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INTRODUCTION |
The molecular mechanisms regulating neutrophil development are not
defined yet. Abnormal neutrophil development is related to several
pathological conditions such as neutropenia, neutrophilia, and
myelogenous leukemia (1-3). The regulation of this developmental process most likely involves the interplay of transcription factors with positive and negative cis-acting elements and cytokines
interacting with their cognate receptors (4, 5). For example,
disrupting either of the murine genes encoding the transcription
factors PU.1 (Spi-1) and CCAAT enhancer-binding protein
impairs
neutrophil development (6-8). Similarly, disruption of the gene
encoding the cytokine granulocyte colony-stimulating factor
(G-CSF)1 inhibits neutrophil
development (3). By contrast, disruption of the murine homolog of the
chemotactic cytokine interleukin-8 (IL-8) receptor gene (CXCR2) causes
neutrophilia (9).
IL-8 receptor subtypes A and B (CXCR1 and CXCR2) are G-protein-coupled
receptors expressed at high levels in a myeloid-specific fashion, in
mature neutrophils and myeloid precursor cells (10-12). IL-8
suppresses the proliferation of myeloid progenitor and precursor cells
via activation of the IL-8 receptor (12, 13). In mature neutrophils,
IL-8 is the major mediator for the recruitment of neutrophils from
circulation to the sites of injury and infection. Despite the
importance of the IL-8 receptor in myeloid development and its
restrictive expression in myeloid cells, the transcriptional mechanisms
regulating myeloid-specific expression of the IL-8 receptor genes are unknown.
Previous studies have shown that CXCR1 and CXCR2 expression is under
transcriptional control (14). The genomic organization and promoter
regions of CXCR1 and CXCR2 have been previously mapped (15, 16). The
CXCR1 gene consists of two exons interrupted by an intron of ~1.7
kilobases. The entire open reading frame is encoded in exon 2. Sprenger
et al. (15) detected promoter activity in the T lymphoma
cell line Jurkat when transfected with the chloramphenicol
acetyltransferase reporter gene driven by 5' flanking sequences of the
CXCR1 gene (
800 to +21 bp). However, identification of regulatory
elements and transcription factors regulating the myeloid
lineage-specific expression of the CXCR1 gene have been precluded by
the lack of suitable myeloid cell lines expressing high levels of CXCR1
or CXCR2. In this work we delineate cis-acting elements that
direct myeloid-specific expression of the CXCR1 promoter and identify
the ets family transcription factor PU.1 as a major
regulator of the CXCR1 promoter.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Cell culture supplies were purchased from Life
Technologies, Inc. 32Dcl3 cells were a gift from Dr. Joel Greenberger
(University of Pittsburgh Medical School, Pittsburg, PA). NIH3T3,
RAW264.7, and CEMT4 cell lines were purchased from the American Type
Culture Collection (Rockville, MD). 32Dcl3 cells were cultured in RPMI 1640 medium supplemented with 15% WEHI-3B conditioned media, 15% fetal bovine serum, and 50 IU/ml penicillin-50 µg/ml streptomycin. RAW264.7 cells were cultured in Dulbecco's modified Eagles's medium supplemented with 10% fetal bovine serum and 50 IU/ml penicillin-50 µg/ml streptomycin. CEMT4 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 50 IU/ml penicillin-50 µg/ml streptomycin. NIH3T3 cells were cultured in Dulbecco's
modified Eagles's medium (1 g/l D-glucose) supplemented
with 10% bovine serum and 50 IU/ml penicillin-50 µg/ml streptomycin.
All cell lines were grown at 37 °C and 5% CO2.
Northern Blot Analysis--
Total RNA was isolated by the RNAzol
procedure (Biotecx Laboratories Inc., Houston, TX). Total RNA (10 µg)
was fractionated on 1% agarose-formaldehyde gels and blotted to nylon
membranes (Micron Separations Inc., Westborough, MA). The blot was
probed with murine PU.1, murine CXCR2, and human
-actin cDNAs
labeled by random priming with 50 µCi of [
-32P]dCTP
(DuPont NEN). The blot was hybridized at 42 °C for 16-20 h in the
following solution: 50% formamide, 5 × SSPE (750 mM
NaCl, 500 mM NaH2PO4, 5 mM EDTA, pH 7.4) 5 × Denhardt's solution, 1% SDS,
and 200 µg/ml sonicated calf thymus DNA. Blots were washed three
times for 10 min with 0.5 × SSC (75 mM sodium
chloride, 7.5 mM sodium citrate) and 1% SDS at 65 °C.
The blot for
-actin was washed two times for 10 min with 0.1 × SSC and 1% SDS at 65 °C.
Plasmid Construction--
Peripheral blood was collected from
healthy human donors and placed in a 1% sodium citrate (anticoagulant)
solution. Genomic DNA was extracted from leukocytes as described (17).
A forward primer corresponding to
800 to
777 bp of CXCR1 and
containing an XhoI site
(5'-CGGCTCGAGGCTAACCAGCCAGACTCTGGGAGT-3'), and a reverse primer
corresponding to +63 to +86 bp of CXCR1 and containing a
HindIII site (5'-GGACACACCTAAGCACCGGCCAGGTGTGTC-3') were
used to amplify an 889-bp 5'-flanking region of CXCR1 (15). Conditions of PCR included a step of 95 °C for 3 min followed by 25 cycles of
95 °C for 1 min 30 s, 65 °C for 1 min 30 s, and
72 °C for 1 min 30 s, and a final extension of 72 °C for 10 min. The amplified PCR product was gel-purified and digested with
XhoI and HindIII, and cloned into
SalI-HindIII sites upstream of a luciferase
cDNA in a promoterless pGEM-3 vector referred to as pLUC. Deletion constructs were made by digestion with unique restriction sites within
the CXCR1 sequence (A2 construct, NdeI; A3 construct,
AccI; and A4 construct, PstI). The A2 mutant
construct was generated by cassette mutagenesis. The sequence (
58 to
+50 bp) was removed at AccI-PstI sites and
replaced by a synthetic double-stranded oligonucleotide in which
guanine nucleotides at positions
13,
12,
3, and
4 bp in the
wild-type sequence were replaced with thymine nucleotides. The pGEM-3
vector containing the SV40 promoter and enhancer upstream of a
luciferase cDNA (pSV40/LUC) was used as a positive control. The
-galactosidase reporter gene driven by the cytomegalovirus promoter
(pCMV/
-gal) was used as an internal control to correct for
differences in transfection efficiency between experiments. The vectors
pSV40/LUC and pCMV/
-gal were provided by Dr. Allan Braiser
(University of Texas Medical Branch, Galveston, TX).
Plasmids were purified by cesium chloride gradient centrifugation as
described by Ausubel et al. (18), and the sequences were verified by
dideoxy sequencing using a Sequenase kit (United States Biochemical
Co., Cleveland, OH).
Transient Transfections--
Log phase 32Dcl3 (14 × 106), RAW264.7 (107), CEMT4 (107),
and NIH3T3 (2.5 × 106) cells were incubated with 20 µg of pIL-8RA/LUC or pSV40/LUC constructs and 2 µg of pCMV/
-gal
for 5 min at room temperature, electroporated using a 300-V, 960-µF
pulse, and then incubated on ice for 15 min. Cells were plated in 5 ml
of complete culture media and incubated at 37 °C with 5%
CO2. Cells were harvested 17 h after electroporation.
Transfected cells were washed three times with phosphate-buffered
saline, resuspended in 100 µl of lysis buffer (25 mM
Tris-PO4, pH 7.8, 2 mM 1,2 diaminocyclo-N,N,N,N'-tetracetic acid, 2 mM
dithiothreitol, 10% glycerol, 1% Triton X-100), vortexed for 2 min,
and stored at
70 °C until ready for reporter gene assays.
Reporter Gene Assays--
Luciferase and
-galactosidase
assays were performed in duplicate with cell lysates (10 µl) from
each transfection. Luciferase assays were performed as described in
Promega Protocols and Applications Guide (19). Lysates were
incubated with a luciferase assay reagent composed of 20 mM
Tricine, 1.07 mM
(MgCO3)4MgOH2, 2.67 mM
MgSO4, 0.1 mM EDTA, 270 µM
coenzyme A, 470 µM luciferin, 530 µM ATP, and 33.3 mM dithiothreitol. (Coenzyme A, luciferin, ATP,
and dithiothreitol were added immediately before each assay.) Purified
luciferase was used as a standard. Emitted light was recorded as
relative light units using an Analytical Luminescence Laboratory
Monolight 2010 luminometer.
-Galactosidase assays were performed as
described by Sambrook et al. (20).
Electrophoretic Mobility Shift Assays (EMSAs)--
Peripheral
blood was collected from healthy human donors and placed in a 1%
sodium citrate (anticoagulant) solution. Neutrophils and monocytes were
separated from other cell types by centrifugation with mono-poly
resolving medium (ICN Pharmaceuticals, Inc., Costa Mesa, CA). Nuclear
extracts were prepared by cell lysis with the detergent Nonidet P-40 as
described by Johnson et al. (21). All extracts were prepared
in the presence of 0.5 mM phenylmethylsulfonyl flouride.
The protease inhibitors leupeptin (1 µM) and aprotinin (0.3 µM) were also used in the preparation of extracts
from neutrophils and monocytes. Protein concentrations were determined
using the Bradford assay (22). The sense strand sequences of the probes are as follows: A2, (
22 to +14 bp),
5'-ATGTGGTTTCCTTATTTCCGTTTATTCATCAAGTGC-3'; M1,
5'-ATGTGGTTTAATTATTTCCGTTTATTCATCAAGTGC-3'; M2,
5'-ATGTGGTTTCCTTATTTAAGTTTATTCATCAAGTGC-3'; M3,
5'-ATGTGGTTTAATTATTTAAGTTTATTCATCAAGTGC-3'; PU.1 (CDllb
promoter), 5'-GCTCAAAGAAGGGCAGAAAAGGAGAAGTAG-3'; and NS
(nonspecific), 5'-CTCGCAGTGACATTAGCATTCCGGTACTGTATCGTA-3'.
Double-stranded oligonucleotides were 5' end-labeled by polynucleotide
kinase using 15 µCi of [
-32P]ATP (DuPont NEN).
Unincorportated [
-32P]ATP was removed from the labeled
probe with nick spin columns-Sephadex G-50 (Amersham Pharmacia Biotech).
DNA probes (0.5 ng) were incubated with 5 µg of nuclear extracts and
2 µg of poly(dI·dC) in binding buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM dithiothreitol, 100 mM NaCl, 10% glycerol) for 30 min at room temperature. A
100-fold molar excess of unlabeled probe was used for competition
experiments. For supershift analyses, 2 µg of a rabbit polyclonal
PU.1 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) or 2 µl
of rabbit preimmune sera were incubated with nuclear extracts for 15 min at 4 °C before addition of probe. Binding reactions were
electrophoresed on 6% acrylamide gels with 0.5 × Tris
borate-EDTA (4.45 mM Tris base, 4.45 mM boric
acid, 1 mM EDTA, pH 8.0) as a running buffer. Gels were
dried and exposed with intensifying screens to x-ray film for 24-48 h
at
70 °C.
Transactivation Assays--
CEMT4 and NIH3T3 cells were
transfected by electroporation with 15 µg of construct A2 (
126 to
+86 bp), 15 µg of an expression vector encoding for murine PU.1
(CMV/pCB6+PU.1) or vector without the PU.1 cDNA (CMV/pCB6+), and 2 µg of pCMV/
-gal. DNA concentrations were adjusted to 42 µg with
the CMV/pCB6+ vector. The vector pCMV/pCB6+PU.1 was generously provided
by Dr. Michael Atchison (University of Pennsylvania, Philadelphia, PA).
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RESULTS |
IL-8 Receptors Are Expressed in Myeloid Precursor Cells--
CXCR1
and CXCR2 are highly expressed in neutrophils and readily detected by
Northern blot analysis. The short life span of neutrophils and the
difficulties of transfecting these cells have precluded the use of
neutrophils as a cellular system to elucidate the transcriptional
regulation of neutrophil-specific genes. On the other hand, most cell
lines of hematopoietic origin express trace amounts of CXCR1 or CXCR2
(23). Recently, we have identified a myeloid cell line, 32Dcl3 (32D),
that increases intracellular calcium in response to IL-8 (12). Northern
blot analysis of murine neutrophils and 32D, NIH3T3 (3T3), CEMT4 (CEM),
and RAW264.7 (RAW) cell lines revealed high levels of murine IL-8
receptor mRNA in 32D cells and neutrophils (Fig.
1). The 32D cell line is a murine
interleukin-3-dependent myeloid precursor cell line that
differentiates into neutrophils in the presence of G-CSF (24). Recently
we found that IL-8 and the related chemokine melanoma
growth-stimulating activity suppress the proliferation of 32D cells
(12). Although previously it was argued that mouse and rat only express
the human homolog of CXCR2, two reports have identified the human
homolog of CXCR1 in rat and mouse (25, 26). These observations indicate
that the murine 32D cell line is a suitable cellular system to identify
the regulatory elements and transcription factors regulating the
myeloid lineage-specific expression of the human CXCR1 gene.

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Fig. 1.
Northern blot analysis of IL-8 receptor
mRNA expression. Total RNA (10 µg) from mouse neutrophils
(lane 1) and 32D (lane 2), 3T3 (lane
3), CEM (lane 4), and RAW (lane 5) cells
were fractionated by electrophoresis on denaturing agarose gels,
blotted into nylon membranes, and probed with a 32P-labeled
mouse IL-8 receptor cDNA and a 32P-labeled human
-actin cDNA as described under "Experimental
Procedures."
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Myeloid Lineage Specificity of the CXCR1 Promoter--
Previous
studies showed that CXCR1 promoter constructs containing the first 800 bp of 5'-flanking DNA directed activation of the reporter gene
chloramphenicol acetyltransferase in hematopoietic cell lines
expressing negligible levels of IL-8 receptors (15, 16). To determine
the specificity of the CXCR1 promoter for the myeloid lineage, myeloid
(32D and RAW) and nonmyeloid (3T3 and CEM) cell lines were transfected
with the
800 to +86 bp/luc promoter construct (A1). As shown in Fig.
2, the A1 promoter construct exhibited
the greatest reporter activities in 32D and RAW cell lines, 200- and
400-fold higher than the promoterless luciferase vector, respectively.
In the nonmyeloid cells CEM and 3T3, the promoter activity of construct
A1 was significantly lower than the activity observed in myeloid cells.
Deletion of the sequence
800 to
126 bp (construct A2) increased
reporter activity 2-fold relative to construct A1. This finding
suggests that the sequence
800 to
126 bp contains negative promoter
elements. Further deletion of the sequence
800 to
58 bp (construct
A3) still exhibited high levels of promoter activity in the myeloid
cells 32D and RAW. Surprisingly, the A3 construct showed higher levels
of promoter activity in 3T3 cells than the A1 and A2 constructs,
suggesting the presence of negative elements between
126 and
58 bp.
Deletion of the
800- to +51-bp region (construct A4) abolished
promoter activity. These data indicate the presence of
cis-acting elements from
126 to +51 bp that direct
positive promoter activity in the myeloid cells 32D and RAW and
elements from
126 to
58 bp that direct negative promoter activity
in the nonhematopoietic cell line 3T3.

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Fig. 2.
Promoter activity of CXCR1 5'-flanking
region. 32D, CEM, RAW, and 3T3 cells were transiently transfected
with reporter gene constructs and assayed for promoter activity as
described under "Experimental Procedures." The SV40 promoter and
enhancer luciferase construct was used as a positive control.
Transfection efficiency was normalized by cotransfection with
CMV/ -galactosidase plasmids. Promoter activity is reported as fold
increase relative to the activity of the promoterlesss luciferase
vector (pLUC). Error bars indicate the S.E. from the results
of at least three separate experiments.
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The Sequence
22 to +14 bp Binds Nuclear Proteins Present in 32D
and RAW Cell Lines--
EMSAs were performed to identify transcription
factors binding to the CXCR1 promoter sequences in a myeloid-specific
fashion. EMSAs were performed with 32P end-labeled DNA
fragments encompassing the
58- to +50-bp sequence, because the
majority of myeloid-specific promoter activity is contained in this
region. Only a fragment corresponding to the sequence
22 to +14 bp
(A2 probe) produced a fast-migrating, myeloid-specific complex (Fig.
3). An excess of unlabeled A2 probe
displaced the binding of the labeled A2 probe to the myeloid-specific
factor (Fig. 3, lanes 3 and 7); however, an
excess of unlabeled nonspecific probe did not displace the binding of
A2 probe (Fig. 3, lanes 11 and 15). A similar
myeloid-specific complex was observed with the A2 probe and nuclear
extracts from human neutrophils and human monocytes (data not shown).
These results suggest that myeloid-specific transcription factors bind
to the
22- to +14-bp fragment.

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Fig. 3.
Formation of a myeloid-specific complex with
the CXCR1 A2 probe (-22 to +14 bp). EMSA conditions are as
described under "Experimental Procedures." Lanes 2-9,
competition analysis of the A2 probe with 100-fold molar excess of
unlabeled A2 probe. Lanes 10-17, competition analysis of
the A2 probe with 100-fold molar excess of unlabeled NS (nonspecific)
probe. The myeloid-specific complex and unbound probe are indicated by
arrows.
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The
22- to +14-bp Fragment Binds to the Hematopoietic
Transcription Factor PU.1--
Analysis of the
22 to +14 bp sequence
revealed consensus binding sequences for transcription factors of the
ets family. PU.1 is a member of the ets family
that is expressed in myeloid cells, including neutrophils and
macrophages, and B cells (27). To determine whether PU.1 binds to the
22- to +14-bp sequence (probe A2), the two putative PU.1 binding
sites were first mutated. Disruption of the two putative PU.1 sites
(M3) or the PU.1 site proximal to the transcription start site (M2) did
not displace the A2 probe bound to the myeloid-specific protein, and M3
and M2 probes did not generate the myeloid-specific complex (Fig.
4). By contrast, disruption of the PU.1
site distal to the transcription start site (M1) effectively displaced
the A2 probe bound to the myeloid-specific protein, and the M1 probe
generated the myeloid-specific complex (Fig. 4). These findings
strongly suggest that the core PU.1 binding motif is located
7 to
4
bp. Second, a fragment corresponding to the PU.1 binding site of the
CD11b promoter was an effective competitor for formation of the
myeloid-specific complex (Fig. 4, lanes 2-5) but not for
nonmyeloid DNA-protein complexes formed with myeloid (Fig. 4,
lanes 2-5) and nonmyeloid extracts (Figs. 4, lanes
6-9, and 5A). Third, the
PU.1 fragment produced a similar myeloid-specific complex as the
22-
to +14-bp fragment A2 with extracts from 32D (Fig. 5A, lane
11) and RAW (Fig. 5A, lane 13), and A2 was an effective
competitor for the formation of the PU.1 complex (Fig. 5A, lanes
12 and 14). Fourth, EMSA in the presence of antibodies
that specifically recognize human and murine PU.1 produced supershifts
of the myeloid-specific complexes generated with the PU.1 fragment
(Fig. 5A, lanes 3 and 6) and the
22 to +14 bp
sequence (probe A2) (Fig. 5B, lanes 10 and 12).
Fifth, Northern blot analysis revealed that PU.1 mRNA is expressed
specifically in the myeloid cells 32D and RAW but not in nonmyeloid
cells CEM and 3T3 (Fig. 6). These results
demonstrate that the myeloid-specific complex is generated by PU.1
binding to the
22 to +14 bp fragment.

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Fig. 4.
Disruption of PU.1 putative sites block
formation of the myeloid-specific complex. EMSA conditions are as
described under "Experimental Procedures." Competition analysis of
the A2 probe was performed with 100-fold molar excess of unlabeled M3
(lane 4), M1 (lane 5), and M2 (lane 6)
probes. Analysis of myeloid complex formation was performed with M1
(lane 7), M2 (lane 8), and M3 (lane 9)
probes. The myeloid-specific complex and unbound probe are indicated by
arrows.
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Fig. 5.
PU.1 binding to the 22- to +14-bp
fragment. EMSA conditions are as described under "Experimental
Procedures." A, competition analysis of A2 probe binding
by PU.1 probe (lanes 1-9) and PU.1 probe binding by A2
probe (lanes 10-16). B, Supershift induced by
PU.1 antibody of the myeloid-specific complexes formed with probes PU.1
and A2. PI, rabbit preimmune sera; Ab, PU.1
rabbit polyclonal antibody. Shifted complexes produced in the presence
of antibody (Ab), myeloid-specific complex, and unbound
probe are indicated by arrows.
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Fig. 6.
Expression of PU.1 mRNA in myeloid
cells. Total RNA (10 µg) from mouse neutrophils (lane
1) and 32D (lane 2), 3T3 (lane 3), CEM
(lane 4) and RAW (lane 5) cells was fractionated
by electrophoresis on denaturing agarose gels, blotted into nylon
membranes, and probed with a 32P-labeled murine PU.1
cDNA and a 32P-labeled human -actin cDNA as
described under "Experimental Procedures."
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PU.1 Binding Site Is Essential for IL-8 Receptor A Promoter
Activity--
To determine whether the PU.1 binding site in the
22-
to +14-bp sequence is functional, myeloid and nonmyeloid cell lines were transfected with the
126 to +86 bp/luc construct mutated at this
site. Disruption of the PU.1 binding site abolished the promoter
activity of the
126 to +86 bp/luc construct (A2) (Fig. 7). This result indicates that the PU.1
site is essential for promoter activity and that compensatory elements
are not present in this construct to drive the expression of the
reporter gene. To directly demonstrate that PU.1 binds and activates
the IL-8RA promoter, the nonmyeloid cell lines that do not express
PU.1, CEM, and 3T3 were cotransfected with the
126 to +86 bp/luc
construct (A2) and an expression vector encoding PU.1. Cotransfections
with PU.1 cDNA increased promoter activity 4-fold in CEM cells
(Fig. 8A) and >16-fold in 3T3
cells (Fig. 8B) compared with vector alone. These findings
strongly suggest that the myeloid-specific expression of the CXCR1 gene
is activated by PU.1 interacting with promoter sequences adjacent to
the transcription start site.

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Fig. 7.
Disruption of PU.1 sites abolished IL-8RA
promoter activity. 32D, CEM, RAW, and 3T3 cells were transfected
with A2 and mutant A2 constructs and assayed for promoter activity as
described under "Experimental Procedures." Transfection efficiency
was normalized by cotransfection with CMV/ -galactosidase plasmids.
Promoter activity is reported as fold increase relative to the activity
of the promoterlesss luciferase vector (pLUC). Error bars
indicate the S.E. from the results of at least three separate
experiments.
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Fig. 8.
Transactivation of the IL-8RA promoter by
PU.1. CEM (A) and 3T3 (B) cells were
cotransfected with the A2 construct ( 126 to +86 bp) and an expression
vector encoding PU.1 (PU.1/pCB6+) or vector alone (pCB6+).
C, 3T3 cells were cotransfected with the A2 construct ( 126
to +86 bp) and increasing concentrations of PU.1/pCB6+. Lysates were
assayed for promoter activity as described under "Experimental
Procedures." Transfection efficiency was normalized by cotransfection
with CMV/ -galactosidase plasmids. Promoter activity is reported as
fold increase relative to the activity of the promoterlesss luciferase
vector (pLUC). Error bars indicate the S.E. from the results
of at least three separate experiments.
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DISCUSSION |
Chemokines are major regulators of the proliferation of myeloid
precursor cells (12, 13, 28). However, little is currently known about
the mechanisms regulating the expression of chemokine receptors during
the commitment and differentiation of progenitor cells toward myeloid
lineages and neutrophil development, in particular. The restrictive
expression of IL-8 receptors in myeloid precursor cells and neutrophils
provides a system to identify the regulatory elements and transcription
factors that may regulate the commitment of progenitor cells to the
neutrophil lineage.
In this study, the promoter activity of the proximal
800 bp of the
CXCR1 gene was analyzed. This sequence was found to contain the
regulatory elements that direct CXCR1 promoter activity in a
myeloid-specific fashion. High levels of promoter activity were detected specifically in 32D (myeloid precursor cells) and RAW (macrophages). Sequences
800 to
126 bp were found to contain negative regulatory elements. This finding is in agreement with that of
Sprenger et al. (15), who suggested the presence of silencer
elements between positions
841 and
280 bp on the basis of
transfection studies with chloramphenicol acetyltransferase reporter
genes in nonmyeloid cell lines. Because most of the promoter activity
is localized within the
56- to +50-bp sequence, myeloid-specific proteins binding in this region were identified. On the basis of EMSA,
the transcription factor PU.1 was shown to bind the
22- to +14-bp
fragment with the common GGAA binding motif at position
7 to
4 bp.
Disruption of the PU.1 binding site abolished the myeloid-specific
transcriptional activity of the CXCR1 promoter. Transfection of
nonmyeloid cell lines CEM (T cells) and 3T3 (fibroblasts) with cDNA
encoding PU.1 increased the promoter activity of the
126 to +86
bp/luc construct. Because PU.1 expression in nonmyeloid cell lines
produced high levels of promoter activity, this suggests that PU.1 does
not require other myeloid-specific factors for activation of the
promoter construct. These data show for the first time the
transcriptional regulation of a chemoattractant G protein-coupled
receptor by the myeloid transcription factor PU.1.
PU.1 has been shown to regulate several myeloid lineage-specific genes,
including granulocyte-macrophage CSF receptor, G-CSF receptor,
macrophage CSF receptor, CD11b, scavenger receptor, Fc
RIIIA,
Fc
R1b, c-fes, interleukin-1
, myeloperoxidase, and neutrophil elastase (8, 29-32). Furthermore, PU.1 has also been found
to control neutrophil development. Studies with PU.1-null mice indicate
that PU.1 is necessary for normal neutrophil development (6-7,
33-35). Neutrophils from mice deficient of PU.1 fail to terminally
differentiate and are functionally incompetent. PU.1
/
mice contain neutrophils that do not respond to IL-8, indicating that
functional receptors are not expressed (33) and further supporting the
view that PU.1 is required for the expression of IL-8 receptors.
Our studies reveal that the regulatory sequences analyzed in this study
do not direct cell-specific expression, because high levels of promoter
activity are demonstrated in both 32D cells, which express IL-8
receptors, and RAW cells, which do not express IL-8 receptors. Similar
findings were observed with the promoter of the eosinophil-specific
IL-5 receptor, in which high levels of promoter activity were obtained
in both myeloid and eosinophilic cell lines (36). The neutrophil
lineage-specific expression of CXCR1 transcripts could possibly be
attributable to post-transcriptional mechanisms or transcription factor
regulatory sites located further upstream or downstream of the
sequences analyzed in this study. Further experiments will be focused
on mapping additional functional elements of the CXCR1 promoter and
identifying the transcription factors that bind to these elements to
elucidate the mechanisms regulating the neutrophil lineage-specific
expression of CXCR1.