Angiotensin II Decreases Endothelial Nitric Oxide Synthase Phosphorylation via AT1R Nox/ROS/PP2A Pathway

Abstract

Increasing evidences suggest that angiotensin (Ang) II participates in the pathogenesis of endothelial dysfunction (ED) through multiple signaling pathways, including angiotensin type 1 receptor (AT₁R) mediated NADPH oxidase (Nox)/reactive oxygen species (ROS) signal transduction. However, the detailed mechanism is not completely understood. In this study, we reported that AngII/AT₁R-mediated activated protein phosphatase 2A (PP2A) downregulated endothelial nitric oxide synthase (eNOS) phosphorylation via Nox/ROS pathway. AngII treatment reduced the levels of phosphorylation of eNOS Ser1177 and nitric oxide (NO) content along with phosphorylation of PP2Ac (PP2A catalytic subunit) Tyr307, meanwhile increased the PP2A activity and ROS production in human umbilical vein endothelial cells (HUVECs). These changes could be impeded by AT₁R antagonist candesartan (CAN). The pretreatment of 10^-8 M PP2A inhibitor okadaic acid (OA) reversed the levels of eNOS Ser1177 and NO content. Similar effects of AngII on PP2A and eNOS were also observed in the mesenteric arteries of Sprague-Dawley rats subjected to AngII infusion via osmotic minipumps for 2 weeks. We found that the PP2A activity was increased, but the levels of PP2Ac Tyr307 and eNOS Ser1177 as well as NO content were decreased in the mesenteric arteries. The pretreatments of antioxidant N-acetylcysteine (NAC) and apocynin (APO) abolished the drop of the levels of PP2Ac Tyr307 and eNOS Ser1177 induced by AngII in HUVECs. The knockdown of p22phox by small interfering RNA (siRNA) gave rise to decrement of ROS production and increment of the levels of PP2Ac Tyr307 and eNOS Ser1177. These results indicated that AngII/AT₁R pathway activated PP2A by downregulating its catalytic subunit Tyr307 phosphorylation, which relies on the Nox activation and ROS production. In summary, our findings indicate that AngII downregulates PP2A catalytic subunit Tyr307 phosphorylation to activate PP2A via AT₁R-mediated Nox/ROS signaling pathway. The activated PP2A further decreases levels of eNOS Ser1177 phosphorylation and NO content leading to endothelial dysfunction.

Keywords: NADPH oxidase; angiotensin II; angiotensin II type 1 receptor; endothelial nitric oxide synthase; protein phosphatase 2A.

Figure 1

Effects of the angiotensin II (AngII)/angiotensin type 1 receptor (AT₁R) pathway on endothelial nitric oxide synthase (eNOS) Ser1177 phosphorylation and nitric oxide (NO) production. (A,B) AngII downregulated phosphorylation levels of eNOS Ser1177 in a dose-dependent manner. Human umbilical vein endothelial cells (HUVECs) were treated with the indicated concentrations of AngII for 12 h, and phosphorylation levels of eNOS Ser1177 were detected by Western Blot analysis (n = 6 independent experiments). (C,D) AngII downregulated phosphorylation levels of eNOS Ser1177 in a time-dependent manner. HUVECs were treated with 10⁻⁷ M AngII for the indicated time, and phosphorylation levels of eNOS Ser1177 were detected by Western Blot analysis (n = 6 independent experiments). (E,F) Candesartan (CAN) remarkably inhibited AngII-mediated downregulation of eNOS Ser1177 in HUVECs. HUVECs were pretreated with CAN (10⁻⁶ M, 3 h) and then stimulated with AngII (10⁻⁷ M, 12 h), and phosphorylation levels of eNOS Ser1177 were detected by Western Blot analysis (n = 6 independent experiments). (G,H) CAN blocked AngII-mediated (10⁻⁷ M, 12 h) downregulation of NO production in HUVECs. HUVECs were incubated with DAF-FM DA (10 μmol/L) for 30 min. The representative images were captured with a fluorescence microscope (200× magnification; n = 4 independent experiments). (I,J) AngII reduced levels of eNOS Ser1177 phosphorylation in rat mesenteric arteries, whereas the AT₁R antagonist, CAN, blocked AngII-mediated downregulation of eNOS Ser1177 phosphorylation (n = 6 rats per group). Rats received AngII infusion via osmotic minipumps for 2 weeks. CAN was administered after minipump implantation by gavage at a dose of 10 mg/kg/day during the AngII infusion period. (K) The content of NO in rat mesenteric arteries of AngII-infused rats. The content of NO was decreased in the mesenteric arteries of AngII-infused rats. CAN blocked AngII-induced downregulation of NO production (n = 3 rats per group). ^* p < 0.05 vs. Control or Sham group; ^# p < 0.05 vs. AngII group.

Figure 2

The AngII/AT₁R pathway reduces the levels of eNOS Ser1177 phosphorylation and NO production by activating protein phosphatase 2A (PP2A). (A) AngII augmented PP2A activity, while CAN blocked AngII-mediated enhancement of PP2A activity. HUVECs were pretreated with 10⁻⁶ M CAN for 3 h or not, then stimulated with 10⁻⁷ M AngII for 12 h (n = 4 independent experiments). (B) PP2A activity was increased in the mesenteric arteries of AngII-infused rats. CAN blocked AngII-induced upregulation of PP2A activity (n = 3 rats per group). (C,D) PP2A inhibitor (OA) blocked the effect of AngII on eNOS Ser1177 in vitro. HUVECs were pretreated with 10⁻⁸ M OA for 1 h or not, then stimulated with 10⁻⁷ M AngII for 12 h (n = 6 independent experiments). (E,F) OA blocked AngII-induced downregulation of NO production in HUVECs (200× magnification; n = 4 independent experiments). ^*p < 0.05 vs. Control or Sham group; ^#p < 0.05 vs. AngII group.

Figure 3

AngII/AT₁R pathway downregulates PP2Ac Tyr307 phosphorylation, which is related with ROS generation. (A,B) CAN blocked AngII-mediated downregulation of PP2Ac Tyr307 phosphorylation in HUVECs. HUVECs were pretreated with 10⁻⁶ M CAN for 3 h or not, then stimulated with 10⁻⁷ M AngII for 12 h (n = 6 independent experiments). (C,D) CAN blocked AngII-mediated downregulation of PP2Ac Tyr307 phosphorylation in rat mesenteric arteries (n = 6 rats per group). (E,F) ROS content was enhanced by AngII (10⁻⁷ M, 12 h) and abolished by pretreatment with CAN (10⁻⁶ M, 3 h). HUVECs were incubated with DCFH-DA (5 μmol/L) for 30 min, and ROS content was measure. The representative images shown were captured using a fluorescence microscope (100× magnification; n = 4 independent experiments). Pretreatment with the antioxidant, (G,H) N-acetylcysteine (NAC; 10⁻³ M, 1 h) and (I,J) Apocynin (APO; 2 × 10⁻⁵ M, 1 h), inhibited AngII/AT₁R-mediated downregulation of PP2Ac Tyr307 and eNOS Ser1177 (n = 6 independent experiments). ^*p < 0.05 vs. Control or Sham group; ^#p < 0.05 vs. AngII group.

Figure 4

AngII downregulating the levels of PP2Ac Tyr307 phosphorylation is dependent on NADPH oxidase (Nox) activation and ROS production in HUVECs. (A,B) AngII (10⁻⁷ M, 12 h) augments p22phox protein expression and pretreatment with CAN (10⁻⁶ M, for 3 h) blocked the effect of AngII (n = 6 independent experiments). (C) Silencing of p22phox protein expression by siRNA abrogated AngII-mediated increase of ROS (100× magnification; n = 4 independent experiments). (D–F) Knockdown of p22phox protein by siRNA reversed the downregulation of PP2Ac Tyr307 and eNOS Ser1177 (n = 6 independent experiments). HUVECs were transfected with control siRNA or p22phox-specific siRNA for 24 h, then treated with 10⁻⁷ M AngII for 12 h. Levels of PP2Ac Tyr307 and eNOS Ser1177 were determined by Western blot analysis. ^*p < 0.05 vs. Control group; ^#p < 0.05 vs. AngII group; ^△p < 0.05 vs. si-control group; ^▲p < 0.05 vs. p22phox siRNA group.

Figure 5

Schematic diagram of AngII-activated PP2A downregulate eNOS phosphorylation via the Nox/ROS signaling pathway.