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, 285 (20), 15346-55

Activation of AMP-activated Protein Kinase alpha1 Alleviates Endothelial Cell Apoptosis by Increasing the Expression of Anti-Apoptotic Proteins Bcl-2 and Survivin

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Activation of AMP-activated Protein Kinase alpha1 Alleviates Endothelial Cell Apoptosis by Increasing the Expression of Anti-Apoptotic Proteins Bcl-2 and Survivin

Chao Liu et al. J Biol Chem.

Abstract

Accumulating evidence suggests that AMP-activated protein kinase (AMPK) activation exerts anti-apoptotic effects in multiple types of cells. However, the underlying mechanisms remain poorly defined. The aim of the present study was to determine how AMPK suppresses apoptosis in endothelial cells exposed to hypoxia and glucose deprivation (OGD). AMPK activity, NF-kappaB activation, and endothelial cell apoptosis were assayed in cultured endothelial cells and mouse common carotid artery with or without OGD treatment. OGD markedly activated AMPK as early as 30 min, and AMPK activity reached maximal at 2 h of OGD. Endothelial apoptosis was not detected until 2 h of OGD but became markedly elevated at 6 h of OGD treatment. Furthermore, AMPK inhibition by Compound C or overexpression of dominant negative AMPK (AMPK-DN) exacerbated, whereas AMPK activation by pharmacologic (aminoimidazole carboxamide ribonucleotide (AICAR)) or genetic means (overexpression of constitutively active AMPK) suppressed endothelial cell apoptosis caused by OGD. Concomitantly, AMPK activation increased the expression of both Bcl-2 and Survivin, two potent anti-apoptotic proteins. Furthermore, AMPK activation significantly enhanced IkappaBalpha kinase activation, NF-kappaB nuclear translocation, and DNA binding activity of NF-kappaB. Consistently, selective inhibition of NF-kappaB, which abolished OGD-enhanced expression of Bcl-2 and Survivin, accentuated endothelial apoptosis caused by OGD. Finally, we found that genetic deletion of the AMPKalpha1, but not AMPKalpha2, suppressed OGD-enhanced NF-kappaB activation, the expression of Bcl-2 and Survivin, and endothelial apoptosis. Overall, our results suggest that AMPKalpha1, but not AMPKalpha2 activation, promotes cell survival by increasing NF-kappaB-mediated expression of anti-apoptotic proteins (Bcl-2 and Survivin) and intracellular ATP contents.

Figures

FIGURE 1.
FIGURE 1.
Time courses of OGD-induced AMPK and apoptosis in endothelial cells. Confluent endothelial cells were subjected to oxygen and glucose deprivation, as described under “Experimental Procedures.” Phosphorylation of both AMPK- Thr-172 and ACC at Ser-79 was monitored in Western blot. A and B, time course of OGD on the phosphorylation of both AMPK and ACC in BAEC is shown. ♣, p < 0.05 versus control; n = 3. C, time-dependent increase of OGD-enhanced TUNEL-positive cells in BAEC is shown. Data are reported as % of TUNEL-positive cells in a microscopic field. ♣, p < 0.05 versus control; n = 5. D, time course effects of OGD on both AMPK at Thr-172 phosphorylation and apoptosis are shown.
FIGURE 2.
FIGURE 2.
Inhibition of AMPK reduces cell viability but increases TUNEL-positive endothelial cells exposed to OGD for 4 h. Cell viability was assayed by using MTT reduction assays. A, AICAR increases, whereas Compound C inhibits AMPK-Thr-172 phosphorylation; n = 3. B, AMPK activation increases, whereas AMPK inhibition decreases endothelial viability in endothelial cells subjected to OGD treatment. ♣, p < 0.05 versus control; †, p < 0.05 versus OGD only; ‡, p < 0.05 versus OGD plus AICAR; n = 3. C, overexpression of constitutively active AMPK (AMPK-CA) increases, whereas overexpression of dominant negative AMPK (AMPK-DN) accentuates the reduction of MTT induced by OGD. ♣, p < 0.05 versus GFP; †, p < 0.05 versus GFP with OGD; ‡, p < 0.05 versus AMPK-CA plus OGD; n = 3. D, AMPK activation suppresses, whereas AMPK inhibition increases OGD-enhanced TUNEL positive staining in endothelial cells. Data are reported as % TUNEL-positive in a microscopic field ± S.E. ♣, p < 0.05 versus control; †, p < 0.05 versus OGD only; ‡, p < 0.05 versus OGD plus AICAR; n = 5. E, AICAR suppresses, whereas compound C increases OGD-enhanced TUNEL positive staining in endothelial cells. Data are reported as % TUNEL-positive in a microscopic field ± S.E. ♣, p < 0.05 versus control; †, p < 0.05 AICAR or compound C plus OGD versus OGD only; ‡, p < 0.05 compound C plus OGD versus OGD only; n = 5.
FIGURE 3.
FIGURE 3.
Inhibition of AMPK increases caspase-3 activity and the cleavage of caspase-3 and PARP in endothelial cells exposed to OGD for 4 h. A, AMPK activation suppresses, whereas AMPK inhibition increases caspase-3 activity enhanced by OGD treatment. ♣, p < 0.05 versus control; †, p < 0.05 versus OGD only; ‡, p < 0.05 versus OGD plus AICAR; n = 3. B, overexpression of constitutively active AMPK (AMPK-CA) suppresses, whereas overexpression of dominant negative AMPK (AMPK-DN) inhibits caspase activity enhanced by OGD. ♣, p < 0.05 versus GFP; †, p < 0.05 versus GFP with OGD; ‡, p < 0.05 versus AMPK-CA plus OGD; n = 3. C, AMPK activation suppresses, whereas AMPK inhibition increases caspase-3 and PARP cleavage enhanced by OGD treatment. The blot is representative of three blots from three independent experiments. D, overexpression of constitutively active AMPK (AMPK-CA) suppresses, whereas overexpression of dominant negative AMPK (AMPK-DN) inhibits caspase-3 and PARP cleavage enhanced by OGD. Both β-actin and histone 3 served as loading controls. The blot is representative of three blots from three independent experiments.
FIGURE 4.
FIGURE 4.
AMPK inhibition reduces the levels of Bcl-2 and Survivin in endothelial cells exposed to OGD for 2 h. Confluent endothelial cells were treated with AICAR or Compound C in the absence or presence of OGD (2 h). β-Actin serves as the loading control. A, AMPK activation increases, whereas AMPK inhibition suppresses Bcl-2 induced by OGD treatment. †, p < 0.05 versus OGD only; ‡, p < 0.05 versus OGD plus AICAR; n = 3. B, AMPK activation increases, whereas AMPK inhibition suppresses Survivin induced by OGD treatment. †, p < 0.05 versus OGD only; ‡, p < 0.05 versus OGD plus AICAR; n = 3.
FIGURE 5.
FIGURE 5.
NF-κB inhibition reduces the levels of Bcl-2 and Survivin increased by AICAR in endothelial cells exposed to OGD for 4 h. Confluent endothelial cells were treated with AICAR, AICAR with NF-κB inhibitor peptide (AICAR+IN), and AICAR with inactive NF-κB inhibitor peptide (AICAR+CIN) in the absence or presence of OGD (4 h). β-Actin serves as the loading control. Data are reported as % TUNEL-positive cells in a microscopic field. A, effects of selective NF-κB inhibition on OGD-enhanced expression of Bcl-2 are shown. ♣, p < 0.05 versus control; †, p < 0.05 versus OGD only; ‡, p < 0.05 versus OGD plus AICAR+CIN; n = 3. B, effects of selective NF-κB inhibition on OGD-enhanced expression of Survivin are shown. ♣, p < 0.05 versus control; †, p < 0.05 versus OGD only; ‡, p < 0.05 versus OGD plus AICAR+CIN; n = 3. C, selective NF-κB inhibition attenuates the protective effects of AICAR in OGD-enhanced apoptosis. Data are reported as % TUNEL-positive cells in a microscopic field ± S.E. ♣, p < 0.05 versus control; †, p < 0.05 versus OGD only; ‡, p < 0.05 versus OGD plus AICAR+CIN; n = 3.
FIGURE 6.
FIGURE 6.
Inhibition of AMPK reduces NF-κB activation in endothelial cells exposed to OGD for 4 h. A, an in vitro kinase assay for glutathione S-transferase-IκBα, with AICAR and compound C treatment with and without OGD (4 h) is shown. IKKβ activity was analyzed by an in vitro kinase assay for glutathione S-transferase-IκBα. BAEC were treated with AICAR and compound C in the absence or presence of OGD for 4 h. n = 3. B, effects of AICAR and Compound C on the phosphorylation of p65 with or without OGD are shown. The blot is representative of three blots obtained from three independent experiments. C, shown are the effects of AICAR and Compound C on the nuclear localization of p65 in endothelial cells with or without OGD. The blot is representative of three blots obtained from three independent experiments. D, effects of AICAR and Compound C on OGD-enhanced NF-κB DNA binding activity in endothelial cells are shown. ♣, p < 0.05 OGD versus control; †, p < 0.05 AICAR versus OGD only; ‡, p < 0.05 Compound C with OGD versus OGD alone; n = 3. E, AICAR enhances, whereas compound C suppresses NF-κB-dependent reporter gene expression enhanced by OGD; data are reported as relative luciferase units ± S.E. ♣, p < 0.05 OGD versus control; †, p < 0.05 AICAR versus OGD only; ‡, p < 0.05 Compound C with OGD versus OGD alone; n = 3. F, overexpression of AMPK-CA increases, whereas overexpression of AMPK-DN suppresses NF-κB DNA binding assay enhanced by OGD for 4 h. ♣, p < 0.05 OGD versus control; †, p < 0.05 AICAR versus OGD only; ‡, p < 0.05 Compound C with OGD versus OGD alone, n = 3.
FIGURE 7.
FIGURE 7.
AMPKα1 depletion reduces OGD-induced NF-κB activation in endothelial cells. A, p65 phosphorylation by Western blot analysis in AMPKα1−/− and -α2−/− with and without OGD (4 h) is shown. p65 phosphorylation and translocation to nuclear was assessed by Western blot in MAEC isolated from WT, AMPKα1−/−, and AMPKα2−/− with and without OGD (4 h). β-Actin and histone 3 served as the respective loading controls; n = 3. B, NF-κB DNA binding activity in WT, AMPKα1−/−, and AMPKα2−/− MAEC with and without OGD (4 h) is shown. ♣, p < 0.05 WT versus AMPKα1−/− without OGD; †, p < 0.05 WT versus WT with OGD; ‡, p < 0.05 WT with OGD versus AMPKα1-KO with OGD; n = 3. C, NF-κB reporter gene assays in WT, AMPKα1−/−, and AMPKα2−/− MAEC with and without OGD (4 h) are shown. Data are reported as relative luciferase units ± S.E. (n = 3). ♣, p < 0.05 WT versus AMPKα1−/− without OGD; †, p < 0.05 WT versus WT with OGD; ‡, p < 0.05 WT with OGD versus AMPKα1-KO with OGD; n = 3. D and E, AMPKα1, but not AMPKα2 depletion, abolishes OGD-enhanced expression of Bcl-2 (D) and Survivin (E) in endothelial cells isolated from WT, AMPKα1−/−, and AMPKα2−/− with or without OGD (4 h). β-Actin serves as the loading control. ♣, p < 0.05 WT versus AMPKα1−/− without OGD; †, p < 0.05 WT versus WT with OGD; ‡, p < 0.05 WT with OGD versus AMPKα1-KO with OGD, n = 3. F, effects of OGD on the contents of ATP in the MAEC isolated from WT, AMPKα1−/−, and AMPKα2 −/−. MAEC were treated with and without OGD for 2 h, and ATP contents were assayed by high performance liquid chromatography as described under “Experimental Procedures.” ♣, p < 0.05 OGD versus w/o OGD; †, p < 0.05 WT with OGD versus AMPKα1−/− with OGD; ‡, p < 0.05 WT with OGD versus AMPKα2-KO with OGD; n = 3.
FIGURE 8.
FIGURE 8.
AMPKα1 depletion increases the cleavage of caspase-3 and PARP in endothelial cells exposed to OGD for 4 h. A, AMPKα1−/− depletion, but not AMPKα2−/−, increases caspase-3 cleavage in isolated MAEC with/without OGD (4 h). Caspase-3 cleavage is reported as % of control ± S.E. β-Actin served as the respective loading controls (n = 3). ♣, p < 0.05 WT with OGD versus WT without OGD; †, p < 0.05 AMPKα1-KO with OGD versus WT with OGD; ‡, p < 0.05 AMPKα1-KO with OGD versus AMPKα2-KO with OGD; n = 3. B, effects of AMPKα1−/− depletion and AMPKα2−/− on OGD-induced PARP cleavage in isolated MAEC with or without OGD (4 h) are shown. Histone 3 is used as a control for nuclear protein. The blot is representative of three blots obtained from three individual experiments. C, shown are the effects of AMPKα1−/− depletion and AMPKα2−/− on OGD-induced apoptosis in isolated MAEC with/without OGD (4 h). Data are reported as % of TUNEL-positive cells in a microscopic field. ♣, p < 0.05 versus control; †, p < 0.05 WT with OGD versus AMPKα1−/− with OGD; n = 5.
FIGURE 9.
FIGURE 9.
AMPKα1 is required for OGD-induced up-regulation of Bcl-2 and Survivin in the endothelial cells in vivo. The common carotid arteries from wild type (C57BL/6), AMPKα1−/−, and AMPKα2−/− were ligated for 4 h, and the expression of Bcl-2 and Survivin was monitored in immunocytochemistry by using specific antibodies. Magnifications = 20×. n = 8–10 mice of each group.
FIGURE 10.
FIGURE 10.
Proposed mechanisms for AMPKα1-mediated anti-apoptotic effects. In this scheme AMPK activation in endothelial cells might delay or prevent endothelial cell apoptosis by two independent pathways; that is, maintaining cellular ATP levels and increasing the expression of anti-apoptotic genes (Bcl-2 and Survivin).

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