doi: 10.1161/ATVBAHA.107.157321
© 2008 American Heart Association, Inc.
Editorials |
Protective Signaling Pathways of Activated Protein C in Endothelial Cells
From the Department of Immunology, The Scripps Research Institute, La Jolla, CA.
Correspondence to Matthias Riewald, MD, Department of Immunology SP30-3040, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail riewald{at}scripps.edu
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Introduction |
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 Top Introduction EPCR-Independent PAR1 Signaling...Role of the Sphingosine-1...References |
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Activated protein C (APC) has been reported to improve survival in patients with severe sepsis.1 Protein C (PC) is physiologically activated on the endothelial cell surface by the key procoagulant enzyme thrombin, and APC downregulates thrombin formation in a negative feedback loop.2 This anticoagulant effect of APC is unlikely to explain its benefit in systemic inflammation because other anticoagulants did not improve survival in septic patients. How the protective effects of APC in systemic inflammation are mediated has thus received a considerable amount of attention in recent years.
See ATVB 2007;2634–2641.
In vitro studies have shown that incubation of cultured endothelial cells with APC leads to a number of potentially antiinflammatory cellular changes. These include downregulation of the expression of adhesion molecules, enhanced barrier function in cell monolayers, and effects on gene expression that result in decreased susceptibility to apoptosis. Most of these cellular responses have been shown to require the binding of APC to endothelial cell protein C receptor (EPCR) and cleavage-mediated activation of protease activated receptor-1 (PAR1).3–6 EPCR may not only enhance activation of PAR1 by APC but also modify downstream responses through other mechanisms.7–9 EPCR is a transmembrane receptor with a very short cytoplasmic domain and an extracellular domain that binds both PC and APC. EPCR recruits and positions PC/APC on the cell surface, and EPCR binding enhances activation of PC. PAR1 is the prototypical thrombin receptor and belongs to a family of 7-transmembrane G protein–coupled receptors. Enzymatic cleavage of PAR1 exposes a new extracellular N terminus that acts as a tethered activation ligand. Importantly, in mouse models of stroke and endotoxemia EPCR-dependent PAR1 signaling is required for protective effects of APC.5,10 However, it is unknown which cellular effects of APC lead to protection in vivo, and it remains possible that mechanisms independent of EPCR-PAR1 also contribute to protective effects of APC.
In the December 2007 issue of Arteriosclerosis, Thrombosis, and Vascular Biology, O’Brien and colleagues11 report that APC downregulates the expression of tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) in endothelial cells dependent on the activation of ERK1/2 and the transcription factor EGR-1. TRAIL is best known for its potential to induce apoptosis specifically in cancer cells but has also been implicated in the regulation of inflammatory responses.
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EPCR-Independent PAR1 Signaling by APC? |
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TopIntroductionEPCR-Independent PAR1 Signaling...Role of the Sphingosine-1...References |
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O’Brien et al report that the downregulation of TRAIL by APC required cleavage of PAR1 but was independent of APC binding to EPCR. Surprisingly, the induction of ERK1/2 phosphorylation and EGR-1 activation by APC–PAR1 signaling were also EPCR independent, whereas previous studies reported the same pathways to be dependent on EPCR.3,12 These divergent results may be explained by the use of different experimental conditions and cell lines. O’Brien et al used specific antibodies blocking the interaction between APC and EPCR and downregulation of EPCR expression by siRNA to establish the role of EPCR in APC-PAR1 signaling. Control experiments demonstrated that effects of APC on the adhesion receptor expression, staurosporine-induced apoptosis, and endothelial barrier integrity were dependent on APC binding to EPCR, which is consistent with previous results. Do these interesting new data establish that there is in fact relevant EPCR independent PAR1 activation by APC (Figure)?
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Studies in purified systems13 and assays using an APC mutant without the EPCR binding domain14 have demonstrated that at high concentrations APC can directly cleave PAR1 even if EPCR binding is not available. EPCR facilitates PAR1 cleavage by lower APC concentrations by recruiting and positioning the protease to specific domains on the plasma membrane for efficient PAR1 cleavage.14 Thus, one possibility is that the EPCR independent downregulation of TRAIL is caused by coreceptor independent activation of PAR1 by APC. The resulting very low rate of PAR1 activation may support TRAIL downregulation because of a high sensitivity of this pathway to PAR1 signaling. In contrast, other downstream pathways such as protection from apoptosis or barrier enhancement may require a higher rate of PAR1 activation. It is also important to keep in mind that antibody blockade of EPCR or siRNA-mediated downregulation are not expected to be complete. It is difficult to rule out that residual availability of EPCR still mediates at least some of the observed effects, especially if higher concentrations of APC are used for prolonged incubation times. These issues could in the future be addressed using EPCR independent activators of PAR1 such as thrombin or variants of APC. APC binds to EPCR through its Gla domain. To ultimately prove that the responses do not require EPCR binding, APC variants with a deleted or mutated Gla domain could be used.14 Furthermore, experiments using cell lines lacking EPCR could be used to definitely establish the role of EPCR in responses to wild-type APC.
Another possibility put forth by the authors is that a not yet identified coreceptor is involved in the PAR1-dependent downregulation of TRAIL by APC. EPCR colocalizes with PAR1 in lipid rafts, and ligand binding to EPCR may modulate its compartmentalization and affect downstream signaling responses.7–9,14 A novel coreceptor may also localize APC in specific microdomains where different signaling complexes are assembled and where PAR1-dependent signaling specifically and efficiently downregulates TRAIL expression. The identification of such novel cofactor for APC-PAR1 signaling will be required to test this model in future studies.
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Role of the Sphingosine-1 Phosphate Pathway in Mediating APC Effects |
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TopIntroductionEPCR-Independent PAR1 Signaling...Role of the Sphingosine-1...References |
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Sphingosine 1-phosphate (S1P) is a biologically active lipid that is generated by cellular sphingosine kinases (SK) and S1P signaling is mediated by the S1P receptor family of seven-transmembrane G-protein-coupled receptors. S1P can induce responses in endothelial cells that resemble APC-mediated responses, including enhanced barrier function, antiapoptotic effects, and downregulation of adhesion molecules.15–17 Indeed, EPCR-dependent protective effects of APC on the barrier integrity of an endothelial cell monolayer have been shown previously to require SK activity and expression of the S1P receptor-1 (S1P1).4,8 The current study by O’Brien et al shows that SK and S1P1 are required for the downregulation of TRAIL by APC. These novel data implicate crossactivation of the S1P pathway in potentially antiapoptotic effects of APC signaling for the first time.
It will be interesting to establish the role of S1P signaling in other responses to APC, including protective effects on staurosporine-induced apoptosis, adhesion molecule expression, and most importantly in beneficial effects of APC in models of systemic inflammation. Infusion of S1P has been shown to be protective in models of endotoxin-induced acute lung injury16 and it is possible that protective in vivo effects of APC require crossactivation of this pathway, including the activation of endothelial cell S1P1. Even if S1P pathway crossactivation is indeed a general requirement for responses to APC in tissue culture, S1P receptor agonists and APC signaling will target different cell populations in vivo. This is because of the fact that in vivo the PC pathway depends on expression of cellular cofactors such as EPCR and thrombomodulin. The relative specificity of PC pathway signaling for endothelial cells may avoid detrimental side effects of S1P receptor activation in other cell types, eg, direct effects on lymphocyte migration, in the treatment of inflammatory conditions.
Clearly, very little is known with regard to the mechanism of S1P receptor crossactivation by APC. How exactly do SK and S1P1 contribute to the signaling? Given that plasma contains large amounts of S1P, it is difficult to explain how the S1P pathway can be relevant for APC signaling. Perhaps autocrine S1P1-dependent signaling of endothelial cell-produced and locally secreted S1P is more efficient compared with plasma S1P, which is expected to be largely bound to plasma proteins. Alternatively, APC has been shown to induce colocalization of EPCR with S1P1, and S1P1 may be activated through other mechanisms that do not necessarily involve S1P binding, eg, cross-phosphorylation events.8
In conclusion, the new results identify TRAIL downregulation as a novel APC-mediated response and they highlight that novel receptors and signaling pathways may be involved in protective APC signaling in endothelial cells. A better mechanistic understanding of how cells sense the proteolytic activity of APC in their microenvironment and how they respond may eventually lead to novel approaches to treat patients with sepsis and other disorders where the inflammatory response plays a key role, including myocardial infarction and stroke.
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Acknowledgments |
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Disclosures
None.
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References |
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TopIntroductionEPCR-Independent PAR1 Signaling...Role of the Sphingosine-1...References |
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Related Article:
Arterioscler Thromb Vasc Biol 2007 27: 2634-2641.
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