VEGF and thrombin induce MKP-1 through distinct signaling pathways: role for MKP-1 in endothelial cell migration.
We have previously reported that MAPK phosphatase-1 (MKP-1/CL100) is a thrombin-responsive gene in endothelial cells (ECs). We now show that VEGF is another efficacious activator of MKP-1 expression in human umbilical vein ECs. VEGF-A and VEGF-E maximally induced MKP-1 expression in ECs; however, the other VEGF subtypes had no effect. Using specific neutralizing antibodies, we determined that VEGF induced MKP-1 specifically through VEGF receptor 2 (VEGFR-2), leading to the downstream activation of JNK. The VEGF-A(165) isoform stimulated MKP-1 expression, whereas the VEGF-A(162) isoform induced the gene to a lesser extent, and the VEGF-A(121) isoform had no effect. Furthermore, specific blocking antibodies against neuropilins, VEGFR-2 coreceptors, blocked MKP-1 induction. A Src kinase inhibitor (PP1) completely blocked both VEGF- and thrombin-induced MKP-1 expression. A dominant negative approach revealed that Src kinase was required for VEGF-induced MKP-1 expression, whereas Fyn kinase was critical for thrombin-induced MKP-1 expression. Moreover, VEGF-induced MKP-1 expression required JNK, whereas ERK was critical for thrombin-induced MKP-1 expression. In ECs treated with short interfering (si)RNA targeting MKP-1, JNK, ERK, and p38 phosphorylation were prolonged following VEGF stimulation. An ex vivo aortic angiogenesis assay revealed a reduction in VEGF- and thrombin-induced sprout outgrowth in segments from MKP-1-null mice versus wild-type controls. MKP-1 siRNA also significantly reduced VEGF-induced EC migration using a transwell assay system. Overall, these results demonstrate distinct MAPK signaling pathways for thrombin versus VEGF induction of MKP-1 in ECs and point to the importance of MKP-1 induction in VEGF-stimulated EC migration.
Kinney
Corttrell M
CM
Department of Cell Biology, Lerner Research Institute and Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio, USA.
Chandrasekharan
Unni M
UM
Mavrakis
Lori
L
DiCorleto
Paul E
PE
D000818:Animals; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D054638:Dual Specificity Phosphatase 1 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D018919:Neovascularization, Physiologic / Q000187:drug effects; D039921:Neuropilins / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D051076:Proto-Oncogene Proteins c-fyn / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013917:Thrombin / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D039921:Neuropilins; D010446:Peptide Fragments; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; C082835:thrombin receptor peptide (42-47); D040301:Vascular Endothelial Growth Factor Receptor-2; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1; C517020:Dusp1 protein, mouse; D013917:Thrombin