(Received for publication, August 31, 1995; and in revised form, March 11, 1996)
From the
These studies examine the molecular basis for increased transcription of tissue factor (TF) in THP-1 cells stimulated with lipopolysaccharide (LPS). DNase I footprinting identified six sites of protein-DNA interaction between -383 and the cap site that varied between control and induced extracts. Four footprints show qualitative differences in nuclease sensitivity. Footprints I (-85 to -52) and V (-197 to -175) are induction-specific and localize to regions of the promoter that mediate serum, phorbol ester, partial LPS response (-111 to +14), and the major LPS-inducible element (-231 to -172). Electrophoretic mobility shift assays with the -231 to -172 probe demonstrate JunD and Fos binding in both control and induced nuclear extracts; however, binding of c-Jun is only detected following LPS stimulation. Antibody inhibition studies implicate binding of Ets-1 or Ets-2 to the consensus site between -192 and -177, a region that contains an induction-specific footprint. The proximal region (-85 to -52), containing the second inducible footprint, binds Egr-1 following induction. These data suggest that LPS stimulation of THP-1 cells activates binding of c-Jun, Ets, and Egr-1 to the TF promoter and implicates these factors in the transcriptional activation of TF mRNA synthesis.
Factor VII/VIIa bound to the cellular receptor tissue factor
(TF), ()initiates both the intrinsic and extrinsic
coagulation pathways by activating Factors IX and X (reviewed in Refs.
1, 2). Constitutive expression of TF is detected in many cell types
that do not normally contact blood, providing a hemostatic barrier to
initiate coagulation following injury(3) . Ischemic tissue
damage following intravascular clotting (Schwartzman reaction) is
produced when LPS-stimulated monocytes are introduced into leukopenic
rabbits(4) . LPS induction of TF expression in monocytes of
patients with sepsis causes disseminated intravascular coagulation,
which has a high mortality rate in humans. In the baboon model,
administration of either anticoagulants (5) or neutralizing
antibodies against TF (6) prevents LPS-induced disseminated
intravascular coagulation.
Monocytes are the only circulating cells
that modulate expression of TF, and synthesis can be up-regulated by a
number of agonists including tumor necrosis factor-(7) ,
lipopolysaccharide (LPS)(8, 9) , and phorbol ester
(PMA, (10) ). TF mRNA is very unstable with a half-life of
60-90 min. In LPS-induced monocytes, the increase in TF mRNA
results from transcriptional activation(8) , whereas, in the
monocytic cell line THP-1, LPS induces both an increase in
transcription and changes in mRNA stability(11) . Functional
studies have demonstrated that promoter sequences between -383 to
+121 support high level constitutive expression(12) . In
the context of the TF promoter, two regions appear to be involved in
maximal induction in LPS-stimulated THP-1 cells (-227 to
-172 and -96 to -33); however, a short
oligonucleotide from the TF promoter (-192 to -172) that
contains an isolated NF-
B/Rel motif is sufficient for LPS
induction. A minimal promoter from -111 to +121 mediates
basal expression and serum induction in Cos-7 cells(13) , serum
and PMA induction in HeLa cells(14) , and tumor necrosis
factor-
induction in endothelial cells(15) . We have also
determined that the -111 to +8 promoter mediates LPS
induction in THP-1 cells and PMA induction in HL-60 cells. (
)
In the present study, we have characterized protein
interactions with the TF promoter using nuclear extracts derived from
control and LPS-induced THP-1 cells. DNase I footprint analysis showed
six regions of nuclease protection and two sites of LPS-induced protein
binding. Electrophoretic mobility shift assays (EMSAs), oligonucleotide
competitions, and antibody supershift studies were used to characterize
the specific proteins that interact with DNA at these sites. Sp1 and
Sp3 interact with the promoter in both control and induced extracts.
Upon induction there is a change in the AP-1 binding from JunD/Fos in
control cells to c-Jun/Fos and JunD/Fos in LPS- induced cells. In
contrast to other reports, we have no evidence for NF-B or Rel
binding to the -192 to -172 site; rather, our data suggest
that either Ets-1 or Ets-2 binds to this sequence following induction.
Finally, in induced nuclear extract, we detect binding of Egr-1 to the
consensus site between -85 and -52. These data suggest that
LPS stimulation of TF expression results from induction of c-Jun, Ets,
and Egr-1 binding to the TF promoter.
Figure 1:
DNase I protection
analysis of pTFPXSma. The 224-bp fragment was labeled at the XbaI site (coding strand, A) or at the EcoRI
site (noncoding strand, B). Footprints are indicated by brackEts and roman numerals. Regions of protected
sequence are indicated by the numbers flanking each footprint
and refer to nucleotides upstream of the TF cap site. G+A, Maxam and Gilbert G + A reaction
sequencing ladder; DNase I, no extract; THP-1 and THP-1(+LPS), 20 µg of nuclear
extract prepared from control THP-1 or LPS-induced THP-1
cells.
Footprints generated with the l67-bp probe (-280 to -113) are shown in Fig. 2. Protein(s) derived from both control and induced extracts generated footprint III (-150 to -120), although induced extracts protected this region of DNA slightly more efficiently. Proteins that protected the nucleotides within footprints IV and VI were present in both control and induced extracts. In contrast, footprint V was only detected when LPS-induced extracts were used. In addition, a strong DNase I-hypersensitive site, located at -197, appeared at the junction of footprints V and VI on the coding strand with the LPS-induced extracts. As summarized in Fig. 3, footprints IV (-175 to -153), V (-197 to -175), and VI (-220 to -197) contain consensus binding sites for numerous proteins. The perturbations in DNase I cleavage visualized when control and induced nuclear extracts are compared suggest that LPS may qualitatively or quantitatively affect the proteins available to interact with these sequences.
Figure 2: DNase I protection analysis of pTFPSma. The 167-bp fragment was labeled at the EcoRI site (coding strand, A) or the XbaI site (noncoding strand, B). Footprints are indicated by brackEts and roman numerals. Regions of protected sequence are indicated by the numbers flanking the brackEts and refer to nucleotides upstream of the TF cap site. G+A, Maxam and Gilbert G + A reaction sequencing ladder; DNase I, no extract; THP-1 and THP-1(+LPS), 20 µg of nuclear extract prepared from control or LPS-induced cells.
Figure 3: Summary of DNase I protection analysis. Footprints are numbered I-VI. The bracketed regions below roman numerals display footprinted regions. Numbers flanking each footprint designate the protected nucleotide sequences relative to the TF cap site. Transcription factor consensus sequences identified by computer-assisted searches are displayed below the relevant footprints. PCR-generated probes used in EMSAs are shown below the binding site homologies.
Oligo 60, which
contains two AP-1 sites and an overlapping NF-B/Rel/Ets site,
interacts with proteins in both control and induced extracts, and there
was a qualitative difference in the pattern of migration of the
complexes (data not shown). In order to improve these binding studies,
Oligo 87 (-231 to -145) was synthesized to extend the
region slightly and include an Sp1 sequence (-167 to -162)
and a CACCC sequence (-157 to -153). Competitive gel shift
analysis performed with Oligo 87 is displayed in Fig. 4.
Specific binding to this probe was detected with both control and
induced extracts, although the induced extract produced a broader area
of protein-DNA complex migration. Binding was specific as indicated by
the effective competition of a 100
molar excess of unlabeled
probe. The addition of a 100
molar excess of an AP-1 consensus
oligo competed for most of the binding activity; an Sp1 consensus oligo
competed for some of the binding activity in the LPS-induced extract,
and the combination of both the AP-1 and Sp1 effectively competed for
binding in both control and LPS-induced extracts. No competition was
observed with the NF-
B or Ets consensus oligos.
Figure 4:
EMSAs with Oligo 87 corresponding to
footprints IV-VI. Control, 5 µg of control nuclear
extract; LPS, 5 µg of LPS-induced nuclear extract.
-, nuclear extract without competitor; Self, 100
molar excess of unlabeled Oligo 87; AP1, NF-
B,
and Sp1, 100
molar excess of each consensus
oligonucleotide. BrackEts to the left and right indicate the mobility of specific protein-DNA complexes. Diagram below EMSA indicates the relative location of consensus
binding sites within the probe.
Most of the
binding to Oligo 60 was also competed with the AP-1 consensus oligo,
although no competition was observed when consensus oligos for
NF-B, Ets, or Sp1 were included in the binding reaction (data not
shown). Antibody supershift assays were performed with Oligos 60 and 87
to characterize the transcription factors interacting with this region (Fig. 5). Induced extract contains an increased level of binding
to these probes, which may contribute to the difference in mobility of
protein-DNA complexes seen when control and induced extracts are
compared. In Fig. 5A, proteins bound to Oligo 60 were
recognized by antibodies against both the Jun and Fos families of
transcription factors since these antibodies supershifted the
protein-DNA complexes (indicated by arrows to the right of the
EMSA). The
-Jun antibody effectively supershifted the entire
binding complex with the control extract and also produced a
supershifted complex with the LPS-induced extract, although this
complex was more diffuse and there was residual binding that was not
completely supershifted. The
-Fos antibody also produced
supershifted complexes with both the control and induced extracts.
These supershifted complexes appeared to be similar in both the control
and induced extracts; however, this complex appeared to slightly more
abundant with the LPS-induced extract.
-Jun and
-Fos
antibodies together supershifted all of the binding complexes that
recognized Oligo 60.
Figure 5: Supershift assays using Oligo 60 and Oligo 87. Control, 5 µg of control nuclear extract; LPS, 5 µg of LPS-induced nuclear extract. -, binding reactions without antibodies. Specific antibodies used are indicated above the lanes. A, complexes formed with Oligo 60; B and C, complexes formed with Oligo 87. Bold dot to the right of C indicates the mobility of a slower migrating complex which forms upon longer incubations of probe with nuclear extract. Arrows to the right indicate the specific supershifted complexes. BrackEts indicate u = uninduced (control) and I = induced complexes. Diagram below each EMSA indicates the relative location of consensus binding sites within the probe.
The binding complexes formed with Oligo 87 were
tested with the same -Jun and
-Fos antisera (Fig. 5B). Although both antibodies produced
supershifted complexes when tested separately and in combination, there
was residual binding activity present in both control and induced
extracts. The protein complexes formed with Oligo 87 were tested with
additional antibodies (Fig. 5C), and the incubations
were extended to 90 min to increase the sensitivity of the assay. The
longer incubation resulted in the appearance of a slower migrating
complex which was much stronger with induced extract (indicated by the dot to the right of the EMSA). Both
-Sp1 and
-Jun
antisera produced a supershifted complex indicated by the arrow at the right of C. Neither
-Rel nor
-NF-
B
(data not shown) antibodies were able to recognize proteins that
interacted with Oligo 87. In contrast, the
-Ets antibody abrogated
the binding of the upper complex which formed upon longer incubations.
This higher mobility complex was also missing in reactions containing
LPS-induced extract and the
-Sp1 and
-Jun antisera.
Since
the TF promoter contains two AP-1 consensus sequences, we were unable
to determine whether or not Jun homodimers, Jun-Fos heterodimers, or a
mixture of both were present in the control and induced nuclear
extracts using either Oligo 60 or Oligo 87 (Fig. 5). Fig. 6demonstrates the results of EMSA with a probe containing a
single AP-1 consensus site. These data suggest that all of the AP-1
binding activity consists of Jun-Fos heterodimers, since the -Fos
antisera supershifts all of the complexes in both control and induced
extracts with both Oligo 60 (Fig. 5A) and a probe
containing a single AP-1 site (Fig. 6). Fig. 6also
demonstrates the activation of c-Jun binding activity in THP-1 cells
induced with LPS. In order to determine whether different Jun family
members were binding to the TF promoter in control and induced nuclear
extracts, a panel of
-Jun antibodies was tested for reactivity
against AP-1 complexes that bound to Oligo 87 (Fig. 7). The
pan-Jun antiserum supershifted complexes in both control and induced
extracts. The
-JunD effectively supershifted the entire complex
with control extracts (indicated by the bracket at the left) but left residual binding with LPS-induced extracts
(indicated by the dotted line at the right).
Incubation of induced extracts with a combination of
-c-Jun and
-JunD effectively supershifted the entire complex.
Figure 6: Supershift assays using AP-1 consensus oligonucleotide. Control, 5 µg of control nuclear extract; LPS, 5 µg of LPS-induced nuclear extract. -, binding reactions without antibodies. Specific antibodies used are indicated above the lanes. Arrow to the right indicates the specific supershifted complexes, and bracket indicates control and induced complexes.
Figure 7: Supershift assays using Oligo 87. Control, 5 µg of control nuclear extract; LPS, 5 µg of LPS-induced nuclear extract. P, probe without nuclear extract; -, binding reactions without antibodies. Specific antibodies used are indicated above the lanes. Solid arrows indicate specific supershifted complexes; open arrow indicates mobility of Sp1 and Sp3 complexes. The bracket on the left indicates complexes in control extract, and the dotted line at the right indicates AP-1-specific complexes in induced nuclear extract. Diagram below EMSA indicates the relative location of consensus binding sites within the probe.
As
summarized in Fig. 3, Oligo 53, which spans the inducible
footprint I, contains consensus sequences for Egr-1/2, AP-2, and two
Sp1 sites, one of which overlaps the Egr-1/2 site. EMSAs with Oligo 53
are shown in Fig. 8. With control extract, two specific bands
are produced indicated by the solid arrows. The lower band was
competed by cold Oligo 53, and an Sp1 consensus oligo competed for
binding to both bands. The induced extract produced an additional
complex that migrated between these two bands, indicated by the open arrow. The addition of cold Oligo 53 effectively competed
for the binding activity and reduced the intensity of this
LPS-inducible complex. As a nonspecific competitor, an oligo for the
Ets binding site was included in the binding reaction and was
ineffective. A combination of the Ets and Sp1 competitor oligos was as
effective as Sp1 alone at titrating the binding to this oligo. Since
one Sp1 consensus site (-78 to -71) overlaps the Egr
consensus site (-81 to -73), an additional competition
experiment was performed to test the effect of increasing the
concentration of cold Sp1 consensus oligo on the binding of the complex
that was observed with LPS induction (Fig. 8B). As the
concentration of cold Sp1 competitor increased from 200 to 400 (lanes 3 and 4), the intensity of this inducible band
appeared to increase. A further increase in concentration of cold Sp1
to 800
actually reduced binding of the inducible band, which
may be due to nonspecific effects of excess DNA in the binding
reaction. Antibody supershifts were performed with Oligo 53 (Fig. 8C).
-Sp1 produced a supershifted complex
with both the control and induced extracts. With LPS-induced extract,
-Egr-1 abrogated the binding of the inducible complex. As a
nonspecific control,
-Ets-1/2 had no effect on any of the
complexes formed with this probe.
Figure 8: EMSA and supershift assay using Oligo 53 corresponding to footprint I. Control, 5 µg of control nuclear extract; LPS, 5 µg of LPS-induced nuclear extract. P, probe without nuclear extract; -, binding reactions without oligonucleotide competitor (A and B) or antibodies (C). A, competitor oligonucleotides are indicated at the top of the panel and are added in 200-fold molar excess of each consensus oligonucleotide. B, competition assays with increasing concentrations of the Sp1 consensus oligo. -, no competitor; 3rd to 5th lanes, 200-, 400-, and 800-fold, respectively, Sp1 consensus oligo. C, antibody supershift assay. Specific antibodies used are indicated above lanes. Solid arrows indicate complexes formed with control and induced nuclear extracts; open arrow indicates specific complex formed with induced extracts. Diagram below each EMSA indicates the relative location of consensus binding sites within the probe.
LPS activation of monocytes stimulates protein tyrosine
kinases, protein kinase C, and protein kinase A(21) . Both LPS
and TPA signal through protein kinase C to activate Raf-1 and mitogen
activated protein kinase(22) , and activation of this pathway
is required for monocyte differentiation and induction of c-Jun and
c-Fos mRNA expression in response to LPS(23, 24) . Jun
and Fos represent families of transcription factors that bind AP-1
sites, 5` TGA G/C TCA 3` (reviewed in (25) ). These
transcription factors share common structural motifs: members dimerize
through interaction at the leucine zipper, they bind to DNA via a basic
region adjacent to the leucine zipper, and they contain additional
modulatory domains(26) . Although the Jun proteins can bind to
DNA as homodimers or heterodimers, the Fos proteins bind DNA only as
obligate heterodimers(27, 28) . Heterodimerization of
Jun and Fos with select members of transcription factor families that
share the leucine zipper motif, i.e. ATF (29) and
C/EBP(30) , has also been reported. We have evaluated
C/EBP binding to the TF promoter and have not detected any (data not
shown).
Phosphorylation of c-Jun on serine and threonine at three
sites in the carboxyl terminus just upstream of the basic region, by
casein-kinase II and glycogen synthase kinase-3, prevents AP-1
binding(31) . In human epithelial cells and fibroblasts,
activation of a phosphatase by protein kinase C results in
dephosphorylation of the latent form of c-Jun within 15-60 min
following incubation with TPA, resulting in an increase in binding to
the TPA response element which is recognized by AP-1(32) .
Changes in phosphorylation of sites in the amino terminus modulate the
function of the A1 domain to mediate transcriptional activation, and
the and
domains that interact with the cell-specific
inhibitory protein IP-1(33, 34) . Serine
phosphorylation by a Ha-Ras-induced c-Jun nuclear kinase causes an
increase in DNA binding(35) , whereas phosphorylation by
mitogen activated protein kinase enhances transcriptional
activation(36) .
Expression of TF in THP-1 cells is rapidly activated within 60 min after LPS addition and is mediated by protein kinase C(12, 37) . The TF promoter contains two AP-1 sites that are required for optimal induction(12) . Since binding studies with a single AP-1 consensus site detect Jun-Fos heterodimers exclusively in both control and induced nuclear extracts, we conclude that AP-1 heterodimers bind to the TF AP-1 consensus sequences (Fig. 6). In addition, these studies demonstrate the induction of c-Jun binding activity in THP-1 cells incubated with LPS. In order to evaluate AP-1 binding in the context of the TF promoter, supershift assays with antibodies that recognize Jun and Fos family members were performed with Oligo 60 (Fig. 5A) and Oligo 87 (Fig. 5, B and C, and Fig. 7). These studies clearly demonstrate that the AP-1 binding in control extracts is due to binding of JunD/Fos heterodimers, whereas, in induced extract, both JunD/Fos and c-Jun/Fos heterodimers interact with the -220 to -197 region of the promoter.
To characterize
the additional binding activity detected with Oligo 87, several
different antibodies were tested (Fig. 5C). In order to
increase the sensitivity of the antibody-protein interaction, the
binding time was increased in these supershift assays and, as a result,
a slower migrating complex appeared with the LPS-induced extract. The
specificity of this complex was confirmed by a self-competition assay
(data not shown). The binding that resulted in the slower migrating
complex was abrogated when incubated with the -Ets antibody that
cross-reacts with Ets-1 and Ets-2. There are also two potential Sp1
binding sites downstream of the Ets site within this region including a
GC box and a CACCC sequence(38) . These studies also
demonstrate Sp1 binding to this probe. Interestingly, both the
-Jun and the
-Sp1 antisera interfered with the formation of
this higher mobility complex. Neither
-c-Rel nor
-NF-
B
antibodies (data not shown) interfered with any of the binding
complexes. In the TF promoter, the Ets consensus core (-181 to
-177) overlaps the NF-
B consensus sequence (-187 to
-179) and is located between the AP-1 and Sp1 binding sites. With
induced extract, interference with either AP-1, Sp1, or Ets binding by
including specific antisera in the reaction prevents formation of the
slower migrating complex, suggesting that there might be some
cooperative interactions among the proteins that interact with Oligo
87.
The Ets family of transcription factors contains many members,
including Ets-1, Ets-2, Elk, Erg, PU.1, and PEA3, that share a common
Ets domain and bind to a number of consensus sequences, all of which
contain a GGAA core (reviewed in Refs. 39, 40). Ets-1 (p39-p51) and
Ets-2 (p56) are nuclear phosphoproteins that are highly conserved
between species(41) . Ets-1 and Ets-2 bind weakly to DNA,
although this binding appears to be strengthened, and transcriptional
activation enhanced, either by the presence of more than one binding
site(42, 43) or by interactions with other adjacent
transcription factors like AP-1, GABP, SRE:SRF, and
Sp1(44, 45, 46, 47) . Synergism
between AP-1 and Ets-2 activates keratin 18 expression in embryonal
carcinoma cells differentiated with retinoic acid(48) . In
activated T-cells, cooperative binding of AP-1 and the Ets-related
protein Elf-1 are required to activate transcription of the granulocyte
macrophage colony-stimulating factor gene through the PB-1
element(49) . Interestingly, intracellular signals that
activate TF expression, such as protein kinase C, and increases in
intracellular Ca
also appear to induce Ets-2 mRNA
synthesis and increase the stability of the
protein(43, 50) . In fact, induction of the macrophage
scavenger receptor in TPA-stimulated THP-1 cells results from
activation of ras and induction of c-Jun, JunB, and Ets-2 binding to
several juxtaposed AP-1/Ets binding sites in the promoter(51) .
We have suggested that the
B site contains an overlapping PEA3
element that might be important for TF function(20) . In fact,
we have recently determined that the -188 to -175 region
contains very strong homology (11/12 nucleotides) to sequences
recognized by Ets-2. (
)In addition, we have demonstrated
that an antibody to Ets-1/Ets-2 interferes with induced nuclear protein
binding to Oligo 87 (Fig. 5). These data suggest that either
Ets-1 or Ets-2 interacts with the TF promoter following LPS stimulation
and may contribute to LPS induction.
The identity of the proteins
that interact with the TF B-like site following LPS induction
remains controversial. It has been well documented that LPS activation
of monocytes induces a rapid translocation of NF
B-related proteins
from the cytoplasm into the
nucleus(12, 20, 52) . Comparison of the TF
sequence with optimal
B/Rel DNA-binding motifs suggests that two
differences from the p50 consensus precludes binding of NF-
B p50;
however, the sequence does conform to the optimal binding sites for
both p65 and c-Rel(53) . We have reported weak
binding(20) , and others (12, 15, 54) have shown that
B/Rel
proteins from induced cells can bind to the TF site when isolated from
the context of the promoter. More importantly, the data from this study
suggest that these proteins do not bind to the site in the context of
the TF promoter. Neither oligonucleotide competition nor antibody
supershift experiments with both p50-specific and p65/c-Rel-specific
antisera have demonstrated any interaction of these proteins with
either Oligo 60 or Oligo 87.
Functional studies have clearly
demonstrated the importance of the -227 to -172 region for
promoter inducibility. Furthermore, the data suggest that cooperative
interactions in this region are required for optimal
induction(12) . When linked to a heterologous promoter, the
-192 to -172 oligonucleotide containing the TF
NFB-like site (-188 to -179) confers LPS induction on
reporter constructs that are 80% of the levels observed with a larger
fragment (-227 to -172) containing the two upstream AP-1
sites and the NF
B-like site. These data are in conflict, however,
with other experiments in the same report that suggest that the
-188 to -179 region is unable to support high level
induction when considered in the context of the TF promoter. In fact,
those studies suggest that the AP-1 motifs (-244 to -194)
are responsible for 75% of the LPS inducibility, and the other 25%
actually resides between -96 and -33. Finally, mutation of
either the AP-1 sites or the
B site dramatically reduces induction
by LPS in monocytes and tumor necrosis factor-
in endothelial
cells(15) . Even a 3-bp mutation introduced into the
B
site of the -227 to -172 fragment (pTFM1) reduces
inducibility by almost 80%. Interestingly, those three nucleotides are
in the critical core sequence (GGAA) common to all Ets-related
consensus sequences. This mutation, which prevents
B-related
binding, would also certainly abrogate Ets binding.
When considered as an independent element, the -192 to -172 sequence may demonstrate a different pattern of protein binding and regulation from this sequence in the context of the TF promoter. The ability of proteins to interact with a consensus sequence can depend on the length of the probe and the effects of neighboring binding sites. For example, AP-1 binding affinity can either be strengthened or weakened depending on the sequences that flanked the binding site(55) . These methodological differences between our analysis and other published reports may account for the differences in results, because our findings suggests that Ets and AP-1 interact with the TF promoter following LPS stimulation of THP-1 cells and mediate activation at this site.
The proximal promoter contains consensus binding sites for several additional proteins including AP-2, Sp1, Egr-1, and Wilms' tumor protein, which has been reported to bind to the Egr-1 consensus(56) . When the Oligo 53 probe was used no AP-2 binding was detected by either oligonucleotide competition or antibody supershift studies and no Wilm's tumor protein binding was detected by antibody supershift studies (data not shown). The TF promoter contains five Sp1 sites between -231 and the cap site and two Sp1 consensus sequences are located in the -80 to -57 region. The zinc finger protein Sp1 is important for expression of a number of genes, including CD11b and CD14 in myeloid cells(57, 58) . Another family member, Sp3, acts as a repressor by binding to both GC and GT boxes and antagonizing Sp1 activation at these sites(59) . Our studies have determined that both Sp1 (Fig. 8) and Sp3 (data not shown) bind to Oligo 53 in both control and induced extracts, although the effect of Sp3 binding on TF expression remains to be resolved.
Finally, we have
identified Egr-1 binding exclusively in induced nuclear extracts. Egr-1
is a member of a gene family of zinc finger-containing nuclear
phosphoproteins that bind to the consensus sequence GCGGGGGCG (reviewed
in (60) ). Expression of Egr-1 is rapidly up-regulated in cells
as an immediate-early response gene(61, 62) .
Expression and DNA binding is stimulated in fibroblasts by serum,
phorbol ester, and the protein phosphatase inhibitor okadaic
acid(63) , and in monocytes by serum and
cytokines(64) . In HL-60 cells, Egr-1 is induced by PMA, and
expression restricts the cells to macrophage
differentiation(65, 66) . In other studies, we have
demonstrated an association between Egr-1 expression and induction of
TF synthesis in HL-60 cells. The proximal promoter
(-111 to +8) linked to the CAT reporter gene is
LPS-inducible in both THP-1 and PMA-inducible in HL-60 cells (data not
shown). In other reports, sequences between -96 to -33
support partial induction at 25% of the level of the fully inducible
promoter fragment (-44 to +121)(12) . These data
suggest that this site, in addition to the upstream region, contributes
to LPS induction of TF in response to LPS. We have identified an
inducible binding activity that interacts between -85 to
-52, a region of the promoter that contains two Sp1 sites, one of
which overlaps an Egr-1 site. EMSA and antibody supershift studies
indicate that both Sp1 and Egr-1 bind to this DNA element; however, the
inducible binding activity that appears in LPS-stimulated cells is
Egr-1.
We have used a combination of DNase I footprinting and EMSA to characterize protein-DNA interactions with the TF promoter, using nuclear extracts prepared from control and LPS-induced THP-1 cells. Sp1 binding is detected in both control and induced extracts. Following induction, we see a conversion in AP-1 binding activity from JunD/Fos in control extract to c-Jun/Fos and JunD/Fos in induced extract. In addition, we detect inducible binding to two regions of the promoter (-197 to -175) and (-85 to -52). Our antibody studies suggest that Ets-1 or Ets-2 interacts with the distal site and Egr-1 interacts with the proximal site. In conclusion, we would like to suggest that AP-1/Ets interactions synergize to enhance expression from the -227 to -172 element. In addition, the binding of Egr-1 to the proximal site makes a significant contribution to the transcriptional activation of TF in activated monocytes.