Thrombomodulin prolongs thrombin-induced extracellular signal-regulated kinase phosphorylation and nuclear retention in endothelial cells.
On endothelial cells, thrombin binds to thrombomodulin (TM), an integral membrane-bound glycoprotein, and to protease-activated receptors (PARs). Thrombin binding to TM modulates endothelial cell and smooth muscle cell proliferation mediated through PAR1. We studied the phosphorylation and nuclear translocation of extracellular signal-regulated kinases (ERKs) 1 and 2 in human umbilical vein endothelial cells activated by thrombin. Thrombin and thrombin receptor-activating peptide (TRAP)-induced DNA synthesis were significantly inhibited by PD98059, an inhibitor of ERK phosphorylation. Immunoblots of phosphorylated ERKs (pERKs) and immunocytochemical studies of pERK localization revealed differences in the signal generated by thrombin and TRAP. After a short activation (15 minutes), the phosphorylation and the intracellular localization of pERKs were the same with the 2 agonists. After 4 hours, however, pERKs were visualized in the nuclei of thrombin-activated cells but barely detectable in TRAP-activated cells. Moreover, after 4 hours, the pERKs were visualized in the nuclei of cells stimulated by TRAP in the presence of a thrombin mutant that bound to TM, whereas they were around the nuclei in cells stimulated by thrombin in the presence of a monoclonal antibody preventing thrombin binding to TM. The results demonstrate that ERKs are involved in human umbilical vein endothelial cell DNA synthesis mediated by PAR agonists, that the duration of pERK nuclear retention is in inverse ratio to the mitogenic response, and that in addition to its role in the regulation of blood coagulation, TM acts as a thrombin receptor that modulates the duration of pERK nuclear retention and cell proliferation in response to thrombin.
Olivot
J M
JM
Unité INSERM 428, Faculté de Pharmacie, Université René Descartes, Paris, France.
Estebanell
E
E
Lafay
M
M
Brohard
B
B
Aiach
M
M
Rendu
F
F
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002384:Catalysis; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000494:pharmacology; D018179:Receptors, Thrombin / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013917:Thrombin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D018180:Thrombomodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013936:Thymidine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D005419:Flavonoids; D011506:Proteins; D018179:Receptors, Thrombin; D018180:Thrombomodulin; D004247:DNA; D020928:Mitogen-Activated Protein Kinases; D013917:Thrombin; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D013936:Thymidine