Biochemical and Biophysical Research Communications
Roles of I2PP2A in the downregulation of eNOS Ser1177 phosphorylation by angiotensin II-activated PP2A
Introduction
Vascular endothelial cells (VECs) constitute a layer of flat epithelial cells that cover the inner surface of the vascular lumen and play critical roles in maintaining circulatory homeostasis [1]. Endothelial cells produce and release endothelium-derived contractile factors (EDCFs) and endothelium-derived relaxing factors (EDRFs); these factors exist in a dynamic equilibrium, and their imbalances can result in endothelial dysfunction (ED) [2]. Nitric oxide (NO) is an important EDRF produced along with l-citrulline from l-arginine in a reaction catalyzed by endothelial nitric oxide synthase (eNOS). eNOS/NO dysfunction is among the important manifestations of ED, and numerous studies have shown that the mechanism underlying ED may be related to a decrease in eNOS activity accompanied by reductions in NO production [3,4].
Angiotensin II (Ang II) is a key component in the renin-angiotensin system (RAS) that participates in a variety of cardiovascular diseases through its specific Ang II type 1 receptor (AT1R) [5,6]. Previous studies have reported that the chronic elevation of Ang II is associated with decreased eNOS activity and the loss of NO production, resulting in ED and hypertension [5,7]. The post-translational phosphorylation modulation of eNOS is an important mechanism to regulate its activity. Studies have shown that eNOS can be dephosphorylated through interactions with protein phosphatase 2 A (PP2A) [[8], [9], [10]].
PP2A, which is a serine/threonine phosphatase, is a heterotrimeric complex that consists of a scaffolding subunit, a regulatory subunit and a catalytic subunit, and its activity regulatory mechanisms include the binding of its inhibitors [11,12]. There are two specific endogenous inhibitors of PP2A, i.e. inhibitor 1 (I1PP2A) and inhibitor 2 (I2PP2A) [13]. Both inhibitors can bind the PP2A catalytic subunit (PP2Ac) and directly inhibit PP2A activity [11,14]. Thus far, knowledge regarding the roles of the PP2A protein inhibitors in eNOS phosphorylation and functional regulation upon Ang II stimulation is limited.
Although studies have reported the roles of eNOS in Ang II-related cardiovascular disease, the mechanism by which how Ang II triggers eNOS dysfunction remains not fully understood. In this study, we sought to explore the roles of PP2A endogenous inhibitor in the Ang II-induced PP2A activation and in the eNOS functional dysregulation.
Section snippets
Materials and reagents
Fetal bovine serum (FBS) (Biological Industries, Connecticut, USA); 0.25% Trypsin EDTA, Pen-Strep Solution, and high-glucose Dulbecco's modified Eagle's medium (DMEM; HyClone, UT, USA); Ang II (Sigma-Aldrich, St. Louis, MO, USA); candesartan (CAN; Selleck, Houston, Texas, USA); a primary antibody against eNOS Ser1177 (Millipore, Billerica, MA, USA); primary antibodies against I2PP2A and PP2Ac (Santa Cruz, CA); and a primary antibody against β-tubulin and secondary antibodies conjugated with
Ang II/AT1R pathway activation downregulates eNOS Ser1177 phosphorylation leading to a reduction in NO production
We carried out experiments with cultured human endothelial cells. We incubated the HUVECs with different concentrations of Ang II for different durations and observed the expression of total eNOS protein and eNOS Ser1177 in each group. After the treatment with Ang II at concentrations of 10−8 M, 10−7 M, 10−6 M, and 10−5 M for 12 h, the phosphorylation levels of eNOS Ser1177 were significantly lower than those after control treatment (Fig. 1-A). The phosphorylation levels of eNOS Ser1177 were
Discussion
According to previous studies, impaired eNOS/NO function is among the most important manifestations of ED [3,4,18]. Moreover, the regulation of eNOS activity is a complex process involving gene transcription, translation and posttranslational modifications. Phosphorylation and dephosphorylation are the most versatile posttranslational regulatory mechanisms of eNOS proteins. The phosphorylation of Ser1177, which is an important phosphorylation site of eNOS, can upregulate eNOS activity [19].
Conflicts of interest
The authors have no conflicts of interest to declare.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (grant No. 31460267), Key Social Development Project of Guizhou Provincial Science and Technology Department (grant SY [2013] 3019), International Cooperation Project of Guizhou Provincial Science and Technology Department (grant G [2010] 7019), and Cooperation Project of Guizhou Provincial Science and Technology Department and Guizhou Medical University (grant LH [2016]7354). YX is supported by the American Diabetes
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These authors contributed equally.