombin (1 U/ml, 1 min). For panels A and B, the degree of threonine 172 phosphorylation of AMPK was determined by Western blot analysis with an anti-phosphospecific threonine 172 antibody. AMPK was stained with an antibody against AMPKα. Representative figures and densitometry data (means ± SEMs) of three independent experiments for each treatment are shown. Phosphospecific signals from ionomycin-stimulated and nonstimulated cells (A) or from cells stimulated with thrombin in the absence or presence of BAPTA-AM (B) were compared. , P < 0.05.

FIG. 4.

Downregulation of CaMKKβ and LKB1 by RNA interference. HUVEC were incubated in the presence of synthetic RNA duplexes targeted to human CaMKKβ or human LKB1 (1 μg/30-mm dish, 72 h). Incubations with transfection reagent only (control) or with a control RNA duplex containing an unrelated sequence (control siRNA) were run in parallel. A. Protein expression was examined in anti-CaMKKβ immune complexes (left panel) or cell lysates (right panel) probed with either anti-CaMKKβ antibody or anti-LKB1 antibody, respectively. Representative Western blots out of three independent experiments for each treatment are shown. B. CaMKKβ or LKB1 activities were determined in anti-CaMKKβ or anti-LKB1 immune complexes isolated from 100 μg cell lysate. Results are plotted as AMPK activity stimulated by the immune complex and measured using the SAMS peptide (U/mg recombinant AMPK, where 1 unit equals 1 nmol ³²PO₄ incorporated into SAMS peptide per min). Means ± SEMs for three independent experiments are shown. , P < 0.05 versus control siRNA.

FIG. 5.

Effect of CaMKKβ inhibition on thrombin-induced AMPK activation. HUVEC were preincubated for 30 min with STO-609 at the indicated concentrations. A. Cells were lysed and subjected to immunoblotting using antibodies against phosphorylated AMPKα (threonine 172) and total AMPKα for counterstaining. A typical experiment and the densitometric analysis of four experiments are shown (means ± SEMs). B. AMPK activity was examined in anti-AMPKα1 immune complexes isolated from 100 μg total protein by phosphorylation of the SAMS peptide. AMPK activity is shown as U/mg lysate protein (mean ± SEM, n = 3), where 1 unit equals 1 nmol ³²PO₄ incorporated into SAMS peptide per min. , P < 0.05 versus untreated thrombin-stimulated controls.

FIG. 6.

Effect of CaMKKβ and LKB1 downregulation on thrombin-induced AMPK activation. Synthetic RNA duplexes (1 μg/30-mm dish) targeted to human CaMKKβ or to human LKB1 were added to HUVEC alone or in combination, and incubations were carried out for 72 h. Treatments with transfection reagent only (control) or with a control RNA duplex containing an unrelated sequence (control siRNA, 1 or 2 μg) were run in parallel. Following siRNA treatment, cells were stimulated with thrombin (1 U/ml, 1 min) or AICAR (2 mM, 2 h) and AMPK activation was monitored by phosphorylation of threonine 172 with a phosphospecific antibody. AMPK was stained with an antibody against AMPKα. A typical blot (A) and the densitometric analysis results (means ± SEMs) of four experiments (B) are presented. , P < 0.05 versus samples pretreated with control siRNA and stimulated with thrombin or AICAR.

FIG. 7.

Effect of thrombin and TFLLR on ACC and eNOS phosphorylation. HUVEC were stimulated with 1 U/ml thrombin (A) or 10 μM TFLLR (B) for the indicated times. ACC and eNOS phosphorylation was determined using specific antibodies against phosphorylated ACC or eNOS phosphorylated at serine 1177. Anti-ACC and anti-eNOS antibodies were used for the respective counterstainings. Representative experiments and densitometry data (means ± SEMs) of three independent experiments for each treatment are shown. , P < 0.05 versus untreated controls.

FIG. 8.

Effect of AMPK inhibition or downregulation on thrombin-induced ACC and eNOS phosphorylation. HUVEC were incubated for 72 h with synthetic siRNA duplexes targeted to CaΜΚΚβ or AMPKα1 or pretreated with inhibitors against either enzyme (STO-609 for CaΜΚΚβ and compound C for AMPK). Subsequently, cells were stimulated with thrombin (1 U/ml, 1 min) and processed for immunoblotting. AMPK, ACC, or eNOS phosphorylation was determined using specific antibodies against phosphorylated AMPK (threonine 172), ACC, or eNOS (serine 1177). Expression of proteins was monitored with anti-AMPKα, anti-ACC, or anti-eNOS antibodies. Representative figures and densitometry data (means ± SEMs) of three (C) or four (A, B, and D) independent experiments for each treatment are shown. A. Cells were incubated for 72 h in the presence of a synthetic siRNA targeted to human CaMKKβ or a nonsilencing control siRNA (1 μg/30-mm dish). Β. HUVEC were pretreated with STO-609 (20 μg/ml, 30 min). C. HUVEC were transfected with an siRNA targeted to human AMPKα1 or a nontargeting control siRNA (2 μg/30-mm dish). D. Cells were pretreated with compound C (20 μM, 30 min). , P < 0.05 versus thrombin-stimulated samples pretreated with control siRNA (A and C) or solvent (B and D).