gms | German Medical Science

24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung

Deutsche Gesellschaft für Arterioskleroseforschung

18.03. - 20.03.2010, Blaubeuren

Regulation of endothelial 5'-AMP-activated protein kinase (AMPK) by VEGF and 2-deoxyglucose

Meeting Contribution

  • corresponding author K. Spengler - Institute for Molecular Cell Biology, Center for Molecular Biomedicine, Friedrich Schiller University of Jena, Germany
  • N. Stahmann - Institute for Molecular Cell Biology, Center for Molecular Biomedicine, Friedrich Schiller University of Jena, Germany
  • S. Krause - Institute for Molecular Cell Biology, Center for Molecular Biomedicine, Friedrich Schiller University of Jena, Germany
  • R. Heller - Institute for Molecular Cell Biology, Center for Molecular Biomedicine, Friedrich Schiller University of Jena, Germany

Deutsche Gesellschaft fr Arterioskleroseforschung e.V.. 24. Jahrestagung der Deutschen Gesellschaft fr Arterioskleroseforschung. Blaubeuren, 18.-20.03.2010. Dsseldorf: German Medical Science GMS Publishing House; 2011. Doc10dgaf20

doi: 10.3205/10dgaf20, urn:nbn:de:0183-10dgaf209

Published: March 23, 2011

© 2011 Spengler et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en). You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.


Abstract

AMP-activated protein kinase (AMPK), a serine/threonine protein kinase, is a potent regulator of cellular energy state and homeostasis. In endothelial cells, AMPK is activated by phosphorylation of its threonine residue 172, which is mediated by the tumor suppressor kinase LKB1 or by calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ). AMPK activation is known to be initiated by various agonists including vascular mediators, hormones and drugs. We now show that vascular endothelial growth factor (VEGF) triggers AMPK phosphorylation and activation in endothelial cells. Using genetic and pharmacological approaches we demonstrate that the VEGF response is mediated by CaMKKβ but not by LKB1. In addition, AMPK is phosphorylated under conditions of energy deprivation induced by 2-deoxyglucose and VEGF and 2-deoxyglucose act synergistically to activate AMPK. We also show that AMPK plays an essential role in VEGF-induced in-vivo angiogenesis by performing Matrigel plug assays in wild type and AMPKα1 knockout mice. Our data demonstrate that AMPK affects VEGF-induced angiogenesis independent from endothelial NO synthase (eNOS). eNOS was not phosphorylated by AMPK regardless of whether VEGF or 2-deoxyglucose was used as a stimulus. Our data suggest that AMPK promotes VEGF-induced angiogenesis and enhances angiogenic effects of VEGF under conditions of energy deprivation.


Introduction

The AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that plays a major role in the maintenance of the intracellular energy state and is involved in the regulation of cellular homeostasis and signalling [1]. AMPK can be activated by phosphorylation at threonine 172 which either occurs in an AMP-dependent way via the tumor suppressor kinase LKB1 or in a calcium-dependent way via the calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) [2], [3]. Once activated AMPK stimulates ATP-generating processes like glycolysis or β-oxidation of fatty acids. At the same time, ATP-consuming pathways such as the synthesis of proteins or fatty acids are inhibited by AMPK [4]. In addition, AMPK is important for the protection from endothelial dysfunction [1].

This study examines the regulation and the function of AMPK in pathways stimulated by vascular endothelial growth factor (VEGF), an important angiogenic stimulus in human umbilical vein endothelial cells (HUVEC). Further, we studied the effects of energy depletion both on basal and VEGF-stimulated AMPK activation.


Methods

Cell culture and experimental incubation: HUVEC were prepared from umbilical cords using 0.01% collagenase and cultured in M199 containing 2.5% human serum, 17.5% fetal calf serum (FCS) and 7.5 g/ml endothelial cell growth supplement (ECGS). Before stimulation with VEGF, cells were starved for 5 h in M199 containing 0.25 % human serum albumin (HSA). Preincubation with inhibitors and cell stimulation was performed in Hepes/HSA buffer (10 mM Hepes (pH 7.4), 145 mM NaCl, 5 mM KCl, 1 mM MgSO4, 10 mM glucose, 1.5 mM CaCl2, 0.25% HSA).

Small interfering RNA (siRNA)-mediated knockdown of CaMKKβ and LKB1: To downregulate the expression of CaMKKβ and LKB1, HUVEC were seeded in 30 mm-diameter dishes 24 h prior to transfection and treated with siRNA for 72 h (1 g specific CaMKKβ-siRNA, specific LKB1-siRNA or non-targeting control-siRNA). Transfections were performed using the amphiphilic delivery system SAINT-RED (Synvolux Therapeutics B.V.).

Western blot analysis: HUVEC lysates were prepared in ice-cold Tris buffer (50 mM Tris (pH 7.4), 2 mM EDTA, 1 mM EGTA, 50 mM NaF, 10 mM Na4P2O7, 1 mM Na3VO4, 1 mM DTT, 1 mM PMSF, 10 l/ml protease inhibitor cocktail (Complete, Roche Diagnostic GmbH), 1% Triton X-100, 0.1% SDS). Blots were subjected to immunostaining with antibodies against phosphorylated AMPK (threonine 172), AMPK, AMPKα1, endothelial NO synthase (eNOS), phosphorylated eNOS and LKB1.

AMPK assay: HUVEC were lysed in Hepes buffer (50 mM Hepes (pH 7.4), 50 mM NaF, 1 mM EDTA, 1 mM DTT, 5 mM Na4P2O7, 0.1 mM PMSF, 0.157 mg/ml benzamidine, 4 mg/ml trypsin inhibitor, 1% Triton X-100, 10% glycerol). AMPK was immunoprecipitated from 100 g of cell lysate protein using a specific antibody against AMPKα1 and protein G-Sepharose. AMPK activity was determined by phosphorylation of the synthetic substrate peptide SAMS (HMRSAMSGLHLVKRR) in the presence of 5 mM MgCl2, 0.2 mM [γ-32P]ATP and 0.2 mM ATP.

CaMKKβ assay: HUVEC were lysed as described above and CaMKKβ was immunoprecipitated from 100 g of cell lysate protein using 2 l of a rabbit sera raised against bacterially expressed CaMKKβ bound to protein A-Sepharose. Activity was measured by the activation of purified recombinant AMPK complexes in the presence of 2 mM CaCl2, 2 M bovine brain calmodulin, and 0.2 mM AMP.

LKB1 assay: LKB1 was immunoprecipitated from 100 g of cell lysate protein using 2 l of rabbit sera raised against bacterially expressed LKB1 bound to protein A-Sepharose. LKB1 activity was measured in the same way as CaMKKβ activity by activation of AMPK but in the absence of CaCl2 and calmodulin.

Matrigel plug assay: 0.5 ml of Matrigel containing 400 ng/ml VEGF and 400 g/ml (12.7 units/ml) heparin or heparin alone was injected subcutaneously into the left and right flanks of 12–15-week-old female wild type and AMPKα1-/- mice. After 7 days the plugs were carefully dissected from the host tissue, fixed overnight in zinc fixative, and subsequently embedded in paraffin. Two 5-m sections were prepared from each plug. After deparaffinization in xylene and rehydration, sections were treated with blocking solution (phosphate-buffered saline containing 0.01% Triton and 10% goat serum) and subjected to immunofluorescence staining using a polyclonal anti-CD31 antibody and a Cy3-labeled goat anti-rabbit secondary antibody. Analysis of CD31-positive cells was performed by fluorescence microscopy and laser scanning microscopy.

Statistical analysis: All data are given as means SEM of three to four independent experiments.


Results

VEGF stimulates AMPK phosphorylation and activation: VEGF (50 ng/ml) led to a time-dependent increase of AMPK phosphorylation at threonine 172. In parallel, AMPK activity in immunoprecipitates was increased with reaction kinetics comparable to threonine 172 phosphorylation (not shown).

VEGF-induced threonine 172 phosphorylation and activation is not mediated by LKB1: To check the involvement of LKB1 in VEGF-stimulated AMPK activation we downregulated LKB1 by approximately 65% using specific siRNA. VEGF-stimulated AMPK phosphorylation at threonine 172 was, however, not affected by LKB1 downregulation (data not shown).

VEGF-stimulated threonine 172 phosphorylation and activation is dependent on CaMKKβ: Specific siRNA against CaMKKβ and STO-609 (5–20 g/ml), an inhibitor of CaMKK, were used to investigate the role of CaMKKβ in VEGF-mediated AMPK phosphorylation. Downregulation of CaMKKβ (remaining activity 22%) led to a significant inhibition of AMPK phosphorylation at threonine 172 and AMPK activation in response to VEGF (88% and 78%, respectively). In addition, AMPK phosphorylation was decreased by STO-609 in a dose-dependent manner up to 100%.

eNOS is not a target of AMPK in VEGF-treated HUVEC: VEGF is able to stimulate eNOS phosphorylation at serine 1177 in a time-dependent manner. To check the role of AMPK in VEGF-induced eNOS phosphorylation we used specific siRNA against AMPKα1. We obtained an 88% downregulation of AMPKα1 expression and a remarkable inhibition of VEGF-induced AMPK phosphorylation (76%, 77% and 69% at 1, 2 and 5 min) but eNOS phosphorylation was not altered under these conditions.

2-deoxyglucose (2-DG) stimulates AMPK phosphorylation in a LKB1- and CaMKKβ-dependent pathway: AMPK phosphorylation at threonine 172 was stimulated by 2-DG (20 mM) in a time-dependent manner and reached a plateau at 10 min. CaMKK inhibition by STO-609 and LKB1 downregulation by specific siRNA reduced 2-DG-mediated AMPK phosphorylation suggesting that both CaMKKβ and LKB were involved. The combination of STO-609 and LKB1-siRNA led to a complete inhibition of threonine 172 phosphorylation (93%).

2-DG does not stimulate eNOS phosphorylation: 2-DG was not able to enhance eNOS phosphorylation at serine 1177 under conditions leading to AMPK phosphorylation.

VEGF and 2-DG act synergistically on AMPK phosphorylation: Treatment of HUVEC with VEGF (50 ng/ml, 2 min) or 2-DG (20 mM, 4 min) increased AMPK phosphorylation to a similar extent. The effect of 2-DG stimulation for 4 min followed by a 2 min VEGF stimulation led to a phosphorylation signal, which was higher than the sum of the two single signals (Figure 1 [Fig.1]).

VEGF-induced in-vivo angiogenesis is mediated by AMPK: To investigate the role of AMPK in in-vivo angiogenesis the matrigel plug assay was applied. Matrigel plugs from wild type mice containing VEGF showed a significantly higher invasion of endothelial cells and an increased formation of capillary-like structures than control plugs. However, in AMPKα1-/- mice no differences in vascularisation between control plugs and VEGF-containing plugs were observed (Figure 2 [Fig.2]).


Discussion

This study shows that VEGF stimulates AMPK phosphorylation at threonine 172 as well as AMPK activity in endothelial cells in a time-dependent manner. VEGF-induced AMPK phosphorylation was not mediated by LKB1. However, siRNA-mediated downregulation of CaMKKβ or inhibition of CaMKK by STO-609 significantly diminished VEGF-stimulated AMPK phosphorylation indicating that CaMKKβ acts as an AMPK upstream kinase in response to VEGF. In addition, we showed that the glycolysis inhibitor 2-deoxyglucose, which leads to an intracellular energy deficit [5], stimulates AMPK phosphorylation. LKB1-siRNA or STO-609 pretreatment of cells induced a partial reduction of 2-DG-induced AMPK phosphorylation when applied alone and an almost complete inhibition when added in combination. These data suggest that under conditions of energy deprivation AMPK activation is not only mediated by an AMP/LKB1-dependent way but also by a calcium/CaMKKβ-dependent pathway. We also found that VEGF and 2-DG act synergistically on AMPK phosphorylation, which is probably due to involvement of different upstream signalling molecules (AMP and calcium). Binding of AMP could reduce dephosphorylation of AMPK by phosphatases and thereby potentiate the effect of the calcium-dependent AMPK kinase CaMKKβ.

Our data suggest that AMPK is involved in VEGF-induced angiogenic effects on endothelial cells and may mediate the potentiation of VEGF effects under energy deprivation and hypoxia. Indeed, we were able to show that VEGF induced vascularization of the injected matrigel plugs in wild type mice but not in AMPKα1-/- mice. These data indicate that in-vivo angiogenesis in response to VEGF is essentially dependent on AMPK. However, AMPK most probably affects VEGF-induced angiogenesis independent from endothelial NO synthase (eNOS). We show that downregulation of AMPKα1 does not affect eNOS phosphorylation and activation in VEGF-treated HUVEC although an almost complete downregulation of AMPKα1 activation was achieved. The phosphorylation of eNOS by AMPK is controversially discussed [6], [7]. Thors et al. [8] suggest that eNOS becomes a target of AMPK only under conditions of energy deprivation. However, we found that 2-DG was not able to induce eNOS phosphorylation although AMPK was stimulated in these cells. These data indicate that eNOS is not a substrate of AMPK under the conditions applied in our study.

In summary, our data reveal AMPK as a relevant mediator of VEGF-induced angiogenesis. Moreover, AMPK may play an important role in the regulation and amplification of VEGF-stimulated angiogenesis under energy deprivation since it is synergistically activated by 2-DG and VEG. The angiogenic function of AMPK may be related to its role in the regulation of the intracellular energy state and metabolism but is most probably not due to eNOS activation.


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