Role of CCAAT/Enhancer-Binding Site in Transcription of Human -1 and -3 Defensin Genes

This information is current as Yuko Tsutsumi-Ishii, Takeshi Hasebe and Isao Nagaoka of September 26, 2021. J Immunol 2000; 164:3264-3273; ; doi: 10.4049/jimmunol.164.6.3264 http://www.jimmunol.org/content/164/6/3264 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Role of CCAAT/Enhancer-Binding Protein Site in Transcription of Human Neutrophil Peptide-1 and -3 Defensin Genes1,2

Yuko Tsutsumi-Ishii, Takeshi Hasebe, and Isao Nagaoka3

The human neutrophil defensins (human neutrophil (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 tran- scriptional levels of HNP-1 and HNP-3 promoters were equivalent and that an ϳ200-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 Downloaded from 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 , 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. The Journal of Immu- nology, 2000, 164: 3264–3273. http://www.jimmunol.org/ eutrophils act as key effectors in host defenses against trophils and comprise 30–50% of azurophil protein (8, 14, microbial infection. They ingest and destroy invading 15). Among HNPs, HNP-1, HNP-2, and HNP-3 have almost iden- N microbes through both oxidative and nonoxidative tical sequences (9). Both HNP-1 and HNP-3 are com- mechanisms (1). The former mechanism depends on reactive ox- posed of 30 amino acid residues, with the first amino acid in ygen intermediates produced by activated neutrophils (2, 3), HNP-1, alanine, substituted by aspartate in HNP-3. HNP-2, 29 whereas the latter is controlled by antimicrobial , peptides, amino acids in length due to lack of the first amino acid, is as- or both in cytoplasmic granules independent of reactive oxygen sumed to be a proteolytic derivative of HNP-1, HNP-3, or both (9). metabolites (4). A number of microbicidal polypeptides have been HNP-1 and HNP-3 are encoded by nearly identical genes on chro- isolated from neutrophils of humans and various animal species mosome 8; notwithstanding this similarity, HNP-1 is more abun- by guest on September 26, 2021 (5–7). The most abundant are low-molecular-weight cationic pep- dant than HNP-3 in neutrophil granules (16–18). The fourth de- tides called “defensins,” which exhibit a broad spectrum of micro- fensin, HNP-4, is the least abundant and except for conserved bicidal activities against Gram-positive and Gram-negative bacte- residues shows low homology with other HNPs (19–21). ria, fungi, parasites, and (8–10). The defensin family of Despite their nearly identical amino acid sequences, HNPs exhibit are widely distributed among many mam- different biological properties in vitro. For example, although mals, insects, and (9–13), and have three or four intramo- HNP-1, HNP-2, and HNP-4 can kill Candida albicans, HNP-3 lecular disulfide bonds which differ in the placement and connec- cannot (20, 22). Interestingly, HNP-1 and HNP-2, but not HNP-3, tivity of their conserved cysteine residues (9–13). The have chemotactic activity for murine and human T cells and mono- defensins consist of two subfamilies, designed ␣ and ␤ defensins cytes (16, 23). Moreover, HNP-1, HNP-2, and HNP-3 have been (10, 11). shown to enhance -dependent immune responses in vivo In humans, ␣ defensins (human neutrophil peptides (HNPs))4 (24). Such diverse functions of HNPs are likely to contribute to are major components of the primary (azurophil) granules of neu- 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 Department of Biochemistry, Juntendo University, School of Medicine, Tokyo, Japan are only expressed in immature bone marrow cells and HL-60 Received for publication July 26, 1999. Accepted for publication January 5, 2000. human promyelocytic leukemia cells (25–28). A more recent re- The costs of publication of this article were defrayed in part by the payment of page port has indicated that PU.1 and an unknown Ets-like factor (IRD) charges. This article must therefore be hereby marked advertisement in accordance are involved in the basal transcription of HNP-1 gene (29). How- with 18 U.S.C. Section 1734 solely to indicate this fact. ever, given that several transcription factors such as CCAAT/en- 1 This work was supported in part by grants from Takeda Science Foundation and the hancer-binding proteins (C/EBPs), c-Myb, and AML1 (PEBP2/ Atopy (Allergy) Research Center, Juntendo University. CBF) cooperatively regulate the efficient transcription of many 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. myeloid-specific genes (30–32), factors other than PU.1 and IRD 3 Address correspondence and reprint requests to Dr. Isao Nagaoka, Department of may also play a role in the transcriptional regulation of HNPs. Biochemistry, Juntendo University, School of Medicine, Hongo 2-1-1, Bunkyo-ku, Furthermore, it is unclear whether the difference in HNP-1 and Tokyo, 113-8421 Japan. E-mail address: [email protected] HNP-3 contents in neutrophil granules is caused by transcriptional 4 Abbreviations used in this paper: HNP, human neutrophil peptide; C/EBP, CCAAT/ regulation, posttranscriptional regulation, or both. Detailed analy- enhancer-binding protein; IRD, increased regulatory element of defensin expression binding during differentiation; EMSA, electrophoretic mobility shift assay; CDP, sis of HNP-1 and HNP-3 genes should therefore provide clues to CCAAT displacement protein. the regulation of HNP expression.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 3265

In the present study, we show that HNP-1 and HNP-3 genes Ϫ58 sense, 5Ј-CGACGCGTCATTAGGACACCTCATCCCA-3Ј (Ϫ58 to have equivalent levels of promoter activity in HL-60 cells and that Ϫ40). Underlining in the sequences indicate the MluI site. Ϫ ⌬ ϳ200 bp of 5Ј flanking sequence identical between HNP-1 and A 133 AML/HNP-Luc plasmid containing a mutant AML1 site from Ϫ100 to Ϫ95 was created by PCR using mutant AML1 primers, where an HNP-3 is sufficient for transcription activity. We further demon- AML1 element (5Ј-ACCACA-3Ј) was replaced by an AgeI restriction site strate that the binding of C/EBP␣ but not c-Myb protein to the (5Ј-ACCGGT-3Ј) (Fig. 4A). PCR reactions were conducted with Ϫ133 overlapping C/EBP and c-Myb sites is important for efficient tran- sense and mutant AML1 antisense (Ϫ83 to Ϫ103; 5Ј-CCATTAAATAAT scription of HNP promoters in HL-60 cells. Our findings suggest ACCGGTGGC-3Ј), and mutant AML1 sense (Ϫ103 to Ϫ83; 5Ј-GCCAC ␣ CGGTATTATTTAATGG-3Ј) and antisense 2 primers. Bold letters indi- that C/EBP acts as a potent positive regulator of the HNP-1 and cate the AgeI site. Amplified PCR products were digested with AgeI and HNP-3 genes. ligated to generate Ϫ133⌬AML/HNP-Luc. All deletion constructs were confirmed by sequencing. Materials and Methods Transfection and luciferase assay Cell culture HL-60 (1 ϫ 107) cells were transfected with 20 ␮g of luciferase reporter Human promyelocytic leukemia HL-60 cells (JCRB0085; Japanese Col- constructs and 10 ␮gof␤-galactosidase expression vector pSV-␤-galac- lection of Research Bioresource, Tokyo, Japan) and T lymphoblastic leu- tosidase (Promega) in 500 ␮l of serum-free RPMI 1640 medium by elec- kemia Jurkat cells (TIB-152; American Type Culture Collection, Manas- troporation at 960 ␮F and 280 V using a Gene Pulser apparatus (Bio-Rad, sas, VA) were maintained in RPMI 1640 medium (Nissui Pharmaceutical, Hercules, CA) as described previously (33). Cells were incubated in 10 ml Tokyo, Japan) supplemented with 10% FBS (Sanko, Tokyo, Japan), 100 RPMI 1640 supplemented with 10% FBS for 6 h posttransfection. U/ml penicillin, and 0.1 mg/ml streptomycin (Sigma, St. Louis, MO) at HeLa cells were plated at 4 ϫ 105 cells/60-mm dish. On the following 37°C in 5% CO2. Cervical carcinoma HeLa cells (JCRB9004) were cul- day, the cells were cotransfected with 8 ␮g reporter plasmids and 2 ␮g tured in DMEM (Nissui Pharmaceutical) with 10% FBS, 100 U/ml peni- pSV-␤-galactosidase with a calcium phosphate transfection system (Life Downloaded from cillin, and 0.1 mg/ml streptomycin at 37°C. Technologies, Rockville, MD) according to the manufacturer’s protocol. Preparation of peripheral blood neutrophils After 24 h, DNA precipitates were removed by washing, and the cells were incubated for another 24 h at 37°C. Peripheral blood obtained from healthy volunteers was heparinized, and To analyze luciferase and ␤-galactosidase activities, cells were lysed in neutrophils (purity Ͼ 95%) were isolated by Ficoll-Conray centrifugation 200 ␮l PicaGene Reporter Lysis Buffer LUC (Toyo Ink, Tokyo, Japan) and after dextran sedimentation of erythrocytes (28). In some experiments, neu- sonicated on ice for 10 s (ultrasonic disrupter; Tomy Seiko, Tokyo, Japan). ϫ 6 trophils were resuspended in RPMI 1640 at a final concentration of 5 10 Protein concentration of cell extracts was determined with a bicinchoninic http://www.jimmunol.org/ cells/ml and stimulated with 1 ␮g/ml LPS (from Escherichia coli O127:B8; acid protein assay kit (Pierce, Rockford, IL). Luciferase activity was mea- Difco, Detroit, MI) or 100 U/ml recombinant human TNF-␣ (Genzyme, sured as relative light units using a PicaGene luciferase assay kit (Toyo Boston, MA) at 37°C for 24 h. Ink) and a Lumat LB9501 luminometer (Berthold, Wildbad, Germany). ␤-Galactosidase activity was measured with a Galacto-Light kit (Tropix, Isolation of HNP-1 and HNP-3 promoters Bedford, MA), and transfection efficiency was normalized to the level of ␤-galactosidase activity. The sequence of the 5Ј flanking region of exon 1 of human HNP-1 and HNP-3 genes was amplified from human genomic DNA by PCR based on Preparation of nuclear extracts published sequences as illustrated in Fig. 1A (18). The first PCR reaction was performed with a primer set of sense 1 and antisense 1 on a thermal Nuclear extracts were prepared as described by Dignam et al. (34), with 8 cycler model 480 (Perkin-Elmer, Norwalk, CT) after incubation at 94°C for minor modifications. Briefly, 1 ϫ 10 cells were washed twice with PBS by guest on September 26, 2021 2 min using 30 cycles of 30 s at 94°C, 30 s at 54°C, and 1 min at 72°C. The and lysed in lysis buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 1 mM final polymerization step was extended by an additional 5 min at 72°C. The EDTA, 1.5 mM MgCl2, 0.5% Nonidet P-40, 1 mM DTT, 1 mM PMSF, 5 second PCR was conducted using the first reaction as a template with a ␮g/ml leupeptin, and 5 ␮g/ml pepstatin) on ice for 10 min. Nuclei pellets primer set of sense 2 and antisense 2. The synthesized 1.2-kbp fragments were washed once with the same buffer except Nonidet P-40. After incu- consisted of the sequences from Ϫ1116 to ϩ61 of HNP genes, and were bation with extraction buffer (10 mM HEPES (pH 7.9), 420 mM NaCl, 1 cloned to a TA-cloning vector pT7Blue(R) (Novagen, Madison, WI). Plas- mM EDTA, 1.5 mM MgCl2, 20% glycerol, 1 mM DTT, 1 mM PMSF, 5 mid inserts were confirmed by sequencing using a Dye Terminator Cycle ␮g/ml leupeptin, and 5 ␮g/ml pepstatin) at 4°C for 20 min, nuclei were Sequencing kit FS and a model 373A DNA autosequencer (PE Applied centrifuged at 12,000 ϫ g for 20 min at 4°C. The resultant nuclear extracts Biosystems Division, Foster City, CA). Base substitutions between HNP-1 were immediately subjected to SDS-PAGE/Western blotting or stored at and HNP-3 promoters are shown in Fig. 1B. Ϫ80°C for gel retardation assay. Protein content in the nuclear extracts was Oligonucleotides used were sense 1 primer (5Ј-AGCCCTGTTA measured with a bicinchoninic acid protein assay kit (Pierce). Ј Ϫ CAGGGGCTGC-3 ; located at 1159 from the transcription start site), Electrophoretic mobility shift assay (EMSA) sense 2 primer containing a MluI site (5Ј-CCGACGCGTCGTCCAAG GCAGGCAACTCAACCC-3Ј;atϪ1116), antisense 1 primer (5Ј-ACGT Nuclear extracts (10 ␮g) were mixed with a 32P-labeled probe (described TCCCTAGCAGGGATTCACCCGC-3Ј;atϩ199), and antisense 2 primer in the next section; 5 ϫ 104 cpm, 10–20 fmol) in 15 ␮l of a binding buffer Ј containing a XhoI site (5 -CCGCTCGAGCGCAGGGTGACCAGAGAG containing 10 mM HEPES (pH 7.9), 50 mM KCl, 5 mM MgCl2,1mM GGCAGA-3Ј;atϩ61). Underlining in the sequences indicate additional EDTA, 5% glycerol, 1 mM DTT, 1 mM PMSF, 1 ␮g/ml leupeptin, 1 ␮g/ml restriction sites. pepstatin, and 2 ␮g poly(dI-dC)⅐poly(dI-dC) (Amersham Pharmacia Bio- tech AB, Uppsala, Sweden) for 20 min on ice. The reaction mixtures were Plasmid construction applied to a native 5% polyacrylamide gel in 0.5ϫ TBE (44.5 mM Tris, The 1.2-kbp MluI-XhoI fragments of HNP-1 or HNP-3 promoter from 44.5 mM boric acid, and 1 mM EDTA (pH 8.3)) at 180 V for 90 min at pT7Blue(R) clones were subcloned into the promoterless luciferase vector 4°C. The gels were dried and exposed to Fuji RX-U x-ray film (Fuji Photo Ϫ pGL3-Basic (Promega, Madison, WI) and named Ϫ1116/HNP1-Luc and Film, Tokyo, Japan) at 80°C. For competition assay, a 20–50-fold molar Ϫ1116/HNP3-Luc, respectively. A series of 5Ј deletion plasmids shown in excess of unlabeled oligonucleotides and PCR products were preincubated ␮ Fig. 2A was constructed as follows. To remove 5Ј upstream sequences of in reaction mixture for 15 min on ice. For Ab supershift experiments, 1 l ␣ ␤ ␤ HNP promoters, Ϫ1116/HNP1-Luc or Ϫ1116/HNP3-Luc plasmids were of rabbit polyclonal Abs to C/EBP [14-AA], C/EBP [C-19], C/EBP ⌬ digested with SmaI (located in the polylinker site of pGL3-Basic) and SspI [ 198], c-Myb [C-19], and PU.1 [T-21] or normal rabbit IgG was added to (position Ϫ293 on the HNP promoters), HincII (Ϫ240), or DraI(Ϫ29). the reaction mixture 20 min before probe addition. All specific Abs (Tras- Each plasmid was recircularized by ligation to generate Ϫ293/HNP-Luc, Cruz Gel Supershift reagents, 1 mg/ml) were obtained from Santa Cruz Ϫ240/HNP-Luc, and Ϫ29/HNP-Luc, respectively. The Ϫ240/HNP-Luc Biotechnology (Santa Cruz, CA). plasmid was utilized as a template for PCR with appropriate sense primers Oligonucleotides for EMSA with MluI sequence at the 5Ј end and antisense 2 primer to construct further deletion derivatives, Ϫ133/HNP-Luc, Ϫ111/HNP-Luc, Ϫ86/HNP-Luc, Synthetic oligonucleotides or PCR products were used as probes for and Ϫ58/HNP-Luc (Fig. 4A). Sequences of sense primers were as follows: EMSA. The oligonucleotides were designed to generate a single 5Ј-G over- Ϫ133 sense, 5Ј-CTCGTACGCGTCCTTCCCAC-3Ј (Ϫ133 to Ϫ124); hang to each end after annealing with their compliments. PCR products Ϫ111 sense, 5Ј-ACCGTACGCGTCTGTCCTTGC-3Ј (Ϫ111 to Ϫ102); were digested with MluI to generate a 5Ј-CGCG overhang. Double- Ϫ86 sense, 5Ј-ACCGTACGCGTATGGACCCA-3Ј (Ϫ86 to Ϫ77); and stranded oligonucleotides and digested PCR fragments were labeled by 3266 TRANSCRIPTIONAL REGULATION OF HNP GENES IN HL-60 CELLS Downloaded from http://www.jimmunol.org/

FIGURE 1. Isolation of 5Ј flanking regions of HNP-1 and HNP-3 genes. A, The 1.2 kbp of the 5Ј 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 ϩ1. 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 ϩ 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 by guest on September 26, 2021 sites are derived from PCR primers. Sequences different from previous reports are indicated, namely, the addition of C at Ϫ589 in both HNP-1 and HNP-3 promoters and substitution of C for G at Ϫ627 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.

filling in the cohesive ends with [␣-32P]dCTP (ICN Biomedicals, Costa Membranes were blocked in Block Ace (Dainippon Pharmaceutical, Mesa, CA) using Klenow fragment. Tokyo, Japan) for1hatroom temperature and probed with appropriate Oligonucleotides used for probes were as follows: EBP/Myb oligonu- rabbit polyclonal Abs in Tris-buffered saline-Tween 20 (TBS-T; 150 mM cleotide, 5Ј-GACCAAATT TCTCAACTGTCCTTGC-3Ј (Ϫ125 to Ϫ102); NaCl, 20 mM Tris-HCl (pH 7.5), and 0.1% Tween 20) for 1 h. After four AML oligonucleotide, 5Ј-GCTTGCCACCACAATTATC-3Ј (Ϫ106 to washes with TBS-T, the membranes were incubated with HRP-conjugated Ϫ90); Ets oligonucleotide, 5Ј-GGACCCAACAGAAAGTAACCCCG goat anti-rabbit IgG (Organon Teknika, Durham, NC) for1hatroom GAAATTAGC-3Ј (Ϫ84 to Ϫ54); and PU.1/GABP␣ consensus oligonu- temperature. Proteins were visualized with the enhanced chemilumines- cleotide, 5Ј-GGGCTGCTTGAGGAAGTATAAGAAC-3Ј (the same se- cence Western blotting detection system (Amersham Pharmacia Biotech). quence as TransCruz Gel Shift Oligonucleotide except for 5Ј-G; Santa Abs used were follows: anti-C/EBP␣ Abs (C103; kindly provided by Dr. Cruz Biotechnology). Primer sets for PCR amplification were as follows: Pernille Rorth, European Molecular Laboratory, Heidelberg, Ger- Ϫ214 sense (5Ј-CGACGCGTCATAGTTGGTTGCTGCCTGGG-3Ј) and many; and 472, the kind gift from Dr. Steven McKnight, University of Ϫ124 antisense (5Ј-GTGGGAAGGTGAGGT TAAAG-3Ј) primers, and Texas, Southwestern Medical Center, Dallas, TX) in a 1:1000 dilution Ϫ146 sense (5Ј-CGACGCGTCTACTTTAACCTCACCTCACCTTC (36–39) and anti-C/EBP␤ (C-19) and anti-c-Myb (C19) Abs at a concen- CCACC-3Ј) and Ϫ64 antisense (5Ј-GGGTTACTTTCTGTTGGGT-3Ј) tration of 0.1 ␮g/ml (Santa Cruz Biotechnology). primers. The underlined MluI sequences have been added to the 5Ј 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 (5Ј- GTTTTCCCAGTCACGAC-3Ј) and reverse (5Ј-CAGGAAACAGCTAT GAC-3Ј) primers (Takara Shuzo, Shiga, Japan). Mutant oligonucleotides Results for competition assay were EBP/Myb mutant oligonucleotide, 5Ј-ACCG Isolation of 5Ј flanking sequences of HNP-1 and HNP-3 genes TACGCGTCTGTCCTTGC-3Ј (Ϫ122 to Ϫ102) and Ets mutant oligonu- cleotide, 5Ј-CGACGCGTCATTAGGACACCTCATCCCA-3Ј (Ϫ67 to HNP-1 and HNP-3 promoters (1.2 kbp) were amplified by PCR Ϫ40). Mutated sequences are indicated by bold letters. from genomic DNAs as shown in Fig. 1A . Thirteen clones con- Western blotting taining either HNP-1 or HNP-3 promoter were isolated, and their sequences were found to be consistent with earlier reports except Nuclear protein extracts (10 ␮g) prepared as described above were boiled for minor differences (18, 29): an additional C was found at posi- for 3 min in SDS-PAGE sample buffer and separated on a 10% or 12% Ϫ polyacrylamide gel (35). Resolved proteins were electrotransferred to Im- tion 589 from the transcription start site in both HNP-1 and mobilon-P membrane (Millipore, Bedford, MA) using a Trans-Blot SD HNP-3 genes, and a HindIII site at Ϫ628 in the HNP-1 promoter apparatus (Bio-Rad). was lost by substitution of C for G at Ϫ627 (Fig. 1B). A total of The Journal of Immunology 3267 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Functional analysis of HNP-1 and HNP-3 promoters. A, A series of 5Ј deletion constructs of HNP-1 and HNP-3 promoters (Ϫ1116/HNP1- Luc, Ϫ1116/HNP3-Luc, Ϫ293/HNP-Luc, Ϫ240/HNP-Luc, and Ϫ29/HNP-Luc) were generated by digestion with appropriate restriction enzymes and by subcloning into the pGL3-Basic luciferase vector. The promoter sequence from Ϫ293 and ϩ1 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 Ϯ SD for 5–10 independent experiments.

15 base differences were located upstream of a SspI site at Ϫ293 into the luciferase reporter vector to form Ϫ1116/HNP1-Luc and between HNP-1 and HNP-3 promoters, and ϳ300 bp of the prox- Ϫ1116/HNP3-Luc, respectively (Fig. 2A). imal region was identical between the two promoters. As shown in As shown in Fig. 2B, Ϫ1116/HNP1-Luc and Ϫ1116/HNP3-Luc Fig. 1B, there were three (HNP-1) or two (HNP-3) CCAAT boxes possessed the same luciferase activities despite having 15 base within the distal region (Ϫ1116 to Ϫ293), whereas one CCAAT box alterations. Luciferase levels with these constructs were Ͼ35-fold and a TATA box were found within the identical region (18, 29). greater than that with the promoterless vector. We further evalu- ated the activities of 5Ј deletional constructs to determine whether Transcription potentials of HNP-1 and HNP-3 promoters in HL- putative CCAAT boxes within the HNP promoters influenced tran- 60 cells scriptional potential (Fig. 2A). Deletion to position Ϫ293 (Ϫ293/ To examine the promoter activity of HNP-1 and HNP-3, the 5Ј HNP-Luc) did not significantly change luciferase activity com- flanking region from Ϫ1116 to ϩ61 of both genes was introduced pared with Ϫ1116/HNP1-Luc and Ϫ1116/HNP3-Luc. Deletion to 3268 TRANSCRIPTIONAL REGULATION OF HNP GENES IN HL-60 CELLS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Putative cis-elements and EMSA with Ϫ243/Ϫ124 and Ϫ146/Ϫ64 fragments of the HNP promoter. A, Sequence of proximal HNP promoter common to HNP-1 and HNP-3 genes. Transcription start site is numbered ϩ1. 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 3Ј end is an artificial sequence used for cloning. Arrows show the 5Ј 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, Ϫ214/Ϫ124 oligonucleotide, and Ϫ146/Ϫ64 oligonucleotide are indicated by brackets. C, Two DNA probes (Ϫ214/Ϫ124 oligonucleotide and Ϫ146/Ϫ64 oligonucleotide) were 32P-labeled and incubated with 10 ␮g nuclear extracts from HL-60 cells. The com- petitors were used in a 50-fold molar excess over labeled probes; Ϫ214/Ϫ124 and Ϫ146/Ϫ64 indicate unlabeled probes, and PUC indicates an unrelated pUC19 fragment. Asterisks indicate specific binding to the probes. position Ϫ240 (Ϫ240/HNP-Luc) showed retained activity, In contrast, in the cervical carcinoma HeLa cell line, which does whereas additional deletion to position Ϫ29, which removed a not express HNP mRNAs, full-length HNP-1 or HNP-3 promoters potential TATA box, decreased promoter activity by Ͼ90%. These displayed only 3-fold stimulation over the promoterless vector results indicate that base substitutions and CCAAT boxes were of (Fig. 2B). Moreover, these promoter activities were not altered by little consequence in the promoter activity of HNP-1 and HNP-3 the deletion to position Ϫ29. genes, and that the proximal promoter region between Ϫ240 and Taken together, these findings suggest that HNP-1 and HNP-3 Ϫ29 contains positive cis-acting element(s) interacting with tran- genes are equally transcribed, and that both HNP genes are tran- scription factors in HL-60 cells. scriptionally regulated in a myeloid-specific manner. The Journal of Immunology 3269 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Luciferase activity of 5Ј 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 Ϫ133⌬AML/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.SDof the mean is indicated by error bars. Data are presented as the percentage of activity of HNP promoter constructs vs that of Ϫ240/HNP-Luc (100%).

Requirement for overlapping C/EBP and c-Myb sites and Ets- AML1 sites was developed, which competed with the excess un- like element for activation of the HNP promoter labeled Ϫ214/Ϫ124 fragment but not with the unrelated pUC19 To localize cis-acting elements within the HNP promoter, the se- fragment (Fig. 3C, left panel). These findings indicate that an ϳ quence from Ϫ240 and Ϫ29 was scanned with the MatInspector 200-bp region of HNP promoter contains multiple binding sites version 2.2 computer program (40, 41). Putative binding sites for for transcription factors. C/EBP, c-Myb, AML1, Ets, and CCAAT displacement protein Next, to elucidate which element(s) were functionally important to Ј (CDP), which are related to myeloid-specific gene expression, HNP promoter activity, successive 5 truncates and a point mutant were contained within the region ϳ200 bp upstream of the TATA were created from Ϫ240/HNP-Luc using PCR (Fig. 4A) and assessed box (Fig. 3A) (30–32, 42). in HL-60 cells (Fig. 4B). Deletion to position Ϫ133 showed a ten- We analyzed whether the nuclear factors bound to the predicted dency to increased promoter activity compared with Ϫ240/HNP-Luc, sites of HNP promoter. EMSA was performed with a ␣-32P-la- suggesting the loss of negative regulatory elements. Deletion to po- beled DNA fragment containing the HNP promoter (Fig. 3B) and sition Ϫ111, which removed overlapping C/EBP and c-Myb sites, nuclear extracts prepared from HL-60 cells. As represented in Fig. resulted in a remarkable decrease in promoter activity. Interestingly, 3C, nuclear protein(s) in HL-60 cells strikingly bound to the Ϫ146/ mutation or deletion of the proximal AML1 site at Ϫ100 Ϫ64 oligonucleotide containing putative C/EBP, c-Myb, and (Ϫ133⌬AML and Ϫ86/HNP-Luc) had little effect on promoter ac- AML1 sites. Formation of the two major complexes was specifi- tivity compared with those of Ϫ133 and Ϫ111/HNP-Luc, respec- cally inhibited by the addition of unlabeled probe, but not by that tively. Additional deletion to position Ϫ58, which removed the bind- of unrelated pUC19 fragment (Fig. 3C, right panel). Weak binding ing site for Ets family proteins, resulted in a further decrease in to the fragment from Ϫ214 to Ϫ124 containing distal Ets and activity. Promoter activity was virtually abolished by deletion of a 3270 TRANSCRIPTIONAL REGULATION OF HNP GENES IN HL-60 CELLS Downloaded from http://www.jimmunol.org/

FIGURE 5. EMSA of the overlapping C/EBP and c-Myb sites on the HNP promoter. A, Sequence of the HNP promoter from Ϫ130 to Ϫ90. Shaded boxes indicate potential transcription factor binding sites for C/EBP and c-Myb. The bracket from Ϫ125 to Ϫ102 shows a region for oligonucleotide probe containing overlapping C/EBP and c-Myb elements (EBP/Myb oligo). B, A total 10 ␮g of nuclear extracts from HL-60, mature neutrophils (resting (rest), LPS activated, or TNF-␣ 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/GABP␣ consensus oligonucleotide (PU), and AML oligo (AML). Supershift assay was performed with 1 ␮g of appropriate Abs: anti-C/EBP␣ (EBP␣), anti-C/EBP␤ (EBP␤), anti-C/EBP recognizing C/EBP␣,-␤, -␦, and -⑀ (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). by guest on September 26, 2021

putative TATA box (at Ϫ29). These results indicate that in addition to clear extracts from mature neutrophils (Fig. 5B, middle panel). the TATA box, the overlapping C/EBP and c-Myb sites and the Ets Activation of neutrophils by inflammatory stimuli including LPS site are required for transcriptional activation of the HNP promoter. and TNF-␣ induced the transcription of mRNAs for cytokines such Moreover, they suggest that the overlapping C/EBP and c-Myb sites as IL-1␤ (data not shown), as reported previously (43, 44). How- seem to be more important than the Ets site in positive regulation of ever, no mRNA for HNPs was detected in neutrophils activated by HNP promoter. LPS or TNF-␣ or in resting neutrophils (data not shown). Further- more, no binding activity was detected using nuclear extracts from ␣ Binding of C/EBP to the C/EBP site on the HNP promoter activated neutrophils (Fig. 5B, middle panel). These observations with HL-60 nuclear extracts indicate the absence of specific factor(s) interacting with the We further investigated which transcription factors could bind to C/EBP site in mature neutrophils. When EMSA was performed the overlapping C/EBP and c-Myb sites in the HNP promoter. using nuclear extracts from HeLa cells, a faint band was found to Nuclear extracts from HL-60 cells, mature neutrophils, and HeLa be supershifted by anti-C/EBP␤ Ab on overexposure of the auto- cells were incubated with a 32P-labeled EBP/Myb oligonucleotide radiogram (Fig. 5B, right panel). These results suggest that spanning Ϫ125 to Ϫ102 (Fig. 5A). As expected, a prominent C/EBP␣ but not c-Myb predominantly interacts with the overlap- DNA-protein complex was detected using HL-60 nuclear extracts, ping C/EBP/c-Myb sites of HNP promoter in HL-60 nuclear ex- and found to be in competition with excess unlabeled EBP/Myb tracts, and that C/EBP␤ is likely to interact with C/EBP/c-Myb oligonucleotide but not with the mutated EBP/Myb oligonucleo- sites in HeLa nuclear extracts. tide or unrelated oligonucleotides such as PU.1 and AML1 (Fig. A recent report has indicated that the interaction of IRDs with 5B, left panel). The addition of Ab against C/EBP␣ (EBP␣) caused an Ets-like site at Ϫ65 is involved in the transcription of the a supershift of the specific complex, whereas the band was only HNP-1 gene (29). Consistent with this, we confirmed the for- weakly shifted by anti-C/EBP␤ Ab (EBP␤). Ab recognizing mation of IRDs and Ets-like complexes using nuclear extracts C/EBP␣,-␤,-␦, and -⑀ (EBPs) markedly abolished the specific from HL-60 cells, which were not affected by either PU.1/ DNA-protein complex. Interestingly, supershift or disappearance GABP␣ consensus oligonucleotide or anti-PU.1 Ab (Fig. 6B, of the specific band was not detected on the addition of anti-c- left panel). In addition, we observed that nuclear proteins from Myb, suggesting that the EBP/Myb oligonucleotide does not in- mature neutrophils could bind to the Ets-like sequence. These teract with c-Myb in HL-60 nuclear extracts. Likewise, neither specific complexes were not destroyed or influenced by the ad- anti-PU.1 Ab nor rabbit IgG had any effect on DNA-binding ac- dition of PU.1/GABP␣ oligonucleotide or anti-PU.1 Ab (Fig. tivity. Of note, no specific DNA binding was detected using nu- 6B, middle panel). Binding activity to the Ets-like sequence was The Journal of Immunology 3271 Downloaded from http://www.jimmunol.org/

FIGURE 6. EMSA of the Ets-like element on the HNP promoter. A, Sequences of the HNP promoter from Ϫ80 to Ϫ20. Shaded boxes indicate the putative Ets-like element and TATA box. A region of oligonucleotide probe (Ets oligo) spanning Ϫ84 to Ϫ54 is shown by a bracket. B, A total 10 ␮gof 32 nuclear extracts from HL-60, mature neutrophils (resting (rest), LPS activated, or TNF-␣ activated), and HeLa cells was incubated with P-labeled Ets oligo by guest on September 26, 2021 probe. Unlabeled oligonucleotides were added in a 25-fold molar excess as a competitor: Ets oligo (Ets), mutated Ets oligo (Mut), and PU.1/GABP␣ oligo (PU1). In supershift experiments, 1 ␮g of anti-PU.1 Ab (PU) or normal rabbit IgG was added to the reaction mixture. Asterisks indicate specific binding to the probes.

decreased in nuclear extracts from neutrophils stimulated with HNP-3 promoters and investigated how each gene is transcription- LPS or TNF-␣ (Fig. 6B, middle panel). Differences in EMSA- ally controlled. binding patterns between HL-60 cells and neutrophils may in- Our data clearly showed that HNP-1 and HNP-3 promoters (1.2 dicate that IRDs include several Ets family proteins. No such kbp) had the same luciferase activities in HL-60 cells despite hav- specific complex with the Ets-like sequence was detected in ing 15 nucleotide differences (Fig. 2B), suggesting that transcrip- nuclear extracts from HeLa cells (Fig. 6B, right panel). tion 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 Expression pattern of transcription factors in HL-60 cells and posttranscriptional and/or posttranslational modifications. One neutrophils possible explanation is that HNP-2 may be selectively produced by We analyzed levels of transcription factors likely to be involved in proteolysis of HNP-3 but not HNP-1, on the basis of earlier reports HNP expression by Western blot analysis (Fig. 7). Both C/EBP␣ indicating that HNP-1 content (50%) in the granules is equal to the and C/EBP␤ proteins were abundantly expressed in HL-60 nuclei sum of those of HNP-2 (30%) and HNP-3 (20%) (16, 24). (Fig. 7A). In contrast, neither C/EBP␣ nor C/EBP␤ were detected Interestingly, promoter activities of HNP-1 and HNP-3 were in nuclear extracts from resting or activated neutrophils (Fig. 7A). markedly low in nonhematopoietic HeLa cells compared with c-Myb protein could not be detected in HL-60 and neutrophil nu- HL-60 cells (Fig. 2B). This result suggests that transcription of clear extracts, although c-Myb was apparent in Jurkat nuclear ex- both HNP genes is regulated in a myeloid-specific manner. None tracts used as a positive control (Fig. 7B). of the putative CCAAT boxes were required for HNP promoter activity, and the 200-bp promoter sequence identical between Discussion HNP-1 and HNP-3, immediately upstream region from the TATA Four different HNPs are exclusively expressed in neutrophils and box, was sufficient for the promoter activity in HL-60 cells. Com- contribute to both innate and acquired immune systems (9, 24). puter analysis of the proximal HNP promoter indicated the puta- Even though HNP-1 and HNP-3 peptides are encoded by nearly tive cis-elements for C/EBP family, c-Myb, AML1, Ets family, identical genes, their activities and contents are considerably dif- and CDP (40, 41). Consistent with the computer prediction, EMSA ferent (8, 16, 18). In the present study, we isolated HNP-1 and revealed that HL-60 nuclear extracts contained multiple nuclear 3272 TRANSCRIPTIONAL REGULATION OF HNP GENES IN HL-60 CELLS

FIGURE 7. Western blot of nuclear extracts from HL-60 cells and mature neutrophils. Nuclear extracts (10 ␮g) from HL-60 cells and neutrophils (resting (rest), and LPS activated, or TNF-␣ activated) were resolved by 12% (A) or 10% (B) SDS-PAGE. Western blotting was performed using anti-C/EBP␣ and anti-C/EBP␤ 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). Downloaded from factors interacting with the sequences of the proximal HNP pro- activity (Fig. 4B), and that IRDs interacted with the Ets-like se- moter (Ϫ214 to Ϫ124 and Ϫ146 to Ϫ64) (Fig. 3C). quence using nuclear extracts from HL-60 cells and mature neu- Sequential deletion from Ϫ240 to Ϫ133 tended to enhance HNP trophils (Fig. 6). Furthermore, we confirmed the presence of PU.1,

promoter activity compared with that of Ϫ240/HNP-Luc (Fig. 4). which is implicated in the basal transcription of HNP genes, in http://www.jimmunol.org/ This suggests the presence of negative regulatory element(s) nuclear extracts from both HL-60 cells and neutrophils (data not within the region. One potent candidate seems to be the CDP- shown and Ref. 25). The finding that mature neutrophils express binding sequence at Ϫ220. CDP is implicated in the transcriptional Ets family factors including IRDs and PU.1 but not HNP mRNAs repression of myeloid-specific genes such as gp91phox and lacto- further support the important role of C/EBP␣ in the transcription ferrin (45–47). It is also possible that other negative element(s) of HNP genes in immature myeloid cells. function within the region, because weak but specific binding was Human ␤ defensins, hBD-1 and hBD-2, have been recently observed using the fragment (Ϫ214 to Ϫ124) lacking the CDP- identified from plasma and various epithelial tissues (11, 50–52). binding sequence (Fig. 3C). Although ␣ defensins (HNPs) and ␤ defensins are encoded by It is notable that the overlapping C/EBP and c-Myb sites (Ϫ122 different genes, these genes share a common evolutionary origin by guest on September 26, 2021 to Ϫ106) are required for both transcription and protein binding in (53, 54). Of note, hBD-2 expression is induced by stimuli such as HL-60 cells (Figs. 4B and 5B). Our data further revealed that and proinflammatory cytokine TNF-␣, thereby evoking C/EBP␣ rather than C/EBP␤ interacted with the C/EBP site of the important functions of hBD-2 in acute inflammation (51, 53). HNP promoter in HL-60 cells (Fig. 5B), despite the fact that both Future investigation of transcriptional regulation of not only HNP C/EBP␣ and C/EBP␤ were detected in HL-60 nuclear extracts but also ␤ defensin genes will afford valuable information about (Fig. 7A). In contrast, neither C/EBP␣ nor C/EBP␤ was contained the role of the defensin family in the innate and acquired immune in nuclear extracts from not only resting but also activated neu- systems. trophils (Fig. 7A). Interestingly, a feeble complex of C/EBP␤ with the C/EBP site was observed using nuclear extracts from HeLa Acknowledgments cells (Fig. 5B), although HeLa cells showed nearly negligible tran- We thank Dr. Pernille Rorth (European Molecular Biology Laboratory) for scriptional activity of the HNP promoter (Fig. 2B). However, providing C103 anti-C/EBP␣ Ab and Dr. Steven L. McKnight (University ␤ Western blot analysis revealed that HeLa cells expressed C/EBP of Texas, Southwestern Medical Center) for 472 C/EBP␣ antiserum. but not C/EBP␣ (data not shown). Taken together, these findings indicate that C/EBP␣ may play an important role in the transcrip- References tion of HNP genes in HL-60 cells, whereas C/EBP␤ is unlikely to 1. Lehrer, R. I., T. Ganz, M. E. Selsted, B. M. Babior, and J. T. Curnutte. 1988. play a role in either HL-60 or HeLa cells despite its expression. Neutrophils and host defense. Ann. Intern. Med. 109:127. Furthermore, c-Myb does not seem to participate in HNP promoter 2. Klebanoff, S. 1992. Oxygen metabolites from . In Inflammation: Basic activity in HL-60 cells, because no c-Myb was detected in nuclear Principles and Clinical Correlates, 2nd Ed. J. I. Gallin, I. M. Goldstein, and R. Snyderman, eds. Raven Press, New York, p. 541. extracts from HL-60 cells or neutrophils (Fig. 7B). 3. Hampton, M. B., A. J. Kettle, and C. C. 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