Department of Physiology, School of Medicine, National Yang-Ming University, Taipei, Taiwan.
J Cell Physiol. 2012 Aug;227(8):3053-62. doi: 10.1002/jcp.23052.
We investigated whether AMP-activated protein kinase (AMPK), a multi-functional regulator of energy homeostasis, participates in the regulation of erythropoietin (EPO)-mediated activation of endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs) and mice. In ECs, treatment with EPO increased the phosphorylation of AMPK, acetyl-CoA carboxylase (ACC), and eNOS, as revealed by Western blot analysis. Inhibition of AMPK activation by compound C or dominant-negative AMPK mutant abrogated the EPO-induced increase in the phosphorylation of AMPK, ACC, and eNOS, as well as nitric oxide (NO) production. Additionally, suppression of AMPK activation abolished EPO-induced EC proliferation, migration and tube formation. Immunoprecipitation analysis demonstrated that AMPK mediated the EPO-induced increase in the phosphorylation of β common receptor (βCR) and the formation of a βCR-AMPK-eNOS complex. In mice, inhibition of AMPK activation by compound C markedly decreased EPO-elicited angiogenesis in Matrigel plugs. Furthermore, the phosphorylation of AMPK and eNOS was significantly higher in aortas from EPO transgenic mice than wild-type mice. Moreover, treatment with EPO neutralizing antibody greatly reduced the exercise training-induced increase in phosphorylation of AMPK and eNOS in aortas of wild-type mice. Taken together, EPO may trigger AMPK-dependent signaling, which leads to enhanced phosphorylation of βCR and eNOS, increased βCR-AMPK-eNOS complex formation, NO production, and, ultimately, angiogenesis.
我们研究了 AMP 激活的蛋白激酶（AMPK）是否参与调节内皮细胞（ECs）和小鼠中促红细胞生成素（EPO）介导的内皮型一氧化氮合酶（eNOS）的激活。在 ECs 中，Western blot 分析显示，EPO 处理可增加 AMPK、乙酰辅酶 A 羧化酶（ACC）和 eNOS 的磷酸化。用化合物 C 或显性负性 AMPK 突变体抑制 AMPK 激活可消除 EPO 诱导的 AMPK、ACC 和 eNOS 磷酸化以及一氧化氮（NO）产生的增加。此外，抑制 AMPK 激活可消除 EPO 诱导的 EC 增殖、迁移和管形成。免疫沉淀分析表明，AMPK 介导了 EPO 诱导的β共受体（βCR）磷酸化增加和βCR-AMPK-eNOS 复合物的形成。在小鼠中，用化合物 C 抑制 AMPK 激活可显著减少 EPO 在 Matrigel 塞中的血管生成。此外，EPO 转基因小鼠的主动脉中 AMPK 和 eNOS 的磷酸化明显高于野生型小鼠。此外，用 EPO 中和抗体处理可大大降低野生型小鼠主动脉中运动训练诱导的 AMPK 和 eNOS 磷酸化的增加。总之，EPO 可能触发 AMPK 依赖性信号通路，导致βCR 和 eNOS 的磷酸化增强，βCR-AMPK-eNOS 复合物形成增加，NO 产生增加，最终导致血管生成。
