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【摘要】
Background— APC is an antithrombotic and antiinflammatory serine protease that plays an important role in vascular function. We report that APC can suppress the proapoptotic mediator TRAIL in human umbilical vein endothelial cells, and we have investigated the signaling mechanism.
Methods and Results— APC inhibited endothelial TRAIL expression and secretion and its induction by cell activation. To explore the mechanism, we examined factors associated with TRAIL regulation and demonstrated that APC increased the level of EGR-1, a transcriptional factor known to suppress the TRAIL promoter. APC also induced a significant increase in phosphorylation of ERK-1/2, required to activate EGR-1 expression. Activation of ERK-1/2 was dependent on the protease activated receptor-1 (PAR-1), but independent of the endothelial protein C receptor (EPCR). Using siRNA, we found that the effect of APC on the EGR-1/ERK signaling required for TRAIL inhibition was dependent on the S1P1 receptor and S1P1 kinase.
Conclusions— Our data suggest that APC may provide cytoprotective activity by activating the ERK pathway, which upregulates EGR-1 thereby suppressing the expression of TRAIL. Moreover, we provide evidence that APC can induce a cell signaling response through a PAR-1/S1P1-dependent but EPCR-independent mechanism.
We report that APC can suppress the proapoptotic mediator TRAIL by activating the ERK pathway to upregulate EGR-1, a negative regulator of TRAIL expression. The effect of APC was PAR-1– and S1P1-dependent, but independent of the endothelial protein C receptor, suggesting a mechanism to suppress injury in cells not expressing this receptor.
【关键词】 endothelial protein C receptor apoptosis protease activated receptor APC TRAIL
Introduction
The TNF family of cytokines mediates apoptosis and a variety of immune/inflammatory responses. The TNF-related apoptosis-inducing ligand (TRAIL) is constitutively expressed in most normal tissues and cell types 1 and is able to induce apoptosis on binding to either TRAIL-R1 or TRAIL-R2 receptors. 2,3 This binding interaction induces oligomerization of intracellular death domains, resulting in cleavage of pro-caspase 8 into its active form, which can activate caspase 3, the key mediator of apoptosis. 4 Early studies suggested that TRAIL induced apoptosis in malignant cells, but had minimal toxicity on normal cells because normal cells expressed high levels of decoy receptors. 5 However, subsequent studies have demonstrated that endothelial cells are susceptible to TRAIL-induced apoptosis. 6,7 Both soluble and membrane-associated TRAIL activate TRAIL-R1 on the cell surface 8 and human endothelial cells display little difference in response to membrane-bound versus soluble ligand. 9 Moreover, several studies have demonstrated both autocrine and paracrine activation of the TRAIL receptors from membrane-associated and soluble TRAIL. 10–13 In addition, recent studies in vivo have suggested a potential proinflammatory and proapoptotic role of TRAIL in vascular injury and atherothrombosis (reviewed in 14 ).
Apoptotic signals induced by TRAIL have been shown to be negatively regulated by the MAPK/ERK pathway, 15,16 and cells become susceptible to apoptosis when ERK is dephosphorylated. 17,18 Recent studies have also suggested that the early transcription factor, early growth response factor –1 (EGR-1), acts as a negative regulator of TRAIL. 19 Of interest, EGR-1 function has been shown to be dependent on ERK1/2 phosphorylation. 20
APC is a serine protease with a well characterized anticoagulant activity. Recent studies have demonstrated that APC has antiinflammatory and cytoprotective activities, which are not attributed to the anticoagulant activity of APC. 21–23 APC can initiate cell-signaling pathways in cells by activating protease-activated receptor 1 (PAR-1). 24 PAR-1 is a member of the 7 transmembrane domain G protein–coupled receptor family, and its activation requires cleavage at a specific site within its extracellular amino terminus. 25 This cleavage produces a new amino terminus, which then acts as its own tethered ligand, but optimal cleavage occurs when APC is juxtaposed to PAR-1. It is suggested that APC must bind to endothelial protein C receptor (EPCR) for it to be juxtaposed to PAR-1. Indeed, many studies have demonstrated antiinflammatory and antiapoptotic activities of APC that are dependent on PAR-1 and EPCR; however, previous studies have not elucidated alternative pathways that are activated by APC. 24,26,27
The present study was undertaken to determine whether APC could modulate the activity of endothelial TRAIL, an important mediator of vascular injury. We show that APC suppresses the expression of TRAIL mRNA by an EPCR-independent mechanism. We demonstrate that APC increases the phosphorylation and activation of ERK and increases the transcriptional activity of EGR-1, which in turn suppresses TRAIL mRNA expression. We also demonstrate that the activation of EGR-1 by APC is dependent both on PAR-1 and sphingosine 1-phosphate receptor (S1P1)-dependent signaling. These results describe a novel pathway by which APC may protect endothelial cells from mediators such as TRAIL and describe an APC-induced cellular response that is EPCR-independent.
Methods
TNF- –Induced TRAIL Expression
Confluent HUVECs were incubated with the EPCR blocking antibody JRK1494 (2.5 µg/mL) for 7 hours in serum-free media, and then concentrations of 8 to 160 nmol/L APC were added overnight. EPCR blocking experiments using RCR-252 were performed at 25 µg/mL for 2 hours. The concentration range was chosen to bracket the known 30 nmol/L Kd for APC interaction with EPCR, although these concentrations are above the therapeutic blood concentration achieved in APC-treated severe sepsis patients. The following morning, 1 ng/mL of TNF- was added to the cells for 1, 6, or 12 hours, then cells were lysed with M-PER and protein concentrations were determined by BCA assay according to the manufacturer?s protocol. The quantity of membrane-associated TRAIL was determined by ELISA, and the standard curve was generated using recombinant human TRAIL. Briefly, 96-well plates were coated with anti-TRAIL antibody overnight. The following day, wells were blocked for 1 hour with 4% bovine albumin in PBS. Samples or recombinant human TRAIL were added to the plate for 1 hour, followed by biotinylated anti-TRAIL Ab for 1 hour. Next, 100 µL of HRP streptavidin was added for 30 minutes. Wells were extensively washed with PBS-Tween between each of these steps. The ELISA was developed by adding TMB substrate solution for 20 minutes and stopped by adding H 2 SO 4. Plates were read at A 450 –A 595.
The effect of blocking EPCR with an siRNA on APC inhibition of TRAIL was performed in confluent HUVECs treated with a nonspecific control or EPCR-specific siRNA for 48 hours before treatment with 80 nmol/L APC. The level of EPCR expression in control and EPCR siRNA-treated cell was determined using a Western blot with S6 ribosomal protein (S6 RP) as a loading control.
Statistics
All experiments were performed at least in triplicate. Statistics were performed using JMP software (SAS Institute). Error bars indicate SEM. For the analysis of TRAIL response genes, comparison were displayed as a heat map generated using hierarchical clustering in JMP 5.1 (Ward method).
Please see supplemental materials, available online at http://atvb.ahajournals.org for additional details on Materials and Methods.
Results
TNF- –Induced TRAIL mRNA Expression Is Decreased by APC
As shown in Figure 1 A, the expression of endothelial TRAIL mRNA was suppressed by APC. Moreover, the induction of TRAIL mRNA by TNF- was blocked by APC treatment. To determine whether the effect of APC was concentration-dependent, cells were stimulated with various concentrations of APC (0 to 160 nmol/L), and the concentration of TRAIL was measured by ELISA. As Figure 1 B demonstrates, APC inhibited the amount of TRAIL secreted from HUVECs in a concentration-dependent manner, with a half-maximal effect at approximately 30 nmol/L. As was observed with the mRNA levels, APC also suppressed the induction of TRAIL secretion induced by TNF- ( Figure 1 C).
Figure 1. Influence of APC on TRAIL expression. A, mRNA levels of TRAIL determined in HUVECs treated with APC or APC plus TNF-. B, Concentration response for effect of APC on TRAIL secretion. C, Effect of APC on TNF- induction of TRAIL. D, Time course for effect of APC on TRAIL.
A time-course study was performed to determine how long APC was effective at reducing TRAIL. As shown in Figure 1 D, TRAIL was unchanged during the first hour after TNF- or APC treatment. At 6 hours, TRAIL was increased by 4.8-fold in TNF- –treated cells; however, TRAIL was reduced by 35% by APC treatment. At 12 hours, TRAIL was further increased by 1.3-fold in TNF- –stimulated cells, but was unchanged in cells that were costimulated with APC. These results suggest that a single exposure of cells to APC reduces TNF- –mediated TRAIL protein levels for at least 12 hours. Overall, our results suggest that APC can modulate TRAIL expression alone or after its induction with inflammatory mediators such as TNF-.
APC Decreases TRAIL Secretion Through an EPCR-Independent Mechanism
Several studies have shown that the antiapoptotic activity of APC can be mediated by the EPCR-dependent activation of PAR-1. To determine whether APC reduced TRAIL secretion via a similar mechanism, cells were treated with APC with or without TNF- administration in the presence of an antibody that prevents APC interaction with EPCR (JRK1494). As shown in Figure 2 A, the ability of APC to inhibit the expression of TRAIL was not affected by blocking the APC-EPCR interaction with anti-EPCR antibody JRK-1494 or with RCR-252 (data not shown). Moreover, blocking the APC-EPCR interaction had no effect on the ability of APC to reduce the TNF-induced secretion of TRAIL ( Figure 2 B). As a control to show that APC interaction with EPCR had in fact been blocked, in parallel experiments we were able to block the reported EPCR-dependent suppression of ICAM 23,25 with the anti-EPCR antibody (supplemental Figure I), and to suppress the known EPCR-dependent effects of APC on staurosporine-induced apoptosis 21,44 (supplemental Figure II) and on thrombin-induced changes in permeability determined by the BSA-Evans blue dye method described by Feistritzer and Riewald 28 (supplemental Figure III). While these data with blocking antibodies strongly suggested EPCR-independence of APC on TRAIL, we further confirmed this by treating cells with an siRNA, which ablated EPCR expression in the cell ( Figure 2 C, inset) but had no effect on the ability of APC to suppress the expression of TRAIL.
Figure 2. Effect of blocking EPCR on APC-mediated TRAIL suppression. Effect of EPCR blocking antibody JRK1494 on APC alone (A) or with TNF- (B). C, Effect of EPCR siRNA on the ability of APC to inhibit TRAIL expression. Error bars indicate SEM, n=3 to 5, * P <0.05 vs Control.
APC Increases the Expression and Functional Activity of EGR-1
Recent studies have suggested that the early transcription factor, EGR-1, acts as a negative regulator of TRAIL. 19 To further explore the mechanism for the inhibition of TRAIL by APC, we examined the relationship between the expression of EGR-1 and TRAIL across untreated and TNF-treated cells. As shown in Figure 3 A, the level of TRAIL expression was negatively associated with the expression of EGR-1. Moreover, we examined the level of EGR-1 and TRAIL at 6 hours after TNF treatment and observed a 53% decrease in EGR-1 expression and corresponding 61% increase in TRAIL expression (data not shown). These data are consistent with the previous report showing that TRAIL is negatively regulated by EGR-1. 19
Figure 3. APC Increases EGR-1 transcriptional activity and ERK-1/2 phosphorylation. A, Relationship of EGR-1 and TRAIL in cells treated with TNF. B, Serum starved HUVECs (30 minutes) were stimulated with 30 nmol/L APC (5 hours), and EGR-1 binding was determined by EMSA (lanes 1 to 2, untreated; 3 to 4, APC-treated, 5, cold probe competed).
To determine whether APC might suppress TRAIL via EGR-1, we analyzed the level of EGR-1 DNA binding activity by gel shift assay in untreated cells and those treated with APC. We observed significantly higher levels of EGR-1 in cells treated with APC ( Figure 3 B). As also shown, the binding was specific as cold binding site probe could completely inhibit the gel shift.
APC Increases pERK1/2 Independent of EPCR
EGR-1 binding activity has been shown to be dependent on ERK1/2 phosphorylation, 20 so we sought to determine whether APC might be affecting EGR-1 by modulating the level of ERK1/2. As shown in Figure 4 A, APC induced a time-dependent increase in the phosphorylation of ERK1/2. There was no change in the level of total ERK1/2 protein in these experiments as determined by Western blot analysis using an antibody to total ERK1/2 (data not shown). If the effect of APC on EGR-1 suppression of TRAIL was dependent on ERK1/2 phosphorylation, we would expect the effect to be EPCR independent as was shown in Figure 2. Therefore, we examined the effect of APC on ERK1/2 phosphorylation in the presence of the anti-EPCR antibody. As shown in Figure 4 B, the increase in pERK induced by APC was not suppressed by blocking the EPCR interaction. However, the effect of APC was completely suppressed by the blocking antibody to PAR-1. These data suggest that APC can induce EGR-1/ERK-1/2 cell signaling in a PAR-1–dependent but EPCR-independent manner.
Figure 4. Effect of APC on ERK-1/2 phosphorylation. A, HUVECs were stimulated with 30 nmol/L APC for various times, and the level of pERK1/2 was determined. B, Confluent HUVECs were incubated with the PAR-1 (ATAP2) or EPCR (RCR-252) blocking antibodies for 2 hours, then stimulated for 15 minutes with APC.
Effect of S1P1 Signaling on APC-Dependent EGR-1 Activation
Recent studies have demonstrated that the S1P1 receptor can effect cell signaling by APC. 28,29 As the effect of APC on TRAIL and the ERK1/2-EGR pathway appears to be PAR-1–dependent but EPCR-independent, we sought to determine the role of S1P1, especially as recent studies have shown that S1P1 signaling occurs with activation of the ERK1/2 pathway. 30–32 As shown in Figure 5 A, a siRNA blocking S1P1 (Edg1), but not a scrambled siRNA, significantly decreased the EGR1-DNA interactions induced by APC. We also examined the effect of SPHK1, the kinase required for generating S1P and activating the S1P1 receptor via PAR-1. As shown, the siRNA against SPHK1 also significantly suppressed the transcriptional induction of EGR-1 by APC. Our proposed model for the EPCR-independent activation of signaling by APC to suppress TRAIL expression, based on the model described by Camerer and Coughlin, 33 is shown in Figure 5 B.
Figure 5. Role of S1P1 signaling on EGR-1 activation. A, HUVECs were pretreated with specific siRNA to block S1P1 and SPHK1, serum starved for 30 minutes, followed by 30 nmol/L APC before determination of EGR-1 binding. B, Proposed pathway for the EPCR-independent signaling of APC to inhibit TRAIL expression.
TRAIL-Response Genes and APC Inhibition
We sought to assess the cellular consequence of APC-mediated TRAIL suppression by examining genes known to be suppressed or induced by TRAIL. For this analysis we examined cyclin-dependent kinase 4 (CDK4), previously shown to be suppressed by TRAIL, 34 and several genes (SOD2, IFI-15K/G1P2, NK4, HLA-A, HLA-C, TFPI-2) shown to be induced by TRAIL. Using both treated and untreated HUVECs, we first examined the relationship between the expression levels of TRAIL and the expression of these genes. Consistent with their known regulation by TRAIL, we observed a highly significant positive correlation between TRAIL and the set of TRAIL-induced genes, and a highly negative correlation between TRAIL and CDK4 ( Figure 6 A). We treated cells with APC to determine whether the APC-mediated reduction in TRAIL resulted in a suppression of TRAIL-induced genes. Figure 6 B shows the level of TRAIL in this experiment. As shown in Figure 6 C, the expression of SOD2, IFI-15K/G1P2, NK4, HLA-A, HLA-C, TFPI-2 were induced as TRAIL levels increased and suppressed by APC coincident with its ability to inhibit TRAIL secretion. Moreover, the TRAIL-suppressed gene CDK4 was increased by APC treatment, coincident with the suppression of TRAIL secretion. These data strongly suggest that the reduction in TRAIL expression by APC alters cellular pathways in HUVECs known to be mediated by TRAIL.
Figure 6. Effect of APC-mediated TRAIL reduction on TRAIL-responsive genes. A, Comparison of relative TRAIL expression levels versus genes modulated by TRAIL from untreated and TNF-treated cells (n=30). Relative level of TRAIL produced (B) and comparison of mRNA expression of genes known to be induced or suppressed by TRAIL (C).
Discussion
Apoptosis plays a major role in pathologic conditions where increased cell death has been associated with organ damage or failure. Indeed, this is true with severe sepsis where immunologic defense mechanisms initiate a cascade of inflammatory events leading to multi-organ failure (reviewed in 22 ). As indicated above, numerous studies have suggested that TRAIL induces apoptosis in cancer cells, 5 but recent studies have demonstrated that normal vascular endothelial cells are susceptible to TRAIL-mediated apoptosis. 6,7 Currently, the only therapy approved for treatment of patients with severe sepsis at high risk of death is recombinant human APC, drotrecogin alfa (activated). 35 Severely septic patients receiving recombinant human APC have better Sequential Organ Failure Assessment scores for cardiovascular and respiratory organ systems than severely septic patients treated with placebo. Although the role of TRAIL in human sepsis has not been defined, the data from the current study suggest that APC can inhibit TRAIL secretion activated by the proinflammatory cytokine TNF-, which is upregulated during sepsis. These observations provide insight into a potential mechanism by which APC might decrease apoptosis and improve organ function in severe sepsis patients. Moreover, the ability of APC to suppress TRAIL-regulated genes associated with proinflammatory and immunomodulatory activity ( Figure 6 ), ie, beyond the apoptosis pathway, suggests additional potential protective mechanisms.
The initial suggestion that APC could block apoptosis and promote cell survival came from a transcriptional profiling experiment, 23 and these results were confirmed in cell-based assays and animal models. 23,25,36 These studies suggested that APC mediates the suppression of apoptosis in a PAR-1/EPCR dependent mechanism by decreasing proapoptotic protein such as Bax, increasing antiapoptotic protein Bcl-2, and inhibiting the activation of caspase 3 and 8. In the current study, we show that APC can decrease the mRNA levels and secretion of the potent apoptosis factor TRAIL, via activation of the EGR-1/ERK-1/2 pathway. In contrast to previous reports, the mechanism of this effect appears to be PAR-1 dependent, but EPCR-independent. To the best of our knowledge, this is the first study to demonstrate that APC may activate cellular pathways in a PAR-1–dependent but EPCR-independent mechanism. Our data open the possibility of another cofactor involved in APC–PAR-1 interaction. A logical candidate would be thrombomodulin (TM); however, we were unable to demonstrate that APC-TM interactions are involved with the cell signaling we have described (data not shown).
Recent studies have suggested that APC signaling via PAR-1 can be transactivated by the S1P-S1P1 receptor interaction. 28,29 PAR-1 is coupled to several G proteins including G -β. The β subunit ultimately leads to the activation of the MAPK and ERK pathways, which can activate several transcription factors that transcribe genes containing a serum response element (SRE). 37 The transcription factor EGR-1 has 5 SREs, and studies have demonstrated that ERK can increase functional EGR-1. 38 Recent studies have also shown that S1P1 signaling activates the ERK1/2 pathway. 30–32 In the current study, we demonstrate that APC induces ERK-1/2 phosphorylation and increases the level of EGR-1 mRNA and functionally active protein. However, unlike previous studies, 28,29 the effect of APC signaling though PAR-1 and S1P1 signaling was independent of EPCR. EPCR has been shown to be expressed primarily on large vessels, but not in the normal microvasculature, and several studies have demonstrated that EPCR is differentially regulated during tissue injury. 39–41 Thus, protective signaling mechanisms independent of EPCR may be important under conditions of little or no EPCR expression.
APC plays an important role in the modulation of vascular function not only through inhibition of thrombin generation, but also by receptor-mediated effects via PAR-1 activation, which results in activation of cytoprotective and antiinflammatory pathways. The requirement of EPCR for APC to signal via PAR-1 has been documented, but its relevance and physiological role have been questioned based on kinetics and tissue distribution. 42 Our data suggest that the activation of protective signaling pathways, such as the ability to activate EGR-1/ERK-1/2 signaling to suppress TRAIL, can occur independent of EPCR. This suggests that conclusions on the role of signaling in the efficacy of APC may not be solely dependent on the tissue expression of this receptor. Recent studies have shown that the components required for the activation of PC and for APC signaling are colocalized to lipid rafts, 43 indicating the complex nature of the microenvironment on the membrane surface involved in the generation of APC and in PAR-1 signaling. Clearly, further studies will be needed to dissect the relative importance and balance of the EPCR-independent versus EPCR-dependent signaling in the in vivo function of APC, and to determine the importance of EPCR-independent signaling during endogenous APC generation versus exogenous APC exposure during therapy. Overall, the results described in this report provide a new mechanistic understanding for the antiapoptotic and antiinflammatory functions of APC and define a novel signaling pathway that is not dependent on EPCR.
Acknowledgments
We thank Michael Flagella for assistance with RNA preparation.
Disclosures
The authors are employees of Eli Lilly and Co, who produces human activated protein C for treatment of severe sepsis.
【参考文献】
Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, Sutherland GR, Smith TD, Rauch C, Smith CA, Goodwin RG. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity. 1995; 3: 673–682.
Pan G, O?Rourke K, Chinnaiyan AM, Gentz R, Ebner R, Ni J, Dixit VM. The receptor for the cytotoxic ligand TRAIL. Science. 1997; 276: 111–113.
Walczak H, Degli-Esposti MA, Johnson RS, Smolak PJ, Waugh JY, Boiani N, Timour MS, Gerhart MJ, Schooley KA, Smith CA, Goodwin RG, Rauch CT, Pan G, O?Rourke K, Chinnaiyan AM, Gentz R, Ebner R, Ni J, Dixit VM. TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. Embo J. 1997; 16: 5386–5397.
Medema JP, Scaffidi C, Kischkel FC, Shevchenko A, Mann M, Krammer PH, Peter ME. FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). Embo J. 1997; 16: 2794–2804.
Gura T. How TRAIL kills cancer cells, but not normal cells. Science. 1997; 277: 768.
Pritzker LB, Scatena M, Giachelli CM. The role of osteoprotegerin and tumor necrosis factor-related apoptosis-inducing ligand in human microvascular endothelial cell survival. Mol Biol Cell. 2004; 15: 2834–2841.
Walczak H, Krammer PH. The CD95 (APO-1/Fas) and the TRAIL (APO-2L) apoptosis systems. Exp Cell Res. 2000; 256: 58–66.
Wajant H, Moosmayera D, Wüest T, Bartke T, Gerlach E, Schönherr U, Peters N, Scheurich P, Pfizenmaier K. Differential activation of TRAIL-R1 and -2 by soluble and membrane TRAIL allows selective surface antigen-directed activation of TRAIL-R2 by a soluble TRAIL derivative. Oncogene. 2001; 20: 4101–4106.
Li J, Kirkiles-Smith N, McNiff J, JS P. TRAIL induces apoptosis and inflammatory gene expression in human endothelial cells. J Immunol. 2003; 171: 1526–1533.
Abadie A, Wietzerbin J. Involvement of TNF-related apoptosis-inducing ligand (TRAIL) induction in interferon gamma-mediated apoptosis in Ewing tumor cells. Ann N Y Acad Sci. 2003; 1010: 117–120.
Martinez-Lorenzo MJ, Anel A, Gamen S, Monlen I, Lasierra P, Larrad L, Pineiro A, Alava MA, Naval J. Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles. J Immunol. 1999; 163: 1274–1281.
Oshima K, Yanase N, Ibukiyama C, Yamashina A, Kayagaki N, Yagita H, Mizuguchi J. Involvement of TRAIL/TRAIL-R interaction in IFN-alpha-induced apoptosis of Daudi B lymphoma cells. Cytokine. 2001; 14: 193–201.
Papageorgiou A, Lashinger L, Millikan R, Grossman HB, Benedict W, Dinney CP, McConkey DJ. Role of tumor necrosis factor-related apoptosis-inducing ligand in interferon-induced apoptosis in human bladder cancer cells. Cancer Res. 2004; 64: 8973–8979.
Martin-Ventura J, Munoz-Garcia B, Egido J, Blanco-Colio L. Trail and vascular injury. Front Biosci. 2007; 12: 3656–3667.
Tran SE, Holmstrom TH, Ahonen M, Kahari VM, Eriksson JE. MAPK/ERK overrides the apoptotic signaling from Fas, TNF, and TRAIL receptors. J Biol Chem. 2001; 276: 16484–16490.
Soderstrom TS, Poukkula M, Holmstrom TH, Heiskanen KM, Eriksson JE. Mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in activated T cells abrogates TRAIL-induced apoptosis upstream of the mitochondrial amplification loop and caspase-8. J Immunol. 2002; 169: 2851–2860.
Mezosi E, Wang SH, Utsugi S, Bajnok L, Bretz JD, Gauger PG, Thompson NW, Baker JR Jr. Interleukin-1beta and tumor necrosis factor (TNF)-alpha sensitize human thyroid epithelial cells to TNF-related apoptosis-inducing ligand-induced apoptosis through increases in procaspase-7 and bid, and the down-regulation of p44/42 mitogen-activated protein kinase activity. J Clin Endocrinol Metab. 2004; 89: 250–257.
Nishida S, Matsuoka H, Tsubaki M, Tanimori Y, Yanae M, Fujii Y, Iwaki M. Mevastatin induces apoptosis in HL60 cells dependently on decrease in phosphorylated ERK. Mol Cell Biochem. 2005; 269: 109–114.
Fu M, Zhu X, Zhang J, Liang J, Lin Y, Zhao L, Ehrengruber MU, Chen YE. Egr-1 target genes in human endothelial cells identified by microarray analysis. Gene. 2003; 315: 33–41.
Ke J, Gururajan M, Kumar A, Simmons A, Turcios L, Chelvarajan R, Cohen D, Wiest D, Monroe J, Bondada S. The role of MAPKs in B cell receptor-induced down-regulation of Egr-1 in immature B lymphoma cells. J Biol Chem. 2006; 281: 39806–39818.
Mosnier L, Zlokovic B, Griffin J. The cytoprotective protein C pathway. Blood. 2007; 109: 3161–3172.
Grinnell BW, Joyce D. Recombinant human activated protein C: a system modulator of vascular function for treatment of severe sepsis. Crit Care Med. 2001; 29: S53–S60.
Joyce DE, Gelbert L, Ciaccia A, DeHoff B, Grinnell BW. Gene expression profile of antithrombotic protein c defines new mechanisms modulating inflammation and apoptosis. J Biol Chem. 2001; 276: 11199–11203.
Riewald M, Petrovan RJ, Donner A, Ruf W. Activated protein C signals through the thrombin receptor PAR1 in endothelial cells. J Endotoxin Res. 2003; 9: 317–321.
Vu TK, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991; 64: 1057–1068.
Cheng T, Liu D, Griffin JH, Fernandez JA, Castellino F, Rosen ED, Fukudome K, Zlokovic BV, Uchiba M, Okajima K, Oike Y, Ito Y, Fukudome K, Isobe H, Suda T, Riewald M, Petrovan RJ, Donner A, Ruf W. Activated protein C blocks p53-mediated apoptosis in ischemic human brain endothelium and is neuroprotective. Nat Med. 2003; 9: 338–342.
Uchiba M, Okajima K, Oike Y, Ito Y, Fukudome K, Isobe H, Suda T, Riewald M, Petrovan RJ, Donner A, Ruf W. Activated protein C induces endothelial cell proliferation by mitogen-activated protein kinase activation in vitro and angiogenesis in vivo. Circ Res. 2004; 95: 34–41.
Feistritzer C, Riewald M. Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation. Blood. 2005; 105: 3178–3184.
Finigan JH, Dudek SM, Singleton PA, Chiang ET, Jacobson JR, Camp SM, Ye SQ, Garcia JG, Feistritzer C, Riewald M. Activated protein C mediates novel lung endothelial barrier enhancement: role of sphingosine 1-phosphate receptor transactivation. J Biol Chem. 2005; 280: 17286–17293.
Jo E, Sanna MG, Gonzalez-Cabrera PJ, Thangada S, Tigyi G, Osborne DA, Hla T, Parrill AL, Rosen H, Liao JJ, Huang MC, Graler M, Huang Y, Qiu H, Goetzl EJ, Waeber C, Blondeau N, Salomone S. S1P1-selective in vivo-active agonists from high-throughput screening: off-the-shelf chemical probes of receptor interactions, signaling, and fate. Chem Biol. 2005; 12: 703–715.
Liao JJ, Huang MC, Graler M, Huang Y, Qiu H, Goetzl EJ, Waeber C, Blondeau N, Salomone S. Distinctive T cell suppressive signals from nuclearized type 1 sphingosine 1-phosphate G protein-coupled receptors. J Biol Chem. 2007; 282: 1964–1972.
Waeber C, Blondeau N, Salomone S. Vascular sphingosine-1-phosphate S1P1 and S1P3 receptors. Drug News Perspect. 2004; 17: 365–382.
Camerer E, Coughlin S. APC signaling:tickling PAR1 for barrier protection? Blood. 2005; 105: 3004–3005.
Lunemann J, Waiczies S, Ehrlich S, Wendling U, Seeger B, Kamradt T, Zipp F. Death ligand TRAIL induces no apoptosis but inhibits activation of human (auto)antigen-specific T cells. J Immunol. 2002; 15: 4881–4888.
Bernard GR, Vincent JL, Laterre PF, LaRosa SP, Dhainaut JF, Lopez-Rodriguez A, Steingrub JS, Garber GE, Helterbrand JD, Ely EW, Fisher CJ Jr. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med. 2001; 344: 699–709.
Guo H, Liu D, Gelbard H, Cheng T, Insalaco R, Fernandez JA, Griffin JH, Zlokovic BV. Activated protein C prevents neuronal apoptosis via protease activated receptors 1 and 3. Neuron. 2004; 41: 563–572.
Whitmarsh AJ, Shore P, Sharrocks AD, Davis RJ. Integration of MAP kinase signal transduction pathways at the serum response element. Science. 1995; 269: 403–407.
Wu SQ, Minami T, Donovan DJ, Aird WC. The proximal serum response element in the Egr-1 promoter mediates response to thrombin in primary human endothelial cells. Blood. 2002; 100: 4454–4461.
Dahlback B, Villoutreix B. The anticoagulant protein C pathway. FEBS Lett. 2005; 579: 3310–3316.
Esmon CT. The protein C pathway. Chest. 2003; 124: 26S–32S.
Gupta A, Berg D, Gerlitz B, Sharma G, Syed S, Richardson M, Sandusky G, Heuer J, Galbreath E, Grinnell B. Role of protein C in renal dysfunction after polymicrobial sepsis. J Am Soc Nephrol. 2007; 18: 860–867.
Esmon CT. Is APC activation of endothelial cell PAR1 important in severe sepsis?: No. J Thromb Haemost. 2005; 3: 1910–1911.
Bae J, Yang L, Rezaie A. Receptors of the protein C activation and activated protein C signaling pathways are colocalized in lipid rafts of endothelial cells. Proc Natl Acad Sci U S A. 2007; 104: 2867–2872.
Mosnier LO, Griffin JH. Inhibition of staurosporine-induced apoptosis of endothelial cells by activated protein C requires protease activated receptor-1 and endothelial cell protein C receptor. Biochem J. 2003; 65–70.
作者单位：Division of Biotechnology Discovery Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Ind.