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(Journal of Leukocyte Biology. 2002;71:154-162.)
© 2002 by Society for Leukocyte Biology

NF-{kappa}B-mediated transcriptional regulation of human ß-defensin-2 gene following lipopolysaccharide stimulation

Yuko Tsutsumi-Ishii and Isao Nagaoka

Department of Biochemistry, Juntendo University, School of Medicine, Tokyo, Japan

Correspondence: Isao Nagaoka, M.D., Ph.D., Department of Biochemistry, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 Japan. E-mail: nagaokai{at}med.juntendo.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ß-Defensins are cationic peptides with broad-spectrum antimicrobial activities that contribute to innate host defense. Among human ß-defensins (hBDs), hBD-2 is up-regulated in epithelial tissues and mononuclear phagocytes in response to bacterial infection and proinflammatory cytokines. However, little is known about the molecular mechanism of hBD-2 gene regulation. Here, we investigated lipopolysaccharide (LPS)-mediated transcriptional regulation of the hBD-2 gene by focusing on the roles of NF-{kappa}B, STAT, and NF-IL-6 sites in mononuclear phagocytes using RAW264.7 cells, which are sensitive to LPS. Luciferase reporter analyses demonstrated that two NF-{kappa}B sites were essential for full LPS responsiveness of the hBD-2 gene. Further, both NF-{kappa}B sites were also crucial for basal transcriptional activity. In contrast, neither the NF-IL-6 nor STAT binding site was required for LPS-induced hBD-2 transcription. Electrophoretic mobility shift assay indicated that in unstimulated cells, NF-{kappa}B p50 homodimer bound to both NF-{kappa}B sites, whereas the p65-p50 heterodimer formed complexes with these sites following LPS stimulation. Together, these observations indicate that NF-{kappa}B plays an important role in the regulation of hBD-2 gene expression in response to LPS.

Key Words: antimicrobial peptide • epithelia • macrophage • NF-IL-6 • innate immunity • host defense


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Defensins, a major family of cationic antimicrobial peptides, are widely distributed among all living species and act as key components of the innate immune system [1 2 3 ]. They exhibit a broad spectrum of microbicidal activities against bacteria, fungi, parasites, and viruses. Mammalian defensins are divided into three subfamilies: {alpha}- and ß-defensins differ in the placement and connectivity of their six conserved cysteine residues, and the third {theta}-defensin, isolated from macaque leukocytes, exhibits a unique circular structure [1 2 3 4 ].

In humans, the {alpha}-defensins are mainly expressed in neutrophils [human neutrophil peptides (HNP)-1 to -4] and in epithelial cells, including those of the small intestine (Paneth cells) and reproductive tract [human defensins (HD)5 and 6] [5 6 7 ]. So far, three ß-defensins [human ß-defensins (hBD)-1 to -3] have been identified and characterized [8 9 10 11 12 ]. hBDs are primarily expressed in various epithelial tissues including skin, lung, and intestine [6 , 11 12 13 14 ]. Further, recent studies have demonstrated that hBDs are also expressed in mononuclear phagocytes such as monocytes and alveolar macrophages [15 , 16 ]. In addition to their striking microbicidal properties, hBD-1 and -2 have chemotactic activity for immature dendritic cells and memory T cells, suggesting that they serve as links between innate and adoptive immunity [17 ]. Recently, we found that hBD-2 promotes histamine release from and prostaglandin D2 (PGD2) production in mast cells, suggesting the involvement of hBD-2 in allergic reactions [18 ].

It is interesting that hBDs show a distinct expression pattern: hBD-1 is constitutively expressed in epithelia [14 , 19 , 20 ], whereas hBD-2 and -3 are induced in response to bacterial infection and proinflammatory cytokines such as tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin 1ß (IL-1ß) [9 , 11 , 12 , 16 , 21 , 22 ]. Their presence as constitutive or inducible components in the epithelial barrier indicates that they play a crucial role in antimicrobial activity in those body surfaces that frequently encounter bacteria. In particular, the evidence that hBD-2 is up-regulated in response to inflammatory stimuli implies an essential role in sites of inflammation.

Over the past few years, a number of ß-defensins have been identified in various animals and found to exhibit constitutive and inducible expression [3 , 6 ]. Among these ß-defensins, bovine tracheal antimicrobial peptide (TAP) has been shown to be up-regulated by lipopolysaccharide (LPS) through a CD14-dependent signaling pathway and the transcription of the TAP gene to be cooperatively regulated by transcription factors such as nuclear factor-{kappa}B (NF-{kappa}B) and nuclear factor for IL-6 expression (NF-IL-6; also referred to as C/EBPß) [23 , 24 ]. As with the TAP gene, the 5' flanking region of the hBD-2 gene has also been shown to include several consensus sequences for NF-{kappa}B and NF-IL-6, suggesting that the cooperative activities of such transcription factors may induce hBD-2 expression [6 , 21 ]. However, the regulatory mechanisms of hBD-2 expression remain unexplored. It is noteworthy that monocytes/macrophages are able to express hBD-2 mRNA in response to low doses of LPS compared with epithelial cells [16 ], because monocytes/macrophages more abundantly express LPS receptors such as CD14 and Toll-like receptors (TLRs) [25 26 27 ]. Thus, it is intriguing to consider how NF-{kappa}B and other transcription factors participate in LPS-mediated hBD-2 gene regulation in mononuclear phagocytes. Detailed analysis of the hBD-2 gene should provide important insights into the molecular mechanisms regulating ß-defensin family expression.

In the present study, we investigated the regulatory mechanisms of LPS-dependent hBD-2 expression in mononuclear phagocytes using a RAW264.7 macrophage cell line. Here, we present data indicating that two tandemly arrayed NF-{kappa}B elements at -577 and -188 are essential to the LPS responsiveness of the hBD-2 gene in mononuclear phagocytes. In contrast, no NF-IL-6 or signal transducer and activator of transcription (STAT) binding sequences within the hBD-2 promoter contributed to LPS-induced hBD-2 transcription. We further demonstrated that the p65-p50 heterodimer and p50 homodimer of NF-{kappa}B can bind to these NF-{kappa}B sequences.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and reagents
Murine macrophage cell line RAW264.7 was obtained from American Type Culture Collection (ATCC; Manassas, VA). Cells were maintained in RPMI-1640 medium (Nissui Pharmaceutical, Tokyo, Japan) with 10% fetal calf serum (FCS; Sanko Junyaku, Tokyo, Japan), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Sigma Chemical Co., St. Louis, MO) at 37°C in 5% CO2. FCS contains <5 pg/100 ml LPS, as certified by the manufacturer. RAW264.7 cells were stimulated with 100 ng/ml LPS (from Escherichia coli O55:B5; purchased from Sigma Chemical Co.) at 37°C for 2.5 h (for preparation of nuclear extracts) or 5 h (for luciferase assay). In some experiments, cells were also stimulated with 100 U/ml recombinant murine interferon {gamma} (IFN-{gamma}; PBL Biomedical Laboratories, New Brunswick, NJ) or 20 ng/ml Chinese hamster ovary (CHO) cell-derived recombinant human IL-6, which was shown to be active in a mouse system (Genzyme, Cambridge, MA), in the absence or presence of 100 ng/ml LPS at 37°C for 5 h and subjected to luciferase assay.

Isolation of the 5' flanking region of the hBD-2 gene
The human hBD-2 promoter sequence between -2274 and +50 was amplified from PstI-digested genomic DNA by polymerase chain reaction (PCR) using oligonucleotide primers -2274-NheI sense (5'-TCGCTAGCCGGACAAGTTTAGCTCCAATGC-3') and +50-XhoI antisense (5'-TTCTCGAGTACAAGACCCTCATGGCTGA-3'). Both primers were designed based on a published sequence [10 ], and additional restriction sites are indicated by underlines (Fig. 1 ). The PCR reaction was performed on a thermal cycler Model 480 (Perkin Elmer, Norwalk, CT) using 30 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C after incubation at 94°C for 2 min. The final polymerization step was extended by an additional 5 min at 72°C. The synthesized 2.3-kbp fragments were cloned to a TA-cloning vector pT7Blue (Novagen, Madison, WI). Plasmid inserts were confirmed by sequencing using a Thermo SequenaseTM II dye terminator cycle sequencing premix kit (Amersham Pharmacia Biotech, Uppsala, Sweden) and halfTERMTM XL dye terminator sequencing reagent (Genpak, Hampshire, UK).



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  		Figure 1. Genomic structure of the 5' flanking region of the hBD-2 gene. A 2.3-kbp 5' flanking region of the hBD-2 gene was amplified by PCR using -2274-NheI sense and +50-XhoI antisense primers based on the published sequence (GenBank accession no. AF040153). Amplified PCR product was subcloned into the luciferase vector pGL3-Basic. Positions of restriction sites and 5' deletion constructs are relative to the transcription initiation site (+1). Arrows indicate the sense and antisense primers used for generation of 5' deletion or mutant constructs. Crosses represent the mutated sequences in the primers. Boxes and ovals indicate the location of putative cis elements in the hBD-2 promoter: black boxes, NF-{kappa}B sites; gray boxes, STAT sites; gray ovals, NF-IL-6 sites; open ovals, AP-1 family sites; and open boxes, TATA and CCAAT boxes. Hatched box shows exon 1 of the hBD-2 gene. NF-{kappa}B binding sites are termed p{kappa}B1 (position -188), p{kappa}B2 (-577), and d{kappa}B (-2187), respectively. Of note, NF-{kappa}B binding motifs are tandemly arrayed in p{kappa}B1 and p{kappa}B2 sites.

 
Plasmid construction
The 2.3-kbp fragments of the hBD-2 promoter with the NheI and XhoI sites were subcloned into the promoterless firefly luciferase vector pGL3-Basic (Promega, Madison, WI), and the resulting hBD2/Luc plasmid was used as a template to generate 5' deletion and mutant promoter constructs. A series of 5' deletion fragments were prepared by digestion with appropriate restriction enzymes (SmaI at -1050, SacI at -412, BglII at -167, and EcoRI at -106) or by PCR using appropriate primers (Fig. 1) . For PCR amplification, the following sense primers containing the restriction site and the +50-XhoI antisense primer were used: -2187 sense (d{kappa}Bmut sense), 5'-ACTTTACGCGTTCCCCAG-3' (-2197 to -2180); -1325 sense, 5'-CCACGCGTCTTGGCTGCACAATATC-3' (-1325 to -1308); -1236 sense, 5'-TAGCTACGTGAAGAGGTCAGGCAGGTCA-3' (-1236 to -1217); -577 sense, 5'-AGACGCGTT TTCACATAAATTTCACCAG-3' (-577 to -558); -208 sense, 5'-GGAAGGGATTTTCTGGGGTTTCCTGA-3' (-208 to -183); and -188 sense, 5'-GAAGGGATTTTCTGGATCCTCCTGAG-3' (-207 to -182). Underlines show the restriction sites for introduction into the vector, corresponding to MluI, NheI, or KpnI. Reporter plasmid constructs containing the 5' deletion fragments were named {Delta}-2187, {Delta}-1325, {Delta}-1236, {Delta}-1050, {Delta}-577, {Delta}-412, {Delta}-208, {Delta}-188, {Delta}-167, and {Delta}-106, respectively (Fig. 2 ).



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  		Figure 2. Effect of 5' deletions of the hBD-2 promoter on basal and LPS-induced luciferase activities. A series of 5' deletion constructs of the hBD-2 promoter were generated by digestion with restriction enzymes or by PCR and introduced to the promoterless vector pGL3-Basic. Symbols represent positions of putative transcription factor binding elements: black boxes, NF-{kappa}B sites; gray boxes, STAT sites; gray ovals, NF-IL-6 sites; open ovals, AP-1 family sites; and open boxes, TATA and CCAAT boxes. Hatched box shows exon 1 of the hBD-2 gene. The transcription start site is numbered +1. Each 5' deletion construct was transfected into the murine macrophage RAW264.7 cell line and incubated for 24 h without LPS stimulation; then, luciferase activities were determined. Basal promoter activity of deletion constructs is expressed as a percentage of luciferase activity for the hBD2/Luc plasmid. For measurement of LPS response, transfected cells were incubated for 19 h in the absence of LPS and then stimulated with 100 ng/ml LPS for 5 h. Luciferase activities are expressed as multiple of induction in LPS-stimulated samples [LPS(+)] relative to unstimulated control (control) for each reporter construct. Values are mean ± SD of at least five independent experiments.

 
Four promoter fragments containing mutant NF-{kappa}B sequences were generated by two-step PCR amplification using mutant NF-{kappa}B primers (Fig. 1) . First, PCR reaction was performed using the -2274-NheI sense primer and a mutant NF-{kappa}B antisense primer (d{kappa}Bmut anti, p{kappa}B2mut anti, or p{kappa}B1mut anti), and a mutant NF-{kappa}B sense primer (d{kappa}Bmut sense, p{kappa}B2mut sense, or p{kappa}B1mut sense) and the +50-XhoI antisense primer. Second, PCR was carried out with the -2274-NheI sense and +50-XhoI antisense primers using first PCR products containing mutated NF-{kappa}B sequences as a template. The amplified PCR products were digested with restriction enzymes, ligated into the pGL3-Basic, and designated as d{kappa}Bmut/Luc, p{kappa}B2mut/Luc, and p{kappa}B1mut/Luc, respectively. For generation of the p{kappa}B1 + 2mut/Luc construct, a 1.8-kbp SacI fragment containing the wild-type p{kappa}B2 sequence was excised from the p{kappa}B1mut/Luc and replaced by another 1.8-kbp SacI fragment containing the mutated p{kappa}B2 sequence derived from the p{kappa}B2mut/Luc (Fig. 3 ). Sequences of primers used for mutagenesis were as follows: d{kappa}Bmut sense (-2187 sense; the same primer used for generation of the 5' deletion fragment), 5'-ACTTTacgcgTTCCCCAG-3' (-2197 to -2180); p{kappa}B2mut sense, 5'-TGctagcTTTCAGacgcgtTTTCACATAAATTCACCAG-3' (-596 to -558); p{kappa}B1mut sense, 5'-GGAAGGacgcgTCTGGtaccTCCTGA-3' (-208 to -183); d{kappa}Bmut anti, 5'-GCTGGGGAAcgcgtAAAGT-3' (-2179 to -2197); p{kappa}B2mut anti, 5'-AAacgcgtCTGAAAgctagCATCTTCCCCAGGGAGAG-3' (-576 to -613); and p{kappa}B1mut anti, 5'-CTCAGGAggtaCCAGAcgcgtCCTTC-3' (-182 to -207). Lowercase letters indicate base substitutions, and underlines indicate restriction sites corresponding to MluI, NheI, or KpnI. All deletion and mutant constructs were sequenced to confirm fidelity.



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  		Figure 3. Mutation analysis of NF-{kappa}B binding sites in the hBD-2 promoter. (A) Schematic representation of the hBD-2 promoter depicting NF-{kappa}B binding sites. Transcription start site is numbered +1. Consensus sequences for NF-{kappa}B are indicated by brackets, and individual NF-{kappa}B sites are at positions -2193/-2182 (d{kappa}B), -596/-583 and -585/-572 (p{kappa}B2), and -205/-194 and -195/-186 (p{kappa}B1). Restriction sites introduced for site-directed mutagenesis are underlined, and mutated bases are indicated by dots above the wild-type sequences. (B) Wild-type or mutant construct was transiently transfected into RAW264.7 cells, and cells were incubated for 24 h without LPS stimulation. Basal promoter activity of mutant NF-{kappa}B constructs is expressed as the percentage of luciferase activity for the wild-type hBD2/Luc. For measurement of LPS response, transfected cells were incubated for 19 h in the absence of LPS and then stimulated with 100 ng/ml LPS for 5 h. Luciferase activities are expressed as multiple of induction in LPS-stimulated samples [LPS(+)] relative to unstimulated control (control) for each reporter construct. Values are mean ± SD of at least five independent experiments.

 
Transfection and luciferase assay
RAW264.7 cells were seeded into 35-mm plates 1 day before transfection at a concentration of 6 x 105 cells/plate. Cells were transiently transfected with 1 µg appropriate reporter constructs and 12.5 ng Renilla luciferase expression vector pRL-SV40 (Promega) using FuGENETM 6 (Roche Diagnostics, Indianapolis, IN), according to the protocol provided by the company. Following transfection, cells were cultured for 19 h and exposed to 100 ng/ml LPS for 5 h in the presence of FCS.

After incubation, cells were harvested, washed, and lysed in 200 µl PicaGene® Dual cell culture lysis reagent (Toyo Ink, Tokyo, Japan). Firefly and Renilla luciferase activities were measured using a PicaGene® Dual SeaPansyTM luminescence kit (Toyo Ink) and Lumat LB9501 luminometer (Berthold Japan, Tokyo, Japan). Protein concentration of cell extracts was determined with a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). Promoter activities were expressed as relative light units (RLU) normalized to Renilla luciferase activity.

Preparation of nuclear extracts
Nuclear extracts were prepared from LPS-unstimulated or -stimulated RAW264.7 cells by minor modification of the procedure of Dignam et al. [28 ]. Briefly, 1 x 108 cells were washed twice in ice-cold phosphate-buffered saline (PBS) and harvested using a rubber policeman. Cells were lysed in 5 ml lysis buffer [10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-KOH, pH 7.9, 10 mM KCl, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 1.5 mM MgCl2, 0.5% Nonidet P-40 (NP-40), 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, and 10 µg/ml pepstatin] on ice for 10 min. Nuclei were washed once in the same buffer except for the exclusion of NP-40. Nuclei pellets were resuspended in 400 µl extraction buffer (10 mM HEPES, pH 7.9, 420 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 25% glycerol, 1 mM DTT, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml pepstatin). After incubation at 4°C for 20 min with gentle rocking, the nuclei were removed by centrifugation at 12,000 g for 20 min at 4°C. The resultant supernatants were collected and stored at -80°C until use for gel retardation assay. Protein concentration in nuclear extracts was measured with a BCA protein assay kit.

Electrophoretic mobility shift assay (EMSA)
Nuclear extracts (4 µg) were mixed with 32P-labeled probe (described in the next section; 5 x 104 cpm, 10–20 fmole) in 15 µl binding buffer containing 20 mM HEPES (pH 7.9), 100 mM NaCl, 1 mM EDTA, 6% glycerol, 1 mM DTT, 1 mM PMSF, 0.25 mg/ml bovine serum albumin, and 2 µg poly(dI-dC) · poly(dI-dC) (Amersham Pharmacia Biotech) for 20 min at room temperature. The reaction mixtures were applied to a native 6% polyacrylamide gel in 0.25 x TBE (22.3 mM Tris, 22.3 mM boric acid, and 0.5 mM EDTA, pH 8.3) at 130 V for 70 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 30-fold molar excess of unlabeled oligonucleotides was preincubated in the reaction mixture for 15 min at room temperature. For antibody supershift experiments, 1 µg polyclonal antibody against NF-{kappa}B p65 (sc-109X), NF-{kappa}B p50 (sc-114X), C/EBPß (sc-150X), or normal rabbit immunoglobulin G (IgG) was added to the reaction mixture 20 min prior to probe addition. All specific antibodies (1 mg/ml TransCruzTM gel supershift reagents) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Oligonucleotides for EMSA
Synthetic oligonucleotides used for EMSA were designed to generate a single 5'-G overhang at each end after annealing with their compliments. Double-stranded oligonucleotides were labeled by filling in the cohesive ends with [{alpha}-32P]dCTP (ICN Biomedicals, Costa Mesa, CA) using a Klenow fragment. Sequences of the double-stranded oligonucleotides were as follows: p{kappa}B1 oligo, 5'-gGAAGGGATTTTCTGGGGTTTCCTGAc-3' (-208 to -183); p{kappa}B1 mutant oligo, 5'-gGAAGACGCGTTCTGGTACCTCCTGAc-3'; p{kappa}B2 oligo, 5'-GAGATGGGGAGTTTCAGGGGAACTTTCACAc-3' (-599 to -571); p{kappa}B2 mutant oligo, 5'-GAGATGCTAGCTTTCAGACGCGTTTTCACAc-3'; NF-{kappa}B consensus oligo, 5'-gGTTGAGGGGACTTTCCCAGGc-3'; and C/EBP consensus oligo, 5'-gTGCAGATTGCGCAATCTGCAc-3'. These consensus oligonucleotides were synthesized based on the sequences of TransCruzTM gel shift oligonucleotides (Santa Cruz Biotechnology). Mutated sequences are indicated by underlines, and additional nucleotides for fill-in reaction are indicated by lowercase letters.

Computer-based analysis
Putative transcription binding sites within the hBD-2, hBD-3, and murine ß-defensin (mBD-2, mBD-3, and mBD-4) promoters were sought with the MatInspector program version 2.2 [29 , 30 ]. The 5' flanking sequences of these genes were aligned using Divide-and-Conquer multiple sequence alignment and the DiAlign 2 computer programs [31 32 33 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation and analysis of the 5' flanking region of the hBD-2 gene
To analyze the promoter region of the hBD-2 gene, we amplified a 2.3-kbp 5' flanking sequence of hBD-2 by PCR and then searched for putative cis elements within the sequence from -2274 to +50 of the hBD-2 gene using MatInspector [29 , 30 ]. As shown in Figure 1 , the hBD-2 promoter contains a TATA box at -29 and a CCAAT box at -76 from the transcription start site. Multiple potential binding motifs for the activator protein 1 (AP-1) family, NF-{kappa}B, NF-IL-6, and STAT, which are involved in the expression of inflammatory responsive genes, were spread over the entire sequence. It is interesting that NF-{kappa}B binding sequences located at -188 and -577 were repeated tandemly [termed proximal {kappa}B1 and {kappa}B2 (p{kappa}B1 and p{kappa}B2, respectively)], although only one NF-{kappa}B sequence was found at -2187 [distal {kappa}B (d{kappa}B)]. Moreover, binding sites for NF-IL-6 were adjacent to the respective NF-{kappa}B site (p{kappa}B1, p{kappa}B2, and d{kappa}B).

To elucidate which element(s) of the hBD-2 promoter is functionally important for LPS-induced transcription in mononuclear phagocytes, a series of 5' truncated fragments of this promoter were introduced to a promoterless luciferase vector, pGL3-Basic. We then transiently transfected these constructs into the RAW264.7 macrophage-like cell line.

As shown in Figure 2 , the transcriptional activity of the 2.3-kbp fragment of the hBD-2 promoter (hBD2/Luc) was enhanced approximately tenfold in response to 100 ng/ml E. coli LPS. Deletion of the d{kappa}B sequence ({Delta}-2187/Luc) did not alter this activity in response to LPS. Deletion from -2187 to -1050 ({Delta}-1325/Luc, {Delta}-1236/Luc, and {Delta}-1050/Luc), which caused the loss of one NF-IL-6 and three STAT sites, showed a moderate decrease in basal activity, although LPS inducibility was retained. However, deletion of tandemly repeated NF-{kappa}B motifs in the p{kappa}B2 site at -577 ({Delta}-577/Luc) decreased basal activity by approximately 90% and LPS responsiveness by 50%. Further deletion between -208 and -188 ({Delta}-208Luc and {Delta}-188/Luc), which resulted in the loss of the tandem repeat of NF-{kappa}B sequences in the p{kappa}B1 site, abolished basal activity and LPS inducibility entirely.

As mentioned above, several binding motifs for potent transcription factor NF-IL-6 were located adjacent to each NF-{kappa}B site, and the AP-1 family binding site lay 3' to the p{kappa}B1 site (Fig. 1) . Therefore, we investigated the requirement of these sites for LPS-induced transcription of the hBD-2 gene using deletion constructs. Surprisingly, LPS responsiveness of the hBD-2 promoter was affected little by deletion of the NF-IL-6-binding sites adjoining individual NF-{kappa}B sites, as shown by comparison of hBD2/Luc versus {Delta}-2187/Luc, {Delta}-577/Luc versus {Delta}-412/Luc, {Delta}-412/Luc versus {Delta}-208/Luc, and {Delta}-188/Luc versus {Delta}-167/Luc (Fig. 2) . Further, no significant differences in promoter activity were found by deletion from -167 to -106, which resulted in the loss of an AP-1 family binding site ({Delta}-167/Luc vs. {Delta}-106/Luc). Consistent with the above data, IL-6 and IFN-{gamma}, which activate NF-IL-6, STAT3, and STAT1 [34 , 35 ], were unable to increase hBD-2 promoter activity in RAW 264.7 cells transfected with the hBD2/Luc plasmid, and no synergistic effects of LPS with IFN-{gamma} or IL-6 on hBD-2 promoter activity were observed (unpublished results). These results indicate that the NF-IL-6, STAT, or AP-1 family-binding element is not essential to the transcriptional regulation of the hBD-2 gene and that the two promoter regions spanning -1050 to -577 and -208 to -188, which contain the p{kappa}B2 and p{kappa}B1 sites, respectively, confer the basal and LPS-induced transcriptional activity of the hBD-2 gene in mononuclear phagocytes.

Roles of NF-{kappa}B consensus sequences in hBD-2 gene expression
Our 5' deletional analysis suggested that the two tandemly repeated NF-{kappa}B sites (p{kappa}B2, from -596 to -572, and p{kappa}B1, from -205 to -186), but not the d{kappa}B site from -2193 to -2182, are crucial to LPS-induced hBD-2 transcription. To further confirm the role of each NF-{kappa}B binding site in the regulation of hBD-2 transcription, we generated four mutant hBD-2 promoter constructs containing mutated NF-{kappa}B sequences and termed them d{kappa}Bmut/Luc (mutated positions from -2192 to -2188), p{kappa}B2mut/Luc (mutations from -594 to -590 and -583 to -578), p{kappa}B1mut/Luc (mutations from -203 to -199 and -192 to -189), and p{kappa}B1 + 2mut/Luc (mutations with the p{kappa}B1 and p{kappa}B2 sites; Fig. 3A ).

Consistent with the data using a deletion construct ({Delta}-2187/Luc; Fig. 2 ), mutation of the d{kappa}B site spanning -2193 to -2182 did not affect basal or LPS-induced promoter activity (Fig. 3B) . In contrast, mutation of the p{kappa}B2 (-596 to -572) or p{kappa}B1 (-205 to -186) site nearly abrogated basal transcription activity, and each p{kappa}B mutant reduced LPS-induced transcription. Reduction was not complete, however, and each mutant construct containing the intact p{kappa}B1 or p{kappa}B2 site still retained LPS responsiveness. It is interesting that mutation of both p{kappa}B sites (pkB1+2mut/Luc) abolished the response to LPS. Together, these results indicate that the p{kappa}B1 and p{kappa}B2 sites are essential for full transcription of the hBD-2 gene induced by LPS in RAW264.7, macrophage-like cells.

Binding NF-{kappa}B p65 and p50 subunits to the p{kappa}B1 and p{kappa}B2 sites from the hBD-2 promoter
We further analyzed whether NF-{kappa}B can interact with the hBD-2 promoter using EMSA by incubating the 32P-labeled oligonucleotides corresponding to the p{kappa}B1 and p{kappa}B2 sequences with nuclear extracts from RAW264.7 cells.

As shown in Figure 4 , specific binding (lower band) was faintly detectable in nuclear extracts from unstimulated cells with the p{kappa}B1 or p{kappa}B2 probe, and an additional specific complex (upper band) was markedly induced by stimulation with LPS. The specific DNA-protein complexes strongly competed with excess unlabeled oligonucleotides containing the p{kappa}B1 or p{kappa}B2 sequence, as well as the NF-{kappa}B consensus sequence. In contrast, the mutated NF-{kappa}B sequences and C/EBP consensus oligonucleotide did not inhibit the formation of these specific complexes.



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  		Figure 4. Analysis of NF-{kappa}B binding to proximal NF-{kappa}B sites in the hBD-2 promoter. EMSA of the p{kappa}B1 (A) and p{kappa}B2 (B) sequences using nuclear extracts from RAW264.7 cells. Top: Nuclear extracts (4 µg) from unstimulated control (Con) or LPS-stimulated (LPS) cells were incubated with 32P-labeled p{kappa}B1 or p{kappa}B2 oligonucleotide. A 30-fold molar excess of unlabeled competitors was added to the reaction mixture: p{kappa}B1 oligo ({kappa}B1), p{kappa}B2 oligo ({kappa}B2), mutated p{kappa}B1 or p{kappa}B2 oligo (m{kappa}B), NF-{kappa}B consensus oligonucleotide ({kappa}B), and C/EBPß (NF-IL-6) consensus oligonucleotide (EBP). Supershift assay was performed with 1 µg appropriate antibodies: anti-NF-{kappa}B p65 (p65), anti-NF-{kappa}B p50 (p50), anti-C/EBPß (EBPß), and normal rabbit IgG as control (IgG). Closed arrowheads indicate specific binding to the probe. Positions of supershifted complexes are denoted by open arrowheads. Bottom: Sequences of wild-type and mutant p{kappa}B oligonucleotides used in EMSA. The sequence positions corresponding to the wild-type oligonucleotides span -208 to -183 (p{kappa}B1) and -599 to -571 (p{kappa}B2). Tandemly repeated NF-{kappa}B motifs are indicated by brackets. Shaded boxes show mutated bases in the m{kappa}B oligonucleotides.

 
To identify which member(s) of the NF-{kappa}B family was involved in complexes with the p{kappa}B1 and p{kappa}B2 probes, nuclear extracts from LPS-stimulated RAW264.7 cells were pretreated with antibody against NF-{kappa}B p65 (p65) or p50 (p50) before the addition of probe DNAs. As shown in Figure 4A , addition of anti-p50 antibody supershifted the upper and lower complexes with the p{kappa}B1 probe, and anti-p65 antibody abolished only the upper complex. Likewise, two specific complexes with the p{kappa}B2 probe were supershifted by anti-p65 or anti-p50 antibody, as observed with the p{kappa}B1 probe (Fig. 4B) . Under our conditions, we could not detect a specific band containing the p65 homodimer, which was shifted by anti-p65 but not anti-p50 antibody. Moreover, neither anti-C/EBPß nor normal rabbit IgG had any effect on the binding activities of the p{kappa}B1 and p{kappa}B2 probes.

These results suggest that the lower band contains the p50 homodimer complex, and the upper band represents the p65-p50 heterodimer complex. Thus, it is possible that in unstimulated RAW264.7 cells, the p50 homodimer forms complexes mainly with p{kappa}B1 and p{kappa}B2 sequences, whereas p65-p50 heterodimer complexes with the p{kappa}B1 and p{kappa}B2 sequences are induced in LPS-stimulated cells.

Alignment of NF-{kappa}B motifs in the proximal promoter regions among the inducible ß-defensin family genes
To date, induction of hBD-2 and -3 and mBD-2 and -3 has been shown [9 , 11 , 12 , 36 , 37 ]. Therefore, we evaluated whether the two critical NF-{kappa}B elements within the hBD-2 promoter are conserved in other inducible ß-defensin promoters. Approximately 1000-bp 5' flanking sequences of these defensin genes were aligned using computer-based alignment programs (DCA and DiAlign 2) [31 32 33 ], and then the locations of regulatory elements were compared [29 , 30 ]. As shown in Figure 5 , the mBD-2 promoter possessed only one conserved NF-{kappa}B binding sequence corresponding to the 3'-p{kappa}B2 site in the hBD-2 promoter, whereas the mBD-3 promoter contained one conserved NF-{kappa}B sequence corresponding to the 3'-p{kappa}B1 site. The fourth gene, mBD-4, which is constitutively expressed, shares high homology with the mBD-3 promoter but lacks the consensus sequences for NF-{kappa}B (unpublished results). Thus, NF-{kappa}B likely plays a role in the induction of mBD-2 and -3 genes. Of interest, neither the p{kappa}B1 nor p{kappa}B2 site is conserved in the hBD-3 promoter (Fig. 5) , although hBD-3 mRNA can be up-regulated in response to TNF-{alpha} and IL-1ß [11 , 12 ]. We speculate that transcription of the hBD-3 gene is controlled by other transcription factors.



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  		Figure 5. Comparison of human- and murine-inducible ß-defensin promoter sequences. Sequences of the two functional promoter regions containing p{kappa}B1 and p{kappa}B2 sites in the hBD-2 gene are compared with those of the corresponding segments of hBD-3, mBD-2, and mBD-3 genes using the DCA and DiAlign 2 computer programs. The sequences containing 5' flanking regions of the hBD-3 gene (accession no. AF202031) and mBD-3 gene (accession no. AF093245) were obtained from the GenBank database using BLAST, and the sequence of mBD-2 gene (Ti no. 1747423) was obtained from NCBI Trace Archive using BLAST. Note that the putative start position (ATG in bold letters) of the open reading frame is numbered +1 in this figure. Nonidentical bases are indicated in lowercase letters, and gaps introduced to optimize the alignment are denoted by dots. Consensus sequences for NF-{kappa}B are enclosed by shaded boxes, whereas NF-IL-6 binding sequences are enclosed by open boxes. Lines above the hBD-2 sequences indicate the p{kappa}B1 and p{kappa}B2 sites that contain, in tandem, repeated NF-{kappa}B motifs (5'- and 3'-p{kappa}B).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The innate immune system is the first line of host defense against invading microorganisms. Antimicrobial peptides expressed in epithelial and phagocytic cells serve as active effector molecules of innate immunity [7 , 38 , 39 ]. In response to microbial infection, production of antimicrobial peptides such as hBD-2 and CAP18/LL-37 is rapidly induced, which in turn leads not only to microbicidal activity but also to recruitment of immune cells that play an important role in acquired immunity [17 , 40 , 41 ]. Such an induction process is thought to be regulated through signaling pathways mediated by several pattern recognition receptors [22 ]. Among these receptors, CD14 and TLRs, which recognize bacterial components such as LPS and peptidoglycan, are involved in the activation of transcription factor NF-{kappa}B [25 , 42 43 44 45 ]. Previous studies have demonstrated that hBD-2 mRNA is up-regulated by LPS not only in airway and intestinal epithelial cells but also in mononuclear phagocytes, which express CD14, TLRs, or both [14 , 16 , 22 , 46 ]. These studies suggest the involvement of NF-{kappa}B in the regulation of hBD-2 transcription. However, the hBD-2 promoter contains several consensus sequences for transcription factors such as NF-IL-6, STAT, and the AP-1 family in addition to NF-{kappa}B. In particular, NF-IL-6 is known as a potent transcription factor implicated in the transcriptional regulation of inflammation-related genes [34 , 47 48 49 ].

In the present study, we evaluated the roles of NF-{kappa}B and other transcription factors in the LPS responsiveness of the hBD-2 promoter in mononuclear phagocytes using a murine macrophage cell line. A 2.3-kb portion of the hBD-2 promoter region includes three NF-{kappa}B binding sites (p{kappa}B1, p{kappa}B2, and d{kappa}B). Among these, the p{kappa}B1 (position at -188) and p{kappa}B2 (position at -577) sites contain tandemly repeated NF-{kappa}B binding motifs and are likely to contribute to LPS-induced transcription of the hBD-2 gene (Figs. 2 and 3) . The requirement for both p{kappa}B sites is evidenced by the following observations: Promoter with deletion or mutation of the p{kappa}B2 site still retained 50% LPS responsiveness compared with the wild-type promoter. Similarly, a mutant construct of the p{kappa}B1 site alone retained apparent LPS inducibility. However, deletion or mutation of the p{kappa}B1 and p{kappa}B2 sites resulted in complete loss of LPS responsiveness, suggesting that these sites are likely to function cooperatively in LPS-mediated hBD-2 transcription (Figs. 2 and 3) . Of interest, deletion or mutation of the p{kappa}B1 or p{kappa}B2 site resulted in the significant loss of the basal transcriptional activity of the gene. In contrast, the d{kappa}B site at -2187 did not appear to be functional in hBD-2 transcription.

Diamond et al. [23 ] have demonstrated that transcription of the bovine ß-defensin TAP gene is cooperatively regulated by NF-{kappa}B and NF-IL-6 in response to LPS. Distinct from the TAP gene, deletion of potent NF-IL-6 binding elements adjacent to individual NF-{kappa}B sites in the hBD-2 promoter had little effect on LPS responsiveness, whereas mutation of two p{kappa}B sites resulted in the loss of LPS-induced transcription activity, as mentioned above (Figs. 2 and 3) . These findings suggest that NF-IL-6 elements are unlikely to participate in the LPS-mediated transactivation of the hBD-2 gene in RAW264.7 cells. This was further supported by the observation that IL-6, which activates NF-IL-6, did not increase hBD-2 promoter activity in RAW264.7 cells (unpublished results). We also noted that stimulation of RAW264.7 cells with IFN-{gamma} did not alter hBD-2 promoter activity (unpublished results), suggesting that the STAT binding motifs are not essential to the LPS responsiveness of the hBD-2 promoter in mononuclear phagocytes.

Taking these findings together, we conclude that the p{kappa}B1 and p{kappa}B2 sites play important roles in the regulation of hBD-2 expression in mononuclear phagocytes. Moreover, we confirmed that up-regulation of hBD-2 promoter activity is also dependent on NF-{kappa}B in epithelial cell lines (HaCaT keratinocytes and A549 lung epithelial cells), based on the observations that TNF-{alpha}, but not IL-6 and IFN-{gamma}, increased hBD-2 promoter activity in epithelial cells and that NF-{kappa}B bound to p{kappa}B sites in TNF-{alpha}-stimulated epithelial cells (unpublished results). Therefore, NF-{kappa}B-mediated transcription appears to be a common regulatory mechanism for hBD-2 expression among different cell types.

LPS stimulation induces activation of multiple forms of NF-{kappa}B, which is composed of homo- or heterodimeric subunits of the NF-{kappa}B/Rel family members [49 50 51 ]. Among these dimers, the p65-p50 heterodimer is the most abundant form of NF-{kappa}B and is known to act as a strong transactivator [51 , 52 ], whereas the p50 homodimer is likely to act on the target genes as a transcriptional suppresser, owing to its lack of a transactivation domain [51 , 53 ]. Consistent with this idea, binding of the p65-p50 heterodimer to the p{kappa}B1 and p{kappa}B2 sites was increased remarkably following LPS stimulation, and this increase correlated well with the enhanced transcriptional activity of the hBD-2 gene by LPS (Fig. 4) . Conversely, the cognate complexes of the p50 homodimer with p{kappa}B1 and p{kappa}B2 sites were mostly observed in unstimulated cells. The low basal level of hBD-2 promoter activity is likely a result of the existence of a low level of p65-p50 heterodimer in unstimulated cells that could not be detected by EMSA. The drastic increase in p65-p50 heterodimers after LPS stimulation is assumed to overcome the suppressive effect of the p50 homodimer, although the p50 homodimer was also induced by LPS (Fig. 4) .

Recently, Wada et al. [54 ] indicated that the transcription of the hBD-2 gene is mediated by NF-{kappa}B in a gastric cancer cell line stimulated with Helicobacter pylori. In contrast to our findings, they have suggested the importance of a single 5'-p{kappa}B1 site at -208/-199 for H. pylori-induced hBD-2 expression and the selective binding of the p65 homodimer to the site under their conditions. However, it is interesting that we found that the p65-p50 heterodimer and p50 homodimer, but not the p65 homodimer, bound to the p{kappa}B1 and p{kappa}B2 sequences using nuclear extracts from TNF-{alpha}-stimulated lung epithelial A549 cells, as observed with RAW264.7 cells (unpublished results). These observations likely suggest that different combinations of NF-{kappa}B subunits may regulate the expression of the hBD-2 gene in cell type-specific, stimulant-specific, or both.

The promoter regions corresponding to the p{kappa}B1 and p{kappa}B2 sites in the hBD-2 gene showed limited homology among the inducible hBD and mBD genes (Fig. 5) . In mBD-2 and -3 genes, a single conserved NF-{kappa} site was found. However, it is unclear whether these NF-{kappa}B sites are sufficient for optimal LPS-mediated transcription of mBD-2 and -3 genes. Moreover, it is interesting that the hBD-3 promoter lacks the NF-{kappa}B sites but contains several binding motifs for NF-IL-6, STAT, and the AP-1 family [12 ]. The transcriptional regulation of other inducible ß-defensin genes may therefore be somewhat different from that of the hBD-2 gene.

Further evaluation of the differential expression and actions of the ß-defensin family may contribute to our understanding of their roles in innate and acquired immune responses in infection and inflammation.


   ACKNOWLEDGEMENTS
 
This work was supported in part by a grant from the Atopy (Allergy) Research Center and High Technology Research Center, Juntendo University, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Received September 1, 2001; accepted September 11, 2001.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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