doi: 10.1161/hh0701.089956
© 2001 American Heart Association, Inc.
Editorial |
Thrombin, Thrombomodulin, and Extracellular Signal–Regulated Kinases Regulating Cellular Proliferation
From the Departments of Pharmacology and Medicine, Georgetown University, Washington, DC.
Correspondence to Jane E. Freedman, MD, Georgetown University, Med-Dent NE 403, 3900 Reservoir Rd NW, Washington, DC 20007. E-mail freedmaj{at}georgetown.edu
Key Words: thrombomodulin • thrombin • mitogen-activated protein kinase
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Introduction |
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 Top Introduction References |
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Thrombin, a coagulation protease, is primarily known for its regulation of hemostasis and thrombosis. However, this enzyme also plays important roles in wound healing and pathological situations, such as inflammation and tumorigenesis. In addition, stimulation of the thrombin receptor signals many cellular events that are associated with the response to vascular injury, including smooth muscle cell proliferation.1 Thrombin activates platelets and regulates the actions of other cells by means of G protein–coupled protease-activated receptors (PARs). PAR1 is activated when thrombin binds to and cleaves the amino terminal extension of its receptor to create a new receptor amino terminus that functions as a tethered ligand (Figure
).2
This new amino terminus then binds intramolecularly to the body of the
receptor to effect transmembrane
signaling.3 In response to
stimulation with thrombin, endothelial cells release
prostaglandin I2 and von
Willebrand factor and undergo cellular
proliferation.
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Thrombomodulin (TM), a membrane-bound
glycoprotein expressed on endothelial
cells, has a high affinity of binding to thrombin and converts thrombin
from a procoagulant to an anticoagulant. TM binds to thrombin and
changes the enzyme’s conformation, allowing thrombin to
activate protein C
(Figure
).
Plasma-soluble TMs are cleaved products of cellular TM that also
have anticoagulant and antifibrinolytic properties, and plasma levels
may reflect the level of endothelial TM
expression.4 Prospective
studies show that high plasma TM levels are associated with a low risk
of developing coronary heart
disease.4 It has also been
shown that overexpression of TM on smooth muscle cells downregulates
proliferation,5 suggesting
that TM could regulate endothelial and smooth muscle
cell proliferation via PAR1.
Cellular proliferation is also regulated by specific mitogen-activated protein kinases (MAPKs) that translocate into nuclei after activation and play critical roles in connecting the signal to gene expression and allowing cell-cycle entry. MAPKs (p42/p44 MAPK, also called extracellular signal–regulated kinase [ERK] 1 and ERK2) are key mediators of signal transduction from the cell surface to the nucleus. Activation of p42/p44 MAPK required for transduction of mitogenic signaling is associated with a rapid nuclear translocation of these kinases.6 Addition of the pharmacological MAPK inhibitor PD 98059 has been shown to block activation of the p42/p44 MAPK pathway, impeding its nuclear accumulation. It is believed that MAPK nuclear translocation requires both activation of the p42/p44 MAPK module and neosynthesis of short-lived proteins thought to be nuclear anchors.7 MAPK modules, composed of three protein kinases activated by successive phosphorylation, are involved in the signal transduction of a wide range of extracellular agents. MAPK is only active when both tyrosyl and threonyl residues are phosphorylated, suggesting that the enzyme functions in vivo to integrate signals from two distinct transduction pathways.8 In mammalian cells, mitogenic stimulation triggers the translocation of p42/p44 MAPK from the cytoplasm to the nucleus, whereas the other protein kinases of the module remain cytosolic. Because MAPK has been shown to phosphorylate and activate nuclear targets, such as the transcription factor Elk1, MAPK nuclear translocation is thought to represent a critical step in signal transduction. Sequestering MAPK in the cytoplasm does not alter its activation or its ability to phosphorylate cytoplasmic substrates of MAPK. However, prevention of MAPK nuclear translocation strongly inhibits Elk1-dependent gene transcription and the ability of cells to reinitiate DNA replication in response to growth factors. Thus, the relocalization of MAPK to the nucleus seems to be an important regulatory step for mitogen-induced gene expression and cell-cycle reentry.9
Stimulation of the MAPK pathway results in nuclear translocation and activation of various transcriptional events leading to cell cycling.9 In addition, cellular stimulation by thrombin can lead to cellular proliferation, and TM may modulate this action. However, the role of the MAPK pathway in regulating the thrombin/TM-induced proliferative response is unknown. The study by Olivot et al10 in this issue of Circulation Research is intriguing, because it suggests a novel mechanism for TM-dependent modulation of cellular proliferation attributable to thrombin stimulation. In this study,10 the authors use thrombin, thrombin receptor–activating peptide (TRAP), a thrombin mutant that binds to TM, and a monoclonal antibody blocking thrombin-TM binding to demonstrate that the interaction of thrombin with TM modulates nuclear retention of phosphorylated ERKs. TRAPs are synthetic peptide agonists for receptor activation even after thrombin cleavage.11 TRAP does not bind to TM; therefore, the differences in activation between TRAP and thrombin can be examined to evaluate thrombin receptor–independent effects. TM is known to bind to PAR, but its action as a thrombin receptor that can regulate the duration of phosphorylated ERK nuclear retention and potentially cellular proliferation in response to thrombin is a potential mechanism for the antiproliferative effects of TM.
Additionally, in the study by Olivot et al,10 the phosphorylation and nuclear translocation of ERK1 and ERK2 in human umbilical vein endothelial cells activated by thrombin were investigated. In this study, as differentiated by experiments using TRAP and thrombin, TM and PAR binding were necessary for sustained nuclear retention of phosphorylated ERK. Again, this finding was confirmed using a thrombin mutant that binds to TM and, conversely, prevented using a monoclonal antibody that prevents thrombin binding to TM. Interestingly, after brief activation, phosphorylation and intracellular localization of phosphorylated ERKs were similar with the two agonists; however, after 4 hours, phosphorylated ERKs were enhanced in the nuclei of thrombin-activated, compared with TRAP-activated, cells. In a previous study of senescent cells, localizing ERK2 in the nuclei restored c-fos transcriptional activity on growth stimuli.12 Furthermore, the nuclear localization of ERK1 and ERK2 potentiated the proliferative activity of the cells, and restoration of nuclear relocalization of active ERK1 and ERK2 was essentially required for the reinitiation of DNA synthesis.12 These findings are consistent with other studies showing that phosphorylation and intracellular localization of phosphorylated ERKs enhance transcriptional activity and hence mitogenic stimulation.9 Therefore, a problem with the present study10 is the finding that the nuclear retention of phosphorylated ERK was enhanced after stimulation with thrombin compared with TRAP. These effects were not seen until 4 hours and were relatively modest; however, as discussed, enhanced phosphorylation of ERK and nuclear retention has been associated with enhanced transcriptional events in many previous studies compared with the inverse relationship seen in this study. This is a curious inconsistency that is not readily explained.
Although most previous studies suggest that the effects of TM are antiproliferative, the data are not entirely consistent. In a study of recombinant human TM, the peptide exhibited mitogenic activity in Swiss 3T3 cells, and in cultured vascular smooth muscle cells, recombinant TM also accelerated [3H]thymidine uptake into DNA in a dose-dependent manner.13 This mitogenic activity was abolished by addition of polyclonal anti-human TM antibody. Finally, recombinant TM treatment increased the level of phosphorylated MAPK in smooth muscle cells. Therefore, in contrast to the previously discussed studies,4 5 these results suggest that TM expression in atherosclerotic lesions may be associated with promotion of atherosclerosis through its mitogenic activity in vascular smooth muscle cells.13 The complexity of the role of TM in the regulation of cellular proliferation is consistent with the conflicting findings of Olivot et al.10
The potential for TM in modulating the proliferative response, therefore, is intriguing but unclear. Because thrombin is a mitogenic mediator of smooth muscle cells in vascular injury, inhibition of its function in vivo could help prevent smooth muscle cell hyperplasia, and the success of additional studies could lead to use of recombinant TM for modulating arterial restenosis.14 It has also been shown that thrombin causes increased contraction and proliferation in smooth muscle cells of saphenous veins compared with the internal mammary artery independent of tethered receptors.15 This suggests differential effects of thrombin in various blood vessels and reinforces the complexity regulating vascular cell proliferation in vivo. Therefore, understanding the mechanisms for the differences in thrombin receptor activation is crucial in the development of therapeutics that can successfully modulate the proliferative response in blood vessels in response to atherogenic and mechanical stimuli.
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Acknowledgments |
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This work was supported by National Institutes of Health Grants RO1HL62267 and AG08226.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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TopIntroductionReferences |
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