title: Characterization of the biological effects of a novel protein kinase D inhibitor in endothelial cells.
abstract: VEGF (vascular endothelial growth factor) 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 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) 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 (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein 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 (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.
authors.affiliation: Department of Medicine, Centre for Cardiovascular Biology and Medicine, The Rayne Institute, University College London, UK. i.evans@ucl.ac.uk
authors.forename: Ian M
authors.initials: IM
authors.lastname: Evans
authors.forename: Azadeh
authors.initials: A
authors.lastname: Bagherzadeh
authors.forename: Mark
authors.initials: M
authors.lastname: Charles
authors.forename: Tony
authors.initials: T
authors.lastname: Raynham
authors.forename: Chris
authors.initials: C
authors.lastname: Ireson
authors.forename: Alexandra
authors.initials: A
authors.lastname: Boakes
authors.forename: Lloyd
authors.initials: L
authors.lastname: Kelland
authors.forename: Ian C
authors.initials: IC
authors.lastname: Zachary
mesh_terms: D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D013379:Substrate Specificity; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
chemical_list: D047428:Protein Kinase Inhibitors; D042461:Vascular Endothelial Growth Factor A; C089212:protein kinase D; D040301:Vascular Endothelial Growth Factor Receptor-2; D011493:Protein Kinase C