1British Heart Foundation Cardiovascular Medicine Unit, Eric Bywaters Centre, Imperial College London, Hammersmith Hospital, London, United Kingdom; 2Institute of Molecular Oncology, Showa University, Tokyo, Japan; and 3Cardiovascular Institute, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois
Submitted 13 October 2004 ; accepted in final form 2 August 2005
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ABSTRACT |
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cytoprotection; proteinase-activated receptor 1
Cell surface receptors for thrombin are members of the proteinase-activated receptor (PAR) family of G protein-coupled, seven-transmembrane receptors activated after proteolytic cleavage and exposure of a tethered ligand (9). Of the four members of the PAR family, only PAR1, PAR3, and PAR4 are cleaved by thrombin. PAR1 is the primary thrombin receptor, although it is also cleaved by factor Xa and activated protein C (54). PAR2, while not cleaved directly by thrombin, can be activated by proteinases, including trypsin and mast cell tryptase. In addition, it has been reported that PAR2 can be transactivated by the thrombin-cleaved, PAR1-tethered ligand, at least in the presence of PAR1 antagonists (47). Expression of PAR2, but not PAR1, is upregulated in ECs by TNF-, IL-1
, and LPS (46). PAR3 is poorly expressed on ECs and PAR4, is undetectable on human umbilical vein ECs (HUVECs), and is expressed by human arterial ECs (15), where expression is regulated by proinflammatory cytokines (17). Although no function has been established in human ECs, PAR4 plays a critical role in human platelet responses to a high concentration of thrombin (23) and in thrombin-induced responses in rodent ECs (26).
PAR-mediated signaling via G proteins activates multiple downstream pathways, including those regulated by MAPKs, PKC, and phosphatidylinositol 3-kinase (PI3-kinase)/Akt, which may act sequentially or in parallel (30, 41). Thrombin induces rapid phosphorylation of ERK1/2, p38 MAPK, and JNK, all of which have been implicated in thrombin-mediated responses (34, 49). Thrombin has been shown to use distinct PKC isozymes, including PKC-, -
, -
, and -
. Thus thrombin-induced intercellular adhesion molecule-1 (ICAM-1) is PKC-
dependent (52), thrombin-induced VCAM-1 requires activation of both PKC-
and -
, and thrombin-mediated changes in vascular permeability follow activation of PKC-
(39). Using specific agonist peptides, EC PAR1 and PAR2 have been reported to use the same signaling pathways. However, functional differences have been identified downstream; for example, only ligation of PAR1 induces monocyte chemoattractant protein-1 (MCP-1) (54) and alters vascular permeability (28), reflecting prolonged activation of RhoA downstream of PAR1 but not PAR2 (66).
Located at the blood-tissue interface, vascular endothelium is continually exposed to autologous bystander injury by complement components, a risk that is enhanced during inflammation. Furthermore, complement activation has an established role in acute coronary syndromes (20) and may also be involved in the pathogenesis of atherosclerosis and myocardial infarction (45, 62). To combat this pathology, both constitutive and inducible cytoprotective mechanisms have evolved to limit complement-mediated injury, including both cell surface and soluble regulatory proteins (32). The membrane-bound complement-inhibitory proteins DAF (CD55), membrane cofactor protein (MCP) (CD46), and cluster of differentiation (CD59) use distinct mechanisms for the regulation of complement and may act cooperatively (32). DAF is a multifunctional, glycosyl phosphatidylinositol-anchored cell surface glycoprotein, which, by acting at the level of the C3 convertase, interferes with the pivotal step of the complement cascade. The cytoprotective importance of DAF is revealed by the increased susceptibility of DAF-deficient mice to glomerulonephritis and ischemia-reperfusion injury (31, 60, 67). In recent EC studies, we have demonstrated the induction of DAF by TNF- and IFN-
(38), thrombin (30), VEGF, and basic FGF (36, 37) and by 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (35). The upregulation of DAF protected ECs against C3 deposition and complement-mediated cell lysis.
Although evidence for the induction of EC cytoprotective genes by thrombin is emerging, the identity of the specific PARs and downstream signaling pathways facilitating these responses remain to be defined completely. However, PAR1 and PAR2 have been linked to protective responses in murine models of colitis, bronchoconstriction, and distal ischemia (7, 13, 16, 40). In this study, we have used thrombin-induced DAF expression in ECs as a means by which to explore the role of the PARs and PKC in thrombin-mediated cytoprotection against complement activation. We provide further evidence in support of the hypothesis that cleaved PAR1 may transactivate PAR2, and we describe a novel, thrombin-activated, PAR2/PKC-/MAPK-cytoprotective pathway in human vascular endothelium.
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MATERIALS AND METHODS |
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Cell isolation and culture. Our human tissue protocols were approved by the hospital Research Ethics Committee. HUVECs were isolated from umbilical cords as described previously (30) and cultured in gelatin-coated tissue culture flasks Costar (Cambridge, MA) in medium-199 (M-199) supplemented with 20% FBS, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine (Invitrogen, Paisley, UK), 10 U/ml heparin, and 30 µg/ml EC growth supplement (ECGS; Sigma). For each experiment, ECs were plated in M-199 containing 10% FBS, 5 U/ml heparin, and 15 µg/ml ECGS for 24 h, and the medium was changed to M-199 containing 5% FBS, 2.5 U/ml heparin, and 7.5 µg/ml ECGS for the duration of the experiment.
Flow cytometry. Flow cytometry was performed as previously described (30). Pharmacological antagonists were added 60 min before the addition of agonist, and samples were analyzed using an Epics XL-MCL flow cytometer (Beckman Coulter), which enabled us to count 5,000 cells/sample. Results are expressed as the relative fluorescence intensity (RFI), which represents the mean fluorescence intensity (MFI) with test MAb divided by the MFI using an isotype-matched irrelevant MAb. To allow the results of multiple experiments to be used, the data in some figures are expressed as the percentage of the RFI for thrombin-treated ECs or for ECs treated with inhibitor alone.
Adenovirus infection.
The generation of adenovirus (Adv) expression vectors for dominant-negative (DN) and constitutively active (CA) PKC isozymes was described previously (19, 59). DN-PKC-, DN-PKC-
, CA-PKC-
, and
-gal-Adv were amplified in human embryonic kidney HEK-293A cells and purified using the BD Adeno-X purification kit (BD Biosciences, Oxford, UK). Viral titers were estimated using the BD Adeno-X Rapid Titer kit. HUVECs were infected at between 50 and 200 infectious units (IFU)/cell in serum-free M-199 for 2 h before the medium was replaced with M-1995% FBS, 2.5 U/ml heparin, and 7.5 µg/ml ECGS. Infected ECs were stimulated with thrombin and PAR peptides 24 h postinfection. Infection of HUVECs with a
-gal control Adv demonstrated a transfection efficiency of 95%.
Western blot analysis. HUVECs were cultured for 2 h in 1% BSA to reduce basal phosphorylation and preincubated with MAPK or PKC antagonists for 1 h before stimulation with thrombin for 7 min and lysis in 4 mM EDTA, 50 mM Tris·HCl, pH 7.4, in 150 mM NaCl with 25 mM sodium deoxycholic acid, 200 µM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 1% Triton X-100, 1 mM PMSF, and 5% protease inhibitor cocktail (Sigma). After being subjected to SDS-PAGE, separated proteins were transferred to Immobilon P membranes (Millipore, Bedford, MA) and blocked for 1 h at room temperature in BSA-0.1% Tween 20 (vol/vol) with 5% milk powder wt/vol. The membrane was incubated with primary Abs overnight at 4°C, washed in TBS-0.1% Tween 20, and incubated for 1 h at room temperature with appropriate peroxidase-labeled secondary Abs developed with an ECL substrate (Amersham Pharmacia Biotech, Little Chalfont, UK), and exposed to autoradiographic film. When appropriate, blots were stripped and reprobed for detection of the nonphosphorylated form of MAPK. Equal loading was achieved by estimating the lysate protein content using the Bio-Rad Dc protein assay (Bio-Rad, Hercules, CA) and Ponceau staining of membranes before analysis. Integrated density values were measured using the ChemiImager 5500 (Alpha Innotech, San Leandro, CA).
Statistics. Differences between results were evaluated by performing ANOVA with the Bonferroni multiple-comparison test using GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA). Differences of P < 0.05 were considered statistically significant.
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RESULTS |
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The importance of PKC- in DAF induction was confirmed by infecting HUVECs with an adenovirus expressing a constitutively active form of PKC-
(CA-PKC-
). CA-PKC-
significantly upregulated DAF expression (Fig. 2), and this effect was inhibited by the presence of the MEK-1 antagonist U0126 (data not shown). The specificity of the response was confirmed using Western blot analysis, which demonstrated that overexpression of PKC-
increased the activated, phosphorylated form of PKC-
in a dose-dependent manner without altering the expression of other PKC isozymes (data not shown). In addition, overexpression of a constitutively active form of PKC-
did not induce DAF expression.
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To investigate the possibility that thrombin-mediated activation of pro-MMP is involved in DAF upregulation (29), further experiments were performed in the presence of the MMP antagonist GM 6001 and its matched negative control. At concentrations up to 1 µM, GM 6001 failed to inhibit thrombin-induced DAF expression with RFI ± SE for untreated ECs, 26.5 ± 1.3; for thrombin-treated ECs, 63.3 ± 3.0; for thrombin with GM 6001, 61.5 ± 2.5; and for thrombin with GM 6001 negative control, 62.0 ± 2.7. The activity of GM 6001 was confirmed by its ability, at all concentrations used, to inhibit EC MMP-2 activation by phorbol dibutyrate as assessed using gelatin zymography (data not shown).
PAR1 and PAR2 use distinct PKC isozymes for DAF upregulation.
Dominant-negative adenoviral constructs were used to further investigate the role of PKC isozymes downstream of PAR1 and PAR2 in DAF regulation. HUVECs were incubated with Adv for 2 h in serum-free medium before culture overnight in HUVEC medium supplemented with TNF- to upregulate PAR2 expression as appropriate. ECs were then exposed to thrombin, SFLLRN, and SLIGKV, alone or in combination, and DAF expression was analyzed using flow cytometry after an additional 20 h. TNF-
alone had no significant effect on DAF expression (data not shown). Thrombin and PAR2-induced DAF upregulation were significantly reduced by DN-PKC-
(Fig. 4A). Surprisingly, however, the induction of DAF by the PAR1 peptide was not significantly inhibited, suggesting the use of an alternative PKC isozyme. In subsequent experiments, we sought to identify the PKC isozymes involved in PAR1-mediated upregulation of DAF. Significant inhibition of this response was observed after expression of the DN-PKC-
construct in HUVECs (Fig. 4B). In contrast, no reduction of PAR2-induced DAF was observed. For all experiments, the data are presented as a percentage of appropriately matched controls to account for any changes in basal DAF expression. In addition, a
-gal control Adv was included that had no consistent effect on either basal or induced DAF expression. The results of these experiments provide further evidence for a role for PAR2, as well as for PAR1, in the regulation of DAF expression by thrombin.
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Thrombin-induced MAPK pathways. Thrombin may activate ERK1/2, p38, and JNK MAPK. However, the relationship between these pathways is complex, may vary between different cell types, and remains to be defined fully (50). In previous work, we demonstrated that thrombin-induced DAF expression is abrogated by pretreatment with pharmacological inhibitors of MEK-1 (PD-98059 or U0126), which prevent ERK1/2 activation, and by SB-202190, which inhibits p38 MAPK (30). Furthermore, as shown in Fig. 6A, pretreatment of HUVECs with pharmacological antagonists of JNK, JNK inhibitor II (SP-600125), and JNK inhibitor I, a cell-permeable peptide inhibitor, significantly inhibited thrombin-induced DAF expression. In addition, the ERK1/2, p38, and JNK MAPK activation inhibitors also abrogated the DAF upregulation observed after treatment with the PAR1 and PAR2 APs (data not shown).
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DISCUSSION |
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The complement-inhibitory protein DAF interferes with the pivotal step of the complement cascade, in which an absolute excess of C3 is required for further activation of the cascade. We have previously shown that expression of DAF on human ECs is increased up to threefold by thrombin, resulting in a significant reduction in C3 deposition and subsequent complement-mediated lysis, thus providing enhanced resistance against complement-mediated injury (30). These observations, and the data presented herein, indicate the presence of an additional protective mechanism activated by thrombin and mediated by the transactivation of PAR2 and signaling via PKC-. However, the net effect of thrombin on the vascular endothelium is influenced by a variety of factors, including the local microenvironment and the balance between proinflammatory and cytoprotective pathways (Fig. 7).
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The importance of the MAPK enzymes ERK1/2, p38, and JNK in thrombin signaling pathways led us to explore further their role in thrombin-induced DAF regulation and their relationship to PKC. Thrombin induced the phosphorylation of these MAPKs in HUVECs, and inhibition of the activation of JNK, ERK1/2, and p38 MAPK prevented DAF induction (30). Cross-inhibition studies revealed interdependence between ERK1/2 and JNK because inhibition of either kinase also prevented the activation of the other by thrombin. Although we cannot completely exclude a lack of specificity, the antagonists of ERK1/2 and JNK activation have not been reported to inhibit other MAPK enzymes significantly at the concentrations used. Moreover, evidence for complex, activating, or inhibitory cross-talk pathways between MAPK enzymes has been identified previously. Inhibition of ERK1/2 also prevents VEGF and thrombin-mediated activation of JNK in ECs (50). Furthermore, cross-talk between JNK and ERK1/2, and between ERK1/2 and p38 MAPK, has been reported in cellular responses to IL-1 and TNF-
, respectively (21, 58).
Our data gathered to date show that inhibition of either the PKC or MAPK pathway is sufficient to prevent thrombin-induced DAF upregulation, suggesting that PKC and MAPK form part of a linear signaling pathway similar to that described for the induction of ICAM-1 by thrombin (52). We show herein that the overexpression of a constitutively active form of PKC- capable of ERK1/2 activation (19) also induces a significant increase in DAF expression, which is inhibited by the MEK-1 antagonist U0126. However, the broad spectrum PKC antagonist GF-109203X failed to inhibit thrombin-induced phosphorylation of the MAPK enzymes. This may reflect the inability of GF-109203X to inhibit atypical isozymes such as PKC-
. Alternatively, PKC and MAPK may not exhibit a simple linear relationship in this setting, and the pathways may integrate downstream or activate distinct transcription factors involved in DAF regulation.
HUVECs express PAR1, PAR2, and PAR3, but not PAR4 (47, 55), which can be detected in human arterial ECs (15). Although PAR1 is the predominant thrombin receptor on vascular endothelium, it remains to be determined whether PAR1 is responsible for all thrombin-mediated signaling in human ECs. In murine ECs, PAR1 and PAR4 may contribute to thrombin signaling (26). However, no role for PAR4 in thrombin-mediated responses has been found in human ECs (15). PAR2, which is expressed in human ECs and upregulated by proinflammatory cytokines (46), does not bind thrombin and may be activated by trypsin and mast cell tryptase. Early studies demonstrating the failure of PAR1-blocking antibodies to completely inhibit thrombin responses suggested the involvement of additional receptors for thrombin (3). Moreover, the response of HUVECs to thrombin can be reduced when PAR2 is first activated with a selective agonist peptide (42, 43). O'Brien et al. (47) subsequently reported that when PAR1 signaling is inhibited, PAR2 may be transactivated by cleaved PAR1, thereby providing a mechanism for indirect activation of PAR2 by thrombin. We sought to extend these findings by looking for evidence of a role for PAR2 transactivation in thrombin-mediated DAF upregulation in HUVECs.
PAR1 and PAR2 APs induced DAF expression in HUVECs, whereas peptides targeting PAR3 (data not shown) and PAR4 had no effect. The relatively high concentration of PAR1 and PAR2 peptides required to induce a response is likely to be a consequence of the long time course of the assay and the presence of associated peptide degradation. The latter hypothesis was confirmed by the ability of the aminopeptidase inhibitor amastatin to reduce substantially the concentration of TFLLR-NH2 required to induce DAF expression. The upregulation of DAF by PAR1 and PAR2 peptides in combination was equivalent to that observed with thrombin alone. In addition, the response to thrombin and the PAR2 peptide was enhanced by pretreating ECs with TNF- to upregulate PAR2 expression before exposure to the AP. These data suggest that PAR2 might play a role in thrombin-induced DAF expression.
In the absence of known antagonists for PAR2, we used the PAR1 signaling antagonists BMS-200261 and SCH79797 to further investigate the hypothesis that transactivation of PAR2 by thrombin-cleaved PAR1 contributes to DAF upregulation. BMS-200261 and SCH79797 prevent binding of the PAR1-tethered ligand to the receptor without interfering with signaling via PAR2 or transactivation of PAR2 by PAR1 (1, 5, 47). As previously reported for thrombin-induced Ca2+ flux in ECs (47), inhibition of PAR1 with BMS-200261 or SCH79797 failed to significantly inhibit thrombin-mediated DAF upregulation while it completely prevented the upregulation by the PAR1 agonists SFLLRN and TFLLR-NH2. In contrast, hirudin, which binds thrombin and inhibits its proteolytic action, and the MAbs WEDE15 and ATAP2, which inhibit thrombin-mediated PAR1 cleavage, completely abrogated thrombin-induced DAF upregulation, whereas the MMP antagonist GM 6001 had no effect. SFLLR-derived peptides at the concentrations used can activate PAR2 in addition to PAR1 (2, 27). We therefore compared the responses observed with SFLLRN to those obtained with TFLLR-NH2, which has more selectivity for PAR1 than for PAR2 (27). SFLLRN and TFLLR-NH2 had directly comparable effects on DAF induction, which were inhibited by the PAR1 antagonists BMS-200261 and SCH79797. In addition, pretreatment of ECs with TNF- to increase PAR2 expression had no effect on the upregulation of DAF by SFLLRN. These data suggest that in this in vitro system, SFLLRN acts in a PAR1-specific manner and that any binding to PAR2 by the soluble peptide is less efficient than that of the orientation adapted by the tethered ligand itself.
Taken as a whole, our data suggest that thrombin cleaves PAR1, which transactivates PAR2, resulting in increased DAF expression to levels that we have previously shown to be capable of enhancing protection against complement-mediated injury (30). Although in the absence of specific PAR2 antagonists we cannot entirely exclude a role for other as yet unidentified EC thrombin receptors requiring PAR1 cleavage for their activation, we suggest that DAF upregulation is a functionally important outcome of the PAR1-mediated transactivation of PAR2 on ECs first proposed by O'Brien et al. (47).
Investigation of the signaling pathways downstream of PAR1 and PAR2 revealed no differences in the utilization of MAPK. However, the predominant PKC isozymes used by these receptors for the induction of DAF expression were different. Inhibition of PKC- with a DN-Adv construct significantly inhibited DAF upregulation by the PAR2 but not the PAR1 APs. In contrast, DN-PKC-
inhibited DAF induction downstream of PAR1 but not downstream from PAR2. Furthermore, the PAR1 signaling antagonists blocked the PAR1/PKC-
-dependent pathway but had no effect on PAR2/PKC-
-induced DAF upregulation. Although PAR1 and PAR2 agonist peptides typically produce similar downstream responses, differences are emerging. Thus MCP-1 mRNA (54) and increased vascular permeability (28, 66) are induced by PAR1 but not PAR2 agonists. This may reflect differences in the ability of PAR1 and PAR2 ligation to activate the PKC-
and Rho-GTPase signaling pathway involved in regulating vascular permeability (28, 39, 66), a hypothesis supported by the recent report that PAR1-induced activation of PKC-
is involved in P-selectin expression on microvascular ECs (14). The link between PAR2 and PKC-
described herein is likely to be important in vascular cytoprotection, and it is relevant that PKC-
has been associated with a variety of other cytoprotective molecules, including endothelial nitric oxide synthase and hemoxygenase-1 (51).
In vascular endothelium, PAR1 activation by thrombin helps to coordinate the response to tissue injury (9). This activation includes proinflammatory actions such as the upregulation of cellular adhesion molecules including E-selectin, VCAM-1, and ICAM-1 and the release of the chemokines IL-8 and MCP-1, which in turn direct leukocyte migration toward sites of inflammation (24, 25, 30, 41). However, PAR1 may also activate a variety of reparative responses, including the release of PDGF, connective tissue growth factor, and vascular remodeling (10, 16, 65). The role of PAR2 in this setting remains not fully understood, and the lack of developmental defects in PAR2-deficient mice suggests that its role may be more restricted than that of PAR1 (10, 65). Ligation of PAR2 can induce neurogenic and cutaneous inflammation (22, 56, 61). Moreover, PAR2-deficient mice are protected against collagen-induced arthritis (12). Notwithstanding this finding, PAR2 may also exert anti-inflammatory, cytoprotective effects in a murine model of colitis and can act to reduce bronchoconstriction and induce angiogenesis, thus accelerating recovery in the ischemic hindlimb (4, 7, 13, 40). This suggests that activation of PAR2, although it is important in the initiation of inflammation, may also facilitate the resolution of inflammation, the initiation of repair, and the protection of surrounding tissues (64).
We propose that thrombin may utilize the transactivation of PAR2 by PAR1 even when PAR1 signaling is intact and subsequently activate a PKC-/MAPK-regulated pathway to increase the expression of the complement-inhibitory protein DAF and thus help to maintain vascular integrity. Moreover, the ability of TNF-
to increase PAR2 expression, thus enhancing DAF upregulation by thrombin, suggests that during inflammation, when the risk of autologous tissue damage by complement is greatest, cytoprotection is maximal. Our data emphasize the importance of establishing a detailed understanding of thrombin- and/or PAR-mediated signaling pathways, which may in turn help to identify novel targets through which vascular inflammation can be controlled and vascular cytoprotection can be preserved.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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