Lipopolysaccharide Induction of Tissue Factor in THP-1 Cells Involves Jun Protein Phosphorylation and Nuclear Factor kappa B Nuclear Translocation*

Adrian J. HallDagger , Hans L. Vos, and Rogier M. Bertina

From the Haemostasis and Thrombosis Research Centre, University Hospital Leiden, Leiden, 2300 RC The Netherlands

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

Tissue Factor (TF) gene expression is transiently induced in human monocytic THP-1 cells by lipopolysaccharide (LPS). We characterized the transcription factor complexes binding to the TF gene promoter LPS response element (LRE) (-220 to -172), which contains binding sites for nuclear factor kappa B (NFkappa B) and activator protein 1 (AP1) transcription factors, and examined the nature of the activation of these factors during a 24-h time course of LPS stimulation. We found proteolysis of the cytoplasmic inhibitory protein Ikappa Balpha and nuclear translocation of the NFkappa B/Rel family proteins p65 and c-Rel, corresponding to the transient binding of a p65/c-Rel heterodimer to the kappa B-like site of the LRE. AP1 binding to the LRE was found to be constitutive, with the majority of the AP1 complexes being JunD/Fra-2 heterodimers. A change in the activation state of the AP1 complexes was, however, found to be transient, as determined by JunD phosphorylation of AP1 bound to the proximal binding site. This directly correlates to the transient activation of Jun N-terminal kinase (SAPK/JNK). These data indicate that LPS induction of TF gene expression in monocytic THP-1 cells is regulated by both the transient phosphorylation of Jun-family proteins and the nuclear translocation and transient binding of NFkappa B/Rel proteins.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Tissue factor (TF)1 is the primary initiator of the serine protease cascade of the coagulation system (1). TF is constitutively expressed in a number of different cell types that do not normally come into contact with blood (2) but is of necessity not usually expressed within the vasculature. However, in various disease states, aberrant TF expression in vascular cells may lead to thrombosis, such as during sepsis, when bacterial endotoxin (lipopolysaccharide) induction of TF in monocytes can lead to disseminated intravascular coagulation (3).

Monocytes are the only circulating cells in which TF expression is subject to inducible regulation (4). Synthesis may be up-regulated by a number of different stimuli including phorbol esters (phorbol 12-myristate 13-acetate) (5), tumor necrosis factor-alpha (6), and bacterial lipopolysaccharide (LPS) (7, 8). In LPS-induced monocytes, TF mRNA levels increase as a result of transcriptional activation (7). In the monocytic cell line THP-1, TF gene expression is induced by LPS in a similar manner with, however, some increase in mRNA stability as well (9).

Functional studies in THP-1 cells identified an enhancer in the TF gene promoter that mediates LPS induction. This 56-base pair region (-227 to -172) is termed the LPS response element (LRE) (10). In addition, a second region (-85 to -52) containing Egr-1 binding sites has been identified that is also subject to inducible binding (11). Our study focused on the LRE that contains two AP-1 sites (a distal, low affinity site and a proximal, high affinity site) and an NFkappa B-like site. Mutation of any of these sites compromises LPS inducibility (10, 12), suggesting that all three are required for optimal LPS induction.

In this study we have analyzed the protein interactions with the LRE binding sites over a 24-h time course of LPS induction in THP-1 cells. We have determined that a number of regulatory mechanisms act to control TF gene transcription in response to LPS stimulation. These include the transient binding of a p65/c-Rel heterodimer from at least 30 min to 2 h, resulting from the proteolysis of Ikappa Balpha and nuclear translocation of p65 and c-Rel and transient phosphorylation of JunD in LRE-bound AP-1 complexes correlating to the activation of Jun N-terminal kinase (SAPK/JNK) from 10 min up to 1 h of LPS treatment. These data suggest multiple mechanisms acting co-operatively at the LRE enhancer element to direct a transient increase in TF mRNA levels in monocytic THP-1 cells.

    EXPERIMENTAL PROCEDURES

Cell Lines-- THP-1 cells (13) were grown in RPMI 1640 with L-glutamine and 25 mM HEPES buffer (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and 100 units/ml penicillin, 100 µg/ml streptomycin. Cells were routinely grown to a density of 1 × 106 cells/ml and induced with 10 µg/ml LPS from Salmonella typhimurium (Sigma) for the times indicated in the figures.

TF Antigen Assay-- THP-1 cells were recovered from suspension and washed in phosphate- buffered saline. Cell extracts were then prepared, and TF antigen levels were determined by enzyme-linked immunosorbent assay as described by Consonni and Bertina (14), using TF 4503 monoclonal antibody (American Diagnostica, Greenwich, CT) as the catching antibody and biotinylated TF 5 monoclonal antibody (Costar) as the tagging antibody. Recomboplastin S/Innovin (Baxter Diagnostica Inc., Deerfield, IL) was used as a standard after calibration against the standard of the Immubind tissue factor enzyme-linked immunosorbent assay kit (American Diagnostica Inc., Greenwich, CT). The final results were expressed as ng TF/106 cells.

RNA Isolation and Northern Blotting-- Total RNA was prepared from THP-1 cells using the TRIzol reagent (Life Technologies, Inc.), a modification of the guanidinium isothiocyanate method (15). 15 µg of total RNA/time point were fractionated on 1% agarose, 0.22 M formaldehyde gels in 20 mM MOPS, 5 mM sodium acetate, pH 7.0, 1 mM EDTA and 0.22 M formaldehyde. After staining with ethidium bromide and visualization of the ribosomal RNA under UV light, RNA was transferred to Hybond-N filters (Amersham Pharmacia Biotech) by standard capillary transfer methods (16). The RNA was then immobilized by UV irradiation on a UV Stratalinker 1800 (Stratagene, San Diego, CA). Filters were prehybridized for 3 h at 42 °C in 50% deionized formamide, 0.5% SDS, 5 × Denhardt's solution, 5 × SSPE (0.9 M NaCl, 50 mM sodium phosphate, pH 7.7, 0.5 mM Na2EDTA), and 100 µg/ml sheared salmon sperm DNA. Filters were then hybridized for 18 h under the same conditions with an [alpha -32P]dCTP (Amersham Pharmacia Biotech)-labeled 601-base pair HhaI-EcoRI TF cDNA probe (17). They were washed in 2 × SSC (1 × SSC = 0.15 M NaCl and 0.015 M sodium citrate), 0.1% SDS for 2 × 15 min at room temperature followed by two 15-min washes in 1 × SSC, 0.1% SDS. Filters were exposed to film at -70 °C with intensifying screens. Equivalence of loading was controlled for by rehybridization of stripped blots with a 32P-labeled glyceraldehyde-3-phosphate dehydrogenase cDNA probe (18).

Electrophoretic Mobility Shift Assays-- Crude nuclear extracts were prepared for use in electrophoretic mobility shift assays (EMSAs) essentially as described previously (11). Modifications include cell shearing by repeated aspiration through a 27-gauge needle rather than a Dounce homogenizer and the addition of phosphatase inhibitors (0.25 mM orthovanadate and 25 mM beta -glycerophosphate) to all solutions. Protein concentrations of all extracts were determined using the Bio-Rad protein assay reagent.

The following oligonucleotides were radiolabeled using T4 polynucleotide kinase (Epicentre Technologies) and [gamma 32P]ATP (Amersham Pharmacia Biotech) before being annealed to oligonucleotides of complementary sequence. TFAP1distal, (-230 to -211) GCGCGGTTGAATCACTGGGG; AP1pkappa B (containing the TF LRE proximal AP1 site and the NFkappa B site), (-213 to -172) GGGTGAGTCATCCCTTGCAGGGTCCCGGAGTTTCCTACCGGG; TFkappa B, (-193 to -174) GGTCCCGGAGTTTCCTACCG; and the NFkappa B consensus, GGGAGGGGACTTTCCGAGAG. Additional double-stranded oligonucleotides used as competitors include the AP1 consensus, GCCGCAAGTGACTCAGCGCGGG, and an Sp1 site from the TF promoter (-96 to -66) used in the current context as a nonspecific competitor, AGTCGGGAGGAGCGGCGGGGGCGGGCGCCGG. Two binding reaction buffers were used; one optimized for AP1 binding (binding buffer 1: 23 mM Hepes, pH 7.9, 113 mM NaCl, 9 mM MgCl2, 0.23 mM EDTA, 19% glycerol, 4.5 mM dithiothreitol, 0.75 mM phenylmethylsulfonyl fluoride, 0.75 µg/ml each leupeptin, aprotinin, antipain, pepstatin A) (11) and a second optimized for NFkappa B binding (binding buffer 2: 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 19% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml each leupeptin, aprotinin, antipain, pepstatin A) (19). 20 µl of binding reactions contained 5 µg of nuclear extract, 0.5 ng of 32P-labeled double-stranded oligonucleotide, and 3 µg poly(dI-dC), unless otherwise stated. When looking specifically for phosphorylated proteins, the binding reactions also contained orthovanadate and beta -glycerophosphate at final concentrations of 0.25 mM and 25 mM, respectively. Reactions were incubated on ice for 30 min before electrophoresis through 6%, 0.5× TGE (1× TGE-25 mM Tris, 190 mM glycine, 10 mM EDTA) polyacrylamide gels. For competition assays and supershift assays, a preincubation at 4 °C for 12-16 h with a 100-fold molar excess of competitor or 1 µg of antibody, respectively, was carried out before the addition of the labeled oligonucleotide. Gels were dried under vacuum and exposed at -70 °C with intensifying screens.

Western Blot Analysis-- Nuclear and cytoplasmic extracts used in the Western blot analyses were prepared essentially as described for EMSAs with the exception of Nonidet P-40 being added to the cells to a final concentration of 0.5% following shearing by aspiration, for extracts used to examine NFkappa B compartmentalization. After centrifugation of the lysed cells, the supernatant was frozen in liquid nitrogen and stored at -70 °C as the cytoplasmic extract. Again, the protein concentrations of all extracts were determined using the Bio-Rad protein assay (a modification of the Bradford protein assay (20)).

20 µg of nuclear extracts or 60 µg of cytoplasmic extracts were fractionated on 10% SDS-polyacrylamide gels and then electrotransferred to a polyvinylidene difluoride membrane (Millipore) in 48 mM Tris, 58.6 mM glycine, 0.1% SDS, 20% methanol at 0.8 V/cm2 for 2.5 h using the Nova Blot system (Amersham Pharmacia Biotech). Western blots were blocked, and immunoreactive products were detected according to the protocol of the Boehringer Mannheim chemiluminescence Western blotting kit. Briefly, blots were blocked in 1% blocking solution overnight at 4 °C then washed in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween 20). The blots were then incubated with the primary antibodies (Santa Cruz, Transcruz antibodies diluted 1:10,000 in 0.5% (w/v) blocking solution and phospho-specific antibodies diluted 1:1,000 in TBST, 5% (w/v) bovine serum albumin). The secondary antibody incubation was in 0.5% blocking solution containing 1:10,000 goat anti-rabbit horseradish peroxidase-conjugated antibody (Bio-Rad) and 1:2,000 anti-biotin antibody (New England Biolabs) to detect the biotinylated protein standard used (New England Biolabs). Comparative blots of nuclear and cytoplasmic proteins were performed simultaneously and exposed to film for the same length of time.

Antibodies-- The polyclonal antibodies used in the EMSA supershift assays and Western blot analyses were as follows: c-Jun/AP-1 (sc-44x); c-Jun/AP-1 (sc-45x); JunB (sc-46x); JunD (sc-74x); ATF-2 (sc-187x); c-Fos (sc-253x); c-Fos (sc-52x); FosB (sc-48x); Fra-1 (sc-605x); Fra-2 (sc-171x); NFkappa B p50 (sc-114x); NFkappa B p52 (sc-298x); NFkappa B p65 (sc-109x); RelB (sc-226x); c-Rel (C) (sc-71x); c-Rel (N) (sc-70x), all obtained from Santa Cruz Biotechnology; phospho-specific c-Jun (Ser-73) (9164); phospho-specific ATF-2 (Thr 71) (9221); SAPK/JNK (9252); phospho-specific SAPK/JNK (Thr-183/Tyr-185) (9251) were all from New England Biolabs. The alpha -tubulin antibody used in the Western blot analysis was a kind gift from Dr. Ingrid Gaemers obtained from Dr. Pavel Draber (21, 22).

    RESULTS

LPS Stimulation of Monocytic THP-1 Cells Produces Transient Increases in the Levels of TF Antigen and mRNA-- To determine their response to LPS, monocytic THP-1 cells were incubated with 10 µg/ml LPS for various times up to 24 h. TF mRNA, analyzed by Northern blot hybridization (Fig. 1), increased by 30 min and reached a peak at 1 h. Levels dropped considerably by 2 h and had returned to preinduction levels at the following time points. Larger transcripts resulting from incomplete splicing (17) can be seen in addition to the mature 2.2-kilobase mRNA. A corresponding increase in the level of TF antigen was also observed, reaching a peak at 2 h then decreasing through to 24 h (data not shown).


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Fig. 1.   Time course of LPS induction of TF in THP-1 cells. THP-1 cells were cultured in the absence or presence of 10 µg/ml LPS for the times indicated. Total RNA was fractionated and transferred to a membrane; TF mRNA levels were determined using Northern hybridization with a TF cDNA probe. 15 µg of total RNA/time point were loaded. Equivalence of loading was examined by rehybridization of the stripped blot with a cDNA probe for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) house-keeping gene. kb, kilobases.

Binding of AP1 and NFkappa B Complexes-- The EMSA studies presented were predominantly carried out using the AP1Pkappa B oligonucleotide (-213 to -172). This region of the TF LRE was chosen as AP1 consists of various dimers of Fos and Jun family proteins, and this oligonucleotide together with TFAP1distal, allowed us to distinguish between AP1 complexes binding to the proximal and distal AP1 sites while maintaining possible effects on complex binding arising from the proximity of the adjacent high affinity proximal AP1 site and the kappa B-like site. We found that variation in the binding reaction conditions had a noticeable effect on the relative intensities of the DNA-protein complexes that were observed (Fig. 2). Under conditions that seemed to favor AP1 binding (binding buffer 1) two complexes became apparent whose intensity appears to be relatively constant throughout the time course (Fig. 2A). A very faint larger complex (I) and a smaller doublet complex (II), which constituted the majority of the binding to this oligonucleotide, are seen. Both complexes I and II could be competed with a 100-fold molar excess of AP1Pkappa B and an AP1 consensus competitor but not with an NFkappa B consensus or a nonspecific competitor (Sp1), indicating that both complex I and II are AP1 complexes. Constitutive binding of an AP1 complex throughout the 24-h time course was also observed using an oligonucleotide containing the distal AP1 site (data not shown).


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Fig. 2.   Transcription factor complexes binding the AP1Pkappa B probe during a time course of LPS induction. Binding activity to the DNA probe containing the LRE proximal AP1 site and the kappa B-like site (-213 to -172) was analyzed in EMSA studies. DNA binding was assayed under three sets of conditions: the first, in binding buffer 1 (panel A); the second, in binding buffer 2 (panel B), both with the DNA probe present in excess; and a third, with binding buffer 1 but with the ratio of nuclear proteins to probe 200-fold higher (20 µg nuclear extract to 0.02 ng of oligonucleotide) (panel C). Under all conditions the effects of specific (AP1Pkappa B, AP1, and NFkappa B) and nonspecific (Sp1) competitors at 100-fold molar excess were analyzed.

Under the conditions of binding buffer 2 (Fig. 2B), we see a much lower intensity of complex II, with complex I no longer visible, whereas complexes III and IV become more obvious. Complexes III and IV begin to appear at 30 min, with binding reaching a peak at 1-2 h. At 4 h and later, their presence is no longer detected. A 100-fold molar excess of AP1Pkappa B or the NFkappa B consensus oligonucleotide both compete complexes III and IV. The AP1 consensus competes complex II (as seen in Fig. 2A), whereas the nonspecific competitor (Sp1) appears to compete only complex IV. These data demonstrate the transient binding of two NFkappa B complexes to the oligonucleotide between 30 min and 2 h, of which only complex III appears to have high affinity for the kappa B-like site.

Under conditions where the ratio of nuclear proteins to oligonucleotide is much higher (200-fold higher, binding buffer 1, Fig. 2C) we see the appearance of a new larger complex (V). This complex is present from 30 min to 2 h, similar to complexes III and IV binding in Fig. 2B. The AP1Pkappa B oligonucleotide and the NFkappa B consensus oligonucleotide compete complex V, as does the AP1 consensus competitor (in addition to complex II). A nonspecific competitor (Sp1) has no effect. These results indicate that complex V is a complex of the oligonucleotide AP1Pkappa B with both AP1 and NFkappa B binding simultaneously.

Characterization of the NFkappa B Complex-- Supershift assays were carried out with the AP1Pkappa B oligonucleotide, the TF kappa B-like site oligonucleotide and the NFkappa B consensus oligonucleotide, and antibodies against specific NFkappa B/Rel proteins. With AP1Pkappa B (Fig. 3, lanes 1-10), supershifts of complex III were observed with an antibody against p65 and to a lesser extent, anti-c-Rel, but not with antibodies specific for other members of the NFkappa B/Rel family. The complex formed with the TFkappa B oligonucleotide (Fig. 3, lanes 11-20) had the same mobility and transient binding characteristics as complex III and was also supershifted by antibodies against p65 and c-Rel. The affinity of protein complexes for the TFkappa B site in isolation was much lower than with the adjacent AP1 proximal site present (lanes 11-20 of Fig. 3 were exposed four times longer than the rest of the assay). The complex formed with the NFkappa B consensus oligonucleotide (Fig. 3, lanes 21-30) was supershifted by antibodies against p50 and p65 but not by antibodies specific for other members of the NFkappa B/Rel family. These data show complex III to be a p65/c-Rel heterodimer.


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Fig. 3.   Characterization of the NFkappa B complexes. NFkappa B complexes were analyzed in supershift assays using antibodies against NFkappa B/Rel proteins (binding buffer 2). Lanes 1-10 show binding to the AP1Pkappa B oligonucleotide; lanes 11-20 to the TF kappa B-like site alone; and lanes 21-30 to an NFkappa B consensus sequence. All data shown are from a single assay; however, the exposition of lanes 11-20 is four times longer than that of lanes 1-10 and 21-30. All oligonucleotides were radiolabeled to similar specific activities. Complexes I to IV, identified in Fig. 2, are shown with the antibody supershifts indicated.

LPS Induces Proteolysis of Ikappa Balpha and Translocation of p65 and c-Rel from the Cytoplasm to the Nucleus-- Nuclear and cytoplasmic extracts from THP-1 cells stimulated with LPS for the times indicated in the figure were examined by Western blot analyses (Fig. 4). Antibodies against p65 and c-Rel, the component proteins of the higher affinity complex III, and also the cytoplasmic inhibitory protein Ikappa Balpha , were used. When nuclear extracts used in the EMSA studies were examined with an antibody against a cytoplasmic protein (alpha -tubulin) in Western blot analysis a certain proportion of cytoplasmic protein was observed. Therefore, to attain a greater degree of nuclear/cytoplasmic separation, an adapted technique of preparation was used (see "Experimental Procedures"). However, rather than single bands corresponding to the p65 and c-Rel proteins, we observe multiple bands after immunostaining of the Western blots, which we would suggest are because of partial degradation of the proteins during this preparation procedure. However, because we are mainly concerned with the localization of these proteins, the degradation was not considered to impede the interpretation of the data. Ikappa Balpha and Fos/Jun family proteins show relatively little degradation on Western blots, indicating that p65 and c-Rel may be particularly susceptible to breakdown with this preparation method. We can see that very little p65 is evident in the nucleus in unstimulated cells. After 10 min of LPS induction, nuclear p65 begins to appear and peak at 1 h, declining again by 2 h. A concomitant decrease in cytoplasmic p65 corresponds to the observed increase in nuclear p65.


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Fig. 4.   Distribution of c-Rel, p65, and Ikappa Balpha in LPS-induced THP-1 cells. Nuclear (panels A and C) and cytoplasmic (panels B, D, and E) extracts were prepared from THP-1 cells treated with LPS for the times indicated and assayed using Western blot analysis for the presence of p65 (panels A and B), c-Rel (panels C and D), and Ikappa Balpha proteins (panel E). M, molecular mass markers.

In the case of c-Rel, again very little protein is evident in the nuclei of unstimulated cells or at 10 min. However, by 30 min to 1 h, nuclear c-Rel has increased to a level that, unlike p65, appears to be maintained up to 24 h. The level of cytoplasmic c-Rel seems relatively constant with only a slight dip observed at 1 h, corresponding to the peak in nuclear c-Rel levels.

The amount of Ikappa Balpha seen in the cytoplasm drops sharply at 30 min, with levels beginning to increase again at 1 h, peaking at 2-4 h, and reaching preinduction levels by 24 h. This peak in Ikappa Balpha expression corresponds to a peak in Ikappa Balpha mRNA levels at 1 h (data not shown). Nuclear levels of Ikappa Balpha in Western analysis were found to be low and constant, confirming breakdown and re-synthesis of cytoplasmic Ikappa Balpha rather than translocation. These data are consistent with the proposal that Ikappa Balpha is proteolyzed after induction of the NFkappa B system, releasing the NFkappa B proteins, which then translocate to the nucleus (10, 19, 23, 24). An NFkappa B element in the Ikappa Balpha promoter (25) directs the increase in Ikappa Balpha levels at later time points.

Characterization of the AP1 Complexes-- Nuclear extracts prepared from THP-1 cells stimulated with LPS through a 24-h time course were used to analyze the identity of complex II in supershift assays (Fig. 5). In stimulated and unstimulated cells, complex II is completely supershifted by an antibody against JunD (Fig. 5A). An unidentified complex that seems to have a slightly higher mobility than complex II becomes evident when complex II is supershifted by anti-JunD. Antibodies specific for c-Jun and ATF-2 have no observable effect on complex II. The effect of an antibody against JunB was also investigated with negative results (data not shown).


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Fig. 5.   Identification of AP1 complexes. Nuclear extracts were prepared from THP-1 cells induced with LPS for the times indicated and subsequently used in supershift analyses with oligonucleotide AP1Pkappa B and antibodies against Jun/ATF/CREB proteins (panel A, binding buffer 1) and Fos proteins (panel B, binding buffer 2).

Further supershifts with antibodies against Fos proteins were also carried out. In the absence of LPS, complex II is completely supershifted by a broadly reactive Fos antibody. Anti-Fra-2, although not producing an observable supershift, does significantly decrease the intensity of complex II. Anti-c-Fos has no effect. This situation remains the same following LPS stimulation of up to 24 h. The effects of antibodies against FosB and Fra-1 were also analyzed with negative results (data not shown).

Our data show complex II to be a JunD/Fra-2 heterodimer both before and after LPS stimulation of up to 24 h. The residual complex II observed in the presence of anti-Fra-2 may either be because of the antibody having a relatively low affinity for Fra-2 or because of the presence of an as yet unidentified Fos-related protein that forms a dimer with JunD.

Supershift assays with the AP1 distal site oligonucleotide demonstrated the binding of both JunD/Fra-2 and JunD/c-Fos heterodimers with no change in the components of the complex through the 24-h time course (data not shown). These data suggest slight differences in binding at the two adjacent AP1 sites.

The Transient Phosphorylation of JunD-- In the absence of a significant quantitative change in binding to the proximal AP1 site through the 24-h time course (see Fig. 2A), we examined whether there was a change in the phosphorylation state of JunD. Supershift assays (Fig. 6) were carried out using anti-JunD alone or in combination with an antibody specific for JunD phosphorylated at Ser-100 (c-Jun phosphorylated at Ser-73, a conserved site, is also recognized). In the absence of LPS, complex II is supershifted with anti-JunD, with the addition of the phospho-specific Jun antibody (alpha -p-Jun) having little effect. At 30 min to 1 h of LPS stimulation, the anti-phospho-Jun antibody caused a further shift in the anti-JunD supershift (supershift I), producing a lower mobility complex (supershift II). The amount of observable "supershift II" is considerably reduced at periods of LPS incubation of 2 h and longer. These data indicate that the majority of the JunD bound to the AP1Pkappa B oligonucleotide is not phosphorylated at its transactivation domain (Ser-100) in the absence of LPS, but with LPS induction of up to 1 h, transient phosphorylation occurs. The presence of phosphorylated JunD at 30 min to 1 h of LPS stimulation was confirmed by Western blot analyses using the phospho-specific Jun antibody (data not shown).


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Fig. 6.   Analysis of the phosphorylation state of JunD bound to the AP1Pkappa B oligonucleotide. A supershift assay was carried out with nuclear extracts prepared from THP-1 cells treated with LPS over a 24-h time course. The supershifts resulting from a single antibody binding (anti-JunD) (supershift I) and dual antibody binding (anti-JunD and anti-phospho-Jun) (supershift II) are indicated.

We also analyzed the activation state of SAPK/JNK, whose downstream targets include JunD. The data indicate no change in the levels of nuclear or cytoplasmic SAPK/JNK (Fig. 7, A and C) but clearly show activation by phosphorylation at the Thr-183/Tyr-185 residues beginning at 10 min and peaking at 30 min to 1 h (Fig. 7, B and D). The activation of SAPK/JNK directly reflects the appearance of phosphorylated JunD.


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Fig. 7.   The phosphorylation state of SAPK/JNK during a 24-h time course of LPS induction. Nuclear extracts prepared from LPS-treated THP-1 cells were assessed for the levels of SAPK/JNK and phosphorylated SAPK/JNK. The two forms of SAPK/JNK, p54 and p46, are indicated. Nuclear extracts (panels A and B) and cytoplasmic extracts (panels C and D) were analyzed. A SAPK/JNK antibody (panels A and C) and a phospho-specific SAPK/JNK antibody recognizing phosphorylation at Thr-183/Tyr-185 were used. M, molecular mass markers.


    DISCUSSION

LPS activation of monocytes and monocytic THP-1 cells produces a transient increase in the levels of TF mRNA and TF antigen and activity (8, 9). Our data charting these levels in THP-1 cells through a 24-h time course show a correlation to previous reports, with a rapid induction of TF mRNA from 30 min to 1 h of LPS stimulation and TF antigen levels peaking at 2 h.

When we examined the TF gene promoter LRE in DNA binding studies, we found the transient binding of an NFkappa B complex and the constitutive binding of AP1 complexes. The NFkappa B complex was observed to bind the LRE site from at least 30 min up to 2 h of LPS stimulation and was found to contain p65 and c-Rel in a heterodimeric complex. The TF LRE NFkappa B-like sequence has been shown to be an optimal site for the binding of p65 and c-Rel but not p50 (26). A number of previous reports studying this NFkappa B complex identified its component proteins as p65 and c-Rel, in agreement with our data (26, 27). A different study, however, suggested that an Ets transcription factor is binding to the core sequence of the kappa B-like site upon LPS stimulation rather than an NFkappa B complex. We found no evidence of Ets proteins binding to this site using an antibody against Ets 1/2 in supershift assays (data not shown).

To confirm the predicted model of NFkappa B activation, we compared nuclear and cytoplasmic extracts for evidence of nuclear translocation. Our data show the proteolysis of the cytoplasmic inhibitory protein Ikappa Balpha and the translocation of both p65 and c-Rel from the cytoplasm to the nucleus, corresponding to the appearance of the NFkappa B complex in gel shift studies. An earlier study charting the nuclear and cytoplasmic levels of the NFkappa B proteins in THP-1 cells (19) found that p65 was present in the nucleus before LPS induction, with levels increasing on stimulation and continuing to be elevated for up to 24 h. This contrasts with our data, where p65 was not detected before stimulation and only translocated to the nucleus for a very short period (up to 2 h), corresponding to the appearance of NFkappa B complexes in the binding studies and the transiently elevated levels of TF gene transcription. This same study, in agreement with our data, observed the rapid nuclear translocation of c-Rel with elevated levels persisting for up to 24 h. The fact that c-Rel is present at these longer periods of LPS stimulation yet does not appear to bind the TF kappa B-like site suggests that either this site is specific for the binding of p65/c-Rel heterodimers and not c-Rel homodimers or that the DNA binding capacity of c-Rel is subject to regulation. A recent study demonstrated that Ikappa Balpha may also regulate the transcriptional activity of c-Rel in the nuclear compartment (28), and other studies have shown that the DNA binding capacity of certain NFkappa B proteins may be controlled by redox mechanisms (29) or at the level of phosphorylation (30-32).

The EMSA data also suggest that the affinity of p65/c-Rel for the kappa B-like site is considerably increased if the proximal AP1 site is present. Although this effect has not been quantitated in our study, comparison of the binding to the AP1Pkappa B oligonucleotide and the isolated TFkappa B oligonucleotide showed that much longer exposure times were required to observe complexes with TFkappa B than with AP1Pkappa B (Fig. 3, lanes 11-20 and 1-10, respectively, probes labeled to similar specific activities). Because under these conditions multiprotein-DNA complexes, which might involve protein-protein interactions, were not observed, the effect appears to lie in the structural influence of the DNA itself. Although this effect may be a result of the size of the oligonucleotides used and the position of the transcription factor binding sites relative to the ends of the oligonucleotides, it may also be a result of adjacent sequence influencing binding characteristics. A recent study demonstrated such a structural role for the AP1 sites in the TF promoter when their replacement by intrinsically bent DNA was able to partially restore LPS induction (12).

The AP1 sites of the LRE are essential for LPS induction, with mutation of either site compromising LPS inducibility (10, 12). AP1 is a dimer of proteins of the Fos/Jun family. Jun proteins are able to bind to DNA as homodimers or as Fos/Jun heterodimers. Fos proteins, however, are obliged to form heterodimers to bind (33, 34). Previous studies analyzing the composition of the LRE-bound AP1 complexes in THP-1 cells report differing binding profiles at these sites. Oeth et al. (12) looked at the two AP1 sites in isolation in THP-1 cells and found c-Jun/c-Fos heterodimers both pre- and post-LPS stimulation, with the amount of protein binding exhibiting some increase after stimulation; these results are consistent with the constitutive expression of c-Jun and c-Fos in human monocytes and their induction in response to LPS (35). A second study in THP-1 cells (11) used a larger oligonucleotide containing the entire LRE with an additional downstream Sp1 site and found LPS induction of c-Jun binding. c-Fos/JunD heterodimers were found preinduction, whereas following LPS stimulation, both c-Fos/JunD and c-Fos/c-Jun complexes were observed. Our data examining AP1 LRE binding through a 24-h time course of LPS induction shows a relatively constant degree of AP1 binding to AP1Pkappa B throughout the 24-h time course, although we have consistently observed a decrease in the amount of complex II at 4 h and a subsequent increase at 24 h (Fig. 2A). Consistent with the study of Groupp and Donovan-Peluso (11), we find the majority of AP1 binding activity to contain JunD. In our study, JunD/Fra-2 bound at the proximal AP1 site (complex II), and both JunD/Fra-2 and JunD/c-Fos bound at the distal AP1 site, both at pre- and post-induction time points.

The Jun proteins may be regulated in their DNA binding and transactivation activities by means of changes in their phosphorylation state. Phosphorylation of c-Jun at the C terminus by casein kinase II or glycogen synthase kinase 3 prevents DNA binding (36), whereas phosphorylation at the N-terminal region (Ser-63/Ser-73) by the mitogen-activated protein kinase homologue SAPK/JNK enhances transcriptional activation. Ser-100 of JunD is a conserved phosphorylation site corresponding to Ser-73 of c-Jun and is also a target for SAPK/JNK (37, 38). Because our data showed no significant change in the degree of AP1 binding or in the composition of the complexes, we examined evidence for the activation states of the Jun proteins by assessing the phosphorylation of the amino acids that influence their transactivation domains. Our data clearly show the transient (30 min to 1 h) phosphorylation of JunD in the JunD/Fra-2 complex bound to the proximal AP1 site (Fig. 6, lanes 6 and 9). The activation of SAPK/JNK, for which JunD is a target, directly correlates to the appearance of phosphorylated JunD. Although only a small proportion of the cellular JunD is phosphorylated when examined by Western blot analysis (data not shown), gel mobility shift assays indicate that a high proportion of the proximal AP1 site-bound JunD is phosphorylated (Fig. 6), raising the question as to whether there may be some form of coordinated regulation of DNA binding and transactivation enhancement. Although we did not assess the phosphorylation state of JunD bound to the distal AP-1 site, it seems probable that it, too, is phosphorylated at the transactivation domain upon LPS stimulation.

Our evaluation of transcription factor binding to the TF gene promoter LRE throughout a 24-h time course of LPS induction demonstrates a number of regulatory features (Fig. 8). These include the transient, inducible binding of a p65/c-Rel heterodimer to the TF kappa B-like site (30 min to 2 h) corresponding to the proteolysis of Ikappa Balpha and the nuclear translocation of p65 and c-Rel as predicted by previously proposed models of NFkappa B activation (10, 19, 23) and the transient phosphorylation (30 min to 1 h) of JunD bound to the proximal AP1 site, reflecting the appearance of phospho-JunD in nuclear extracts. JunD is a target for SAPK/JNK, which we have demonstrated is activated by phosphorylation for this same period. The transient nature of the NFkappa B translocation and binding of p65/c-Rel to the TFkappa B-like site and of the activation of SAPK/JNK and phosphorylation of target AP1 proteins directly correlate with the observed transient increases in TF mRNA in LPS-induced THP-1 cells through the 24-h time course.


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Fig. 8.   Proposed model of events at the TF gene promoter LRE after LPS induction. Our data suggest that LPS stimulation leads to the activation of SAPK/JNK (JNK) and subsequent phosphorylation of Jun proteins binding the AP1 sites. In addition, Ikappa Balpha proteolysis releases p65/c-Rel, which translocate from the cytoplasm to the nucleus and bind the LRE kappa B-like site. Ub, ubiquitin; LBP, LPS binding protein; P, phosphate group.


    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by a Travelling Research Fellowship from The Wellcome Trust (045087/Z/95/Z). To whom correspondence should be addressed: Div. of Molecular and Genetic Medicine, Floor M, Royal Hallamshire Hospital, Sheffield S10 2JF, UK. Tel.: +44 (0)114 271 3750; Fax: +44 (0)114 272 1104; E-mail: a.j.hall{at}sheffield.ac.uk.

    ABBREVIATIONS

The abbreviations used are: TF, tissue factor; LPS, lipopolysaccharide; LRE, LPS response element; SAPK/JNK, Jun N-terminal kinase; AP1, activator protein 1; NFkappa B, nuclear factor kappa B; MOPS, 4-morpholinepropanesulfonic acid; EMSA, electrophoretic mobility shift assays..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
REFERENCES
  1. Edgington, T. S., Mackman, N., Brand, K., and Ruf, W. (1991) Thromb. Haemostasis 66, 67-79[Medline] [Order article via Infotrieve]
  2. Drake, T. A., Morrisey, J. H., and Edgington, T. S. (1989) Am. J. Pathol. 134, 1087-1097[Abstract]
  3. Osterud, B., and Flaestad, T. (1983) Thromb. Haemostasis 49, 5-7[Medline] [Order article via Infotrieve]
  4. Mackman, N. (1997) Thromb. Haemostasis 78, 747-754[Medline] [Order article via Infotrieve]
  5. Lyberg, T., and Prydz, H. (1981) Biochem. J. 194, 699-706[Medline] [Order article via Infotrieve]
  6. Conkling, P. R., Greenberg, C. S., and Weinberg, J. B. (1988) Blood 72, 128-133[Abstract]
  7. Niemetz, J., and Morrison, D. C. (1977) Blood 49, 947-956[Abstract]
  8. Gregory, S. A., Morrisey, J. H., and Edgington, T. S. (1989) Mol. Cell. Biol. 9, 2752-2755[Medline] [Order article via Infotrieve]
  9. Brand, K., Fowler, B. J., Edgington, T. S., and Mackman, N. (1991) Mol. Cell. Biol. 11, 4732-4738[Medline] [Order article via Infotrieve]
  10. Mackman, N., Brand, K., and Edgington, T. S. (1991) J. Exp. Med. 174, 1517-1526[Abstract]
  11. Groupp, E. R., and Donovan-Peluso, M. (1996) J. Biol. Chem. 271, 12423-12430[Abstract/Free Full Text]
  12. Oeth, P., Parry, G. C. N., and Mackman, N. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 365-374[Abstract/Free Full Text]
  13. Tsuchiya, N., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T., and Tada, K. (1980) Int. J. Cancer 26, 171-176[Medline] [Order article via Infotrieve]
  14. Consonni, R., and Bertina, R. M. (1995) Thromb. Haemostasis 74, 904-909[Medline] [Order article via Infotrieve]
  15. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve]
  16. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  17. van der Logt, C. P. E., Reitsma, P. H., and Bertina, R. M. (1992) Thromb. Haemostasis 67, 272-276[Medline] [Order article via Infotrieve]
  18. Tso, J. Y., Sun, X. H., Kao, T., Reece, K. S., and Wu, R. (1985) Nucleic Acids Res. 13, 2485-2502[Abstract]
  19. Cordle, S. R., Donald, R., Read, M. A., and Hawiger, J. (1993) J. Biol. Chem. 268, 11803-11810[Abstract/Free Full Text]
  20. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
  21. Vicklicky, V., Draber, P., Hasek, J., and Bartek, J. (1982) Cell Biol. Int. Rep. 6, 725-731[Medline] [Order article via Infotrieve]
  22. Draber, P., Draberova, E., Linhartova, I., and Vicklicky, V. (1989) J. Cell Sci. 92, 519-528[Abstract]
  23. Donovan-Peluso, M., George, L. D., and Hassett, A. C. (1994) J. Biol. Chem. 269, 1361-1369[Abstract/Free Full Text]
  24. Beg, A. A., and Baldwin, A. S., Jr. (1993) Genes Dev. 7, 2064-2070[CrossRef][Medline] [Order article via Infotrieve]
  25. Le Bail, O., Schmidt-Ullrich, R., and Israel, A. (1993) EMBO J. 15, 5043-5049
  26. Oeth, P. A., Parry, G. C. N., Kunsch, C., Nantermet, P., Rosen, C. A., and Mackman, N. (1994) Mol. Cell. Biol. 14, 3772-3781[Abstract]
  27. Parry, G. C., and Mackman, N. (1995) Arterioscler. Thromb. Vasc. Biol. 15, 612-621[Abstract/Free Full Text]
  28. Luque, I., and Gelinas, C. (1998) Mol. Cell. Biol. 18, 1213-1224[Abstract/Free Full Text]
  29. Suzuki, Y. J., Mizuno, M., Tritschler, H. J., and Packer, L. (1995) Biochem. Mol. Biol. Int. 36, 241-246[Medline] [Order article via Infotrieve]
  30. Naumann, M., and Scheidereit, C. (1994) EMBO J. 13, 4597-4607[Abstract]
  31. Hayashi, T., Sekine, T., and Okamoto, T. (1993) J. Biol. Chem. 268, 26790-26795[Abstract/Free Full Text]
  32. Bird, T. A., Schooley, K., Dower, S. K., Hagen, H., and Virca, G. D. (1997) J. Biol. Chem. 272, 32606-32612[Abstract/Free Full Text]
  33. Vogt, P. K., and Bos, T. J. (1990) Adv. Cancer Res. 55, 1-35[Medline] [Order article via Infotrieve]
  34. Ransone, L. J., and Verma, I. M. (1990) Annu. Rev. Cell Biol. 6, 539-557[CrossRef]
  35. Dokter, W. H. A., Esselink, M. T., Halie, M. R., and Vellenga, E. (1993) Blood 81, 337-343[Abstract]
  36. Nikolakaki, E., Coffer, P. J., Hemelsoet, R., Woodgett, J. R., and Defize, L. H. (1993) Oncogene 8, 833-840[Medline] [Order article via Infotrieve]
  37. Derijard, B., Hibi, M., Wu, I. H., Barrett, T., Su, B., Deng, T., Karin, M., and Davis, R. J. (1994) Cell 76, 1025-1037[Medline] [Order article via Infotrieve]
  38. Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E. A., Ahmad, M. F., Avruch, J., and Woodgett, J. R. (1994) Nature 369, 156-160[CrossRef][Medline] [Order article via Infotrieve]


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