A novel lipopolysaccharide response element in the Bombyx mori cecropin B promoter.

Cecropin B is one of the major antibacterial peptides in the silkworm, Bombyx mori. Transcription of the cecropin B gene (CecB) occurs rapidly after bacterial invasion. Using 235 base pairs (bp) of the CecB promoter region, a kappaB-related protein and two additional DNA-binding complexes (designated F2BPI and F4BP) were identified in nuclear extracts from immunized larval fat body by the electrophoretic mobility shift assay (EMSA) (1). Further EMSA analyses indicated that the F2BPI-binding site was CATTA, and that F2BPI translocated from the cytoplasm to the nucleus after infection. In a recently established B. mori cell line, NISES-BoMo-DZ, 235 bp of CecB promoter linked to a reporter luciferase was activated 6-fold by stimulation with lipopolysaccharide (LPS), which is a major trigger of CecB expression in larvae. Truncation of the F2BPI-binding site from the promoter reduced the activation 2-fold. Deletion of either of two kappaB motifs also reduced promoter activation 2-fold. Elimination of both the F2BPI-binding site and the kappaB motifs resulted in the complete loss of LPS inducibility. These results indicate that the F2BPI-binding site is an LPS-responsive cis-element that is necessary for full activation of CecB.

Cecropin B is one of the major antibacterial peptides in the silkworm, Bombyx mori. Transcription of the cecropin B gene (CecB) occurs rapidly after bacterial invasion. Using 235 base pairs (bp) of the CecB promoter region, a B-related protein and two additional DNAbinding complexes (designated F2BPI and F4BP) were identified in nuclear extracts from immunized larval fat body by the electrophoretic mobility shift assay (EMSA) (1). Further EMSA analyses indicated that the F2BPIbinding site was CATTA, and that F2BPI translocated from the cytoplasm to the nucleus after infection. In a recently established B. mori cell line, NISES-BoMo-DZ, 235 bp of CecB promoter linked to a reporter luciferase was activated 6-fold by stimulation with lipopolysaccharide (LPS), which is a major trigger of CecB expression in larvae. Truncation of the F2BPI-binding site from the promoter reduced the activation 2-fold. Deletion of either of two B motifs also reduced promoter activation 2-fold. Elimination of both the F2BPI-binding site and the B motifs resulted in the complete loss of LPS inducibility. These results indicate that the F2BPI-binding site is an LPS-responsive cis-element that is necessary for full activation of CecB.
Insects have developed an effective innate immune system consisting of humoral and cellular responses. Rapid induction of several antimicrobial peptides in the hemolymph is a major humoral defense against microorganisms (2,3). Five different peptides have been isolated from the hemolymph of Bombyx mori larvae immunized by bacterial injection: cecropin A, cecropin B, cecropin D, lebocin, and moricin (4,5). The expression of these genes occurs simultaneously in fat body cells and hemocytes within a few hours of bacterial injection (6,7). Triggers activating the antibacterial cecropin B gene (CecB) 1 have been characterized in detail (6). Various species of LPS, Lipid A (the lipid part of the LPS core), 2-keto-3-deoxyoctonate (a saccharide in the LPS core), peptidoglycan (PG), and lipoteicoic acid (LTA) from bacteria induced the gene expression strongly. However, laminarin, zymozan, and scyzophillan, all of which contain mainly ␤-1,3-glucan, or spores of Beauveria bassiana never induced any gene expression. ␤-1,3-glucan is a common cell wall component of fungi. These observations indicate that B. mori distinguishes bacteria from fungi and expresses CecB in a bacteria-specific response.
The structure of CecB has been analyzed to elucidate the bacteria-specific gene activation mechanism (1). At least four copies of the genes exist in each individual. Two cloned genes, CecB1 and CecB2, revealed 90% identity with the upstream region spanning 800 bp, suggesting that the genes are regulated by the same transcription factors. In the proximal region of the promoters, two B-like decamer motifs, three GATA, sites and one mammalian type II interleukin-6 response element (IL-6RE) were found. The electrophoretic mobility shift assay (EMSA) identified three different DNA-binding proteins that bind to 235 bp of the CecB1 promoter. One of the proteins is probably a B-related factor because competition with a B-like sequence inhibited the binding (1). We designated the other proteins F2BPI and F4BP. B motifs and a GATA site have been identified in most insect immune-inducible protein genes (8,9). The induction mechanisms of antimicrobial peptide gene expression have been well studied in Drosophila melanogaster. NF-B-related factors of the Rel family (Dorsal, Dif, and Relish) (10 -12) and a GATA site-binding protein (Serpent) (13) play major roles in the induction. Several other mammalian cis-elements have been reported in insect immune-inducible promoters, such as both type I and type II IL-6REs and the interferon response element (14,15). The presence of the nuclear factors for these elements was suggested in the D. melanogaster diptericin promoter using the DNase I footprinting assay (16). However, the function of the elements has not been verified.
In this study, we characterized one of the DNA-binding proteins, F2BPI. We found that the F2BPI-binding site consists of two CATTA in the CecB promoter region. Furthermore, we explored a B. mori cell line that responds to bacterial cell wall components. Transfection assays using these cells revealed that the F2BPI-binding site is necessary for full activation of the CecB1 promoter by LPS.

EXPERIMENTAL PROCEDURES
Insects, Nuclear and Cytoplasmic Extracts, and EMSA-B. mori larvae (Tokai ϫ Asahi strain) were reared on an artificial diet (Nohon Nosanko) at 26°C and were used at day 3 of the fifth instar. The fat bodies were dissected from control larvae, or from larvae injected with autoclaved Escherichia coli (10 7 cells/larva), and incubated at 26°C for 4, 6, or 12 h. A nuclear extract was prepared by the method of Kobayashi et al. (17), and a cytoplasmic extract was prepared as described (18). EMSA using 32 P-labeled F2 or F2S DNA (8 fmol) and competitor (800 fmol) was done by essentially the same method as described previously (1).
Oligonucleotide Probes and Competitors-Oligonucleotides were synthesized in a DNA synthesizer (model 392, Applied Biosystems). F2 DNA probe was prepared by annealing of two oligonucleotides, 5Ј-AA-TCCTCATTAACTGGGAGAGCATTAATTGAGGGGATTAACTTTTA-3Ј and 5Ј-TAAAAGTTAATCCCCTC-3Ј, and then the gap was filled using Klenow fragment in the presence of [␣-32 P]dATP, dCTP, dGTP, and dTTP. F2S DNA probe was prepared by annealing two oligonucleotides, 5Ј-TCATTAACTGGGAGAGC-3Ј and 5Ј-TAATGCTCTCCCAGT-3Ј, and then the gap was filled by the same method described above. Four competitors were prepared by annealing two complementary oligonucleotides. The sequences of the upper strands of the competitors are shown in Fig. 1A.
Cell Lines and Triggers-B. mori BmN4 and NISES-BoMo-DZ (19) and Spodoptera frugiperda Sf9 were provided by Dr. Shigeo Imanishi, Department of Insect Genetics and Breeding, of our institute. D. melanogaster Schneider line 2 (SL2) cells were a gift from Dr. Kumiko Tei, Nippon Medical School. These cells were maintained at 26°C. The culture medium was EX-CELL (JRH Biosciences) for BmN4, SF900II (Life Technologies, Inc.) for NISES-BoMo-DZ and Sf9, and Shields and Sang M3 (Sigma) for SL2 cells. All media were supplemented with 5-10% fetal bovine serum (TRACE Scientific Ltd.), 100 units/ml of penicillin, and 0.1 mg/ml of streptomycin. LPS from E. coli 0111:B4 was purchased from DIFCO. Laminarin from Laminaria digitata and LTA from Staphylococcus aureus were from Sigma. PG from Micrococcus luteus was obtained from Wako Pure Chemical.
Reporter Assay-All reporter plasmids were constructed using pGL3-Basic vector (Promega). A minimum promoter region (Ϫ79 to ϩ10) from the B. mori actin A3 gene (20) was fused with the CecB1 promoter region and then inserted upstream from the luciferase gene in the vector. Various lengths of CecB1 promoter regions (790, 479, 235, 200, 151, and 123 bp), which were from immediately upstream of the TATA box to further upstream, were amplified by polymerase chain reaction using pBlueScript-CecB1 (1) as a template. The resulting plasmids were pC790, pC479, pC235, pC200, pC200, pC151, and pC123, respectively. Deletion mutant plasmids (pD1, pD2, pDG1, and pDG2) were created using a GeneEditor in vitro site-directed mutagenesis system (Promega) using pC235 as a template. To remove a 72-bp region (pDFG), two 6-bp sequences in pC235 were mutated to SalI sites, and the plasmid was then digested with SalI followed by ligation. The nucleotide sequences of the mutants are shown in Fig. 3. One day before transfection, the cells were seeded at 3 ϫ 10 5 /well in 24-well plates (Falcon) and transfected with 500 ng of the plasmid/well mediated by a cationic ion lipid, Tfx10 (Promega). To measure the relative transfection efficacy, the plasmid, pA3-LacZ (a gift from Dr. Pierre Couble) (20), which encodes LacZ under the A3 promoter was co-transfected at 500 ng/well. The transfected cells were either incubated with 100 g/ml of LPS (final concentration) or left without LPS for 24 h before the luciferase activity was measured using a luciferase assay system (Promega) and Lumicounter-700 (Microtec Co., Ltd., Funabashi, Japan). The relative ␤-gal activity was measured using a chemiluminescent assay system, Galacto-Light Plus (TROPIX, Inc).

RESULTS
Determination of the Binding Sequence of F2BP-Two nuclear proteins with differing migration patterns were bound to the F2 DNA fragment in EMSA (Fig. 1A, lane 2). F2 contains a B-like motif, GGGATTAACT, and an IL-6RE, CTGGGA. The fast migrating band is probably the B-like protein described previously. We designated the slow migrating complex F2BPI (F2-binding protein I). To determine the F2BPI binding sequence, we used four different competitors in EMSA. Competitor F2S, which is a partial sequence of F2 (Fig. 1A, lane 4), inhibited F2BPI binding. Competitor M1, which has two point mutations in IL-6RE, also inhibited F2BPI binding (Fig. 1A,  lane 5). Thus, the binding site of F2BPI is not identical to IL-6RE. In competitor M2, two CATTA were replaced by GC-CGG and CAACG, respectively. As shown in lane 6, M2 could not inhibit the formation of the F2BPI complex. Therefore, CATTA is an important sequence for F2BPI binding. The C oligo, which contains a B-like motif from the CecB promoter region, did not inhibit F2BPI binding, but it inhibited the formation of the fast migrating complex (Fig. 1A, lane 7).
Cellular Localization of F2BPI-The cellular localization of  2-7). The upper strand of the competitors is depicted below the EMSA photograph. Bold letters indicate IL-6RE, italics indicate the B-like motifs, and the probable F2BPI-binding sites are underlined. B, F2BPI localizes to the cytoplasm of the fat body. EMSAs were done using the F2S probe and nuclear (N12) and cytoplasmic (C12) extracts from larval fat body of 12-h immunization. The specific competitor was mixed at the indicated excess level. C, localization and time course of F2BPI. EMSAs were done using the F2S probe and nuclear (N0, N4, N6, and N12) or cytoplasmic (C0 and C12) extracts. The numbers after N and C indicate hours after immunization.

Novel LPS Response Element in Cecropin B Gene 13180
F2BPI was analyzed by EMSA. Using F2S probe, one distinct band shift was observed in both nuclear and cytoplasmic extracts from 12-h immunized larvae (Fig. 1B). This band was inhibited by a specific competitor, F2S. In the cytoplasmic extract, another faint band was observed at a higher position than F2BPI, and F2S also inhibited this band. Similarly, two bands were observed in normal cytoplasmic extract (Fig. 1C,  C0). No band shift was observed in normal nuclear extract (Fig.  1C, N0). The intensity of F2BPI was similar in nuclear extracts from 4 and 6 h, but it declined slightly at 12 h. These results indicate that F2BPI is localized in the cytoplasm and then translocates to the nucleus upon infection.
NISES-BoMo-DZ Respond to Various CecB Triggers-No other B. mori cell line that responds to bacterial challenge has been reported. To develop a promoter assay system of immunerelated genes using culture cells, we screened four cell lines, NISES-BoMo-DZ, BmN4, Sf9, and SL2 cells. To test the cell lines, a reporter plasmid (pC235) carrying luciferase under the control of 235 bp of the CecB1 promoter was transfected and analyzed with and without LPS stimulation. After incubation with LPS, luciferase activity increased 6-fold in NISES-BoMo-DZ and 2-fold in Sf9 cells (Fig. 2A). The luciferase activities in BmN4 and SL2 cells were not changed after LPS addition. The induced activity of the CecB1 promoter in NISES-BoMo-DZ increased in an LPS dose-dependent manner (Fig.  2B). The other triggers for CecB expression in larvae, PG, and LTA also increased luciferase activity in a dose-dependent manner (Fig. 2B). On the other hand, the cells did not respond to laminarin, in which ␤-1,3-glucan is a major component (Fig.  2B). As laminarin cannot induce CecB expression in larvae, the response of NISES-BoMo-DZ is similar to that of larvae. The responses suggest that this cell line can be used as a model system to analyze gene regulation of antibacterial peptides in B. mori.
F2BPI-Binding Site Is Necessary for Full Activation of CecB1 Promoter-To examine the functions of the F2BPI site, B motifs, and GATA sites in the activation of the CecB1 promoter, we transfected NISES-BoMo-DZ cells with the reporter plasmids containing different lengths of wild-type and mutated CecB1 promoters (Fig. 3). As shown in Fig. 4, the luciferase activity generated by pC790, pC479, pC235, pC200, pC151, pDG1, and pDG2 (all of these plasmids contain the F2BPI site, B motifs, and two GATA sites) did not differ significantly. Induction of luciferase activity by LPS was raised 4 -6-fold. The induction with pC123 lacking the F2BPI site was increased only 2-fold. Deletion of either of the B motifs also decreased the level of induced activity. The induction with pD1 and pD2 was 2-fold. Deletion of both B motifs further reduced the induction level to 1.8-fold. Elimination of both the F2BPI site and the B motifs (pDFG) resulted in the complete loss of LPS inducibility of the promoter. Most of the plasmids except pD1, pDG, and pDFG produced basal activity that was about 10-fold the activity of the control vector without CecB1 promoter. DISCUSSION We identified NISES-BoMo-DZ as a useful cell line for assaying promoter activity of immune-inducible genes. We tested whether endogenous CecB is expressed in the cells by Northern blot analysis and found that the cells were incapable of expressing CecB with or without LPS (data not shown). Nevertheless, this cell line recognizes bacterial cell wall components and possesses the cellular signaling pathway(s) to activate exogenous CecB promoter constructs. Using this cell line, the F2BPI site was identified as a cis-element necessary for the full response to LPS (compare pC151 with pC123, Fig. 4). In addition, both B motifs are functional and required for full activation of the CecB promoter. The plasmid (pDG) without B motifs still has LPS inducibility, suggesting that the F2BPI site is capable of inducing the CecB promoter independently of the B motifs. F2BPI and the B motifs seem to contribute equally to LPS inducibility. The basal activities of the promoters with the F2BPI site and the B motifs suggest that NISES-BoMo-DZ cells are constitutively stimulated in medium without the addition of bacterial factors. No other site spanning 790 bp of the promoter region was identified as important for LPS inducibility. In our system, deletion of any one GATA site did not affect promoter activity at all.
We determined that the probable F2BPI site consists of two CATTA, although we did not identify the binding sequence precisely. Further experiments should determine whether both CATTA are required or whether one is enough for CecB1 promoter activation. The CATTA appears as a mammalian immune-related cis-element in CLEO, the conserved lymphokine element 0, and in the promoter of IL-4, IL-5, and human granulocyte/macrophage colony-stimulating factor (GM-CSF) (21,22). The function of the CATTA has been demonstrated using the GM-CSF promoter (23). In this case, CATTT was also functional. The CATT(A/T) repeat in the promoter was required for gene expression in T-lymphocytes and several leukemia cells. Because GM-CSF gene expression is induced by LPS in macrophages (24), CATT(A/T) also could be an LPS response element of this gene. If so, CATT(A/T) is a common LPS response cis-element in mammals and insects, in addition to the B motifs and GATA site.
We examined the 5Ј upstream region of other LPS-inducible genes for the presence of the CATT(A/T) motif and found that most insect immune-inducible genes contain this motif. In B. mori, two CATTA are conserved in CecB1 and CecB2, and one is conserved in CecA1 and CecA2. Other B. mori genes and other genes from four different insect species contain one to seven copies of CATT(A/T) on both or either strand in the proximal promoter region, although the number and position vary ( Table I). The wide distribution of CATT(A/T) suggests that this sequence is a common LPS response element in insect immune-inducible genes.               Novel LPS Response Element in Cecropin B Gene 13182