©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanism of the Tumor Necrosis Factor -mediated Induction of Endothelial Tissue Factor (*)

(Received for publication, March 7, 1995; and in revised form, July 31, 1995)

Angelika Bierhaus (1) Youming Zhang (1) Youhua Deng (1) Nigel Mackman (2) Peter Quehenberger (1) Michael Haase (3) Thomas Luther (3) Martin Müller (3) Hubert Böhrer (1) Johannes Greten (1) Eike Martin (1) Patrick A. Baeuerle (4) Rüdiger Waldherr (1) Walter Kisiel (5) Reinhard Ziegler (1) David M. Stern (6) Peter P. Nawroth (1)(§)

From the  (1)Departments of Medicine, Pathology, and Anesthesiology, University of Heidelberg, Heidelberg 69115, Germany, the (2)Department of Immunology, Scripps Research Institute, La Jolla, California 92037, the (3)Department of Pathology, Technical University of Dresden, Dresden 01307, Germany, the (4)Institute for Biochemistry, Freiburg 75106, Germany, the (5)Department of Pathology, University of New Mexico, Albuquerque, New Mexico 87131, and the (6)Department of Physiology, Columbia University, New York, New York 10032

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

This study examines the regulation of the human tissue factor (TF) promotor in vitro and in vivo. Transient transfections were performed in bovine aortic endothelial cells to investigate the role of two fundamentally different AP-1 sites and a closely located NF-kappaB site in the human TF promotor. The NF-kappaB site is functionally active, since overexpression of NF-kappaB(p65) resulted in induction of TF mRNA and activity. Promotor analysis showed that NF-kappaB induction was dependent on the integrity of the region from base pair -188 to -181. Overexpression of Jun/Fos resulted in TF induction of transcription and protein/activity. Functional studies revealed that the proximal AP-1 site, but not the distal, was inducible by Jun/Fos heterodimers. The distal AP-1 site, which has a G A switch at position 4, was inducible by Jun homodimers. Electrophoretic mobility shift assays, using extracts of tumor necrosis factor alpha (TNFalpha)-stimulated bovine aortic endothelial cells, demonstrated TNFalpha-inducible binding to the proximal AP-1 site, comprising JunD/Fos heterodimers. At the distal AP-1 site, only minor induction of binding activity, characterized as proteins of the Jun and ATF family, was observed. Consistently, this site only marginally participates in TNFalpha induction. Functional studies with TF promotor plasmids confirmed that deletion of the proximal AP-1 or the NF-kappaB site decreased TNFalpha-mediated TF induction to a higher extend than loss of the distal AP-1 site. However, integrity of both AP-1 sites and the NF-kappaB site was required for optimal TNFalpha stimulation. The relevance of these in vitro data was confirmed in vivo in a mouse tumor model. Expression plasmids for a dominant negative Jun mutant or I-kappaB were packaged in liposomes. When either mutated Jun or I-kappaB were injected intravenously 48 h before TNFalpha, a reduction in TNFalpha-mediated TF expression in the tumor endothelial cells was observed. Simultaneously, fibrin/fibrinogen deposition decreased and free blood flow could be restored. Thus, TNFalpha-induced up-regulation of endothelial cell TF depends on a concerted action of members of the bZIP and NF-kappaB family.


INTRODUCTION

Unstimulated endothelial cells express no tissue factor (TF)^1in vitro and in vivo(1, 2, 3, 4, 5, 6) . Recent studies show that TF expression in vitro can be induced by TNFalpha(7, 8, 9, 10, 11) . In vivo, however, expression of TF has only been shown in selected vascular beds: in the splenic endothelium in a septicemia model (12) and in tumors such as Meth-A and Karposi sarcomas(13, 14) . The human TF promotor has been characterized, and its function has been extensively studied in monocytes/macrophages(15, 16, 17, 18) . The porcine TF promotor has been described in endothelial cells(11) . The data available from the human TF promotor suggest that two AP-1 and the NF-kappaB site are central in the endotoxin-dependent regulation of TF expression(16, 17, 18) . In contrast, the study of the porcine TF promotor shows that mainly the induction of NF-kappaB is responsible for lipopolysaccharide- and TNFalpha-mediated induction of endothelial TF expression(11) .

The discrepancy in the role of AP-1 in the human and porcine TF promotor might be due to differences in the sequence composition of the AP-1 binding sites of the various species. Sequence alignments revealed important differences between the AP-1 sites of the different species (11) . The porcine TF promotor has two non-canonical AP-1 sites that differ from the defined AP-1 consensus sequence in one central base (11) . Non-canonical AP-1 sites have been reported of being weak binding sites(19) . In contrast, the proximal AP-1 site in the human (and the mouse) TF promotor contains the core of the consensus sequence (11, 15, 16) and thereby represents a high affinity site for AP-1 binding. This indicates that different AP-1-like proteins may be involved in the regulation of TF expression in different species. If the porcine model (11) would be relevant for human disease, then blocking of NF-kappaB activation would provide a powerful way to prevent excess TF expression in human disease. If the human model (15, 16, 17, 18) is relevant, then inhibition of NF-kappaB activation might lead to increased c-Fos transcription (20, 21) and thereby to AP-1-mediated TF induction. To resolve this issue, we studied the role of both AP-1 sites and their cooperative action with the NF-kappaB site in the human TF promotor.

A number of homo- and heterodimers can recognize AP-1 sites(22, 23, 24, 25) but exhibit different affinities for different motifs(26) . These proteins have been termed bZIP family (27) due to their ability to dimerize via an alpha-helical leucine ``zipper.'' The members of the Jun subfamily c-Jun, JunB, and JunD are highly homologous in their dimerization and binding domains and can compete for the same AP-1 sites(28) . The transactivating capacities of c-Jun and JunD are dramatically increased in combination with c-Fos(29, 30) . The functional homologues of c-Fos, Fra-1, Fra-2, and FosB can also dimerize with Jun proteins(30, 31, 32, 33) . In addition, members of the ATF/CREB family like ATF-2, ATF-3, and ATF-4 (but not ATF-1) are capable of binding to proteins of the AP-1 transcription factor family (25, 26, 34) . Thus, it was our hypothesis that the distal non-canonical AP-1 site in the human TF promotor and the proximal canonical AP-1 site bind different members of the AP-1/bZIP family.


MATERIALS AND METHODS

Reagents

Reagents were obtained as follows: DMEM, RPMI 1640, HEPES buffer solution, L-glutamine, penicillin-streptomycin mixture, and PBS, pH 7.4, were from BioWhittaker, Walkersville, MD. FCS, DOTAP, DNase I (RNase-free), proteinase K, RNase A, and the DIG nucleic acid detection kit were from Boehringer, Mannheim, Germany. Barbital buffer was obtained from Behring, Marburg, Germany. S2222 was purchased from KabiVitrum, Stockholm, Sweden. [alpha-P]dCTP (3000 Ci/mmol at 10 Ci/ml), [-P]dATP (3000 Ci/mmol at 10 Ci/ml), [alpha-P]UTP (3000 Ci/mmol at 10 Ci/ml), [S]methionine (>10,000 Ci/mmol), Hybond-N nylon filter, and Hyperfilm x-ray films were obtained from Amersham, Braunschweig, Germany. Poly(dI-dC) was purchased from Sigma, Deisenhofen, Germany. Rabbit reticulocyte lysate and recombinant AP-1 (c-Jun, human) were purchased from Promega. Anti-p50 (sc-114X), anti-p65 (sc-109X), and anti-c-Rel (sc-70X) polyclonal antibodies, anti-c-Jun (sc-45X; specific for c-Jun, non-cross-reactive with JunB or JunD), anti-pan-Jun (sc-44X; recognizing the C termini of c-Jun, JunB, JunD), anti-JunD (sc-74X; no c-Jun or JunB cross-reactivity), anti-c-Fos (sc-52X; specific for c-Fos p62, non-cross-reactive with FosB, Fra-1, Fra-2) and anti-ATF-2 (sc-187X; non-cross-reactive with other ATF/CREB proteins) polyclonal antibodies, an anti-ATF-1 (sc-243X; non-cross-reactive with other ATF/CREB proteins) monoclonal antibody, and recombinant ATF-2-bZIP(350-505) were obtained from Santa Cruz Inc., Santa Cruz, CA. TNFalpha (10^8 units/mg) was a gift from Knoll AG, Ludwigshafen, Germany.

Plasmids

The SV-40-driven luciferase control plasmid pGL2-control, the promotorless plasmid pGL2-basic, the beta-galactosidase control plasmid pSV-beta-Gal, and the chloramphenicol transferase control vector pCAT-control were obtained from Promega. pSPT18 was purchased from Boehringer Mannheim, Germany. The TF promotor mutants pHTF(-278)Luc (A1-A2-N), pHTFM2(-278)Luc (A2-N), pHTFM3(-278)Luc (A1-N), and pHTF(-111)Luc have been described previously ( (15) and (16) ; see Table 1). The plasmid pHTFO(-441)Luc (A1-A2) was prepared by inserting the polymerase chain reaction product of region bp -441 to bp -195 into the SmaI site of pHTF(-111)Luc (Table 1). The clones A1, A2, and N were constructed by subcloning oligonucleotides, spanning 24 bp of the respective binding site into the SmaI site of pHTF(-111)Luc (Table 1). All plasmids were characterized by DNA sequencing. pSV-c-Jun(35) , pBK28(c-Fos)(35) , T7-Jun, and T7-Fos (36, 37) were generously provided by Dr. I. Verma (San Diego, CA). NFkappaB(p65) (RC-CMV-p65) and I-kappaB (RC/CMVMAD-3wt) have been described(38) . The mutated Jun plasmid pDB7, a derivate of the point mutant Mut14(39) , was obtained from Dr. D. Bohmann (EMBL, Heidelberg, Germany). The human TF cDNA probe HTF8 was generously provided by Dr. E. Sadler (Washington University, St. Louis, MO)(40) . Meth-tRNA was a gift of Dr. Xanthopoulus (Karolinska Institute, Stockholm, Sweden); other plasmids mentioned were obtained from ATCC.



Tissue Culture

Bovine aortic endothelial cells (BAEC) were cultured in DMEM supplemented with 10% FCS as described previously (41, 42) and characterized as endothelial cells by the expression of von Willebrand factor, thrombomodulin, low basal levels of TF, and morphologic features. Cells were passaged every 8-10 days without showing gross morphological changes until passage 15. Passages 4-8 were used. All experiments were performed with cells that had been confluent for 3-5 days. For transfection experiments, cells growing in the logarithmic phase were used.

Methylcholanthrine-A-induced (Meth-A) sarcoma cells were a gift of Dr. D. Männel (DKFZ, Heidelberg, Germany) and were cultured in RPMI 1640, 10% FCS, 100 units/ml penicillin, 100 units/ml streptomycin as described elsewhere(14) .

Determination of Tissue Factor (TF) Activity by One-stage Clotting Assay

One-stage clotting assays were performed as described(1) . After washing, cells were removed non-enzymatically by scraping in barbital buffer, pH 7.6, collected by centrifugation for 5 min at 1000 rpm, and resuspended in 100 µl of the same buffer. After addition of 100 µl of citrated bovine plasma 100 µl of citrated bovine plasma, and 100 µl of 25 mM CaCl(2) solution (Behring, Marburg, Germany) the samples were incubated at 37 °C. The time from addition of CaCl(2) to the first defined fibrin strand was determined. TF activity was calculated by comparing the measured clotting time with a standard curve made with known amounts of TF(1) . The measured amount of TF was expressed as picograms of TF/10^6 cells ± S.D. All experiments were performed at least three times, and each experiment was done in triplicates.

Determination of Tissue Factor (TF) Activity by Monitoring the Hydrolysis of S2222 Synthetic Peptide Substrate

TF activity was also assessed by monitoring the hydrolysis of the synthetic peptide substrate Bz-Ile-Glu-Gly-Arg-p-nitroanilide (S2222, KabiVitrum, Stockholm, Sweden) as described previously(43) . Cells were harvested in PBS, pH 7.4, and incubated with 30 µl of human factor VII (final concentration 35 µg/ml) (44) and 10 µl of factor X (final concentration 200 µg/ml) (44) in the presence of 10 mM CaCl(2). Aliquots (30 µl) were added to 470 µl of 50 mM Tris, pH 7.9, 175 mM NaCl, 5 mM EDTA, and 0.5 mg/ml BSA. Factor Xa formation was assessed using 100 µl of S2222 added to the entire 0.5-ml sample. Hydrolysis was monitored at room temperature by measuring the change in absorbance at 405 nm, using a Beckmann DU 7400 spectrophotometer (Beckmann, Dreieich, Germany). Factor Xa (final concentration 20 µg/ml) (44) served as control. Each experiment was repeated three times.

Statistical Analysis

Data were analyzed with the aid of SIGMA PLOT software (Jandel Scientific). Levels of significance were determined by Student's t test. Any p value of 0.05 and below was considered to be significant. For calculation of p values in Fig. 1c, the Mann-Whitney U test was used.


Figure 1: Overexpression of c-Jun/Fos(AP-1), NF-kappaB(p65), or c-Jun/Fos(AP-1) and NF-kappaB(p65) induces TF transcription, activity, and antigen. a, nuclei were extracted from BAEC transiently transfected with CAT (= mock control), c-Jun, c-Fos, and/or NF-kappaB(p65) overexpressing plasmids. Nuclear run-on experiments were performed as described under ``Materials and Methods'' to allow in vitro synthesis of [alpha-P]UTP-labeled mRNA. This was hybridized against filters onto which cDNAs for TF (top), Meth-tRNA (bottom), pSPT18, and TNFalpha (negative controls, data not shown) had been fixed. b, activation of factor X by BAEC transfected with CAT (= mock control), c-Jun, c-Fos, and/or NF-kappaB(p65). Cells were transiently transfected with the respective plasmids, harvested after 42 h, and assayed for factor X activation in the presence of factor VII (35 µg/ml). The generation of factor Xa was measured spectrophotometrically over a period of 15 min. S2222 served as substrate as described under ``Materials and Methods.'' Two experiments were performed in triplicates with identical results. One typical experiment is shown. c, BAEC were transiently transfected with CAT (= mock control), c-Jun, c-Fos, and/or NF-kappaB(p65), harvested 42 h after transfection, and assayed for procoagulant activity as described under ``Materials and Methods.'' TF activity of each sample was determined based on comparison with a standard curve established with known amounts of recombinant TF. The data ± S.D. represent the mean of three independent experiments performed in triplicate (p values: control versus Jun/Fos = 0.008; control versus NF-kappaB = 0.009; control versus Jun/Fos/NF-kappaB = 0.009)



Nuclear Run-on Transcription Assay

Nuclear run-on transcription assays were performed essentially according to the procedure of Greenberg and Ziff (45) as described elsewhere(46, 47, 48) . In brief, nuclei were harvested from 2 times 10^7 cells after 42 h of transfection. Run-on reactions were performed in 0.7 M KCl, 50 mM MgCl(2), 50 mM Tris-HCl, pH 8.0, 25 mM DTT, 1 mM EDTA in the presence of 250 µCi of [alpha-P]UTP (3000 Ci/mmol) and incubated for 30 min at 30 °C. The synthesized mRNA was incubated with DNase I for 5 min at 30 °C, treated with proteinase K (10 mg/ml), and extracted with 0.45-µm Millipore filters (type HA). The RNA was collected by EtOH precipitation and redissolved in 300 µl of DEPC-H(2)O, 1 µl was counted, and equal numbers of Cerenkov counts were made up to 2 ml of hybridization solution and added to the previously prepared slot blot filters. Hybridization was performed without prehybridization in 50% formamide, 5 times SSC, 5 times Denhardt's solution, 1% SDS for 4 days at 42 °C. Filters were washed three times at room temperature for 10 min in 2 times SSC and once for 10 min at 60 °C in 1 times SSC. Blots were exposed to Amersham Hyperfilms for 1-4 weeks at -80 °C with intensifying screens. The density of autoradiographic signals was quantitated using a Beckman DU 7400 densitometer (Beckman, Dreieich, Germany)(46, 47, 48) . For preparation of filters, 3 µg of human TF plasmid DNA or the respective household and control plasmids (GAPDH, Meth-tRNA, pSPT18, p19-Luc, TNFalpha cDNA) were applied to Hybond-N membranes using a slot blot apparatus (Schleicher & Schüll, Dassel, Germany) as described previously(46, 47, 48) .

Northern Blot Analysis

Northern blots were performed essentially as described previously(46, 47) . Tumor tissue was broken under liquid nitrogen and homogenized in an Ultrathurax (Wheaton, Millville, NJ), and total RNA was purified by the guanidine isothiocyanate-cesium chloride method(49) . RNA concentrations were determined spectrophotometrically, and 20 µg of RNA/lane were separated onto an 1.1% agarose-formaldehyde gel and transferred to Hybond-N-Nylon membranes (Amersham, Braunschweig, Germany) according to standard methods(50) . A mouse tissue factor cDNA fragment (bp 721 to bp 1043) (14, 48) was labeled with [alpha-P]dCTP to a specific activity >10^8 cpm/µg DNA by the random prime technique (51) . Filters were prehybridized for 1 h and hybridized for 12-16 h at 65 °C in 50 mM PIPES, pH 6.8, 200 mM NaCl, 20 mM Na(2)PO(4), 30 mM NaHPO4, 1 mM EDTA, 5% SDS, 50 µg of salmon sperm/ml, 50 µg of yeast tRNA/ml(47) , washed 3 times 15 min in 1.3 times SSC, 5% SDS, and exposed to Hybond-N Hyperfilms (Amersham, Braunschweig, Germany) for 5 days.

Electrophoretic Mobility Shift Assay

For electrophoretic mobility shift assays (EMSA), nuclear proteins were harvested by the method of Andrews (52) as described previously(47) . Approximately 2 times 10^7 were harvested in cold PBS, pH 7.4, and pelleted at 1500 rpm for 5 min. The pellet was resuspended in 400 µl of cold buffer A (10 mM HEPES-KOH, pH 7.9 at 4 °C, 1.5 mM MgCl(2), 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF), and incubated for 10 min on ice. The samples were centrifuged for 10 s at highest speed, the supernatant was discarded and the pellet was resuspended in 100 µl of cold buffer C (20 mM HEPES-KOH pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl(2), 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF) and incubated on ice for 20 min. After centrifugation (2 min, 4 °C, highest speed) the supernatant was quick-frozen at -80 °C. Protein concentration was determined using a colorimetric assay based on Bradford(53) . When organ tissue was used, the above protocol was modified according to Deryckere and Gannon(54) . Large pieces of tissue (0.1 times 0.2 cm) were frozen in liquid nitrogen, broken mechanically with a hammer, and transferred to a 50-ml Falcon tube containing 5 ml of cold buffer A (10 mM Hepes-KOH, pH 7.9, at 4 °C, 10 mM KCl, 1.5 mM MgCl(2), 0.5 mM DTT, 1 mM EDTA, 0.2 mM PMSF, 0.6% Nonidet® P-40). The tissue was homogenized in an Ultrathurax (Wheaton, Millville, NJ) for 1 min, transferred to an 15-ml tube, and centrifuged for 30 s at 2000 rpm, 4 °C to remove tissue debris. The supernatant was incubated on ice for 10 min and centrifuged for 5 min at 8000 rpm at 4 °C. The supernatant was discarded, and the nuclear pellet was resuspended in 100 µl of buffer B (25% glycerol, 20 mM Hepes-KOH, pH 7.9 at 4 °C, 420 mM NaCl, 1.5 mM MgCl(2), 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 2 mM benzamidine, 5 mg/ml leupeptin) and incubated on ice for 20 min. Cellular debris was removed by 2 min of centrifugation at 4 °C and the supernatant was quick-frozen at -80 °C. Protein concentrations were determined as above.

Oligonucleotides, listed in Table 2, were synthesized on a Gene Assembler Plus (Pharmacia, Freiburg, Germany) and purified on histidine gels(55) . They were labeled by kinasing to a specific activity >5 times 10^7 cpm/µg DNA. Binding of AP-1 was performed in 25 µl of 10 mM HEPES, pH 7.9, 0.1 mM EDTA, 75 mM NaCl, 4 mM MgCl(2), 2 mM DTT, 17.5% glycerol, 1 mg/ml BSA (DNase-free) in the presence of 0.01 µg/µl poly(dI-dC)(47) . For organ preparations poly(dI-dC) was scaled up to 0.15 µg/µl. When recombinant proteins produced in rabbit reticulocyte lysates (Promega) were included in the reaction, the poly(dI-dC) concentration was increased to 0.05 µg/µl. When recombinant human c-Jun (Promega) was used, poly(dI-dC) was replaced by 0.01 µg/µl AP-3 oligonucleotides (Promega) according to the manufacturer's instruction. NF-kappaB binding was performed in 10 mM HEPES, pH 7.5, 0.1 mM EDTA, 100 mM NaCl, 1 mM ZnCl(2), 4 mM MgCl(2), 2 mM DTT, 17.5% glycerol, 1 mg/ml BSA (DNase-free), and 0.1 µg/µl poly(dI-dC) in a total of 25 µl(47) . For organ preparations, the poly(dI-dC) concentration was increased to 0.3 µg/µl. 8-10 µg of nuclear extract were incubated on ice for 20 min in the appropriate binding buffer before adding approximately 1 ng of labeled oligonucleotide. A typical binding reaction contained 50,000 cpm (Cerenkov). The samples were incubated at room temperature for and additional 15 min. Protein-DNA complexes were separated from the free DNA probe by electrophoresis through 4% (AP-1) or 5% (NF-kappaB) native polyacrylamide gels containing 2.5% glycerol and 0.5 times TBE buffer(47) . The gels run at room temperature with 30 mA for approximately 2.5 h. Gels were dried under vacuum on Whatmann D-81 paper (Schleicher and Schüll, Dassel, Germany) and exposed for 12-48 h to Amersham Hyperfilms at -80 °C with intensifying screens. Specificity of binding was ascertained by competition with a 160-fold molar excess of cold consensus oligonucleotides. For supershifting experiments 2.5 µg of the respective antibody were applied to the reaction mixture at the time the labeled oligonucleotide was added.



Synthesis of Recombinant Proteins

c-Jun and c-Fos RNA were synthesized under the control of the T7 promotor according to the method of Melton and Krieg(56) . Yeast inorganic pyrophosphatase was included in the reaction to increase the yield of RNA obtained(57) . The size of in vitro transcribed RNA was assessed by 5% polyacrylamide gels. About 3.6 µg of c-Jun or c-Fos RNA were added to 70 µl of rabbit reticulocyte lysate (Promega) to translate proteins in vitro by the method of Sassone-Corsi(36) . When c-Jun and c-Fos were cotranslated to form heterodimers, 2 µg of each RNA was used. Efficient translation was monitored in a parallel reaction using [-S]Methionine as substrate. 0.3-10 µl (approximately 0.15-5 µg) of in vitro translated proteins were used in EMSA. Unprogrammed lysate served as control.

Transient Transfection of Endothelial Cells

Logarithmically growing endothelial cells were transfected as described by Lee(58, 59) with minor modifications(47) . Cells were grown in DMEM containing 10% FCS to 70% confluence. 1.4 µg of the appropriate plasmids/ml of medium were transfected by the calcium phosphate (CaPO(4)) method. Cells were exposed to the precipitate for 6 h (HUVEC) to 8 h (BAEC). Medium was changed, and cells were incubated for 42 h. Cells were harvested in the appropriate buffers.

Plasmid DNA used in transfections was isolated by alkaline lysis, followed by CsCl equilibrium centrifugation(50) . For promotor studies 0.5 µg of luciferase promotor constructs/ml of medium were transfected. To correct for variability in transfection efficiency 0.15 µg of pSV-beta-Gal plasmid/ml of medium were included. For transactivation experiments, 0.25 µg/ml pSV-c-Jun, pBK28(c-Fos), NF-kappaB(p65), I-kappaB, or mutated Jun were cotransfected with 0.5 µg of luciferase containing promotor constructs. Reactions were filled up with pCAT-control (serving as mock control) to give the final DNA concentration of 1.4 µg/ml medium. Cell extracts were prepared by lysis in 25 mM Tris phosphate, pH 7.8, 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N,N`-tetraacetic acid, 10% glycerol, 1% Triton X-100 and assayed directly for luciferase activity (60) . beta-Galactosidase activity was determined in the same lysis buffer(61) . Luciferase and beta-galactosidase activity were determined for each sample. The ratio of luciferase activity to beta-galactosidase activity served as a measure for normalized luciferase activity. For each experiment the normalized luciferase activity of the promotorless luciferase plasmid was subtracted from this quotient. The result was multiplied by 1000. To compare different transfections for each series of experiments, the relative Luc units of the triplicate were divided by those of the control construct pGL2-control. The quotient was multiplied by 100 and expressed as percentage of pGL2-control. In addition relative luciferase units were calculated as percentage of pHTF(-278)Luc basal expression(47) . Each experiment was performed in triplicate. The data presented are the mean of at least three independent transfections performed. Standard deviations are given as vertical error bars.

Intravenous Somatic Gene Transfer

25-30 µg of plasmid DNA were dissolved in 150 µl of DMEM, added to 100 µg of DOTAP/150 µl of DMEM, and incubated at room temperature for 20 min(14) . 300 µl of this solution were injected intravenously via the tail vein of adult female C(3)H mice (18-20 g) at the day, Meth-A sarcoma (10^6 cells/animal) were implanted. A second DNA:DOTAP injection was performed 2 days before application of TNFalpha. As soon as the tumors reached an average diameter of 0.5 cm (12-14 days after planting), TNFalpha (5 µg/animal) was injected intravenously. Mice were sacrificed at the times indicated in the figure legend. Before harvesting the tumor tissues, mice were perfused with 30-40 ml of PBS by intracardiac injection of PBS into the left ventricle(14) .

Determination of Blood Flow

5 times 10^5 of colored microspheres (10 mM, E-Z-Trac Ultraspheres(TM), E-Z-Trac Inc., Los Angeles, CA) were injected for 10-20 s into the left ventricle of the anesthetized mouse before the mice was sacrificed. Tumors were harvested, weighted, and hydrolyzed in sodium hydroxide-SDS solution (E-Z-Trac Inc., Los Angeles, CA) at 80 °C overnight. Microbeads were isolated according to the manufacturer's instructions, counted microscopically, and expressed as microspheres per gram of tumor (± S.D.)(14, 48) .

Immunohistochemistry

Tumors were fixed in 4% formaldehyde. After cutting, sections were incubated with anti-mouse TF antibodies (48) for 2 h at room temperature as described previously(13, 14, 48) . After three washes with 150 mM NaCl, 100 mM Tris-HCl, pH 7.5, sections were incubated for 1 h at room temperature with a peroxidase-conjugated second antibody. After washing color development was performed with 3-amino-9-ethylcarbazole and H(2)O(2)(13, 14, 48) . Negative controls included omission of first or second antibodies and the substitution of the first antibody by nonspecific antibodies (data not shown). For immunofluorescence staining anti-fibrin-fibrinogen antibodies (fluorescine conjugate, Cappel, West Chester, PA) were used in a 1:8 dilution(14) . Sections were incubated with the antibody for 45 min at room temperature, followed by three washes with PBS. 10 cuts from at least three different experiments were analyzed independently.^2

In Situ Hybridization

In situ hybridization was performed as described before(13, 14) . In brief, tumor sections were fixed in 4% paraformaldehyde, 0.5% glutaraldehyde in PBS, pH 7.4, and incubated with proteinase K (100 µg/ml) for 10 min. Hybridization was performed for 16 h at 50 °C against a digoxigenin-labeled TF antisense riboprobe, synthesized from a mouse tissue factor cDNA fragment (bp 721 to bp 1043). Hybridization solution was 0.6 M NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.6, 10% dextran sulfate, 1 times Denhardt's, 50% formamide, 0.25% SDS, 100 mM DTT, 50 µg/ml salmon sperm DNA, 100 µg/ml tRNA, and 5-10 ng of labeled RNA probe. At the end of hybridization, samples were incubated for 30 min at 37 °C with RNase A (20 µg/ml) and washed once at 37 °C for 30 min with 2 times SSC, 50% formamide and twice for 30 min at 50 °C with 0.2 times SSC. Detection was performed immunologically with the DIG nucleic acid detection system according to the manufacturer's instruction. A TF sense riboprobe served as negative control (data not shown).


RESULTS

Tissue Factor Is Induced by Overexpression of Proteins of Jun/Fos and NF-kappaB(p65)

Cultured bovine aortic endothelial cells (BAEC) were transiently transfected with plasmids overexpressing c-Jun and c-Fos or NF-kappaB(p65). Transient transfection with c-Jun/Fos(AP-1), NF-kappaB(p65), or both transcription factors together resulted in enhanced expression of TF compared to mock transfected cells (expressing the bacterial CAT). TF was studied at several levels.

(i) Nuclear run-on assays (Fig. 1a) revealed that induction of transcription of the TF gene occurred.

(ii) The biological activity was characterized by its factor VII-dependent activation of factor X (synthetic substrate assay; Fig. 1b) and by a coagulant assay (one-stage clotting time; Fig. 1c). Since almost no factor X activation was observed in the absence of factor VII (data not shown), most of the coagulant activity induced is TF. But it cannot be absolutely excluded that other proteins involved in activation of coagulation are also inducible by overexpression of AP-1 or NF-kappaB(p65). When BAEC were cotransfected with AP-1 and NF-kappaB(p65), an additive effect in TF induction was observed in all systems tested (Fig. 1).

Structural analysis of the human TF promotor has demonstrated two linked AP-1 sites, a proximal canonical site at -210 (A2), a distal non-canonical site at -223 (A1), and an NF-kappaB site(N) at position -188(15, 16, 17, 18) . The availability of promotor constructs (Table 1) allows for definition of the areas involved in activation of TF by AP-1 or NF-kappaB(p65) (Fig. 2). The plasmids containing both AP-1 sites responded to c-Jun/Fos overexpression in the presence or absence of the NF-kappaB site (Fig. 2a). However, deletion of the NF-kappaB binding site reduced the induction by c-Jun/Fos (Fig. 2a), suggesting that endogenously present proteins (presumably p65/c-Rel) capable to bind to this site, act in concert with AP-1. To test whether optimal induction by c-Jun/Fos was dependent on the presence of endogenous NF-kappaB, we cotransfected BAEC with plasmids overexpressing I-kappaB. Overexpression of I-kappaB reduced the c-Jun/Fos induction as long as the NF-kappaB site was present (Fig. 2c).


Figure 2: Functional analysis of the TF promotor demonstrates activation of TF expression by c-Jun/Fos(AP-1) and NF-kappaB(p65). BAEC cells were cotransfected with the TF promotor plasmids [A1-A2-N], [A1-A2], or [N] (1 µg/ml medium) (see ``Materials and Methods'') and c-Jun, c-Fos, NF-kappaB(p65), mutated Jun, and/or I-kappaB (0.5 µg each/ml medium) and cultivated for 42 h. After harvest luciferase activity was determined in each sample and normalized for transfection efficiency to the amount of beta-galactosidase expressed by the plasmid pSV-beta-gal (Promega). The normalized data are expressed as relative Luc units and represent the mean of three independent experiments ± S.D. performed in triplicate. a, transactivation of the TF promotor by Jun/Fos(AP-1); Jun/Fos(AP-1) overexpression induced TF as long as AP-1 sites were present in the TF promotor constructs. b, transactivation of the TF promotor by NF-kappaB(p65); overexpression of NF-kappaB(p65)-induced TF as long as the NF-kappaB site was present in the TF promotor constructs. c, the TF promotor plasmid [A1-A2-N] was cotransfected with c-Jun, c-Fos, or NF-kappaB(p65) and the specific inhibitor of the opposite transcription factor; the Jun/Fos(AP-1)-dependent transactivation could be partly suppressed by the NF-kappaB-specific inhibitor I-kappaB, while the AP-1-specific inhibitor mutated Jun partly suppressed transactivation by NF-kappaB(p65).



When BAEC were transfected with a plasmid overexpressing NF-kappaB(p65) (Fig. 2b), induction was maximal, when both AP-1 sites and the NF-kappaB site were present. NF-kappaB(p65) inducibility was reduced, but not lost, when the AP-1 sites were deleted (Fig. 2b). Deletion of the NF-kappaB site resulted in loss of inducibility by NF-kappaB(p65). To test, whether optimal NF-kappaB induction was equally dependent on the presence of endogenous AP-1, we cotransfected BAEC with plasmids overexpressing NF-kappaB(p65) and the Jun-specific inhibitor mutated Jun. This negatively dominant c-Jun point mutant (39) is able to dimerize with other members of the AP-1/bZIP family; however, it is unable to bind to the DNA recognition site. Since c-Fos does not bind to DNA at all, due to its failure to homodimerize(31) , overexpression of mutated Jun leads to a significant reduction of Jun/Fos heterodimer binding. When NF-kappaB(p65) and mutated Jun were cotransfected, a reduction of the NF-kappaB(p65)-mediated stimulation was seen (Fig. 2c). Thus AP-1 and NF-kappaB(p65) act in concert in inducing TF ( Fig. 1and Fig. 2), which seems to be dependent on the presence of DNA sequences previously described as binding sites for AP-1 and the NF-kappaB subunits c-Rel and p65(11, 15, 16, 17, 18, 62) .

Both AP-1 Sites Differ in Their Affinity for Jun/Fos Heterodimers and Jun Homodimers

The human TF promotor contains a distal non-canonical and a proximal canonical AP-1 binding site. Therefore EMSA were performed to compare the binding capacity of both AP-1 sites for Jun/Fos heterodimers (AP-1; Fig. 3). Decreasing amounts of c-Jun/Fos programmed rabbit reticulocyte lysate were incubated with either the proximal (A2; Fig. 3a) or the distal (A1; Fig. 3b) AP-1 site. The proximal canonical AP-1 site (A2) demonstrated the expected high affinity binding of c-Jun/Fos heterodimers detectable down to 2.5 µl of programmed lysate (Fig. 3a). However, the distal non-canonical AP-1 site (A1) showed only very weak, nearly undetectable binding of c-Jun/Fos heterodimers (Fig. 3b). More rapidly migrating bands on gel shift assays were observed in all samples and represented binding not inhibited by excess unlabeled AP-1 oligonucleotides (data not shown) and are therefore marked as ``nonspecific lysate bands.'' These observations were confirmed by functional promotor studies looking at the inducibility of mutants containing either the proximal (A2) or the distal (A1) AP-1 site (Fig. 3c). EMSA were performed to compare the binding capacity for both AP-1 sites for Jun homodimers. Decreasing amounts of recombinant Jun homodimers were incubated with either the canonical proximal AP-1 site (A2) (Fig. 4a) or the non-canonical distal AP-1 site (A1) (Fig. 4b). Binding to the proximal AP-1 site was detectable only down to 0.55 µg of recombinant c-Jun homodimers, while binding was still detectable down to 2.5 µl (= 1.25 µg) of Jun/Fos programmed lysate (Fig. 4a, lane 1). At the distal AP-1 site (A1), prominent binding of c-Jun homodimers, even at only 0.1 µg, was detected (Fig. 4b). Our observations using EMSA were extended by transient transfection studies of BAEC using TF promotor mutants. Mutants containing only the distal AP-1 site (A1) were more strongly induced by c-Jun overexpression than mutants containing only the proximal AP-1 site (A2) (Fig. 4c). Therefore, the distal AP-1 site (A1) has properties of a Jun site rather than an AP-1 site.


Figure 3: The proximal AP-1 site (A2) preferentially binds Jun/Fos heterodimers (AP-1), while the distal AP-1 (A1) site is only very weakly recognized and not induced by AP-1: a and b, radiolabeled oligonucleotide probes containing the canonical proximal (a) or the non-canonical distal (b) AP-1 site were incubated with decreasing amounts of Jun/Fos programmed rabbit reticulocyte lysate. The amount of programmed lysate, added to the binding reaction, is given above the lanes. The mobility of the formed complexes was analyzed on 4% nondenaturing polyacrylamide gels. Arrows indicate the specific AP-1 binding. Nonspecific lysate reactions are marked by brackets. c, BAEC were cotransfected with the TF promotor plasmids [A1], [A2], or [A1-A2] (1 µg) (Table 1) and a control plasmid (CAT = ``mock'') or c-Jun and c-Fos (0.5 µg each) and cultivated for 42 h. After harvest, luciferase activity was determined and normalized for transfection efficiency to the amount of beta-galactosidase expressed by the plasmid pSV-beta-gal (Promega). The normalized data are expressed as relative Luc units and represent the mean of three independent experiments ± S.D. performed in triplicate. The inducibility of the various TF promotor plasmids by c-Jun/Fos heterodimers is shown. The level of basal expression (transfected with CAT as control) is indicated with Basal.




Figure 4: The distal AP-1 site (A1) preferentially binds Jun homodimers, while the binding capacity for Jun homodimers is lower at the proximal AP-1 site (A2). a and b, radiolabeled oligonucleotide probes containing the proximal (a) or the distal (b) AP-1 site were incubated with recombinant Jun, produced as inclusion body in E. coli. The amount of recombinant Jun, included in the binding reaction, is shown above the lanes (control lane 1 shows 2.5 µl (approximately 1.25 µg) of c-Jun/Fos programmed lysate). The mobility of the formed complexes was analyzed on 4% nondenaturing polyacrylamide gels. Arrows indicate the specific AP-1 binding. Nonspecific reactions are marked by brackets. c, BAEC were cotransfected with the TF promotor plasmids [A1], [A2], or [A1-A2] (1 µg) (Table 1) and a control plasmid (CAT = ``mock'') or c-Jun (0.5 µg) and cultivated for 42 h. After harvest, luciferase activity was determined and normalized for transfection efficiency to the amount of beta-galactosidase expressed by the plasmid pSV-beta-gal (Promega). The normalized data are expressed as relative Luc units and represent the mean of three independent experiments ± S.D. performed in triplicates. The inducibility of the various TF promotor plasmids by c-Jun homodimers is shown. The level of basal expression (transfected with CAT as control) is indicated with Basal.



Characterization of the Proteins Binding to Both AP-1 Sites

When BAEC were transfected with mutated Jun, AP-1 binding to the proximal AP-1 (A2) site was inhibited in unstimulated cells (Fig. 5a, lane 1 versus lane 2), in TNFalpha-induced cells (Fig. 5a, lane 3 versus lane 4) and in cells cotransfected with Jun/Fos(AP-1) (Fig. 5a, lane 6 versus lane 7). Specificity of binding was demonstrated, since binding of c-Jun/Fos programmed lysate resulted in a similar shift (Fig. 5a, lane 8). In addition, cold consensus AP-1 oligonucleotides inhibited the shift seen after TNFalpha stimulation (Fig. 5a, lane 5).


Figure 5: TNFalpha induces binding of different proteins to the proximal (A2) and the distal AP-1 site (A1) of the human TF promotor: BAEC were transiently transfected with CAT (= ``mock''), mutated Jun, or c-Jun and c-Fos (AP-1) overexpressing plasmids and cultivated for 42 h. Where indicated, TNFalpha (1 nM) was added 1 h before harvest. Nuclear extracts (10 µg/binding reaction) were prepared as described under ``Materials and Methods'' and assayed in EMSA for binding to the proximal (a) or the distal (b) AP-1 site. To confirm AP-1 binding, TNFalpha-induced nuclear extract was competed with a 160-fold molar excess of cold consensus AP-1 (lane 5). In addition, a parallel binding reaction was performed with 10 µl of Jun/Fos programmed lysate (lane 8). a, the AP-1 complex binding to the canonical proximal AP-1 site is indicated with an arrow. b, the complexes binding to the distal noncanonical AP-1 site are termed I, II, and III and indicated by arrows (see ``Results'').



In contrast, in nuclear extracts of BAEC three complexes (marked I, II, and III) were observed at the non-canonical distal AP-1 (Fig. 5b). When exposition of the films was extended for up to 8 days, a weak band, marked as complex I, occurred (Fig. 5b). The weak binding and its migration in the gel confirmed the results shown in Fig. 3b, i.e. this band is due to weak binding of Jun/Fos heterodimers.

Complex II (Fig. 5b) does not represent AP-1 heterodimers based on the following criteria. (i) The bands in control extracts (Fig. 5b, lane 1) and extracts from cells overexpressing Jun/Fos (Fig. 5b, lane 6) were only weakly (Fig. 5b, lanes 2 and 7) inhibited in the presence of mutated Jun. (ii) Complex II seen after TNFalpha induction (Fig. 5b, lane 3) was not competed by a 160-fold molar excess of cold AP-1 consensus oligonucleotides (Fig. 5b, lane 5) or by mutated Jun (Fig. 5b, lane 4), suggesting differences between control and TNFalpha-stimulated cells. These data further indicate that complex II does not contain c-Fos or Fos-related proteins; however, they do not exclude the involvement of other members of the bZIP family, i.e. members of the ATF family (see Fig. 6b and Table 2), which are able to bind DNA and therefore are only minorly influenced by mutated Jun. (iii) Complex II migrated more rapidly (Fig. 5b) than the complex formed with c-Jun homodimers (Fig. 4b).


Figure 6: Characterization of complexes I, II, and III, formed at the distal AP-1 site (A1). a, initial characterization of the nuclear complexes I, II, and III formed at the distal AP-1 site (Table 2) derived from the human TF promotor indicates that complex I and complex II contain members of the Jun and ATF family; complex III did not react with the antibodies used and was defined as nonspecific (see ``Results''). Characterization was performed six times, using three different nuclear extract preparations, with identical results. One typical experiment is shown. 10 µg of nuclear extract were included in each binding reaction: lane 1, 1 nM TNFalpha (1 nM, 2 h); lane 2, 1 nM TNFalpha + 2.5 µg of anti-pan-Jun antibodies; lane 3, 1 nM TNFalpha + 2.5 µg of anti-c-Jun antibodies; lane 4, 1 nM TNFalpha + 2.5 µg of anti-JunD antibodies; lane 5, 1 nM TNFalpha + 2.5 µg of anti-c-Fos antibodies; lane 6, 1 nM TNFalpha + 2.5 µg of anti-ATF-1 antibodies; lane 7, 1 nM TNFalpha + 2.5 µg anti-ATF2 antibodies; lane 8, 0.3 µg of recombinant c-Jun; lane 9, 10 µl of c-Jun/Fos programmed lysate. Antibodies were added directly before addition of the P-labeled distal AP-1 oligonucleotides. Complexes I and II and the nonspecific complex III are indicated by arrows. b, binding of recombinant Jun, recombinant ATF-2, recombinant Jun/ATF-2 heterodimers, and Jun/Fos programmed lysate to the distal non-canonical AP-1 site compared to binding of TNFalpha-induced nuclear proteins (10 µg/reaction). The experiment was performed three times with identical results. One typical experiment is shown: lanes 1 and 2 represent cellular extract from control (lane 1) or TNFalpha (1 nM, 2 h) treated cells (lane 2). Lanes 3-6 represent EMSA of the distal AP-1 site (A1) incubated with various recombinant members of the AP-1/bZIP family. Lane 1, control; lane 2, 1 nM TNFalpha (1 nM, 2 h); lane 3, 0.5 µg of Jun homodimers; lane 4, 0.5 µg of ATF-2 homodimers; lane 5, 0.25 µg of Jun and 0.25 µg of ATF-2, coincubated for 30 min at room temperature before addition to the binding reaction; lane 6, 8 µl of Jun/Fos programmed lysate; lane 7, 1 nM TNFalpha + 500-fold molar excess of unlabeled AP-1 consensus oligonucleotides. The nuclear extract-derived complexes I, II, and III are indicated by arrows (left). The complexes formed by recombinant Jun homodimers, ATF-2 homodimers, and Jun/ATF-2 heterodimers are marked with filled triangles (right). c, time course of the TNFalpha-inducible complex II, binding to the distal AP-1 site (A1). Nuclear extracts were prepared from BAEC induced for various times (0-6 h) with TNFalpha (1 nM) (lanes 1-8). To demonstrate that the observed bands were not due to the oligonucleotide preparation used, a reaction without nuclear extract was included (lane 9). 10 µg of nuclear extract were used in each binding reaction. DNA-protein complexes were analyzed on native 4% polyacrylamide gels. The very weak binding of complex I, the TNFalpha-inducible complex II, and the nonspecific complex III are indicated by arrows.



Since complex III (Fig. 5b) was not at all competed by unlabeled consensus AP-1 oligonucleotides (see below) and also present in unprogrammed rabbit reticulocyte lysate (Fig. 3b), it is regarded as nonspecific as depicted in Fig. 3b.

To analyze the complexes I and II that were observed at the distal non-canonical AP site (Fig. 5b), characterization with supershifting antibodies was performed (Fig. 6a). The upper gel shift band (complex I; Fig. 6a, lane 1) was reduced in the presence of pan-Jun antibodies (Fig. 6a, lane 2) and anti-JunD antibodies (Fig. 6a, lane 4) and suppressed in the presence of anti-c-Jun (Fig. 6a, lane 3) and anti-ATF-2 antibodies (Fig. 6a, lane 7). In addition, anti-c-Fos (Fig. 6a, lane 5) and anti-ATF-1 antibodies (Fig. 6a, lane 6) slightly decreased the shift. Complex II was suppressed when anti-ATF-2 antibodies were included in the reaction (Fig. 6a, lane 7) and reduced in the presence of anti-pan-Jun and anti-c-Jun antibodies (Fig. 6a, lanes 2 and 3), while anti-JunD, anti-c-Fos, and anti-ATF-1 antibodies did not affect binding (Fig. 6a, lanes 4-6). Thus complex I and II also consist of different members of the AP-1/bZIP family. Intensity of the lower band (complex III), previously characterized as nonspecific (Fig. 5b), was not affected by any of the antibodies.

To further confirm this hypothesis, recombinant Jun and recombinant ATF-2 (0.5 µg each), produced as inclusion bodies in Escherichia coli and able to heterodimerize, were used in binding reactions and their migration was compared to the complexes seen in extracts of control and TNFalpha stimulated cells (Fig. 6b). Consistent with the above data Jun homodimers bound to the distal non-canonical AP site (Fig. 6, panel a, lane 8, and panel b, lane 3) forming complexes that migrated in the gel at the same position as complex I (Fig. 6, panel a, lane 1, and panel b, lane 2). The faster migrating ATF-2 homodimers demonstrated stronger binding to the distal non-canonical AP-1 site (Fig. 6b, lane 4) than Jun homodimers (Fig. 6b, lane 3). When equimolar amounts of recombinant ATF-2 and recombinant c-Jun were coincubated in the binding reaction, a slightly faster migrating complex was observed (Fig. 6b, lane 5). No significant binding was observed, when programmed Jun/Fos lysate was included in the binding reaction (Fig. 6b, lane 6). The observed binding, seen in lane 6, is also present in unprogrammed lysate (Fig. 3b and Fig. 4b) and therefore regarded as nonspecific. To further characterize the TNFalpha-inducible complexes, a 500-fold molar excess of unlabeled AP-1 oligonucleotides (instead of 160-fold; Fig. 5b, lane 5) was included in the binding reaction with TNFalpha stimulated nuclear extract (Fig. 6b, lane 7). This unusual high excess of AP-1 competitor abolished binding of complexes I and II, but not of complex III. Since unlabeled oligonucleotides did not compete binding of complex III that was also detected in unprogrammed lysate (Fig. 3b and 4b), we defined complex III as nonspecific. The data shown in Fig. 6(a and b) indicate that the distal non-canonical AP-1 site forms complexes with members of the Jun and ATF family. However, more detailed studies have to be performed to elucidate the nature and the functional significance of the proteins involved in complex I and II.

Complex II was the major specific binding observed after TNFalpha stimulation. Therefore the time course of complex II induction by TNFalpha was studied (Fig. 6c). TNFalpha-mediated binding of complex II to the distal AP-1 site was biphasic, with a fast initial response at approximately 5 min and a slower response, maximal between 2 and 6 h (Fig. 6c). No signal was observed in the absence of nuclear extracts (Fig. 6c, lane 9).

At the proximal canonical AP-1 site TNFalpha induced time-dependent (Fig. 7a) and dose-dependent (Fig. 7b) induction of proteins, which reached a maximum between 30 min and 2 h. These proteins were characterized as AP-1 by (i) competing the binding with an excess of cold AP-1 consensus oligonucleotides (Fig. 7, panel a, lane 8 and panel b, lane 7) and (ii) by reducing binding activity by overexpression of mutated Jun (Fig. 5a, lane 4). For the characterization using polyclonal antibodies, extract of TNFalpha-stimulated cells (Fig. 7c, lane 1) was incubated with the antibodies (Fig. 7c, lanes 2-7). Migration was compared to the shift observed with recombinant Jun homodimers (Fig. 7c, lane 8) and Jun/Fos programmed lysate (Fig. 7c, lane 9). Pretreatment with anti-pan-Jun (Fig. 7c, lane 2) or anti-JunD antibodies (Fig. 7c, lane 4) resulted in supershifted bands; pretreatment with anti-c-Fos antibodies abolished the observed binding (Fig. 7c, lane 5). No reaction was observed with antibodies directed against members of the ATF family (Fig. 7c, lanes 6 and 7). Consistent with the above results (Fig. 4a), no binding was observed with 0.3 µg of Jun homodimers (Fig. 7c, lane 8). Therefore, the TNFalpha-induced binding to the proximal canonical AP-1 site comprises JunD/Fos heterodimers. Although c-Jun-specific antibodies did not result in supershifted or reduced binding, c-Jun might contribute to the observed complexes, since it might be possible that the anti-c-Jun antibody fails to recognize c-Jun in Jun/Fos heterodimers.^3


Figure 7: TNFalpha induces time- and dose-dependent binding of AP-1 to the proximal AP-1 site (A2) of the human TF promotor. a, time course of AP-1 binding to the proximal AP-1 site (A2) of the human TF promotor. Nuclear extracts were prepared from BAEC induced for various times (0-3 h) with TNFalpha (1 nM) (lanes 1-7). 10 µg of nuclear extract were included in each binding reaction. DNA-protein complexes were analyzed on 4% native polyacrylamide gels. EMSA detected AP-1 binding to the proximal AP-1 site (Table 2) derived from the human TF promotor. The TNFalpha-inducible AP-1 complex is indicated with an arrow. Specificity of binding was ascertained by competing with 160-fold molar excess of cold AP-1 consensus oligonucleotides (Table 2) included in the binding reaction (lane 8). b, dose response of AP-1 binding to the proximal AP-1 site (A2). BAEC were stimulated with various doses of TNFalpha (0 pM to 1000 pM) for 30 min (lanes 1-6). Nuclear extracts were prepared, and 10 µg of this extract were included in each binding reaction and analyzed as above. The TNFalpha-inducible AP-1 complex (JunD/Fos) is indicated with an arrow. Specificity of binding was ascertained by competing with 160-fold molar excess of cold AP-1 consensus oligonucleotides (Table 2) included in the binding reaction (lane 7). c, characterization of the complex bound to the proximal AP-1 site (A2) after TNFalpha induction. Characterization was performed two times, using two different nuclear extract preparations, with identical results. One typical experiment is shown. 10 µg of nuclear extract were included in each binding reaction: lane 1, 1 nM TNFalpha (1 nM, 1 h); lane 2, 1 nM TNFalpha + 2.5 µg of anti-pan-Jun antibodies; lane 3, 1 nM TNFalpha + 2.5 µg of anti-c-Jun antibodies; lane 4, 1 nM TNFalpha + 2.5 µg of anti-JunD antibodies; lane 5, 1 nM TNFalpha + 2.5 µg of anti-c-Fos antibodies; lane 6, 1 nM TNFalpha + 2.5 µg of anti-ATF-1 antibodies; lane 7, 1 nM TNFalpha + 2.5 µg of anti-ATF2 antibodies; lane 8, 0.3 µg of recombinant c-Jun; lane 9, 10 µl of c-Jun/Fos programmed lysate. Antibodies were added directly before addition of the P-labeled proximal AP-1 oligonucleotide. The TNFalpha-inducible complex is indicated with an arrow.



TNFalpha-induced Binding of p65/c-Rel to the TF-derived NF-kappaB Site

Stimulation with TNFalpha resulted in time- and dose-dependent binding of two distinct protein DNA-complexes to the TF derived NF-kappaB site, which were identified as NF-kappaB (data not shown). Consistent with previous studies, binding of the slower migrating complex was characterized as NF-kappaB(p65/c-Rel) complex(11, 18, 62, 63) , while in the faster migrating band only NF-kappaB(p65) seemed to participate in the binding complex (11) (data not shown). Binding of NF-kappaB(p65/c-Rel) was already detected after 5 min, reached its maximum after 10 min, and decreased to basal levels after 1 h (data not shown). The p65-containing faster migrating complex was induced between 5 min and 3 h (data not shown). Taken together, these data indicate that activation of the human TF promotor follows a kinetic that includes first activation of NF-kappaB(p65/c-Rel), followed by activation of JunD/Fos heterodimers that bind to the proximal AP-1 site. While NF-kappaB(p65/c-Rel) activation declines, the distal AP-1 site exhibits maximal binding of AP-1/bZIP proteins, characterized to contain Jun and ATF proteins. This might explain, why TF transcription is still enhanced after 4-6 h ( (9) and (10) and data not shown), even TNFalpha-induced NF-kappaB(p65/c-Rel) activation has already dropped to base-line level.

TNFalpha Mediates Induction of Endothelial TF by a Concerted Action of the AP-1 and NF-kappaB Sites

To confirm that different members of the AP-1/bZIP and NF-kappaB family functionally act on the human TF promotor and contribute to TF induction by TNFalpha, transient transfections of BAEC with TF promotor mutants (Fig. 8) were performed. When BAEC were stimulated with TNFalpha, promotor activity compared to basal expression was highest, when the proximal AP-1 (A2) and the NF-kappaB site were present (Fig. 8, a and b). Loss of the proximal AP-1 site or the NF-kappaB site resulted in significantly decreased TF induction (Fig. 8, a and b). Loss of the non-canonical distal AP-1 site had a less prominent effect, as expected (Fig. 8, a and b). Promotor mutants with only the proximal AP-1 site (A2) cloned in front of the minimal promotor were still inducible by TNFalpha, while the distal AP-1 site (A1) alone was unable to confer TF induction (Fig. 8, a and b). The proximal AP-1 significantly contributed to TF basal expression (Fig. 8a). Therefore mutants that contained the NF-kappaB, but not the proximal AP-1 site, were more inducible by TNFalpha than mutants comprising the proximal AP-1 site (Fig. 8b); however, the overall TF expression of mutants without proximal AP-1 was lower (Fig. 8a). Highest TF expression was observed only when both AP-1 sites and the NF-kappaB site were intact, consistent with participation of all three sites in regulation of TF transcription (Fig. 8a). The TNFalpha-induced activity of the construct A1-A2-N, spanning the complete promotor, is higher (396 Luc units) than the added activity of the constructs containing each element alone (A1 = 50, A2 = 125, N = 106 Luc units; total 281 Luc units). Thus, these elements act in concert on the human TF promotor in mediating TNFalpha-induced TF transcription.


Figure 8: TNFalpha induces tissue factor expression by a concerted action of AP-1/bZIP- and NF-kappaB-like proteins. Functional analysis of TF expression in unstimulated and TNFalpha-induced BAEC. BAEC were transfected with various TF promotor plasmids (Table 1; for detail see ``Materials and Methods'') for 36 h before TNFalpha (1 nM) was added for 6 h, where indicated. After harvest luciferase activity was determined in the cell lysates and normalized for transfection efficiency to the amount of beta-galactosidase activity expressed by the control plasmid pSV-beta-Gal (Promega). Corrected values were expressed as relative luciferase units. The results represent the mean of at least three independent experiments ± S.D. that were performed in triplicate. a, functional analysis of TF expression in unstimulated BAEC compared with TF expression in TNFalpha stimulated BAEC; the mean of six independent experiments ± S.D. performed in triplicate is shown. b, the inducibility by TNFalpha relating to basal expression is shown. The level of basal expression is indicated with B. c, to directly demonstrate the role of NF-kappaB(p65) in TNFalpha-mediated TF induction, various TF promotor plasmids (Table 1) were cotransfected with plasmids overexpressing CAT (= mock) or the NF-kappaB(p65) specific inhibitor I-kappaB and cultivated for 36 h, before TNFalpha (1 nM) was added to the cells for 6 h. After harvest luciferase activity and transfection efficiency were determined as above. The data represent the mean of three different experiments performed in triplicate. d, to directly demonstrate the role of JunD/Fos (AP-1) in TNFalpha-mediated TF induction, various TF promotor plasmids (Table 1) were cotransfected with plasmids overexpressing CAT (= mock) or mutated Jun and cultivated for 36 h before TNFalpha (1 nM) was added to the cells for 6 h. Data were obtained from three different experiments ± S.D. performed in triplicate.



The concept that both AP-1 sites and the NF-kappaB site act in concert was further supported by studies overexpressing specific inhibitors of NF-kappaB(p65)(I-kappaB) or AP-1/bZIP (mutated Jun). Overexpression of I-kappaB reduced TNFalpha-mediated TF induction as long as the NF-kappaB site was present (Fig. 8c). Overexpression of mutated Jun reduced TF induction by TNFalpha as long as the proximal AP-1 site was present (Fig. 8d).

TNFalpha-mediated TF Induction Is Dependent on AP-1/bZIP and NF-kappaB Proteins in Vivo

No data are available to support the concept of AP-1/bZIP- and NF-kappaB-mediated TF induction in vivo. Since in the human and the mouse TF promotor the same sequences are found for the distal and the proximal AP-1, as well as for the NF-kappaB recognition motif, we used a mouse model to study the dependence of TNFalpha-mediated TF expression of endothelial cells on Jun and NF-kappaB in vivo. In this model, TNFalpha induces the expression of TF and fibrin formation on endothelial cells of the tumor vasculature(14, 64) . 3 h after intravenous injection of TNFalpha TF expression was induced, based on in situ hybridization (Fig. 9a) and immunohistochemistry (Fig. 9b). TF was expressed by subendothelial structures including tumor cells and also by endothelial cells. With respect to the topic of this study, we focused on endothelial cells. More than 150 vessels were evaluated in this part of the study.


Figure 9: AP-1/bZIP proteins and NF-kappaB control TF expression in vivo. Meth-A sarcoma (10^6 cells/animal) were implanted into C(3)H mice. After the tumors reached an average size of 0.5 cm, intravenous somatic gene transfer was performed with plasmids overexpressing a vector control, mutated Jun, or I-kappaB. 12 days after planting the tumors, mice received PBS (control) or 5 µg of TNFalpha/animal for 3 h. Mice were sacrificed and perfused with 30-40 ml of PBS by intracardiac injection of PBS into the left ventricle. Tumors were harvested and TF transcription (a, in situ hybridization), TF antigen (b, immunohistochemistry), and fibrin/fibrinogen deposition (c; immunofluorescence) was evidenced in the tissue. a, in situ hybridization with a mou se TF-specific riboprobe (see ``Materials and Methods'') in control (top) and TNFalpha (bottom) treated animals, transfected with vector control (left), mutated Jun (middle), or I-kappaB (right). Magnification, times 160. b, immunohistochemistry using an anti-mouse TF antibody (see ``Materials and Methods'') in control (top) and TNFalpha (bottom) treated animals, transfected with vector control (left), mutated Jun (middle) or I-kappaB (right). Magnification, times 160. c, immunofluorescence of fibrin/fibrinogen deposition in control (top) and TNFalpha (bottom) treated animals, transfected with vector control (left), mutated Jun (middle), or I-kappaB (right). Magnification, times 40.



When animals were treated by intravenous somatic gene transfer with mutated Jun 24 h prior to TNFalpha injection, a decrease in the endothelial response to TNFalpha was observed by in situ hybridization (Fig. 9a) and immunohistology (Fig. 9b) compared to vector-transfected animals. Thus, by blocking the interaction of Jun with other members of the bZIP family by somatic gene transfer with a plasmid overexpressing mutated Jun, endothelial TF induction could be partially reduced (Fig. 9, a and b). Similar data were obtained when a plasmid overexpressing I-kappaB was used (Fig. 9, a and b). Mutated Jun and I-kappaB both reduced the inducibility of TF in endothelial cells in this tumor model in vivo. The tumor model was further used to examine the functional effect of TF; when the fibrin/fibrinogen deposition in response to TNFalpha was studied in animals perfused with 30-40 ml of PBS (see ``Materials and Methods''), a reduction by mutated Jun and I-kappaB was demonstrated in some, but not all vessels (Fig. 9c). Thus TF expression in vivo is under the control of AP-1/bZIP and NF-kappaB-like proteins.

Successful transfection with mutated Jun or I-kappaB was monitored in EMSA of tumor tissue (Fig. 10). Tumors derived from animals transfected with vector DNA prior to TNFalpha had a stronger AP-1 binding activity than tumors derived from animals transfected with mutated Jun (Fig. 10, top left). Consistently, tumors from I-kappaB transfected animals demonstrated reduced NF-kappaB binding activity at the TF derived NF-kappaB site (Fig. 10, top right) compared to vector controls. In addition, Northern blot of mRNA, derived from whole tumors, showed decreased TF mRNA levels, when the animals had been transfected with mutated Jun or I-kappaB prior to TNFalpha application (Fig. 10, bottom). However, the suppression obtained was only partial, since (i) members of the Jun and ATF family are less responsive to inhibition by overexpression of mutated Jun than c-Fos, (ii) the in vivo involvement of other transcription factors can not be excluded, and (iii) transfection did not reach all cells.


Figure 10: Efficiency of intravenous somatic gene transfer. Transfection efficiency was monitored in EMSA (top) and Northern blot (bottom). Top I, EMSA of tumor nuclear extracts, derived from mice transfected with vector control (left) or mutated Jun (middle) before application of TNFalpha EMSA were performed with the proximal AP-1 site (A2) of the human TF promotor. AP-1 binding was confirmed by suppressing the observed shift in TNFalpha-induced vector controls by a 160-fold molar excess of unlabeled AP-1 consensus competitor (right). Top II, EMSA of tumor nuclear extracts, derived from mice transfected with vector control (left) or I-kappaB (middle) before application of TNFalpha. EMSA were performed with the TF-derived NF-kappaB site. NF-kappaB binding was confirmed by suppressing the observed shift in TNFalpha-stimulated vector controls by an 160-fold molar excess of unlabeled NF-kappaB consensus competitor (right). Bottom I, Northern blot: total mRNA of tumors from TNFalpha treated animals, transfected with vector control (left, 1) or mutated Jun (right, 3) was hybridized against tissue factor (TF; top) or GAPDH (bottom) specific DNA probes. Bottom II, Northern blot: total mRNA of tumors from TNFalpha-treated animals, transfected with vector control (left, 1) or I-kappaB (right, 3) was hybridized against tissue factor (TF; top) or GAPDH (bottom) specific DNA probes.



To give a picture of the overall efficiency of transfection, microbeads were used for measuring blood flow of the whole organ, avoiding potential artifacts due to selection of a single area in histological studies. When microbeads were injected into animals, a high number of beads was present in tumors of animals not treated with TNFalpha (Fig. 11). The number of beads reflecting tumor perfusion was clearly decreased after TNFalpha injection with previous somatic gene transfer with vector DNA (Fig. 11). This indicated that TNFalpha treatment resulted in loss of free blood flow, potentially due to TF-mediated microvascular thrombosis. Therefore this method adds to the histological study by providing data about the effect of I-kappaB and mutated Jun on the whole organ. Gene transfer with I-kappaB or mutated Jun partially reversed this effect of TNFalpha (Fig. 11). Hence blocking TF on the transcriptional level reduced not only TF induction by TNFalpha, but also reduced the fibrin/fibrinogen deposition and restored free blood flow.


Figure 11: Somatic gene transfer with mutated Jun or I-kappaB restores the free blood flow in tumors treated with TNFalpha. 10^6 Meth-A sarcoma cells were planted intracutaneously into mice. Somatic gene transfer and TNFalpha application was performed as described in Fig. 9. 3 h after intravenous injection of 5 µg of TNFalpha, mice were anesthetized, microspheres were injected into the left ventricle for 10-20 s (see ``Materials and Methods''), and mice were sacrificed thereafter. Tumor tissue was harvested and microbeads counted microscopically (see ``Materials and Methods''). The free blood flow is shown, evidenced by the number of latex particles per gram of tumor tissue. A, tumors before TNFalpha application; B, tumors after TNFalpha, pretreated with mutated Jun; C, tumors after TNFalpha, pretreated with I-kappaB; D, tumors after TNFalpha, pretreated with mutated Jun and I-kappaB; E, tumors after TNFalpha.




DISCUSSION

Tissue factor (TF) is a potent initiator of the coagulation cascade (1, 2, 4, 65, 66) and normally is not expressed by quiescent endothelial cells(1, 3, 6, 11, 12) . In vitro data showed induction of TF synthesis in endothelial cells by inflammatory mediators such as endotoxin, phorbol esters, oxygen-free radicals, or cytokines(7, 8, 9, 10, 11, 67, 68, 69, 70) . Recently members of the NF-kappaB and the AP-1/bZIP family have been reported to be involved in the lipopolysaccharide- and cytokine-mediated TF induction in monocytes (15, 16, 17, 18, 62) and porcine endothelial cells(11) . It has been more difficult to show endothelial TF in vivo(3, 67) ; however, recent studies demonstrate that in selected areas of the vascular bed activators of the host response or TNFalpha lead to the synthesis and expression of TF(12, 13, 14, 71) . This study addresses the molecular mechanisms that underlie the regulation of the human TF promotor in response to the proinflammatory cytokine TNFalpha.

We used bovine aortic endothelial cells (BAEC), which exhibit lower basal AP-1 and NF-kappaB activity than porcine (PAEC) or human (HUVEC) endothelial cells, and the human TF promotor. A striking difference between the human and the porcine TF promotor is seen at the proximal AP-1 site, which resembles a canonical high affinity site in the human TF promotor(15, 16) , while it is a low affinity non-canonical site in the porcine promotor, due to a G A switch at position 4 of the AP-1 heptamer(11) . Thus, the proximal AP-1 site of the human promotor is more prominently involved in the TNFalpha-mediated up-regulation of TF than it is in the porcine promotor and, as a consequence, in addition to NF-kappaB activation, AP-1 activation may be relevant for human disease.

In transient transfection studies, the highest TNFalpha inducibility was only observed when the NF-kappaB(p65/c-Rel) site was present in the TF promotor plasmids. Enhanced NF-kappaB(p65/c-Rel) binding to its TF-derived motif was detectable within 5 min after TNFalpha stimulation and rapidly down-regulated after 1 h (data not shown). This fast activation of NF-kappaB(p65/c-Rel) reflects that TF mRNA can be rapidly induced in the absence of protein synthesis; therefore, TF has been classified as an immediate early gene(72) . The dependence of TF induction by inflammatory mediators on NF-kappaB(p65/c-Rel) activation may insure that this induction is transient, since it has been recently reported that increased NF-kappaB levels lead to increased expression of the inhibitor I-kappaB(73, 74) , followed by NF-kappaB inactivation. This might prompt the activated endothelial cells to return to a quiescent state.

Therefore, the existence of increased TF mRNA levels in HUVEC and BAEC 4-6 h after TNFalpha stimulation (9, 10) cannot be explained solely on the basis of NF-kappaB activation and demands the involvement of other inducible transcription factors. Functional studies demonstrated that optimal TF induction by TNFalpha was also mediated by both AP-1 sites in the human TF promotor (Fig. 8). EMSA revealed that the TNFalpha-inducible complex bound to the canonical proximal AP-1 site of the human TF promotor consisted mainly of JunD/Fos (Fig. 7c); however, it cannot be excluded that other members of the Jun family are also involved. Highly vascularized organs (spleen, lung, intestine, ovary, and brain) express high levels of JunD(30) . While the expression of c-Jun and Jun B is rapidly up-regulated by various stimuli, JunD is only modestly induced by growth factors and phorbol esters(26, 30, 75, 76) . Transactivation by JunD homodimers is significant lower than by c-Jun homodimers(30) . In cooperation with c-Fos, however, JunD has transactivation capacities similar to those of c-Jun(29, 30) . The results displayed here demonstrate that in cultured endothelial cells TNFalpha induces JunD/Fos heterodimers that recognize the proximal AP-1 site of the human TF promotor and thereby enhance TF transcription. In this respect the human and the porcine system differ significantly. Binding of JunD/Fos-containing complexes to the proximal AP-1 site is already detected 30 min after TNFalpha stimulation. This rapid response excludes newly synthesized JunD or Fos and indicates the rapid activation of preexisting proteins. This availability of JunD/Fos heterodimers therefore is a limiting factor. Consistently, the canonical proximal AP-1 alone was not able to confer high TNFalpha-mediated induction in transient transfection experiments and required the presence of the NF-kappaB(p65/c-Rel) site(11, 15, 16, 18) for optimal TF expression. These data imply that the disposal of JunD/Fos heterodimers is not sufficient for maximal induction by TNFalpha and need to recruit NF-kappaB (p65/c-Rel) nuclear binding activity. Since NF-kappaB(p65/c-Rel) translocation into the nucleus precedes JunD/Fos activation only by 20 min, one might speculate that binding of one transcription factor facilitates binding of the other.

The proximal high affinity AP-1 site of the human TF promotor, which is missing in the porcine TF promotor, is of particular importance with respect to therapeutic interventions. A great variety of antioxidative agents has been reported to suppress activation of NF-kappaB in vitro and in vivo(77) and therefore might potentially be used for reducing TF activity under certain pathophysiological conditions. However, recent studies elucidated that changes in the cellular redox system by radical scavengers suppress very fast NF-kappaB, but at the same time induce time-dependent AP-1 activation (Jun/Fos)(20, 21) . Antioxidative conditions strongly induce c-Fos, which can form reactive heterodimers with preexisting Jun homodimers (20, 21) . As pointed out before, tissues with high endothelial portions contain constitutively high amounts of JunD homodimers (30) and are therefore primed to generate large amounts of JunD/Fos heterodimers under antioxidative therapy.

To define the role of the non-canonical distal AP-1 site in human TF regulation is more difficult. This site is a low affinity site for AP-1 binding and resembles the two non-canonical AP-1 sites of the porcine TF promotor(11) . In accordance with these data, several independent approaches demonstrated that Jun homodimers, but not Jun/Fos heterodimers, bind to this site (Fig. 3, Fig. 4, and Fig. 6). Differences in the structure of the DNA binding domains for Jun homodimers and Jun/Fos heterodimers have been described(78) ; therefore, it seems likely that a G A switch at position 4 of this site facilitates Jun binding and excludes significant Jun/Fos binding. Furthermore, specific properties of the regions outside the defined AP-1 binding sites might be responsible for preference in binding of the various homo- and heterodimer complexes(79) . EMSA demonstrated (Fig. 6, a-c) that TNFalpha also induced protein complexes that were different from Jun homodimers. These complexes have been characterized to contain Jun and ATF family proteins (Fig. 6, a-c). In contrast, Moll et al.(11) recently reported constitutive binding of c-Jun, JunD, and possibly Fra2 complexes to the porcine TF promotor-derived non-canonical AP-1 sites. Since basal AP-1 binding activity is low in BAEC compared to PAEC), this might explain why the study presented here detected TNFalpha-inducible binding at the non-canonical distal AP-1 site of the human TF promotor. Consistent with our observations, Donovan-Peluso and co-workers mentioned that in THP-1 cells large differences between the distal and the proximal AP-1 site were detected in EMSA, which indicate the involvement of different heterodimers(17) . This finding differs from previous observations in HUVEC, where Jun homodimer and Jun/Fos heterodimer binding occurs at the distal and the proximal AP-1 site(47, 63) . This might be due to (i) a greater availability of Jun homodimers, (ii) to a different composition of the complexes induced, or (iii) to species differences in HUVEC versus BAEC. The low affinity distal AP-1 site of the human TF promotor only marginally participates in TNFalpha-induced TF expression, consistent with the data described for the two low affinity AP-1 sites in the porcine TF promotor(11) . However, the non-canonical AP-1 site significantly supports NF-kappaB-mediated TF induction, even when the proximal AP-1 is deleted (Fig. 8). Since recently a cooperative action of ATF proteins and NF-kappaB family members has been demonstrated(80) , one might speculate that proteins bound to the distal AP-1 site support and facilitate NF-kappaB activity. Therefore a set of different transcription factors has to be activated at the same time before endothelial TF is successfully induced.

The in vivo data presented (Fig. 9-11) support this concept. Intravenous somatic gene transfer with plasmids overexpressing I-kappaB or mutated Jun reduce TF induction in vascular endothelial cells of the tumor. They also decrease deposition of fibrin/fibrinogen. The antibody used does not discriminate between fibrin and fibrinogen. Therefore, the animals were perfused with 30-40 ml of PBS (see ``Materials and Methods'') prior to harvest of the organs to remove non-clotted material. The reactive material represents at least in part fibrin, since we observed striking differences in fibrin/fibrinogen deposition between the different animal groups corresponding to the perfusion studies with microbeads (the later ones giving a better view of the overall efficiency of I-kappaB and mutated Jun). However, in these experiments cells other than endothelial cells may be affected. Nevertheless, the in situ hybridization and immunohistochemical studies (Fig. 9) showed that endothelial cell expression of TF is under control of NF-kappaB and AP-1 in the animal model used. The incomplete suppression of TF and fibrin/fibrinogen deposition can be explained (i) by the expected low to moderate transfection efficiency, (ii) by local differences in endothelial cells (capillaries still growing versus already grown vessels, dividing vessels versus non-dividing endothelial cells), (iii) by the involvement of other transcription factors than AP-1 and NF-kappaB, and (iv) other EC genes influenced by cytokines. Hence the TNFalpha-mediated activation of endothelial TF transcription occurs in vitro and in vivo by members of the NF-kappaB and AP-1/bZIP family.


FOOTNOTES

*
This work was in part supported by Deutsche Forschungsgemeinschaft Grant Na-138/2-2 (to P. P. N.), Mildred Scheel Stiftung Grant W 3/90/Na 1 (to P. P. N.), and a grant from the Verein zur Förderung der Krebsforschung in Deutschland (to P. P. N.). Part of this work was presented at the ISTH Meeting, July, New York, NY (Bierhaus, A., Nachman, N., Haase, M., Ziegler, R., Edgington, Th., Stern, D., and Nawroth, P.(1993) Thromb. Haemost.69, 1683). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
A Heisenberg Scholar of the Deutsche Forschungsgemeinschaft. To whom correspondence should be addressed: Dept. of Medicine I, Bergheimer Str. 58, D69115 Heidelberg, Germany. Tel.: 49-6221-568606; Fax: 49-6221-564696.

(^1)
The abbreviations used are: TF, tissue factor; TNFalpha, tumor necrosis factor alpha; BAEC, bovine aortic endothelial cells; PAEC, porcine aortic endothelial cells; HUVEC, human umbilical vein endothelial cells; Meth-A sarcoma, methylcholanthrine-A-induced sarcoma cells; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethylsulfate; TBE, Tris-borate-EDTA; EMSA, electrophoretic mobility shift assay; NF-kappaB, nuclear factor kappaB; AP-1, activator protein 1; ATF-2, activating transcription factor-2; CAT, chloramphenicol acetyltransferase; beta-Gal, beta-galactosidase; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; bp, base pair(s); DEPC, diethyl pyrocarbonate; BSA, bovine serum albumin; CAT, chloramphenicol acetyltransferase.

(^2)
Y. Zhang and Y. Deng, unpublished results.

(^3)
N. Mackman, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. D. Bohmann for the cDNA of mutated Jun, Dr. J. E. Sadler for the human tissue factor cDNA probe, and Dr. I. Verma for T7-Jun, T7-Fos, pSV-c-Jun, and pBK28(c-Fos) expression plasmids. TNFalpha was a generous gift of Knoll AG, Ludwigshafen, Germany. Meth-A sarcoma cells were kindly provided by Dr. D. Männel.


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