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INTRODUCTION |
Monocyte chemoattractant protein-1
(MCP-1)1 is a member of the
C-C chemokine family that mediates leukocyte chemotaxis. We initially
isolated it from the human malignant glioma cell line U-105 MG as a
specific chemoattractant for monocytes (1, 2). Physiologically, MCP-1
is produced by endothelial cells, smooth muscle cells, and macrophages
in response to a variety of mediators including platelet-derived growth
factor, tumor necrosis factor
, interleukin 1, epidermal growth
factor, and interferon-
(3). The expression of MCP-1 is also
increased under pathological conditions with monocyte-rich inflammatory
processes such as atherosclerosis (4), rheumatoid arthritis (5), and
certain malignant tumors (6, 7).
The cDNA of the specific receptor for human MCP-1, which is
designated hCCR2, was recently cloned and shown to belong to
seven-transmembrane domain receptor families (8) that couple via
heterotrimeric G-proteins to affect cellular responses. The activation
of hCCR2 is blocked by pertussis toxin, suggesting that hCCR2 couples
to G
i-class G-proteins (9). The hCCR2 gene has been
cloned, and the mechanism of how it produces two alternatively spliced
variants that differ only in their carboxyl-terminal tail has been
elucidated (10). Human CCR5, one of the members of the C-C chemokine
receptor family, acts as an essential cell surface co-receptor with CD4 in cell entry by macrophage-tropic human immunodeficiency virus type 1 strains (11, 12). In addition, hCCR2 and hCCR3 have been implicated as
human immunodeficiency virus type 1 co-receptors in certain cell types
(13, 14).
We previously reported that tumor-associated macrophages attracted by
MCP-1 inhibit the growth of transplanted rat tumors in vivo
(15). We posited that if we could stimulate the expression of MCP-1 in
tumors and the expression of hCCR2 in monocytes, we would be able to
obtain a greater inhibitory effect by a larger number of infiltrated
macrophages against the growth of MCP-1-producing tumors such as human
malignant glioma. The treatment of malignant tumors by the enhancement
of an intrinsic immune system may be possible. The promoter region of
the human MCP-1 gene has already been analyzed; it contains
a distal nuclear factor
B binding site for induction by interleukin
1, tumor necrosis factor
, and a proximal GC box for basal
transcriptional activity that is important for the transcriptional
activation of MCP-1 (16). However, the promoter region for the
hCCR2 gene remains to be characterized.
To elucidate the molecular mechanisms that regulate hCCR2, we cloned
the hCCR2 gene, sequenced approximately 1.7 kbp of the 5'-flanking region, and mapped the transcription initiation site. We
also assessed the promoter activity of the 5'-flanking region using
luciferase (Luc) assays and gel mobility shift assays. We found that
the Oct-1 binding sequence located 36 bp upstream from the TATA box and
the tandem CAAT/enhancer-binding protein (C/EBP) binding sequences
located at +50 to +77 within the 5'-untranslated region (UTR) are
essential for the transcriptional activation and the tissue
specificity of hCCR2 expression.
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EXPERIMENTAL PROCEDURES |
Cells and Cell Cultures--
Two human monocytic leukemia cell
lines, THP-1 and J-111, were obtained from the Japanese Cancer Research
Resources Bank (Tokyo, Japan). The human malignant glioma cell line
U-251 MG was obtained as described previously (7). THP-1 cells and
U-251 MG cells were grown in RPMI 1640 medium supplemented with 10%
fetal calf serum and maintained at 37 °C in 5% CO2.
J-111 cells were grown in Minimum Essential Medium with nonessential
amino acids (Life Technologies, Inc., Gaithersburg, MD) supplemented
with 10% fetal calf serum and maintained at 37 °C in 5%
CO2.
DNAs Used as Hybridization Probes--
The cDNA encoding the
hCCR2 type B (hCCR2b) was isolated by reverse
transcription-polymerase chain reaction as described previously (17) from a larger number of infiltrated macrophages, using a
set of primers (5'-CATCGGATCCATGCTGTCCACATCTCGTTCTCG-3' and 5'-GCTCAAGCTTTTATAAACCAGCCGAGACTTCCT-3') that contain BamHI
and HindIII cloning sites, respectively, at their 5'-ends.
The 1.1-kbp PCR product containing the entire coding sequence for
hCCR2b was double-digested with BamHI and HindIII
and subcloned into the pGEX-2TH vector (18) to generate the plasmid
pGEX-2TH-CCR2b. The sequence of the insert was confirmed to be
identical to the previously published sequences (8). To prepare the
hCCR2b cDNA probe, the pGEX-2TH-CCR2b insert was double-digested
with BamHI and HindIII, purified by low-melting
point agarose gel electrophoresis, and labeled with
[
-32P]dCTP using a random primer labeling kit (Takara,
Otsu, Japan). We also used a 5'-end-labeled oligonucleotide probe
(5'-AAAACGATCAGAGTAGTGGTATTTCACCG-3') complementary to the 28 S
ribosomal RNA to standardize the amount of RNA.
Isolation and Characterization of Clones Containing the hCCR2
Gene--
A human genomic DNA library packaged in the phage vector
EMBL3 SP6/T7 was purchased from (Palo
Alto, CA). Plaques (1 × 106) from this library were
screened by the plaque hybridization technique (19), using
32P-labeled hCCR2b cDNA as a probe. Positive clones
were re-screened at least twice, and a restriction enzyme mapping of
the cloned DNA was carried out.
DNA Sequence Analysis--
The nucleotide sequence of the
5'-flanking region and that of the entire exon-intron boundary were
determined by the dideoxynucleotide chain termination method using the
ABI PRISM dye terminator cycle sequencing ready reaction kit
(Perkin-Elmer, Foster City, CA) and a PE 373A sequencer (Perkin-Elmer).
Sequence data were analyzed using the MatInspector V.2.1 computer
program via the publicly available World Wide Web server
() to
find specific sequences, including some transcriptional factor binding elements.
Northern Blot Analysis--
Total cellular RNA (10 µg)
extracted by the guanidinium thiocyanate-phenol chloroform extraction
method (20) was subjected to 1% agarose gel electrophoresis and
blotted onto a nitrocellulose filter (Schleicher & Schuell, Keene, NH).
After baking at 80 °C for 2 h, the filters were hybridized with
a hCCR2 probe and a 28 S RNA oligonucleotide probe at 42 °C in 50%
formamide, 5× SSC, 1× Denhardt's solution, 50 mM sodium
phosphate buffer (pH 7.0), and 100 µg/ml heat-denatured salmon sperm
DNA. After hybridization, the filter was washed twice for 10 min with
2× SSC and 0.1% SDS at room temperature and washed once for 45 min
with 0.1× SSC and 0.1% SDS at 56 °C. The filters were subsequently
exposed to Fuji RX film at
80 °C for 24-72 h.
Primer Extension--
The primer extension reaction was
conducted using the avian myeloblastosis virus reverse transcriptase
primer extension system (Promega, Madison, WI). For the primer,
5'-end-labeled synthetic 30-nucleotide-long DNA
(5'-CTTATGCAACCTTGAGTGTGAGTCAGGCAA-3') that corresponds to the
anti-coding strand covering nucleotides
57 to
86 downstream from
the TATA box was used. Polyadenylated RNA (1 µg) extracted from THP-1
cells and 100 fmol of 32P-labeled primer were annealed
using 50 mM Tris-HCl (pH 8.3), 50 mM KCl, 10 mM dithiothreitol, 1 mM each deoxynucleotide
triphosphate, and 0.5 mM spermidine, and the primer
extension reaction was started by adding 1 unit of reverse
transcriptase. The reaction mixture was incubated for 30 min at
42 °C. The primer-extended product was analyzed by electrophoresis
on a 6% denaturing acrylamide gel. Autoradiography was performed for
24 h at
80 °C.
Construction of the Luc Reporter Plasmid--
A 6-kbp
HindIII/XbaI fragment of the hCCR2
gene containing the 5'-flanking region and the first exon was subcloned
into pBluescriptSK(
). We generated a series of 5'-nested deletion
mutants by PCR using this construct as a template. The following sense
primers contained a KpnI site at their 5'-ends:
pGL3-5K, 5'-GTGAGGTACCAGTGTCAGGGAATATGCTGGTGG-3'; pGL3-0.5K,
5'-TAGTGGTACCGAATTCTGTATATATAAATAGCT-3'; pGL3-0.3K, 5'-TCCAGGTACCCACTGAAACCAGCACACATGATC-3'; pGL3-0.26K,
5'-TCCA-GGTACCCCCTTTATTCTCTGGAACATGAA-3'; pGL3-0.23K,
5'-TCCAGGTAC-CGTGCTCATATCATGCAAATTATC-3'; pGL3-0.2K, 5'-TCCAGGTACCGAGA-GCAGAGAGTGGAAATGTTC-3'; and pGL3-0.17K,
5'-ATCAGGTACCCAGGTATA-AAGACCCACAAGATA-3'. The common antisense primer
was 5'-TCATAAGCTTCAGGAACATTGTACATTGGGTTG-3', which contained a
HindIII site at the 5'-end. A series of 3'-nested deletion
mutants was also generated by PCR. The following antisense primers
contained a HindIII site at their 5'-ends: pGL3-0.18K
E, 5'-TCATAAGCTTATTTTGAAATCTTGCTTATGCAA-3'; pGL3-0.14K
E,
5'-TCATAAGCTTGCAAACCCTGTACATCTGCTCCT-3'; pGL3-0.12K
E,
5'-TCATAAGCTTTCTGAGCTTCTTTATCTTGTGGG-3'; and pGL3-0.1K
E, 5'-TCATAAGCTTTTTATACCTGGAACATTTCCACT-3'. The sense primer for pGL3-0.18K
E and pGL3-0.14K
E was the same as that for pGL3-0.23K, and the sense primer for pGL3-0.12K
E and pGL3-0.1K
E was the same
as that for pGL3-0.26K. These PCR products were double-digested with
KpnI and HindIII and ligated into the
KpnI/HindIII sites of the promoterless Luc vector
pGL3-Basic (Promega) immediately upstream from the firefly
Luc gene. For the construction of other deletion mutants, the
pGL3-5K was double-digested with KpnI and PvuII
(pGL3-3.1K), SacI (pGL3-1.8K), SmaI (pGL3-1.7K),
and PstI (pGL3-1.4K), blunt-ended with T4 DNA polymerase,
and self-ligated. To obtain the mutated constructs pGL3-OM,
pGL3-CM1, pGL3-CM2, and pGL3-CMd, site-specific mutagenesis was carried
out by PCR with the following mutated primers: for pGL3-OM, sense
primer 5'-TCCAGGTACCGTGCTCATATCATGCAAGCTATCACTAGTAGGAGAGCAGAG-3'; for pGL3-CM1, antisense primer
5'-TCATAAGCTTCAGGAACATTGTACATTGGGTTGAGGTCTCCAGAATAGGATTAATTTTGAAATCTGACTAGTCGACTAGTGAGTGTGAG-3'; for pGL3-CM2, antisense primer
5'-TCATAAGCTTCAGGAACATTGTACATTGGGTTGAGGTCTCCAGAATAGGAT
GACTAGTCGACTAGTGCTTATGCAACCTTGAG-3'; and for pGL3-CMd, antisense primer
5'-TCATAAGCTTCAGGAACATTGTACATTGGGTTGAGGTCTCCAGAATAGGATGACTAGTCGACTAGTCGACTAGTCGACTTGAGTGTGAG-3'. The sequence changes are indicated above in bold type, and the putative transcriptional factor binding sites are underlined. The
antisense primer for pGL3-OM and the sense primer for other constructs
were the same as those for pGL3-0.23K. All plasmid DNAs were isolated
using reagents from Qiagen (Chatsworth, CA). The entire sequences of
DNA fragments generated by PCR were confirmed to be identical to the
original 5'-flanking sequences.
Transient Transfection and Luc Assays--
Cell lines grown to
roughly 70% confluence were transfected with the DEAE-dextran method
as described previously (21). Each deletion construct (5 µg) was
dissolved with 5 µg of pRL-SV40 vector (Promega) as an internal
control in 1 ml of 500 µg/ml DEAE-dextran in suspension Tris-buffered
saline solution containing 25 mM Tris-HCl (pH 7.4), 137 mM NaCl, 5 mM KCl, 0.6 mM
Na2HPO4, 0.7 mM CaCl2, and 0.5 mM MgCl2. The DNA/DEAE-dextran solution
was added to the cells, and they were then incubated at room
temperature for 1 h before treatment with dimethyl sulfoxide
(DMSO) at room temperature for 2 min. Cell extracts were prepared
48 h after transfection using reporter lysis buffer (Promega).
Each lysate (20 µl) was used for the dual luciferase reporter assay,
as recommended by the manufacturer (Promega). Luc activities were
measured in a lumicounter 700 (NITI-ON, Funabashi, Japan). After
normalization to the Renilla Luc control, the
transactivation activity of each construct was calculated relative to
the pGL3-Basic vector, the activity of which was arbitrarily defined as
1. All transfections were carried out in duplicate and repeated more
than twice, and the relative promoter activities shown are mean values.
Gel Mobility Shift Assay--
Nuclear extracts from THP-1 cells,
J-111 cells, and U-251 MG cells were prepared according to the method
of Dignam et al. (22). The probe and competitor sequences
used are shown in Figs. 6A and 7A. Complementary
single-stranded oligonucleotides were synthesized, mixed with TE,
heated briefly, and cooled to room temperature for more than 1 h.
Probes were end-labeled using 50 mCi of [
-32P]ATP and
T4 polynucleotide kinase (Takara); unincorporated radiolabel was
removed by Sephadex G-25 chromatography. Nuclear extracts (4 µg) were
premixed with or without unlabeled competitor (used in 100-fold molar
excess over the radiolabeled probe) in gel shift binding buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM dithiothreitol, 0.5 mM EDTA, 1 mM MgCl2, 4% (v/v) glycerol, and 50 µg/ml
poly(dI·dC) ·poly(dI·dC)) for 10 min at room temperature.
Radiolabeled probe (175 fmol) was added to this mixture and incubated
for an additional 20 min before the binding reaction was loaded onto a
native 4% polyacrylamide gel, dried under a vacuum, and exposed to
x-ray film. For the supershift assay, 3 µg of each specific
polyclonal antibody against Oct-1, Oct-2, C/EBP
, C/EBP
, and
C/EBP
(Santa Cruz Biotechnology, Santa Cruz, CA) were added to the
binding reaction, and the mixture was incubated at room temperature for 10 min before the addition of radiolabeled probe.
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RESULTS |
Isolation and Genomic Organization of the Human CCR2
Gene--
Using hCCR2b cDNA as a probe, we screened 1 × 106 plaques from a human genomic DNA library and isolated
two independent positive clones designated
MCP-1R1 and
MCP-1R2.
Most of the genomic organization of the hCCR2 gene was
determined by the phage clone
MCP-1R1, which covers the entire
coding sequence of the hCCR2b cDNA but lacks the carboxyl-terminal
intracellular domain for hCCR2a (Fig. 1).

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Fig. 1.
Schematic representation of the hCCR2
gene and two kinds of alternatively spliced cDNA.
A, restriction map of the 8-kbp fragment in the genomic
clone. E, EcoRI; H,
HindIII; X, XbaI; Sc,
SacI; Sm, SmaI; T,
Tth111I; A, ApaI. B, DNA
region included in MCP-1R1. C, Structure of the
hCCR2 gene and two types of hCCR2 cDNA produced by
alternative splicing of the second intron. , the noncoding regions
of the exons. , the common coding regions of the exons. , the
sequences corresponding to the carboxyl-terminal intracellular domain
of hCCR2 type A and B. Thick bars show the intron and the
flanking regions. The exon-intron boundary sequences were consistent
with the GT/AG rule. The structures of the two types of hCCR2 cDNA
produced by alternative splicing of the second intron are shown at the
top (type B) and bottom (type A). The sites for
translation initiation (ATG) and termination (TAA and TAG) are
indicated. The sites for polyadenylation signals are indicated by
asterisks.
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The hCCR2 gene consists of three exons divided by two
introns, spanning a minimum of 8 kbp (Fig. 1). All splice junction
sequences for the donor and the acceptor are in agreement with the
GT/AG rule (data not shown). The sequence of the entire coding region was identical to that reported previously (8). The first exon encodes a
5'-untranslated region, and the second exon encodes the amino-terminal
extracellular domain, the seven-transmembrane domain, and the hCCR2b
carboxyl-terminal intracellular domain. The third exon contains the
carboxyl-terminal intracellular domain of hCCR2a. The second intron is
optionally used to create the hCCR2a isoform, whereas the first intron
is essentially spliced.
Structure of the 5'-Flanking Region and Transcription Initiation
Site of the CCR2 Gene--
Several features of the nucleotide sequence
1.7 kbp upstream from the transcription initiation site of the
hCCR2 gene are notable (Fig.
2). First, the putative promoter region
contains the typical mammalian promoter consensus element TATAA at
31 to
27, and CCAAT at
128 to
124 (numbering the transcription initiation site as +1). Second, we found clusters of several well-known tissue-specific transcriptional factor binding sites, i.e.
seven GATA consensus binding sites, three consensus C/EBP binding
motifs, and three Oct-1 binding sequences. Third, the region lacks Sp1 and nuclear factor
B binding sites, which are important for the regulation of the MCP-1 gene.

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Fig. 2.
Nucleotide sequences of the 5'-flanking
region of the hCCR2 gene. Noncoding first exon
sequences are shown in lowercase letters. Intron and
flanking sequences are in uppercase letters. The
transcription initiation site is boxed and assumed to be at
position +1. A TATA box, a CAAT box, C/EBP binding sequences, Oct-1
binding sequences, and GATA consensus sequences are
underlined.
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The transcription initiation sites of the hCCR2 gene were
determined in a primer extension analysis. A single prominent band was
observed 26 bp downstream from the TATA box; this is commonly observed
in nonhousekeeping mammalian genes (Fig.
3).

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Fig. 3.
Primer extension analysis of the hCCR2
gene. Yeast tRNA (lane 1; 10 µg) and
polyadenylated RNA extracted from THP-1 (lane 2; 10 µg)
were subjected to a primer extension analysis with the oligonucleotide
primer complementary to nucleotides 57 to 86 downstream from the
TATA box. The band specific for polyadenylated RNA extracted from THP-1
cell is indicated by an arrowhead. The size markers are
shown to the left. The position of the primer is indicated
to the right.
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Functional Analysis of the 5'-flanking Region of the hCCR2
Gene--
To determine which cell line(s) expressed hCCR2 mRNA, we
performed a Northern blot analysis using extracts of total RNA prepared from THP-1, J-111, and U-251 MG cells. The expression of hCCR2 mRNA
was clearly detectable only in THP-1 cells; it was at undetectable levels in J-111 and U-251 MG cells (Fig.
4).

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Fig. 4.
Northern blot analysis of hCCR2 in THP-1,
J-111, and U-251 MG cells. Total RNA (10 µg) isolated from human
monocytic leukemia cell lines (THP-1 and J-111) and a human malignant
glioma cell line (U-251 MG) was subjected to electrophoresis on an
agarose gel and transferred onto nitrocellulose filters. Hybridization
was performed with either a 32P-labeled human CCR2 cDNA
probe (top panel) or an oligonucleotide probe complementary
to 28 S ribosomal RNA (bottom panel) to standardize the
total amount of RNA.
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To define the minimal promoter region important for the basal and
cell-specific expressions of the hCCR2 gene, we subcloned a
series of 5'- and 3'-nested deletion mutants of the hCCR2
promoter into the Luc reporter vector pGL3-Basic for transient
transfection assays using the three cell lines. Remarkable Luc activity
was observed only in THP-1 cells; little activity was found across these constructs in J-111 cells and U-251 MG cells (Table
I). This result was consistent with the
Northern blot analysis, suggesting that these regions contained
essential cis-regulatory elements important for the
cell-specific expression of the hCCR2 gene.
Deletion from
429 to
185 (pGL3-0.5K versus pGL3-0.3K)
reduced the level of expression by nearly 50% (Fig.
5). Further truncation to
122 restored
the Luc activity to the previous level, suggesting the possible
presence of inhibitory sequences that contain the CAAT box in this
region. Although deletion from
122 to
89, which removed the distal
Oct-1 binding sequence, did not change the Luc activity, deletion to
58 reduced the level of expression by nearly 60%. This suggests that
the region from
89 to
59 that contains the proximal Oct-1 binding
site is important for the transcriptional activation of hCCR2.

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Fig. 5.
Localization of the hCCR2
regulatory elements. A, scheme of hCCR2
promoter constructs used in the transfection experiments. The
restriction enzyme map of the 5'-flanking region of the
hCCR2 gene is illustrated at the top. ,
5'-untranslated region; , 5'-flanking region; vertical
bars, potential cis-regulatory elements. B,
the Luc activity (relative Luc activity) from THP-1 cells transfected
with each construct described in A. Chimeric firefly Luc
promoter constructs containing various hCCR2 promoter
fragments were co-transfected with Renilla Luc control
vector pRL-SV40 into THP-1 cells. A dual luciferase assay was
performed, and the activity of firefly Luc was normalized to that of
Renilla Luc. The relative promoter activities were
calculated by arbitrarily defining the activity of the pGL3-Basic
vector as 1. Columns indicate the mean ratio of the Luc activities.
Error bars, the standard deviation.
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Curiously, pGL3-0.17K, which consisted of only a TATA box downstream
from a TATA box and a part of the 5'-UTR, retained the cell-specific
promoter activity at a 6-fold increase relative to the activity of the
pGL3-Basic vector. To determine which region functioned as a
cell-specific cis-regulatory element, we constructed a
series of 3' deletion mutants. Deletion from +118 to +75 (pGL3-0.23K versus pGL3-0.18K
E) reduced the level of expression by
nearly 60%. Further deletion of more than 40 bp (pGL3-0.14K
E,
pGL3-0.12K
E, and pGL3-0.1K
E) completely abolished the Luc
activity. These observations suggest that the region from +36 to +118
contained tissue-specific cis-regulatory elements and that
the sequence near position +75 is responsible. Finally, highly probable
C/EBP binding sites were identified in tandem at residues +50 to +63 (site 1) and +64 to +77 (site 2) by consensus search.
Binding of Octamer Factors to the hCCR2 Promoter--
The
identification of the 31-bp region (
89 to
59) adjacent to the TATA
box and the 83-bp region (+36 to +118) within the 5'-UTR prompted us to
determine which nuclear factors could bind to these sites, because the
former region contained a fully matched octamer sequence, ATGCAAAT, and
the latter region retained C/EBP binding sequences, i.e.
AGGTTGCATAAGCA (site 1; +50 to +63) and AGATTTCAAAATTA (site 2; +64 to
+77). First, we used the gel mobility shift assay to test nuclear
extract from THP-1 cells for its ability to bind to a 30-bp
radiolabeled probe corresponding to nucleotides
89 to
60 of the
hCCR2 promoter (Fig.
6A). At least three
protein-DNA complexes (B1, B2, and B3) representing specific binding to
this 30-bp probe could be detected (Fig. 6B, lane 2). B1 and
B2 are efficiently competed by the same unlabeled oligonucleotide or the oligonucleotide composed of the octamer sequence itself (Fig. 6B, lanes 3 and 4). B3 was reduced even when
nonspecific competitor was used (Fig. 6B, lane 5),
suggesting that this band represents a nonspecific DNA-protein complex.
The addition of a specific antibody against Oct-1 to the reaction
abolished B1 and resulted in the formation of a supershifted complex
(Fig. 6C, lane 3). In contrast, the inclusion of a specific
antibody against Oct-2 resulted in the formation of a small amount of
supershifted complex but apparently did not change the intensity of any
of the bands. (Fig. 6C, lane 4). Thus, this octamer sequence
is recognized mainly by Oct-1 in THP-1 cells, and Oct-1 may
constitutively activate the expression of hCCR2 to the basal level.

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Fig. 6.
Binding of octamer factors to the hCCR2
promoter. A, sequence and position of the probe and
competitor DNAs used in the gel mobility shift assay. B,
results of a gel mobility shift assay using a radiolabeled probe that
covers 89 to 60 (O1) and nuclear extracts from THP-1
cells, as described under "Experimental Procedures." Unlabeled
competitors, which were present at a 100-fold molar excess, are as
indicated in A (lanes 3-5). Complex
co-migrations with bands B1, B2, and B3 formed on the O1 probe are
indicated. C, antibodies against Oct-1 and Oct-2 were
preincubated with nuclear extracts from THP-1 cells before the addition
of radiolabeled O1. Antibody against C/EBP was used for the negative
control.
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Binding of the C/EBP family to the 5'-UTR of hCCR2 Gene--
To
investigate the possible transcriptional factor(s) that interact with
the 5'-UTR of the hCCR2 gene, we subjected a 73-bp radiolabeled probe covering the region from +46 to +118 to gel mobility
shift assays. Three retarded complexes (C1, C2, and C3) were observed
with nuclear extracts from THP-1 cells (Fig.
7B, lane 2). The formation of
all complexes was abolished by unlabeled competitors that contain two
C/EBP binding sites (N1 and N6), but not by competitor containing the
3' part of the 5'-UTR (K2) (Fig. 7B, lanes 3, 4, and
6). The formation of complexes at the C/EBP-specific site
was confirmed using oligonucleotides containing an alternative C/EBP
recognition sequence (CP) as a competitor; its sequence is totally
different from that found in the 5'-UTR of the hCCR2 gene
(Fig. 7B, lane 5). All three complexes were abolished by the
addition of CP, suggesting that the formation of these complexes
requires the transcriptional factors that recognize the C/EBP binding
motifs.

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Fig. 7.
Binding of C/EBP family to the 5'-UTR of the
hCCR2 gene. A, sequence and position of the
probe and competitor DNAs used in the gel mobility shift assay.
B, results of a gel mobility shift assay using a
radiolabeled probe that covers +46 to +118 (N1) and nuclear
extracts from THP-1 cells, as described under "Experimental
Procedures." Unlabeled competitors, which were present at a 100-fold
molar excess, are as indicated in A (lanes 3-6).
Complex co-migrations with bands C1, C2, and C3 formed on the N1 probe
are indicated. C, antibodies against C/EBP , C/EBP , and
C/EBP were preincubated with nuclear extracts from THP-1 cells
before the addition of radiolabeled N1. Antibody against Oct-1 was used
for the negative control.
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To further corroborate the presence of C/EBP family members in the
respective complexes, we performed antibody perturbation experiments.
Antibodies against C/EBP
, C/EBP
, and C/EBP
were tested for
their ability to interact with the complexes described above. The
presence of C1 was due to C/EBP
binding, because anti-C/EBP
antibody decreased C1, resulting in a supershifted band (Fig. 7C,
lane 3). The presence of C2 was due to C/EBP
recognition, and
the presence of C3 was due to C/EBP
recognition, because they were
reduced by anti-C/EBP
or anti-C/EBP
antibodies, respectively, resulting in supershifted bands (Fig. 7C, lanes 4 and
5).
Roles of Oct-1 Binding and C/EBP Binding in the Expression of
hCCR2--
To determine whether the cell-specific expression of CCR2
is actually dependent on the expression of Oct-1 or the C/EBP family, we performed a gel mobility shift assay using nuclear extracts from
CCR2-negative J-111 and U-251 MG cells with a radiolabeled O1 probe or
N1 probe (Figs. 6A and 7A). These transcriptional factors were almost absent in U-251 MG cells and were considerably decreased in J-111 cells (Fig. 8). It was
particularly interesting that C/EBP
(indicated by the C1 complex)
was not detected in either cell line. These results suggest that the
expression of hCCR2 is regulated by the tissue-specific expression of
Oct-1 and the C/EBP family.

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Fig. 8.
Differential expressions of octamer factors
and the C/EBP family in THP-1 cells, J-111 cells, and U-251 MG
cells. Results of gel mobility shift assays using nuclear extracts
from THP-1 cells, J-111 cells, and U-251 MG cells, as described under
"Experimental Procedures." Equal amounts (4 µg) of nuclear
extracts from each cell line were mixed with either radiolabeled O1
probe or N1 probe to confirm the binding of octamer factors and the
C/EBP family.
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The role of the particular octamer sequence and the two C/EBP binding
sites was confirmed by the Luc assay using promoter reporter constructs
containing single or multiple mutations of each motif (Fig.
9). Alteration of the octamer sequence by
substitution decreased promoter activity by 68%, which is consistent
with the results of deletion analysis (Fig. 5).

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Fig. 9.
Inhibition of the hCCR2 promoter activity by
the introduction of mutations at the octamer factor binding sequence
and C/EBP binding sequences. Schematic representation of
hCCR2 reporter constructs. The TATA box, proximal octamer
factor binding site ( 81 to 68), and two C/EBP binding sites
(Site 1 and Site 2) are indicated by closed box,
open oval, and closed ovals, respectively. The transcription initiation
site is indicated by a bent arrow. These sites were mutated
(indicated by X) in some of the constructs. These constructs
were transfected into THP-1 cells as indicated in Fig. 5, and the
relative promoter activities were calculated by arbitrarily defining
the activity of pGL3-Basic vector as 1. Columns indicate the mean ratio
of the Luc activities. Error bars, the standard
deviation.
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The introduction of mutations at site 1 or both C/EBP binding sites
completely abolished the Luc activity, whereas the mutagenesis of site
2 reduced the promoter activity by 66%. Accordingly, the Oct-1 binding
site and both C/EBP binding sequences are actually active in the
transcriptional regulation of hCCR2 in THP-1 cells. Among them, site 1 was the most important for basal and tissue-specific transcriptional activity.
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DISCUSSION |
In the present work, we isolated and functionally characterized
the 5'-flanking region of the hCCR2 gene to elucidate its regulatory mechanism. The structural analysis disclosed that the proximal 5'-flanking region of this gene contained a classic TATA box,
a CAAT box, and clustered consensus sequences for tissue-specific cis-regulatory elements, i.e. the GATA binding
sequence, Oct-1 binding sequence, and C/EBP binding motifs. This is
compatible with the fact that the hCCR2 gene is not a
housekeeping gene and is regulated in a cell-specific manner.
Among the members of the CCR family, only the promoter region for hCCR5
has been isolated and functionally characterized to date (23). The
transcription of the hCCR5 gene was initiated by two
different promoters, and both proximal and distal promoters lack the
canonical TATA or CAAT box. Instead, they contain the consensus
sequence for several transcriptional factors such as Oct-1 and multiple
motifs for GATA-1, which are also observed in the hCCR2
gene. It is interesting that these closely related genes have rather
different transcriptional initiations, although they are thought to
share the same ancestral gene.
The Luc assay using various deletion mutants of the hCCR2
promoter region demonstrated that at least four regions are important for the constitutive expression of hCCR2 in THP-1 cells: (a)
429 to
186 for activation, (b)
185 to
123 for
repression, (c)
89 to
59 for activation, and
(d) +36 to +118 for cell specificity and activation. Among
these four regions, we focused on the latter two, because the deletion
mutant containing only these regions (
89 to +118) was sufficient for
the basal expression and tissue specificity.
The gel mobility shift assay and mutagenesis demonstrated that Oct-1
bound mainly to the octamer consensus sequence (ATGCAAAT) located in
the third region (from
89 to
59) and was probably responsible for
the trans-activation of this region. Although Oct-1 is
ubiquitously expressed (24), it is reduced or under the detectable
level in CCR2-negative cell lines (J-111 and U-251 MG cells) (Fig. 8).
This suggests that Oct-1 plays a certain role in the tissue-specific
expression of hCCR2.
The most important cis-acting element was located at a
rather unusual position, the 5'-UTR of the hCCR2 gene (+36
to +118). It has been reported that the 5'-UTR could regulate the
expression of genes in two different ways, one of which is that the
5'-UTR can work as a tissue-specific active translational enhancer
taking a stable stem and loop structure; examples are the human
-glutamyl transferase gene (25), the thymidine kinase gene (26), the glutathione peroxidase gene (27), and the ornithine decarboxylase gene
(28). Stable secondary structures were predicted between +12 and +118
of the hCCR2 gene, presenting the free energy formation of
43.6 kcal/mol. However, the present Northern blot analysis indicated
that the hCCR2 gene is regulated at least at the
transcriptional level in various cell lines (Fig. 4). Therefore, we
should focus on the other mechanism, the transcriptional activity of
the 5'-UTR, which has been demonstrated in the human plasminogen gene
(29) and the A
-globin gene (30), indicating that the
transcriptional factor bound to a specific region within 5'-UTR.
We particularly noted the position near +75, where two putative C/EBP
binding sequences were located in tandem, because pGL3-0.18K
E, which
partially disrupted the C/EBP binding consensus, reduced the Luc
activity by nearly 60%. The gel mobility shift assay using a 73-bp
fragment containing tandem C/EBP binding sequences as a probe detected
three prominent bands. Competition using the C/EBP consensus sequence
and the supershift assay demonstrated that each band was related to
C/EBP
, C/EBP
, or C/EBP
. Finally, the introduction of mutation
in each motif disclosed that site 1 was the most important site for the
tissue-specific expression of hCCR2.
The C/EBP family of transcriptional factors is involved in
tissue-specific gene expression in adipocytes, hepatocytes, and monocyte/macrophages (31). In particular, target genes for C/EBP include acute phase response genes in liver cells and cytokine genes in
monocytes/macrophages (31). Our present results demonstrate that
CCR2-negative cells (J-111 and U-251 cells) had considerably reduced
expression of the C/EBP family (Fig. 8). Therefore, it is reasonable to
speculate that the C/EBP binding sequence located in the 5'-UTR can
regulate the expression of hCCR2 in a monocyte-specific manner.
It is interesting that C/EBP
and C/EBP
contribute to the
lipopolysaccharide response of MCP-1, the ligand of CCR2 (32). MCP-1
and CCR2 are probably coordinately regulated at the transcriptional level by the C/EBP family for effective immune responses. The tumor
cytotoxicity of macrophages from C/EBP
knockout mice was severely
impaired (33). Our previous study showed that the growth of
MCP-1-producing tumors was inhibited by the infiltration of tumor-associated macrophages (15), indicating that MCP-1 and CCR2 may
play an important role in the cytotoxicity of macrophages in cancer.
Although we were unable to identify the stimulation that effectively
induces the expression of hCCR2, our present findings provide a useful
foundation for further studies.