Gene transcription of fgl2 in endothelial cells is controlled by Ets-1
and Oct-1 and requires the presence of both Sp1 and Sp3
Mingfeng Liu
1
, Julian L. Leibowitz
2
, David A. Clark
1,4
, Michael Mendicino
1
, Qin Ning
1
, Jin Wen Ding
1
,
Cheryl D’Abreo
3
, Laisum Fung
1
, Philip A. Marsden
3
and Gary A. Levy
1
1
Multi Organ Transplant Program, Toronto General Hospital and The University of Toronto, Canada;
2
Department of Pathology and
Laboratory Medicine, Texas A & M University System College of Medicine, USA;
3
Renal Division and Department of Medicine,
St. Michael’s Hospital and University of Toronto, Canada;

PRD. These results clearly demonstrate that multiple cis
DNA elements in a clustered region work cooperatively to
regulate constitutive fgl2 expression and interact with indu-
cible elements to regulate viral-induced fgl2 expression in
endothelial cells.
Keywords: fibroleukin; fgl2; hepatitis; immune coagulant;
transcription regulation.
Activation of the coagulation system represents an import-
ant facet of immune and inflammatory reactions, account-
ing for the fibrin deposition that is commonly observed in
these reactions [1]. Cellular procoagulants and the soluble
factors of the coagulation cascade are important partici-
pants in a number of human diseases including allograft
rejection [2,3], glomerulonephritis [4,5], septic shock [6,7],
and bacterial and viral infections [8]. For example, tissue
factor (TF) [9], the transmembrane receptor of factor VII, is
the major procoagulant of the classical extrinsic pathway of
coagulation resulting in thrombin generation, and subse-
quent fibrin deposition. Furthermore, TF has important
roles in the regulation of angiogenesis in cancer, embryonic
blood vessel development, and intracellular signaling
[10–12]. Thrombin is involved in many biological processes.
In addition to its function in blood coagulation and wound
healing, thrombin has diverse functions in many different
cell types. For instance, thrombin induces proliferation of
fibroblasts and smooth muscle cells, and neurite retraction
and synapse reduction in neurons [12]. Moreover, thrombin
is a chemotactic agent for inflammatory cells such as
macrophages and neutrophils [12].
fgl2/fibroleukin was originally described as a fibrinogen-

virus strain 3 (MHV-3) induced fulminant hepatic failure
[21,22]. Administration of neutralizing antibodies against
fgl2 conferred protection against MHV-3 induced fulmi-
nant hepatic failure in susceptible BALB/cJ mice [23].
Humanfgl2wasclonedandshowntobeexpressedin
endothelial cells and macrophages of livers from patients
with hepatitis [20,24]. Expression of fgl2 during both
mouse and human fulminant hepatitis correlates with
fibrin deposition, a typical feature of fulminant hepatic
failure [20,21,25,26].
As a multifunctional protein, fgl2 has a transcription
regulation mechanism that seems to reflect its function in
different cells and developmental stages. Fgl2 mRNA is
constitutively expressed in some cell types [13,20,26],
but transcription can also be robustly induced by viruses
and cytokines, such as interferon-c (IFN-c) [26–28]. Con-
stitutive activity of the fgl2 promoter suggests that fgl2
functions as a matrix/adhesion protein, possibly necessary
for development of a normal fetus and in regulating
immune responses within the intestine. Induction of fgl2
in macrophages in response to viruses results in the
production of a potent coagulant activity with the charac-
teristics of a prothrombinase. This combination of consti-
tutive expression and induction may permit a rapid response
to inflammation. However, the mechanisms regulating fgl2
expression, either constitutive or induced, remain unclear. A
comprehensive understanding of the general transcription
machinery on the native fgl2 promoter is necessary for
developing further insight into the TF-independent coagu-
lation pathway in diseased states. We report here the

Sequencing
Plasmid DNA fragments were sequenced by cycle sequen-
cing on an automated DNA sequencer (Model 377, Applied
Biosystems, Foster City, CA) using dideoxy dye terminator
chemistry and primer-directed strategies. Sequences were
determined on both DNA strands.
Primer extension analysis
A 30 base oligonucleotide (5¢-CCTCCACCGCTCGGCA
GGCAGCGAGGACGG-3¢) complementary to nucleo-
tides 1363–1392 (GenBank Accession AF025817) at the
5¢ end of the mouse fgl2 coding sequence was purified by
polyacrylamide gel electrophoresis and used for the primer
extension reaction. End labelling and primer extension
reactions were performed as described previously [31].
Labelled oligonucleotide (100 000 c.p.m.) was hybridized
with 50 lg of total RNA from MHV-3 infected (6 h)
BALB/cJ mouse peritoneal macrophages. Nucleic acids
were recovered by ethanol precipitation and reverse tran-
scribed with 100 U of MMLV reverse transcriptase
(MMLV-RT). The primer extension products were extrac-
ted with phenol/chloroform, precipitated with ethanol and
analyzed in 8% sequencing gel. The same primer was used
to create a dideoxynucleotide chain termination-sequencing
ladder from a double stranded genomic DNA fragment
loaded adjacent to the primer extension sample.
5¢-RACE analysis
5¢-RACE analysis was performed as previously described
with minor modifications [31,32]. Briefly, 0.5 lgoftotal
RNA extracted from MHV-3 treated (at 10 pfu for 24 h)
Balb/cJ liver and peritoneal macrophages infected MHV-3

with restriction endonucleases SacII and EcoRI. The
fragment was subcloned into the pBluescript plasmid
(Strategen). The transformants were subjected to DNA
sequence analysis.
RT-PCR detection of fgl2
Expression of fgl2 mRNA was assessed using RT-PCR.
Total RNA from BALB/cJ macrophages, macrophages
infected with MHV-3, SVE-10 murine endothelial cells, and
murine small intestine RNA (Clontech, Palo Alto, CA) were
used to synthesize the first strain cDNA. PCR was then
performed in 50-lL reactions using 1-lL portions of cDNA
and the primers fgl2318 (5¢-TGCCCACGCTGACCATC
CA-3¢) corresponding to nucleotides 318–336 of BALB/cJ
fgl2 cDNA (M15761) and fgl21224 (5¢-GAGACAAC
GATCGGTACCCCT-3¢) corresponding to nucleotides
1224–1244 of BALB/cJ fgl2 cDNA. (M16238), which
yield a 906-base pair band in 1% agarose DNA gel after
30 cycles of PCR reaction. Amplification products were not
obtained when reverse transcriptase was omitted (data not
shown). RT-PCR for b-actin was set upas an internal control
to ensure equal loading and first strand synthesis with
forward primer, 5¢-ATGTTTGAGACCTTCAACAC-3¢,
and reverse primer, 5¢-CACGTCACACTTCATGAT
GGA-3¢.
DNA constructs
AsingleSalI site was introduced into pM166 after the
initiating ATG by site directed mutagenesis. The restriction
fragment extending 3.5 kb upstream from the introduced
SalI site was excised by digestion with SalIandSmaI
(utilizing the SmaI contained in the pBluescript vector) and

amplified from a pM166 template using a common sense
primer 5¢-GAATAAGGAGGGCAGGGTGAA-3¢ (posi-
tions )1320 to )1302 in GenBank accession no. AF025817),
and the antisense primers 5¢-TAGTGGGGAAAGA
GTTGGAACG-3¢ for fragment )1320/)274. The PCR
products representing this promoter fragment was sub-
cloned into PCR2.1 and transferred into pGL2-Basic using
the KpnIandXhoI sites in the vector and KpnIandSalIsites
in the PCR2.1 clones of the promoter fragments. All
promoter constructs were sequenced to confirm their
orientation and to verify their sequences.
pCR3.1A59 MHV nucleocapsid protein (N protein)
expression vector was reported previously [33]. The N gene
fragments were subcloned into the expression vector
pCR3.1 (Invitrogen), under the control of the cytomegalo-
virus promoter and bovine growth hormone 3¢-processing
signals.
Drosophila Eukaryotic Expression Constructs Expression
cassettes for Sp1, Sp3 variants, and Ets-1 were based upon
pPacUO, a transient episomal vector which contains the
2.6-kb Drosophila actin 5C promoter, a 0.7-kb 5¢-UTR
Ultrabithorax (Ubx) internal ribosome entry site, the first
eight codons of the Ubx open reading frame and 1.1 kb of
3¢-UTR from the actin 5c gene. These constructs have been
reported previously [30].
Linker scanning analysis
Seven site-directed mutants were created within a 70-bp
region ()119 to )41) of the fgl2 promoter in the
plasmid pfgl2()1320/+9)Luc using primers listed in
Table 1. Each mutant contains the sequence GGTACC,

ted as described [34]. For EMSA, double stranded oligo-
nucleotide probes (Table 2) were 5¢-end-labelled with
[c-P
32
]ATP (Amersham) using T4 polynucleotide kinase.
ForeachEMSAreaction2–5lg of nuclear extracts were
incubatedfor15minonicein20lL of binding buffer.
1 · 10
4
d.p.m. of probe was added to each reaction and the
mixtures were incubated at room temperature for 30 min.
For supershifts, 2 lL of antibodies were incubated with
nuclear extracts for 45 min before adding DNA probe.
Anti-Sp1, Sp3, Stat3, Oct-1, Oct-2, Ets1/2, and PU.1 Ig are
from Santa Cruz Biotechnology Inc. (Santa Cruz, CA,
USA). For competition, 100 · cold oligo were added to the
reaction. Consensus double stranded oligos Oct-1, 5¢-TGT
CGAATGCAAATCACTAGAA-3¢; Sp1, 5¢-ATTCGATC
GGGGCGGGGCGAGC-3¢; Ets/Pea3, 5¢-GATCTCGAG
CAGGAAGTTCGA-3¢; Ets (PU.1), 5¢-GGGCTGCTTG
AGGAAGTATAAGAAT-3¢;Stat3,5¢-GATCCTTCTG
GGAATTCCTAGATC-3¢; and C/EBP, 5¢-TGCAGATT
GGGCAATCTGCA-3¢ are from Santa Cruz Biotechno-
logy. The binding reactions were size-fractionated on a
nondenaturing, 5% acrylamide gel, run at 150 V at room
temperature for 2 h in 1 · Tris/borate/EDTA buffer.
Results
Mapping the transcription start site of
fgl2
gene

fgl2 )85/)66 AAT GCG CCC GCC CTT TTC TG
fgl2 m)85/)66 AAT GCG CCA GGT ACC TTC TG
fgl2 )97/)77 GAC TGT GAT GCA AAT GCG CCC
fgl2 m)97/)77 GAC TGT GAT GCG GTA CCT CCC
fgl2 )76/)56 GCC CTT TTC TGG GAA CTC AGA
fgl2 m)76/)56 GCC CTT TTG AGG TAC CTC AGA
Table 1. Primer pairs used to construct linker-scanning fgl2 promoter mutants. Underlined are actually mutated sequences.
Name Primer sequences
Mu-S)108/)99
CTC CTC CTG TAA GGT ACC ACA GAC TGT GAT GC
Mu-AS)108/)99 GCA TCA CAG TCT GTG GTA CCT TAC AGG AGG AG
Mu-S)97/)91 GTG GCG TCT GAG GTA CCA ATG CAA ATG CGC
Mu-AS)97/)91 GCG CAT TTG CAT TGG TAC CTC AGA CGC CAC
Mu-S)87/)80 GAG ACT GTG ATG CGG TAC CTC CCG CCC TTT C
Mu-AS)87/)80 GAA AAG GGC GGG AGG TAC CGC ATC ACA GTC TC
Mu-S)77/)71 GCA AAT GCG CCA GGT ACC TTC TGG GAA CTC
Mu-AS)77/)71 GAG TTC CCA GAA GGT ACC TGG CGC ATT TGC
Mu-S)68/)59 CCG CCC TTT TGA GGT ACC AGA GAA CGC CTG
Mu-AS)68/)59 CAG GCG TTC TCT GGT ACC TCA AAA GGG CGG
Mu-S)57/)50 CTG GGA ACT CAT GGT ACC ACA GTC AGG CGG
Mu-AS)57/)50 CCG CCT GAC TGT GGT ACC ATG AGT TCC CAG
Mu-S)50/)44 CTC AGA ACG CCA GGT ACC CGC GGC GGT GGC
Mu-AS)50/)44 GCC ACC GCC GCG GGT ACC TGG CGT TCT GAG
Ó FEBS 2003 fgl2 expression regulation in endothelial cells (Eur. J. Biochem. 270) 2277
MHV-3 infected macrophages and transcription was shown
to initiate at a site consistent with our 5¢-RACE results
(lane T, corresponding to a nucleotide A at that position).
Mapping the region essential for fgl2 transcription
in endothelial cells
Recently, BALB/cJ mouse endothelial cells have been

potential cis-acting elements, including Sp1, Ets, Stat3,
Octamer [35] binding sites and AP1 sites (Fig. 3) and these
motifs might be critical for fgl2 expression. Together, the
5¢-deletion and 3¢-deletion analyses indicate that sequences
from )119 through +9 contain sequences that are sufficient
for constitutive fgl2 transcription.
Linker-scanning and EMSA analyses
To gain further insight into the regulation of the fgl2
promoter, the region between )119 to )41 bp, just
upstream of the putative TATA box (Fig. 4A), was
subjected to systematic site-directed mutagenesis [30]. Seven
constructs were created in pfgl2()1320/+9)LUC so that
each construct contained an 8–10 bp mutation (Table 1).
These constructs were transfected into SVE-10 cells and
luciferase activity was assessed. Mutation of position )108
to )99 and )97 to )91 had no significant impact on fgl2
promoter activity. Mutation of positions )87 bp to )80
(containing an Octamer motif 5¢-ATGCAAAT-3¢)
Fig. 1. Mapping of the transcription start site
of fgl2 mRNA. (A) 5¢-RACE of fgl2 mRNA
from macrophages. Stars indicate the 5¢-ter-
minus of 33 clones isolated after 5¢-RACE. (B)
Primer extension analysis of fgl2 mRNA. The
left side of the figure is the sequence ladder
using the same primer and the pM166 clone as
template. Arrow indicates the nucleotide that
is matched to the band present in the primer
extension reaction on the right. The sequence
of the primer is 5¢-CCTCCACCGCTCGG
CAGGCAGCGAGGACGG-3¢. The lanes

not with mutant oligonucleotides (fgl2 m-97/)77, m-85/
)66, and m-76/)56, Table 2). As both probes contain the
Sp1 motif, we predict that DNA-protein complexes could
be formed with Sp1 and Sp3 transcription factors as seen
Fig. 2. Functional analysis of fgl2 promoter. (A) Analysis of fgl2 RNA by semiquantitative RT-PCR. RT-PCR was performed with 4 lgofeach
RNA sample. M, macrophages; M + MHV-3, macrophages infected with MHV-3; endothelial cells, RNA from murine endothelial SVE-10 cells;
intestine, total mouse intestinal RNA. (B) Fgl2 promoter-luciferase constructs. The numbers give the 5¢-ending and 3¢-ending nucleotide of each
construct. (C) Relative luciferase activity of 5¢-truncations of fgl2 promoter constructs in endothelial cells. Activity of pGl2()1320/+9)Luc is set at
100%. (D) Luciferase activity of 3¢-truncation of fgl2 promoter construct in endothelial cells. The data is the average of at least three separate
experiments each performed in triplicate. The data was corrected for variations in transfection efficiency by normalization to the activity of a
cotransfected plasmid expressing b-galactosidase.
Fig. 3. Schematic drawing representing the proximal promoter of the
mouse fgl2 gene. Positive regulatory region (PRD) and functional cis-
regulatory DNA elements are shown. Identified cis elements are
underlined. Boxes indicate mutated regions. Probes used in EMSA are
also shown as underline with specific numbers.
Ó FEBS 2003 fgl2 expression regulation in endothelial cells (Eur. J. Biochem. 270) 2279
in the case of eNOS in vascular endothelial cells [30]. To
test this hypothesis, we performed an EMSA experiment
with a probe containing the fgl2 Sp1 binding site (probe
)85/)66, Table 2 and Fig. 3). As shown in Fig. 5, at least
four nucleoprotein complexes were seen using endothelial
cell nuclear extracts (lane 2). These complexes were
competed away by the same unlabelled probe (lane 3)
but not by an unrelated nonspecific probe (lane 4). The
addition of anti-Sp1 IgG
1
shifted the slowest migrating
complex to a higher position in the gel (lane 5) while anti-
Sp3 antibody detected two forms of Sp3 complexes, the

interaction of Oct-1 with the fgl2 Octamer DNA motif. The
Fig. 4. Linker-scanning analysis of important cis elements in fgl2 pro-
moter. (A) Linker scanning mutations of fgl2 promoter. All mutant
constructs were made using pfgl2()1320/+9)Luc as template. The
numbered boxes indicate the specific nucleotides mutated which were
also shown in Table 1 and Fig. 5. (B) Luciferase activity of wild-type
and mutated fgl2 promoter in SVE-10 endothelial cells. Luciferase
activity was analyzed as described in Fig. 2.
Fig. 5. EMSA analysis of cis elements overlapping the 38 bp identified
in Fig. 4 of the fgl2 promoter. Nuclear extracts (NE) from SVE-10
endothelial cells were incubated with probes shown in Table 2 and
Fig. 5. Arrows indicate the bands that are interacting with these
probes. All the lanes have labelled probes. The adding of extracts and
cold probe are indicated in the top of the panel. Supershift, ss-Sp1
and ss-Sp3, were shown in presence of anti-Sp1 and anti-Sp3 Ig. Cold
and mutated competitors are indicated at the top of the figure.
2280 M. Liu et al.(Eur. J. Biochem. 270) Ó FEBS 2003
nucleocomplex B and C in panel A could represent binding
of the Sp1 protein family, given that the probe contains a
half site of a Sp1 motif (Fig. 3). Consistent with this view, a
consensus Sp1 oligonucleotide blocked the formation of
these complexes (Fig. 6A, lane 12). To assess this possibility,
we performed a supershift assay using antibodies against
Sp1 and Sp3. We did not see a supershift or blocking of the
complexes B and C (data not shown). Thus, it can be
concluded that these complexes are not formed by the
binding of Sp1 or Sp3. However, we cannot rule out a Sp1
related transcription factor or an unknown factor at this
time. Future studies are required to address this issue.
We next examined whether the motifs located between

plex upon the 39 bp PRD region and functionally contri-
bute to the constitutive expression of fgl2, especially in
vascular endothelial cells. To further evaluate this hypo-
thesis, we employed the Drosophila Schneider cell line
(SL2), which is deficient in constitutive Sp1, Sp3, and Ets-1
transcription factors [36,37]. As shown in Fig. 8A, cotrans-
fection of the pfgl2LUC()1320/+9) promoter, along with
increasing amounts of Sp1 expression cassette resulted in a
concentration-dependent increase in functional fgl2 pro-
moter activity (22-fold maximum effect). Increasing
amounts of a Sp3 expression cassette also increased fgl2
promoter activity in a concentration-dependent fashion,
though to a lesser extent (Figs 8B, fivefold increase).
However, cotransfection of increasing amounts of an Ets-1
expression cassette alone had no important effect on fgl2
promoter activity (Fig. 8C). To examine whether Sp1, Sp3,
and Ets-1 exert an interactive effect on fgl2 promoter
activity, we cotransfected combinations of the varied
transcription factors. As shown in Fig. 9A, cotransfection
Fig. 6. EMSA analysis of the function of Octamer motif in the fgl2 promoter. Lanes 2–5 of panel A and lanes 2–5 of panel B, NE were incubated with
labelled consensus Oct-1. Lanes 6–12 of panel A and lanes 6–8 of panel B, NE were incubated with
32
P-labelled fgl2 fragment )97/)77. All lanes
contained hot probes. Addition of NE, cold probe competitors, and antibodies are indicated at the top of the panel. (A) Cross competition with
consensus Oct-1 and fgl2–97/)77. (B) Antibody analysis of Oct-1 binding in the presence of antiOct-1 and antiOct-2 antibody. Arrows indicate the
specific Oct-1 DNA-protein complexes. All probes are shown in Table 2 and Fig. 5.
Ó FEBS 2003 fgl2 expression regulation in endothelial cells (Eur. J. Biochem. 270) 2281
of increasing amounts of Sp3 along with 50 ng of Sp1 (half-
maximum amount) demonstrated increased activity of fgl2
promoter compared to Sp1 or Sp3 alone (Fig. 9A). In

competition of Stat3 DNA cis element with its binding protein. Consensus Stat3 was labelled and competes with cold fgl2 oligonucleotide
)76/)57 (Fig. 5).
Fig. 8. Sp1, Sp3, and Ets-1 transactivate mouse fgl2 promoter/reporter
luciferase constructs in Drosophila Schneider cells. Assay of pro-
moter activity of pfgl2LUC()1320/+9) promoter/reporter luciferase
construct cotransfection with increasing amounts of pPacUSp1
(10–250 ng, panel A), pPacUSp3 (10–250 ng, panel B),and pPacUEts-1
(25–250 ng, panel C). Luciferase activity was analyzed as described in
Fig. 2.
2282 M. Liu et al.(Eur. J. Biochem. 270) Ó FEBS 2003
the nucleocapsid (N) protein of MHV-3 can induce
transcription of fgl2 and implicated regions of the fgl2
promoter located )372 to )306 bp upstream of the
transcription initiation site [33]. Therefore, we undertook
studies to determine the contribution of the PRD at
nucleotide )87 to nucleotide )49 that is implicated in the
constitutive expression of fgl2 to the induced transcription of
fgl2 in response to MHV-3. We examined whether a
mutation of the PRD would abolish the induction of fgl2
by MHV-3 nucleocapsid protein. As shown in Fig. 10, we
cotransfected plasmid pfgl2()1320/+9) with a constitu-
tively active nucleocapsid protein expression plasmid
(pCR3.1A59) into endothelial cells. Co-transfection of the
N gene expressed by pCR3.1A59 induced a 5.5-fold activa-
tion of the fgl2 promoter relative to that observed with
cotransfection with empty vector (pCR3.1). Mutation of the
Sp1/Sp3 (mut4) site completely abolished this activation.
Discussion
Evidence indicates that inducible expression of fgl2 correlates
with the vascular thrombosis of the liver seen in fulminant

–/–
mice, in
which the fgl2 coding region is replaced with b-galactosidase
(unpublished data). The constitutively expressed fgl2 might
reflect functional contributions of fgl2 as a matrix/adhesion
protein, possibly necessary for development of a normal
fetus.
In this study we have functionally characterized the
5¢-flanking region of the mouse fgl2 gene. We mapped the
5¢-terminal region of the fgl2 mRNA using primer extension
analysis and 5¢-RACE. Sequence analysis revealed a TATA
box (TATTAAA) located 33 bp upstream of the major
transcription start site. The results of transient transfection
with a 3¢-deletion construct that removed this major
transcription start site and TATA box ()1320/)274)
provided functional evidence for this conclusion.
When we performed functional analysis of the 5¢-flanking
region of the fgl2 gene, we demonstrated in murine
endothelial cells that a region 119 bp upstream from the
transcriptional start site and 9 bp downstream are sufficient
for the basal expression of the fgl2 gene. This finding is
consistent with the observation that fgl2 is constitutively
expressed in cultured endothelial cells, as well as in the
primary endothelial cells in low level. Although the maximal
activity we observed with the fgl2 promoter is approxi-
mately 10% of the strong SV-40 promoter/enhancer, this
is not an unusual finding. For example, the promoter of the
constitutively expressed human endothelial NOS promoter
evidences approximately 10% of the activity of the SV-40
promoter/enhancer [30].

and an endothelial cell-restricted cofactor [40]. We also
identified that the Ets/Stat3 motifs are functional and
contribute to fgl2 constitutive expression in vascular endo-
thelial cells (Fig. 7). Ets proteins have been shown to be
promiscuous in their binding to the same GGAA core
sequence [41]. Consistent with reports from others, we did
not detect supershifts with antiEts1/2 and antiPU.1 anti-
body. However, we did demonstrate that both Ets1/Pea3
consensus and PU.1 consensus oligonucleotides were able to
inhibit the formation of nucleoprotein complexes with the
fgl2 GGAA core.
It is known that Ets family members generally serve as
coactivators for the transcriptional activator Sp1 and
synergistically regulate gene expression [42]. The presence
of an Ets site immediately proximal to the Sp1 site in the fgl2
promoter may explain the constitutive expression of fgl2.
With our EMSA results (Fig. 7) suggesting the involvement
of Ets family members in the regulation of fgl2 promoter,
we found that Ets-1, a major Ets family member in
endothelial cells, participated in activation of the fgl2
promoter. Our results suggested that Ets-1 can cooperate
with Sp1/Sp3 in a positive manner in Drosophila cells that
areknowntobedeficientinconstitutivelyexpressedEtsand
Sp1 protein family members (Figs 8 and 9). Removing any
one of these factors from the system or mutating any of
these two activator recognition sites found in the PRD
resulted in a marked decrease in functional promoter
activity.
Another motif, TTCTGGGAACT that overlaps the Ets
binding site, differed by one nucleotide from the consensus

115, 80 and 78 kDa that are abundantly expressed in a
broad number of mammalian cells. In EMSA, the two
shorter forms comigrate as a fast protein–DNA complex,
while the longer form migrates as a slower protein–
DNA complex [47]. In addition, Sp1 has also been
established to be responsible for induced expression of
IL-10 [48]. Our data from endothelial cells and the
Drosophila system, supports the contention that both Sp1
and Sp3 positively affect fgl2 constitutive expression.
Importantly, the presence of both Sp1 and Sp3 is required
for Ets-1 function.
Taken together, our data suggest that DNA regions
spanning 39 bp, from )87 to )49 of the fgl2 promoter,
represent functional cis-DNA regulatory elements that
interact with Oct-1, Ets-1, and Sp1/Sp3. As fgl2 expression
is tightly controlled [49], the interaction of transcription
factors Sp1/Sp3 with the fgl2 promoter form a core to
recruit both Oct-1 and Ets-1 to form a multicomponent
PRD that is sufficient to confer basal level expression of fgl2
in endothelial cells. In addition, this PRD is required to
interact with inducible elements upon activation to enhance
fgl2 expression. This mechanism may explain how fgl2 can
be both constitutively transcribed and rapidly responsive to
proinflammatory stimuli. Although the observations pre-
sented in this study suggest that this PRD is necessary for
the inducible expression of fgl2, we plan to undertake
additional studies aimed at more precisely elucidating
specific mechanisms regulating induction of fgl2 promoter
activity in response to proinflammatory stimuli such as
IFN-c or other viral factors.

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