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

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.

[Palmitic acid decreases phosphorylation of eNOS Ser1177 by activating protein phosphatase 2C (PP2C) of human umbilical vein endothelial cells].

Objective To investigate the roles of protein phosphatase 2C (PP2C) activated by palmitic acid (PA) in the phosphorylation modulation of endothelial nitric oxide (eNOS) at the site of serine 1177 (eNOS Ser1177) in human umbilical vein endothelial cells (HUVECs). Methods HUVECs were randomly divided into control group, PA group, specific PP2C inhibitor sanguinarine (San) combined with PA group and PP2Cα specific small interference RNA (siRNA) transfection group. The protein expression of total eNOS, phosphorylated eNOS Ser1177 and PP2Cα were detected by Western blotting. The intracellular NO content was measured by DAF-FM DA. The co-localization of eNOS protein and PP2C protein was observed by co-immunoprecipitation. Results Compared with the control group, the phosphorylation levels of eNOS Ser1177 and NO content decreased significantly in the PA group. The PP2C inhibitor sanguinarine reversed PA-induced decrease of eNOS Ser1177 phosphorylation level and NO content. The phosphorylation levels of eNOS Ser1177 were enhanced dramatically after PP2Cα protein was knocked down by specific siRNA. The eNOS protein and PP2C protein co-localized in endothelial cells. Conclusion PA reduces the phosphorylation level of endothelial eNOS Ser1177 in HUVECs by activating PP2C.

Roles of I2PP2A in the downregulation of eNOS Ser1177 phosphorylation by angiotensin II-activated PP2A.

The chronic elevation of angiotensin II (Ang II) is an important cause of endothelial dysfunction (ED). The Ang II/type 1 receptor (AT1R) signaling pathway can cause endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) dysfunction through various mechanisms leading to ED. The modulation of eNOS phosphorylated at Ser1177 is an important mechanism upregulating eNOS activity. Protein phosphatase 2 A (PP2A) has been reported to dephosphorylate eNOS at Ser1177. The PP2A inhibitor 2 protein (I2PP2A) is a specific endogenous inhibitor that binds the catalytic subunit of PP2A and directly inhibits PP2A activity. Therefore, we hypothesized that Ang II might attenuate I2PP2A expression to activate PP2A, which downregulates eNOS Ser 1177 phosphorylation, leading to eNOS dysfunction. In our study, we used Ang II-treated human umbilical vein endothelial cells (HUVECs) and, found that the eNOS Ser1177 phosphorylation levels were downregulated, the activity of PP2A was increased, and I2PP2A expression was decreased. Furthermore, these effects were blocked by candesartan (CAN). The phosphorylation levels of eNOS Ser1177 were decreased after I2PP2A was knocked down by specific siRNA but increased after I2PP2A overexpression. We also found that the Ang II treatment decreased the association of I2PP2A with PP2A but increased the association between PP2A and eNOS. Taken together, our results suggest that Ang II activates PP2A by downregulating the I2PP2A expression through the AT1R signaling pathway leading to the loss of eNOS Ser1177 phosphorylation and ED.