Department of Physiology, National Yang-Ming University, Taipei, Taiwan.
J Cell Physiol. 2011 Dec;226(12):3330-9. doi: 10.1002/jcp.22678.
Erythropoietin (EPO), the key hormone for erythropoiesis, also increases nitric oxide (NO) bioavailability in endothelial cells (ECs), yet the definitive mechanisms are not fully understood. Increasing evidence has demonstrated that β common receptor (βCR) plays a crucial role in EPO-mediated non-hematopoietic effects. We investigated the role of βCR in EPO-induced endothelial NO synthase (eNOS) activation in bovine aortic ECs (BAECs) and the molecular mechanisms involved. Results of confocal microscopy and immunoprecipitation analyses revealed that βCR was colocalized and interacted with EPO receptor (EPOR) in ECs. Inhibition of βCR or EPOR by neutralizing antibodies or small interfering RNA abolished the EPO-induced NO production. Additionally, blockage of βCR abrogated the EPO-induced increase in the phosphorylation of eNOS, Akt, Src, or Janus kinase 2 (JAK2). Immunoprecipitation analysis revealed that treatment with EPO increased the interaction between βCR and eNOS, which was suppressed by inhibition of Src, JAK2, or Akt signaling with specific pharmacological inhibitors. Furthermore, EPO-induced EC proliferation, migration, and tube formation were blocked by pretreatment with βCR antibody and Src, JAK2, or PI3K/Akt inhibitors. Moreover, in vivo experiments showed that EPO increased the level of phosphorylated eNOS, Src, JAK2, and Akt, as well as βCR-eNOS association in aortas and promoted the angiogenesis in Matrigel plug, which was diminished by βCR or EPOR neutralizing antibodies. Our findings suggest that βCR may play an integrative role in the EPO signaling-mediated activation of eNOS in ECs.
促红细胞生成素（EPO）是红细胞生成的关键激素，也能增加内皮细胞（ECs）中一氧化氮（NO）的生物利用度，但确切的机制尚不完全清楚。越来越多的证据表明，β共同受体（βCR）在 EPO 介导的非造血作用中发挥着关键作用。我们研究了βCR 在 EPO 诱导的牛主动脉 ECs（BAECs）内皮型一氧化氮合酶（eNOS）激活中的作用及其相关的分子机制。共聚焦显微镜和免疫沉淀分析的结果表明，βCR 与 ECs 中的 EPO 受体（EPOR）共定位并相互作用。用中和抗体或小干扰 RNA 抑制βCR 或 EPOR 可消除 EPO 诱导的 NO 产生。此外，阻断βCR 可消除 EPO 诱导的 eNOS、Akt、Src 或 Janus 激酶 2（JAK2）磷酸化增加。免疫沉淀分析表明，EPO 处理增加了βCR 与 eNOS 之间的相互作用，而 Src、JAK2 或 Akt 信号的特异性药理学抑制剂的抑制作用则抑制了这种相互作用。此外，用βCR 抗体和 Src、JAK2 或 PI3K/Akt 抑制剂预处理可阻断 EPO 诱导的 EC 增殖、迁移和管形成。此外，体内实验表明，EPO 增加了磷酸化 eNOS、Src、JAK2 和 Akt 的水平以及主动脉中βCR-eNOS 的结合，并促进了 Matrigel 塞中的血管生成，而用βCR 或 EPOR 中和抗体则减弱了这一作用。我们的研究结果表明，βCR 可能在 EPO 信号转导介导的 ECs 中 eNOS 的激活中发挥整合作用。
