title: Norisoboldine suppresses VEGF-induced endothelial cell migration via the cAMP-PKA-NF-κB/Notch1 pathway.
abstract: 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.
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Lu
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, China.
authors.forename: Bei
authors.initials: B
authors.lastname: Tong
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, China.
authors.forename: Yubin
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Department of Pediatrics, Nanjing Maternal and Child Health Hospital of Nanjing Medical University, Nanjing, China.
authors.forename: Li
authors.initials: L
authors.lastname: Sha
authors.affiliation: Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
authors.forename: Guixin
authors.initials: G
authors.lastname: Chou
authors.affiliation: Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
authors.forename: Zhengtao
authors.initials: Z
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, China.
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Xia
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Dai
mesh_terms: D000470:Alkaloids / Q000494:pharmacology*; D015153:Blotting, Western; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D016328:NF-kappa B / Q000378:metabolism*; D051881:Receptor, Notch1 / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: D000470:Alkaloids; D016328:NF-kappa B; D051881:Receptor, Notch1; D042461:Vascular Endothelial Growth Factor A; C464745:norisoboldine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases