ZBP-89, Sp1, and nuclear factor-kappa B regulate epithelial neutrophil-activating peptide-78 gene expression in Caco-2 human colonic epithelial cells.

We reported previously that human colonic epithelial cells produce the C-X-C chemokine epithelial neutrophil-activating peptide-78 (ENA-78) and that its expression is up-regulated in ulcerative colitis. The aim of this study was to investigate the transcriptional regulation of ENA-78 gene expression in Caco-2 intestinal epithelial cells. Reporter gene transfection and electrophoretic mobility shift assay studies demonstrated that cooperation between two regions of the ENA-78 promoter were required for maximal gene expression in interleukin-1beta-stimulated Caco-2 cells. Binding of activated p50/p65 nuclear factor-kappaB to nucleotides -82 to -91 was essential for interleukin-1beta-dependent gene transcription, whereas binding of constitutively expressed zinc-requiring nuclear factors to nucleotides -125 to -134 (site A) was required for basal gene expression. Scanning mutagenesis of site A demonstrated overlapping binding elements at this locus. One site (CTCCCCC) bound Sp1 and Sp3, and overexpression of Sp1 (but not Sp3) up-regulated basal ENA-78 transcription. Another site (CCCCTCCCCC) was found to bind the zinc finger nuclear factor ZBP-89, and overexpression of this protein significantly repressed ENA-78 reporter gene activity. This study demonstrates that ENA-78 gene expression in Caco-2 intestinal epithelial cells is subject to complex regulation involving the coordinate binding of ZBP-89, Sp1, and nuclear factor-kappaB to the ENA-78 promoter.

Leukocyte recruitment to areas of inflammation is a multistep event that includes endothelial cell activation, expression of leukocyte adhesion molecules, and the production of chemoattractant cytokines or chemokines that direct leukocyte migration to the inflammatory focus (1)(2)(3). To date, more than 30 members of the chemokine family have been identified, and they have been divided into four subfamilies according to the number and arrangement of conserved cysteine residues (C, C-C, C-X-C, or C-X 3 -C) (2, 4 -7). Epithelial neutrophil-activating peptide-78 (ENA-78) 1 is a C-X-C chemokine that is structurally and functionally similar to IL-8. Like IL-8, it contains the neutrophil-activating amino acid motif glutamic acidleucine-arginine (ELR) and is thus a potent neutrophil-activating agent, inducing chemotaxis, cytoskeletal reorganization, and modulation of neutrophil adhesion molecule expression (8,9). Recent studies indicate that ELR-bearing C-X-C chemokines are also potent angiogenic factors that regulate endothelial cell proliferation and chemotaxis (10,11).
A wide variety of cell types can release IL-8 if stimulated appropriately; however, it is produced mainly by activated monocytes/macrophages and endothelial cells and to a lesser extent by neutrophils and epithelial cells (2,10,(12)(13)(14). ENA-78 appears to have a more restricted tissue distribution, being produced predominantly by epithelial cells and activated monocytes (9,15,16). Recent studies from our laboratory and others have shown that intestinal epithelial cells are the major site of ENA-78 production in human colon and that production of this chemokine is up-regulated in ulcerative colitis. Furthermore, we and others find that ENA-78 immunoreactivity in the human colon is confined mainly to intestinal epithelial cells, particularly the crypt epithelium (16,17). Chemokines produced by activated epithelial cells are capable of binding to sulfated polysaccharides present in the extracellular matrix, thereby creating a fixed chemotactic gradient (18,19). Thus, ENA-78 production may be especially important in the recruitment of neutrophils to the epithelial layer.
The production of chemokines is regulated largely at the level of gene transcription through the binding of activated transcription factors to specific gene promoter regulatory elements (2). Sequence analysis of the ENA-78 gene promoter indicates potential binding sites for the nuclear factors NF-B, AP-2, C/EBP, interferon regulatory factor-1, and cAMP response element (20,21). The NF-B site was found to be a functionally important regulatory element for ENA-78 gene expression in human embryonic kidney 293 cells (21). The contribution, if any, of the other potential nuclear factor binding sites has not been determined. In this study we have used luciferase reporter gene transfections and electrophoretic mobility shift assays to elucidate the molecular mechanisms responsible for ENA-78 gene expression in human Caco-2 intestinal epithelial cells. Our studies confirm that the NF-B binding site on the ENA-78 promoter plays a major role in regulating IL-1␤-induced gene transcription in Caco-2 cells. Our findings also identified a second 5Ј-regulatory element (nucleotides Ϫ127 to Ϫ136) that is required for maximal gene expression in Caco-2 cells. We demonstrate that ZBP-89 and Sp1 bind to overlapping sites in this region of the ENA-78 promoter and also regulate ENA-78 gene transcription.

MATERIALS AND METHODS
Cell Culture-Caco-2 ileocecal cells (American Type Culture Collection, Rockville, MD) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin G sodium, 100 g/ml streptomycin sulfate, and nonessential amino acids (Sigma) at 37°C in an atmosphere of 5% CO 2 and 95% air. Drosophila SL2 cells (American Type Culture Collection) were maintained in Schneider's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 40 units/ml penicillin G sodium, 40 g/ml streptomycin sulfate, and 2 mM L-glutamine at 24°C in a normal atmosphere.
Construction of Luciferase Reporter Genes-A 721-base pair fragment (containing nucleotides Ϫ706 to ϩ15) of the 5Ј-promoter region of the ENA-78 gene (20,21) was prepared by polymerase chain reaction amplification of human genomic DNA using a sense primer containing a KpnI restriction site and an antisense primer containing a BamHI restriction site. The polymerase chain reaction product was purified by agarose gel electrophoresis, extracted from gel slices (QIAquick; Qiagen Inc., Chatsworth, CA), and cloned into the pCR II vector (Invitrogen, San Diego, CA). After restriction digestion with KpnI and BamHI the ENA-78 promoter fragment was cloned directionally into the pGL2-Basic firefly luciferase expression vector (Promega, Madison, WI) between unique KpnI and BglII sites to generate a "full-length" ENA-78 reporter construct. Reporter genes containing sequentially truncated fragments (nucleotides Ϫ529, Ϫ399, Ϫ216, Ϫ154, Ϫ126, Ϫ78 to ϩ15) of the ENA-78 5Ј-promoter region were prepared in a similar manner using sense primers containing KpnI restriction sites and the antisense primer used to generate the full-length ENA-78 reporter construct (see Fig. 1).
Mutant full-length ENA-78 reporter constructs (see Table I) containing either deletions or substitutions in the NF-B binding site (nucleotides Ϫ81 to Ϫ92), the C/EBP binding site (nucleotides Ϫ107 to Ϫ114), or each of three putative AP-2 binding sites (20, 21) (nucleotides Ϫ127 to Ϫ136, designated site A; nucleotides Ϫ166 to Ϫ176, designated site B; or nucleotides Ϫ236 to Ϫ246, designated site C) were prepared using an oligonucleotide-directed in vitro mutagenesis system (QuikChange; Stratagene, La Jolla, CA).
Transient Transfections-ENA-78 reporter constructs were transfected into Caco-2 cells by calcium phosphate-DNA coprecipitation using a modification of the method of Hauck and Stanners (22). Briefly, cells were plated 3 days before transfection at a density of 1 ϫ 10 5 /well on a 12-well tissue culture dish (Corning Costar, Cambridge, MA) and transfected with 3 g of ENA-78 reporter gene plasmid DNA or equimolar amounts of ENA-78 reporter constructs containing truncated promoter sequences. To correct for variations in DNA uptake by the cells, each test construct was cotransfected with either 2 g of pCAT 3-Control vector (Promega) or 0.2 g of pRL-TK Renilla luciferase control vector (Promega). Transfections using pGL2-Basic vector without an insert were used as a negative control. For experiments investigating the effect of Sp1, Sp3, and ZBP-89 overexpression on ENA-78 reporter gene activity Caco-2 cells were cotransfected with 1 g of full-length ENA-78 reporter gene and 3 g of pCMV-Sp1 or pCMV-Sp3 (kindly provided by Dr G. Suske, Marburg, Germany), or 3 g of pSPORT-ZBP-89. After transfection for 15 h the precipitate was removed and replaced with normal growth medium. Caco-2 cells were washed twice with sterile phosphate-buffered saline and incubated with serum-free medium for 16 h to reduce background luciferase levels prior to stimulation with 25 ng/ml IL-1␤ for 8 h. The firefly luciferase activity of the cells was determined using a commercially available kit (Luciferase Assay System, Promega). The chloramphenicol acetyltransferase activity of the cells was assessed using a commercially available kit (CAT Enzyme Assay System, Promega), that measured the conversion of [ 14 C]chloramphenicol to n-butyryl [ 14 C]chloramphenicol. In experi-ments using the pRL-TK control vector, firefly and Renilla luciferase activities were measured simultaneously in each sample using the Dual-Luciferase Reporter Assay System according to the manufacturer's instructions (Promega).
Drosophila SL2 cells were transfected using a modification of the method of Han et al. (23). Briefly, 24 h prior to transfection, cells were plated onto 12-well dishes at a density of 1 ϫ 10 6 /well and transfected by calcium phosphate-DNA coprecipitation. Each well received an equal amount of DNA that included 2.5 g of the full-length ENA-78 reporter gene and variable amounts of pcDNA3-p50NF-B, Rc-CMV-p65NF-B (both kindly provided by Simos Simeonidis, Division of Gastroenterology, Beth Israel Deaconess Medical Center, Boston), pCMV-Sp1, and pBK-ZBP-89 overexpression contructs (or their respective control plasmids). 48 h after transfection the cells were washed twice with phosphate-buffered saline, and the firefly luciferase activities were determined. Luciferase activities were normalized to total lysate protein concentration.
Electrophoretic Mobility Shift Assay (EMSA)-To examine the activation of transcription factors EMSA was performed using nuclear extracts from nontransfected Caco-2 cells as described by Ladias et al. (24). Briefly, Caco-2 cells were seeded onto 100-mm tissue culture dishes (Becton Dickinson, Bedford, MA) and grown until confluent. After overnight incubation with serum-free medium, cells were stimulated with 25 ng/ml IL-1␤ for up to 6 h and washed twice with ice-cold phosphate-buffered saline. Cells were removed from tissue culture plates by scraping into ice-cold buffer containing 40 mM Tris/HCl (pH 7.5), 150 mM NaCl, and 1 mM EDTA, and spun in a microcentrifuge at 20,000 ϫ g for 5 s. Cells were then resuspended in a buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, and 0.5 mM phenylmethylsulfonyl fluoride, and incubated for 10 min on ice. Nonidet P-40 was added to achieve a final concentration of 0.5% (v/v), and the cell suspension was incubated for a further 2 min on ice. After centrifugation at 20,000 ϫ g for 5 s, the supernatants were removed, and the pellets were gently resuspended in a buffer containing 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 0.1 mM phenylmethylsulfonyl fluoride, and incubated for 30 min on ice. Extracts were then centrifuged at 20,000 ϫ g for 2 min and the supernatants frozen on dry ice and stored at Ϫ80°C until use.
Single-stranded complementary oligonucleotides bearing either wild type or mutant ENA-78 binding elements (i.e. NF-B and site A) were prepared by custom oligonucleotide synthesis (Genosys Biotechnologies). Double-stranded oligonucleotides containing consensus binding sites for AP-2␣ and Sp1 were obtained commercially (Santa Cruz Biotechnology). After annealing, 100 ng of the double-stranded oligonucleotide was labeled using either DNA polymerase I large (Klenow) fragment (Promega) in the presence of [␣-32 P]dCTP (PerkinElmer Life Sciences) or polynucleotide kinase (Promega) in the presence of [␥-32 P]ATP (PerkinElmer Life Sciences). Labeled probes were then purified on a Sephadex G-25 spin column (Amersham Pharmacia Biotech). Binding reactions for NF-B EMSAs were performed as described by Ferrari et al. (25). Site A EMSAs were performed according to the method of Wang et al. (26) except that 100 M ZnSO 4 was added to the binding reactions. Caco-2 cell nuclear extracts, purified transcription factors (AP-2␣ and Sp1; Promega), or recombinant rat ZBP-89 (produced by in vitro transcription and translation using a pcDNA3-ZBP-89-FLAG plasmid (27)) were preincubated for 10 min at room temperature in the reaction mixture after which the probe DNA was added and the incubation continued for another 30 min. Probeprotein complexes were then separated from free probe using 5% native polyacrylamide gels. Dried gels were exposed to x-ray film at Ϫ80°C to visualize the probe-protein complexes. To confirm the specificity of the binding reactions supershift assays were performed using antibodies to AP-2␣, AP-2␤, AP-2␥, Sp1, Sp2, Sp3, Sp4, and the NF-B components p50, p52, p65, RelB, and c-Rel (Santa Cruz Biotechnology). Supershift experiments were also performed using antibodies directed against the entire sequence of rat ZBP-89 (28). In some experiments binding specificity was also determined by competition with excess unlabeled probe. Antibodies or competing probe were added to the binding reactions at the start of the 30-min incubation period.
Data Analysis-Statistical analyses were performed using Sigma-Stat for Windows version 2.0 (Jandel Scientific Software, San Rafael, CA). Analysis of variance followed by protected t tests were used for intergroup comparisons, except where otherwise stated.

RESULTS
ENA-78 Luciferase Reporter Gene Activity in IL-1␤-stimulated Caco-2 Intestinal Epithelial Cells-Previous studies have shown that the production of chemokines is regulated largely via increases in gene transcription (29 -31). To obtain a more complete understanding of the factors that control ENA-78 gene expression in Caco-2 intestinal epithelial cells, a series of luciferase reporter constructs containing sequentially truncated fragments of the ENA-78 promoter was created (Fig. 1). After transfection of Caco-2 cells with equimolar amounts of each construct the cells were incubated for 8 h with or without 25 ng/ml IL-1␤, and the luciferase activities were determined.
Cytokine-stimulation of Caco-2 cells transfected with an empty luciferase expression vector or an ENA-78 reporter construct containing only a TATA binding site (nucleotides Ϫ78 to ϩ 15) did not significantly elevate luciferase activity compared with nonstimulated cells. In contrast, luciferase activity in stimulated Caco-2 cells transfected with an ENA-78 reporter construct containing NF-B and C/EBP binding sites (nucleotides Ϫ126 to ϩ15) was increased 4.4-fold compared with nonstimulated cells and was increased ϳ26-fold compared with cells transfected with a reporter gene containing no promoter elements. The basal luciferase activity of Caco-2 cells transfected with this construct was also increased 9.3-fold compared with cells transfected with the promoterless construct. Stimulation of Caco-2 cells transfected with a reporter construct containing site A in addition to the NF-B and C/EBP binding sites (nucleotides Ϫ154 to ϩ15) further increased luciferase activity 4-fold compared with the reporter gene containing only NF-B and C/EBP binding sites. Again, the basal luciferase activity of this construct was increased 2.7-fold compared with the construct containing nucleotides Ϫ126 to ϩ15 of the ENA-78 promoter. Transfection of Caco-2 cells with ENA-78 reporter genes containing additional putative transcription factor binding sites did not significantly increase IL-1␤-stimulated luciferase activity. Taken together, these results suggest that nucleotides Ϫ154 to ϩ15 of the ENA-78 promoter are sufficient for responses to IL-1␤ stimulation as well as basal activity, whereas regulatory elements upstream of this region (nucleotides Ϫ706 to Ϫ155) are not required for gene expression.
The ENA-78 Promoter B Element Binds p50/p50 and p50/ p65 NF-B and Is Required for IL-1␤-induced Gene Expression-To define further the relative contributions of the NF-B and C/EBP regulatory elements to IL-1␤-stimulated ENA-78 gene expression, mutant full-length ENA-78 reporter constructs containing either a targeted deletion or nucleotide substitutions within each putative transcription factor binding site were prepared by oligonucleotide-directed in vitro mutagenesis (Table I).
Under nonstimulated conditions deletion or substitution of the NF-B binding site reduced the luciferase activity of transfected Caco-2 cells ϳ 50% (p Ͻ 0.01) compared with the fulllength ENA-78 reporter construct ( Fig. 2A). In contrast, deletion or substitution of the C/EBP binding site had no significant effect on basal Caco-2 luciferase activity. In Caco-2 cells transfected with the full-length ENA-78 reporter gene exposure to 25 ng/ml IL-1␤ induced a 10-fold increase in luciferase activity compared with nonstimulated cells ( Fig. 2A). Deletion or substitution of the NF-B binding site reduced the luciferase activity of stimulated Caco-2 cells by ϳ95 and 90% respectively (p Ͻ 0.01 for each) compared with the wild type construct. Moreover, the NF-B deletion or substitution mutants were largely unresponsive to IL-1␤ stimulation. In contrast, deletion or substitution of the C/EBP binding site had no significant effect on IL-1␤-stimulated reporter gene activity. These findings indicate that NF-B is the major regulator of ENA-78 gene expression in response to IL-1␤ stimulation in Caco-2 intestinal epithelial cells.
We next performed EMSAs in nontransfected Caco-2 cells to determine the species of NF-B which binds the ENA-78 promoter B element (Fig. 2B). Nuclear extracts prepared from control Caco-2 cells showed very little binding of NF-B to the ENA-78-specific probe. After stimulation with IL-1␤ for 1 h Caco-2 nuclear extracts showed markedly increased levels of a slowly migrating NF-B complex (band 1) as well as elevated levels of a second faster migrating NF-B band (band 2). To investigate the composition of the two NF-B complexes binding to the ENA-78 probe, supershift studies using antibodies directed against various Rel proteins were performed. As shown in Fig NF-B binding site appears to bind two species of activated NF-B: p50/p50 homodimers and p50/p65 heterodimers.
The Site A Region of the ENA-78 Promoter Regulates Basal Gene Expression and Binds Constitutively Expressed Nuclear Factors-As shown in Fig. 1, sequential truncations of the ENA-78 promoter indicated that in addition to the NF-B binding site region, functionally important regulatory element(s) were also likely to be located within nucleotides Ϫ154 to Ϫ126 of the promoter. A previous study (21) has reported that this region of the ENA-78 promoter contains a putative AP-2 binding site (designated site A; see Fig. 1). To define the contribution, if any, of site A to IL-1␤-stimulated ENA-78 gene expression, a mutant full-length ENA-78 reporter construct containing a targeted deletion of this potential binding site was prepared. Deletion of site A reduced basal and IL-1␤-stimulated ENA-78 reporter gene activity ϳ75% ( Fig. 3A; p Ͻ 0.01 versus wild type). However, in contrast to deletion or substitution of the NF-B site, site A deletion did not prevent cytokinestimulated ENA-78 reporter gene activation (9.6-fold induction with IL-1␤). Mutant ENA-78 reporter genes containing deletions of two other potential AP-2 binding sites (site B or site C; see Fig. 1), which by promoter truncation analysis did not contribute to Caco-2 luciferase activity, were prepared also. As shown in Fig. 3, targeted deletion of either site B or site C had no significant effect on basal or IL-1␤-induced luciferase activity. Taken together with the data presented in Fig. 2, these findings suggest that NF-B is a critical regulator of the ENA-78 gene expression in response to IL-1␤ stimulation, whereas site A appears to control the overall activity of ENA-78 gene transcription but is not required for responses to IL-1␤.
EMSA using a site A probe (nucleotides Ϫ146 to Ϫ118 of the ENA-78 promoter; Fig. 3B) revealed the presence of two major complexes (designated NF-1 and NF-2) and two faster migrating minor complexes (designated NF-3 and NF-4) in nuclear extracts from control Caco-2 cells. Binding of all bands to the site A probe was reduced significantly upon competition with a 200-fold molar excess of unlabeled site A probe (shown in Fig.  4B), indicating specific binding to the probe. Exposure of Caco-2 cells to IL-1␤ did not appear to alter levels of any complex over the 6-h time course of the experiment. To determine whether these complexes contained AP-2, supershift assays were performed using antibodies to AP-2␣, AP-2␤, and AP-2␥; however, none of the complexes was supershifted with any of the antibodies tested (data not shown). Thus, contrary to the report of Chang et al. (21), the site A region of the ENA-78 promoter does not appear to contain a functional binding element for known members of the AP-2 family of transcription factors.
Sp1 and Other Zinc-requiring Nuclear Factors Bind to Site A of the ENA-78 Promoter-Further analysis of the site A region for homology to known transcription factor binding elements (using the TransFac data base) indicated potential binding sites for members of the zinc finger family of transcription factors, in particular, Sp1. To determine whether binding of NF-1, -2, -3, or -4 to the site A probe required zinc, we next performed EMSAs in the presence of the zinc-chelating agent EDTA. As shown in Fig. 4A, the addition of increasing amounts of EDTA to the EMSA reaction mixture dose dependently decreased the binding of all four complexes to the site A probe. The addition of 1.25 mM EDTA to the binding reaction com-  pletely inhibited the formation of complex NF-4 but had little effect on the binding of complexes NF-1, -2, or -3. Increasing the EDTA concentration to 2.5 mM partially inhibited the formation of complexes NF-1, -2, and -3, whereas addition of 5 mM EDTA to the EMSA reaction mixture prevented the binding of all complexes to the site A probe. As shown in Fig. 4A, titration of varying amounts of Zn 2ϩ (1-4 mM) to EMSA reactions containing 5 mM EDTA restored the binding of complexes NF-1, -2, -3, and -4 to the site A probe. In a separate experiment, 1 mM 1,10-phenanthroline, another divalent metal ion chelator, also completely abolished complex formation (data not shown). These findings clearly demonstrate that Caco-2 nuclear factors NF-1, -2, -3, and -4 require zinc for binding to site A of the ENA-78 promoter.
To test whether any of the complexes that bound to the site A probe contained Sp family proteins, supershift assays were performed using Caco-2 cell nuclear extracts and antibodies to various Sp1 family members (Fig. 4B). Interestingly, the most slowly migrating complex, NF-1, appeared, in these experiments, to consist of two closely migrating bands (see also Fig.  8A). The upper part of this complex was completely supershifted by an antibody to Sp1, whereas the lower part was supershifted by an Sp3 antibody. One of the faster migrating complexes, NF-3, was also supershifted by the Sp3 antibody. However, antibodies directed against Sp2 or Sp4 were unable to shift any of the complexes. A 200-fold molar excess of a consensus Sp1 oligonucleotide probe (which binds Sp1 and Sp3) competed for the binding of NF-1 and NF-3 but not the other complexes. In a separate experiment, the site A probe also bound to purified recombinant Sp1 and the Sp1 antibody shifted this complex (data not shown).
The Site A Binding Site for NF-1 and NF-3 (Sp1/Sp3) (CTC-CCCC) Overlaps That for NF-2 (CCCCTCCCCC) and NF-4 (GCCCCCCTCCCCC)-To characterize further the site A region we next performed scanning mutagenesis of the ENA-78 promoter between nucleotides Ϫ142 and Ϫ118 to define a specific nuclear factor-binding element(s). As shown in Fig. 5A, no difference in Caco-2 nuclear factor binding was observed when mutant site A oligonucleotide probes 1, 7, and 8 were subjected to EMSA analysis. In contrast, site A scanning mutants 4, 5, and 6 were unable to bind NF-1 or NF-3 (Sp1/Sp3); however, these factors were able to bind to mutant oligonucleotide 3. An identical pattern of binding to that seen with NF-1 was observed when EMSAs were performed with recombinant Sp1 instead of Caco-2 nuclear extracts (Fig. 5B). Interestingly, in addition to being unable to bind to mutant probes 4, 5, and 6, NF-2 was also unable to bind to mutant probe 3, whereas NF-4 was unable to bind mutant probes 2 or 3. These data indicate that the nucleotide sequence CTCCCCC comprises the NF-1 and NF-3 (Sp1/Sp3) site A binding element, whereas factors NF-2 and NF-4 appear to bind the overlapping nucleotide sequences CCCCTCCCCC and GCCCCCCTCCCCC, respectively.
To determine whether the nuclear factor binding sites identified by scanning mutagenesis within the site A region could functionally regulate ENA-78 gene expression we next pre- pared a reporter construct carrying a targeted substitution of nucleotides Ϫ128 to Ϫ129 of the ENA-78 promoter (see Table  I). The 2-base pair substitution was chosen because this mutation has been shown to prevent the interaction of Sp1 with its binding element (32). Importantly, the nucleotides involved in this mutation were removed from the reporter gene carrying the "site A deletion" (see Table I). As shown in Fig. 6A, ENA-78 reporter genes carrying substitutions at nucleotides Ϫ128 to Ϫ129 showed a 75% reduction in basal and IL-1␤-induced gene expression (p Ͻ 0.01 for each). Moreover, similar to our findings with the site A deletion reporter gene, this construct was still capable of responding to IL-1␤ stimulation (8.4-fold induction). To complement this reporter gene experiment, a mutant site A probe substituted at positions Ϫ128 to Ϫ129 was constructed, and an EMSA was performed using Caco-2 cell nuclear extracts. As shown in Fig. 6B, nucleotide substitution at positions Ϫ128 to Ϫ129 prevented the binding of all complexes to the site A probe. Thus, the observed reduction in ENA-78 reporter gene activity resulting from substitutions within site A appears to correlate with reduced binding of nuclear factors to site A as determined by EMSA.
Basal ENA-78 Gene Expression Is Up-regulated by Overexpression of Sp1 but Not Sp3-As shown in Figs. 4B and 5B, the site A region of the ENA-78 promoter can form a stable complex with the zinc finger transcription factors Sp1 and Sp3 (NF-1/ NF-3). To investigate whether either or both of these nuclear factors could transactivate the ENA-78 promoter we next examined the effect of Sp1 and Sp3 overexpression in Caco-2 cells transfected with the full-length ENA-78 reporter construct. As shown in Fig. 7, transfection of Caco-2 cells with an Sp1 expression construct (pCMV-Sp1) increased both basal and IL-1␤-stimulated ENA-78 luciferase reporter gene activity ϳ3-fold compared with cells transfected with a control construct (p Ͻ 0.01 for both). In contrast, basal and IL-1␤-stimulated ENA-78 reporter gene activity was unaltered when Caco-2 cells were transfected with an Sp3 expression construct (pCMV-Sp3). Because Sp1 overexpression had little effect upon IL-1␤-stimulated reporter gene activation (2.0-fold) compared with control cells (2.4-fold) these findings indicate that Sp1 regulates basal ENA-78 gene expression in Caco-2 intestinal epithelial cells.

Caco-2 Nuclear Factor NF-2 Comprises the Zinc Finger Transcription
Factor ZBP-89 -Analysis of the nucleotide sequence of the NF-2 and NF-4 binding motifs using the TransFac data base indicated putative binding sites for a number of nuclear factors including the recently described zinc finger transcription factor ZBP-89. To examine whether complexes NF-2 or NF-4 represent ZBP-89 binding activity in Caco-2 nuclear extracts a supershift EMSA was performed. As shown in Fig. 8A, an antibody directed against rat ZBP-89 supershifted complex NF-2 but had no effect upon the binding of complex NF-1, NF-3, or NF-4. To confirm this finding we examined the binding of recombinant full-length in vitro transcribed and translated rat ZBP-89 to the site A probe. As shown in Fig. 8B, recombinant ZBP-89 was capable of forming a stable complex with the wild type site A probe. Moreover, this complex was supershifted by the ZBP-89 antibody similarly to complex NF-2 present in Caco-2 nuclear extracts. Recombinant ZBP-89 was also able to bind to a NF-ODC probe (which contains a ZBP-89 binding site from the human ornithine decarboxylase promoter). In contrast, in vitro transcribed and translated ZBP-89 was unable to bind to a consensus Sp1 probe or a mutant NF-ODC probe (which does not bind ZBP-89).
Overexpression of ZBP-89 Represses ENA-78 Gene Expression-To determine the functional importance of ZBP-89 binding to the site A regulatory region full-length ENA-78 reporter constructs were cotransfected into Caco-2 cells with either a ZBP-89 overexpression construct (pSPORT-ZBP-89) or empty vector to serve as a control. As shown in Fig. 9A, in the presence of the control construct (pSPORT) IL-1␤-induced ENA-78 reporter gene activity was increased ϳ4-fold compared with nonstimulated Caco-2 cells. Interestingly, compared with the control construct, overexpression of ZBP-89 had little effect on basal luciferase activity (1.3-fold induction). In contrast, ZBP-89 overexpression repressed ENA-78 reporter gene activation by 30% in IL-1␤-stimulated Caco-2 cells.
Given the known function of ZBP-89 as a strong repressor, the modest repression by ZBP-89 in Caco-2 cells is likely caused by the opposing effect of endogenous Sp1. Therefore, to study the full functional effects of ZBP-89 on ENA-78 reporter gene activity we also performed experiments using Drosophila SL2

FIG. 5. The site A binding site for NF-1 and NF-3 (Sp1/Sp3) (CTCCCCC) overlaps that for NF-2 (CCCCTCCCCC) and NF-4 (GCCCCCTCCCCC).
To determine the ENA-78 promoter site A binding element(s), scanning mutagenesis of nucleotides Ϫ118 to Ϫ142 was performed. A, EMSAs were performed using nuclear extracts from Caco-2 cells treated with 25 ng/ml IL-1␤ for 30 min and either a wild type 32 P-labeled ENA-78 site A probe or one of eight sequentially substituted 32 P-labeled site A gel shift probes. The upper portion of the autoradiogram containing the probe-protein complexes is presented. B, EMSAs were performed using recombinant Sp1 and either a wild type 32 P-labeled ENA-78 site A probe or one of eight sequentially substituted 32 P-labeled site A gel shift probes. The upper portion of the autoradiogram containing the probe-protein complexes is presented. The boxed nucleotide sequence represents the binding site for NF-1 and NF-3 (Sp1/Sp3). The nucleotide sequence marked with a solid line represents the binding site for NF-2. The nucleotide sequence marked with a dashed line represents the binding site for NF-4. cells, which do not express either ZBP-89 or Sp1 (33,34). As shown in Fig. 9B, Sp1 overexpression in SL2 cells significantly elevated ENA-78 gene transcription (2.4-fold). Interestingly, ZBP-89 overexpression by itself had little effect on reporter gene activity, whereas Sp1-mediated increases in ENA-78 gene transcription were almost completely inhibited (95%) by overexpression of ZBP-89. To determine the functional effect of ZBP-89 on NF-B-regulated ENA-78 gene expression SL2 cells were also transfected with plasmids encoding p50 NF-B and p65 NF-B. As expected, overexpression of p50/p65 NF-B in SL2 cells significantly increased ENA-78 reporter gene activity (4-fold; Fig. 9C). Under these experimental conditions, overexpression of ZBP-89 alone or in combination with Sp1 inhibited NF-B-mediated increases in ENA-78 gene transcription (by 97 and 69% respectively). In keeping with our Caco-2 cell data, Sp1 was able to blunt ZBP-89 repression in the SL2 cells. In contrast, luciferase activity levels in SL2 cells transfected with Sp1 and NF-B were not significantly different form cells transfected with NF-B alone. These data indicate that ZBP-89 acts as a functional repressor of basal and inducible ENA-78 gene expression.

DISCUSSION
The aim of this study was to characterize the cis-regulatory elements and trans-activating factors that regulate basal and IL-1␤-induced expression of the ENA-78 gene in human intestinal epithelial cells. Using ENA-78 reporter constructs and EMSA analysis we demonstrate that two regions of the ENA-78 promoter are required for maximal gene expression in Caco-2 cells: an NF-B binding site spanning nucleotides Ϫ82 to Ϫ91 and a novel regulatory element (site A) located between nucleotides Ϫ125 and Ϫ134. We found that NF-B binding is essential for ENA-78 gene activation because deletion or substitution of the B binding site significantly reduced basal luciferase reporter gene activity and completely prevented responses to IL-1␤. Binding of p50/p65 heterodimers to the ENA-78-specific B site was observed 30 min-6 h after IL-1␤ treatment, in keeping with our previous report of the time course of mRNA and protein production (16). In contrast, deletion or substitution of the site A element reduced basal luciferase activity but had little effect upon IL-1␤-mediated reporter gene up-regulation. Scanning mutagenesis of the site A region indicated that several overlapping binding sites were contained within this element. One of these sites (CTCCCCC) bound Sp1 and Sp3, and overexpression of Sp1 (but not Sp3) increased basal ENA-78 reporter gene activity. Another site (CCCCTCCCCC) bound the zinc finger transcription factor ZBP-89. Moreover, overexpression of ZBP-89 in Drosophila SL2 cells repressed both basal and NF-B-mediated ENA-78 reporter activity. Thus, production of ENA-78 by Caco-2 cells appears to result from the coordinated activation and binding of multiple nuclear factors to the ENA-78 promoter.
An important finding of the present study is that the site A region of the ENA-78 promoter contains a binding site for Sp1/Sp3. Whereas Sp1 and Sp3 were each capable of binding the site A probe in vitro, overexpression of each protein in Caco-2 cells suggested that only Sp1 could transactivate ENA-78 gene expression. Sp1 overexpression increased the overall activity of the reporter gene but had no effect upon inducible responses to IL-1␤, indicating that this nuclear factor can regulate basal ENA-78 gene expression. To our knowledge, this is the first demonstration that other transcription factors in addition to NF-B can functionally regulate ENA-78 production. Interestingly, functional Sp1 binding elements have been identified in the promoters of several other chemokine genes including Gro-␣, MCP-1, and RANTES (32,35,36). Similar to the findings of this study, the Sp1 binding site in each of these promoters is required for the maintenance of basal gene expression, whereas a NF-B binding element is required for maximal cytokine-induced activity. Moreover, Ping et al. (37) have recently shown that Sp1 binding is an absolute requirement for promoter assembly and activation of the MCP-1 gene in response to stimulation by tumor necrosis factor-␣. The exact mechanism whereby Sp1 regulates ENA-78 gene expression is unclear. However, previous reports have demonstrated that Sp1 can interact directly with several elements of the general transcription apparatus including TATA box-binding protein (38) and the TATA box-binding protein-associated factors hTAFII130 and hTAFII55 (39,40). Moreover, Ryu et al. (41) have shown recently that Sp1 and the general transcription machinery can be linked indirectly via a multiprotein coactivator termed CRSP. A direct interaction between Sp1 and the p65 subunit of NF-B has also been reported (42). Which, if any, of these mechanisms regulates ENA-78 gene expression in Caco-2 intestinal epithelial cells remains to be established.
Although Sp1 can bind to site A and transactivate ENA-78 gene expression, our EMSA experiments indicated that two other Caco-2 nuclear factors, designated NF-2 and NF-4, were also capable of interacting with this regulatory element. Initial characterization indicated that like Sp1, binding of each of these factors to the site A EMSA probe was zinc-dependent. Several lines of evidence, however, indicated that the site A binding motifs for NF-2 and NF-4 are distinct from that of Sp1. First, in EMSA experiments antibodies to Sp1 only supershifted the upper portion of complex NF-1 in Caco-2 nuclear extracts, and recombinant Sp1 migrated to the same position on EMSA gels as the NF-1 complex. These findings indicate that the upper part of complex NF-1 contains only Sp1 and suggest that binding of nuclear factors to the Sp1 motif and the NF-2/NF-4 motifs is likely to be mutually exclusive. Second, scanning mutant oligonucleotide 3 was able to bind NF-1 and NF-3 (Sp1/Sp3), but not NF-2 or NF-4. Furthermore, complex NF-2 (but not NF-4) was able to bind scanning mutant oligonucleotide 2. These data suggest the presence of other site A binding elements for NF-2 (CCCCTCCCCC) and NF-4 (GC-CCCCCTCCCCC) spanning nucleotides Ϫ125 to Ϫ137. Interestingly, binding elements similar to that observed for NF-2 have been reported previously. In particular, several recent studies have reported that ZBP-89, a kruppel-like zinc finger protein, binds to the consensus nucleotide sequence gccCCtC-CxCC (33,43). To test whether ZBP-89 may constitute NF-2 or NF-4 binding activity, supershift analysis was performed using a polyclonal antibody directed against rat ZBP-89. Our data clearly demonstrate that site A complex NF-2, but not NF-4, was recognized by the antibody. Finally, overexpression of human ZBP-89 in Drosophila SL2 cells was found to repress both basal and NF-B-induced ENA-78 reporter gene activity significantly. Taken together, these data strongly suggest that ZBP-89 comprises ENA-78 site A binding factor NF-2.
Binding elements for ZBP-89 have been identified recently in the promoters of a variety of genes (27,33,(43)(44)(45)(46)(47). Functional studies indicate that depending upon the promoter, ZBP-89 can either activate or repress gene transcription. For example, overexpression of ZBP-89 can increase human stromolysin reporter gene activity (46). Moreover, ZBP-89 can potentiate butyrate-induced p21 WAF1 gene expression by human HT-29 colonic epithelial cells (27). In contrast, and similar to the findings of this study, binding of ZBP-89 to GC-rich elements in the human gastrin (44), ornithine decarboxylase (33) and vimentin (43) promoters represses the transcriptional activation of these genes. In each of these cases the ZBP-89 element either overlaps or is adjacent to a binding site for Sp1. In some instances ZBP-89 and Sp1 binding have been shown to be mutually exclusive as appears to be the case for the ENA-78 promoter (33,44). Several studies indicate that post-translational modifications to Sp1 (e.g. phosphorylation and O-glycosylation) regulate both its abundance and binding activity in cells (48,49). Moreover, Bai and Merchant (27) have recently shown that in HT-29 cells ZBP-89 can recruit the coactivator protein p300, whereas Sp1 cannot. Thus, the relative binding affinities and abundance of proteins competing for binding to Sp1 and ZBP-89 elements, and Sp1 and ZBP-89 themselves, may be important mechanisms regulating ENA-78 gene expression. Interestingly, recent coimmunoprecipitation studies by Wieczorek et al. (43) have shown that Sp1 can form heterodimers with ZBP-89 bound to the proximal silencer element of the vimentin gene. A direct interaction between Sp1 and ZBP-89 has also been reported by Bai and Merchant (27). These findings suggest that Sp1 and ZBP-89 may also regulate target gene transcription in a cooperative manner.
These studies indicate that regulation of ENA-78 transcription is more complex than reported previously. A model of ENA-78 gene regulation in Caco-2 cells which incorporates these new observations is presented in Fig. 10. Overexpression   FIG. 8. The site A region of the ENA-78 promoter binds the zinc finger transcription factor ZBP-89. A, to examine whether site A of the ENA-78 promoter can bind the zinc-requiring transcription factor ZBP-89, nuclear extracts from Caco-2 cells treated with 25 ng/ml IL-1␤ for 2 h were incubated with either a polyclonal antibody (Ab) directed against rat ZBP-89, excess unlabeled consensus Sp1 oligonucleotide (to remove complexes NF-1 and NF-3), or the ZBP-89 antibody in combination with excess cold Sp1 oligonucleotide then subjected to EMSA. Supershifted ZBP-89-probe-antibody complexes are indicated by the arrow. B, to determine whether recombinant ZBP-89 can bind to site A from the ENA-78 promoter, full-length in vitro transcribed and translated ZBP-89 protein (see "Materials and Methods") was incubated with a wild type (WT) site A probe, a consensus Sp1 probe, a wild type NF-ODC probe (which contains a ZBP-89 binding site from the ornithine decarboxylase promoter), or a mutant NF-ODC probe and then subjected to EMSA. Binding specificity was assessed using a ZBP-89 antibody and excess unlabeled wild type site A probe. Supershifted ZBP-89-probe-antibody complexes are indicated by the arrow. studies using Caco-2 and Drosophila SL2 cells demonstrate that ZBP-89 represses gene expression, whereas Sp1 activates transcription in Caco-2 cells. Moreover, our EMSA data suggest that because of their overlapping binding sites, each factor likely competes for mutually exclusive binding to site A region. Taken together, these findings indicate that site A acts as a transcriptional switch and that the equilibrium of binding between ZBP-89 and Sp1 at site A regulates basal ENA-78 gene expression in vitro. Interestingly, we have reported previously (16) that significant levels of ENA-78 protein are produced by normal noninflamed colonic epithelial cells, which presumably reflects the in vivo balance between Sp1 and ZBP-89 binding to site A.
In keeping with the study of Chang et al. (21), we find that cytokine-inducible gene expression requires NF-B activation. However, maximal gene expression in Caco-2 cells is secondary to the coordinated activation and binding of Sp1 and p50/p65 NF-B (Fig. 10). Increased levels p65 NF-B have been reported in epithelial cells and lamina propria macrophages from patients with active inflammatory bowel disease (50,51). Moreover, p65 antisense treatment can ameliorate intestinal inflammation in mice (52). Previous studies from our laboratory have shown that in chronic ulcerative colitis ENA-78 mRNA and protein levels are significantly elevated compared with noninflamed colonic tissue (16). Interestingly, the findings of this study indicate that ZBP-89 inhibits both basal and inducible reporter activity and therefore may act as a global repressor of ENA-78 gene expression. A similar function has recently been  9. Overexpression of ZBP-89 represses ENA-78 gene expression. A, Caco-2 cells were cotransfected with the full-length ENA-78 reporter gene and either a pSPORT-ZBP-89 expression vector or control vector (pSPORT) as described under "Materials and Methods." Caco-2 cells were also transfected with the pRL-TK control vector to correct for variations in transfection efficiency. 48 h later the cells were treated with 25 ng/ml IL-1␤ for 8 h, and the firefly and Renilla luciferase activities of the cells were determined. The activity of each construct is presented relative to the nonstimulated activity of the wild type ENA-78 reporter gene and corrected for transfection efficiency. Data are expressed as the mean Ϯ S.E. (n ϭ 12). B, Drosophila SL2 cells were cotransfected as described under "Materials and Methods" with the fulllength ENA-78 reporter gene and either a pCMV-Sp1 expression vector (or its vector control) or a pBK-ZBP-89 expression vector (or its vector control) as indicated. 48 h later the cells were washed, and the firefly luciferase activities were determined. Luciferase activities are presented relative to the control activity of the ENA-78 reporter gene and corrected for extract protein concentration. Data are expressed as the mean Ϯ S.E. (n ϭ 6). C, Drosophila SL2 cells were cotransfected as described under "Materials and Methods" with the full-length ENA-78 reporter gene and the NF-B expression vectors pcDNA3-p50 and Rc-CMV-p65 or their vector control plasmids. SL2 cells were also cotransfected with either a pCMV-Sp1 expression vector (or its vector control) or a pBK-ZBP-89 expression vector (or its vector control) as indicated. 48 h later the cells were washed, and the firefly luciferase activities were determined. Luciferase activities are presented relative to the control activity of the ENA-78 reporter gene and corrected for extract protein concentration. Data are expressed as the mean Ϯ S.E. (n ϭ 6). attributed to Oct-1 repression of IL-8 transcription in Caco-2 cells (53). Thus, it will be important to characterize further the factors that interact with NF-B, Sp1, and ZBP-89 to provide a more detailed understanding of the molecular mechanisms that regulate ENA-78 gene expression both in normal and in inflamed human colonic epithelial cells.