State Key Laboratory of Natural Medicines, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, China.
Department of Pediatrics, Nanjing Maternal and Child Health Hospital of Nanjing Medical University, Nanjing, China.
PLoS One. 2013 Dec 9;8(12):e81220. doi: 10.1371/journal.pone.0081220. eCollection 2013.
The migration of endothelial cells has been regarded as a potential target for the treatment of angiogenesis-related diseases. Previously, we demonstrated that norisoboldine (NOR), an alkaloid compound isolated from Radix Linderae, can significantly suppress synovial angiogenesis by selectively inhibiting endothelial cell migration. In this study, we evaluated the importance of various pathways in VEGF-induced endothelial cell migration using specific inhibitor. VEGF-induced endothelial cell migration and sprouting were significantly inhibited by H-89 (an inhibitor of protein kinase A (PKA)) but not by inhibitors of other pathways. NOR markedly suppressed VEGF-induced intracytoplasmic cAMP production and PKA activation and thereby down-regulated the activation of downstream components of the PKA pathway, including enzymes (src, VASP and eNOS) and the transcription factor NF-κB. Moreover, the transcription activation potential of NF-κB, which is related to IκBα phosphorylation and the disruption of the p65/IκBα complex, was reduced by NOR. Meanwhile, NOR selectively inhibited the expression of p-p65 (ser276) but not p-p65 (ser536) or PKAc, indicating that PKAc participates in the regulation of NF-κB by NOR. Co-immunoprecipitation and immunofluorescence assays confirmed that NOR inhibited the formation of the PKAc/p65 complex and thereby decreased p65 (ser276) phosphorylation to prevent p65 binding to DNA. Docking models indicated that the affinity of NOR for PKA was higher than that of the original PKA ligand. Moreover, the fact that H-89 improved Notch1 activation, but DAPT (an inhibitor of Notch) failed to affect PKA activation, suggested that PKA may act on upstream of Notch1. In conclusion, the inhibitory effects of NOR on endothelial cell migration can be attributed to its modulation of the PKA pathway, especially on the processes of p65/IκBα complex disruption and PKAc/p65 complex formation. These results suggest that NOR inhibit VEGF-induced endothelial cell migration via a cAMP-PKA-NF-κB/Notch1 signaling pathway.
内皮细胞的迁移已被视为治疗血管生成相关疾病的潜在靶点。此前，我们证明，从乌药中分离得到的生物碱化合物去甲乌药碱（NOR）可通过选择性抑制内皮细胞迁移，显著抑制滑膜血管生成。在这项研究中，我们使用特定抑制剂评估了 VEGF 诱导的内皮细胞迁移中各种途径的重要性。H-89（蛋白激酶 A（PKA）抑制剂）显著抑制了 VEGF 诱导的内皮细胞迁移和发芽，但对其他途径的抑制剂没有抑制作用。NOR 显著抑制了 VEGF 诱导的细胞内 cAMP 产生和 PKA 激活，并由此下调了 PKA 途径下游成分的激活，包括酶（src、VASP 和 eNOS）和转录因子 NF-κB。此外，与 IκBα磷酸化和 p65/IκBα复合物的破坏有关的 NF-κB 的转录激活潜能也被 NOR 降低。同时，NOR 选择性地抑制了 p-p65（ser276）的表达，而不是 p-p65（ser536）或 PKAc 的表达，表明 PKAc 参与了 NOR 对 NF-κB 的调节。免疫共沉淀和免疫荧光分析证实，NOR 抑制了 PKAc/p65 复合物的形成，从而降低了 p65（ser276）的磷酸化，防止了 p65 与 DNA 的结合。对接模型表明，NOR 与 PKA 的亲和力高于原始 PKA 配体。此外，H-89 改善了 Notch1 激活，但 DAPT（Notch 的抑制剂）未能影响 PKA 激活的事实表明，PKA 可能作用于 Notch1 的上游。总之，NOR 对内皮细胞迁移的抑制作用可归因于其对 PKA 途径的调节，特别是对 p65/IκBα 复合物破坏和 PKAc/p65 复合物形成过程的调节。这些结果表明，NOR 通过 cAMP-PKA-NF-κB/Notch1 信号通路抑制 VEGF 诱导的内皮细胞迁移。
