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Department of Biochemistry, Juntendo University, School of Medicine, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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200-bp
region identical between promoters was sufficient for transcriptional
activity. Furthermore, overlapping CCAAT/enhancer-binding protein
(C/EBP) and c-Myb sites in the region were found to be required for
efficient transcription. Gel mobility shift assay demonstrated that
C/EBP
predominantly bound to the C/EBP/c-Myb sites using HL-60
nuclear extracts. No specific binding to C/EBP/c-Myb sites was observed
in nuclear extracts from mature neutrophils, which expressed neither
C/EBP protein nor HNP mRNAs. Taken together, these findings suggest
that the difference in the amounts of HNP-1 and HNP-3 peptides in
neutrophils is caused by posttranscriptional regulation and that
C/EBP plays an important role in the transcription of
HNP genes in immature myeloid
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß defensins (10, 11).
In humans, defensins (human neutrophil peptides
(HNPs))4 are major
components of the primary (azurophil) granules of neutrophils and
comprise 30–50% of azurophil granule protein (8, 14, 15). Among HNPs, HNP-1, HNP-2, and HNP-3 have almost identical
amino acid sequences (9). Both HNP-1 and HNP-3 are
composed of 30 amino acid residues, with the first amino acid in HNP-1,
alanine, substituted by aspartate in HNP-3. HNP-2, 29 amino acids in
length due to lack of the first amino acid, is assumed to be a
proteolytic derivative of HNP-1, HNP-3, or both (9). HNP-1
and HNP-3 are encoded by nearly identical genes on chromosome 8;
notwithstanding this similarity, HNP-1 is more abundant than HNP-3 in
neutrophil granules (16, 17, 18). The fourth defensin, HNP-4,
is the least abundant and except for conserved cysteine residues shows
low homology with other HNPs (19, 20, 21). Despite their
nearly identical amino acid sequences, HNPs exhibit different
biological properties in vitro. For example, although HNP-1, HNP-2, and
HNP-4 can kill Candida albicans, HNP-3 cannot (20, 22). Interestingly, HNP-1 and HNP-2, but not HNP-3, have
chemotactic activity for murine and human T cells and monocytes
(16, 23). Moreover, HNP-1, HNP-2, and HNP-3 have been
shown to enhance T cell-dependent immune responses in vivo
(24). Such diverse functions of HNPs are likely
to contribute to both innate and acquired host defenses.
In contrast to the abundance of information on the function of these peptides, their gene regulation is little known. HNP mRNAs are only expressed in immature bone marrow cells and HL-60 human promyelocytic leukemia cells (25, 26, 27, 28). A more recent report has indicated that PU.1 and an unknown Ets-like factor (IRD) are involved in the basal transcription of HNP-1 gene (29). However, given that several transcription factors such as CCAAT/enhancer-binding proteins (C/EBPs), c-Myb, and AML1 (PEBP2/CBF) cooperatively regulate the efficient transcription of many myeloid-specific genes (30, 31, 32), factors other than PU.1 and IRD may also play a role in the transcriptional regulation of HNPs. Furthermore, it is unclear whether the difference in HNP-1 and HNP-3 contents in neutrophil granules is caused by transcriptional regulation, posttranscriptional regulation, or both. Detailed analysis of HNP-1 and HNP-3 genes should therefore provide clues to the regulation of HNP expression.
In the present study, we show that HNP-1 and
HNP-3 genes have equivalent levels of promoter activity
in HL-60 cells and that 200 bp of 5' flanking sequence identical
between HNP-1 and HNP-3 is sufficient for
transcription activity. We further demonstrate that the binding of
C/EBP but not c-Myb protein to the overlapping C/EBP and c-Myb sites
is important for efficient transcription of HNP
promoters in HL-60 cells. Our findings suggest that C/EBP acts as a
potent positive regulator of the HNP-1 and
HNP-3 genes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human promyelocytic leukemia HL-60 cells (JCRB0085; Japanese Collection of Research Bioresource, Tokyo, Japan) and T lymphoblastic leukemia Jurkat cells (TIB-152; American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% FBS (Sanko, Tokyo, Japan), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Sigma, St. Louis, MO) at 37°C in 5% CO2. Cervical carcinoma HeLa cells (JCRB9004) were cultured in DMEM (Nissui Pharmaceutical) with 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37°C.
Preparation of peripheral blood neutrophils
Peripheral blood obtained from healthy volunteers was
heparinized, and neutrophils (purity > 95%) were isolated by
Ficoll-Conray centrifugation after dextran sedimentation of
erythrocytes (28). In some experiments, neutrophils were
resuspended in RPMI 1640 at a final concentration of 5 x
106 cells/ml and stimulated with 1 µg/ml LPS
(from Escherichia coli O127:B8; Difco, Detroit, MI) or 100
U/ml recombinant human TNF- (Genzyme, Boston, MA) at 37°C for
24 h.
Isolation of HNP-1 and HNP-3 promoters
The sequence of the 5' flanking region of exon 1 of human
HNP-1 and HNP-3 genes was amplified from
human genomic DNA by PCR based on published sequences as illustrated in
Fig. 1
A (18). The first PCR reaction was
performed with a primer set of sense 1 and antisense 1 on a thermal
cycler model 480 (Perkin-Elmer, Norwalk, CT) after incubation at 94°C
for 2 min using 30 cycles of 30 s at 94°C, 30 s at 54°C,
and 1 min at 72°C. The final polymerization step was extended by an
additional 5 min at 72°C. The second PCR was conducted using the
first reaction as a template with a primer set of sense 2 and antisense
2. The synthesized 1.2-kbp fragments consisted of the sequences from
-1116 to +61 of HNP genes, and were cloned to a
TA-cloning vector pT7Blue(R) (Novagen, Madison, WI). Plasmid inserts
were confirmed by sequencing using a Dye Terminator Cycle Sequencing
kit FS and a model 373A DNA autosequencer (PE Applied Biosystems
Division, Foster City, CA). Base substitutions between
HNP-1 and HNP-3 promoters are shown in
Fig. 1B.
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Plasmid construction
The 1.2-kbp MluI-XhoI fragments of
HNP-1 or HNP-3 promoter from pT7Blue(R)
clones were subcloned into the promoterless luciferase vector
pGL3-Basic (Promega, Madison, WI) and named -1116/HNP1-Luc and
-1116/HNP3-Luc, respectively. A series of 5' deletion plasmids shown
in Fig. 2A was constructed as follows. To remove 5' upstream
sequences of HNP promoters, -1116/HNP1-Luc or -1116/HNP3-Luc plasmids
were digested with SmaI (located in the polylinker site of
pGL3-Basic) and SspI (position -293 on the
HNP promoters), HincII (-240), or
DraI (-29). Each plasmid was recircularized by ligation to
generate -293/HNP-Luc, -240/HNP-Luc, and -29/HNP-Luc, respectively.
The -240/HNP-Luc plasmid was utilized as a template for PCR with
appropriate sense primers with MluI sequence at the 5' end
and antisense 2 primer to construct further deletion derivatives,
-133/HNP-Luc, -111/HNP-Luc, -86/HNP-Luc, and -58/HNP-Luc (Fig. 4A). Sequences of sense primers were as follows: -133
sense, 5'-CTCGTACGCGTCCTTCCCAC-3' (-133 to -124); -111
sense, 5'-ACCGTACGCGTCTGTCCTTGC-3' (-111 to -102); -86
sense, 5'-ACCGTACGCGTATGGACCCA-3' (-86 to -77); and -58
sense, 5'-CGACGCGTCATTAGGACACCTCATCCCA-3' (-58 to -40).
Underlining in the sequences indicate the MluI
site.
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AML/HNP-Luc plasmid containing a mutant AML1 site from -100
to -95 was created by PCR using mutant AML1 primers, where an AML1
element (5'-ACCACA-3') was replaced by an AgeI restriction
site (5'-ACCGGT-3') (Fig. 4
A). PCR reactions were conducted
with -133 sense and mutant AML1 antisense (-83 to -103;
5'-CCATTAAATAAT ACCGGTGGC-3'), and mutant AML1 sense (-103
to -83; 5'-GCCACCGGTATTATTTAATGG-3') and antisense 2
primers. Bold letters indicate the AgeI site. Amplified PCR
products were digested with AgeI and ligated to generate
-133AML/HNP-Luc. All deletion constructs were confirmed by
sequencing. Transfection and luciferase assay
HL-60 (1 x 107) cells were transfected with 20 µg of luciferase reporter constructs and 10 µg of ß-galactosidase expression vector pSV-ß-galactosidase (Promega) in 500 µl of serum-free RPMI 1640 medium by electroporation at 960 µF and 280 V using a Gene Pulser apparatus (Bio-Rad, Hercules, CA) as described previously (33). Cells were incubated in 10 ml RPMI 1640 supplemented with 10% FBS for 6 h posttransfection.
HeLa cells were plated at 4 x 105 cells/60-mm dish. On the following day, the cells were cotransfected with 8 µg reporter plasmids and 2 µg pSV-ß-galactosidase with a calcium phosphate transfection system (Life Technologies, Rockville, MD) according to the manufacturer’s protocol. After 24 h, DNA precipitates were removed by washing, and the cells were incubated for another 24 h at 37°C.
To analyze luciferase and ß-galactosidase activities, cells were lysed in 200 µl PicaGene Reporter Lysis Buffer LUC (Toyo Ink, Tokyo, Japan) and sonicated on ice for 10 s (ultrasonic disrupter; Tomy Seiko, Tokyo, Japan). Protein concentration of cell extracts was determined with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Luciferase activity was measured as relative light units using a PicaGene luciferase assay kit (Toyo Ink) and a Lumat LB9501 luminometer (Berthold, Wildbad, Germany). ß-Galactosidase activity was measured with a Galacto-Light kit (Tropix, Bedford, MA), and transfection efficiency was normalized to the level of ß-galactosidase activity.
Preparation of nuclear extracts
Nuclear extracts were prepared as described by Dignam et al. (34), with minor modifications. Briefly, 1 x 108 cells were washed twice with PBS and lysed in lysis buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 1 mM EDTA, 1.5 mM MgCl2, 0.5% Nonidet P-40, 1 mM DTT, 1 mM PMSF, 5 µg/ml leupeptin, and 5 µg/ml pepstatin) on ice for 10 min. Nuclei pellets were washed once with the same buffer except Nonidet P-40. After incubation with extraction buffer (10 mM HEPES (pH 7.9), 420 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 20% glycerol, 1 mM DTT, 1 mM PMSF, 5 µg/ml leupeptin, and 5 µg/ml pepstatin) at 4°C for 20 min, nuclei were centrifuged at 12,000 x g for 20 min at 4°C. The resultant nuclear extracts were immediately subjected to SDS-PAGE/Western blotting or stored at -80°C for gel retardation assay. Protein content in the nuclear extracts was measured with a bicinchoninic acid protein assay kit (Pierce).
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts (10 µg) were mixed with a
32P-labeled probe (described in the next section;
5 x 104 cpm, 10–20 fmol) in 15 µl of a
binding buffer containing 10 mM HEPES (pH 7.9), 50 mM KCl, 5 mM
MgCl2, 1 mM EDTA, 5% glycerol, 1 mM DTT, 1 mM
PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 2 µg
poly(dI-dC)·poly(dI-dC) (Amersham Pharmacia Biotech AB, Uppsala,
Sweden) for 20 min on ice. The reaction mixtures were applied to a
native 5% polyacrylamide gel in 0.5x TBE (44.5 mM Tris, 44.5 mM boric
acid, and 1 mM EDTA (pH 8.3)) at 180 V for 90 min at 4°C. The gels
were dried and exposed to Fuji RX-U x-ray film (Fuji Photo Film, Tokyo,
Japan) at -80°C. For competition assay, a 20–50-fold molar excess
of unlabeled oligonucleotides and PCR products were preincubated in
reaction mixture for 15 min on ice. For Ab supershift experiments, 1
µl of rabbit polyclonal Abs to C/EBP [14-AA], C/EBPß [C-19],
C/EBPß [198], c-Myb [C-19], and PU.1 [T-21] or normal rabbit
IgG was added to the reaction mixture 20 min before probe addition. All
specific Abs (TrasCruz Gel Supershift reagents, 1 mg/ml) were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA).
Oligonucleotides for EMSA
Synthetic oligonucleotides or PCR products were used as probes
for EMSA. The oligonucleotides were designed to generate a single 5'-G
overhang to each end after annealing with their compliments. PCR
products were digested with MluI to generate a 5'-CGCG
overhang. Double-stranded oligonucleotides and digested PCR fragments
were labeled by filling in the cohesive ends with
[-32P]dCTP (ICN Biomedicals, Costa Mesa, CA)
using Klenow fragment.
Oligonucleotides used for probes were as follows: EBP/Myb
oligonucleotide, 5'-GACCAAATT TCTCAACTGTCCTTGC-3' (-125 to -102); AML
oligonucleotide, 5'-GCTTGCCACCACAATTATC-3' (-106 to -90); Ets
oligonucleotide, 5'-GGACCCAACAGAAAGTAACCCCGGAAATTAGC-3' (-84 to
-54); and PU.1/GABP consensus oligonucleotide,
5'-GGGCTGCTTGAGGAAGTATAAGAAC-3' (the same sequence as TransCruz Gel
Shift Oligonucleotide except for 5'-G; Santa Cruz Biotechnology).
Primer sets for PCR amplification were as follows: -214 sense
(5'-CGACGCGTCATAGTTGGTTGCTGCCTGGG-3') and -124 antisense
(5'-GTGGGAAGGTGAGGT TAAAG-3') primers, and -146 sense
(5'-CGACGCGTCTACTTTAACCTCACCTCACCTTC CCACC-3') and -64
antisense (5'-GGGTTACTTTCTGTTGGGT-3') primers. The underlined
MluI sequences have been added to the 5' end of sense
primers for labeling. As an unrelated sequence, a 123-bp fragment from
the plasmid pUC19 was amplified by PCR using M13 forward
(5'-GTTTTCCCAGTCACGAC-3') and reverse (5'-CAGGAAACAGCTATGAC-3')
primers (Takara Shuzo, Shiga, Japan). Mutant oligonucleotides for
competition assay were EBP/Myb mutant oligonucleotide,
5'-ACCGTACGCGTCTGTCCTTGC-3' (-122 to -102) and Ets
mutant oligonucleotide, 5'-CGACGCGTCATTAGGACACCTCATCCCA-3'
(-67 to -40). Mutated sequences are indicated by bold letters.
Western blotting
Nuclear protein extracts (10 µg) prepared as described above were boiled for 3 min in SDS-PAGE sample buffer and separated on a 10% or 12% polyacrylamide gel (35). Resolved proteins were electrotransferred to Immobilon-P membrane (Millipore, Bedford, MA) using a Trans-Blot SD apparatus (Bio-Rad).
Membranes were blocked in Block Ace (Dainippon Pharmaceutical, Tokyo,
Japan) for 1 h at room temperature and probed with appropriate
rabbit polyclonal Abs in Tris-buffered saline-Tween 20 (TBS-T; 150 mM
NaCl, 20 mM Tris-HCl (pH 7.5), and 0.1% Tween 20) for 1 h. After
four washes with TBS-T, the membranes were incubated with
HRP-conjugated goat anti-rabbit IgG (Organon Teknika, Durham, NC)
for 1 h at room temperature. Proteins were visualized with the
enhanced chemiluminescence Western blotting detection system (Amersham
Pharmacia Biotech). Abs used were follows: anti-C/EBP Abs (C103;
kindly provided by Dr. Pernille Rorth, European Molecular Biology
Laboratory, Heidelberg, Germany; and 472, the kind gift from Dr. Steven
McKnight, University of Texas, Southwestern Medical Center, Dallas, TX)
in a 1:1000 dilution (36, 37, 38, 39) and anti-C/EBPß (C-19)
and anti-c-Myb (C19) Abs at a concentration of 0.1 µg/ml (Santa
Cruz Biotechnology).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HNP-1 and HNP-3 promoters (1.2 kbp)
were amplified by PCR from genomic DNAs as shown in Fig. 1A . Thirteen clones
containing either HNP-1 or HNP-3 promoter
were isolated, and their sequences were found to be consistent with
earlier reports except for minor differences (18, 29): an
additional C was found at position -589 from the transcription start
site in both HNP-1 and HNP-3 genes, and a
HindIII site at -628 in the HNP-1 promoter
was lost by substitution of C for G at -627 (Fig. 1B). A
total of 15 base differences were located upstream of a
SspI site at -293 between HNP-1 and
HNP-3 promoters, and 300 bp of the proximal region
was identical between the two promoters. As shown in Fig. 1B, there were three (HNP-1) or two
(HNP-3) CCAAT boxes within the distal region
(-1116 to -293), whereas one CCAAT box and a TATA box were found
within the identical region (18, 29).
Transcription potentials of HNP-1 and HNP-3 promoters in HL-60 cells
To examine the promoter activity of HNP-1 and
HNP-3, the 5' flanking region from -1116 to +61 of both
genes was introduced into the luciferase reporter vector to form
-1116/HNP1-Luc and -1116/HNP3-Luc, respectively (Fig. 2A).
As shown in Fig. 2B, -1116/HNP1-Luc and -1116/HNP3-Luc
possessed the same luciferase activities despite having 15 base
alterations. Luciferase levels with these constructs were >35-fold
greater than that with the promoterless vector. We further evaluated
the activities of 5' deletional constructs to determine whether
putative CCAAT boxes within the HNP promoters influenced
transcriptional potential (Fig. 2A). Deletion to position
-293 (-293/HNP-Luc) did not significantly change luciferase activity
compared with -1116/HNP1-Luc and -1116/HNP3-Luc. Deletion to position
-240 (-240/HNP-Luc) showed retained activity, whereas additional
deletion to position -29, which removed a potential TATA box,
decreased promoter activity by >90%. These results indicate that base
substitutions and CCAAT boxes were of little consequence in the
promoter activity of HNP-1 and HNP-3
genes, and that the proximal promoter region between -240 and -29
contains positive cis-acting element(s) interacting with
transcription factors in HL-60 cells.
In contrast, in the cervical carcinoma HeLa cell line, which does not
express HNP mRNAs, full-length HNP-1 or
HNP-3 promoters displayed only 3-fold stimulation over
the promoterless vector (Fig. 2B). Moreover, these promoter
activities were not altered by the deletion to position -29.
Taken together, these findings suggest that HNP-1 and HNP-3 genes are equally transcribed, and that both HNP genes are transcriptionally regulated in a myeloid-specific manner.
Requirement for overlapping C/EBP and c-Myb sites and Ets-like element for activation of the HNP promoter
To localize cis-acting elements within the
HNP promoter, the sequence from -240 and -29 was
scanned with the MatInspector version 2.2 computer program (40, 41). Putative binding sites for C/EBP, c-Myb, AML1, Ets, and
CCAAT displacement protein (CDP), which are related to myeloid-specific
gene expression, were contained within the region 200 bp upstream of
the TATA box (Fig. 3A)
(30, 31, 32, 42).
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-32P-labeled DNA fragment containing the HNP
promoter (Fig. 3B) and nuclear extracts prepared from HL-60
cells. As represented in Fig. 3C, nuclear protein(s) in
HL-60 cells strikingly bound to the -146/-64 oligonucleotide
containing putative C/EBP, c-Myb, and AML1 sites. Formation of the two
major complexes was specifically inhibited by the addition of unlabeled
probe, but not by that of unrelated pUC19 fragment (Fig. 3C,
right panel). Weak binding to the fragment from -214 to
-124 containing distal Ets and AML1 sites was developed, which
competed with the excess unlabeled -214/-124 fragment but not with
the unrelated pUC19 fragment (Fig. 3C, left
panel). These findings indicate that an 200-bp region of HNP
promoter contains multiple binding sites for transcription factors.
Next, to elucidate which element(s) were functionally important
to HNP promoter activity, successive 5' truncates and a
point mutant were created from -240/HNP-Luc using PCR (Fig. 4A) and assessed in HL-60
cells (Fig. 4B). Deletion to position -133 showed a
tendency to increased promoter activity compared with -240/HNP-Luc,
suggesting the loss of negative regulatory elements. Deletion to
position -111, which removed overlapping C/EBP and c-Myb sites,
resulted in a remarkable decrease in promoter activity. Interestingly,
mutation or deletion of the proximal AML1 site at -100 (-133AML
and -86/HNP-Luc) had little effect on promoter activity compared with
those of -133 and -111/HNP-Luc, respectively. Additional deletion to
position -58, which removed the binding site for Ets family proteins,
resulted in a further decrease in activity. Promoter activity was
virtually abolished by deletion of a putative TATA box (at -29). These
results indicate that in addition to the TATA box, the overlapping
C/EBP and c-Myb sites and the Ets site are required for transcriptional
activation of the HNP promoter. Moreover, they suggest
that the overlapping C/EBP and c-Myb sites seem to be more important
than the Ets site in positive regulation of HNP
promoter.
Binding of C/EBP to the C/EBP site on the HNP promoter with
HL-60 nuclear extracts
We further investigated which transcription factors could
bind to the overlapping C/EBP and c-Myb sites in the HNP
promoter. Nuclear extracts from HL-60 cells, mature neutrophils, and
HeLa cells were incubated with a 32P-labeled
EBP/Myb oligonucleotide spanning -125 to -102 (Fig. 5A). As expected, a prominent
DNA-protein complex was detected using HL-60 nuclear extracts, and
found to be in competition with excess unlabeled EBP/Myb
oligonucleotide but not with the mutated EBP/Myb oligonucleotide or
unrelated oligonucleotides such as PU.1 and AML1 (Fig. 5B,
left panel). The addition of Ab against C/EBP (EBP)
caused a supershift of the specific complex, whereas the band was only
weakly shifted by anti-C/EBPß Ab (EBPß). Ab recognizing
C/EBP, -ß, -
, and -
(EBPs) markedly abolished the specific
DNA-protein complex. Interestingly, supershift or disappearance of the
specific band was not detected on the addition of anti-c-Myb,
suggesting that the EBP/Myb oligonucleotide does not interact with
c-Myb in HL-60 nuclear extracts. Likewise, neither anti-PU.1 Ab nor
rabbit IgG had any effect on DNA-binding activity. Of note, no specific
DNA binding was detected using nuclear extracts from mature neutrophils
(Fig. 5B, middle panel). Activation of
neutrophils by inflammatory stimuli including LPS and TNF- induced
the transcription of mRNAs for cytokines such as IL-1ß (data not
shown), as reported previously (43, 44). However, no mRNA
for HNPs was detected in neutrophils activated by LPS or TNF- or in
resting neutrophils (data not shown). Furthermore, no binding activity
was detected using nuclear extracts from activated neutrophils (Fig. 5B, middle panel). These observations indicate
the absence of specific factor(s) interacting with the C/EBP site in
mature neutrophils. When EMSA was performed using nuclear extracts from
HeLa cells, a faint band was found to be supershifted by
anti-C/EBPß Ab on overexposure of the autoradiogram (Fig. 5B, right panel). These results suggest that
C/EBP but not c-Myb predominantly interacts with the overlapping
C/EBP/c-Myb sites of HNP promoter in HL-60 nuclear
extracts, and that C/EBPß is likely to interact with C/EBP/c-Myb
sites in HeLa nuclear extracts.
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consensus oligonucleotide or anti-PU.1 Ab (Fig. 6B, left panel). In
addition, we observed that nuclear proteins from mature neutrophils
could bind to the Ets-like sequence. These specific complexes were not
destroyed or influenced by the addition of PU.1/GABP oligonucleotide
or anti-PU.1 Ab (Fig. 6B, middle panel).
Binding activity to the Ets-like sequence was decreased in nuclear
extracts from neutrophils stimulated with LPS or TNF- (Fig. 6B, middle panel). Differences in EMSA-binding
patterns between HL-60 cells and neutrophils may indicate that IRDs
include several Ets family proteins. No such specific complex with the
Ets-like sequence was detected in nuclear extracts from HeLa cells
(Fig. 6B, right panel).
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We analyzed levels of transcription factors likely to be involved
in HNP expression by Western blot analysis (Fig. 7). Both C/EBP and C/EBPß proteins
were abundantly expressed in HL-60 nuclei (Fig. 7A). In
contrast, neither C/EBP nor C/EBPß were detected in nuclear
extracts from resting or activated neutrophils (Fig. 7A).
c-Myb protein could not be detected in HL-60 and neutrophil nuclear
extracts, although c-Myb was apparent in Jurkat nuclear extracts used
as a positive control (Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data clearly showed that HNP-1 and
HNP-3 promoters (1.2 kbp) had the same luciferase
activities in HL-60 cells despite having 15 nucleotide differences
(Fig. 2B), suggesting that transcription of
HNP-1 and HNP-3 genes is equal. Thus, the
difference in peptide contents between HNP-1 and HNP-3 appears to be
due to posttranscriptional and/or posttranslational modifications. One
possible explanation is that HNP-2 may be selectively produced by
proteolysis of HNP-3 but not HNP-1, on the basis of earlier reports
indicating that HNP-1 content (50%) in the granules is equal to the
sum of those of HNP-2 (30%) and HNP-3 (20%) (16, 24).
Interestingly, promoter activities of HNP-1 and
HNP-3 were markedly low in nonhematopoietic HeLa cells
compared with HL-60 cells (Fig. 2B). This result suggests
that transcription of both HNP genes is regulated in a myeloid-specific
manner. None of the putative CCAAT boxes were required for
HNP promoter activity, and the 200-bp promoter sequence
identical between HNP-1 and HNP-3,
immediately upstream region from the TATA box, was sufficient for the
promoter activity in HL-60 cells. Computer analysis of the proximal
HNP promoter indicated the putative
cis-elements for C/EBP family, c-Myb, AML1, Ets family, and
CDP (40, 41). Consistent with the computer prediction,
EMSA revealed that HL-60 nuclear extracts contained multiple nuclear
factors interacting with the sequences of the proximal
HNP promoter (-214 to -124 and -146 to -64) (Fig. 3C).
Sequential deletion from -240 to -133 tended to enhance
HNP promoter activity compared with that of
-240/HNP-Luc (Fig. 4). This suggests the presence of negative
regulatory element(s) within the region. One potent candidate seems to
be the CDP-binding sequence at -220. CDP is implicated in the
transcriptional repression of myeloid-specific genes such as
gp91phox and lactoferrin (45, 46, 47).
It is also possible that other negative element(s) function within the
region, because weak but specific binding was observed using the
fragment (-214 to -124) lacking the CDP-binding sequence (Fig. 3C).
It is notable that the overlapping C/EBP and c-Myb sites (-122 to
-106) are required for both transcription and protein binding in HL-60
cells (Figs. 4B and 5B). Our data further
revealed that C/EBP rather than C/EBPß interacted with the C/EBP
site of HNP promoter in HL-60 cells (Fig. 5B), despite the fact that both C/EBP and C/EBPß were
detected in HL-60 nuclear extracts (Fig. 7A). In contrast,
neither C/EBP nor C/EBPß was contained in nuclear extracts from
not only resting but also activated neutrophils (Fig. 7A).
Interestingly, a feeble complex of C/EBPß with the C/EBP site was
observed using nuclear extracts from HeLa cells (Fig. 5B),
although HeLa cells showed nearly negligible transcriptional activity
of the HNP promoter (Fig. 2B). However,
Western blot analysis revealed that HeLa cells expressed C/EBPß but
not C/EBP (data not shown). Taken together, these findings indicate
that C/EBP may play an important role in the transcription of
HNP genes in HL-60 cells, whereas C/EBPß is unlikely
to play a role in either HL-60 or HeLa cells despite its expression.
Furthermore, c-Myb does not seem to participate in HNP
promoter activity in HL-60 cells, because no c-Myb was detected in
nuclear extracts from HL-60 cells or neutrophils (Fig. 7B).
AML1 proteins facilitate the transcriptional activity of myeloid
promoters via interaction with other adjacent transcription factors
(30, 48, 49). In the HNP promoter, the consensus AML1
sequence (at -102) lies between the C/EBP and Ets-like sites. However,
disruption of the AML1 site had little effect on the promoter activity
of HNP genes in HL-60 cells (Fig. 4B). Thus,
the AML1 site is unlikely to be involved in the regulation of
HNP gene expression.
Ma et al. (29) have reported the importance of IRD binding
to the Ets-like site at -65 on the HNP promoter . We
observed that deletion of this site caused a further 30% decrease in
promoter activity (Fig. 4B), and that IRDs interacted with
the Ets-like sequence using nuclear extracts from HL-60 cells and
mature neutrophils (Fig. 6). Furthermore, we confirmed the presence of
PU.1, which is implicated in the basal transcription of
HNP genes, in nuclear extracts from both HL-60 cells and
neutrophils (data not shown and Ref. 25). The finding that
mature neutrophils express Ets family factors including IRDs and PU.1
but not HNP mRNAs further support the important role of C/EBP in the
transcription of HNP genes in immature myeloid
cells.
Human ß defensins, hBD-1 and hBD-2, have been recently identified
from plasma and various epithelial tissues (11, 50, 51, 52).
Although defensins (HNPs) and ß defensins are encoded by
different genes, these genes share a common evolutionary origin
(53, 54). Of note, hBD-2 expression is induced by stimuli
such as bacteria and proinflammatory cytokine TNF-, thereby evoking
the important functions of hBD-2 in acute inflammation (51, 53). Future investigation of transcriptional regulation of not
only HNP but also ß defensin genes will
afford valuable information about the role of the defensin family in
the innate and acquired immune systems.
|
Acknowledgments |
|---|
Ab and Dr. Steven L. McKnight
(University of Texas, Southwestern Medical Center) for 472 C/EBP
antiserum. |
Footnotes |
|---|
2 The sequences of the HNP-1 and HNP-3 promoters have been deposited in the GenBank database under accession numbers AB025231 and AB025232, respectively.
3 Address correspondence and reprint requests to Dr. Isao Nagaoka, Department of Biochemistry, Juntendo University, School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo, 113-8421 Japan. E-mail address:
4 Abbreviations used in this paper: HNP, human neutrophil peptide; C/EBP, CCAAT/enhancer-binding protein; IRD, increased regulatory element of defensin expression binding during differentiation; EMSA, electrophoretic mobility shift assay; CDP, CCAAT displacement protein.
Received for publication July 26, 1999. Accepted for publication January 5, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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