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INTRODUCTION |
Leukotrienes, products of the 5-lipoxygenase
(5-LO)1 pathway, are potent
inflammatory mediators involved in many diseases, such as asthma,
allergic rhinitis, glomerulonephritis, rheumatoid arthritis, and
inflammatory bowel disease (1-4). The soluble enzyme, 5-LO, and the
integral membrane protein, 5-lipoxygenase activating protein (FLAP),
are required for the cellular synthesis of leukotrienes in intact cells
(5). 5-LO translocates to the nuclear envelope in response to a variety
of stimuli and catalyzes the oxygenation of arachidonic acid to 5-HPETE
and the subsequent dehydration of 5-HPETE to leukotriene (LT)
A4. LTA4 can be metabolized to LTB4
by LTA4 hydrolase or to the cysteinyl leukotrienes
LTC4, LTD4, and LTE4, by the action
of LTC4 synthase (6, 7).
FLAP is a member of the membrane-associated proteins in eicosanoid and
glutathione metabolism family of proteins (8). Other members of
this family include LTC4 synthase, prostaglandin
E2 synthase, and the microsomal
glutathione-S-transferases (8). The exact function of the
FLAP enzyme remains controversial, but previous studies suggest that it
acts as an arachidonic acid transfer protein for 5-LO (9). In support
of this role, MK-886, a specific inhibitor of FLAP, has been shown to
inhibit the binding of arachidonic acid to FLAP and the resultant
synthesis of leukotrienes (9).
In several types of inflammatory cells, FLAP mRNA can be induced by
dexamethasone, interleukin (IL) 3, and granulocyte-monocyte colony-stimulating factor (10-12), suggesting that gene expression is
regulated. Cloning of FLAP gene by Kennedy et al. and
computer-assisted analysis of this sequence suggested the existence of
a possible TATA box 22 bp upstream of the transcription start site, as
well as AP-2, NF
B, and glucocorticoid receptor binding sites (13). A
promoter analysis, using a FLAP gene promoter-chloramphenicol acetyltransferase (CAT) reporter gene construct in the mouse macrophage cell line, P388 D1, indicated the presence of enhancer elements and
cell-specific activity (13). They also found a restriction site
polymorphism in the second intron and that this polymorphism is present
in the normal population at a fairly high frequency (13). A FLAP gene
polymorphism in the proximal 5'-UTR region has been identified 94 bp
upstream of the transcription start site (14). They found that a
hetero- or homozygous poly(A) sequence of 21 bp in the proximal FLAP
promoter is present in a higher frequency in asthmatics (73.2%), as
compared with normal subjects (54.9%) (14). The functional
significance of this finding is unclear.
The CCAAT/enhancer-binding protein (C/EBP) family members, of which six
have been identified, are transcription factors that regulate cellular
differentiation and the inflammatory response (15, 16). C/EBP family
members have been identified as mediating IL-6 signaling and are known
to bind to promoter elements within the genes of cyclooxygenase-2,
tumor necrosis factor
(TNF
), IL-8, granulocyte-colony
stimulating factor, CD14, and inducible nitric oxide synthase (15,
17-21). While C/EBP
,
, and
are expressed in liver and lung,
C/EBP
expression is believed to be confined to cells of myeloid and
lymphoid lineage (15). C/EBP
expression has also been reported in
peripheral blood mononuclear cells (15). The C/EBP
,
,
, and
proteins are similar in their C-terminal DNA-binding basic region
and leucine zipper dimerization domains, with a higher degree of
diversity in their N-terminal transactivation domain (15, 22, 23). The
dimerization domain is highly conserved and is believed to be required
for DNA binding (15). C/EBP family members form homo- and heterodimers,
the formation of which is required for DNA binding (24). Cell-specific gene regulation by C/EBP proteins has been shown to be dependent upon
interactions with other transcription factors, including NF
B, Sp1,
and Fos/Jun family members (25, 26).
In this report, we investigate the role of cis-acting
elements that are located in the first 134 bp of the FLAP promoter that are important in the transcriptional regulation of the FLAP gene in
inflammatory cells. Our findings indicate that the
,
, and
members of the C/EBP family of transcription factors bind to these
elements and that the
and
isoforms, at least, function to
up-regulate FLAP gene expression in mononuclear phagocytes.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
THP-1 and HeLa cells were obtained from
American Type Culture Collection (Manassas, VA). The monocyte-like cell
line, THP-1, constitutively expresses 5-LO and FLAP and has been
extensively utilized as a model to study leukotriene metabolic pathways
(11, 27). THP-1 cells were grown at 37 °C with 5% CO2
in RPMI 1640 medium (BioWhittaker, Walkersville, MD), supplemented with
10% heat-treated fetal calf serum (FCS), 100 units/ml penicillin, 100 µg/ml streptomycin, and 100 µg/ml gentamicin. HeLa cells were grown
in Eagle's minimum essential medium, supplemented with 10% FCS, 100 µg/ml penicillin, 100 µg/ml streptomycin, and 1% non-essential amino acids. The media were changed every two to three days for all experiments.
Construction of Luciferase Promoter-Reporter Constructs--
A
3.4-kb segment of the FLAP 5'-UTR, ligated into a pCAT vector (Promega,
Madison, WI), was generously provided by Dr. Brian Kennedy (Merck
Frosst, Kirkland, Québec, Canada). The -3368FLAP-pGL3 construct
was created by release of the 3.4 kb-segment from the pCAT vector by
restriction digest with SacI, with subsequent ligation into
the firefly luciferase pGL3 Basic vector (Promega, Madison, WI). The
-3368FLAP-pGL3 construct was used as a PCR template using primers with
a common NheI restriction site engineered on the 3' end
(5'-ccgctagcggaaggggaagtggagc-3'). The following forward primers were
used to create the designated deletion promoter-reporter constructs:
5'-gaataccaggcagccac-3' (-965FLAP-pGL3), 5'-atgccactctgtctgac-3' (-371FLAP- pGL3), 5'-gacacactgaaccacag-3' (-134FLAP-pGL3), and 5'-ctgaaagagygcaagctctcacttccccttccg-3' (-19FLAP-pGL3). Thirty six cycles of PCR were performed, with each cycle consisting of denaturation at 94 °C for 45 s, annealing at 57 °C for
30 s, and extension at 72 °C for 60 s. The promoter
segment for the -1192FLAP-pGL3 construct was created by restriction
digest of the 3.4-kb pCAT construct with RsaI. All fragments
were subsequently subcloned into the pGEM-T vector (Promega). The FLAP
promoter segments were released by restriction digest with
SacI and NheI and directionally subcloned into
the pGL3 Basic vector. Mutant constructs (containing mutations at the
C/EBP consensus sites) were created using the QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA). Sequences of the wild-type
and mutant constructs were subsequently confirmed by the dideoxy chain
termination method. All constructs were purified using the EndoFree
Maxi-prep kit (Qiagen; Chatsworth, CA).
Transient Transfection--
THP-1 cells (1 × 106) were transiently co-transfected with 450 ng of the
FLAP promoter-pGL3 firefly luciferase vector and 50 ng of the pRL-TK
Renilla luciferase vector (Promega) or a
pCMV-
-galactosidase vector (generously provided by Dr. Kenneth
Chien, University of California, San Diego, CA) using Effectene
transfection reagent (Qiagen), as has been previously described (27).
For overexpression experiments, DNA mixtures consisted of 225 ng of the
wild-type or mutant -134FLAP-pGL3 construct, 50 ng of the
Renilla luciferase pRL-TK construct, and 225 ng of the
C/EBP
,
,
, and
expression vectors to a total of 500 ng of
DNA per condition. For TNF
experiments, the cells were subsequently
treated with TNF
(at 10 ng/ml) for 24 h. HeLa cells were
transfected using Lipofectin reagent (Invitrogen) per the
manufacturer's instructions. The cells were incubated for 24 h at
37 °C with 5% CO2 in their respective media
supplemented with 10% FCS.
Luciferase activities were measured by the Dual Luciferase Assay
Reporter System (Promega) per the manufacturer's instructions.
-Galactosidase activity was measured using the Tropix
-galactosidase assay system (Tropix, Bedford, MA) per the
manufacturer's instructions. Measurements were made using an Optocomp
I luminometer (MGM Instruments, Hamden, CT). Firefly luciferase values
were normalized to either Renilla luciferase values or
-galactosidase values to control for transfection efficiency. Data
are expressed as values normalized to the activity of the promoter less
pGL3 Basic vector or to the activity of the SV40-driven pGL3 Control
vector (Promega).
DNase I Footprint Analysis--
Nuclear extracts were prepared
from THP-1 and HeLa cells as previously described (28). The promoter
region of the FLAP gene from
134 to +12 bp (with respect to the
transcription start site) was prepared by restriction digest of the
-134FLAP-pGL3 construct with KpnI and NcoI. The
probe was labeled on the 3' end with [32P]ATP using T4
polynucleotide kinase (Promega). DNase I footprinting was performed in
100-µl reaction volumes, with 4 ng (~1 × 104 cpm)
of labeled probe and 40 µg of nuclear extract. The reaction conditions consisted of 5% glycerol, 10 mM HEPES, 50 mM KCl, 1 mM dithiothreitol, 1 µg of
poly(dI-dC) (Amersham Biosciences), and 1 µg of bovine serum
albumin. After a 20-min incubation at room temperature,
CaCl2 (1 mM) and MgCl2 (0.5 mM) were added and the reaction was incubated for 1 min.
DNase I (1 unit) (Promega) was added, and the reaction was digested for
60 or 120 s. The reaction was inactivated, and the DNA was
extracted by phenol:chloroform, followed by ethanol precipitation. The
DNA was then analyzed on an 8% PAGE and 50% urea gel. A G + A ladder
was prepared by using the same end-labeled fragment and was run on the
same gel.
Electrophoretic Mobility Shift Assays (EMSAs)--
EMSA
reactions were performed in 20 µl final volumes under the identical
conditions as described for footprinting. The probe was labeled with
[32P]ATP using T4 polynucleotide kinase (Promega). Each
reaction contained 10 µg of nuclear protein extracts from control or
TNF
-conditioned THP-1 cells and ~3 × 104 cpm of
duplexed, labeled nucleotide probe. The reactions were incubated at
room temperature for 20 min. Supershift analyses were conducted with
antibodies against C/EBP family proteins (Santa Cruz Biotechnology,
Santa Cruz, CA) added 5 min before the addition of the radiolabeled
probe. The samples were subsequently electrophoresed on a 5%
nondenaturing acrylamide gel containing 1% glycerol.
Northern Blot Analysis--
THP-1 cells were treated with TNF
(10 ng/ml) or transiently transfected with expression vectors for
C/EBP
,
,
, and
(450 ng × 106 cells) as
has been previously described (27). Following 24 h of incubation,
total cellular RNA was extracted and subjected to electrophoresis on a
1% agarose/2.2 M formaldehyde gel. The RNA was then
blotted overnight onto a Zeta-Probe nylon membrane (Bio-Rad). The blot
was probed with a 32P-labeled full-length cDNA probe
for FLAP, washed under high-stringency conditions, and exposed to
autoradiographic film. Loading equivalency and transfer efficiency were
assessed by probing with a 32P-labeled full-length cDNA
probe for
-actin (Clontech, Palo Alto, CA).
Chromatin Immunoprecipitation (ChIP) Assays--
ChIP assays
were performed by a modification of a previously described method (29).
Briefly, THP-1 cells (at 1 × 107 cells/ml) were
treated with 1% formaldehyde for 30 min at 37 °C and lysed in SDS
buffer (50 mM Tris-HCl/1% SDS/10 mM EDTA, pH
8.1). The chromatin samples were sonicated for 5-10 s (to reduce the
DNA length to ~200-500 bp) and were precleared with protein A-agarose beads to minimize nonspecific binding of proteins in protein
A-agarose. Antibodies (2 µg) were added to the chromatin samples, and
the samples were incubated overnight at 4 °C. To minimize
nonspecific binding of DNA to protein A-agarose, salmon sperm DNA and
protein A-agarose beads were incubated in 1% BSA for 4 h at
4 °C. These beads were then washed repeatedly and resuspended in
binding buffer. Subsequently, 50 µl of the treated beads were added
to the chromatin samples and incubated for 6 h at 4 °C. The
beads were then washed, eluted, and cross-linking-reversed by protease
digestions using proteinase K as previously described (30). After
proteinase K treatment, the DNA was extracted with phenol-chloroform
and precipitated with ethanol. The DNA was dissolved in 20 µl of
Tris-EDTA buffer, and 5 µl of the DNA was used for the PCR
reaction. PCR was performed using a pair of primers (forward
134 bp
to
118 bp; reverse
9 bp to
25 bp, sequence as noted above) that
generated a product spanning
134 to
9 bp of the FLAP promoter (126 bp). PCR was performed for 30 cycles under the following conditions:
denaturation at 94 °C for 60 s, annealing at 55 °C for
60 s, and extension at 72 °C for 45 s. The PCR products were electrophoresed through an agarose gel and visualized by ethidium
bromide staining.
Materials--
FCS, penicillin, streptomycin, and gentamicin
were obtained from the Cell Culture Facility, University of California.
RPMI 1640 medium was obtained from BioWhittaker (Walkersville, MD). All
restriction enzymes were obtained from Invitrogen. All synthesized oligonucleotides and primers were obtained from Operon Technologies, Inc. (Alameda, CA). The C/EBP
expression vector was obtained from
Dr. Steve McKnight, University of Texas Southwestern Medical Center
(Dallas, TX) (31). The C/EBP
expression vector was obtained from Dr.
Mario Chojkier, Veterans Affairs San Diego Healthcare System (San
Diego, CA) (32, 33). The C/EBP
expression vector was obtained from
Dr. Jan Trapman, Erasmus University (Rotterdam, Holland) (34). The
C/EBP
expression vector was obtained from Dr. Julie Lekstrom-Himes,
NIAID National Institutes of Health (Bethesda, MD). TNF
was obtained
from Calbiochem (La Jolla, CA). Autoradiographic film was purchased
from Eastman Kodak Co. (Rochester, NY). The Qiagen-tip 500 column was
purchased from Qiagen. All other reagents were from Sigma and were of
the finest grade available.
Data Analysis--
Data are expressed as the mean ± S.E.
in all circumstances where mean values are compared. Data were analyzed
by unpaired Student's t test (InStat, version 2.03, GraphPad Software, San Diego, CA). Differences were considered
significant when p < 0.05.
 |
RESULTS |
Functional Analysis and Cell-specific Activity of the FLAP
Promoter--
The full-length -3368FLAP-pGL3 construct was assessed
for promoter activity in THP-1 cells. Following transient transfection, the cells were assayed for luciferase activity. The -3368FLAP-pGL3 construct exhibited 22-fold higher activity than that of the
promoterless pGL3 Basic vector (Fig. 1).
In the non-inflammatory cell lines, HeLa and COS-1, which do not
constitutively express FLAP protein, the -3368FLAP-pGL3 construct
exhibited minimal promoter activity (data not shown). These results are
consistent with the known cell-specific pattern of FLAP gene expression
in inflammatory cells.

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Fig. 1.
Functional analysis of the FLAP
promoter. Schematic representation of FLAP promoter-luciferase
reporter constructs that possess a common 3' end at +12 bp from
transcription start site and unique 5' ends as indicated. FLAP promoter
constructs and the pCMV- -galactosidase construct were co-transfected
into THP-1 cells, and luciferase activities were normalized to
-galactosidase activity. Data are presented as the percentage of the
SV40-driven pGL3 control vector (n = 3, ± S.E.). The
first 134 bp of the FLAP 5'-UTR mediate almost all of the promoter
activity observed with the full-length (3368 bp) FLAP
promoter.
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|
A serial deletion analysis demonstrated that the first 134 bp of the
FLAP promoter (as represented by the activity of the -134FLAP-pGL3
construct) mediated a 5-fold increase in promoter activity over that of
the minimal promoter construct, -19FLAP-pGL3 (Fig. 1). Notably, the
-134FLAP-pGL3 construct accounted for almost all of the observed
full-length (3.4 kb) promoter activity.
THP-1 Nuclear Extract Proteins Bind to the FLAP Promoter--
Because the -134FLAP-pGL3 construct demonstrated maximal
promoter activity, we attempted to identify the transacting factors that bind to this region of the FLAP promoter. DNase I footprint analysis using a probe from
134 to +12 bp demonstrated that THP-1 nuclear extract binds to the FLAP promoter region corresponding to
approximately
30 to
10 bp (Fig.
2A, lane 3). In
similar DNase I footprint assays, HeLa nuclear extract did not
demonstrate binding to the FLAP promoter (Fig. 2B,
lane 3). Data base analysis revealed that this region of the
FLAP promoter contains a proximal C/EBP consensus site (located at
25
to
12 bp), a distal C/EBP consensus site (located at
36 to
28
bp), and an Octomer-1 consensus site (located at
29 to
15 bp).

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Fig. 2.
THP-1 nuclear extract proteins bind to the
FLAP promoter. The FLAP promoter segment (from 134 to +12 bp)
was radiolabeled with [32P]ATP on the 3' end and used as
a probe in DNase I footprint analysis. A, a G + A (C + T)
ladder (lane 1), control reaction without THP-1 nuclear
extract (lane 2), and reaction with 40 µg of THP-1 nuclear
extract (lane 3) are displayed. The proximal and distal
consensus binding sites for C/EBP family transcription factors are
denoted (boxed). B, similar DNase I footprint
experiment using HeLa nuclear extract.
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|
C/EBP
,
, and
Proteins Bind to the Proximal
C/EBP Consensus Site in the FLAP Promoter--
Wild-type
and mutant duplexed oligonucleotide probes (Fig.
3) were synthesized for EMSAs to identify
the transcription factor(s) that bind to the FLAP promoter region that
demonstrated binding in the prior footprint assay. In the presence of
nuclear extract from unconditioned THP-1 cells, the EMSA probe from
25 to
9 bp, containing the wild-type proximal C/EBP consensus site
(located at
25 to
12 bp), exhibited a 3-band complex (Fig.
4A, lane 1). Supershifted bands were observed in the conditions using antibodies against C/EBP
(lane 2),
(lane 4), and
(lane 5). Our data indicate the binding of C/EBP
,
,
and
to the proximal C/EBP consensus site in unconditioned THP-1
cells.

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Fig. 3.
FLAP promoter EMSA probes. Duplexed
oligonucleotide probes were synthesized and utilized for EMSAs. A 17-bp
probe (from 25 to 9 bp of the FLAP promoter) containing either the
wild-type (WT) or mutant (Mut) proximal C/EBP
consensus site (underlined) was used. A 20-bp probe (from
39 to 20 bp of the FLAP promoter) containing either the wild-type
(WT) or mutant (Mut) distal C/EBP consensus site
(underlined) was used. Mutated bases within the probes are
denoted in lowercase.
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Fig. 4.
EMSA analysis of the proximal C/EBP consensus
site of the FLAP promoter. Nuclear extract (10 µg) from THP-1
cells was incubated with a [32P]ATP-labeled duplexed
segment of the FLAP promoter (from 25 to 9 bp) containing the
proximal C/EBP site (located at 25 to 12 bp). A, EMSA
and supershift assays were performed with wild-type probe using THP-1
nuclear extract (lane 1) and nuclear extract with
antibodies against C/EBP proteins , (lane 2), (lane 3), (lane 4), and (lane
5). B, EMSA and supershift assays were performed with a
probe containing a mutation of the proximal C/EBP site, using THP-1
nuclear extract (lane 2) and nuclear extract with antibodies
against C/EBP (lane 3) and (lane 4).
C, EMSA and supershift assays were performed with wild-type
probe using HeLa nuclear extract (lane 1) and nuclear
extract with antibodies against C/EBP (lane 3), (lane 4), (lane 5), and (lane
6).
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Mutation of the Proximal C/EBP Consensus Site in the
FLAP Promoter Abolishes C/EBP Protein Binding--
To
identify the critical base pairs within the proximal C/EBP consensus
binding site, we used the
25 to
9 bp EMSA probe containing a 4-bp
mutation within the proximal C/EBP site at
15 to
12 bp (Fig. 3). An
EMSA performed with the probe containing a mutation of the C/EBP site
demonstrated a loss of specific binding (Fig. 4B, lane
2), and antibodies against C/EBP
and
failed to produce
supershifted bands (Fig. 4B, lanes 3 and 4).
These results confirmed that the proximal consensus site bound C/EBP proteins in a site-specific manner.
HeLa Nuclear Extract Does Not Bind to the Proximal C/EBP
Consensus Site in the FLAP Promoter--
When similar EMSAs were
performed using HeLa nuclear extract and the wild-type
25 to
9-bp
EMSA probe, only nonspecific gel-shifted bands were observed.
Supershift assays performed with this probe, HeLa nuclear extract, and
C/EBP antibodies against
,
,
, and
(Fig. 4C,
lanes 3-6) failed to produce supershifted bands, suggesting that these proteins were not constitutively present or did not demonstrate binding in the HeLa cell line.
C/EBP
and
Proteins Bind to the Distal
C/EBP Consensus Site in the FLAP Promoter--
To identify
the transcription factors that bind to the distal C/EBP consensus site
(located at
36 to
28 bp), EMSAs were performed using a probe
extending from
39 to
20 bp. Nuclear extracts from unconditioned
THP-1 cells produced multiple bands on this EMSA (Fig.
5A, lane 2).
C/EBP
and
antibodies produced supershifted bands (Fig.
5A, lanes 3 and 6), but C/EBP
and
did not (Fig. 5A, lanes 4 and 5).
A 3-bp mutation of the distal C/EBP binding site abolished the
supershifted bands observed with antibodies of C/EBP
and
. (Fig.
5B, lanes 3-7). These data demonstrate that
C/EBP
and
bind to the distal C/EBP consensus site in
unconditioned THP-1 cells.

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Fig. 5.
EMSA analysis of the distal C/EBP consensus
site of the FLAP promoter. Nuclear extract (10 µg) from THP-1
cells was incubated with a [32P]ATP-labeled duplexed
segment of the FLAP promoter (from 39 to 20 bp) containing the
distal C/EBP site (located at 36 to 28 bp). A, EMSA and
supershift assays were performed with wild-type probe, using THP-1
nuclear extract (lane 2) and nuclear extract with antibodies
against C/EBP (lane 3), (lane 4), (lane 5), (lane 6), and / (lane
7). B, EMSA and supershift assays were performed with
probe containing a mutation of the distal C/EBP site using THP-1
nuclear extract (lane 2) and nuclear extract with antibodies
against C/EBP (lane 3), (lane 4), (lane 5), (lane 6), and / (lane
7).
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Proximal and Distal C/EBP Consensus Sites Mediate
Constitutive FLAP Promoter Activity--
To elucidate the functional
importance of the proximal and distal C/EBP consensus sites for FLAP
promoter activity, these specific sequences were mutated by
site-directed mutagenesis within the -134FLAP-pGL3 construct (at the
sequences designated in Fig. 3). In transiently transfected THP-1
cells, mutation of the distal and proximal C/EBP sites reduced FLAP
promoter activity by 33 and 81%, respectively (Fig.
6A). Moreover, mutation of
both consensus sites further reduced luciferase activity by 89% (Fig.
6A). These results demonstrate that the C/EBP sites function
as positive constitutive regulatory elements, with the proximal site
serving a critical role.

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Fig. 6.
Proximal and distal C/EBP consensus binding
sites mediate constitutive FLAP promoter activity. A,
the -134FLAP-pGL3 constructs, consisting of wild-type
(Control) or mutated at the proximal (Proximal
Mut) and/or distal (Distal Mut) C/EBP consensus sites
were transiently co-transfected with the pRL-TK Renilla
luciferase vector into THP-1 cells. Luciferase activities were measured
and expressed relative to the activity of the pGL3 Basic vector.
Mutation of the proximal and distal C/EBP sites results in loss of
constitutive FLAP promoter activity. Data represent mean ± S.E.,
n = 3. B, wild-type -134FLAP-pGL3
(closed bars) and combined proximal and distal C/EBP mutant
-134FLAP-pGL3 constructs (open bars) were transiently
co-transfected with the pRL-TK Renilla luciferase vector and
expression vectors for C/EBP , , , and into THP-1 cells.
Luciferase activities were measured, expressed relative to the activity
of the pGL3 Basic vector, and normalized to the luciferase activity of
wild-type -134FLAP-pGL3 construct. C/EBP , , and differentially induce FLAP promoter activity. Overexpression of
C/EBP had no effect on promoter activity. Induction by C/EBP ,
, and is abolished in the presence of combined mutations of the
proximal and distal C/EBP consensus sites. Data represent mean ± S.E., n = 3.
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Overexpression of C/EBP Transcription Factors Enhances
FLAP Promoter Activity--
To further define the role of the C/EBP
family members in modulating gene transcription through the proximal
and distal consensus sites, C/EBP expression vectors for
,
,
,
and
were transiently co-transfected with the wild-type
-134FLAP-pGL3 construct into THP-1 cells. Overexpression of the
C/EBP
,
, and
proteins up-regulated FLAP gene activity by 5-, 5.2-, and 14.2-fold, respectively (Fig. 6B). Overexpression
of C/EBP
had no effect on promoter activity (Fig. 6B).
Mutation of both the proximal and distal C/EBP sites of the
-134FLAP-pGL3 construct essentially abolished the C/EBP-induced enhancement of promoter activity (Fig. 6B). Co-transfection
of the C/EBP
expression vector into HeLa cells did not significantly induce promoter activity (data not shown), consistent with the lack of
FLAP gene expression in this cell line.
Overexpression of C/EBP
,
, and
Enhances FLAP mRNA Accumulation--
To further define the role of
the C/EBP family members in modulating native gene transcription
through the proximal and distal FLAP promoter consensus sites, THP-1
cells were transiently transfected with expression vectors for
C/EBP
,
,
, and
, incubated for 24 h, and subjected to
Northern blot analysis. The results demonstrate that C/EBP
,
, and
can up-regulate FLAP mRNA expression by 6.6-, 6.2-, and
3.3-fold, respectively (Fig. 7,
lanes 1-5). Overexpression of C/EBP
had no effect on
FLAP mRNA expression (Fig. 7, lanes 6 and
7).

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Fig. 7.
Overexpression of C/EBP , , and enhances FLAP mRNA accumulation. THP-1 cells were transiently
co-transfected with expression vectors for C/EBP , , , and .
Total RNA was extracted and subjected to Northern blot analysis.
A, representative Northern blots probed for FLAP and
-actin with their respective empty vector controls. B,
densitometric analysis of Northern blots for FLAP, relative to
-actin and normalized to their control. Overexpression of C/EBP ,
, and increases native FLAP mRNA accumulation.
Overexpression of C/EBP has no effect on FLAP mRNA.
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|
TNF
Induces FLAP mRNA Accumulation--
Prior research has
demonstrated that TNF
can induce the expression of 5-LO activity
(35) and FLAP (36). To examine the role of C/EBP in TNF
-induced
expression of FLAP we conditioned THP-1 cells with TNF
, followed by
Northern blot analysis. The results demonstrate that TNF
up-regulates FLAP mRNA expression more than 6-fold (Fig.
8, B and C).

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Fig. 8.
TNF induces FLAP
mRNA accumulation and promoter activity. A, THP-1
cells were conditioned with TNF (10 ng/ml) for 24 h. Total RNA
was extracted and subjected to Northern blot analysis. Representative
Northern blot probed for FLAP and -actin. B,
densitometric analysis of Northern blot for FLAP, relative to -actin
and normalized to control. TNF treatment increases native FLAP
mRNA accumulation. Data represent mean ± S.E.,
n = 4. C, THP-1 cells were transiently
transfected with the -3368FLAPpGL3 construct and were subsequently
conditioned with TNF for 24 h. Luciferase activities were
measured and expressed relative to the activity of the pGL3 Basic
vector. TNF treatment induces FLAP promoter activity. Data represent
mean ± S.E., n = 3.
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TNF
Induces FLAP Promoter Activity--
To determine whether
TNF
induces FLAP promoter activity, THP-1 cells were transiently
transfected with the -3368FLAP-pGL3 construct and were conditioned with
TNF
for 24 h. In the presence of TNF
, promoter activity was
increased, as compared with control (60.4 ± 5.9 versus 21.4 ± 1.0, respectively; mean ± S.E.,
n = 3) (Fig. 8C). The results demonstrate
that the increase in FLAP mRNA induced by TNF
is mediated, at
least in part, by an increase in FLAP gene transcription.
TNF
Induces C/EBP Family Member Binding to the
Proximal Consensus Site in the FLAP Promoter--
To determine the
role of TNF
in the induction of C/EBP family member binding to the
FLAP promoter, THP-1 cells were conditioned with TNF
(for various
time periods), and the nuclear extracts were used to perform EMSAs
using the proximal FLAP promoter probe (
25 to
9 bp). In the
presence of nuclear extracts obtained from TNF
-treated THP-1 cells,
the proximal EMSA probe exhibited a time-dependent
increased binding pattern, with peak binding occurring at 4-8 h (Fig.
9, lanes 1-5). Supershift
assays identified the bands as being due to C/EBP
(lane
6),
(lane 8), and
(lane 9). Our data
indicate that TNF
induces increased binding of C/EBP
,
, and
to the proximal C/EBP consensus site in the FLAP promoter.

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Fig. 9.
TNF -induces C/EBP
family member binding to the proximal consensus site of the FLAP
promoter. TNF -treated (10 ng/ml) nuclear extract (10 µg) from
THP-1 cells was incubated with a [32P]ATP-labeled
duplexed segment of the FLAP promoter (from 25 to 9 bp) containing
the proximal C/EBP site (located at 25 to 12 bp). A,
EMSAs were performed using THP-1 nuclear extracts treated with TNF
for periods ranging from 0 to 8 h (lanes 1-5). TNF
induces time-dependent binding of C/EBP , , and to
the proximal FLAP promoter consensus site. B, EMSAs were
performed using THP-1 nuclear extracts treated with TNF for 2 h
and antibodies against C/EBP proteins (lane 6), (lane 7), (lane 8), and (lane
9). C/EBP , , and antibodies supershift these proteins
present in the TNF -induced complexes.
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TNF
Induces C/EBP Family Member Binding to the Distal
Consensus Site in the FLAP Promoter--
To determine the role of
TNF
in the induction of C/EBP family member binding to the FLAP
promoter, THP-1 cells were conditioned with TNF
(for various time
periods), and the nuclear extracts were used to perform EMSAs using the
distal FLAP promoter probe (
39 to
20 bp). In the presence of
nuclear extracts obtained from TNF
-treated THP-1 cells, the proximal
EMSA probe exhibited a time-dependent increase in binding
pattern, with peak binding occurring at 2-4 h (Fig.
10, lanes 1-5). Supershift
assays identified the bands as being due to C/EBP
(lane
6),
(lane 8), and
(lane 9). Our data
indicate that TNF
induces increased binding of C/EBP
and
to
the distal C/EBP consensus site in the FLAP promoter and also induces
the new binding of C/EBP
to this site.

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Fig. 10.
TNF induces C/EBP
family member binding to the distal consensus site of the FLAP
promoter. TNF -treated (10 ng/ml) nuclear extract (10 µg) from
THP-1 cells was incubated with a [32P]ATP-labeled
duplexed segment of the FLAP promoter (from 39 to 20 bp) containing
the distal C/EBP site (located at 36 to 28 bp). A, EMSAs
were performed using THP-1 nuclear extracts treated with TNF for
periods ranging from 0 to 8 h (lanes 1-5). TNF
induces time-dependent binding of C/EBP , , and to
the distal FLAP promoter consensus site. B, EMSAs were
performed using THP-1 nuclear extracts treated with TNF for 2 h
and antibodies against C/EBP proteins (lane 6), (lane 7), (lane 8), and (lane
9). C/EBP , , and antibodies supershift these proteins
present in the TNF -induced complexes.
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Chromatin Immunoprecipitation (ChIP) Assays Demonstrate Binding of
C/EBP
,
, and
to the FLAP Promoter--
To
determine whether C/EBP isoforms are bound to native chromatin in THP-1
cells, we performed ChIP assays on chromatin obtained from THP-1 cells
(Fig. 11A). When the PCR
reaction was run in the absence of a template or in the presence of an
irrelevant antibody (
actin), no product was observed. When the PCR
reaction was run in the presence of FLAP cDNA corresponding to the
DNA sequence of interest containing the two C/EBP sites (
134 to
9)
an appropriate product was generated (126 bp). Likewise, when chromatin
was incubated with antibodies to C/EBP
,
, and
, a PCR
product corresponding to this segment was also detected indicating that
these isoforms of C/EBP were bound to the C/EBP consensus binding
sites. However, when PCR reactions were run on immunoprecipitates
generated using the C/EBP
antibody, no cDNA product was
detected, indicating the absence of binding of C/EBP
to either C/EBP
site (Fig. 11A). To further clarify whether C/EBP
could
bind to this segment of the FLAP 5'-UTR, C/EBP
was overexpressed in
THP-1 cells transiently transfected with a C/EBP
expression
construct (Fig. 11B). No PCR product (no binding) was
detected when immunoprecipitation was performed with C/EBP
antibody.
To determine whether activation with TNF
would modify this outcome,
THP-1 cells transfected with C/EBP
were activated with 10 ng/ml of
TNF
for 2 h. Again, no PCR product was detected when chromatin
was immunoprecipitated with C/EBP
antibody (Fig.
11B).

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Fig. 11.
C/EBP family member binding to native
chromatin as demonstrated by ChIP assay. THP-1 cells were fixed
with 1% formaldehyde, lysed with SDS, and subjected to
immunoprecipitation. DNA was released from immunoprecipitates by
proteinase K treatment and the DNA was amplified by PCR using primers
corresponding to 134 to 9 of the FLAP gene. A,
lane 1, 100 bp ladder. Lane 2, PCR reaction mix,
negative control. Lane 3, cDNA template corresponding to
region of interest, positive control. Lane 4, ChIP performed
on THP-1 cells with -actin antibody as an irrelevant control
antibody. Lanes 5-8, ChIP assay performed on THP-1 cells
with antibody to C/EBP , , , and , respectively. When
fixed chromatin was treated with an irrelevant antibody ( -actin), no
product was detected. Fixed chromatin immunoprecipitated with
antibodies to C/EBP , , and yielded a PCR product
corresponding to this segment of the FLAP gene, but when using
antibodies to C/EBP no product was detected. B, to
further examine the role of C/EBP , THP-1 cells were studied further.
Lane 1, ladder. Lane 2, negative control.
Lane 3, positive control. Lane 4, irrelevant
antibody ( -actin). Lane 5, ChIP assay performed on THP-1
cells with C/EBP antibody. Lane 6, ChIP with THP-1 cells
transfected with an expression construct encoding for C/EBP .
Lane 7, ChIP with THP-1 cells transfected with an expression
construct encoding for C/EBP and conditioned with TNF (10 ng/ml)
for 2 h. Even under these highly induced conditions, binding of
C/EBP to native chromatin could not be detected.
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DISCUSSION |
Our findings demonstrate that C/EBP family members play a role in
constitutive expression of the FLAP gene in the human monocyte-like cell line, THP-1. Our findings identify the presence of
cis-acting regulatory elements that are contained within the
first 134 bp region of the 5'-UTR of this gene. This region accounts
for the majority of FLAP promoter activity and appears to confer gene expression in THP-1 cells. Expression of FLAP in inflammatory cells can
be attributed, at least in part, to the proximal and distal C/EBP sites
contained in this segment of the promoter. Moreover, these sites also
play a role in TNF-induced expression of FLAP.
FLAP is a pivotal protein in the 5-LO pathway of leukotriene
metabolism. Studies indicate that the concomitant expression of FLAP is
required for 5-LO activity in stimulated, intact inflammatory cells
(5). Moreover, a variety of studies have suggested that levels of the
FLAP protein are regulated and that modulation of its expression may
play a key role in altering the capacity of inflammatory cells for
5-lipoxygenation of arachidonic acid. The original description of the
FLAP gene suggested that it may possess a TATA box in its proximal
promoter, and a deletion analysis was performed in a mouse macrophage
cell line (13). These studies suggested that the promoter region
contained probable enhancer elements further upstream of the
134 bp
position (13), although specific characterization of these sites was
not attempted. In the current study, performed in a human monocyte-like
cell line, we demonstrate that the full-length FLAP promoter
(consisting of 3368 bp) mediates a 22-fold increase in activity over
that of the promoterless pGL3 Basic construct. Our findings demonstrate that almost all of the observed constitutive activity is mediated by
the first 134 bp of the promoter. This region contains the proximal and
distal C/EBP consensus sites and confers gene expression in THP-1
cells. The noted differences between our findings and those reported
previously could be explained by the use of more sensitive luciferase
constructs in our experiments and the use of a human, rather than
mouse, monocytic cell line.
We demonstrate that C/EBP
,
, and
bind to the proximal
consensus site and that C/EBP
and
bind to the distal consensus site within the FLAP promoter under constitutive conditions. C/EBP
does not appear to bind to the FLAP proximal or distal promoter sequences in THP-1 cells, consistent with the findings of other investigators that this protein is not expressed in unstimulated cells
(20). Previous studies suggest that C/EBP
,
,
, and
are
similar in their C-terminal basic region and leucine zipper domains,
accounting for the interaction of these proteins with virtually
identical DNA sequences (22, 23, 37). However, our finding of
differential binding of the C/EBP
protein to the proximal but not
the distal site under constitutive conditions may be accounted for by
the fact that the C/EBP proteins can also bind in a sequence-specific
fashion. This has been reported to occur for adjacent C/EBP sites
within the Clara cell secretory protein gene promoter (38). The
differential binding of C/EBP family members may be related to DNA
bending, known to be induced by C/EBP
or
or other basic
region-leucine zipper (bZIP) proteins that bind to adjacent sequence
elements (38-40).
The C/EBP family members are known to exhibit a variety of
protein-protein interactions. C/EBP proteins interact with each other
to form homo- and heterodimers, and this dimerization is believed to be
required for DNA binding (24). The individual C/EBP dimer components
may function to synergistically activate gene transcription (38). The
binding of multiple C/EBP proteins, likely present as complexed dimers,
occurs at both the proximal and distal domains in the FLAP promoter.
The possibility that other transcription factor proteins may be present
in this complex cannot be specifically excluded based upon our data.
Previous studies indicate that cell-specific gene regulation by C/EBP
proteins may also involve interactions with other transcription
factors, including NF
B, Sp1, and Fos/Jun (25, 26). The binding of C/EBP members to consensus sites that exist in close association with
NF
B sites has been reported for multiple genes (16). Notably, the
FLAP gene possesses an NF
B consensus site located adjacent to the
C/EBP consensus sites at
43 to
34 bp. Interactions between C/EBP
proteins and NF
B proteins may enhance or antagonize gene transcription (16), depending on promoter structure and the cellular
milieu. Exploration of the role of potential interactions between
proteins binding to the adjacent C/EBP and NF
B consensus sites in
the FLAP promoter remains a subject for future investigation.
The functional consequence of C/EBP
and
protein binding to
consensus sites within the FLAP promoter is to stimulate gene transcription. Oddly, C/EBP
binds to the distal site only when cells
are conditioned with TNF
. We have not found that C/EBP
independently plays a role in FLAP transcription, although it is
present and binds to both proximal and distal sites. Moreover, the
overexpression of C/EBP
can increase promoter activity and induce increased expression of native mRNA encoding for FLAP. The
nuclear extracts from control or TNF
-conditioned THP-1 cells do not
contain C/EBP
. More importantly, C/EBP
does not bind to native
chromatin corresponding to FLAP in these regions under any
circumstances, as shown by ChIP assay. These data indicate that C/EBP
may play an indirect role in regulating the FLAP gene but has no direct effect.
Mutation of both the proximal and distal C/EBP sites results in an
approximate 90% decrement in promoter activity, suggesting that these
two consensus sites account for the vast majority of the observed
function within the first 134 bp of the FLAP promoter. C/EBP family
members have been described to function as positive and negative
regulators of gene transcription (16). Our findings also suggest that
the C/EBP family members exhibit differential capabilities to induce
promoter activity. This difference in transactivation potential has
been previously reported (22, 38), with C/EBP
being a more potent
transcriptional activator than C/EBP
(41). The observed differences
in the potential for activation by each of these proteins in our
studies may be related to differential binding affinity, cooperative or
antagonistic interaction with other transcription factors, the
differential expression of C/EBP proteins in the THP-1 cell line, or
differential phosphorylation of the C/EBP proteins (41). In addition,
C/EBP
and
mRNAs can give rise to truncated forms of the
proteins, which may act as inhibitors or weak activators of gene
transcription (42, 43). The findings from Northern blot analyses
establish the biological relevance of our findings by demonstrating
that overexpression of the C/EBP
and
proteins can positively
regulate native gene expression in intact mononuclear phagocytes.
Although C/EBP
also induces mRNA encoding for FLAP, this is not
a direct effect mediated through binding of the FLAP 5'-UTR.
The constitutive or induced expression of C/EBP family members may
account for the differential expression of genes in which C/EBP
proteins play a regulatory role. The C/EBP
and
proteins are
known to be constitutively expressed in mononuclear cells (15, 44-47),
and C/EBP
expression has been described in myeloid cell lines (48).
Although prior studies suggest that C/EBP
is not expressed in THP-1
cells or in the premonocytic cell line, U937, under unstimulated
conditions (20), this factor is believed to be involved in macrophage
activation (49) and has been constitutively detected in RAW264 murine
macrophages (17). Our study specifically addresses the TNF
-induced
binding of C/EBP proteins to the FLAP promoter. While previous studies
indicate that TNF
induces the expression of C/EBP
(50) and
(51), we have found that TNF
induces the binding of C/EBP
,
,
and
, but not
to the both the proximal and distal FLAP promoter
consensus sites. This is confirmed by our ChIP assays. Notably, TNF
treatment results in induction of C/EBP
binding to the distal
consensus site, which was not present in the absence of TNF
conditioning. These findings suggest that TNF
induction of FLAP gene
expression is mediated by a transcriptional mechanism.
Prior research in our laboratory has demonstrated that FLAP expression
can be induced by corticosteroids (11). This is in sharp contrast to
the knowledge that the 5-LO pathway is a potent proinflammatory
mediator pathway and the finding that FLAP can be induced by
proinflammatory cytokines like TNF
. However, the induction of C/EBP
family members may represent a common mediating pathway for these
disparate stimuli. Glucocorticoids are known to increase the expression
of C/EBP
(52) and C/EBP
(47, 53, 54). In addition, the induction
of expression or activation of C/EBP
may occur in response to IL-1,
IL-6, and lipopolysaccharide (15, 18, 55, 56). Similarly, C/EBP
expression also is induced by lipopolysaccharide (17, 56). The
induction of C/EBP family members by inflammatory stimuli, as has been
demonstrated for other mediator genes (15, 17-21), may serve to
further modulate FLAP gene transcription. In addition to the
established effects of C/EBP family members on inflammatory mediator
expression and cell differentiation, our data further define their role
in the constitutive and induced expression of a gene of the 5-LO
pathway of leukotriene metabolism.
In conclusion, we demonstrate that transcriptional expression of the
FLAP gene is constitutively regulated by members of the C/EBP family of
transcription factors in the monocyte-like cell line, THP-1. Our data
indicate a critical role for C/EBP proteins in modulating expression of
this gene in the 5-LO pathway in mononuclear phagocytes. The induction
of C/EBP family member binding to the FLAP promoter by TNF
(and
potentially other inflammatory stimuli) contributes to the modulation
of FLAP gene expression.