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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:365-374

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:365-374.)
© 1997 American Heart Association, Inc.

Articles

Regulation of the Tissue Factor Gene in Human Monocytic Cells

Role of AP-1, NF-{kappa}B/Rel, and Sp1 Proteins in Uninduced and Lipopolysaccharide-Induced Expression

Paul Oeth; Graham C.N. Parry; Nigel Mackman

Departments of Immunology and Vascular Biology, 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. E-mail nmackman@scripps.edu.


*    Abstract
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 *Abstract
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Tissue factor (TF) expression by peripheral blood monocytes during sepsis initiates intravascular thrombosis. Bacterial lipopolysaccharide (LPS) rapidly induces TF gene transcription in monocytes. The human TF promoter contains binding sites for the transcription factors AP-1, c-Rel/p65, Egr-1, and Sp1. NF-{kappa}B/Rel proteins have been shown to physically interact with both AP-1 and Sp1 proteins. In this study, we investigated the role of these transcription factors in uninduced and LPS-induced TF gene expression in human monocytic THP-1 cells. Deletional analysis indicated that five Sp1 sites mediated basal expression in uninduced cells. The two AP-1 sites bound c-Fos/c-Jun heterodimers in both unstimulated and LPS-stimulated cells. Maximal LPS induction of the TF promoter required the two AP-1 sites and the {kappa}B site within the LPS response element. Disruption of the conserved spacing between the proximal AP-1 site and the {kappa}B site abolished LPS induction. Replacement of the two AP-1 sites with intrinsically bent DNA partially restored LPS induction, suggesting an additional structural role for the AP-1 sites. Synergistic transactivation of the LPS response element in Drosophila Schneider cells by coexpression of c-Fos, c-Jun, c-Rel, and p65 or c-Jun and p65 required the transactivation domains of c-Jun and p65. These data indicated that c-Fos/c-Jun, c-Rel/p65, and Sp1 regulate TF gene expression in human monocytic cells.


Key Words: thrombosis • tissue factor • gene regulation • transcription factors


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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Exposure of blood to TF rapidly initiates the coagulation serine protease cascades.1 Aberrant TF expression within the vasculature leads to thrombosis in patients with a variety of diseases, including septic shock, chronic unstable angina, atherosclerosis, and cancer.2 3 4 5 LPS induction of the TF gene in monocytes and monocytic cells is mediated by preexisting transcription factors.6 7 The human TF promoter contains binding sites for AP-1, NF-{kappa}B/Rel, Egr-1, and Sp1 proteins.8 9 Our functional studies using human monocytic THP-1 cells indicated that transcriptional activation of the TF promoter requires a 56-bp LRE, which contains two AP-1 sites and a {kappa}B site.10 Similar studies indicate that these AP-1 and {kappa}B sites in the human and porcine TF promoters regulate induction of the TF gene in endothelial cells.11 12 13 14 Nuclear extracts from monocytic and endothelial cells protected the human and porcine TF LREs from DNase I digestion,11 15 indicating that these regions bind nuclear proteins. In addition, analysis of the porcine TF promoter revealed several DNase I "footprints" spanning Sp1-like sites downstream from the LRE.11 LPS stimulation of human monocytes and monocytic cells induces the nuclear translocation of c-Rel/p65 heterodimers that selectively bind to the nonconsensus {kappa}B site within the TF LRE.16 Our recent studies demonstrated that inhibition of the nuclear translocation of c-Rel/p65 by protease inhibitors and salicylates abolishes LPS induction of TF gene expression in human monocytes.17 18

LPS rapidly activates NF-{kappa}B/Rel proteins in human monocytes and monocytic cells.19 This family of transcription factors includes p50 (NFKB1), p65 (RelA), and c-Rel.20 Dissociation of inhibitor protein I{kappa}B{alpha} from cytoplasmic NF-{kappa}B(p50/p65) and c-Rel/p65 complexes allows nuclear translocation of these transcription factors and induction of gene expression.21 Human monocytes constitutively transcribe the c-fos and c-jun genes and express AP-1 binding activity.22 23 24 LPS increases AP-1 binding activity via de novo synthesis of c-fos and c-jun.22 The AP-1 family of transcription factors is divided into two groups: the fos-related genes that include c-fos, fos B, fra-1, and fra-2 and the jun family that includes c-jun, jun B, and jun D.25 Homodimers and heterodimers of Jun proteins can bind to DNA, whereas Fos proteins must heterodimerize with Jun proteins to bind DNA.26 Sp1 is a constitutively expressed, sequence-specific transcription factor found in a wide variety of cells. It recognizes GC-rich binding sites referred to as GC "boxes".27 The DNA binding specificity of Sp1 is conferred by three zinc "fingers," each of which interacts with separate nucleotide triplets.28 Transcriptional activation by Sp1 is determined by four domains (A through D) that are distinct from the zinc fingers.29 30 Sp1 mediates transcription of numerous genes containing multiple GC boxes in their promoters.27

Activation of Sp1 appears to involve cofactors associated with the TFIID component of the basal transcription complex.31 p50, p65 (RelA), and c-Rel have been shown to physically associate with other transcription factors, including ATF proteins,32 C/EBP proteins,33 34 and the basal transcriptional complex.35 36 Cooperative interactions between NF-{kappa}B and NF–IL-6 regulate activation of the IL-6 and IL-8 genes.37 38 39 40 In addition, studies of the virus-inducible enhancer element in the human interferon-ß gene have demonstrated that functional interactions between ATF2/c-Jun heterodimers and NF-{kappa}B are required for transcriptional activation.41 42 Recent studies have reported physical interaction between the transactivation domains of p65 and c-Jun or c-Fos and synergistic transactivation of multimerized AP-1 or {kappa}B sites.43 In addition, an interaction between the DNA binding domains of p65 and Sp1 mediates activation of the HIV type 1 long-terminal repeat.44 45

At present, the functional interactions between AP-1, NF-{kappa}B/Rel, Egr-1, and Sp1 proteins bound to the TF promoter in monocytic cells have not been defined. In this study, we determined that Sp1 regulated basal expression of the TF gene in unstimulated human monocytic THP-1 cells, but that c-Rel/p65 and Sp1 alone were not sufficient to mediate LPS induction. LPS-induced transcriptional activation of the TF gene required functional interaction between c-Fos/c-Jun and c-Rel/p65 heterodimers bound to the AP-1 and {kappa}B sites within the LRE.


*    Methods
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 *Methods
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Cell Culture and Transfection
The human monocytic THP-1 cell line was obtained from the American Type Culture Collection, Rockville, Md, and cultured in RPMI-1640 medium supplemented with 7% fetal calf serum, 2 mmol/L L-glutamine, 50 µmol/L 2-mercaptoethanol, and 10 mmol/L HEPES-buffered saline, pH 7.3. Cells were routinely grown to a density of 2 to 5x105/mL and stimulated with 10 µg/mL of LPS (Escherichia coli serotype O111:B4; Calbiochem, La Jolla, Calif). For transient transfection of THP-1 cells, 2x107 cells were transfected with DEAE-dextran and cultured for 46 hours before a 5-hour exposure to LPS.10 pUHG15.1 and pUHG10.3CAT (kindly provided by R. Hooft) were used to assess transfection efficiencies.46 Chloramphenicol acetlytransferase activity was determined with a diffusion-based assay10 and exhibited <16% variation between samples (data not shown). Drosophila Schneider line-2 (SL2) cells47 were grown in Schneider's Drosophila medium (GIBCO Laboratories) containing 10% heat-inactivated fetal calf serum at 25°C. The SL2 cells were transfected with 10 µg of pTFSV-LUC, 2.5 µg of each expression plasmid, and pUC18 to a total of 20 µg, with calcium phosphate from the CellPhect Transfection Kit (Pharmacia). Transfected cells were incubated for 48 hours at 25°C. Luc activity was determined with a Luc assay system (Promega Corp) and a Monolight 2010 luminometer (Analytical Luminescence Inc).

Plasmids
The eukaryotic expression plasmids pCMVp65 (kindly provided by C. Kunsch) and pCMVc-Rel (kindly provided by N. Rice) have been described.21 48 pCMVp65(1-309) (kindly provided by C. Kunsch) expresses a truncated form of p65 that contains only the Rel homology domain and lacks the C-terminal transactivation domain.39 pCMVp65mLZ, which expresses p65 containing a mutated Leu zipper–like structure,48 and pCMVc-Jun({Delta}1-194), which expresses a truncated form of c-Jun with the first 194 amino acids deleted,43 were kindly provided by B. Stein. The expression vectors pRSVc-Jun, pRSVc-Fos, and pPACSp1 were provided by A. McLachlan. Plasmids containing various 5' deletions of the human TF promoter cloned upstream from the luciferase reporter gene have been described previously.10 pTF(-111)Sp1MLUC has been described.49 Plasmids pTF(-227)LUC and pTF(-227)Mut1-4 were made by cloning the wild-type and mutated TF promoters (-227 to +121 bp) into pGL2-Basic (Promega). The distal AP-1 site was mutated from 5'-TGAATCA-3' to 5'-ACAACAA-3', the proximal AP-1 site was mutated from 5'-TGAGTCA-3' to 5'-ACAACAA-3', and the {kappa}B site was mutated from 5'-CGGAGTTTCC-3' to 5'-CGGAGTTAGA-3'. The wild-type (-227 to -172 bp) and mutated LREs were cloned upstream from the minimal simian virus 40 promoter expressing the LUC reporter gene in pGL2-Promoter (Promega) to create pTFSV-LUC and pTFSVMut1-3. Plasmids pTF(+8)LUC and pTF(+20)LUC were created by inserting 8 and 20 bp, respectively, into the Ava II site between the proximal AP-1 site and the {kappa}B site. Plasmids containing intrinsically bent DNA50 in place of the two AP-1 sites were created by cloning various oligonucleotides (Table 2) into pTF(-190)LUC in the forward (+) or reverse (-) orientation. All plasmids were characterized by DNA sequencing.

EMSAs
EMSAs were performed as described.51 Nuclear extracts were prepared as described.16 Double-stranded oligonucleotides containing binding sites for Sp1 and AP-2 (Promega) were used for competition studies. All other double-stranded oligonucleotides (Table 1) were purchased from Operon Technologies Inc and were labeled with either [{gamma}-32P]ATP (5000 Ci/mmol, ICN) or [{alpha}-32P]dCTP (>3000 Ci/mmol, ICN).16 An oligonucleotide containing an Egr-1 site has been reported previously.49

Antibodies
The affinity-purified rabbit polyclonal antibodies p65-NAb (sc-109), c-Jun-Ab (sc-45), Jun B-NAb (sc-46), Jun D-Ab (sc-74), c-Fos-Ab (sc-52), Fos B-Ab (sc-102), AP-2 CAb (sc-184), and Sp1-Ab (sc-059) were purchased from Santa Cruz Biotechnologies. Polyclonal antibodies against c-Jun and c-Fos were kindly provided by P. Vogt.


*    Results
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 up arrowMethods
 *Results
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Role of Sp1 Sites in Basal Expression of the Human TF Promoter in Unstimulated THP-1 Cells
The human TF promoter (-244 to +121 bp) contains AP-1, {kappa}B, Egr-1, and Sp1 sites. The role of these sites in basal expression of the TF promoter in human monocytic THP-1 cells was determined by transient transfection of a series of plasmids containing different amounts of 5' flanking sequence. Deletion of the two AP-1 sites in a region between -244 and -195 bp did not affect basal expression (Fig 1ADown). Deletion of a region (-194 to -154 bp) containing the {kappa}B site and Sp1 site V reduced basal expression. The {kappa}B site does not bind protein in unstimulated THP-1 cells,16 suggesting that the reduced expression in pTF(-153)LUC was due to deletion of the Sp1 site. A stepwise reduction of basal expression was also observed after deletion of three DNA fragments containing Sp1 sites IV, III, and II (Fig 1A). No promoter activity was expressed by pTF(-67)LUC, which contained a single Sp1 site. Our recent studies with HeLa cells had indicated that mutation of Sp1 sites III, II, and I within pTF(-111)LUC abolished TF promoter activity.49 Similar results were observed when THP-1 cells were used (data not shown). To further examine the functional role of Sp1 in basal TF promoter activity, we cotransfected Drosophila SL2 cells, which do not contain endogenous Sp1,29 52 with a TF promoter plasmid and a plasmid expressing Sp1. The wild-type TF promoter was transactivated by Sp1, whereas mutation of the three Sp1 binding sites in pTF(-111)Sp1MLUC dramatically reduced transactivation (Fig 1BDown).




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  		Figure 1. Basal expression of the TF promoter. A, Luc activity expressed by various TF promoter plasmids in unstimulated THP-1 cells. Seven plasmids containing truncations of the TF promoter are shown. DNA recognition sites for AP-1 (stipled), {kappa}B (stripped), Egr-1 (open), and Sp1 (filled) transcription factors are indicated by boxes. The bent arrow represents the start site of transcription upstream from the LUC reporter gene. Total Luc activity (light units) from transfected THP-1 cells was measured in several independent experiments and normalized for the amount of DNA uptake by using pRSVCAT as an internal control. Luc activity of these plasmids was expressed as a percentage (mean±SD) of the activity expressed by pTF(-244)LUC, which was defined as 100%. Numbers in brackets indicate the number of individual experiments. B, Activation of the TF promoter by Sp1 in Drosophila SL2 cells. Luc activity expressed by pTF(-111)LUC and pTF(-111)Sp1MLUC49 are shown with and without the Sp1 expression plasmid PACSp1. Mean (±SD) from three independent experiments is shown.

EMSAs were performed to examine Sp1 binding to the five predicted Sp1 sites. A single, major complex was formed with the five radiolabeled oligonucleotides Sp1I to Sp1V (Table 1Down), each of which contained an Sp1 site (Fig 2ADown). Protein binding was not increased by LPS stimulation (Fig 2ADown). The complex formed with Sp1V specifically competed with an unlabeled oligonucleotide containing an Sp1 site and was supershifted with anti-Sp1 antiserum, indicating that it represented binding of Sp1 protein (Fig 2BDown). Similar results were observed with the other four Sp1 sites as probes (data not shown). Taken together, these data indicated that Sp1 bound to five GC boxes in the TF promoter mediated basal TF expression in unstimulated human monocytic cells.


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  		Table 1. Oligonucleotides Used in EMSA




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  		Figure 2. Characterization of protein binding to the Sp1 sites. A, EMSAs were performed in nuclear extracts from unstimulated and LPS-stimulated monocytic THP-1 cells incubated with radiolabeled oligonucleotides Sp1I, Sp1II, Sp1III, Sp1IV, and Sp1V (Table 1Up). Stimulated cells were treated for 1 hour with LPS (10 µg/mL). The position of the Sp1 complex formed with the various probes is indicated. B, Characterization of the nuclear complex bound to Sp1V. Nuclear extracts from LPS-stimulated cells were incubated with radiolabeled Sp1V probe (lanes 1-5). Lane 1 was incubated without competitor or antibodies. A 50-fold molar excess of an oligonucleotide containing an Sp1 site (lane 2) or an oligonucleotide containing an AP-2 site (lane 3) was incubated with the nuclear extract before addition of the Sp1V probe. Antibody supershift experiments were performed with 2.5 µL of an anti-Sp1 antiserum (lane 4) or an anti–AP-2 antiserum (lane 5). Positions of the Sp1 and supershift complexes are indicated. C, Egr-1 binding to the human TF promoter. EMSAs were performed in nuclear extract from uninduced cells, cells stimulated with LPS (10 µg/mL) for 2 hours or cells stimulated with PMA (100 ng/mL) for 2 hours. Nuclear extracts were incubated with the radiolabeled oligonucleotide Sp1IIA (Table 1Up) or an oligonucleotide containing an Egr-1 site.49 Positions of the Sp1 and Egr-1 complexes are shown.

Role of Egr-1 in Uninduced and LPS-Induced Expression of TF Promoter in Monocytic Cells
In HeLa cells, we have shown that three Egr-1 sites overlapping with Sp1 sites I, II, and III mediated serum and PMA induction of the TF promoter.49 To determine whether Egr-1 played a role in uninduced and LPS-induced expression of the TF promoter in THP-1 cells, we examined Egr-1 expression by EMSA. Egr-1 was not detected in the uninduced or LPS-induced THP-1 cells in this study (Fig 2CUp). Furthermore, functional studies with these cells showed that pTF(-190)LUC, which contains three Egr-1 sites, was not induced by LPS (see Fig 4Down). In contrast, our previous studies with a different THP-1 subline showed a low level of LPS induction of the proximal TF promoter (-111 to +121 bp) without the LRE.10 Analysis of other THP-1 sublines revealed LPS induction of Egr-1 (Fig 2CUp). Taken together, these studies suggest that further experiments are necessary to examine the role of Egr-1 in low-level induction of the proximal TF promoter in LPS-stimulated monocytic cells.



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  		Figure 4. Insertion of DNA between the AP-1 and {kappa}B sites abolishes LPS induction. The spacing between the proximal AP-1 and {kappa}B sites was increased by insertion of 8 or 20 bp (see "Methods"), which are indicated by vertical lines. DNA binding sites for AP-1 (stipled), {kappa}B (stripped), Egr-1 (open), and Sp1 (filled) proteins are indicated by boxes. THP-1 cells transfected with these plasmids (4 µg) were stimulated with LPS (10 µg/ml) for 5 hours. LPS induction of Luc activity (mean±SD) from three independent experiments is shown. Transfection efficiencies exhibited <16% variation between samples.

Characterization of Nuclear Proteins That Bind to the Distal and Proximal AP-1 Sites
The proximal AP-1 site (AP-1P) 5'-TGAGTCA-3' matches the AP-1 consensus 5'-TGAC/GTCA-3',26 whereas the distal AP-1 site (AP-1D) 5'-TGAATCA-3' contains a nonconsensus adenine at position 4. To examine binding of nuclear proteins to the distal and proximal AP-1 sites, EMSAs were performed by incubating the radiolabeled oligonucleotides AP-1D and AP-1P (Table 1Up) with nuclear extracts from THP-1 cells. A single complex was observed when nuclear extracts from unstimulated cells were incubated with either AP-1D or AP-1P (Fig 3ADown), suggesting that these cells constitutively expressed AP-1 binding activity. Larger amounts of protein bound to AP-1P than to AP-1D, indicating that the consensus proximal AP-1 site was a high-affinity site whereas the nonconsensus distal AP-1 site was a low-affinity site. To determine the composition of the protein complexes that bound to AP-1D and AP-1P, supershift experiments were performed using antibodies specific for various members of the AP-1 family, including c-Jun, Jun B, Jun D, c-Fos, and Fos B. Complexes formed with nuclear extracts from unstimulated THP-1 cells and AP-1D or AP-1P were supershifted with c-Jun– and c-Fos–specific antibodies but were not recognized by anti–Jun B, anti–Jun D, anti–Fos B, or anti-p65 antibodies (Fig 3ADown). Similar results were observed with nuclear extracts from LPS-stimulated cells (Fig 3BDown). These results established that both the distal and proximal AP-1 sites bound c-Fos/c-Jun heterodimers in unstimulated and LPS-stimulated THP-1 cells.






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  		Figure 3. Characterization of protein binding to distal and proximal AP-1 sites. Protein composition of complexes of nuclear extracts from unstimulated (A) and LPS-stimulated (B) cells. Antibody supershift experiments were performed with anti–c-Jun, anti–Jun B, anti–Jun D, anti–c-Fos, anti–Fos B, and anti-p65 antisera. These antibodies (5 µL) were incubated with nuclear extracts before addition of radiolabeled AP-1D (lanes 1-7) and AP-1P (lanes 8-14) (Table 1Up). Supershift complexes are indicated. C, EMSAs were performed in nuclear extracts from unstimulated and LPS-stimulated THP-1 cells incubated with the radiolabeled oligonucleotides AP-1D (lanes 1 and 2) and AP-1P (lanes 3 and 4). Stimulated cells were treated for 1 hour with LPS (10 µg/mL). D, LPS induction of AP-1 binding activity requires de novo protein synthesis. Nuclear extracts were prepared from LPS-stimulated THP-1 cells with or without a 15-minute pretreatment with CHX (10 µg/mL; Sigma). AP-1 binding activity was measured by EMSA using the AP-1P probe. Position of the AP-1 complex is indicated.

LPS stimulation increased the amount of AP-1 binding to AP-1D and AP-1P (Fig 3CUp). Our previous studies with an inhibitor of protein synthesis indicated that LPS induction of the TF gene in THP-1 cells is mediated by preexisting transcription factors.7 It has been shown that c-fos and c-jun are constitutively expressed in human monocytes and that gene expression is increased by LPS.22 To determine whether the increase in AP-1 binding activity in LPS-stimulated THP-1 cells was due to de novo protein synthesis of c-Fos and c-Jun or increased binding of preexisting c-Fos/c-Jun heterodimers, we performed experiments in the presence of the protein synthesis inhibitor CHX. Treatment of THP-1 cells with CHX for 15 minutes before LPS stimulation abolished increases in AP-1 binding activity (Fig 3DUp), indicating that de novo protein synthesis was required for the increase in AP-1 binding activity in LPS-stimulated monocytic THP-1 cells.

Functional Assembly of c-Fos/c-Jun and c-Rel/p65 on the LRE Is Dependent on the Spacing Between the Proximal AP-1 and {kappa}B Sites
Our previous studies had indicated that binding of both AP-1 and NF-{kappa}B/Rel proteins to the LRE was required for LPS induction of the human TF promoter in monocytic THP-1 cells.10 Conservation of the 15-bp spacing between the proximal AP-1 site and the {kappa}B site in the human and murine TF promoters53 suggested that this separation might optimize the functional interaction between c-Fos/c-Jun and c-Rel/p65 bound to the LRE. To examine the effect of changing this spacing, we made two plasmids that contained additional DNA sequences between the proximal AP-1 site and the {kappa}B site. We inserted 8 bp to change the relative positions of the AP-1 and {kappa}B sites by 0.8 helical turns and inserted 20 bp to increase the separation of the sites without changing the relative orientation of the sites. Both insertions abolished LPS induction of the TF promoter (Fig 4Up), indicating that the 15-bp separation between these sites was required for the functional activity of the LRE. The distal and proximal AP-1 sites are separated by 6 and 4 bp in the human and murine TF promoters, respectively,53 suggesting that the spacing between these two AP-1 sites is not critical for the functional activity of the LRE. LPS failed to induce pTF(-190)LUC, which does not contain the two AP-1 sites, indicating that c-Rel/p65 heterodimers and Sp1 alone were not sufficient to mediate LPS induction (Fig 4Up).

LPS Induction of Plasmids Containing Intrinsically Bent DNA
Recent studies demonstrated that c-Fos/c-Jun heterodimers induce DNA bending,54 55 suggesting that the two AP-1 sites in the TF promoter may play an additional structural role in the regulation of TF expression. The NF-ELAM1/ATF element in E-selectin can be functionally replaced by intrinsically bent DNA.56 To examine whether DNA bending induced by c-Fos/c-Jun contributes to LPS induction, we replaced the two AP-1 sites with a series of intrinsically bent DNA. Because it is unclear in which plane c-Fos/c-Jun heterodimers bend DNA when bound to the TF promoter, we inserted a series of well defined oligonucleotides,50 listed as curves A through E in Table 2Down. These molecules all carry an intrinsic bend of about 100° but differ by 72° in the plane of the bend.50 56 Curve A was arbitrarily set at 0° for the bend plane. pTF(0°+)LUC was induced threefold by LPS, whereas the other plasmids exhibited no significant LPS induction (Fig 5).Down These data showed that intrinsically bent DNA, oriented in a distinct spatial plane, partially restored LPS induction of the TF promoter lacking the two AP-1 sites. It is very unlikely that LPS induction of pTF(0°+)LUC containing curve A results from binding of additional transcription factors because curves A through E are repetitive and highly similar in sequence (Table 2Down). In addition, no DNA-protein complexes were detected by EMSA using these oligonucleotides (data not shown). These results suggest that DNA bending induced by binding of c-Fos/c-Jun to the two AP-1 sites may provide structural information that is required for optimal assembly of transcription factors on the TF promoter.


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  		Table 2. Oligonucleotides Used for Cloning Intrinsically Bent DNA



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  		Figure 5. LPS induction of plasmids containing intrinsically bent DNA. LPS induction of pTF(-227)LUC (positive control) and pTF(-190)LUC (negative control) was compared with a series of plasmids containing intrinsically bent DNA cloned upstream from the {kappa}B site in pTF(-190)LUC. LPS induction of Luc activity (mean±SD) from three independent experiments is shown. Asterisk indicates that LPS induction of pTF(0°+)LUC was significantly greater than that of of pTF(-190)LUC using a Student t test (P=.01).

Synergistic Transactivation of the TF LRE by c-Fos/c-Jun and c-Rel/p65 in Drosophila SL2 Cells Requires Both AP-1 Sites and the {kappa}B Site
To determine the role of c-Fos, c-Jun, c-Rel, and p65 in the transcriptional activation of the TF promoter, we examined expression of the TF LRE in Drosophila SL2 cells, which lack mammalian AP-1 and NF-{kappa}B/Rel proteins.29 52 A previous study using F9 embryonal carcinoma cells showed synergistic transactivation of either a duplicated {kappa}B enhancer or a pentad of AP-1 sites linked to a minimal promoter by coexpression of p65 with c-Fos or c-Jun.43 We wished to determine whether transactivation of the TF LRE by c-Fos, c-Jun, c-Rel, and p65 required both AP-1 sites and the {kappa}B site. The wild-type TF LRE present in pTFSV-LUC and mutated derivatives of the LRE present in pTFSVMut1-3 were cotransfected with plasmids expressing c-Fos, c-Jun, c-Rel, and p65. The wild-type TF LRE was strongly transactivated by these proteins, whereas mutation of either of the AP-1 sites or the {kappa}B site in the LRE abolished transactivation (Fig 6).Down Similar results were observed with pTF(-227)LUC, which contains the homologous TF promoter, and corresponding mutations of the AP-1 and {kappa}B sites (data not shown). These results demonstrated that transactivation required protein binding to both AP-1 sites and the {kappa}B site of the TF LRE.



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  		Figure 6. Transactivation of wild-type and mutated TF LRE. Plasmids (10 µg) containing the wild-type LRE (pTFSV-LUC) and those containing mutations in the AP-1 or {kappa}B sites (pTFSVMut1-3) were cotransfected with plasmids (2.5 µg each) expressing c-Fos, c-Jun, c-Rel, and p65. The fold induction of Luc activity expressed by these plasmids in the presence of the expression plasmids is shown (mean±SD) from three independent experiments relative to the levels of Luc activity expressed in the absence of the expression plasmids.

To determine the relative contribution of c-Fos, c-Jun, c-Rel, and p65 to the transactivation of the TF LRE, pSVTF-LUC was cotransfected into SL2 cells with various combinations of these expression plasmids. c-Fos alone did not transactivate the TF LRE, whereas c-Jun, c-Rel, and p65 weakly transactivated the LRE (Fig 7Down, lanes 1 through 4). Coexpression of c-Fos with c-Jun, c-Jun with c-Rel, and c-Rel with p65 slightly increased the level of transactivation, whereas coexpression of c-Jun with p65 synergistically transactivated the LRE (Fig 7Down, lanes 5 through 10). Similarly, coexpression of c-Fos, c-Jun, c-Rel, and p65 also synergistically transactivated the TF LRE (Fig 7Down, lane 11).



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  		Figure 7. Transactivation of the TF LRE in Drosophila SL2 cells. c-Fos, c-Jun, c-Rel, or p65 expression plasmids were cotransfected into Drosophila SL2 cells with pTFSV-LUC (10 µg), which contains the TF LRE cloned upstream from the minimal simian virus 40 promoter. Each expression plasmid (2.5 µg) was added together with pUC18 to a total of 10 µg. Plasmids expressing mutant proteins pCMVp65(1-309), which expresses a deleted C-terminal transactivation domain; pCMVp65mLZ, which expresses a mutated mini-Leu zipper and pCMVc-Jun({Delta}1-194), which expresses a deleted transactivation domain, were also examined. The fold induction of Luc activity expressed by pTFSV-LUC with different combinations of these expression plasmids is shown (mean±SD) from three independent experiments relative to the levels of Luc activity expressed by pTFSV-LUC without the expression plasmids.

Regions of c-Jun and p65 that were involved in functional interactions were identified by replacing the wild-type proteins with mutated proteins that did not contain the transactivation domains or that contained a mutation in the mini-leucine zipper of p65, which is required for full transcriptional activity.43 Proteins containing a deletion of the transactivation domain of p65 or a mutation of the mini-leucine zipper of p65 failed to synergistically transactivate the TF LRE in the presence of wild-type c-Fos, c-Jun, and c-Rel (Fig 7,Up lanes 12 and 13). In addition, a c-Jun protein lacking the transactivation domain failed to synergistically transactivate the TF LRE in the presence of wild-type c-Fos, c-Rel, and p65 (Fig 7Up, lane 14). These results indicated that synergistic transactivation of the LRE by c-Fos, c-Jun, c-Rel, and p65 or c-Jun and p65 required the transactivation domains of c-Jun and p65.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
This study investigated functional interactions between AP-1, NF-{kappa}B/Rel, and Sp1 transcription factors assembled on the human TF promoter. Low-level uninduced expression of the TF gene in human monocytic THP-1 cells was mediated by binding of Sp1 to five GC boxes between -170 and -59 bp. Maximal LPS induction of the human TF promoter required two AP-1 sites and the {kappa}B site within the LRE (-224 and -180). Disruption of the spacing between the proximal AP-1 site and the {kappa}B site abolished LPS induction. Replacement of the AP-1 sites with intrinsically bent DNA partially restored LPS induction. In addition, synergistic transactivation of the TF LRE by coexpression of c-Fos, c-Jun, c-Rel, and p65 or c-Jun and p65 required the transactivation domains of c-Jun and p65.

Sp1 and NF-{kappa}B have been shown to function in a cooperative manner to activate transcription of type 1 HIV via a physical association between Sp1 and p65.44 45 In our studies, EMSAs identified binding of Sp1 to five distinct GC boxes in the human TF promoter. Nevertheless, functional interaction between c-Rel/p65 and Sp1 was not sufficient to mediate a high level of LPS induction of pTF(-190)LUC, which contains the {kappa}B site and five Sp1 sites but lacks the two AP-1 sites. Furthermore, our previous studies indicated that c-Fos/c-Jun and c-Rel/p65 mediated LPS induction in the absence of Sp1 sites.10 However, in LPS-stimulated cells, we cannot exclude the possibility that the p65 bound to the {kappa}B site as a c-Rel/p65 heterodimer may physically interact with Sp1 bound to one or more of the downstream GC boxes. In addition, recruitment of coactivators and components of the basal transcription complex by Sp131 may contribute to full transcriptional activation of the TF gene in LPS-stimulated cells.

LPS did not induce Egr-1 expression in the THP-1 subline used in this study. Moreover, pTF(-190)LUC, which contains three Egr-1 sites, was not induced by LPS. In contrast, our previous studies with a different THP-1 subline showed low-level induction of pTF(-111)LUC, which contains the three Egr-1 sites.10 Indeed, we observed LPS induction of Egr-1 in several THP-1 sublines (Fig 2CUp). Recently, we demonstrated that Egr-1 mediates induction of the TF promoter in HeLa cells.49 Taken together, these studies suggest that Egr-1 may mediate low-level induction of the proximal TF promoter in LPS-stimulated monocytic cells.

EMSAs demonstrated that c-Fos/c-Jun heterodimers were present in unstimulated human monocytic THP-1 cells and bound to both the low-affinity distal and the high-affinity proximal AP-1 site. AP-1 binding was increased by LPS stimulation. However, the composition of the c-Fos/c-Jun complex bound to the two AP-1 sites was not changed by LPS stimulation. Similarly, c-Fos/c-Jun heterodimers present in unstimulated human umbilical vein endothelial cells bound to AP-1D and AP-1P, and the composition of the complexes was unaffected by IL-1ß stimulation.12 In contrast to our studies with human cells, AP-1D and AP-1P have been reported to bind c-Jun/ATF and c-Fos/Jun D heterodimers, respectively, using nuclear extracts of bovine aortic endothelial cells.14 In addition, the two nonconsensus AP-1 sites in the porcine TF promoter, which are identical to AP-1D, bind c-Jun/Jun D heterodimers present in porcine aortic endothelial cells.11 Moreover, a recent report indicated that serum induction of the murine TF gene in murine fibroblasts is mediated by c-Fos/Jun D heterodimers and requires de novo synthesis of c-Fos.57 These results suggest that distinct members of the AP-1 family may regulate TF gene expression in endothelial cells from different mammalian species and in different cell types.

In our studies with human monocytic cells, LPS induction of AP-1 binding activity was abolished by inhibition of de novo protein synthesis. We previously demonstrated that the TF gene was regulated by preexisting transcription factors.7 It seems likely, therefore, that pre-existing c-Fos/c-Jun heterodimers rather than newly synthesized c-Fos/c-Jun heterodimers mediate activation of the TF gene. However, in view of the fact that c-Jun is phosphorylated in response to growth factors (with a concurrent increase in transcriptional activity58 ), the c-Fos/c-Jun heterodimers bound to the TF promoter in unstimulated cells may be modified by phosphorylation after exposure to LPS to increase their transcriptional activity.

LPS induction of the TF gene was sensitive to nucleotide spacing between the proximal AP-1 and {kappa}B sites, because insertions of 8 and 20 bp disrupted LPS induction. Conservation of this 15-bp spacing in the human, murine, and porcine promoters (Fig 8Down) may be required for physical association between c-Fos/c-Jun and c-Rel/p65 heterodimers. In contrast, the spacing between the distal and proximal AP-1 sites is not conserved (Fig 8Down). The crystal structure of DNA-bound c-Fos/c-Jun suggests that a flexible fork structure allows the Fos-Jun pair to interact with transcription factors bound at adjacent sites on the DNA.59 Alternatively, the conserved spacing between the AP-1 and {kappa}B sites may be important in allowing interaction of c-Fos/c-Jun and c-Rel/p65 with the basal transcriptional "machinery."



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  		Figure 8. Alignment for DNA sequences (-230 to +9 bp) spanning the human,8 murine,53 and porcine11 TF promoters. Numbering is from the human TF sequence.8 Shaded boxes show conservation of the AP-1D, AP-1P, {kappa}B, and Sp1 sites (I through IV) and the TATA box in the three mammalian species. Bent arrow indicates the start site of transcription.

Binding of c-Fos/c-Jun heterodimers induces DNA bending,54 55 which could facilitate functional interaction between AP-1 and other transcription factors. DNA bending may also permit the transactivation domains of c-Jun, c-Rel, and p65 to interface with the TATA box binding protein and TFIIB within the basal transcriptional machinery. Previous studies have shown that these transcription factors can physically interact with components of the basal transcription complex.35 36 60 61 62 We found that replacing the AP-1 sites with DNA containing a defined bend and oriented in a particular plane partially restored LPS induction of the TF promoter. Full restoration of LPS induction would have suggested that binding of c-Fos/c-Jun to the AP-1 sites played only a structural role, similar to that of the binding of ATF2/c-Jun heterodimers in the E-selectin promoter.56 However, we have shown that binding of c-Fos/c-Jun to their recognition sites is necessary for maximal LPS induction and requires the transactivation domain of c-Jun. Therefore, in addition to a role for AP-1 in protein-protein interactions, DNA bending induced by the binding of c-Fos/c-Jun to the two AP-1 sites appears to play a structural role in the regulation of the TF gene.

Independent functional analysis with Drosophila SL2 cells showed that synergistic transactivation of the LRE by coexpression of c-Fos, c-Jun, c-Rel, and p65 or c-Jun and p65 required the transactivation domains of both c-Jun and p65. Synergistic transactivation of the TF promoter was dependent on the presence of both AP-1 sites and the {kappa}B site. Stein and colleagues43 showed synergistic transactivation of either a duplicated {kappa}B enhancer or a pentad of AP-1 sites by coexpression of p65 with c-Fos or c-Jun, suggesting that multimerization of either {kappa}B or AP-1 sites permitted functional interaction of the transcription factors in the absence of both types of binding site. Our results indicated that functional interaction between c-Fos/c-Jun and c-Rel/p65 required the binding of these transcription factors to each of their respective DNA sites for transcriptional activation of the TF gene.

The DNA sequence of the TF promoter in the region adjacent to the TATA box is highly conserved in the human, murine, and porcine TF genes (Fig 8Up). Four of the five Sp1 sites (II, III, IV, and V) show a high degree of conservation in sequence and spacing, consistent with a functional role for Sp1 in the regulation of TF gene expression. DNase I footprinting studies have shown protection of Sp1V in nuclear extracts from human monocytic cells15 and protection of sites in the porcine TF promoter equivalent to Sp1V, Sp1IV, and Sp1III in nuclear extracts from porcine endothelial cells.11 Furthermore, Sp1 from porcine endothelial cells bound to an oligonucleotide spanning two putative Sp1 sites in the porcine TF promoter,11 one of which is equivalent to Sp1V. The LRE also shows strong conversation of both AP-1 sites and the {kappa}B site. DNase I footprinting studies of nuclear extracts from human monocytic cells and porcine endothelial cells have shown protection of the AP-1 sites and the {kappa}B site in the human and porcine TF promoters, respectively.11 15 The C-to-T substitution at position 1 of {kappa}B in the porcine TF promoter does not alter the ability of c-Rel/p65 heterodimers to selectively bind to this nonconsensus {kappa}B site.51

Maximal induction of the human TF promoter in monocytic and endothelial cells requires both AP-1 sites and the {kappa}B site,9 11 12 13 suggesting that the mechanism of transcriptional activation of the TF gene is conserved in both cell types. Figure 9Down shows a model for uninduced and induced expression of the TF gene in monocytes and endothelial cells. In unstimulated cells, c-Fos/c-Jun heterodimers and Sp1 are bound to their respective sites and may bend the TF promoter, facilitating the formation of a higher-order complex necessary for transcriptional activation. LPS stimulation of monocytes and LPS or cytokine stimulation of endothelial cells induces nuclear translocation of c-Rel/p65 heterodimers, which bind to the {kappa}B site within the LRE. Functional interactions between c-Fos/c-Jun and c-Rel/p65 heterodimers in the presence of Sp1 and the basal complex mediate the transcriptional activation of the TF gene.



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  		Figure 9. Model for basal and LPS-induced expression of the human TF gene in monocytic cells. DNA binding sites for AP-1 (stipled), {kappa}B (stripped), and Sp1 (filled) are shown. In unstimulated monocytic cells, c-Fos/c-Jun binds to the AP-1 sites and is proposed to bend the DNA but does not contribute to uninduced expression, whereas Sp1 binding and interaction with proteins within the basal complex mediates uninduced expression. In LPS stimulated monocytic cells and endothelial cells, occupation of the {kappa}B site by c-Rel/p65 and functional interaction with c-Fos/c-Jun activates transcription of the TF gene.


*    Selected Abbreviations and Acronyms
 
AP = activator protein
CHX = cycloheximide
EMSA(s) = electrophoretic mobility shift assay(s)
IL = interleukin
LPS = lipopolysaccharide
LRE = LPS response element
LUC/Luc = luciferase
NF = nuclear factor
PMA = phorbol 12-myristate 13-acetate
TF = tissue factor


*    Acknowledgments
 
This work was supported by National Institutes of Health grant HL-48872 (to N.M.). This work was performed during the tenure of an Established Investigatorship from the American Heart Association (to N.M.). This is manuscript No. 9161-IMM from The Scripps Research Institute. We acknowledge H. McClary, M. Smith, and Y. Ko for technical assistance; and Jenny Robertson and Barbara Parker for preparing the manuscript.

Received January 24, 1996; revision received April 12, 1996;
*    References
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 *References
 

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