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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:612-621

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© 1995 American Heart Association, Inc.

Articles

Transcriptional Regulation of Tissue Factor Expression in Human Endothelial Cells

Graham C. N. Parry; Nigel Mackman

From the Department of Immunology, The Scripps Research Institute, La Jolla, Calif.

Correspondence to Nigel Mackman, PhD, The Scripps Research Institute, 10666 N Torrey Pines Rd, IMM-17, La Jolla, CA 92037.


*    Abstract
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 *Abstract
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Abstract Tissue factor (TF) expression by endothelial cells is implicated in thrombotic episodes in patients with a variety of clinical disorders. In a baboon model of lethal sepsis, TF is expressed by endothelial cells in the splenic microvasculature. In vitro, endothelial cells are induced to express TF in response to tumor necrosis factor–{alpha} (TNF-{alpha}), interleukin-1ß (IL-1ß), and bacterial endotoxin (lipopolysaccharide [LPS]). Here, we identified cis-acting regulatory elements that control TF gene transcription in primary human endothelial cells. Functional studies showed that the TF promoter contained a 56-bp enhancer (-227 to -172 bp), which included two activator protein–1 (AP-1) sites and a {kappa}B-like site, that mediated induction by TNF-{alpha}, IL-1ß, and LPS. Electrophoretic mobility shift assays demonstrated that endothelial cells contained constitutive AP-1 binding activity, whereas the {kappa}B-like site, 5'-CGGAGTTTCC-3', bound an inducible nuclear complex composed of c-Rel–p65 heterodimers. Taken together, our data suggest that induction of TF gene transcription in endothelial cells is mediated by functional interactions between Fos-Jun and c-Rel–p65 heterodimers.


Key Words: endothelium • tissue factor • gene expression


*    Introduction
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 *Introduction
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Tissue factor (TF) expression within the vasculature activates the coagulation protease cascades and promotes thrombotic episodes in patients with a variety of clinical disorders, including cancer, atherosclerosis, and septic shock (reviewed in References 1 and 21 2 ). Under normal physiological conditions, endothelial cells present an anticoagulant surface by expressing such proteins as thrombomodulin.3 However, in vitro, bacterial endotoxin (lipopolysaccharide [LPS]) and the inflammatory cytokines tumor necrosis factor–{alpha} (TNF-{alpha}) and interleukin-1ß (IL-1ß) change endothelial cells to a procoagulant state by inducing TF expression.4 5 6 7 8 9 During endotoxemia, endothelial TF may be induced directly by LPS or indirectly by TNF-{alpha} and IL-1ß.10 11 In experimental sepsis models, endothelial TF may play a role in disseminated intravascular coagulation.12 13 Indeed, TF expression is induced in endothelial cells of the splenic microvasculature in a baboon model of lethal Escherichia coli sepsis.14

Induction of TF gene expression in human endothelial cells exposed to LPS, TNF-{alpha}, phorbol 12-myristate 13-acetate (PMA), and oxidized LDL is controlled at the level of transcription.4 9 15 However, posttranscriptional stabilization mechanisms also contribute to TF mRNA accumulation in human umbilical vein endothelial cells (HUVECs) exposed to LPS,9 although this mechanism was not detected in LPS-stimulated human cardiac valve endothelial cells.15 To date, cis-acting regulatory DNA elements within the TF promoter that control TF gene expression in endothelial cells have not been identified.

We have shown that a 56-bp enhancer (-227 to -172), which includes two activator protein–1 (AP-1) sites and a {kappa}B-like site, in the 5' flanking region of the TF gene mediates LPS induction in human monocytic cells.16 The AP-1 transcription factor family includes fos-related antigens and jun proteins that form homodimers and heterodimers, which specifically bind to sites matching the consensus AP-1 recognition sequence, 5'-TGA(C/G)TCA-3'.17 Similarly, members of the nuclear factor (NF)–{kappa}B/Rel family specifically recognize a decameric consensus sequence, 5'-GGGRNNYYCC-3',18 where R is A or G, Y is T or C, and N is any nucleotide. The NF-{kappa}B/Rel family includes p50 (NFKB1), p65 (RelA), and c-Rel, which also form various homodimers and heterodimers.18 Our recent studies using nuclear extracts from LPS-stimulated human monocytic cells demonstrate that the TF promoter contains a {kappa}B-like site, 5'-CGGAGTTTCC-3', that selectively binds c-Rel–p65 heterodimers.19 In unstimulated endothelial cells, NF-{kappa}B/Rel family complexes are retained in the cytoplasm by the binding of inhibitor proteins, including I{kappa}B{alpha}. Cellular activation dissociates I{kappa}B{alpha} and allows translocation of the transcription factors to the nucleus, where they bind to target sites to regulate gene expression.20

In this study, we showed that induction of TF gene transcription in primary cultures of human endothelial cells exposed to TNF-{alpha}, IL-1ß, and LPS was mediated by the 56-bp enhancer in the TF promoter. The two AP-1 sites bound AP-1 proteins constitutively, whereas the {kappa}B-like site bound an inducible complex composed of c-Rel–p65 heterodimers, suggesting that binding of these two distinct families of transcription factors regulates TF gene transcription.


*    Methods
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 *Methods
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Reagents
LPS (E coli serotype O111:B4) was obtained from Calbiochem, human recombinant TNF-{alpha} and IL-1ß were obtained from Collaborative Biomedical Products, and PMA was obtained from Sigma Chemical Co.

Cell Culture
Primary cultures of human endothelial cells obtained from collagenase-digested umbilical veins21 were cultured in gelatin-coated flasks in medium 199 supplemented with 20% fetal bovine serum (Gemini Bioproducts Inc), 90 µg/mL porcine intestinal heparin (Sigma), 30 µg/mL endothelial cell growth supplement (H-Neurext, Upstate Biotechnology Inc), 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin (Irvine Scientific), and 25 mmol/L HEPES. Culture media contained <12.5 pg/mL endotoxin as determined by Limulus amebocyte lysate assay (BioWhittaker). All experiments used HUVECs between passages 3 and 5.

TF Activity
Cell pellets were solubilized with 15 mmol/L octyl-ß-D-glucopyranoside at 37°C for 15 minutes, and lysates were assayed for TF activity in a one-stage clotting assay.22 Briefly, equal volumes of prewarmed human citrated plasma and 20 mmol/L CaCl2 were added to the test samples, and the clotting times were determined manually. Functional TF was quantified by using a standard curve established with phospholipid-reconstituted, affinity-purified TF from human brain.22 A pool of neutralizing anti–human TF monoclonal antibodies (TF8-5G9, TF8-6B4, and TF9-9C3) demonstrated that the observed procoagulant activity was due to TF protein.

Analysis of TF mRNA
Total cellular RNA (8 µg) isolated from HUVECs was subjected to denaturing electrophoresis in 1.2% agarose-formaldehyde gels23 and transferred to a GeneScreen membrane (DuPont–New England Nuclear). Membranes were hybridized with a TF cDNA fragment labeled using [{alpha}-32P]dCTP (>3000 Ci/mmol, Amersham) as described.24 To control for variations in RNA loadings, membranes were rehybridized with a radiolabeled cDNA fragment from the human glucose-6-phosphate dehydrogenase gene. Membranes were exposed to Kodak XAR film at -80°C.

Transfections
HUVECs were transfected using DEAE-dextran.25 Briefly, medium from subconfluent monolayers was replaced with 4 mL of RPMI 1640 medium (BioWhittaker) containing 10% Nu-Serum I (Collaborative Biomedical Products) and 2 mmol/L L-glutamine before incubation for 4 hours with 200 µg/mL DEAE-dextran. Two micrograms and 10 µg of plasmid DNA were used for six-well and 10-cm dishes, respectively. Cells were cultured in complete medium for 20 hours before a 5-hour stimulation. Cell lysates were assayed for luciferase activity as described26 using a monolight 2010 luminometer (Analytical luminescence Laboratory). To control for variation in transfection efficiencies, cells were cotransfected with a control plasmid, pRSVCAT (2 µg),27 which expresses the chloramphenicol acetyltransferase reporter gene. Levels of chloramphenicol acetyltransferase activity were determined as described28 and exhibited <20% variation between samples (data not shown).

Plasmids
Plasmids used in these studies have been described previously.16 19 The cytomegalovirus (CMV) promoter–containing plasmid soCMVIN was used for eukaryotic expression of I{kappa}B{alpha}, p65, p50, and the chimeric proteins p50TA65 and c-RelTA65, which contain the Rel homology domain of p50 and c-Rel, respectively, fused to the transactivation domain of p65.29 30 These plasmids were the generous gift of Drs C. Rosen, S. Ruben, and C. Kunsch. pCMV–c-Rel contains the c-Rel cDNA cloned into the pRc/CMV vector and was kindly provided by Dr N. Rice.

Electrophoretic Mobility Shift Assay (EMSA)
Nuclear and cytoplasmic extracts were prepared from 5x106 HUVECs as described.31 Protein concentrations in nuclear extracts were 1 to 5 mg/mL, as determined by BCA protein assay (Pierce). The following oligonucleotides were obtained from Operon Technologies Inc: TF, 5'-GTCCCGGAGTTTCCTACCGGG-3'; TFm, 5'-GTCCCGGAGTTAGATACCGGG-3'; and Ig{kappa}, 5'-CAGAGGGACTTTCCGAGA-3'. The {kappa}B and {kappa}B-like sites are underlined, and the mutated TF (TFm) site contains a 3-bp substitution. Oligonucleotides were radiolabeled using [{alpha}-32P]dCTP (>3000 Ci/mmol, Amersham) as described.19 Analysis of NF-{kappa}B/Rel family protein binding was performed as follows. Nuclear extracts (1 to 2 µg) were incubated with radiolabeled DNA probes ({approx}10 ng; 1x106 cpm) for 20 minutes at room temperature in a 10-µL binding reaction containing 20 mmol/L HEPES, pH 7.9, 50 mmol/L KCl, 0.5 mmol/L EDTA, 5% glycerol, 1 mmol/L DTT, 1 mg/mL bovine serum albumin, 0.1% NP40, and 25 µg/mL poly(dI:dC). Protein-DNA complexes were separated from free DNA probe by electrophoresis through 6% nondenaturing acrylamide gels (Novex) in 0.5x Tris/borate/EDTA. AP-1 binding activity was analyzed with the Gel Shift System (Promega Corp) using the following double-stranded oligonucleotides containing a prototypic AP-1 site (underlined) or AP-1 sites from the TF promoter: AP-1 consensus (AP-1C), 5'-CGCTTGATGAGTCAGCCGGAA-3'; distal TF AP-1 site (AP-1D), 5'-CGCGGTTGAATCACTGGGG-3'; and proximal TF AP-1 site (AP-1P), 5'-CTGGGGTGAGTCATCCCTT-3'. For competition and antibody supershift experiments, binding reactions were incubated with unlabeled double-stranded oligonucleotides or monospecific antibodies for 20 minutes before the addition of the radiolabeled oligonucleotide.

Antibodies
Anti-p65, anti–c-Rel, anti–c-Fos, anti–Jun B, anti–Jun D, and anti–Fos B rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology. In addition, anti–c-Jun antisera were provided by Dr P. Vogt, and anti-p50 and anti-I{kappa}B{alpha} antisera were kindly provided by Dr. W. Greene.

Western Blot Analysis
Cytoplasmic extracts from HUVECs stimulated with IL-1ß (20 ng/mL) for various times were electrophoresed on 8% to 16% SDS-polyacrylamide gels (Novex) and transferred to Hybond-ECL (Amersham Corp). I{kappa}B{alpha} protein was detected according to the ECL protocol (Amersham Corp) using a 1:2500 dilution of an I{kappa}B{alpha} antiserum.32


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Functional Analysis of the Human TF Promoter in HUVECs
Exposure of HUVECs to TNF-{alpha} (20 ng/mL), IL-1ß (20 ng/mL), LPS (1 µg/mL), or PMA (50 ng/mL) rapidly induced TF mRNA and TF activity (Fig 1Down). These concentrations were found to maximally induce TF expression (data not shown). Similar results were obtained with LPS-stimulated human cardiac valve endothelial cells and IL-1ß–stimulated human dermal microvascular endothelial cells (data not shown). To localize cis-acting DNA elements that regulate TF gene transcription, HUVECs were transfected with plasmids containing sequential 5' truncations of the TF promoter between -2106 and -21 bp cloned upstream of a luciferase reporter gene (Fig 2ADown). After 20 hours, the cells were stimulated for 5 hours to determine the fold induction of luciferase activity. Plasmids containing at least -244 bp of the TF promoter, including the 56-bp enhancer (-227 to -172 bp), were all induced by TNF-{alpha}, whereas luciferase activity expressed by pTF(-153)LUC and pTF(-111)LUC was not increased by TNF-{alpha} stimulation (Fig 2BDown). pTF(-21)LUC did not express any promoter activity. These results suggest that the 56-bp enhancer may mediate TNF-{alpha} induction of the TF promoter in HUVECs. Similarly, luciferase activity expressed by pTF(-278)LUC, which contained the enhancer, was induced by IL-1ß and LPS, but these agonists failed to induce the enhancerless plasmid pTF(-111)LUC (Fig 2CDown). PMA induction of pTF(-278)LUC was significantly greater than the induction of pTF(-111)LUC, suggesting that the 56-bp enhancer may mediate, at least in part, PMA induction of the TF promoter. PMA induction of pTF(-111)LUC, although weak, suggested the presence of a second PMA response element in the TF promoter.



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  		Figure 1. Induction of tissue factor (TF) activity and TF mRNA in human umbilical vein endothelial cells (HUVECs). A, HUVECs were stimulated for 5 hours with tumor necrosis factor–{alpha} (TNF-{alpha}, 20 ng/mL), interleukin-1ß (IL-1ß, 20 ng/mL), lipopolysaccharide (LPS, 1 µg/mL), or phorbol 12-myristate 13-acetate (PMA, 50 ng/mL). Total TF activity was measured in triplicate samples in a one-stage clotting assay. Results of a typical experiment (mean±SD) are shown. B, HUVECs were stimulated as described above for 1 hour before total RNA (8 µg per lane) was isolated, and TF mRNA levels were determined by Northern blotting. Glyceraldehyde-6-phosphate dehydrogenase (G6PDH) mRNA levels were used to assess RNA loading.



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  		Figure 2. Functional analysis of the human TF promoter in HUVECs. A, The 5' boundaries of seven plasmids containing various truncations of the TF promoter are shown schematically. Positions of the TATA box, activator protein–1 (AP-1) sites, {kappa}B-like site, and enhancer are indicated. The bent arrow represents the start site of transcription upstream of the luciferase reporter gene (LUC). B, HUVECs transfected with each of these plasmids (10 µg) were cultured for 20 hours before a 5-hour stimulation with TNF-{alpha} (20 ng/mL). Basal expression of luciferase activity (light units) of a typical experiment was pTF(-2106)LUC, 3889; pTF(-383)LUC, 5181; pTF(-278)LUC, 8356; pTF(-244)LUC, 4927; pTF(-153)LUC, 1397; and pTF(-111)LUC, 1000. Induction of luciferase activity (mean±SD) from three independent experiments is shown. C, HUVECs transfected with either pTF(-278)LUC, containing the 56-bp enhancer, or pTF(-111)LUC, which lacks the enhancer, were stimulated for 5 hours with either TNF-{alpha} (20 ng/mL), IL-1ß (20 ng/mL), LPS (10 µg/mL), or PMA (50 ng/mL). Induction of luciferase activity (mean±SD) from three independent experiments is shown. *Statistically significant difference between luciferase activity expressed by pTF(-278)LUC and pTF(-111)LUC (P<.05 by Student's t test). See the legend to Fig 1Up for explanation of abbreviations.

To determine whether the two AP-1 sites and the {kappa}B-like site within the enhancer mediated TNF-{alpha} induction, HUVECs were transfected with plasmids containing mutations in these sites. Mutation of each of these three sites independently abolished TNF-{alpha} induction of the TF promoter, suggesting that all three sites were required for inducible expression (Fig 3ADown). To further examine the role of the two AP-1 sites and the {kappa}B-like site in TNF-{alpha} induction, HUVECs were transfected with a plasmid containing the 56-bp TF enhancer cloned upstream of a heterologous promoter. The 56-bp enhancer present in pTF(227/172)PAI-LUC conferred TNF inducibility to the minimal promoter (Fig 3BDown). Plasmid pTF(227/189)PAI-LUC, which contained the two AP-1 sites alone, was not inducible by TNF-{alpha}, whereas plasmid pTF(192/172)PAI-LUC, which contained a single copy of the {kappa}B-like site alone, was induced by TNF-{alpha}. Similar results were obtained with IL-1ß, LPS, and PMA (data not shown). The function of the TF {kappa}B-like site also was examined by using plasmids containing four tandem copies of either the TF {kappa}B-like site or a mutated TF {kappa}B-like site cloned upstream of a minimal SV40 promoter expressing the luciferase reporter gene. Luciferase activity expressed by p(TF)4LUC, which contained four copies of the wild-type site, was strongly induced by TNF-{alpha}, IL-1ß, LPS, and PMA (Fig 3CDown). In contrast, luciferase activity expressed by p(TFmut)4LUC, which contained four copies of the mutated TF {kappa}B-like site, or the parental plasmid pSVLUC was not induced by these agonists.



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  		Figure 3. Functional analysis of the 56-bp TF enhancer in HUVECs. A, Mutations in the two activator protein–1 (AP-1) sites or {kappa}B-like site are indicated by filled boxes. HUVECs transfected with these plasmids (10 µg) were stimulated with TNF-{alpha} as described in the legend to Fig 2Up. Induction of luciferase activity (mean±SD) from three independent experiments is shown. B, Plasmids containing the complete 56-bp enhancer (-227 to -172), the AP-1 sites alone (-227 to -189), or the {kappa}B-like site alone (-192 to -172), cloned upstream of the minimal plasminogen activator inhibitor–1 (PAI-1) promoter expressing the luciferase reporter gene (LUC), were transfected into HUVECs and stimulated with TNF-{alpha} as described in the legend to Fig 2Up. Induction of luciferase activity (mean±SD) from three experiments is shown. C, HUVECs were transfected with pSVLUC, p(TF)4LUC, or p(TFmut)4LUC19 and cultured for 20 hours before a 5-hour stimulation with agonists as described in the legend to Fig 2Up. The fold induction of luciferase activity (mean±SD) from three independent experiments is shown. See the legend to Fig 1Up for explanation of abbreviations.

AP-1 Binding Activity in HUVECs
To assess the binding of nuclear proteins to the two AP-1 sites in the TF promoter, EMSAs were performed with oligonucleotides that contained the distal (AP-1D) or proximal (AP-1p) AP-1 sites from the TF promoter (see "Methods"). Nuclear extracts from unstimulated HUVECs formed a prominent protein-DNA complex with both the distal and proximal TF AP-1 sites, as well as with an oligonucleotide containing a prototypic AP-1 site (Fig 4ADown). Competition studies using unlabeled oligonucleotides containing either an AP-1 site or an Sp1 site demonstrated that the two AP-1 sites in the TF promoter and the prototypic AP-1 site specifically bound AP-1 proteins (Fig 4ADown). To determine whether stimulation of HUVECs increased AP-1 binding activity, radiolabeled oligonucleotides were incubated with nuclear extracts from HUVECs exposed to TNF-{alpha}, IL-1ß, LPS, or PMA for 1 hour. The intensity of the protein-DNA complex formed with nuclear extracts from unstimulated cells was similar to that observed with nuclear extracts from stimulated cells (Fig 4BDown).





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  		Figure 4. Activator protein–1 (AP-1) binding activity in HUVECs. A, Radiolabeled DNA probes containing an AP-1 consensus site (AP-1c), the distal TF AP-1 site (AP-1D), or the proximal TF AP-1 site (AP-1p) were incubated with nuclear extracts from unstimulated HUVECs. Protein-DNA complexes were analyzed on 6% polyacrylamide gels, and the position of the protein-DNA complexes is indicated. To demonstrate the specificity of protein binding, a 100-fold molar excess of cold competitor oligonucleotides containing either the prototypic AP-1 site or an Sp1 site were used. B, AP-1 binding activity in stimulated HUVECs. Nuclear extracts from unstimulated HUVECs and cells stimulated with IL-1ß, TNF-{alpha}, LPS, or PMA for 1 hour as described in the legend to Fig 2Up were incubated with radiolabeled DNA probes, and protein-DNA complexes were analyzed by electrophoretic mobility shift assay. C, Composition of complexes formed with nuclear extracts from unstimulated cells and IL-1ß–stimulated cells was determined by antibody supershift experiments. Antibodies (5 µL) were added 20 minutes before addition of the radiolabeled probe containing AP-1p. Supershift complexes migrate more slowly. Position of the AP-1 complexes is indicated. See the legend to Fig 1Up for explanation of abbreviations.

Monospecific antibodies to various members of the AP-1 family were used to determine the composition of the AP-1 complexes. The AP-1 complex formed between AP-1p and nuclear extracts from unstimulated cells or IL-1ß–stimulated cells was not recognized by antisera against Jun B, Jun D, Fos B, and p65 (Fig 4CUp). In contrast, this complex was supershifted with anti–c-Fos and anti–c-Jun antisera (Fig 4CUp). Similar results were observed with AP-1D (data not shown). Increasing the amount of c-Fos or c-Jun antisera or adding both antisera simultaneously did not completely remove the residual complex, suggesting that it did not represent binding of Fos-Jun heterodimers or c-Jun homodimers. At present the identity of this residual complex is unknown, but it may represent binding of other bZIP proteins. These data suggest that in unstimulated HUVECs the two AP-1 sites in the TF promoter constitutively bind Fos-Jun heterodimers. Stimulation of the cells with various agonists did not change the composition of the complex or increase binding of the Fos-Jun heterodimers.

The TF {kappa}B-like Site Binds c-Rel–p65 Heterodimers
EMSAs were performed to examine the binding of nuclear proteins to an oligonucleotide containing the TF {kappa}B-like site, 5'-CGGAGTTTCC-3'. A constitutive complex was observed with nuclear extracts from both unstimulated and IL-1ß–stimulated HUVECs, whereas an additional inducible complex (TF complex) was observed with nuclear extracts from IL-1ß–stimulated cells (Fig 5ADown, lanes 1 and 2). To determine the specificity of these protein-DNA complexes, competition studies were performed using unlabeled oligonucleotides containing either the TF {kappa}B-like site (TF) or a mutated TF {kappa}B-like site (TFm). Both complexes were competed with an oligonucleotide containing the TF {kappa}B-like site (Fig 5ADown, lane 4), whereas an oligonucleotide containing the mutated TF {kappa}B-like site only competed with the faster-migrating complex (Fig 5ADown, lane 5). These data indicated that the inducible TF complex represented specific protein binding to the {kappa}B-like site and the faster-migrating complex represented nonspecific protein binding. The TF complex was also observed with nuclear extracts from HUVECs stimulated for 1 hour with TNF-{alpha}, LPS, or PMA (Fig 5BDown, lanes 7 through 10). For comparison, we examined the binding of NF-{kappa}B (p50-p65) to a prototypic {kappa}B site from the mouse light-chain enhancer (Ig{kappa}). The low levels of p50-p65 heterodimers present in nuclear extracts from unstimulated HUVECs were dramatically increased upon stimulation with each of the four agonists (Fig 5BDown, lanes 1 through 5). Significantly, the TF complex migrated more slowly than the Ig{kappa} complex (Fig 5BDown), suggesting that it represented binding of NF-{kappa}B/Rel proteins distinct from p50-p65 heterodimers.




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  		Figure 5. The TF {kappa}B-like site binds c-Rel–p65 heterodimers. Nuclear extracts from unstimulated HUVECs or cells stimulated for 1 hour with TNF-{alpha} (20 ng/mL), IL-1ß (20 ng/mL), LPS (10 µg/mL), or PMA (50 ng/mL) were incubated with radiolabeled oligonucleotides containing the TF {kappa}B-like site (TF) or the prototypic {kappa}B site (Ig{kappa}). Protein-DNA complexes were analyzed on a low-ionic-strength 6% polyacrylamide gel. A, Nuclear extracts from unstimulated (-) or IL-1ß–stimulated (+) cells were analyzed with an oligonucleotide containing the TF {kappa}B-like site. Position of the inducible TF complex is indicated. Complex formation was competed with a 100-fold molar excess of cold competitor oligonucleotides containing either the TF {kappa}B-like site (TF) or a mutated TF {kappa}B-like site (TFm). B, Nuclear extracts from unstimulated and stimulated HUVECs were incubated with oligonucleotides containing the Ig{kappa} site (lanes 1-5) or the TF {kappa}B-like site (lanes 6-10). Positions of the nuclear factor (NF)–{kappa}B (p50-p65) complex and the TF complex (arrow) are indicated. C, Mobilities of TF and Ig{kappa} complexes were compared with those of various translated homodimeric and heterodimeric complexes as indicated. Nuclear extracts from IL-1ß–stimulated cells (N.E.), various programmed lysates, and DNA probes are shown above. D, The TF complex (arrow) formed with nuclear extracts from TNF-{alpha}–stimulated HUVECs was analyzed with monospecific antibodies that specifically recognize p50, p65, or c-Rel. Antibody (2 µL) was added 20 minutes before addition of 32P-labeled DNA probe containing the TF {kappa}B-like site. See the legend to Fig 1Up for explanation of abbreviations.

To assess the protein composition of the nuclear complex that bound to the TF {kappa}B-like site, the mobility of the TF complex was compared with that of translated p50, p65, and c-Rel homodimers as well as p50-p65 and c-Rel–p65 heterodimers. The TF complex comigrated with c-Rel–p65 heterodimers and migrated independently of p65 and c-Rel homodimers (Fig 5CUp, lanes 4 through 8). As expected, the Ig{kappa} complex comigrated with the p50-p65 heterodimers (Fig 5CUp, lanes 2 and 3). These data suggest that the TF complex may represent binding of c-Rel–p65 heterodimers.

Monospecific antibodies to p50, p65, and c-Rel were used to independently analyze the identity of the TF complex. Anti-p65 or anti–c-Rel antibodies abolished formation of the TF complex, whereas an anti-p50 antibody had no effect, indicating that the TF complex represented binding of c-Rel–p65 heterodimers (Fig 5DUp). These antibodies did not recognize the faster-migrating nonspecific complex. The low levels of c-Rel–p65 heterodimers in HUVECs prohibited analysis of this complex by UV cross-linking. c-Rel–p65 heterodimers also were activated in LPS-stimulated human cardiac valve endothelial cells and IL-1ß–stimulated human dermal microvascular endothelial cells (data not shown). Taken together, these data demonstrated that stimulation of HUVECs with four diverse agonists activated c-Rel–p65 heterodimers, which specifically bound to the novel {kappa}B-like site in the TF promoter.

Transactivation of the TF {kappa}B-like Site in HUVECs by Expression of NF-{kappa}B/Rel Proteins
To investigate in vivo binding of NF-{kappa}B/Rel family members to the TF {kappa}B-like site, HUVECs were cotransfected with p(TF)4LUC and plasmids expressing p50, p65, or the chimeric proteins p50TA65 and c-RelTA65, which contained the transactivation domain of p65 fused to the DNA binding portions of p50 and c-Rel, respectively. To eliminate ambiguities created by transactivation domains with different activities, all proteins contained an identical transactivation domain. p50 does not contain a transactivation domain and therefore was used as a negative control. Transactivation by these NF-{kappa}B/Rel proteins was measured by increased expression of luciferase activity. p(TF)4LUC was transactivated by p65 and c-RelTA65 but not by p50 or p50TA65 (Fig 6ADown). In contrast, p(Ig{kappa})4LUC was transactivated by p65 and p50TA65 but not by p50 or c-RelTA65. These NF-{kappa}B/Rel proteins did not transactivate pSVLUC, which contained the minimal SV40 promoter alone, or p(TFmut)4LUC, which contained the mutated TF {kappa}B-like site (data not shown).




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  		Figure 6. Transactivation of the TF {kappa}B-like site by nuclear factor (NF)–{kappa}B/Rel proteins. A, HUVECs were transfected with p(TF)4LUC or p(Ig{kappa})4LUC (10 µg) alone or in the presence of plasmids (5 µg) expressing either p50, p65, or the chimeric proteins p50TA65 and c-RelTA65 (see "Methods"). Cells were harvested 24 hours after transfection and luciferase activity was determined. Results from a representative experiment with triplicate samples are shown and are expressed as fold induction of luciferase activity (mean±SD). Similar results were observed in two independent experiments. The vector alone, pSVLUC, or a plasmid containing four copies of a mutated {kappa}B-like site, p(TFmut)4LUC, were not transactivated under these conditions. B, HUVECs were cotransfected with p(TF)4LUC (2 µg) and 1 µg each of plasmids expressing p50, p65, or c-Rel as indicated. Total plasmid DNA was equalized to 5 µg per transfection using pUC18 DNA. Transactivation by each combination of plasmids was expressed relative to the transactivation by p65 alone (100%) ±SD. C, Cells were transfected with p(TF)4LUC (2 µg) alone or cotransfected with a plasmid expressing I{kappa}B{alpha}. Data are shown from three independent experiments (mean±SD). See the legend to Fig 1Up for explanation of abbreviations.

To examine transactivation by p65 or c-Rel homodimers as well as by c-Rel–p65 or p50-p65 heterodimers, cells were cotransfected with p(TF)4LUC in the presence of plasmids expressing p50, p65, or c-Rel alone or in combination. c-Rel homodimers were weak transactivators compared with p65 homodimers (Fig 6BUp), as reported previously.33 Next, the cells were transfected with plasmids expressing c-Rel and p65, or p50 and p65, at a 1:1 ratio to generate c-Rel–p65 and p50-p65 heterodimers, with the assumption that there is approximately equal expression from each plasmid. These results indicated that c-Rel–p65 heterodimers transactivated the TF {kappa}B-like site, whereas formation of p50-p65 heterodimers abolished transactivation, presumably by eliminating p65 homodimers (Fig 6BUp). These results strongly suggested that c-Rel–p65 heterodimers regulated the TF {kappa}B-like site in human endothelial cells. To determine whether c-Rel–p65 heterodimers were regulated by I{kappa}B{alpha}, HUVECs were cotransfected with p(TF)4LUC and a plasmid that expresses I{kappa}B{alpha}. TNF-{alpha} induction of luciferase activity mediated by the binding of c-Rel–p65 heterodimers to the TF {kappa}B-like site was abolished by expression of I{kappa}B{alpha}, indicating that binding of exogenously expressed I{kappa}B{alpha} to endogenous c-Rel–p65 heterodimers prevented nuclear translocation (Fig 6CUp).

Activation of c-Rel–p65 Heterodimers Precedes TF mRNA Induction in HUVECs
TF mRNA was induced rapidly after TNF-{alpha} or IL-1ß stimulation of HUVECs (Fig 1Up). As shown in Fig 7Down (upper panel), stimulation of HUVECs resulted in a rapid and transient increase in TF mRNA levels that were maximal at 60 minutes. To examine the kinetics of activation of c-Rel–p65 heterodimers in HUVECs, nuclear extracts were prepared from cells at various times after stimulation. c-Rel–p65 heterodimers were rapidly activated within 15 minutes and declined after 30 minutes (Fig 7Down, middle panel). To determine whether translocation of c-Rel–p65 to the nucleus was associated with loss of the inhibitor protein I{kappa}B{alpha}, cytoplasmic extracts from stimulated cells were analyzed by Western blotting and an anti-I{kappa}B{alpha} antibody. I{kappa}B{alpha} protein was present in unstimulated HUVECs but was rapidly and transiently reduced after stimulation (Fig 7Down, lower panel). Maximal reduction of I{kappa}B{alpha} protein was observed at 15 minutes, which corresponded to maximal levels of c-Rel–p65 heterodimers in the nucleus. I{kappa}B{alpha} protein remained low at 30 minutes before increasing after 1 hour. Taken together, these data indicated that activation of c-Rel–p65 heterodimers was regulated by I{kappa}B{alpha}. The rapid activation and nuclear translocation of c-Rel–p65 heterodimers within 15 minutes preceded increases in TF mRNA levels at 40 minutes, consistent with a role for this transcription factor in the induction of TF gene transcription.



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  		Figure 7. Nuclear translocation of c-Rel–p65 heterodimers to the nucleus precedes TF mRNA induction. HUVECs were stimulated for the times indicated with either TNF-{alpha} (20 ng/mL) or IL-1ß (20 ng/mL). Total RNA was prepared from TNF-{alpha}–stimulated cells, and TF mRNA levels were determined by Northern blotting (upper panel). To examine activation of c-Rel–p65 heterodimers, nuclear extracts prepared from cells stimulated with IL-1ß for the times indicated were incubated with a radiolabeled oligonucleotide containing the TF {kappa}B-like site (middle panel). Cytoplasmic extracts were electrophoresed on 8% to 16% SDS polyacrylamide gels (Novex) and transferred to Hybond-ECL (Amersham Corp). I{kappa}B{alpha} protein was detected according to the ECL protocol (Amersham Corp) using a 1:2500 dilution of I{kappa}B{alpha} antiserum. See the legend to Fig 1Up for explanation of abbreviations.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
This study elucidated a transcriptional regulatory mechanism that coordinated induction of TF expression in primary cultures of human endothelial cells exposed to TNF-{alpha}, IL-1ß, and LPS. Functional analysis of the human TF promoter demonstrated that a 56-bp enhancer (-227 to -172), containing two AP-1 sites and a {kappa}B-like site, mediated induction in response to these agonists. Mutational analysis indicated that all three sites were required for induction of the homologous TF promoter. EMSAs revealed that the two AP-1 sites constitutively bound Fos-Jun heterodimers, whereas the {kappa}B-like site bound an inducible nuclear complex, which was composed of c-Rel–p65 heterodimers. Nuclear extracts from LPS-stimulated human cardiac valve endothelial cells and IL-1ß–stimulated human dermal microvascular endothelial cells also contained c-Rel–p65 heterodimers (data not shown). Nuclear translocation of c-Rel–p65 heterodimers preceded induction of TF mRNA, consistent with a positive regulatory role for this transcription factor in TF gene transcription. Taken together, these data suggest that Fos-Jun and c-Rel–p65 heterodimers regulate TF gene expression in human endothelial cells exposed to TNF-{alpha}, IL-1ß, and LPS.

Comparable levels of TF activity and TF mRNA were induced in HUVECs exposed to optimal doses of TNF-{alpha}, IL-1ß, and LPS, whereas PMA was a more potent agonist (Fig 1Up). The apparent discrepancy between increases in the steady-state levels of TF mRNA and the magnitude of induction of the TF promoter expressing the luciferase reporter gene suggested that posttranscriptional control mechanisms contributed to TF mRNA accumulation in these cells, as reported previously.9 Nevertheless, studies of TF gene regulation in human endothelial cells have consistently reported that LPS, TNF-{alpha}, and PMA increase the rate of TF gene transcription,4 9 15 indicating that TF expression is controlled, in part, at the level of transcription. Our studies with transfected HUVECs were consistent with the changes in the rate of transcription of the endogenous TF gene in LPS-stimulated HUVECs.4 9 However, we cannot discount the possibility that DNA elements not present between -2106 and +121 bp also regulate TF gene expression in HUVECs, which may explain why PMA did not strongly induce the cloned TF promoter despite a dramatic induction of TF mRNA. Our studies indicated the presence of two PMA response elements in the cloned TF promoter: the TF enhancer (-227 to -172 bp) and a region (-111 to +121 bp) that contains Sp1 and EGR-1 binding sites.34 TNF-{alpha} induction of the TF promoter in HUVECs was mediated by the 56-bp enhancer alone because it was abolished by deletion of the enhancer and by individual mutations of the two AP-1 sites and the {kappa}B-like site. Previously, we showed similar results in LPS-stimulated monocytic THP-1 cells,16 suggesting that a common mechanism regulates TF gene expression in both human monocytes and endothelial cells.

A recurring theme in the regulation of gene expression in eukaryotic cells is the binding of multiple transcription factors.35 Here, we showed that in stimulated endothelial cells, Fos-Jun heterodimers bound to the two AP-1 sites and c-Rel–p65 heterodimers bound to an adjacent {kappa}B-like site in the 56-bp TF enhancer. The binding of Fos-Jun heterodimers was unaffected by stimulation of the cells, and the composition of the complex was not changed upon stimulation. NF-{kappa}B/Rel family proteins cooperate with AP-1 proteins as well as with NF-IL6, SRF, and Sp1 to activate both cellular and viral genes.36 37 38 39 40 Recent studies by Stein and colleagues40 demonstrated that the bZIP regions of Fos and Jun proteins from the AP-1 family directly interacted with the Rel homology domain of p65 from the NF-{kappa}B/Rel family to form a functionally active transcription factor complex. Thus, in a similar manner, binding of Fos-Jun heterodimers to the TF enhancer may cooperate with c-Rel–p65 heterodimers to facilitate activation of TF gene transcription.

Endothelial cell activation induces the rapid expression of many genes involved in inflammation, which contain {kappa}B and {kappa}B-like sites in their promoters.20 Here, we have shown that the TF {kappa}B-like site plays a central role in regulating TF gene transcription in endothelial cells. The TF {kappa}B site, 5'-CGGAGTTTCC-3', specifically bound c-Rel–p65 heterodimers. The nonconsensus C at position 1 is conserved in the mouse TF promoter and is a T in the porcine TF promoter.41 42 Other genes that are inducibly expressed in endothelial cells, including IL-8 and intercellular adhesion molecule–1,43 44 45 contain {kappa}B-like sites with a T at position 1 and bind c-Rel–p65 heterodimers,46 suggesting that a distinct subset of genes are regulated by c-Rel–p65 heterodimers.

LPS, TNF-{alpha}, IL-1ß, and PMA all activate c-Rel–p65 heterodimers, which activation represents a common mechanism for induction of TF expression in HUVECs. This conclusion is supported by a recent study demonstrating that both activation of NF-{kappa}B/Rel family proteins and induction of TF mRNA expression in HUVECs exposed to LPS are dependent on the presence of soluble CD14 receptors.31 LPS stimulation of cells induces the rapid phosphorylation of I{kappa}B{alpha} and subsequent translocation of NF-{kappa}B/Rel complexes to the nucleus.47 TNF-{alpha} induction mediated by the TF {kappa}B-like site was abolished by overexpression of I{kappa}B{alpha}, indicating that c-Rel–p65 heterodimers are regulated by binding of I{kappa}B{alpha}. Thus, despite the binding of LPS, TNF-{alpha}, and IL-1ß to distinct cell-surface receptors, intracellular signaling pathways converge to activate c-Rel–p65 heterodimers, which interact with Fos-Jun heterodimers to coordinate induction of TF expression in endothelial cells.


*    Acknowledgments
 
This work was supported by a Research Fellowship (Dr Parry) from the American Heart Association, California Affiliate, San Diego County Chapter, and by National Institutes of Health grants HL-16411 and HL-48872 (Dr Mackman). This is publication IMM-8547 from The Scripps Research Institute. We wish to thank P. Nantermet, H. Larson, and P. Oeth for expert technical assistance and D. Nielsen for the preparation of the manuscript. We thank P. Vogt for anti–c-Jun antibodies; W. Greene for the anti-p50 and anti-I{kappa}B{alpha} antibodies; N. Rice for the c-Rel expression vector; C. Rosen, C. Kunsch, and S. Ruben for the soCMVIN expression vectors; D. Loskutoff and K. Roegner for endothelial cells; T. Drake for human cardiac valve endothelial cells; and S. Cordle and M. Read for advice on EMSA. We thank L. Curtiss and C. Banka for critical review of this manuscript.

Received October 6, 1994; accepted February 17, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
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 *References
 

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