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Originally published online as doi:10.1189/jlb.0803384 on November 21, 2003

Published online before print November 21, 2003
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(Journal of Leukocyte Biology. 2004;75:332-341.)
© 2004 by Society for Leukocyte Biology

{alpha}-Defensin expression during myelopoiesis: identification of cis and trans elements that regulate expression of NP-3 in rat promyelocytes

Cindy M. Yamamoto, Niaz Banaiee, Nannette Y. Yount, Bindi Patel and Michael E. Selsted1

Departments of Pathology & Microbiology & Molecular Genetics, College of Medicine, University of California, Irvine

1 Correspondence: Department of Pathology, College of Medicine, University of California, D440 Med. Sci. I, Irvine, CA 92697-4800. E-mail: meselste{at}uci.edu


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ABSTRACT
 
{alpha}-Defensins are antimicrobial peptides that contribute to innate-immune functions of neutrophils and intestinal Paneth cells. Transcription of {alpha}-defensin genes occurs early in neutrophilic myelopoeisis. To examine the mechanisms that regulate {alpha}-defensin gene expression, we analyzed transcription of rat neutrophil {alpha}-defensin NP-3 in D4 cells, a subclone of the promyelocytic cell line IPC-81. Northern blot analysis showed that D4 cells express fivefold higher levels of {alpha}-defensin mRNA than the parental cell line in a manner relatively independent of passage number. Increased levels of steady-state mRNA in D4 cells correlated with markedly elevated peptide levels detected by immunocytochemical staining. To identify the cis-acting DNA elements involved in tissue-specific expression, D4 cells were transfected with luciferase reporter constructs containing NP-3 gene 5'-flanking sequences. Analyses of transfected D4 cells demonstrated that the proximal 87 base pair (bp) sequence contained cis-acting DNA elements necessary for optimal promoter activity. Mutational analyses within the 87-bp region suggested the involvement of the CAAT box and a putative polyoma enhancer-binding protein 2/core-binding factor (PEBP2/CBF) site in defensin gene transcription. Transient transfection analyses using tandem repeats of oligonucleotides containing these sequences demonstrated that proximity of the CAAT box and PEBP2/CBF site was important for defensin promoter activity. Electrophoretic mobility shift assays indicated that PEBP2/CBF or a PEBP2/CBF-related protein was involved in a specific protein-DNA interaction occurring within a DNA fragment containing the CAAT and PEBP2/CBF sequences. These data identify functional trans- and cis-elements that regulate rat defensin gene expression in high defensin-expressing promyelocytic cells.

Key Words: neutrophils • mRNA • cellular differentiation • gene regulation


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INTRODUCTION
 
{alpha}-Defensins are microbicidal peptides stored in the primary granules of neutrophils of many mammalian species [1 , 2 ] as well as Paneth cells of the small intestine [3 4 5 ], rabbit kidney [6 ], and human vaginal epithelium [7 ]. Structurally related ß- and {theta}-defensins are also expressed in a variety of myeloid and/or epithelial cells [8 9 10 11 12 13 14 ]. Myeloid {alpha}-defensins are 29–35 amino acids in length and have broad-spectrum antimicrobial activities against gram-negative and gram-positive bacteria, fungi, giardia, and enveloped viruses [15 16 17 18 19 20 ]. Given their potent antimicrobial activities in vitro, their storage in neutrophil azurophil granules, and their delivery to phagosomes [21 ], it is likely that defensins contribute to oxygen-independent killing of phagocytosed microorganisms.

Neutrophil {alpha}-, ß-, and {theta}-defensins are transiently expressed during myelopoeisis in bone marrow [12 , 14 , 22 ]. In situ hybridization of rat bone marrow cells demonstrated the highest levels of {alpha}-defensin mRNA in promyelocytes, and no detectable transcripts were present in metamyelocytes, bands, or mature neutrophils [22 ]. In contrast, the {alpha}-defensin peptide was immunologically detected at all stages of neutrophilic myelopoiesis [12 , 22 ], indicating that the mature peptides are stored in the granule compartment after transcription ceases. A similar relationship between transcription and translation is observed for expression of leukocyte-derived ß- and {theta}-defensins during myelopoiesis [12 , 14 ].

Azurophil granule proteins such as myeloperoxidase (MPO) and elastase are expressed during myelopoiesis in a similar pattern to defensins [23 , 24 ]. Studies using the human promyelocytic cell line HL-60 have shown that transcriptional and post-transcriptional mechanisms regulate MPO, elastase, and neutrophil defensin gene expression [25 26 27 ]. These studies indicate that myeloid transcription factors, such as polyoma enhancer-binding protein 2 (PEBP2) and CCAAT1 enhancer-binding protein (C/EBP)-{alpha}, may regulate expression of these genes.

To facilitate studies on {alpha}-defensin gene expression, we isolated subclone D4 from the rat promyelocytic cell line, IPC-81. As compared with the parental cell line, D4 cells were much more homogeneous and expressed higher levels of {alpha}-defensin mRNA and peptide. Using D4 cells, we performed experiments to characterize the promoter of the rat neutrophil defensin gene, NP-3, cloned from a rat genomic library. Transient transfections with luciferase reporter constructs containing NP-3 5'-flanking sequences, as well as deletions, tandem repeats, and mutations thereof, revealed that cis-response elements located in the proximal 5'-flanking region are required for optimal promoter activity.


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MATERIALS AND METHODS
 
Cell culture
The rat promyelocytic cell line IPC-81 was a generous gift from Michelle Lanotte (Unite INSERM 301, Hopital St. Louis, Paris, France). Cells were maintained in RPMI 1640 (Mediatech, Herndon, VA), supplemented with 10% heat-inactivated horse serum (HS; Gibco-BRL, Grand Island, NY) at 37°C and 5% carbon dioxide.

Cellular subcloning
IPC-81 cells were diluted to approximately one cell per well and were grown in 96-well Falcon plates (Becton Dickinson, Franklin Lakes, NJ). Growth of cells was monitored by inverted microscopy. Single cell-derived subclones were grown in one part IPC-81 conditioned medium (CM), two parts RPMI-1640/10% HS. The IPC-81 CM was obtained by isolating the supernatant of IPC-81 cells grown for 48 h. Two subclones, termed D4 and D5 and characterized as described below, were maintained in identically prepared CM-supplemented RPMI-1640/10% HS.

Antibody production
RatNP-3 was purified to homogeneity from acid extracts of casein-induced neutrophils lavaged from the peritoneum of Sprague-Dawley rats as described [28 ]. Immunogen was prepared by glutaraldehyde conjugation of ratNP-3 to ovalbumin [22 ]. Antibody was produced by repeated immunization of a New Zealand white rabbit, and the immunoglobulin G (IgG) fraction was obtained by diethylaminoethyl Econo-Pac chromatography (Bio-Rad Laboratories, Richmond, CA) of the antiserum, as recommended by the manufacturer.

Immunocytochemistry
Cytocentrifuge preparations of IPC-81, D4, and D5 cells were fixed in 95% ethanol for 10 min at room temperature and stored in 70% ethanol at 4°C. Fixed cells were rehydrated with phosphate-buffered saline (PBS) and incubated overnight at 4°C with a 1:5 dilution of rabbit anti-ratNP-1 IgG [22 ], 1:10 dilution of rabbit anti-ratNP-3 IgG, or rabbit normal (preimmune) serum IgG. Immunoreactivity was detected using biotinylated goat anti-rabbit IgG followed by avidin:biotinylated glucose oxidase complex, visualized with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP), per the manufacturer’s instructions (Vector Laboratories, Burlingame, CA). Slides were counterstained with nuclear fast red.

In situ hybridization
In situ hybridization was performed as described previously with minor modifications [22 ]. Briefly, cytocentrifuge preparations of parental IPC-81, D4, and D5 cells were fixed in 4% paraformaldehyde for 10 min and dehydrated by sequential immersion in 70%, 95%, and 95% ethanol. Sense and antisense riboprobes were transcribed from linearized plasmids containing ratNP-3 cDNA [22 ] using Sp6 (sense) or T7 (antisense) RNA polymerases. Slides were incubated for 1 h at 37°C in prehybridization solution [7% sodium dodecyl sulfate (SDS), 4x Denhardt’s solution, 5x SSPE (1xSSPE is 0.15 M NaCl, 0.05 M NaH2PO4, 5.0 mM EDTA, pH 7.4), 0.1 mg/ml polyadenylic acid] and were then hybridized with 10 µg/ml digoxigenin–uridine triphosphate-labeled riboprobe for 18 h at 42°C. RNA–RNA hybrids were detected with alkaline phosphatase-conjugated antidigoxigenin antibody and developed with NBT/BCIP using the Genius system (Boehringer Mannheim, Indianapolis, IN). Cells were evaluated for RNA–RNA hybridization by light microscopy.

Genomic library screening
A {lambda} EMBL3 rat genomic library (Clontech, Palo Alto, CA) was screened using polymerase chain reaction (PCR)-amplified products corresponding to the coding regions of the ratNP-1–4 cDNAs. Approximately 8.0 x 105 recombinant phages (approximately four genomic equivalents) were transferred to Nytran membranes (Magna Nytran 0.22 mm, MSI, Westborough, MA) and were baked at 80°C for 1 h. PCR products for library screening were generated with the following ratNP-1–4 primers: ratNP-1 and -2 (5'-TGTGTCTCTTGCTCTGC-3'; 5'-AAAACCACAGCGTGTGCT-3'), ratNP-3 and -4 (5'CTTCTCCTGCTGGCCCTCCA-3'; 5'-AGAGTCGGTAGATGCGGCCATT-3'). The PCR products were radiolabeled with [32P] {alpha}-deoxycytidine triphosphate (dCTP; NEN/DuPont, Boston, MA) by random priming. Membranes were incubated in prehybridization solution (4x Denhardt’s solution, 5x SSPE, 7% SDS, 0.1 mg/ml polyadenylic acid) for 6 h at 42°C. Membranes were hybridized in the prehybridization solution containing 40% formamide and the radiolabeled probe at a concentration of 1.0 x 106 cpm/ml for 16 h at 42°C. Membranes were washed (0.5x SSPE and 0.1% SDS) at room temperature for 5 min, 42°C for 20 min, and 55°C for 20 min. After primary screening, recombinant phages were replated at lower densities and rescreened until pure populations were obtained.

Subcloning
{lambda}-Phage DNA was purified using polyethylene glycol precipitation followed by phage-head disruption (Magic Lambda Preps, Promega, Madison, WI). Single and double digests of phage DNA were performed with restriction enzymes (Boehringer Mannheim). DNA samples were size-fractionated by agarose gel electrophoresis and transferred to Nytran membranes for Southern blot analysis. Southern blots were probed with random prime-labeled PCR products for ratNP-1–4, as described above. Hybridizing genomic fragments were gel-purified from agarose (Sea Plaque, FMC Bioproducts, Rockland, ME) and subcloned into the pUC18 vector. Recombinant clones were used to transform INV{alpha}-F' (Invitrogen, San Diego, CA)-competent Escherichia coli cells. Subcloned fragments were sequenced using the dideoxy chain-termination method and analyzed by the Intelligenetics Geneworks 2.1 software package (Intelligenetics, Mountainview, CA). Both strands of DNA were sequenced for all clones described.

Northern blot analysis
Total RNA was isolated from frozen cell pellets with RNA Stat-60 (Tel-Test "B," Inc., Friendswood, TX) per the manufacturer’s recommended protocol or by centrifugation through a CsCl cushion. For Northern blots, 10 µg total RNA was separated on 1.25% agarose-formaldehyde gels, transferred to 0.22 µm Nytran membranes (Micron Separations, Westboro, MA), and probed with random, prime-labeled, PCR-generated products corresponding to nucleotides 188–378 of ratNP-1, 117–348 of ratNP-3, and 114–336 of ratNP-4 as described previously [22 ]. When examination of NP-3-specific mRNA expression was desired, blots were probed with random, prime-labeled, PCR-generated products corresponding to nucleotides 117–348 of ratNP-3. Membranes were washed in 0.5x SSPE, 0.1% SDS at 50°C, and exposed to Reflection films (NEN/Dupont) for 8 h to 4 days at -70°C. To verify the integrity and amount of RNA loaded, membranes were stripped and hybridized with a random, prime-labeled, 441-base pair (bp) rat ß-actin probe generated by PCR amplification of rat bone marrow cDNA. Hybridizing bands were sized using a 0.155- to 1.77-kb RNA molecular weight marker (Gibco-BRL). Autoradiographs were scanned with an LKB 2202 Ultrascan laser densitometer. {alpha}-Defensin mRNA signals were normalized to that of ß-actin in each sample.

Plasmid construction
A pUC18 plasmid containing a genomic subclone of the ratNP-3A gene was digested with SmaI and XhoI. The resulting, smaller fragment was ligated into SmaI/XhoI-digested pGL2 basic (Promega) to yield NP-3(p18/0)luc. This construct contains ~2500 bp NP-3 5'-flanking sequence. Luciferase constructs containing smaller regions of NP-3 5'-flanking sequence [NP-3(37)–NP-3(687)] were generated by PCR amplification of the NP-3(p18/0)luc plasmid using sense and antisense primers containing MluI and XhoI sites. PCR products were digested with MluI and XhoI and ligated into MluI/XhoI-digested pGL2 basic.

The NP-3(87)luc plasmid contains the proximal 87 bp of the NP-3 promoter and was the template for PCR amplification of products used in the generation of luciferase constructs containing dinucleotide mutations in CAAT and PEBP2 sequences. Dinucleotide mutations in the CAAT box [NP-3(87/CAAT)luc; conversion of AA to GG at -74/-75] were generated by PCR with primers A (5'-AAACTCGAGCAGACAGGTTCTACAAAG-3') and B (5'-AAAACGCGTGTTTCAGGTATCCGGTAGAAA-3'). Dinucleotide mutations in the PEBP2 site [NP-3(87/PEBP)luc; conversion of AA to TC at -64/-65] were generated using primers A and C (5'-AAAACGCGTTTTCAGGTATCCAATAGAAATTTCCCACAA-3'). PCR mutagenesis with primers A and D, an oligonucleotide containing dinucleotide mutations in both sites (5'-AAAACGCGTTTTCAGGTATCCGGTAGAAATTTCCCACAA-3'), was used to generate the double CAAT/PEBP2 mutant, NP-3(87/C+P)luc.

Luciferase constructs containing one and four tandem repeats of the CAAT box [NP-3(CAAT/TR1) and NP-3(CAAT/TR4)] and one and three tandem repeats of the PEBP2/core-binding factor (CBF) sequence [NP-3(PEBP/TR1) and NP-3(PEBP/TR3)] were constructed by ligation of the annealed, kinased oligonucleotides (15–18 bp) containing the CAAT (5'-CCAGGTATCCAATAGAAATGAGCT-3', 5'-CATTTCTATTGGATACCTGGAGCT-3') and PEBP2/CBF (5'-CGAAATTAACCACAAAAAAAGAGCT-3', 5'-CTTTTTTGTGGTTAATTTCGAGCT-3') sequences to the SacI-digested NP-3(87)luc construct. NP-3(87/TR1) contains a tandem repeat of the proximal 87-bp region and was generated by PCR amplification using the following primers (5'-AAAGGCGCGCCTTTCAGGTATCCA-3', 5'-AAAACGCGTGAAACATCTT-3') with 55E plasmid as template. PCR products were digested with AscI/MluI and ligated into an MluI-digested NP-3(87)luc construct.

To aid in the verification of a repressor element within the -487/-87 region, a construct was produced containing this sequence ligated into an MluI/XhoI site located upstream of a Simian virus 40 (SV40) promoter containing luciferase reporter construct (pGL2 promoter vector, Promega). The luciferase activity of this construct was compared with activity observed with pGL2 promoter vector alone. Sequences of all constructs were confirmed by standard sequence analysis. All sequence analyses, including DNA alignments of human and rat promoters, were performed using Geneworks 2.4 (Intelligenetics). A cytomegalovirus promoter-driven luciferase construct (a generous gift from Timothy Osborne, University of California, Irvine, CA) was used as a control plasmid for transfections performed with D4 cells.

Transient transfections
D4 cells grown to 70–80% confluence were suspended in RPMI 1640 to 1 x 107 cells/ml. The suspension (0.5 ml) was electroporated (Bio-Rad Gene Pulser II) with 10 µg sample plasmid and 2 µg ß-galactosidase control plasmid in RPMI 1640 at 960 µF and 300 V. Cells were then incubated for 15 min on ice and cultured for 4 h in one part IPC-81 CM and two parts RPMI-1640/10% HS. The cells were harvested by centrifugation in 0.25 ml reporter lysis buffer (Promega). Cell lysates were assayed for luciferase activity using the Promega luciferase assay system. ß-Galactosidase activities and protein concentration were measured using the ß-galactosidase enzyme assay system (Promega) and bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL), respectively. Luciferase activity in the lysates was normalized to ß-galactosidase activity and protein concentration.

Nuclear extracts
Nuclear extracts were prepared by a modification of the method of Dignam et al. [29 ]. D4 cells were washed in ice-cold PBS, incubated in ice-cold lysis buffer (80 mM Tris-HCl, pH 7.4, 10 mM KCl, 3 mM MgCl2), and then lysed by addition of 0.1% (v/v) Nonidet P-40. Nuclei were washed in 1 vol lysis buffer, recovered by centrifugation at 234 g for 10 min, and resuspended in 0.1 ml storage buffer (40% glycerol, 50 mM Tris-HCl, pH 8.3, 5 mM MgCl2, 0.1 mM EDTA). Nuclei were then lysed by the addition of 300 mM NaCl. After incubation on ice for 30 min, the extracts were centrifuged at 22,500 g for 20 min, and the supernatants were aliquoted and stored at -70°C.

Electrophoretic mobility shift assays (EMSA)
The PCR-generated DNA fragment containing the proximal 87 bp of the 5'-flanking region was [{alpha}32P]-dCTP-labeled (NEN/DuPont) by a fill-in reaction using Klenow enzyme. Probes (5x104–1x105 cpm/µl) were purified by centrifugation using Bio-Spin 30 columns (Bio-Rad). For EMSA, 8–10 µg D4 nuclear extract protein, estimated by the BCA reagent, was incubated on ice for 10 min with or without unlabeled competitor or noncompetitor DNA. The labeled DNA fragment (1 ng) was then added, and incubation was continued on ice for 20 min. For competition studies, unlabeled, annealed oligomers containing the C/EBP consensus sequence (5'-ATGAATTCTGCAGATTGCGCAATCTGCAGGATCCT-3', 3'-ACTTAAGACGTCTAACGCGTTAGACGTCCTAGGAT-5'), the PEBP2/CBF consensus sequence (5'-AATTCGAGTATTGTGGTTAATACG-3', 3'-GCTCATAACACCAATTATGCTTAA-5'), a 102-bp PCR-amplified fragment corresponding to nucleotides 173–274 of rat cryptdin-1 cDNA, or the indicated NP-3 promoter region were added to the binding reaction. For supershift analyses, 1 µl acute myeloid leukemia (AML)-1 antiserum [30 ] (a generous gift from Scott Hiebert, Vanderbilt University, Nashville, TN) or 1 µl C/EBP-{alpha} rabbit polyclonal IgG (100 µg/0.1 ml; Santa Cruz Biotechnology, Santa Cruz, CA) was added to the binding reactions. The mixtures were then resolved on a 4% acrylamide gel run at 100 V in Tris-boric acid-EDTA buffer at 8°C for 3 h. Gels were dried and exposed to X-ray film for 1–24 h at -70°C.


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RESULTS
 
{alpha}-Defensin expression in IPC-81, D4, and D5 cell lines
The IPC-81 cell line, originally isolated by primary culture of Brown Norway rat myelocytic leukemia cells, was determined by electron microscopy and histochemical analysis to be promyelocytic [31 ]. As abundant {alpha}-defensin mRNA is found in rat bone marrow promyelocytes [22 ], IPC-81 cells were expected to express myeloid {alpha}-defensin genes. To confirm this, the levels of {alpha}-defensin mRNA from bone marrow and IPC-81 cells were analyzed by Northern blotting using PCR-generated probes designed to hybridize with mRNA for rat neutrophil {alpha}-defensins 1–4 (Fig. 1A ). When normalized to ß-actin hybridization, the level of {alpha}-defensin transcripts in IPC-81 was surprisingly low as compared with the signal obtained from adult rat bone marrow. To determine the homogeneity and level of mRNA in the IPC-81 cell population, in situ hybridization was performed using a ratNP-3-specific riboprobe. Only a small fraction (4.5%±0.5%) of the IPC-81 cells was positive for {alpha}-defensin mRNA (Fig. 1B) . In a parallel experiment, the immunoreactivity of IPC-81 cells was evaluated using anti-ratNP-1 antibody [22 ]. Similar to the in situ hybridization result, only a minor fraction (3.3%±0.5%) of the cells was immunopositive for {alpha}-defensin peptide (Fig. 1C) , and the low percentage of cells expressing {alpha}-defensin mRNA and peptide correlated with the relatively low signal obtained by Northern analysis. Moreover, the heterogeneity of {alpha}-defensin expression in IPC-81 cells suggested that this line would probably not be suitable for further studies of {alpha}-defensin gene regulation.



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  		Figure 1. {alpha}-Defensin mRNA and peptide expression in IPC-81 cells. (A) Northern blot analysis of steady state {alpha}-defensin mRNA levels in IPC-81 cells and rat bone marrow cells. Total RNA (10 µg) was hybridized to random prime-labeled PCR products corresponding to rat neutrophil defensins NP-1–4. A rat ß-actin probe was used to normalize the quantity of RNA loaded. (B) In situ hybridization to defensin mRNA in IPC-81 cells used a defensin riboprobe generated from full-length ratNP-3 cDNA. (C) Immunocytochemical staining of defensin peptide in IPC-81 cells was performed with rabbit anti-ratNP-1 IgG.

Evaluation of the anti-ratNP-1 immunocytochemical pattern of IPC-81 cells obtained from different passages suggested that the cells were composed of a mixture of phenotypes. To address this, we performed dilutional subcloning with different media containing varied compositions of RPMI 1640, serum, and IPC-81 CM. The most effective subcloning mixture (one part IPC-81 CM:two parts RPMI-1640/10% HS) supported the clonal expansion of two distinct cell lines, D4 and D5 clones, which were further characterized.

Northern blot analysis demonstrated that D4 cells expressed approximately fivefold higher steady-state {alpha}-defensin mRNA levels compared with IPC-81 (Fig. 2 ). D4 cells also demonstrated a sixfold increase (26.5%±1.5%) in cells positive for {alpha}-defensin transcripts, as assessed by in situ hybridization (Fig. 3A ) and a tenfold increase in the percentage of cells immunopositive for ratNP-1 peptide (32.1%±2.1%; Fig. 3B ). Immunostaining with anti-ratNP-3 antibody gave similar results (data not shown). In contrast to D4 cells, D5 cells had much lower levels of steady-state {alpha}-defensin mRNA and peptide than even the IPC-81 parental cells (Figs. 2 and 3C and 3D ). None of the D5 cells contained detectable {alpha}-defensin transcripts by in situ hybridization (Fig. 3C) , and fewer than 1% of the cells was immunopositive for ratNP-1 peptide (Fig. 3D) . Taken together, these data demonstrate that D4 cells are a high {alpha}-defensin-expressing IPC-81 subclone.



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  		Figure 2. {alpha}-Defensin mRNA levels in IPC-81, D4, and D5 cells. Northern blot analysis of 10 µg total RNA isolated from each cell source using the same NP-1–4 probe used in Figure 1 . Steady-state mRNA levels were normalized to the ß-actin control.



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  		Figure 3. Defensin in situ hybridization and immunolocalization in D4 and D5 subclones. (A) In situ hybridization and (B) immunohistochemical analysis were conducted on D4 cells as described for analysis of expression in IPC-81 cells. Parallel (C) in situ hybridization and (D) immunocytochemical analyses demonstrate the very low levels of mRNA and peptide in D5 cells.

Isolation of ratNP-3 and -4 genes
A {lambda} EMBL3 rat genomic library was screened with PCR-amplified products corresponding to the full-length ratNP-1–4 cDNAs. Five positive clones were selected for characterization by Southern analysis, and hybridizing restriction fragments were subcloned and sequenced. Upon sequencing, two independently derived 6.5-kb EcoRI fragments were found to correspond to two different ratNP-3 genes, termed ratNP-3A (GenBank accession #U50353) and ratNP-3B (U50354). The ratNP-4 gene (GenBank accession # U50355) was contained in a 5.0-kb XbaI/SalI fragment.

NP-3 promoter analysis
The transcriptional start site for ratNP-3A, -3B, and -4 was determined by circularized RNA mapping and confirmed by Southern and Northern blot analysis. Sequencing data indicated that the 5'-flanking sequences of these genes contain several putative myeloid response elements including C/EBP, PEBP2/CBF, c-myb, and PU.1 (Fig. 4 ), all of which have been shown to regulate myeloid-specific genes [32 33 34 35 36 37 38 39 ].



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  		Figure 4. cis-Regulatory elements found in the ratNP-3 promoter. Consensus-binding sequences for several myeloid response elements were used to identify the following underlined putative-binding sites within the proximal 688 bp of the ratNP-3 promoter. -1, The nucleotide immediately flanking the transcriptional start site.

As a step toward identifying functional cis-elements, we performed deletion analyses of the NP-3 gene 5'-flanking region. Luciferase reporter constructs containing 5' sequences ranging from -37 to -2500 bp were analyzed by transfection of D4 cells. Compared with the longest construct, NP-3(p18/0), promoter activity was only modestly affected by deletion of most of the 2.5-kb of the 5'-flanking sequence, as there was no more than a twofold difference in promoter activity among constructs containing approximately 2500, 687, and 487 bp of upstream sequence (Fig. 5 ). These results suggested that the region between -2500 and -487 does not contain major regulatory sequences for NP-3 expression. Furthermore, the first intron of NP-3 did not appear to contain functional cis-acting elements, as the intron-containing construct, NP-3(687/I), had the same activity as the construct lacking the intron. The region between -487 and -87, however, may contain a repressor element as a result of the relative increase (fourfold) in luciferase activity observed with construct NP-3(87) compared with NP-3(487). Linking this region upstream to an SV40 promoter-driven luciferase construct decreased basal activity by nearly 50% (data not shown), confirming the presence of a negative regulatory element in the 400-bp region.



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  		Figure 5. Transient expression analysis of the ratNP-3 promoter. The data are represented as percent fold stimulation relative to the activity of the NP-3(p18/0)luc construct, which contains ~2.5 kb of the NP-3 5'-flanking region. NP-3(687/I)luc contains the proximal 687 bp of NP-3 promoter and the first exon and intron of the NP-3 gene. The other constructs contain the proximal 37–687 bp of the NP-3 promoter and are appropriately labeled. pGL2 basic does not contain any promoter sequences upstream of the luciferase gene. The activities were measured as corrected relative light units (RLUs; normalized against ß-galactosidase activity and protein concentration). The average of the activities obtained by the NP-3(p18/0)luc construct was set at 100%. The average and SD of four to eight experiments are shown on the right.

The activity of NP-3(87), which was 15-fold over background, was the highest of any of the constructs produced, indicating that the proximal 87-bp region contained one or more critical NP-3 response elements important for driving gene expression. Truncation of the 5' end by an additional 11 bp [NP-3(76)] led to a 40% reduction in promoter activity, and reduction of the upstream sequence to less than 66 bp resulted in nearly complete abrogation of promoter function (Fig. 5) . The sequences of two putative response elements, a CAAT box and a PEBP2/CBF site, are present within the proximal 87-bp region of the 5'-flanking sequence. To more selectively test for the potential function of these cis-elements, constructs were generated that contained dinucleotide mutations in one or both sites. Individual mutations in the CAAT box and PEBP2/CBF site decreased promoter activity fivefold and twofold, respectively (Fig. 6 ). Dinucleotide mutations in both sites reduced promoter activity to background levels, demonstrating the requirement for CAAT and PEBP2 sites in NP-3 expression (Fig. 6) .



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  		Figure 6. Mutational analysis of the ratNP-3 promoter in D4 cells. The average activities obtained by the NP-3(p18/0)luc construct were set at 100%, and data are represented as percent fold stimulation relative to NP-3(p18/0)luc. Activity is measured as RLUs normalized to ß-galactosidase activity and protein content. NP-3(p18/0)luc and NP-3(87)luc contain 2500 and 87 bp of the NP-3 promoter. As described in Materials and Methods, NP-3(87/CAAT)luc, NP-3(87/PEBP)luc, and NP-3(87/C+P)luc constructs contain the proximal 87 bp of the ratNP-3 promoter with dinucleotide mutations in the CAAT box, PEBP2/CBF site, and both sites, respectively. The data are based on four to eight individual experiments.

The possible enhancer function of the CAAT box and PEBP2 site was examined by transfecting D4 cells with constructs containing sequences encoding one or more tandem repeats of the individual elements. The results of these experiments, summarized in Figure 7 , demonstrated that constructs containing one to four tandem repeats of the CAAT or PEBP2 elements were no more active than NP-3(87). However, the production of a single tandem repeat of the 87-bp sequence resulted in a threefold increase in luciferase activity compared with unmodified NP-3(87). These data demonstrate an enhancer function within the 87-bp region that requires the CAAT box and PEBP2 site and suggests that proximity of the two sequences (PEBP2 at -64 to -58; CAAT at -76 to -72) may be necessary for this response.



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  		Figure 7. Transient transfection analysis of constructs containing tandem repeats of the CAAT, PEBP2/CBF, and proximal 87-bp sequences of the ratNP-3 promoter. All constructs contain the proximal 87 bp of the NP-3 5'-flanking region. Constructs NP-3(CAAT/2) and NP-3(CAAT/5) contain one and four tandem repeats of the CAAT box, respectively. Constructs NP-3(PEBP/2) and NP-3(PEBP/4) contain one and three tandem repeats of the PEBP2/CBF site, respectively. Construct NP-3(87/2) contains the proximal 87-bp region of the NP-3 promoter in tandem upstream of the luciferase reporter gene. The data are presented as percent fold stimulation relative to the activity of the NP-3(87) construct. The activity of the NP-3(87) construct was represented as corrected RLUs (normalized against ß-galactosidase activity and protein concentration) and was arbitrarily set at 100%. The data shown are based on two to four individual experiments.

EMSA using the proximal 87-bp region of the NP-3 promoter
To identify possible protein-DNA interactions occurring within the proximal 87-bp region, gel mobility-shift assays were performed with D4 nuclear extracts. Analysis of protein-DNA interactions occurring within the CAAT- and PEBP2/CBF-containing fragment revealed two specifically shifted bands (Fig. 8 ). The shifted bands were effectively competed by a 200-fold molar excess of nonradiolabeled template but not by a noncompetitor DNA sequence. EMSA also was performed with 32P-labeled 87-bp probes containing dinucleotide mutations in the CAAT box, in the PEBP2/CBF site, and in the CAAT and PEBP2/CBF sites. These three probes did not bind any specific nuclear proteins from the promyelocytic cells, providing further support for the importance of these two sites in regulating NP-3 promoter activity (data not shown).



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  		Figure 8. Specific binding to ratNP-3 promoter sequences determined by EMSA. The proximal 87-bp region of the ratNP-3 promoter was labeled with 32P-dCTP by a fill-in reaction and incubated in the absence of nuclear protein (lane 1) or in the presence of 10 µg D4 nuclear extract (lane 2). Lanes 3 and 4 show the absence of binding in the presence of a 50 and 200 molar excess of unlabeled competitor DNA (87-bp fragment). Lanes 5 and 6 indicate binding in the presence of a 50 and 200 molar excess of unlabeled, noncompetitor DNA (rat cryptdin-1 partial cDNA). Shorter exposure times reveal two distinct complexes. Here, the shifted complexes are labeled A and B.

To determine whether C/EBP-{alpha} and/or PEBP2/CBF are involved in the specific protein-DNA interactions observed, gel mobility-shift assays with an excess of cold C/EBP-{alpha} and PEBP2/CBF consensus oligonucleotides were performed (Fig. 9 ). The addition of the PEBP2/CBF oligonucleotide caused a decrease in the amount of shifted species (A and B), but the addition of the C/EBP-{alpha} oligonucleotide did not cause any change in the amount of shifted species observed with the proximal 87-bp fragment. Antiserum (AML1B) raised against one of the human homologues of PEBP2 was used for super-shift studies. AML1B antiserum binds to PEBP2 and proteins that bind to the PEBP2 consensus sequence 5'-PuACCPuCA-3' [36 ]. The inclusion of AML1B antiserum caused the appearance of two specifically super-shifted bands (Fig. 10 ). This suggests that PEBP2 or a PEBP2-related protein binds to this region.



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  		Figure 9. Characterization of specific binding to the ratNP-3 promoter. Lanes 1 and 2 are the radiolabeled, proximal, 87-bp region of the ratNP-3 promoter in the absence and presence of 10 µg D4 nuclear extract. Lanes 3 and 4 indicate binding in the presence of 100 ng double-stranded oligonucleotides, corresponding to consensus C/EBP-{alpha}- or PEBP2/CBF-binding sites. The shifted complexes are labeled A and B.



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  		Figure 10. PEBP2 or a PEBP2-related protein binds a ratNP-3-promoter sequence. The radiolabeled, proximal, 87-bp region of the ratNP-3 promoter was incubated in the absence of nuclear protein (lane 1) or in the presence of 10 µg D4 nuclear extract (lane 2). Lanes 3 and 4 demonstrate super-shifts performed with 1 µl AML1B (PEBP2) antiserum in the absence and presence of 100 ng PEBP2 consensus oligonucleotide. *, Super-shifted bands.

Comparison of human neutrophil defensin (HNP) and rat neutrophil defensin promoters
A relatively small region of the HNP (HNP-1 and HNP-3) promoters controls transcriptional activity [26 , 27 ]. Putative cis-elements such as C/EBP, c-myb, and PU.1 are situated in human and rat promoter sequences in similar positions (Fig. 11 ). Although a functional PEBP2 site at -64 to -58 exists in the ratNP-3 promoter, the human counterpart, AML, does not play a role in HNP transcriptional activity in HL-60 cells. Additional Ets sites are also found along the HNP promoter but not in the ratNP-3 promoter.



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  		Figure 11. Schematic representation of putative cis-elements located on the HNP and ratNP-3 promoters. A 200-bp region identical between HNP-1 and HNP-3 promoters sufficient for transcriptional activity is shown for HNP aligned with putative elements identified in the ratNP-3 promoter. The transcription start site is numbered +1, and the TATA box is shown. Colored boxes indicate the relative position of cis-elements identified by computer analysis.


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DISCUSSION
 
Previously, Yount et al. [22 ] demonstrated that rat neutrophil {alpha}-defensin transcripts are most abundant in bone marrow promyelocytes, consistent with studies on guinea pig granulocytes [40 , 41 ]. To further investigate the regulation of myeloid {alpha}-defensin gene expression, we initially analyzed {alpha}-defensin expression in IPC-81, a rat promyelocytic cell line, to assess its usefulness in such studies. Compared with rat bone marrow cells, IPC-81 cells expressed surprisingly low levels of steady-state {alpha}-defensin mRNA. Analysis of {alpha}-defensin expression in IPC-81 cells by in situ hybridization and immunocytochemistry revealed a high degree of heterogeneity in the population. Furthermore, {alpha}-defensin mRNA levels in IPC-81 cells, assessed by Northern blots, were unaltered by treatment with all-trans-retinoic acid (ATRA), phorbol 12-myristate 13-acetate, granulocyte-colony stimulating factor (G-CSF), and interleukin-1 (data not shown). To circumvent the experimental limitations of this heterogeneous cell line, we sought to isolate from IPC-81 cells a more uniform cell phenotype by dilutional subcloning. Expansion of subclone D4 provided a useful cell line for studies on defensin gene regulation reported here.

Compared with the IPC-81 parental line, D4 cells were much more homogeneous in their morphology, had elevated mean levels of {alpha}-defensin expression, and consisted of a higher percentage of defensin-positive cells. The level of {alpha}-defensin mRNA and the fraction of immunopositive cells in D4 cells, assessed with anti-ratNP-1 and anti-ratNP-3 antibodies, have remained stable for more than 3 years. Although NP-3 promoter activity in transiently transfected IPC-81 cells was quite low, a finding similar to that reported for human defensin HNP-1 promoter activity in HL-60 cells [26 ], transiently transfected D4 cells supported nearly fivefold higher levels of promoter activity compared with the parental line. In addition, {alpha}-defensin expression in D4 cells but not IPC-81 cells was responsive to 30 ng/ml granulocyte macrophage (GM)-CSF (decreased NP-1–4 mRNA) and 10 µM ATRA (increased NP-1–4 mRNA; data not shown).

The ratNP-3 promoter is contained within the proximal 100 bp of the 5'-flanking region, similar to other myeloid-specific genes such as the M-CSF, G-CSF, and GM-CSF receptors, and is similar in several respects to the HNP-1 promoter. Like NP-3, a short sequence (-83 to +82) is sufficient to drive HNP-1 transcription in HL-60 cells [26 ]. The proximal 87 bp of the ratNP-3 promoter shares 61% identity with the corresponding region of the HNP-1 promoter, and the HNP-1 5'-flanking region contains several CAAT sequences and putative PEBP2/CBF sites. In addition, the HNP-1 promoter contains negative regulatory elements in the -416/-191 region similar to the -487/-87 region identified in the ratNP-3 promoter. The -487/-87 region in the NP-3 promoter contains a weak repressor, as indicated by the fourfold increase in luciferase activity with deletion of this region. Thus, human and rat neutrophil {alpha}-defensin expression appears to be dependant on a small region of a myeloid-specific promoter, although additional elements may be necessary for position-independent, high-level expression in a chromosomal locus [38 ].

The ratNP-3 promoter is contained within the proximal 100 bp of the 5'-flanking region, similar to other myeloid-specific genes such as the M-CSF, G-CSF, and GM-CSF receptors, and is similar in several respects to the HNP-1 and HNP-3 promoters. Like NP-3, a short sequence is sufficient to drive HNP transcription in HL-60 cells [26 , 27 ]. The proximal 87 bp of the ratNP-3 promoter shares 61% identity with the corresponding region of the HNP promoter, and the HNP 5'-flanking region contains several CAAT sequences and putative PEBP2/CBF sites. In addition, the HNP-1 promoter contains negative regulatory elements in the -416/-191 region similar to the -487/-87 region identified in the ratNP-3 promoter. Negative regulatory element(s) within the region of -240 to -133 have been identified in the HNP-3 promoter sequence [27 ]. The -487/-87 region in the NP-3 promoter contains a weak repressor, as indicated by the fourfold increase in luciferase activity with deletion of this region. Thus, human and rat neutrophil {alpha}-defensin expression appears to be dependent on a small region of a myeloid-specific promoter, although additional elements may be necessary for position-independent, high-level expression in a chromosomal locus [38 ].

The HNP and NP-3 promoters also differ in certain respects. In contrast to the NP-3 promoter, two functional ETS-like sequences (GGAA core sequence) regulate HNP-1 promoter activity [26 ]. PU.1 has been shown to be essential for regulation of myeloid-specific genes such as GM-CSF and M-CSF receptor and neutrophil elastase [33 34 35 ]. In the HNP-1 promoter, the proximal ETS-like element at -22/-19 binds the PU.1 transcription factor in vitro, and the distal site at -62/-59 interacts in vitro with an unidentified factor, distinct from PU.1, ETS-1, polyoma enhancer activator 3, and ELK-1 [26 ]. In contrast, the PU.1 site at -28/-18 of the ratNP-3 5'-flanking sequence was not essential to promoter activity.

Transfection of D4 cells with various promoter constructs demonstrated that the proximal 87 bp of the NP-3 promoter contains sequences required for optimal promoter activity. Mutational analyses suggested that the CAAT box at -72/-76 and a PEBP2/CBF site at -58/-64 regulate neutrophil defensin transcription. Data from EMSA indicated that PEBP2/CBF or a PEBP2/CBF-related protein in D4 extracts bounds to NP-3 promoter sequences. These data are in accord with results from a previous study [42 ], which demonstrated the binding of overexpressed AML1B (human homologue of PEBP2/CBF) to the NP-3 promoter.

Although C/EBP-{alpha} did not transactivate NP-3(87), electrophoretic mobility shift studies by Westendorf et al. [42 ] have demonstrated that AML1B and C/EBP-{alpha} (overexpressed in COS-7 cells) bind to the proximal 87-bp region. However, C/EBP-{alpha} did not bind this region of the promoter in D4 cells. Further, D4 cells were immunopositive (data not shown) for C/EBP-{alpha}, indicating the presence of C/EBP-{alpha} in D4 cells. The failure to detect binding of C/EBP-{alpha} may have been a result of inhibitors in nuclear extracts, mutations in C/EBP-{alpha} in D4 cells, or proteins other than C/EBP-{alpha} that bind the CAAT box.

Genes expressed specifically in immature myeloid cells have been found to be regulated by four factors: PEBP2/CBF, C/EBP, PU.1, and c-myb. We speculate that for rat neutrophil {alpha}-defensin NP-3 expression, factors binding to the CAAT box interact with PEBP2/CBF to activate transcription. Further studies are required to determine whether other factors such as CAAT-binding proteins and a possible repressor element in the region between -487 and -87 may also be involved in defensin gene regulation. The differential responses of D4 cells and defensin-deficient D5 cells to differentiating agents may also provide additional insights into defensin gene regulation.


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ACKNOWLEDGEMENTS
 
This work was supported in part by NIH awards AI22931 and GM07311 and a grant from Large Scale Biology (Vacaville, CA). We thank Drs. Helen Ross, Timothy Osborne, and Jennifer Westendorf for valuable suggestions on experimental protocols, Heather J. Inoue for excellent technical assistance, and Karen Reiser for assistance in preparation of the manuscript.

Received August 17, 2003; revised September 30, 2003; accepted October 1, 2003.


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