Characterisation of biological effects of a novel protein kinase D inhibitor in endothelial cells
Résumé
Vascular endothelial growth factor (VEGF) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2/KDR. Recent studies indicate that VEGF activates protein kinase D (PKD) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor, CRT5, in HUVECs. The activity of PKD isoforms 1 and 2 were blocked by this inhibitor as indicated by reduced phosphorylation at serines 916 and 876, respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB and heat shock protein 27 serine 82 (HSP27), was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 serine 82 phosphorylation. Furthermore, phosphorylation of HSP27 at serine 78, which occurs solely via the p38 MAP kinase pathway was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA. CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.
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