©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of the Tissue Factor Promoter in Endothelial Cells
BINDING OF NFkappaB-, AP-1-, AND Sp1-LIKE TRANSCRIPTION FACTORS (*)

(Received for publication, May 12, 1994; and in revised form, November 14, 1994)

Thomas Moll (1) Malgorzata Czyz (1) Harry Holzmüller (1) Renate Hofer-Warbinek (1) Ernst Wagner (3) Hans Winkler (2) Fritz H. Bach (1) (2) Erhard Hofer (1)(§)

From the  (1)Department of Transplantation Immunology, Vienna International Research Cooperation Center, Brunnerstrasse 59, A-1230 Vienna, Austria, the (2)Sandoz Center for Immunobiology, New England Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02215, and (3)Bender and Company GesmbH, Dr. Boehringergasse 5-11, A-1121 Vienna, Austria

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tissue factor is up-regulated on endothelial cells and monocytes in response to cytokines and endotoxin and is the main trigger of the extrinsic pathway of the coagulation cascade. We have isolated the porcine tissue factor gene and studied the regulation of the promoter, which has not been investigated previously in endothelial cells. Comparison of the promoter sequences with the respective human and murine genes reveals short stretches of homology, which encompass potential binding sites for AP-1, NFkappaB, and Sp1 transcription factors. Using DNase I footprinting, we detect binding of nuclear factors to these promoter elements. Transfection experiments demonstrate that a 300-base pair fragment containing the conserved elements can mediate induced transcription and that the NFkappaB-like element is essential. In accordance, electrophoretic mobility shift assays show a strong increase in the binding of factors to the NFkappaB-like site following induction. We further provide evidence that RelA (p65), c-Rel, and possibly novel polypeptides bind to the tissue factor NFkappaB element. In addition, we show constitutive binding of members of the Fos/Jun and Sp1 families to the AP-1 and Sp1 sites, respectively. We propose a concerted action of AP-1-, NFkappaB-, and Sp1-like factors in transcription from the tissue factor promoter in endothelial cells.


INTRODUCTION

The transmembrane glycoprotein tissue factor (TF) (^1)serves as the high affinity receptor and essential cofactor for the plasma serine protease VII/VIIa and plays a central role in the activation of the extrinsic pathway of the coagulation cascade (for review, see (1) and (2) ). The relatively high constitutive levels of TF in certain extravascular cells are thought to represent a hemostatic barrier preventing excessive bleeding following tissue injury. Endothelial cells (EC) normally function to maintain an anti-coagulant environment and inhibit the formation of thrombi(3, 4, 5) . Upon activation, EC change the balance of anti- and procoagulation toward blood clotting through the up-regulation of TF (1, 6, 7, 8) . In many situations of inflammation, septic shock, various thromboembolic disorders, and several forms of disseminated intravascular coagulation, aberrant TF expression can result in life-threatening diseases(2, 9) . In the special situation of vascular rejection of xenotransplants, EC activation and consequent TF expression has also been implicated as one of the key mechanisms leading to blood clotting and organ death(10, 11) .

It was previously shown that TF is up-regulated in response to endotoxin and inflammatory cytokines in EC and macrophages. Control of expression is largely at the transcriptional and mRNA level. Exposure of EC or monocytes to LPS or TNF-alpha transiently induces TF gene transcription(2, 12, 13, 14) ; in addition, TF mRNA may be stabilized in response to endotoxin(15, 16) . The determinants in the 5`-flanking region of the human gene that contribute to LPS-mediated activation in a monocytic leukemia cell line have previously been analyzed. A 56-bp region between -227 and -172 of the promoter, which contains AP-1 and NFkappaB sites, is necessary for transcriptional up-regulation(17, 18) . Recently, Oeth et al.(19) have described the binding of c-Rel/p65 heterodimers to the TF NFkappaB site and provided evidence that this site confers LPS inducibility to a heterologous promoter in reporter gene assays using monocytic cells. A central role for NFkappaB in the up-regulation of genes in endothelial cells during inflammation has been demonstrated by several laboratories(20, 21, 22, 23) , including ours (24, 25) . In view of this, we have now investigated a potential involvement of the TF promoter region containing the NFkappaB and the two neighboring AP-1 sites in the regulation of endothelial TF expression.

We report here for the first time the importance of this region also for EC-specific TF up-regulation. Using DNase I footprinting, we detect binding of nuclear factors to the AP-1 and NFkappaB as well as the Sp1-like promoter elements. Reporter gene assays were used to show the functional importance of this region and to demonstrate that the NFkappaB-like site is essential for TF gene transcription in EC. By electrophoretic mobility shift assays with extracts from unstimulated and induced EC, we demonstrate inducible NFkappaB and constitutive AP-1 and Sp1 binding. In a biochemical analysis of the factors binding to the TF promoter, we show binding of Rel-, Fos/Jun-, and Sp1-family members to the respective sites.


MATERIALS AND METHODS

Culture of Porcine Aortic Endothelial Cells and Assay for Procoagulant Activity

Endothelial cells were isolated from pig aorta and cultured as described(26) . Cells were used for experiments up to passage number 12. Recombinant human IL-1alpha and TNF-alpha used for endothelial cell activation were purchased from Genzyme Inc. Cellular procoagulant activity was tested in an amidolytic assay as described previously(27) . In short, the cells were incubated with a chromogenic thrombin substrate (tosyl-Gly-Pro-Arg-p-nitroanilide acetate) and fibrinogen depleted plasma as a source of factors VII, X, V, and prothrombin. Absorbance at 405 nm was recorded in parallel to a standard curve with rabbit brain thromboplastin obtained from Sigma.

Isolation and Sequencing of the Porcine TF Promoter

A cDNA library (24) in ZAPII (Stratagene) was first screened with a human TF cDNA probe(28) . The isolated 1800-bp partial porcine TF cDNA^2 was then used to screen a porcine genomic library in EMBL3 obtained from Clontech according to published procedures(29) . From the clone two isolated XhoI fragments of about 5.5 and 3.6 kilobase pairs were subcloned into pSK (Stratagene) by standard techniques(29) . The two subclones were shown by sequencing to comprise approximately 4 kilobase pairs of 5`-flanking region and the 5`-part of the gene including the first two exons and a large part of the second intron.

Taq cycle sequencing was performed on an Applied Biosystem 373A DNA Sequencer according to the manuals provided by the manufacturer. The complete sequence of the promoter up to -1100 bp and of the transcribed region down to the beginning of the second intron was established on both strands. Sequence alignments were performed with the Pileup program of the Genetics Computer Group Sequence Analysis Software Package at default values(30) .

DNase I Footprinting

DNase I footprinting analysis was performed on an end-labeled XhoI-DraI fragment from the porcine TF promoter (position -329 to -27) as described(31, 32) . Approximately 50 pmol of the probe were preincubated with nuclear extract corresponding to 50 µg of protein in a total volume of 200 µl in reaction buffer (10 mM Hepes-KOH, pH 7.9, 50 mM KCl, 0.1 mM EDTA, 5 mM MgCl(2), 2 mM CaCl(2), 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10% glycerol, 0.01% Nonidet P-40, 2 µg/ml poly(dIbulletdC) for 10 min at room temperature. Then, DNase I was added (Boehringer Mannheim, 2 units), and the reaction stopped after 30 s. The DNA was then precipitated and analyzed on a 6% sequencing gel(29) .

Transfection of Primary Aortic Endothelial Cells

24 h prior to transfection, PAEC were seeded at near confluency in six-well tissue culture plates. Transfection of the cells was carried out using psoralen- and UV-inactivated biotinylated adenovirus and streptavidine-poly-L-lysine as vectors for DNA delivery to the cells essentially as described ( (33, 34, 35) and references therein). Briefly, confluent EC were incubated for 2 h with adenovirus-poly-L-lysine complexes and 3 µg of plasmid DNAs (including a cytomegalovirus-promoter-beta-Gal construct as internal control) in 1 ml of Dulbecco's modified Eagle's medium, 10% fetal calf serum. After 2 h, the medium was replaced, and the cells incubated for another 48 h prior to LPS induction (1 µg/ml for 8 h) and harvesting. We have omitted transferrin-poly-L-lysine conjugates during the transfection procedure since at least for primary porcine EC the addition of transferrin-poly-L-lysine did not enhance transfection efficiences. These findings suggest that EC may possess sufficient adenovirus receptors to mediate efficient internalization of the conjugates. (^3)

Reporter Gene Constructs and Assays

Fragments of the porcine TF promoter (-330 to +34 and -330 to +118) were synthesized by polymerase chain reaction according to standard procedures and cloned into a luciferase expression vector (pUBT-luc, Ref.36) with a destroyed XhoI site. The XhoI restriction enzyme fragment encompassing the sequences from about -4000 to -330 was then inserted into the XhoI site at the 5`-end of the -330/+34 construct to give the -4000/+34 construct. The NFkappaB and AP-1 deletion fragments were obtained from polymerase chain reaction fragments of the -330/+118 basal TF promoter, resulting in the substitution of the sequences from position -161 to -137 or from -209 to -156 with an XbaI site sequence, respectively. All constructs were partially sequenced to show the fidelity of the polymerase chain reaction and subcloning procedures. Luciferase assays were performed with cellular lysates of transfected cells as described (37) . Between 10^3 and 10^4 luciferase units were obtained with the basal TF promoter constructs using about 10^5 cells/well for the transfections.

Nuclear Extract Preparation and Electrophoretic Mobility Shift Assays

Nuclear extracts from confluent endothelial cell cultures were prepared essentially as described(38) , except that a modified buffer C was used during nuclear extraction (20 mM Hepes-KOH, pH 7.9, 420 mM NaCl, 400 mM (NH(4))(2)SO(4), 1.5 mM MgCl(2), 0.2 mM EDTA, 0.5 mM dithiothreitol, 25% glycerol). Protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 25 µMN-p-tosyl-L-lysine chloromethyl ketone, 50 µMN-tosyl-L-phenylalanine chloromethyl ketone) were added at all steps during extract preparation. For the electrophoretic mobility shift assays, 2.5-5 µg of nuclear protein was incubated with 0.3-1.0 ng of radioactively labeled oligonucleotide (10^5 cpm/ng) in binding buffer (20 mM Hepes-KOH, pH 7.9, 1 mM EDTA, 5 mM MgCl(2), 50 mM KCl, 1 mM dithiothreitol, 10% glycerol) for 20-30 min at room temperature. Where indicated, 2 µg of polyclonal rabbit anti-peptide antibodies, all obtained from Santa Cruz Biotechnology, or 2 µl of polyclonal Fos and Jun antisera were added to the binding reactions for 30 min at 4 °C prior to the addition of radioactive probe. Protein-DNA complexes were resolved on 5% polyacrylamide gel electrophoresis in 0.5 times Tris borate/EDTA(29) .

The double-stranded synthetic oligonucleotides were radioactively labeled by filling in the overhangs with Klenow enzyme in the presence of [alpha-P]dATP and subsequently purified over a 7% polyacrylamide gel. The sequences of the probes used were as follows: TFkappaB, 5`-AATTCTTGGAGTTTCCTACGGG-3`; TFmkappaB, 5`-AATTCTTGCAGTTTAGTACGGG-3`; hIgkappaB, 5`-AATTCAGAGGGGGATTTCCCAGAGG-3`; ECI6/BS2, 5`-AATTCGGCTTGGAAATTCCCCGAGCG-3`; Coll-AP-1, 5`-GCATAAAGCATGAGTCAGACACCTC-3`; TFAP-1, 5`-AATTGGGTTGAATCACGGGTGAATCAGCCCTTGCAGG-3`; mut1, 5`-AATTGGGTTCTAGAACGGGTGAATCAGCCCTTG-3`; mut2, 5`-AATTGGGTTGAATCACGGGTCTAGAAGCCCTTG-3`; mut1/2, 5`-AATTGGGTTCTAGAACGGGTCTAGAAGCCCTTGC-3`; TFSp1, 5`-AATTCGGGGGCGGGACCAGGGCGGGGCCTCG-3`; cons. Sp1, 5`-AATTCGGGGCGGGGCGATCGGGGCGGGGCG-3`; nonspecific oligo, 5`- AATTCCGAATTCTTTG-3`.

In Vitro Transcription-Translation

In vitro transcription-translation reactions were carried out using a wheat germ-coupled transcription-translation system (TNT, Promega) according to the manufacturer's instructions. Reactions including [S]methionine were carried out in parallel, and the correct size of the translated products was confirmed by SDS-polyacrylamide gel electrophoresis (data not shown). T7 expression vectors for p65, p50, and c-Rel were obtained from Drs. P. Bäuerle and W. Greene, respectively. A T3 expression vector containing murine p52 was a kind gift of Drs. B. Sha and D. Baltimore.

UV Cross-linking

UV cross-linking experiments were performed as described previously(39) . Briefly, the oligonucleotides were body-labeled on the top strand with [alpha-P]dATP using Klenow enzyme and short oligonucleotides as primers (TFkappaB, 5`-CCCTTGCGATATCG-3`; ECI-6/BS2, 5`-TAGCGAATTC-3`). The reaction was carried out in the presence of bromodeoxyuridine triphosphate. In the case of the TFkappaB probe the double-stranded oligonucleotide was then digested with EcoRV, resulting in an oligonucleotide essentially identical to the TFkappaB oligo used for electrophoretic mobility shift assays but with a completely bromodeoxyuridine-substituted top strand. 10 µg of nuclear extract was then incubated with 1,5-2 times 10^6 cpm of gel-purified radioactive probe. After native gel electrophoresis, the protein-DNA complexes were covalently cross-linked by UV irradiation (302 nm, 12 min) in the gel. The bands containing the complexes of interest were excised, and the gel slice was boiled for 5 min in approximately one volume of 2 times SDS sample buffer. Subsequently both (gel slice and sample buffer) were loaded onto a 7.5% polyacrylamide gel and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography.


RESULTS

Induction of Procoagulant Activity on Endothelial Cells

To characterize the induction of procoagulant activity on primary PAEC, cultures were treated with endotoxin and cytokines. Procoagulant activity was strongly induced, starting 2-4 h after addition of LPS (Fig. 1A). A similar strong induction was observed with IL-1alpha or TNF-alpha after 6 h (Fig. 1B). The cellular procoagulant activity is thought to be due to the triggering of the extrinsic coagulation pathway by TF expressed on the surface of the cells(27) . Our own data using factor VII-deficient plasma and neutralizing anti-factor VII antibodies support this conclusion(11) . In accordance with these data, we find that TF mRNA is induced following induction of PAEC (data not shown), which is in agreement with the findings reported by others on the induction of TF mRNA in response to cytokines as well as endotoxin(1, 2, 12, 13, 14, 40) .


Figure 1: Induction of cellular procoagulant activity. A, PAEC incubated with LPS for 1-10 h were analyzed in an amidolytic thrombin assay. Cells were treated with 10 ng/ml (circle), 100 ng/ml (box), or 1000 ng/ml (up triangle) of LPS. Background values obtained with untreated samples were subtracted. B, cells were treated with increasing amounts of LPS, IL-1alpha, or TNF-alpha for 6 h, and then the assay was performed. The average value obtained with untreated samples is shown with an openbar (U). LPS was used in concentrations of 1, 10, 100, and 1000 ng/ml, IL-1alpha in 10, 100, 1000, and 10,000 units/ml; TNF-alpha was used in concentrations of 1, 10, 100, and 1000 units/ml, and the results are shown for increasing concentrations from left to right. The mean of four independent samples and the standard deviations are given.



Sequence of the Porcine TF Promoter

A porcine TF cDNA was isolated and used to select genomic clones from a porcine genomic library in EMBL3. The region from 1100 bp upstream to 1362 bp downstream of the transcription initiation site was sequenced (deposited at EMBL nucleotide data base, accession Z46238). A sequence alignment of the region from -366 to +36 of the putative transcription start site with the corresponding regions of the human and murine genes (17, 41) identified a stretch of largely conserved sequence motifs that extends up to position -203 in Fig. 2. A deletion of 33 nucleotides in the porcine promoter corresponding to position -68 to -36 of the human homologue suggests that this part of the immediate upstream sequence may not be essential for the function of the TF promoter. The region encompassing conserved motifs contains an almost completely conserved EGR1 as well as four Sp1 sites. The following NFkappaB site and the unique dimeric AP-1 site differ by only one nucleotide between the different species. In the flanking regions of the consensus binding sites, additional conserved nucleotides are present, which might contribute to the specificity of the binding of nuclear factors. It is noteworthy that in all three species the NFkappaB and AP-1-like binding sites do not completely correspond to the respective consensus sequences but differ in one position. In the case of the NFkappaB-like site, there is a T (or C) instead of a G on the first position of the decamer; in the case of the AP-1 like elements, there is an A instead of a G or C in the middle position of the distal heptamer(17, 41) . This base is also modified in the proximal AP-1 site of the porcine gene.


Figure 2: Sequence of the porcine TF promoter and alignment with the respective human and murine promoter sequences. The sequence of the the porcine TF promoter (Por) from -366 to +122 including the 5`-untranslated region of the gene and a sequence alignment with the corresponding regions of the human (Hum) and murine (Mur) TF gene is shown. Identical nucleotides in the sequences of all three species are shown in capital letters and as a consensus sequence (Con). Residues not conserved in all three species(-) and gaps (bullet) in the individual sequences introduced for optimal alignment to the sequences of the other species are indicated. The two AP-1-like sites, the NFkappaB-like site, 4 Sp1 elements, the EGR-1 site, and the TATA box are underlined; the presumed transcription initiation site and the translation initiation codon are shown in boldface. The bases are numbered according to their relative position to the presumed transcription initiation site on the porcine TF promoter.



DNase I Footprinting Analysis of the TF Promoter

To determine potentially important sites of protein-DNA interactions in the most conserved part of the TF promoter, a restriction fragment encompassing the sequence from -329 to the TATA box was radioactively labeled on the coding strand and used as a probe for DNase I footprinting. Nuclear extracts from uninduced and LPS-treated endothelial cells were incubated with this fragment to reveal the sites potentially involved in induced TF gene transcription. The resulting protection pattern, shown in Fig. 3, identifies the footprint regions 1 (-320 to -300), 2 (-186 to -178), 3 (-174 to -167), 4 (-154 to -142), 5 (-136 to -115), 6 (-110 to -91), and 7 (-79 to -65).


Figure 3: DNaseI footprint analysis of the porcine TF promoter. The XhoI-DraI fragment (position -329 to -27) from the TF promoter was end-labeled on the coding strand and incubated with nuclear extracts from either unstimulated (U) or LPS-treated (I) cells. For unstimulated cells, footprints obtained from two independent preparations of nuclear extracts are shown. Protected regions are outlined by bars, and the position on the TF promoter relative to the transcriptional start site is given on the left. G, Maxam/Gilbert G reaction sequencing ladder; -, no extract added.



Footprint 1 maps to a region of the promoter that contains a potential Sp1 binding site. Regions 2 and 3 contain the distal and proximal AP-1 sites, implying that the TF AP-1 sites are bound by nuclear factors in endothelial cells. A similar situation is indicated for the TF NFkappaB element (region 4), which also shows DNase I protection. The four potential Sp1 sites downstream of the TFkappaB element are preferentially occupied in nuclear extracts from induced cells (footprints 5-7). These results clearly demonstrate that all of the conserved promoter elements located in the footprint region are occupied at a time point when the TF gene is actively transcribed, suggesting that these sites contribute in one or the other way to transcription.

The TF NFkappaB-like Site Is Essential for Induced Transcription

To investigate the functional competence of the TF promoter to mediate induced gene transcription, we have transfected luciferase reporter genes containing various parts of the porcine promoter into PAEC. Primary aortic EC are notoriously difficult to transfect, and we consistently found that conventional transient transfection procedures tested on porcine EC yielded a transfection efficiency at least 2-3 orders of magnitude below fibroblast cell lines and thus did not allow reasonable transcription from the very weak TF promoter.^3 To circumvent this problem, we have adapted a method using adenovirus-polylysine conjugates for cellular DNA delivery(25, 33, 34) . This procedure is highly efficient, and depending on the cell type used up to 100% of the cells can be transiently transfected(34) . In accordance, using porcine primary EC, we have reproducibly obtained transfection rates of about 10% as determined with a beta-galactosidase expression vector (data not shown).

Initial transfection studies were carried out with luciferase reporter constructs containing the TF promoter from -330 to +34, approximately -4000 to +34 and -330 to +118. The average results obtained from at least two independent experiments with two independent isolates of PAEC each are shown in Fig. 4. Inducibility varied somewhat between individual experiments and PAEC isolates, but LPS stimulation of luciferase expression was reproducibly between 1.3-3-fold for all three constructs. This relative induction rate may not completely reflect the extent of up-regulation of the TF gene in quiescent EC since the relatively high basal levels of expression are consistent with the EC being already partially activated following the transfection procedure. Similar effects were observed for other endotoxin-induced promoters, and partial activation is indicated by increased levels of TF activity on transfected cells (data not shown). However, we assume that our transfection data faithfully reflect induced transcription levels, suggesting that the major regulatory elements mediating induced transcription rates in response to LPS in EC are contained within -330 and +34 of the TF promoter. Sequences upstream -330 and between +34 and +118 appeared to increase efficiency of transcription, but relative inducibility did not change when these regions were present.


Figure 4: Functional analysis of the porcine TF promoter in EC by transient transfection. Fragments of the porcine TF promoter were fused to a luciferase gene as indicated and transfected into primary aortic endothelial cells using adenovirus-polylysine conjugates for DNA delivery. The cells were then cultured for 48 h prior to the addition of 1 µg/ml LPS, and noninduced(-) and LPS-treated (+) cells were harvested after 8 h. Luciferase activity as determined in cell lysates from transfected cells was normalized using expression from a cotransfected cytomegalovirus-promoter-beta-Gal expression vector as internal control. Basal luciferase activity of pTF(-330/+118) was arbitrarily set to 100% to emphasize the complete loss of transcriptional activity from the TFkappaB mutant promoter (pTF(-330/+118)mutkappaB).



Given the role of the NFkappaB- and AP-1-like elements in TF expression in a monocytic cell line(18, 19) , we have tested the contributions of these elements for LPS-inducible expression in EC. To this end, the respective sites in the -330 to +118 luciferase promoter construct were substituted with an XbaI linker sequence. Strikingly, deletion of the NFkappaB element results in a complete abrogation of transcription from the TF promoter, comparable with a promoterless control construct (Fig. 4). Deletion of the two AP-1 elements has a more moderate effect, which is somewhat variable for different cell isolates. Nevertheless, LPS-inducibility of transcription appears to be reduced.

Complex Formation on the TF NFkappaB Site Is Inducible and Involves p65 (RelA), c-Rel, and Possibly Novel Polypeptides

Given both the importance of the TF NFkappaB site in driving transcription and its unusual structure, we have focused on the nuclear factors binding to this motif. Using electrophoretic mobility shift assays, there is only little, if any, binding of proteins in nuclear extracts from unstimulated PAEC (Fig. 5A, lane1). In contrast, nuclear extracts prepared from cultures stimulated for 2 h with either LPS, TNF-alpha, or IL-1alpha (Fig. 5A, lanes2 and 6, data not shown for IL-1alpha) contain a strongly induced TF NFkappaB binding activity. It is noteworthy that upon incubation of endothelial proteins with the TF NFkappaB oligonucleotide, two distinct major protein-DNA complexes are formed. The specificity of these protein-DNA interactions was confirmed by complete competition of the binding with unlabeled oligonucleotides and the lack of competition with a mutated form thereof.


Figure 5: Binding of nuclear proteins to the TF NFkappaB site. A, binding on the TF NFkappaB site is inducible. An oligonucleotide containing the NFkappaB site of the porcine TF promoter was used for electrophoretic mobility shift assays with extracts from untreated PAEC (lane1) or cells induced for 2 h with 100 ng/ml LPS (lanes2-5) or 100 units/ml TNF-alpha (lanes6-9). For competition studies, a 50-fold excess of the TFkappaB oligonucleotide (TFkappaB, lanes3 and 7), a mutated form thereof (mutkappaB, lanes5 and 9) or a kappaB oligonucleotide derived from the human immunoglobulin kappa light chain enhancer (hIgkappaB, lanes4 and 8) were added to the binding reactions. f.p., free probe. The triangles indicate the position of the two major protein-DNA complexes formed. B, the complexes binding to the TF NFkappaB element contain p65 and c-Rel. Electrophoretic mobility shift assays with the TF NFkappaB site were performed with nuclear extracts isolated from untreated PAEC (lane1) or cells treated with LPS (lanes2-7) for 2 h. The respective rabbit polyclonal anti-human (p50, p52, p65) and anti-mouse (c-Rel, RelB) peptide antibodies were added to the binding reactions at a concentration of 0.2 µg/ml as indicated (lanes3-7). Identical results were obtained with TNF-alpha-treated cells.



Polyclonal rabbit peptide antibodies against known subunits of NFkappaB (p52, p50, p65(RelA), c-Rel, and RelB; for review, see (42) ) were used to probe the nuclear TF NFkappaB binding complexes for the presence of the corresponding proteins (Fig. 5B). The addition of anti-p65 antibodies leads to the formation of slower migrating complexes and the intensity of both TFkappaB binding complexes is strongly reduced. Interestingly, c-Rel antibodies interact specifically with the upper complex. We conclude that p65 is contained within both complexes, whereas c-Rel is a constituent only of the upper complex. In contrast, there was no significant reactivity of p50 or p52 antibodies with the TF NFkappaB complexes, whereas the p50 antibodies reacted well with the proteins binding to the BS2-NFkappaB site of the porcine IkappaB promoter(25) . RelB antibodies did not show any reactivity with the TF NFkappaB complexes.

These findings are in agreement with earlier observations (43, 44) that substitution of the first G in the NFkappaB consensus recognition site to a C or T, as in the TF NFkappaB site, will prevent p50 from binding to this element, although p65 and c-Rel still interact. When analyzing the binding of in vitro translated NFkappaB subunits, p50, p52, p65, as well as c-Rel are able to interact with the BS2-NFkappaB element (Fig. 6, lanes18-21). In contrast, only p65 and c-Rel bind to the wild-type TF NFkappaB site (lanes4 and 6). No binding of either p50 or p52 could be detected to this element (lanes2 and 3), even in the presence of p65 or c-Rel (lanes8-11). Interestingly, substitution of the T in position one of the TF NFkappaB element to a G creates a kappaB site that interacts with p50, although still no binding of p52 (lanes13 and 14) is detected.


Figure 6: DNA-binding of in vitro translated members of the NFkappaB family. p50, p52, p65, and c-Rel were produced by in vitro translation in a wheat germ extract, and the resulting products were assayed in electrophoretic mobility shift assays for binding to either the TF NFkappaB element (TFkappaB, left panel, lanes 1-11), to a modified TF NFkappaB site (TFkappaB-G, middle panel, lanes 12-16) or to the BS2-NFkappaB site from the porcine ECI-6 promoter (ECI-6/BS2, right panel, lanes 17-21) as indicated. Antibodies against p65 (lane5) or against c-Rel (lane7) were added as described in Fig. 5. WGE, control wheat germ extract; f.p., free probe.



To further investigate the subunit composition of the nuclear TF NFkappaB binding complexes, photoreactive bromodeoxyuridine-substituted TF NFkappaB and BS2-NFkappaB oligonucleotides were used for protein-DNA complex formation and covalent cross-linking (Fig. 7). In agreement with our earlier observations (25) two proteins of approximately 75 and 55-60 kDa, presumably p65 (RelA) and p50(45) , respectively, were found to interact with BS2-NFkappaB. Both proteins appear to migrate as doublets, which may mean either that the subunits are differentially modified or that the smaller forms represent partially degraded proteins. On the TF NFkappaB site, a doublet at approximately 75 kDa is seen, which comigrates with p65. In addition, there is a weak but reproducible band at 84 kDa, which is the expected molecular mass of cross-linked c-Rel(45) . Furthermore, two additional polypeptides of approximately 63 and 55 kDa are found associated with the TFkappaB oligo. Whereas no 63 kDa band could be detected with the BS2-NFkappaB site, the smaller 55 kDa band migrates similar to p50. Based on the binding data from the in vitro translated proteins (Fig. 6) and the antibody experiments (Fig. 5B), we nevertheless consider it unlikely that this cross-linked protein actually represents p50.


Figure 7: UV cross-linking of nuclear proteins to the ECI-6/BS2 and the TF NFkappaB elements. Photoreactive bromodeoxyuridine-substituted ECI-6/BS2 and TFkappaB oligonucleotides were synthesized by primer extension with Klenow DNA polymerase in the presence of [alphaP]dATP. The obtained oligonucleotides were used for a preparative electrophoretic mobility shift assay. Following separation of the NFkappaB/DNA complex on a native polyacrylamide gel the DNA was cross-linked to the proteins by UV irradiation, and the retarded material was loaded onto a 7.5% SDS-polyacrylamide gel electrophoresis gel. The relative molecular masses of the cross-linking adducts formed on either the TFkappaB (middle) or on the BS2-NFkappaB (right) elements are given. The sizes of the radioactive molecular mass marker proteins in kDa are given to the left.



Binding to the Dimeric TF AP-1 Site Is Constitutive

The structure of the TF promoter is somewhat remarkable in that two potential AP-1 sites are found in close proximity not only to each other but also close to the TF NFkappaB binding site (Fig. 2). Since both AP-1 sites are protected in the DNase I footprints, we have investigated the protein-DNA interactions with the AP-1 like elements, using an oligonucleotide spanning both sites. Strong binding to AP-1 with nuclear extracts from untreated as well as LPS- or TNF-alpha-induced cultures is observed (Fig. 8A, lanes1, 5, and 9). Similar results were also obtained with IL-1alpha (data not shown). Complex formation is specific since it can be competed with a 100-fold excess of nonradioactively labeled specific probe (Fig. 8A, lanes2, 6, and 10) but not with an unrelated oligonucleotide (lanes4, 8, and 12). Furthermore protein-DNA interactions appear to involve the AP-1 elements, as the binding is competed with an AP-1 site containing, but otherwise divergent, oligonucleotide from the human collagenase promoter (46, 47) (lanes3, 7, and 11).




Figure 8: Binding of nuclear proteins to the TF AP-1 elements. A, binding to the TF AP-1 element is constitutive. An oligonucleotide containing both of the AP-1 sites of the porcine TF promoter was used for electrophoretic mobility shift assays with nuclear extracts from untreated PAEC (nonind., lanes1-4) and cells induced with 100 ng/ml LPS (lanes5-8) or 100 units/ml of TNF-alpha (lanes9-12) for 2 h. Competition was performed with a 100-fold excess of the TF AP-1 oligonucleotide (TF AP-1, lanes 2, 6, and 10), an oligonucleotide containing a consensus AP-1 motif derived from the human collagenase promoter (CollAP-1, lanes3, 7, and 11), and with a control oligonucleotide (nonspec. comp., lanes4, 8, and 12). f.p., free probe. B, contribution of individual AP-1 sites. Electrophoretic mobility shift assays were performed with nuclear extracts from LPS-treated EC and with either the dimeric TF AP-1 element (WT, lanes1, 7-14) or with oligonucleotides where either the 5` (mut1, lanes2 and 3), the 3` (mut2, lanes4 and 5), or both AP-1 sites (mut1/2, lane 6) had been mutated. Competition studies were carried out with an excess of non radioactively labeled oligonucleotides as indicated (lanes3, 5, 7-14). Identical results were obtained with nuclear extracts from unstimulated endothelial cells (data not shown). C, the TF AP-1 binding complex contains both Jun and Fos family members. Electrophoretic mobility shift assays were performed with the TF AP-1 element and nuclear extracts from LPS-treated EC both in the absence (lane1) or presence of 0.2 µg/ml peptide rabbit antibodies to mouse c-Jun, JunB, JunD, human c-Fos, mouse FosB, human Fra1, and chicken Fra2 as indicated (lanes2-8). In addition antibody binding studies were carried out with two rabbit antisera broadly reactive with Jun (lane9) and Fos (lane10) antigens, respectively. Identical results were obtained with nuclear extracts from unstimulated endothelial cells (data not shown).



In order to determine whether binding to the two neighboring AP-1 sites was cooperative or would occur independently of each other, oligonucleotides were used in which either the distal (mut1), the proximal (mut2), or both (mut1/2) sites were individually substituted with an XbaI linker sequence. As is shown in Fig. 8B, substitution of either the distal (lane2) or the proximal (lane4) AP-1 site has little effect on complex formation, although binding is somewhat reduced. In contrast, substitution of both elements leads to a complete loss of binding (lane6). In addition, both single mutants efficiently compete for complex formation on the dimeric wild-type AP-1 element (lanes9-12). We conclude that binding to the individual AP-1 sites is not cooperative. There was no qualitative difference in the protein-DNA complexes formed with either a single or a double AP-1 element. This suggests that under the conditions used (oligonucleotide in excess over protein), only a single site is occupied on the oligo containing a dimeric AP-1 element and further supports the notion of independent binding to either AP-1 site in the TF promoter. In some experiments (see for example Fig. 8A), formation of a small amount of a second slower migrating complex was seen, which was only observed with the dimeric AP-1 element (data not shown). It is conceivable that this complex represents some double occupancy of the TF AP-1 element.

In an initial analysis of the transcription factors constituting the TF AP-1 binding complex, antibody supershift experiments were performed (Fig. 8C). Identical results were obtained with nuclear extracts from both uninduced and LPS-treated cells, and only the results for activated endothelial cells are shown. The addition of rabbit antisera broadly reactive with either Jun or Fos antigens^4 (lanes9 and 10) leads to an almost complete prevention of complex formation or to the formation of slower migrating complexes, suggesting that both Jun and Fos family members are present in the TF AP-1 binding complex. Using peptide antibodies against individual members of the Jun/Fos family of transcription factors (lanes2-8), reactivity is seen both with an anti-c-Jun and an anti-JunD antibody. None of the anti-Fos peptide antibodies reacts strongly with the porcine TF AP-1 binding complex, although some weak reactivity with an anti-Fra2 antibody is observed.

Footprint Region 5 Is Bound by an Sp1 Transcription Factor

A striking feature of the DNase I footprint analysis is the occupancy of a number of regions in the TF promoter (Fig. 3, regions1 and 5-7), which contain consensus recognition sites for the transcription factor Sp1. Gel retardation analysis with nuclear extracts and an oligonucleotide spanning footprint region 5 detects a constitutive binding activity that is specifically competed with both an excess of nonradioactively labeled oligonucleotide or with a consensus Sp1 oligonucleotide (48) (Fig. 9). In addition, complex formation is reduced in the presence of anti-Sp1 antibodies, and a slower migrating complex is formed instead (lane5). Purified Sp1 also binds to this element (data not shown). Recently, a number of Sp1-related transcription factors with very similar binding specificities have been identified (see for example (49) and (50) ). Although it is possible that several of these factors will interact with the TF promoter, the antibody supershift experiment nevertheless suggests that Sp1 is part of the activity that interacts with region 5. We conclude that the TF promoter region from -136 to -115, which contains two potential Sp1 elements, is recognized and bound by an Sp1-like transcription factor in endothelial cells. Although we have not tested the binding of nuclear factors to footprint regions 1, 6, and 7 in gel retardation assays, it seems reasonable to predict that Sp1 or an Sp1-related factor will also bind to these sequences within the TF promoter.


Figure 9: Binding of nuclear proteins to the Sp1 containing footprint region 5. An oligonucleotide spanning footprint region 5 and containing two potential Sp1 binding sites was used for electrophoretic mobility shift assays with nuclear extracts from noninduced PAEC (lanes1-6) and cells induced with 100 ng/ml LPS (lane7) or 100 units/ml of TNF-alpha (lane8) for 2 h. For competition, a 100-fold excess of nonradioactive specific oligonucleotide (lane2), of a consensus Sp1 (lane3), or a nonspecific oligonucleotide (lane4) was added. In lanes5 and 6, 0.2 µg/ml of a rabbit polyclonal peptide antibody against Sp1 or a control IgG were added during the binding reaction, respectively. f.p., free probe.




DISCUSSION

TF synthesis is subject to tight control in EC and monocytes, presumably since any slight perturbation of the delicate balance between anti- and procoagulation can result in intravascular thrombosis (1, 2, 52) . On the cells surrounding the endothelial cell layer, constitutive expression has been documented, and the expression of TF may therefore be controlled by distinct mechanisms in these cells(3, 4) . Inappropriate expression of TF in the vasculature can be the cause of life-threatening diseases(9) . An understanding of the principles of TF gene control is therefore of great importance for the design of novel therapeutic approaches including gene therapy. In this context, the porcine system provides both a well established primary EC culture, where EC can be obtained in large quantities for in vitro studies, as well as an experimental animal, which might also serve as a donor of organs for xenotransplantation(10) .

For these reasons we have isolated the porcine homologue of the TF gene and have performed an analysis of the porcine TF promoter in primary aortic endothelial cells. A triple alignment of the promoter with the corresponding human and murine sequences reveals several well conserved regions with homology to previously characterized transcription factor binding motifs. The part of the promoter defined recently by Edgington and co-workers (18) to function as an LPS-inducible element in monocytic cells is among the most conserved. This element contains a dimeric AP-1 site close to a single NFkappaB site. Using DNaseI footprint analysis of the TF promoter, these, in addition to several Sp1 sites, are found to be the major occupied elements. In the porcine TF promoter, the NFkappaB and both AP-1 sites differ from the main consensus for the individual motifs by one nucleotide. Considering in addition the conserved bases in the flanking regions of these sites, a pattern of transcription factor binding unique to the TF NFkappaB and AP-1 sites seems possible.

Using reporter gene constructs in which up to 4 kilobase pairs of the porcine TF promoter were fused to the luciferase gene, we observe an up to 3-fold induction of promoter activity following treatment of the transiently transfected primary endothelial cells with endotoxin. Since the transfection procedure appears to partially induce the cells, we assume that the basal transcription level in these experiments is higher than in normal unstimulated cells, but the induced levels should faithfully reflect the transcription rate in LPS-treated cells. LPS inducibility is preserved in a construct that contains TF promoter sequences from -330 to +34, suggesting that the LPS responsive promoter elements required for transcriptional up-regulation of the TF gene in EC are present in this part of the promoter. Mutation of the NFkappaB element demonstrates that this element plays an essential role in TF gene transcription in EC. Deletion of the dimeric AP-1 element appears to reduce the LPS inducibility, suggesting that this element is involved in LPS responsiveness.

Previous analyses of the TF promoter and its regulation in response to endotoxin have been restricted to a monocytic cell line(18, 52) . These studies have suggested the binding of nuclear factors to the TF NFkappaB and AP-1 sites. Whereas in an earlier work binding to both sites was described as inducible(18) , more recently a constitutive interaction of transcription factors with the AP-1 elements was reported(52) . Our analysis extends these studies to endothelial cells, demonstrates that in our system the AP-1 binding is constitutive, and includes an extended analysis of the nuclear factors binding to both the NFkappaB and AP-1 sites.

The data obtained for the NFkappaB complex with nuclear extracts from primary PAEC demonstrate a strong induction of binding following treatment of the cells with endotoxin or inflammatory cytokines. We further observe that p65 (RelA) as well as c-Rel participates in the binding complexes. In addition, UV cross-linking experiments of the TFkappaB binding complexes reveal the presence of two additional bands of 63 and 55 kDa, respectively, which could be two differentially modified forms of a novel subunit or two separate proteins. In a recent study, Oeth et al.(19) have demonstrated the presence of p65 and c-Rel in the monocyte TF NFkappaB complex. These authors have, however, not detected any other proteins binding to this element. This could be accounted for because of a differential availability of cross-linkable bromodeoxyuridine residues in the specific oligos used. On the basis of the cross-linking data, we cannot completely exclude the possibility that the 63- and 55-kDa polypeptides we detect are degradation products of c-Rel or p65 that retain DNA binding activity. Clearly more detailed studies will be required to verify the nature of these proteins.

The binding of proteins to the dimeric AP-1 site follows a different pattern from that of the NFkappaB site in endothelial cells. In this case we see constitutive binding with extracts from unstimulated EC as well as with extracts from cells induced with LPS or TNF-alpha. Although a slight increase in the amount of complex formed is seen after induction, we have not observed qualitative changes in the complexes formed on the AP-1 elements. It is possible that the contribution of the AP-1 binding factors to the regulation of the TF gene in EC may be exerted by modifications of the proteins not grossly changing the binding capabilities of the factors to DNA.

Recently, examples have been published where a direct interaction of AP-1 or AP-1-related factors with NFkappaB proteins was demonstrated. In the case of the human E-selectin promoter, ATF family members were found to cooperate with NFkappaB in the cytokine-dependent activation of the gene. Furthermore, a direct physical association between ATF family members and p50 or p65, respectively, was shown in vitro(53) . Similarly, physical interactions between c-Jun, c-Fos, and p65 and a resulting functional synergism have also been suggested to occur in vivo by reporter gene assays(54) .

In addition to NFkappaB and AP-1-like factors, the footprint analysis suggests a potential importance of Sp1 binding factors for the transcription of the TF gene. This seems even more significant given the almost complete conservation of the Sp1 sites, even in their relative position to the transcriptional start site in the TF promoter. Functional interactions between inducible and constitutive cellular transcription factors like Sp1 have been described. A well documented example is the human immunodeficiency virus, type 1 enhancer, which is thought to be regulated by a cooperative interplay of NFkappaB and Sp1 transcription factors(55, 56) . It could very well be that a comparable situation exists on the TF promoter. On the basis of our results, we propose a concerted action of AP-1, NFkappaB, and Sp1 transcription factors in the regulation of the tissue factor promoter in primary endothelial cells.


FOOTNOTES

*
This work was supported in part by FWF Grant P10436-MED from the Austrian Science Foundation (to E. H.). 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z46238[GenBank].

§
To whom correspondence should be addressed. Tel.: 43-1-86634-308; Fax: 43-1-86634-623.

(^1)
The abbreviations used are: TF, tissue factor; EC, endothelial cells; LPS, lipopolysaccharide; TNF-alpha, tumor necrosis factor alpha; bp, base pair(s); IL-1alpha, interleukin 1alpha; PAEC, porcine aortic endothelial cells.

(^2)
H. Holzmüller, B. Wohlwend, F. H. Bach, and E. Hofer, manuscript in preparation.

(^3)
T. Moll, M. Czyz, H. Holzmüller, R. Hofer-Warbinek, E. Wagner, H. Winkler, F. H. Bach, and E. Hofer, unpublished observations.

(^4)
P. Angel, personal communication.


ACKNOWLEDGEMENTS

We acknowledge the generous gift of a human TF cDNA by Drs. N. Mackman and T. S. Edgington. Furthermore we are grateful to Bettina Wohlwend, Erni Schwarzinger and Veronika Kummer for excellent technical assistance. We thank Drs. P. Angel and P. Herrlich for Fos and Jun antibodies, Drs. B. Sha and D. Baltimore for a p52 expression plasmid, Drs. P. Bäuerle and W. Greene for p65, p50, c-Rel expression plasmids and Dr. F. J. Werner for help with the sequence analysis program.


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