Plasma saturated free fatty acid (FFA)-induced endothelial dysfunction (ED) contributes to the pathogenesis of atherosclerosis and cardiovascular diseases. However, the mechanism underlying saturated FFA-induced ED remains unclear. This study demonstrated that palmitic acid (PA) induced ED by activating the NADPH oxidase (NOX)/ROS signaling pathway to activate protein phosphatase 4 (PP4) and protein phosphatase 2A (PP2A), thereby reducing endothelial nitric oxide synthase (eNOS) phosphorylation at Ser633 and Ser1177, respectively. Okadaic acid (OA) and fostriecin (FST), which are inhibitors of PP2A, inhibited the PA-induced decreases in eNOS phosphorylation at Ser633 and Ser1177. The antioxidants -acetylcysteine (NAC) and apocynin (APO) or knockdown of gp91phox or p67phox (NOX subunits) restored PA-mediated downregulation of PP4R2 protein expression and eNOS Ser633 phosphorylation. Knockdown of the PP4 catalytic subunit (PP4c) specifically increased eNOS Ser633 phosphorylation, while silencing the PP2A catalytic subunit (PP2Ac) restored only eNOS Ser1177 phosphorylation. Furthermore, PA dramatically decreased the protein expression of the PP4 regulatory subunit R2 (PP4R2) but not the other regulatory subunits. PP4R2 overexpression increased eNOS Ser633 phosphorylation, nitric oxide (NO) production, cell migration and tube formation but did not change eNOS Ser1177 phosphorylation levels. Coimmunoprecipitation (Co-IP) suggested that PP4R2 and PP4c interacted with the PP4R3α and eNOS proteins. In summary, PA decreases PP4R2 protein expression through the Nox/ROS pathway to activate PP4, which contributes to ED by dephosphorylating eNOS at Ser633. The results of this study suggest that PP4 is a novel therapeutic target for ED and ED-associated vascular diseases.

中文翻译：

---

蛋白磷酸酶 4 通过降低 HUVEC 中丝氨酸 633 的 eNOS 磷酸化来介导棕榈酸诱导的内皮功能障碍


血浆饱和游离脂肪酸（FFA）诱导的内皮功能障碍（ED）有助于动脉粥样硬化和心血管疾病的发病机制。然而，饱和 FFA 诱发 ED 的机制仍不清楚。这项研究表明，棕榈酸（PA）通过激活NADPH氧化酶（NOX）/ROS信号通路激活蛋白磷酸酶4（PP4）和蛋白磷酸酶2A（PP2A），从而减少内皮一氧化氮合酶（eNOS）的磷酸化而诱导ED。分别为 Ser633 和 Ser1177。冈田酸 (OA) 和磷三星 (FST) 是 PP2A 抑制剂，可抑制 PA 诱导的 eNOS Ser633 和 Ser1177 磷酸化降低。抗氧化剂乙酰半胱氨酸 (NAC) 和罗布麻素 (APO) 或 gp91phox 或 p67phox（NOX 亚基）的敲低可恢复 PA 介导的 PP4R2 蛋白表达和 eNOS Ser633 磷酸化的下调。敲低 PP4 催化亚基 (PP4c) 会特异性增加 eNOS Ser633 磷酸化，而沉默 PP2A 催化亚基 (PP2Ac) 仅恢复 eNOS Ser1177 磷酸化。此外，PA 显着降低了 PP4 调节亚基 R2 (PP4R2) 的蛋白质表达，但不影响其他调节亚基。 PP4R2 过表达增加 eNOS Ser633 磷酸化、一氧化氮 (NO) 产生、细胞迁移和管形成，但不改变 eNOS Ser1177 磷酸化水平。免疫共沉淀 (Co-IP) 表明 PP4R2 和 PP4c 与 PP4R3α 和 eNOS 蛋白相互作用。总之，PA 通过 Nox/ROS 途径降低 PP4R2 蛋白表达，激活 PP4，从而通过使 eNOS Ser633 去磷酸化而导致 ED。这项研究的结果表明，PP4 是 ED 和 ED 相关血管疾病的新治疗靶点。