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INTRODUCTION |
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-
(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 NF
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 I
B
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.
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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 [
-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
-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
[
32P]ATP (Amersham Pharmacia Biotech) before being
annealed to oligonucleotides of complementary sequence.
TFAP1distal, (
230 to
211)
GCGCGGTTGAATCACTGGGG; AP1p
B (containing the
TF LRE proximal AP1 site and the NF
B site), (
213 to
172)
GGGTGAGTCATCCCTTGCAGGGTCCCGGAGTTTCCTACCGGG;
TF
B, (
193 to
174) GGTCCCGGAGTTTCCTACCG; and the
NF
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 NF
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
-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 NF
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); NF
B p50 (sc-114x); NF
B p52 (sc-298x);
NF
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
-tubulin antibody used in the Western
blot analysis was a kind gift from Dr. Ingrid Gaemers obtained from Dr.
Pavel Draber (21, 22).
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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.
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Binding of AP1 and NF
B Complexes--
The EMSA studies
presented were predominantly carried out using the AP1P
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
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
AP1P
B and an AP1 consensus competitor but not with an
NF
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
AP1P B probe during a time course of LPS induction.
Binding activity to the DNA probe containing the LRE proximal AP1 site
and the 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 (AP1P B, AP1, and NF B) and nonspecific
(Sp1) competitors at 100-fold molar excess were analyzed.
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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 AP1P
B or the NF
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 NF
B complexes to the
oligonucleotide between 30 min and 2 h, of which only complex III
appears to have high affinity for the
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 AP1P
B
oligonucleotide and the NF
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
AP1P
B with both AP1 and NF
B binding simultaneously.
Characterization of the NF
B Complex--
Supershift assays were
carried out with the AP1P
B oligonucleotide, the TF
B-like site oligonucleotide and the NF
B consensus oligonucleotide, and antibodies against specific NF
B/Rel proteins. With AP1P
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 NF
B/Rel family.
The complex formed with the TF
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 TF
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 NF
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 NF
B/Rel family. These data show
complex III to be a p65/c-Rel heterodimer.

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Fig. 3.
Characterization of the NF B
complexes. NF B complexes were analyzed in supershift assays
using antibodies against NF B/Rel proteins (binding buffer 2).
Lanes 1-10 show binding to the AP1P B
oligonucleotide; lanes 11-20 to the TF B-like site
alone; and lanes 21-30 to an NF 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.
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LPS Induces Proteolysis of I
B
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 I
B
, were used. When nuclear
extracts used in the EMSA studies were examined with an antibody
against a cytoplasmic protein (
-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. I
B
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 I B 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 I B proteins
(panel E). M, molecular mass markers.
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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 I
B
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 I
B
expression corresponds to a peak in I
B
mRNA levels at 1 h (data not shown). Nuclear levels of I
B
in Western analysis were
found to be low and constant, confirming breakdown and re-synthesis of
cytoplasmic I
B
rather than translocation. These data are consistent with the proposal that I
B
is proteolyzed after
induction of the NF
B system, releasing the NF
B proteins, which
then translocate to the nucleus (10, 19, 23, 24). An NF
B element in
the I
B
promoter (25) directs the increase in I
B
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 AP1P B and antibodies against
Jun/ATF/CREB proteins (panel A, binding buffer 1) and Fos
proteins (panel B, binding buffer 2).
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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 (
-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 AP1P
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 AP1P 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.
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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.
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 |
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 NF
B complex and the constitutive
binding of AP1 complexes. The NF
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
NF
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 NF
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
B-like site upon LPS stimulation rather than an
NF
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 NF
B activation, we compared
nuclear and cytoplasmic extracts for evidence of nuclear translocation. Our data show the proteolysis of the cytoplasmic inhibitory protein I
B
and the translocation of both p65 and c-Rel from the cytoplasm to the nucleus, corresponding to the appearance of the NF
B complex in gel shift studies. An earlier study charting the nuclear and cytoplasmic levels of the NF
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
NF
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
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 I
B
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 NF
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
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 AP1P
B oligonucleotide and the isolated TF
B oligonucleotide showed that much longer exposure times were required to observe complexes with TF
B than with
AP1P
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
AP1P
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
B-like site (30 min to 2 h) corresponding
to the proteolysis of I
B
and the nuclear translocation of p65 and
c-Rel as predicted by previously proposed models of NF
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 NF
B translocation and binding of p65/c-Rel to the TF
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, I B proteolysis releases p65/c-Rel, which translocate
from the cytoplasm to the nucleus and bind the LRE B-like site.
Ub, ubiquitin; LBP, LPS binding protein;
P, phosphate group.
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