Thromboxane receptor activates the AMP-activated protein kinase in vascular smooth muscle cells via hydrogen peroxide

Abstract

Thromboxane A2 receptor (TPr) stimulation induces cellular hypertrophy in vascular smooth muscle cells (VSMCs); however, regulation of VSMC hypertrophy remains poorly understood. Here we show that TPr stimulation activates AMP-activated kinase (AMPK), which in turn limits TPr-induced protein synthesis in VSMCs. Exposure of cultured VSMCs to either TPr agonists, IBOP and U46619, or exogenous hydrogen peroxide (H2O2) caused time- and dose-dependent AMPK activation, as evidenced by increased phosphorylation of both AMPK-Thr172 and acetyl-coenzyme A carboxylase-Ser79, a downstream enzyme of AMPK, whereas SQ29548, a selective TPr antagonist, significantly attenuated TPr-enhanced AMPK activation. In parallel, both IBOP and U46619 significantly increased the production of reactive oxygen species such as H2O2. Furthermore, adenoviral overexpression of catalase (an H2O2 scavenger) abolished, whereas superoxide dismutase (which catalyzes H2O2 formation) enhanced, IBOP-induced AMPK activation, suggesting that TPr-activated AMPK was mediated by H2O2. Consistently, exposure of VSMCs to either TPr agonists or exogenous H2O2 dose-dependently increased the phosphorylation of LKB1 (at serines 428 and 307), an AMPK kinase, as well as coimmunoprecipitation of AMPK with LKB1. In addition, direct mutagenesis of either Ser428 or Ser307 of LKB1 into alanine, like the kinase-dead LKB1 mutant, abolished both TPr-stimulated AMPK activation and coimmunoprecipitation. Finally, genetic inhibition of AMPK significantly accentuated IBOP-enhanced protein synthesis, whereas adenoviral overexpression of constitutively active AMPK abolished IBOP-enhance protein synthesis in VSMCs. We conclude that TPr stimulation triggers reactive oxygen species-mediated LKB1-dependent AMPK activation, which in return inhibits cellular protein synthesis in VSMCs.

Figure 1

TxA₂ mimetics activate AMPK in cultured VSMCs. Confluent VSMCs were treated with the TxA₂ mimetics IBOP and U46619 at the concentrations indicated for 10 minutes. Both phosphorylated AMPK-Thr172 and ACC-Ser79 were detected in Western blots by using specific antibodies (a and c). Exposure of VSMCs to IBOP (b) and U46619 (d) increased both AMPK-Thr172 and ACC-Ser79 in a dose-dependent manner. Data are means±SEM (b, n=4; d, n=3). ♣P< 0.05; ‡P<0.01, treated vs untreated control cells.

Figure 2

TxA₂ mimetics activate AMPK via TPr in VSMCs. TPr activation causes a time-dependent AMPK activation in VSMCs (a and b). VSMCs were treated with either IBOP (1 µmol/L) or U46619 (1 µmol/L) for 5, 15, and 30 minutes. Data are means±SEM (n=4). ♣P<0.05, ‡P<0.01, treated vs control cells. VSMCs were treated with either IBOP (1 µmol/L) or U46619 (1 µmol/L) for 24 hours (c). TPr was detected at 55 and 64 kDa (Cayman’s TPr polyclonal antibody detects the TPr receptor at 55 and 64 kDa according to the production information) via Western blotting (d). The TPr antagonist SQ29548 abolishes TPr-induced AMPK activation. Confluent VSMCs were preincubated with or without the TPr antagonist SQ29548 at 1 µmol/L or 10 µmol/L for 30 minutes before being exposed to IBOP (1 µmol/L) for either 5 or 15 minutes. The blot is representative of 3 blots from 3 individual experiments.

Figure 3

TxA₂-induced AMPK activation is mediated by ROS production. TPr stimulation increases ROS production in VSMCs (a). ROS were determined by measuring DCF after incubation with either IBOP (1 µmol/L) or U46619 (1 µmol/L) for 10 minutes. Data are means±SEM (a, n=4 [♣P<0.05]; b, n=3 [♣P<0.05]). Infection of VSMCs with adenoviruses encoding catalase or SOD1 increased the expression of both catalase and SOD1 in VSMCs, respectively (b). The blot is representative of at least 3 blots of 3 independent experiments. Adenoviral overexpression of catalase increased catalase activity in VSMCs (b). Catalase activity was examined after 48 hours of infection with adenoviral GFP or catalase. Catalase activity is shown in nanomoles per minute per milliliter (means±SEM, n=3). ‡P<0.01 catalase vs GFP controls. Adenoviral overexpression of catalase suppresses IBOP-induced (1 µmol/L, 10 minutes) AMPK activation, whereas overexpression of SOD1 (Cu/Zn SOD) increases AMPK activation (c). The blot is representative of 3 blots from 3 individual experiments. Exogenous H₂O₂ at concentrations indicated for 10 minutes increases AMPK activation in VSMCs (d). Data are means±SEM (n=3). ♣P<0.05; ‡P<0.01, treated vs control cells.

Figure 4

TPr stimulation enhances LKB1 phosphorylation in cultured VSMCs. Time-dependent effects of IBOP (1 µm) or U46619 (1 µmol/L) on LKB1-Ser428 phosphorylation in VSMCs (a and b). Data as means±SEM (a, n=5; b, n=3). ♣P<0.05; ‡P<0.01, treated vs untreated cells. Time-dependent effects of IBOP (1 µm) on LKB1-Ser307 phosphorylation in VSMCs (c). Data are means±SEM (n=3). ♣P<0.05, treated vs untreated cells.

Figure 5

TPr-induced LKB1-Ser428 phosphorylation is mediated by ROS production. Effect of catalase and SOD1 overexpression on IBOP-induced (1 µmol/L, 10 minutes) LKB1-Ser428 phosphorylation (a). The blot is representative of 3 blots from 3 independent assays. Exogenous H₂O₂ dose-dependently increases LKB1 phosphorylation at the 10-minute time point (b). The blot is representative of 3 blots from 3 independent assays.

Figure 6

TPr-induced AMPK activation requires LKB1. Activation of AMPK by IBOP is LKB1-dependent (a). LKB1-deficient A549 cells were transfected for 24 hours with LKB1 wild-type (WT) or LKB1 mutants (S307A, S428A, and D194A). The plasmid encoding LacZ was used as a control. Transfected cells were stimulated with 1 µmol/L IBOP or with vehicle for 10 minutes. Data are means±SEM (n=3). ♣P<0.05, treated vs untreated cells. Adenoviral overexpression of LKB1 mutants abolishes IBOP-induced AMPK phosphorylation in VSMCs (b). VSMCs were infected with adenoviruses encoding LKB1 mutants D194A, S307A, and S428A, respectively. Cells were stimulated with 1 µmol/L IBOP for 10 minutes. Data are means±SEM (n=3). ♣P<0.05; ‡P<0.01, treated vs untreated cells. IBOP enhances the coimmunoprecipitation of LKB1 and AMPK in VSMCs but not in A549 cells transfected with LKB1 mutants (c). LKB1 was immunoprecipitated after IBOP treatment (1 µmol/L, 10 minutes) from VSMCs or A549 cells transfected with S307A, S428A, or D194A mutant LKB1 plasmids, and AMPK was then detected in Western blots. Data are means±SEM (n=3). ‡P<0.01, treated vs untreated cells. Effect of CaMKKβ on TPr-induced AMPK activation (d). VSMCs were preincubated for 6 hours with 1 µg/mL (2.6 µmol/L) STO-609 or transfected by CaMKKβ small interfering RNA to knockdown CaMKKβ expression; then the cells were treated with 1 µmol/L IBOP for 10 minutes. The blot is representative of 3 blots obtained from 3 independent experiments. Data are means±SEM (n=3). ♣P<0.05, treated vs untreated cells.

Figure 7

Activation of AMPK by IBOP limits cellular protein synthesis in VSMCs. a, AMPK-dependent inhibition of protein synthesis in 5-hour IBOP-treated VSMCs. Forty-eight hours after having being infected with GFP, AMPK-DN, or AMPK-CA adenoviral vectors, VSMCs were treated for 5 hours with IBOP (1 µmol/L) or vehicle. Protein synthesis was assayed by [³H]leucine incorporation, as described in Materials and Methods. At same time, the cell number was counted using a hemocytometer. VSMC protein synthesis was calculated by dividing the total [³H]leucine (cmp) by the number of cells in each well and the vehicle treated GFP was used as 100%. Two-way ANOVA indicated significant effect of IBOP (P<0.01) for leucine incorporation and effect of AMPK mutation (P<0.01) for leucine incorporation. Interaction effect between IBOP and AMPK mutation approached significance (P<0.05). Data are means±SEM (n=4). ♣P<0.01, GFP vs GFP plus IBOP; ‡P<0.01, AMPK-DN vs AMPK-DN plus IBOP; †P<0.05, AMPK-DN plus IBOP vs GFP plus IBOP; #P<0.05, AMPK-CA plus IBOP vs GFP plus IBOP. b, Inhibition of AMPK by AMPK-DN enhanced IBOP-induced protein synthesis in VSMCs. Confluent VSMCs infected with adenovirus of either GFP or AMPK-DN for 48 hours were treated for 48 hours with IBOP (1 µmol/L) or vehicle. Two-way ANOVA indicated significant effect of IBOP treatment (P<0.01) and AMPK-DN transfection (P<0.01) for leucine incorporation. Interaction effect between IBOP and AMPK-DN transfection approached significance (P<0.01) for leucine incorporation. Data are means±SEM (n=3). ♣P<0.01, GFP vs GFP plus IBOP; ‡P<0.01, AMPK-DN vs AMPK-DN plus IBOP; †P<0.01, AMPK-DN plus IBOP vs GFP plus IBOP.