Transactivation of fetal liver kinase-1/kinase-insert domain-containing receptor by lysophosphatidylcholine induces vascular endothelial cell proliferation.
Lysophosphatidylcholine (LPC), a major lipid component of oxidized low-density lipoprotein, is a bioactive lipid molecule involved in numerous biological processes including the progression of atherosclerosis. Recently orphan G protein-coupled receptors were identified as high-affinity receptors for LPC. Although several G protein-coupled receptor ligands transactivate receptor tyrosine kinases, LPC-stimulated transactivation of receptor tyrosine kinase has not yet been reported. Here we observed for the first time that LPC treatment of human umbilical vein endothelial cells (HUVECs) induces tyrosyl phosphorylation of vascular endothelial growth factor receptor 2 [fetal liver kinase-1/kinase-insert domain-containing receptor, Flk-1/KDR)]. Flk-1/KDR transactivation by LPC was inhibited by vascular endothelial growth factor receptor tyrosine kinase inhibitors, SU1498 and 4-[(4'-chloro-2'-fluoro) phenylamino]6,7-dimethoxyquinazoline (VTKi) in immunoprecipitation. Furthermore, we examined the effects of the Src family kinases inhibitors, herbimycin A and 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2), on LPC-induced Flk-1/KDR transactivation. Results from Western blots, c-Src is involved in LPC-induced Flk-1/KDR transactivation because herbimycin A and PP2 inhibited this transactivation. Kinase-inactive (KI) Src transfection also inhibited LPC-induced Flk-1/KDR transactivation. In addition, results from Western blots, ERK1/2 and Akt, which are downstream effectors of Flk-1/KDR, were also activated by LPC, and this was inhibited by SU1498, VTKi, herbimycin A, PP2, and KI Src transfection in HUVECs. LPC-induced stimulation of HUVEC proliferation was shown to be secondary to transactivation because it was suppressed by SU1498, VTKi, herbimycin A, PP2, and KI Src transfection in dimethylthiazoldiphenyltetra-zoliumbromide assay. These findings suggest that LPC-induced Flk-1/KDR transactivation via c-Src may have important implications for the progression of atherosclerosis.
Fujita
Yoshiko
Y
Department of Pharmacology, The University of Tokushima Graduate School of Medical Sciences, 3-18-15 Kuramoto, Tokushima 770-8503, Japan. fujita56@ri.ncvc.go.jp
Yoshizumi
Masanori
M
Izawa
Yuki
Y
Ali
Nermin
N
Ohnishi
Hideki
H
Kanematsu
Yasuhisa
Y
Ishizawa
Keisuke
K
Tsuchiya
Koichiro
K
Tamaki
Toshiaki
T
D050197:Atherosclerosis / Q000473:pathology; D016227:Benzoquinones; D015153:Blotting, Western; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D002934:Cinnamates / Q000494:pharmacology; D018450:Disease Progression; D042783:Endothelial Cells / Q000166:cytology*; D004730:Endothelium, Vascular / Q000166:cytology*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D047029:Lactams, Macrocyclic; D008024:Ligands; D008244:Lysophosphatidylcholines / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D015533:Transcriptional Activation; D014162:Transfection; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics* / Q000378:metabolism*; D019061:src-Family Kinases
C412373:AG 1879; D016227:Benzoquinones; C508740:CB676475; D002934:Cinnamates; D047029:Lactams, Macrocyclic; D008024:Ligands; D008244:Lysophosphatidylcholines; D011743:Pyrimidines; D011799:Quinazolines; D011809:Quinones; C483044:SU 1498; D017828:Rifabutin; C020754:herbimycin; D011505:Protein-Tyrosine Kinases; D040301:Vascular Endothelial Growth Factor Receptor-2; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human