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• W1978082123 abstract "Tissue factor is critically important for initiating the activation of coagulation zymogens leading to the generation of thrombin. Quiescent endothelial cells do not express tissue factor on their surface, but many stimuli including cytokines and coagulation proteases can elicit tissue factor synthesis. We challenged human endothelial cells simultaneously with tumor necrosis factor α (TNFα) and thrombin because many pathophysiological conditions, such as sepsis, diabetes, and coronary artery disease, result in the concurrent presence of circulating inflammatory mediators and activated thrombin. We observed a remarkable synergy in the expression of tissue factor by thrombin plus TNFα. This was due to altered regulation of the transcription factors c-Jun and c-Fos. The activation of c-Jun was greater and more sustained than that obtained with either thrombin or TNFα alone. Thrombin-stimulated expression of c-Fos was both enhanced and prolonged by the concurrent presence of TNFα. These changes support the increased availability of c-Jun/c-Fos AP-1 complexes for mediating transcription at the tissue factor promoter. Transcription factors downstream of the extracellular signal-regulated kinases as well as changes in NFκB regulation were not involved in the synergistic increase in tissue factor expression by thrombin and TNFα. Thus, concurrent exposure of vascular endothelial cells to cytokines and procoagulant proteases such as thrombin can result in greatly enhanced tissue factor expression on the endothelium, thereby perpetuating the prothrombotic phenotype of the endothelium. Tissue factor is critically important for initiating the activation of coagulation zymogens leading to the generation of thrombin. Quiescent endothelial cells do not express tissue factor on their surface, but many stimuli including cytokines and coagulation proteases can elicit tissue factor synthesis. We challenged human endothelial cells simultaneously with tumor necrosis factor α (TNFα) and thrombin because many pathophysiological conditions, such as sepsis, diabetes, and coronary artery disease, result in the concurrent presence of circulating inflammatory mediators and activated thrombin. We observed a remarkable synergy in the expression of tissue factor by thrombin plus TNFα. This was due to altered regulation of the transcription factors c-Jun and c-Fos. The activation of c-Jun was greater and more sustained than that obtained with either thrombin or TNFα alone. Thrombin-stimulated expression of c-Fos was both enhanced and prolonged by the concurrent presence of TNFα. These changes support the increased availability of c-Jun/c-Fos AP-1 complexes for mediating transcription at the tissue factor promoter. Transcription factors downstream of the extracellular signal-regulated kinases as well as changes in NFκB regulation were not involved in the synergistic increase in tissue factor expression by thrombin and TNFα. Thus, concurrent exposure of vascular endothelial cells to cytokines and procoagulant proteases such as thrombin can result in greatly enhanced tissue factor expression on the endothelium, thereby perpetuating the prothrombotic phenotype of the endothelium. Many diseases with a pronounced inflammatory component, such as sepsis, type II diabetes, coronary artery disease, heparin-induced thrombocytopenia, and anti-phospholipid syndrome, also exhibit enhanced activation of coagulation enzymes (1Lindmark E. Wallentin L. Siegbahn A. Thromb. Res. 2001; 103: 249-259Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 2Merrill J.T. Curr. Rheumatol. Rep. 2001; 3: 293-300Crossref PubMed Scopus (9) Google Scholar, 3Levi M. ten Cate H. van der Poll T. Crit. Care Med. 2002; 30: S220-S224Crossref PubMed Scopus (146) Google Scholar, 4Reilly M.P. McKenzie S.E. Curr. Opin. Hematol. 2002; 9: 395-400Crossref PubMed Scopus (21) Google Scholar, 5Gonzalez M.A. Selwyn A.P. Am. J. Med. 2003; 115: 99S-106SAbstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). These serine proteases, most notably thrombin, stimulate protease-activated receptors (PARs) 1The abbreviations used are: PAR, protease-activated receptor; DPBS, Dulbecco's phosphate buffered saline; ERK, extracellular signal-regulated kinase; HUVEC, human umbilical vein endothelial cells; JNK, c-Jun NH2-terminal kinase; MAP, mitogen-activated protein; MAPK, MAP kinase; TF, tissue factor; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester).1The abbreviations used are: PAR, protease-activated receptor; DPBS, Dulbecco's phosphate buffered saline; ERK, extracellular signal-regulated kinase; HUVEC, human umbilical vein endothelial cells; JNK, c-Jun NH2-terminal kinase; MAP, mitogen-activated protein; MAPK, MAP kinase; TF, tissue factor; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). on the surface of endothelial cells. Signaling through the PARs shifts the endothelium toward a prothrombotic phenotype, exacerbating the initiating pathophysiological condition. Our interest in thrombin signaling by endothelial cells (6Woolkalis M.J. DeMelfi Jr., T.M. Blanchard N. Hoxie J.A. Brass L.F. J. Biol. Chem. 1995; 270: 9868-9875Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 7Ukropec J.A. Hollinger M.K. Salva S.M. Woolkalis M.J. J. Biol. Chem. 2000; 275: 5983-5986Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) led us to question how inflammatory mediators such as TNFα might alter thrombin-initiated signaling events and thereby bolster the prothrombotic phenotype of the endothelium. Normally, the apical surface of endothelial cells is designed to minimize interactions with circulating coagulation zymogens and blood cells. Inflammatory stimuli (cytokines, lipopolysaccharide, vascular endothelial cell growth factor, sphingosine 1-phosphate, thrombin (8Bevilacqua M.P. Pober J.S. Majeau G.R. Fiers W. Cotran R.S. Gimbrone Jr., M.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4533-4537Crossref PubMed Scopus (841) Google Scholar, 9Pendurthi U.R. Williams J.T. Rao L.V. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3406-3413Crossref PubMed Scopus (114) Google Scholar, 10Zucker S. Mirza H. Conner C.E. Lorenz A.F. Drews M.H. Bahou W.F. Jesty J. Int. J. Cancer. 1998; 75: 780-786Crossref PubMed Scopus (261) Google Scholar, 11Eto M. Kozai T. Cosentino F. Joch H. Luscher T.F. Circulation. 2002; 105: 1756-1759Crossref PubMed Scopus (325) Google Scholar, 12Takeya H. Gabazza E.C. Aoki S. Ueno H. Suzuki K. Blood. 2003; 102: 1693-1700Crossref PubMed Scopus (55) Google Scholar)) can alter the anticoagulatory phenotype of the endothelium by promoting the expression of adhesive molecules, receptors, and chemoattractants. Expression of tissue factor (TF), the coagulation Factor VII receptor, on the surface of endothelial cells serves as an excellent marker for an activated endothelium with an inflamed and procoagulant phenotype (13Luther T. Mackman N. Trends Cardiovasc. Med. 2001; 11: 307-312Crossref PubMed Scopus (29) Google Scholar, 14Versteeg H.H. Semin. Hematol. 2004; 41: 168-172Crossref PubMed Scopus (24) Google Scholar). Binding of Factor VII to tissue factor can then initiate the consecutive activation of Factor X and prothrombin, supporting fibrin generation and localized clot formation within the vasculature. Thus, understanding the regulation of endothelial cell tissue factor expression has been an area of intense research (15Bierhaus A. Zhang Y. Deng Y. Mackman N. Quehenberger P. Haase M. Luther T. Muller M. Bohrer H. Greten J. Martin E. Baeuerle P.A. Waldherr R. Kisiel W. Ziegler R. Stern D.M. Nawroth P.P. J. Biol. Chem. 1995; 270: 26419-26432Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 16Parry G.C. Mackman N. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 612-621Crossref PubMed Scopus (200) Google Scholar, 17Wu S.Q. Minami T. Donovan D.J. Aird W.C. Blood. 2002; 100: 4454-4461Crossref PubMed Scopus (44) Google Scholar).In the current study, we made the novel observation that exposure of human endothelial cells to thrombin plus TNFα resulted in a marked synergistic increase in the expression of tissue factor. Thrombin and TNFα receptors are known to signal through many of the same pathways, including the mitogen-activated protein (MAP) kinase cascades. We investigated whether any of the MAP kinase cascades served as the signaling conduit(s) responsible for the synergistic increase in tissue factor expression by TNFα and thrombin. We found that the temporal activation of the MAP kinase c-Jun NH2-terminal kinase (JNK), and its downstream target c-Jun was elevated and sustained when endothelial cells were exposed to both thrombin and TNFα when compared with either stimulus presented alone. c-Fos expression, which was dependent upon thrombin-induced calcium mobilization, increased both in quantity and duration in cells that were co-incubated with TNFα. Thus, the augmented, prolonged availability of c-Jun/c-Fos heterodimers, the most effective AP-1 complex for binding to the tissue factor promoter, provides a mechanism for the observed synergistic stimulation of tissue factor expression by TNFα and thrombin.EXPERIMENTAL PROCEDURESAntibodies—Antibodies used were goat anti-human tissue factor (American Diagnostica, Inc., Stamford, CT); mouse anti-human NFκB p65, mouse anti-human IκBα, mouse anti-human pc-Jun, rabbit anti-human c-Fos (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); rabbit anti-active MAPK (extracellular signal-regulated kinase (ERK)), p38, and JNK (Promega Corp., Madison, WI); and rabbit anti-MAPKAPK-2 (Cell Signaling Technology, Beverly, MA). All secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).Cell Culture—Human umbilical vein endothelial cells (HUVEC) were isolated and characterized as described by Jaffe et al. (18Jaffe E.A. Nachman R.L. Becker C.G. Minick C.R. J. Clin. Invest. 1973; 52: 2745-2756Crossref PubMed Scopus (5984) Google Scholar). Cells were grown in Medium 199 containing 10% fetal calf serum, 1 mm glutamine, 12 units/ml heparin, 100 μg/ml crude endothelial cell growth supplement, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C on fibronectin-coated tissue culture dishes. Confluent, quiescent HUVEC were used at passages 1–4.Cell Treatment—Where indicated, cultures were preincubated for 1 h in human endothelial serum-free medium (Invitrogen) with 10 μm U0126, 10 μm SB202190, 5 μm BAPTA/AM (Biomol Research Laboratories, Inc., Plymouth Meeting, PA) or 25 μm SP600125 (Calbiochem). Cultures were incubated without or with 5 or 15 ng/ml TNFα (R&D Systems, Inc., Minneapolis, MN), 0.5 or 2 units/ml thrombin (Enzyme Research Laboratories, South Bend, IN), 100 μm TFLLR (Bachem Biosciences, Inc., Plymouth Meeting, PA), or 100 nm ionomycin (Biomol Research Laboratories, Inc.), as indicated.Cell Extraction—HUVEC monolayers were washed with ice-cold Dulbecco's PBS containing 0.7 mm CaCl2 and 0.5 mm MgCl2 (DPBS) prior to scraping in extraction buffer A: 1% Triton X-100, 60 mm octyl glucoside, 10 mm Tris-HCl, pH 7.6, 50 mm NaCl, 30 mm Na4P207,50 mm NaF, 1 mm Na3VO4, 2 mm CaCl2, 0.2 mm H2O2, plus mammalian protease inhibitor mixture (Sigma). After solubilization on ice for 15 min with intermittent vortexing, the extract was microcentrifuged for 10 min and the supernatant recovered.Nuclear and Cytosolic Fractionation—Nuclear and cytosolic fractions were prepared by a protocol modified from the procedure of Dignam et al. (19Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9140) Google Scholar, 20Edmead C. Kanthou C. Benzakour O. Anal. Biochem. 1999; 275: 180-186Crossref PubMed Scopus (12) Google Scholar). HUVEC monolayers were washed three times with DPBS, then scraped in DPBS and centrifuged at 1500 × g for 10 min. The cells were resuspended in hypotonic buffer (10 mm Hepes, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, 0.1% Nonidet P-40) and allowed to swell for 30 min on ice and briefly vortexed. Nuclei were pelleted through a 10% sucrose layer. The pelleted nuclei were lysed in extraction buffer A for 10 min on ice, microcentrifuged for 15 min at 4 °C, and the supernatant recovered.Immunoblotting—Samples containing equal amounts of protein (5–20 μg) were solubilized in Laemmli sample buffer (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205955) Google Scholar), separated by SDS-PAGE, and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA). Immunoblots were blocked with 5% milk, 0.1% Tween 20 in Tris-buffered saline, pH 7.5, and probed sequentially with primary and secondary antibodies diluted in the milk-containing buffer. Detection was by enhanced chemiluminescence (PerkinElmer Life Sciences). Analysis of scanned images was performed using Kodak 1D™ 3.6.2 software.RESULTSHUVEC monolayers were challenged without or with 15 ng/ml TNFα, 2 units/ml thrombin, or the combination of TNFα plus thrombin for 5 h, an optimal time for tissue factor expression in endothelial cells. In the absence of any stimulus, tissue factor was almost undetectable (Fig. 1). TNFα was a potent stimulus for TF expression, while thrombin produced a more modest response, as has been observed by others (9Pendurthi U.R. Williams J.T. Rao L.V. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3406-3413Crossref PubMed Scopus (114) Google Scholar). We were surprised, though, by the pronounced synergy in tissue factor expression (6.2-fold ± 0.8 S.E.) induced by thrombin plus TNFα. Similar results were obtained when HUVEC were treated with 5 ng/ml TNFα and 0.5 unit/ml thrombin. The thrombin receptor-activating peptide TFLLR also potentiated TNFα-stimulated TF expression (data not shown), consistent with thrombin initiating the signaling through PARs and not through other potential thrombin receptors on the surface of endothelial cells.Signaling through the different MAP kinase families is known to regulate the expression of tissue factor in a stimulus and cell context dependent manner (11Eto M. Kozai T. Cosentino F. Joch H. Luscher T.F. Circulation. 2002; 105: 1756-1759Crossref PubMed Scopus (325) Google Scholar, 22Xuereb J.M. Sie P. Boneu B. Constans J. Thromb. Haemostasis. 2000; 84: 129-136Crossref PubMed Scopus (13) Google Scholar, 23Mechtcheriakova D. Schabbauer G. Lucerna M. Clauss M. De Martin R. Binder B.R. Hofer E. FASEB J. 2001; 15: 230-242Crossref PubMed Scopus (178) Google Scholar, 24Guha M. Mackman N. J. Biol. Chem. 2002; 277: 32124-32132Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). Thrombin and TNFα alone or in combination activate the ERKs as well as the stress-activated kinases p38 and JNK in human endothelial cells. To determine whether signaling through any of the MAP kinase pathways was responsible for the synergistic interactions of thrombin and TNFα on endothelial cell TF expression, we utilized selective pharmacological inhibitors for each family of MAP kinases. When confluent HUVEC were pretreated with 10 μm U0126, a concentration of inhibitor that completely blocks ERK activation in HUVEC, and then exposed to TNFα alone or TNFα plus thrombin, TF expression was not significantly inhibited (Fig. 2). Thrombin-stimulated TF expression was attenuated by U0126 by about 35%, although significant variability was observed between experiments. Thus, although some diminution of thrombin-stimulated tissue factor expression was observed when ERK activation was blocked, signaling through the ERK pathway was not important for the synergistic increase in tissue factor expression elicited by the combination of TNFα plus thrombin.Fig. 2Tissue factor expression was greatly attenuated by selective inhibitors of p38 and JNK.A, HUVEC were preincubated 1 h without or with 10 μm U0126 (an ERK inhibitor), 10 μm SB202190 (a p38 inhibitor), 25 μm SP600125 (a JNK inhibitor), or 10 μm SB202190 plus 25 μm SP600125. The cultures were then incubated for 5 h without or with TNFα, thrombin, or TNFα plus thrombin. Immunoblots are representative of 3 independent experiments with each inhibitor. B, collated data from 3 independent experiments, each bar representing percent inhibition of TF expression ± S.E. for cultures pretreated with inhibitor relative to cultures incubated without inhibitor. Con, control.View Large Image Figure ViewerDownload (PPT)Inhibition of either MAP kinase p38 with 10 μm SB202190 or JNK with 25 μm SP600125 resulted in significant attenuation of TF expression elicited by TNFα and thrombin, individually or in combination (Fig. 2). Pretreatment of HUVEC with both SB202190 and SP600125 almost completely ablated synthesis of tissue factor stimulated by TNFα and thrombin. Clearly, activation of both p38 and JNK is critical for TNFα- and thrombin-stimulated tissue factor expression in human endothelial cells.The temporal patterns of activation of the MAP kinases affect the regulation of downstream signaling molecules, both at the level of transcription and post-translational modification (25Murphy L.O. Smith S. Chen R.H. Fingar D.C. Blenis J. Nat. Cell Biol. 2002; 4: 556-564Crossref PubMed Scopus (754) Google Scholar, 26Murphy L.O. MacKeigan J.P. Blenis J. Mol. Cell. Biol. 2004; 24: 144-153Crossref PubMed Scopus (264) Google Scholar). Based on the inhibitor studies described above, we next examined the activation of p38 and JNK over a time course of 5 h using immunoblot analysis with phosphospecific antibodies for the active kinases. For both thrombin and TNFα, peak activation of p38 occurred between 5 and 15 min, followed by a drop in activity that was sustained above the basal level through 5 h (Fig. 3A). TNFα was a more potent initiating stimulus than thrombin. The combination of thrombin plus TNFα exhibited a similar pattern of p38 activation. Activation of a downstream target of p38, MAPKAPK-2, closely mimicked the pattern of p38 activation (data not shown). In contrast, the pattern of JNK activation was markedly different in endothelial cells stimulated either with TNFα or thrombin (Fig. 3B). TNFα stimulation of JNK resulted in a sharp peak of JNK activity at 15 min, followed by a precipitous drop almost to basal levels by 30–60 min. After this, JNK activity steadily increased over the next 4 h. Thrombin-stimulated activation of JNK gradually increased to a peak at 1 h and then dropped slightly to a plateau. Thus the time of peak thrombin-stimulated JNK activity occurred at the nadir of TNFα-stimulated JNK activation. In endothelial cells challenged with both thrombin and TNFα, JNK activity peaked dramatically at 15 min, then dropped to a plateau well above baseline that was maintained through 5 h. The phosphorylation of c-Jun, an immediate downstream target of activated JNK and a component of AP-1 transcription factor dimers, also was studied (Fig. 4). The patterns of c-Jun activation in thrombin- or TNFα-stimulated cells mimicked those observed for JNK, with peak activation by thrombin occurring at 1 h when TNFα activation of c-Jun was minimal. Interestingly, when the endothelial cells were stimulated with the combination of TNFα plus thrombin, c-Jun phosphorylation increased steadily, reaching a peak at 1 h that diminished very gradually over the next 4 h. Thus, the combined stimulus of thrombin plus TNFα resulted in the continuous, enhanced activation of c-Jun, allowing sustained c-Jun homodimer and heterodimer interaction at AP-1 sites on the TF promoter for periods ≥4 h.Fig. 3Time courses of p38 and JNK activation by TNFα, thrombin, or TNFα plus thrombin. HUVEC were incubated without or with TNFα, thrombin, or TNFα plus thrombin for the indicated times. Extracts were prepared and processed by immunoblot analysis for active p38 (A) and active JNK (B) using phosphospecific antibodies. The immunoblots are representative of ≥4 independent experiments for each condition. C, control.View Large Image Figure ViewerDownload (PPT)Fig. 4The magnitude and temporal duration of c-Jun activation differed when human endothelial cells were exposed to TNFα alone, thrombin alone, or TNFα plus thrombin. Only exposure to TNFα plus thrombin supported the sustained activation of c-Jun. A, HUVEC were incubated without or with TNFα, thrombin, or TNFα plus thrombin for 15 min, 1 h, and 5 h. Extracts were prepared and processed by immunoblot analysis for active c-Jun (pc-Jun) using a phosphospecific antibody. These blots are representative of 2 independent experiments. B, HUVEC were incubated without or with TNFα, thrombin, or TNFα plus thrombin for the indicated times. Extracts were prepared and processed by immunoblot analysis for phosphorylated c-Jun. The values ± S.E. for c-Jun activation were collated from 4 independent experiments for each condition (thrombin, TNFα, TNFα plus thrombin) that had been normalized to values obtained at the 1 h time points. C, control.View Large Image Figure ViewerDownload (PPT)An important component of AP-1 transcription factor heterodimers in endothelial cells is c-Fos (27Mackman N. Thromb. Haemostasis. 1997; 78: 747-754Crossref PubMed Scopus (246) Google Scholar). In HUVEC, very little c-Fos was expressed by cells treated with TNFα alone (Fig. 5, A and B). Thrombin stimulated an increase in c-Fos expression within 30 min, peak expression occurred at 1 h, and c-Fos was barely detectable 2 h after stimulation (Fig. 5C). The combined presence of TNFα with thrombin both prolonged the time course of c-Fos expression and significantly enhanced the quantity of c-Fos expressed.Fig. 5c-Fos expression was induced by thrombin and TNFα plus thrombin but not by TNFα alone. TNFα increased the magnitude and duration of thrombin-stimulated c-Fos expression. A, HUVEC were incubated for 1 h without or with TNFα, thrombin, or TNFα plus thrombin. Cells were extracted and immunoblotted for c-Fos. C, control. B, analysis of c-Fos expression from 3 independent experiments. Values ± S.E. are relative to the value for c-Fos expression elicited by thrombin, which was designated as 1. C, control. C, HUVEC were incubated for 0, 0.5 h, 1 h, 2 h, or 3 h with thrombin or TNFα plus thrombin. Cells were extracted and immunoblotted for c-Fos.View Large Image Figure ViewerDownload (PPT)Thrombin is a potent mediator of calcium mobilization in endothelial cells (6Woolkalis M.J. DeMelfi Jr., T.M. Blanchard N. Hoxie J.A. Brass L.F. J. Biol. Chem. 1995; 270: 9868-9875Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 28Goligorsky M.S. Menton D.N. Laszlo A. Lum H. J. Biol. Chem. 1989; 264: 16771-16775Abstract Full Text PDF PubMed Google Scholar), a second messenger that is important for c-Fos expression (29Lampugnani M.G. Colotta F. Polentarutti N. Pedenovi M. Mantovani A. Dejana E. Blood. 1990; 76: 1173-1180Crossref PubMed Google Scholar). We hypothesized that thrombin-stimulated calcium mobilization was important for the synergistic stimulation of tissue factor expression by thrombin plus TNFα. To test this hypothesis, we pretreated HUVEC with the cell-permeable calcium chelator BAPTA/AM for 1 h prior to challenge with thrombin plus TNFα. Tissue factor expression was significantly attenuated (∼70%) by chelation of intracellular calcium, as was c-Fos expression (Fig. 6). Phosphorylation of c-Jun, in contrast, was unaffected by the presence of the intracellular calcium chelator.Fig. 6Chelation of intracellular calcium attenuated expression of TF and c-Fos but had no effect upon the phosphorylation of c-Jun elicited by TNFα plus thrombin.A, HUVEC were preincubated for 1 h without or with 5 μm BAPTA/AM. Cultures were then incubated with TNFα plus thrombin for the indicated times before cell extraction. Samples were immunoblotted for tissue factor, c-Fos, and pc-Jun. Results are representative of ≥3 independent experiments.View Large Image Figure ViewerDownload (PPT)To confirm our hypothesis that the potentiation of TNFα-stimulated tissue factor expression by thrombin in endothelial cells is related to the capacity of thrombin to increase intracellular calcium and, thereby, c-Fos expression, we used the calcium ionophore ionomycin in place of thrombin. Ionomycin treatment alone resulted in detectable TF expression (∼25% relative to TNFα alone). When HUVEC were treated with both ionomycin and TNFα, there was a significant increase (3-fold) in tissue factor expression above that obtained with TNFα alone (Fig. 7). This correlated with detectable c-Fos expression, which was observed when HUVEC were treated with TNFα plus agents that mobilize calcium, ionomycin (Fig. 7) or thrombin (Fig. 5), but not in cells treated with TNFα alone (Figs. 5 and 7).Fig. 7Calcium mobilization by ionomycin in cultures concurrently exposed to TNFα induced c-Fos expression and potentiated TF synthesis. HUVEC were incubated with TNFα alone or in combination with 100 nm ionomycin for the times indicated. The cultures were extracted, and samples were immunoblotted for tissue factor and c-Fos. Results are representative of ≥3 independent experiments.View Large Image Figure ViewerDownload (PPT)In considering altered regulation of transcription factors important for TF synthesis, we also questioned whether NFκB was playing a role in the synergistic increase in TF expression by TNFα plus thrombin (9Pendurthi U.R. Williams J.T. Rao L.V. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3406-3413Crossref PubMed Scopus (114) Google Scholar, 15Bierhaus A. Zhang Y. Deng Y. Mackman N. Quehenberger P. Haase M. Luther T. Muller M. Bohrer H. Greten J. Martin E. Baeuerle P.A. Waldherr R. Kisiel W. Ziegler R. Stern D.M. Nawroth P.P. J. Biol. Chem. 1995; 270: 26419-26432Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 16Parry G.C. Mackman N. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 612-621Crossref PubMed Scopus (200) Google Scholar). We examined NFκB mobilization to the nucleus after stimulating endothelial cells for 1 h, a time of maximal degradation of the cytosolic regulator of NFκB, IκB. Thrombin proved to be a very poor stimulus compared with TNFα for promoting the nuclear translocation of NFκB (Fig. 8). The combination of thrombin plus TNFα did not significantly increase the nuclear localization of NFκB when compared with TNFα alone. In contrast, the TNFα-plus thrombin-mediated increases in c-Jun activation and c-Fos expression were clearly apparent in the same experiment. Thus, the synergistic increase in TF expression by thrombin plus TNFα is not dependent upon altered NFκB nuclear localization.Fig. 8Thrombin did not affect TNFα-stimulated mobilization of NFκB to the nucleus. HUVEC were incubated for 1 h without or with TNFα, thrombin, or TNFα plus thrombin. Nuclear and cytosolic extracts were prepared from the cultures and immunoblotted for NFκB, pc-Jun, and c-Fos. Immunoblots are representative of 4 independent experiments. C, control.View Large Image Figure ViewerDownload (PPT)DISCUSSIONWe have discovered a novel synergy between thrombin and TNFα that results in greatly enhanced tissue factor expression by endothelial cells. Knowledge of the mechanisms that underlie this synergy are likely to be crucial for understanding the potentially life-threatening thrombotic events that can occur in individuals with inflammatory pathologies.When we observed the synergistic increase in tissue factor expression in endothelial cells exposed to the combination of thrombin plus TNFα, we hypothesized that the synergy was due to selective differences in signaling by the two mediators. Thrombin activates G protein-coupled receptors, the PARs, enlisting a variety of G protein subtypes that can signal through many different networks. TNFα clusters and activates TNF receptors, which recruit a distinctive set of adapter proteins (TRADD, RIP, TRAF2) to mediate TNFα signaling (30Madge L.A. Pober J.S. Exp. Mol. Pathol. 2001; 70: 317-325Crossref PubMed Scopus (259) Google Scholar, 31MacEwan D.J. Cell. Signal. 2002; 14: 477-492Crossref PubMed Scopus (519) Google Scholar). Thrombin and TNFα, although they initiate signaling by different subsets of proteins, both activate many of the same signaling networks. Therefore, we looked for temporal or functional changes in signaling pathways that were utilized by both thrombin and TNFα (MAP kinase cascades) or signaling pathways that were preferentially activated by either thrombin (calcium mobilization) or TNFα (NFκB nuclear localization).Tissue factor expression is regulated by the transcription factors Egr-1, AP-1, and NFκB, some of which are immediate early gene products (15Bierhaus A. Zhang Y. Deng Y. Mackman N. Quehenberger P. Haase M. Luther T. Muller M. Bohrer H. Greten J. Martin E. Baeuerle P.A. Waldherr R. Kisiel W. Ziegler R. Stern D.M. Nawroth P.P. J. Biol. Chem. 1995; 270: 26419-26432Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 16Parry G.C. Mackman N. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 612-621Crossref PubMed Scopus (200) Google Scholar, 32Cui M.Z. Parry G.C. Oeth P. Larson H. Smith M. Huang R.P. Adamson E.D. Mackman N. J. Biol. Chem. 1996; 271: 2731-2739Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Recent studies (25Murphy L.O. Smith S. Chen R.H. Fingar D.C. Blenis J. Nat. Cell Biol. 2002; 4: 556-564Crossref PubMed Scopus (754) Google Scholar, 26Murphy" @default.
• W1978082123 created "2016-06-24" @default.
• W1978082123 creator A5040962843 @default.
• W1978082123 creator A5051377664 @default.
• W1978082123 creator A5084359914 @default.
• W1978082123 date "2004-08-01" @default.
• W1978082123 modified "2023-09-26" @default.
• W1978082123 title "Thrombin and Tumor Necrosis Factor α Synergistically Stimulate Tissue Factor Expression in Human Endothelial Cells" @default.
• W1978082123 cites W1584398295 @default.
• W1978082123 cites W1904917208 @default.
• W1978082123 cites W1964304127 @default.
• W1978082123 cites W1966756337 @default.
• W1978082123 cites W1969507427 @default.
• W1978082123 cites W1969719203 @default.
• W1978082123 cites W1973787501 @default.
• W1978082123 cites W1976523980 @default.
• W1978082123 cites W1993243592 @default.
• W1978082123 cites W1998078787 @default.
• W1978082123 cites W2001182083 @default.
• W1978082123 cites W2007010087 @default.
• W1978082123 cites W2010817360 @default.
• W1978082123 cites W2018099403 @default.
• W1978082123 cites W2052426854 @default.
• W1978082123 cites W2053669774 @default.
• W1978082123 cites W2063230936 @default.
• W1978082123 cites W2068425507 @default.
• W1978082123 cites W2069681479 @default.
• W1978082123 cites W2072861616 @default.
• W1978082123 cites W2078320770 @default.
• W1978082123 cites W2086783008 @default.
• W1978082123 cites W2089156917 @default.
• W1978082123 cites W2100837269 @default.
• W1978082123 cites W2106780768 @default.
• W1978082123 cites W2111688914 @default.
• W1978082123 cites W2130939448 @default.
• W1978082123 cites W2149637615 @default.
• W1978082123 cites W2157603347 @default.
• W1978082123 cites W2168592519 @default.
• W1978082123 cites W2240149772 @default.
• W1978082123 cites W2245299166 @default.
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