The human neutrophil defensins (human neutrophil peptides (HNPs)), major components of azurophilic granules, contribute to innate and acquired host immunities through their potent antimicrobial activities and ability to activate T cells. Despite being encoded by nearly identical genes, HNP-1 is more abundant in the granules than HNP-3. We investigated the regulation of HNP-1 and HNP-3 expression at the transcriptional level using a promyelocytic HL-60 cell line. Luciferase analysis showed that transcriptional levels of HNP-1 and HNP-3 promoters were equivalent and that an ∼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/EBPα 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/EBPα 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/EBPα plays an important role in the transcription of HNP genes in immature myeloid cells.

Neutrophils act as key effectors in host defenses against microbial infection. They ingest and destroy invading microbes through both oxidative and nonoxidative mechanisms (1). The former mechanism depends on reactive oxygen intermediates produced by activated neutrophils (2, 3), whereas the latter is controlled by antimicrobial proteins, peptides, or both in cytoplasmic granules independent of reactive oxygen metabolites (4). A number of microbicidal polypeptides have been isolated from neutrophils of humans and various animal species (5, 6, 7). The most abundant are low-molecular-weight cationic peptides called “defensins,” which exhibit a broad spectrum of microbicidal activities against Gram-positive and Gram-negative bacteria, fungi, parasites, and viruses (8, 9, 10). The defensin family of antimicrobial peptides are widely distributed among many mammals, insects, and plants (9, 10, 11, 12, 13), and have three or four intramolecular disulfide bonds which differ in the placement and connectivity of their conserved cysteine residues (9, 10, 11, 12, 13). The vertebrate defensins consist of two subfamilies, designed α and β defensins (10, 11).

In humans, α 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/EBPα 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/EBPα acts as a potent positive regulator of the HNP-1 and HNP-3 genes.

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.

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 × 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-α (Genzyme, Boston, MA) at 37°C for 24 h.

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. 1 B.

FIGURE 1.

Isolation of 5′ flanking regions of HNP-1 and HNP-3 genes. A, The 1.2 kbp of the 5′ flanking regions of HNP-1 and HNP-3 genes were amplified by nested PCR (1st PCR and 2nd PCR). The transcription initiation site is numbered +1. Arrows indicate oligonucleotide sense and antisense primers. MluI and XhoI restriction sites were added to sense 2 and antisense 2 primers so that the PCR products could be subcloned into the luciferase vector pGL3-Basic. B, Structure of 1.2-kbp promoter regions of HNP-1 and HNP-3 genes. Position of the restriction site is relative to +1. MluI and XhoI restriction sites are derived from PCR primers. Sequences different from previous reports are indicated, namely, the addition of C at −589 in both HNP-1 and HNP-3 promoters and substitution of C for G at −627 in the HNP-1 promoter. Positions of base substitutions between HNP-1 and HNP-3 promoters are indicated by gray vertical lines. Filled bars indicate CCAAT and TATA boxes. Striped boxes indicate exon 1 sequences.

FIGURE 1.

Isolation of 5′ flanking regions of HNP-1 and HNP-3 genes. A, The 1.2 kbp of the 5′ flanking regions of HNP-1 and HNP-3 genes were amplified by nested PCR (1st PCR and 2nd PCR). The transcription initiation site is numbered +1. Arrows indicate oligonucleotide sense and antisense primers. MluI and XhoI restriction sites were added to sense 2 and antisense 2 primers so that the PCR products could be subcloned into the luciferase vector pGL3-Basic. B, Structure of 1.2-kbp promoter regions of HNP-1 and HNP-3 genes. Position of the restriction site is relative to +1. MluI and XhoI restriction sites are derived from PCR primers. Sequences different from previous reports are indicated, namely, the addition of C at −589 in both HNP-1 and HNP-3 promoters and substitution of C for G at −627 in the HNP-1 promoter. Positions of base substitutions between HNP-1 and HNP-3 promoters are indicated by gray vertical lines. Filled bars indicate CCAAT and TATA boxes. Striped boxes indicate exon 1 sequences.

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Oligonucleotides used were sense 1 primer (5′-AGCCCTGTTACAGGGGCTGC-3′; located at −1159 from the transcription start site), sense 2 primer containing a MluI site (5′-CCGACGCGTCGTCCAAGGCAGGCAACTCAACCC-3′; at −1116), antisense 1 primer (5′-ACGTTCCCTAGCAGGGATTCACCCGC-3′; at +199), and antisense 2 primer containing a XhoI site (5′-CCGCTCGAGCGCAGGGTGACCAGAGAGGGCAGA-3′; at +61). Underlining in the sequences indicate additional restriction sites.

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. 2,A 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. 4 A). 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.

FIGURE 2.

Functional analysis of HNP-1 and HNP-3 promoters. A, A series of 5′ deletion constructs of HNP-1 and HNP-3 promoters (−1116/HNP1-Luc, −1116/HNP3-Luc, −293/HNP-Luc, −240/HNP-Luc, and −29/HNP-Luc) were generated by digestion with appropriate restriction enzymes and by subcloning into the pGL3-Basic luciferase vector. The promoter sequence from −293 and +1 is common between HNP-1 and HNP-3 genes. Vertical lines indicate base substitutions between HNP-1 and HNP-3 sequences. Filled bars indicate CCAAT and TATA boxes. HNP-3 promoter lacks one CCAAT box as shown by (CCAAT). B,HNP promoter constructs were transfected into promyelocytic HL-60 and cervical carcinoma HeLa cell lines. pGL3 promoter was used as a positive control. Relative luciferase activity of each construct is shown in comparison to the promoterless pGL3-Basic. The value represents the mean ± SD for 5–10 independent experiments.

FIGURE 2.

Functional analysis of HNP-1 and HNP-3 promoters. A, A series of 5′ deletion constructs of HNP-1 and HNP-3 promoters (−1116/HNP1-Luc, −1116/HNP3-Luc, −293/HNP-Luc, −240/HNP-Luc, and −29/HNP-Luc) were generated by digestion with appropriate restriction enzymes and by subcloning into the pGL3-Basic luciferase vector. The promoter sequence from −293 and +1 is common between HNP-1 and HNP-3 genes. Vertical lines indicate base substitutions between HNP-1 and HNP-3 sequences. Filled bars indicate CCAAT and TATA boxes. HNP-3 promoter lacks one CCAAT box as shown by (CCAAT). B,HNP promoter constructs were transfected into promyelocytic HL-60 and cervical carcinoma HeLa cell lines. pGL3 promoter was used as a positive control. Relative luciferase activity of each construct is shown in comparison to the promoterless pGL3-Basic. The value represents the mean ± SD for 5–10 independent experiments.

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FIGURE 4.

Luciferase activity of 5′ deletion and point mutation constructs of HNP promoter in HL-60 cells. A, Successive deletion constructs of putative cis-elements and a mutant of the AML1 site were generated by PCR and by subcloning to the pGL3-Basic vector. Shaded boxes indicate the location of putative cis-elements on the HNP promoter. The crossed box of −133ΔAML/HNP-Luc shows a mutated AML1 element. B, Deletion and point mutation constructs were transiently transfected into HL-60 cells. Average luciferase activities were generated from 5 to 10 separate experiments. SD of the mean is indicated by error bars. Data are presented as the percentage of activity of HNP promoter constructs vs that of −240/HNP-Luc (100%).

FIGURE 4.

Luciferase activity of 5′ deletion and point mutation constructs of HNP promoter in HL-60 cells. A, Successive deletion constructs of putative cis-elements and a mutant of the AML1 site were generated by PCR and by subcloning to the pGL3-Basic vector. Shaded boxes indicate the location of putative cis-elements on the HNP promoter. The crossed box of −133ΔAML/HNP-Luc shows a mutated AML1 element. B, Deletion and point mutation constructs were transiently transfected into HL-60 cells. Average luciferase activities were generated from 5 to 10 separate experiments. SD of the mean is indicated by error bars. Data are presented as the percentage of activity of HNP promoter constructs vs that of −240/HNP-Luc (100%).

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A −133ΔAML/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 −133ΔAML/HNP-Luc. All deletion constructs were confirmed by sequencing.

HL-60 (1 × 107) cells were transfected with 20 μg of luciferase reporter constructs and 10 μg of β-galactosidase expression vector pSV-β-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 × 105 cells/60-mm dish. On the following day, the cells were cotransfected with 8 μg reporter plasmids and 2 μg pSV-β-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 β-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). β-Galactosidase activity was measured with a Galacto-Light kit (Tropix, Bedford, MA), and transfection efficiency was normalized to the level of β-galactosidase activity.

Nuclear extracts were prepared as described by Dignam et al. (34), with minor modifications. Briefly, 1 × 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 × 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).

Nuclear extracts (10 μg) were mixed with a 32P-labeled probe (described in the next section; 5 × 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 0.5× 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/EBPα [14-AA], C/EBPβ [C-19], C/EBPβ [Δ198], 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).

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 [α-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/GABPα 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.

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/EBPα 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/EBPβ (C-19) and anti-c-Myb (C19) Abs at a concentration of 0.1 μg/ml (Santa Cruz Biotechnology).

HNP-1 and HNP-3 promoters (1.2 kbp) were amplified by PCR from genomic DNAs as shown in Fig. 1,A . 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. 1,B). 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. 1 B, 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).

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. 2 A).

As shown in Fig. 2,B, −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. 2 A). 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. 2 B). 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.

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. 3 A) (30, 31, 32, 42).

FIGURE 3.

Putative cis-elements and EMSA with −243/−124 and −146/−64 fragments of the HNP promoter. A, Sequence of proximal HNP promoter common to HNP-1 and HNP-3 genes. Transcription start site is numbered +1. Open boxes indicate putative cis-elements for transcription factors C/EBP, c-Myb, AML1 (AML), Ets family, and CDP. Of note, the C/EBP and c-Myb elements overlap each other. The TATA box is shown as a shaded box. Restriction sites for HincII, DraI, and XhoI are underlined. GCTCGAG containing the XhoI site at the 3′ end is an artificial sequence used for cloning. Arrows show the 5′ ends of deletion constructs. B, Schematic representation of cis-elements on the HNP promoter. Shaded boxes indicate the location of cis-elements. The probe regions for EMSA, −214/−124 oligonucleotide, and −146/−64 oligonucleotide are indicated by brackets. C, Two DNA probes (−214/−124 oligonucleotide and −146/−64 oligonucleotide) were 32P-labeled and incubated with 10 μg nuclear extracts from HL-60 cells. The competitors were used in a 50-fold molar excess over labeled probes; −214/−124 and −146/−64 indicate unlabeled probes, and PUC indicates an unrelated pUC19 fragment. Asterisks indicate specific binding to the probes.

FIGURE 3.

Putative cis-elements and EMSA with −243/−124 and −146/−64 fragments of the HNP promoter. A, Sequence of proximal HNP promoter common to HNP-1 and HNP-3 genes. Transcription start site is numbered +1. Open boxes indicate putative cis-elements for transcription factors C/EBP, c-Myb, AML1 (AML), Ets family, and CDP. Of note, the C/EBP and c-Myb elements overlap each other. The TATA box is shown as a shaded box. Restriction sites for HincII, DraI, and XhoI are underlined. GCTCGAG containing the XhoI site at the 3′ end is an artificial sequence used for cloning. Arrows show the 5′ ends of deletion constructs. B, Schematic representation of cis-elements on the HNP promoter. Shaded boxes indicate the location of cis-elements. The probe regions for EMSA, −214/−124 oligonucleotide, and −146/−64 oligonucleotide are indicated by brackets. C, Two DNA probes (−214/−124 oligonucleotide and −146/−64 oligonucleotide) were 32P-labeled and incubated with 10 μg nuclear extracts from HL-60 cells. The competitors were used in a 50-fold molar excess over labeled probes; −214/−124 and −146/−64 indicate unlabeled probes, and PUC indicates an unrelated pUC19 fragment. Asterisks indicate specific binding to the probes.

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We analyzed whether the nuclear factors bound to the predicted sites of HNP promoter. EMSA was performed with a α-32P-labeled DNA fragment containing the HNP promoter (Fig. 3,B) and nuclear extracts prepared from HL-60 cells. As represented in Fig. 3,C, 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. 3,C, 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. 3 C, 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. 4,A) and assessed in HL-60 cells (Fig. 4 B). 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 (−133ΔAML 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.

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. 5,A). 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. 5,B, left panel). The addition of Ab against C/EBPα (EBPα) caused a supershift of the specific complex, whereas the band was only weakly shifted by anti-C/EBPβ Ab (EBPβ). Ab recognizing C/EBPα, -β, -δ, and -ε (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. 5,B, middle panel). Activation of neutrophils by inflammatory stimuli including LPS and TNF-α induced the transcription of mRNAs for cytokines such as IL-1β (data not shown), as reported previously (43, 44). However, no mRNA for HNPs was detected in neutrophils activated by LPS or TNF-α or in resting neutrophils (data not shown). Furthermore, no binding activity was detected using nuclear extracts from activated neutrophils (Fig. 5,B, 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/EBPβ Ab on overexposure of the autoradiogram (Fig. 5 B, right panel). These results suggest that C/EBPα 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/EBPβ is likely to interact with C/EBP/c-Myb sites in HeLa nuclear extracts.

FIGURE 5.

EMSA of the overlapping C/EBP and c-Myb sites on the HNP promoter. A, Sequence of the HNP promoter from −130 to −90. Shaded boxes indicate potential transcription factor binding sites for C/EBP and c-Myb. The bracket from −125 to −102 shows a region for oligonucleotide probe containing overlapping C/EBP and c-Myb elements (EBP/Myb oligo). B, A total 10 μg of nuclear extracts from HL-60, mature neutrophils (resting (rest), LPS activated, or TNF-α activated), and HeLa cells was incubated with 32P-labeled EBP/Myb oligo. Thirty-fold molar excess of unlabeled competitors was added to the reaction mixture: unlabeled probe (EBP/Myb), mutated EBP/Myb oligo (Mut), PU.1/GABPα consensus oligonucleotide (PU), and AML oligo (AML). Supershift assay was performed with 1 μg of appropriate Abs: anti-C/EBPα (EBPα), anti-C/EBPβ (EBPβ), anti-C/EBP recognizing C/EBPα, -β, -δ, and -ε (EBPs), anti-PU.1 (PU), and normal rabbit IgG as a control (Ig). Asterisks indicate specific binding to the probes. Positions of supershifted complexes are denoted by a filled arrow (HL-60) and an open arrow (HeLa).

FIGURE 5.

EMSA of the overlapping C/EBP and c-Myb sites on the HNP promoter. A, Sequence of the HNP promoter from −130 to −90. Shaded boxes indicate potential transcription factor binding sites for C/EBP and c-Myb. The bracket from −125 to −102 shows a region for oligonucleotide probe containing overlapping C/EBP and c-Myb elements (EBP/Myb oligo). B, A total 10 μg of nuclear extracts from HL-60, mature neutrophils (resting (rest), LPS activated, or TNF-α activated), and HeLa cells was incubated with 32P-labeled EBP/Myb oligo. Thirty-fold molar excess of unlabeled competitors was added to the reaction mixture: unlabeled probe (EBP/Myb), mutated EBP/Myb oligo (Mut), PU.1/GABPα consensus oligonucleotide (PU), and AML oligo (AML). Supershift assay was performed with 1 μg of appropriate Abs: anti-C/EBPα (EBPα), anti-C/EBPβ (EBPβ), anti-C/EBP recognizing C/EBPα, -β, -δ, and -ε (EBPs), anti-PU.1 (PU), and normal rabbit IgG as a control (Ig). Asterisks indicate specific binding to the probes. Positions of supershifted complexes are denoted by a filled arrow (HL-60) and an open arrow (HeLa).

Close modal

A recent report has indicated that the interaction of IRDs with an Ets-like site at −65 is involved in the transcription of the HNP-1 gene (29). Consistent with this, we confirmed the formation of IRDs and Ets-like complexes using nuclear extracts from HL-60 cells, which were not affected by either PU.1/GABPα consensus oligonucleotide or anti-PU.1 Ab (Fig. 6,B, 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/GABPα oligonucleotide or anti-PU.1 Ab (Fig. 6,B, middle panel). Binding activity to the Ets-like sequence was decreased in nuclear extracts from neutrophils stimulated with LPS or TNF-α (Fig. 6,B, 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. 6 B, right panel).

FIGURE 6.

EMSA of the Ets-like element on the HNP promoter. A, Sequences of the HNP promoter from −80 to −20. Shaded boxes indicate the putative Ets-like element and TATA box. A region of oligonucleotide probe (Ets oligo) spanning −84 to −54 is shown by a bracket. B, A total 10 μg of nuclear extracts from HL-60, mature neutrophils (resting (rest), LPS activated, or TNF-α activated), and HeLa cells was incubated with 32P-labeled Ets oligo probe. Unlabeled oligonucleotides were added in a 25-fold molar excess as a competitor: Ets oligo (Ets), mutated Ets oligo (Mut), and PU.1/GABPα oligo (PU1). In supershift experiments, 1 μg of anti-PU.1 Ab (PU) or normal rabbit IgG was added to the reaction mixture. Asterisks indicate specific binding to the probes.

FIGURE 6.

EMSA of the Ets-like element on the HNP promoter. A, Sequences of the HNP promoter from −80 to −20. Shaded boxes indicate the putative Ets-like element and TATA box. A region of oligonucleotide probe (Ets oligo) spanning −84 to −54 is shown by a bracket. B, A total 10 μg of nuclear extracts from HL-60, mature neutrophils (resting (rest), LPS activated, or TNF-α activated), and HeLa cells was incubated with 32P-labeled Ets oligo probe. Unlabeled oligonucleotides were added in a 25-fold molar excess as a competitor: Ets oligo (Ets), mutated Ets oligo (Mut), and PU.1/GABPα oligo (PU1). In supershift experiments, 1 μg of anti-PU.1 Ab (PU) or normal rabbit IgG was added to the reaction mixture. Asterisks indicate specific binding to the probes.

Close modal

We analyzed levels of transcription factors likely to be involved in HNP expression by Western blot analysis (Fig. 7). Both C/EBPα and C/EBPβ proteins were abundantly expressed in HL-60 nuclei (Fig. 7,A). In contrast, neither C/EBPα nor C/EBPβ were detected in nuclear extracts from resting or activated neutrophils (Fig. 7,A). 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. 7 B).

FIGURE 7.

Western blot of nuclear extracts from HL-60 cells and mature neutrophils. Nuclear extracts (10 μg) from HL-60 cells and neutrophils (resting (rest), and LPS activated, or TNF-α activated) were resolved by 12% (A) or 10% (B) SDS-PAGE. Western blotting was performed using anti-C/EBPα and anti-C/EBPβ Abs (A) or anti-c-Myb Ab (B). Nuclear extracts from Jurkat cells were used as positive control in B. Positions of molecular size markers are shown in kDa on the right. Specific bands are indicated by arrows with molecular mass (kDa).

FIGURE 7.

Western blot of nuclear extracts from HL-60 cells and mature neutrophils. Nuclear extracts (10 μg) from HL-60 cells and neutrophils (resting (rest), and LPS activated, or TNF-α activated) were resolved by 12% (A) or 10% (B) SDS-PAGE. Western blotting was performed using anti-C/EBPα and anti-C/EBPβ Abs (A) or anti-c-Myb Ab (B). Nuclear extracts from Jurkat cells were used as positive control in B. Positions of molecular size markers are shown in kDa on the right. Specific bands are indicated by arrows with molecular mass (kDa).

Close modal

Four different HNPs are exclusively expressed in neutrophils and contribute to both innate and acquired immune systems (9, 24). Even though HNP-1 and HNP-3 peptides are encoded by nearly identical genes, their activities and contents are considerably different (8, 16, 18). In the present study, we isolated HNP-1 and HNP-3 promoters and investigated how each gene is transcriptionally controlled.

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. 2 B), 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. 2,B). 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. 3 C).

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. 3 C).

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. 4,B and 5B). Our data further revealed that C/EBPα rather than C/EBPβ interacted with the C/EBP site of HNP promoter in HL-60 cells (Fig. 5,B), despite the fact that both C/EBPα and C/EBPβ were detected in HL-60 nuclear extracts (Fig. 7,A). In contrast, neither C/EBPα nor C/EBPβ was contained in nuclear extracts from not only resting but also activated neutrophils (Fig. 7,A). Interestingly, a feeble complex of C/EBPβ with the C/EBP site was observed using nuclear extracts from HeLa cells (Fig. 5,B), although HeLa cells showed nearly negligible transcriptional activity of the HNP promoter (Fig. 2,B). However, Western blot analysis revealed that HeLa cells expressed C/EBPβ but not C/EBPα (data not shown). Taken together, these findings indicate that C/EBPα may play an important role in the transcription of HNP genes in HL-60 cells, whereas C/EBPβ 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. 7 B).

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. 4 B). 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. 4,B), 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/EBPα in the transcription of HNP genes in immature myeloid cells.

Human β defensins, hBD-1 and hBD-2, have been recently identified from plasma and various epithelial tissues (11, 50, 51, 52). Although α defensins (HNPs) and β 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-α, thereby evoking the important functions of hBD-2 in acute inflammation (51, 53). Future investigation of transcriptional regulation of not only HNP but also β defensin genes will afford valuable information about the role of the defensin family in the innate and acquired immune systems.

We thank Dr. Pernille Rorth (European Molecular Biology Laboratory) for providing C103 anti-C/EBPα Ab and Dr. Steven L. McKnight (University of Texas, Southwestern Medical Center) for 472 C/EBPα antiserum.

1

This work was supported in part by grants from Takeda Science Foundation and the Atopy (Allergy) Research Center, Juntendo University.

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.

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.

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