Transcriptional regulation of mouse TREM-1 gene in RAW264.7 macrophage-like cells

Life Sciences 89 (2011) 115–122

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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e

Transcriptional regulation of mouse TREM-1 gene in RAW264.7 macrophage-like cells Hiroshi Hosoda a, Hiroshi Tamura b, Satoshi Kida c, Isao Nagaoka a,⁎ a b c

Department of Host Defense and Biochemical Research, Juntendo University, Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan Seikagaku Biobusiness Corporation, 1-17-24 Arakawa, Chuo-ku, Tokyo 104-0033, Japan Department of Bioscience, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan

a r t i c l e

i n f o

Article history: Received 17 August 2010 Accepted 18 May 2011 Keywords: Lipopolysaccharide Sepsis Endotoxin shock Transcription factor

a b s t r a c t Aims: Triggering receptor expressed on myeloid cells (TREM)-1 is expressed in macrophages, and functions as an amplifying molecule in inflammatory responses. TREM-1 is constitutively expressed in macrophage, and upregulated by bacterial components, such as lipopolysaccharide (LPS). In this present study, we investigated the regulatory mechanism for the basal and LPS-induced transcription of mouse TREM-1 gene in mononuclear cells using RAW264.7 macrophage-like cells. Main methods: To elucidate the potential role of cis-acting elements in the basal and LPS-induced transcription of mouse TREM-1 gene, the luciferase vector containing the promoter with 5′ deletion and adenine substitution mutants was transfected into RAW264.7 cells and incubated in the absence or presence of LPS. To further identify the transcription factor(s), gel shift/supershift analysis was performed. Key findings: The CRE (cAMP response element) and NF-κB-1 (a distal NF-κB site) in the mouse TREM-1 promoter are positively and negatively regulating the basal TREM-1 transcription via the interaction with C/EBPα and NF-κB p50/p50 homodimer, respectively. In addition, the CRE and NF-κB-1 likely participate in the LPS-induced upregulation of TREM-1 promoter activity possibly via the interaction with phosphorylated CREB and NF-κB p65/p50 heterodimer. Furthermore, the AP-1-1 (a distal AP-1 site) is likely to be involved in the LPS-induced TREM-1 transcription via the interaction with phosphorylated c-fos/c-jun. Significance: The present study has demonstrated for the first time the detailed mechanism for the basal and LPS-induced expression of TREM-1, an amplifying molecule in inflammation. © 2011 Elsevier Inc. All rights reserved.

Introduction Triggering receptor expressed on myeloid cells (TREM)-1 is a recently identified molecule that is expressed on neutrophils and monocytes/macrophages. It has been reported that the expression of TREM-1 in monocytes/macrophages is upregulated by gram negative and positive bacterial components, such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA) (Bouchon et al., 2001). Although TREM-1 ligands have not been identified, flow cytometrical analyses of the binding of labeled-recombinant TREM-1 suggest that TREM-1 ligands are expressed on neutrophils (Gibot et al., 2006b) and platelets (Haselmayer et al., 2007). Activation of TREM-1 using an agonistic monoclonal antibody elicits the interaction of TREM-1 with DAP12, a transmembrane adaptor molecule, thereby inducing the production of pro-inflammatory cytokines, such as IL-8, monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-α, and IL-1β (Bouchon et al., 2000; Bleharski et al., 2003; Dower et al., 2008). On the other

⁎ Corresponding author. Tel.: + 81 3 5802 1033; fax: + 81 3 3813 3157. E-mail address: [email protected] (I. Nagaoka). 0024-3205/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2011.05.007

hand, silencing of TREM-1 gene expression in LPS-stimulated macrophages suppresses the expression of inflammatory cytokines (such as IL-1 β, MCP-1, IL-10 and IL-2) (Ornatowska et al., 2007). Furthermore, silencing or blocking of TREM-1 represses the cytokine production (IL-1 β, TNF-α and IL-6) and prolongs the survival of mice or rats with bacterial sepsis (Bouchon et al., 2001; Gibot et al., 2006a; Gibot et al., 2007). These observations indicate that TREM-1 plays role in inflammatory responses as an amplifying molecule, which likely modulates the production of cytokines. Zeng et al. have suggested that the transcription of TREM-1 is positively and negatively regulated by NF-κB and PU.1, respectively, in LPS-stimulated RWA264.7 cells, based on the findings that an NF-κB inhibitor (BMS-345542) suppresses the increase of TREM-1 mRNA level, whereas silencing of PU.1 increases TREM-1 mRNA level in LPSstimulated cells (Zeng et al., 2007). However, they could not confirm the involvement of NF-κB and PU.1 sites in the LPS-induced TREM-1 promoter activity by the reporter assay, because the deletions of NF-κB and PU.1 sites did not affect the TREM-1 promoter activity. Thus, the mechanism for the LPS-stimulated promoter activity of TREM-1 gene remains controversial. Furthermore, TREM-1 is constitutively expressed in monocytes/macrophages; however, transcription factors controlling

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the TREM-1 gene expression in unstimulated cells have not been identified. In this present study, to elucidate the regulatory mechanism for the basal and LPS-induced TREM-1 expression in mononuclear cells, we investigated the TREM-1 promoter activities (by luciferase reporter assay), the transcription factors associated with the TREM1 promoter sequences (by gel shift assay) and the expression of transcription factors (by western blotting) in resting and LPSstimulated RAW264.7 murine macrophage-like cells. Material and methods Regents and antibodies LPS (from Escherichia coli serotype O111:B4) was purchased from Sigma Chemical Co. (St Louis, MO, USA); 5′-FITC labeled or unlabeled oligonucleotides from Operon Biotechnology, Tokyo, Japan; rabbit anti-C/EBPα polyclonal antibody (sc-61), rabbit anti-c-jun polyclonal antibody (sc-1694) and rabbit anti-c-fos polyclonal antibody (sc-253) from SantaCruz Biotechnology (Santa Cruz, CA, USA); rabbit antiCREB polyclonal antibody (AB3006) from Chemicon International (Temecala, CA, USA); rabbit anti-NF-κB p65 polyclonal antibody (3034) from Cell Signaling Technology (Beverly, MA, USA); rabbit anti-NF-κB p50 antiserum (06-886) from Upstate Biotechnology (Lake Placid, NY, USA). Cell culture RAW264.7 murine macrophage cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were grown and maintained in Dalbeco's modified Eagle's medium (DMEM: Sigma Chemical Co.) containing 10% fetal calf serum (FCS, endotoxin level b10 EU/ml; Cell Culture Technologies, Herndon, VA, USA), penicillin (100 U/ml) and streptomycin (0.1 mg/ml) at 37 °C in a 5% CO2 incubator. Plasmid construction Transcription initiation site of murine TREM-1 was estimated using murine the sequence of chromosome 17 and EST database (NCBI reference sequence numbers: NT_039649.6 and BY192784). A 1.2-kbp fragment of murine TREM-1 promoter (− 1200 to + 54) was amplified from RAW264.7 genomic DNA by polymerase chain reaction (PCR) using KOD -Plus- polymerase (Toyobo Co., Ltd., Osaka, Japan) and a set of oligonucleotide primers; -1200 sense primer, 5′-GGGACGCGTGTGTGATGGAGTGTGTCCAG-3′ and +54 antisense primer, 5′-GGGAGATCTCCTTCAAGCTCAGCTCCAAC-3′ (underlines indicate the restriction sites MluI and BglII, respectively). PCR products were digested with MluI and BglII, and then subcloned into a promoterless firefly luciferase expression plasmid pGL3-Basic (Promega, Madison, WI, USA) to generate a −1200 plasmid. The cis-acting motifs were investigated using database TFSEARCH in the 5′ upstream region (− 1200–+ 54) of mouse TREM-1 promoter. A series of 5′ deletion fragments of TREM-1 promoter was amplified by PCR using −1200 plasmid (as a template), KOD -Plus- polymerase and appropriate sets of sense and antisense primers containing MluI and BglII restriction sites (indicated by underlines), respectively; -1000 sense primer (5′-GGGACGCGTTGTATGTGGGCAAATGTAGTG-3′), -800 sense primer (5′-GGGACGCGTAAGTCAGGACTGGAAATTAAG-3′), -600 sense primer (5′-GGGACGCGTCTCATGGAGGCATTTCCTCA-3′), -400 sense primer (5′-GGGACGCGTCCCAGGCAGGACCGAATG-3′), -200 sense primer (5′-GGGACGCGTAAATTATTACAATACAAAAGAAAAT-3′), -100 sense primer (5′-GGGACGCGTCTGATGTCAGCCCGCAGG-3′), -50 sense primer (5′-GGGACGCGTTGGCCTCACATCCTGTTGTG-3′) and +54 antisense primer (5′-GGGAGATCTCCTTCAAGCTCAGCTCCAAC-3′). The inserts were confirmed by sequencing with a BigDye® Terminator v3.1

sequencing kit and a 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The promoter sequences of NF-κB-1 (−743 to −730), NF-κB-2 (−651 to − 638), NF-κB-3 (− 593 to − 584), SP1-2 (−244 to − 235), AP-1-1 (−907 to − 898), AP-1-2 (− 266 to −257), and CRE (−99 to −92) deduced with a database TFSEARCH were substituted by adenine nucleotides by PCR-based site directed mutagenesis using − 1200 plasmid (as a template), KOD -Plus- polymerase and appropriate oligonucleotide sense and antisense primers shown in Table 1. Synthesized blunt-ended PCR products were purified with a MiniElute Gel extraction kit (QIAGEN, Valencia, CA, USA), phosphorylated with polynucleotide kinase, and then self-ligated with T4 DNA ligase. Mutated cis-acting motifs were confirmed by sequencing. Transfection and luciferase assay RAW264.7 cells (1 × 10 5) in a 24-well plate were transfected with series of reporter plasmids containing the TREM-1 promoter with 5′ deletions or adenine substitutions (500 ng) and renilla luciferase expression plasmid phRL-TK (1 ng, an internal control) (Promega) using FuGENE®HD regent (Roche Applied Science, Mannheim, Germany) and incubated for 20 h. Thereafter, cells were stimulated with LPS (250 ng/ml) for 8 h, washed twice with PBS, and lysed in Passive Lysis Buffer (Promega). Then, firefly and renilla luciferase activities were measured using a Dual-luciferase® Reporter Assay System (Promega) and a microplate luminometer (SpectraMax® L, Molecular Devices, Sunnyvale, CA, USA). Promoter activities were normalized with renilla luciferase activity, and expressed as a ratio relative to the firefly luciferase activity of −1200 plasmid-transfected cells incubated without LPS. Preparation of nuclear extracts RAW264.7 cells (1 × 10 8) in a 150 mm plate were incubated with or without LPS (250 ng/ml) for 1 h, and then nuclear extracts were prepared from the cells as described previously (Dignam et al., 1983; Tsutsumi-Ishii and Nagaoka, 2002). In brief, after the incubation, cells were washed twice with PBS, harvested using 0.1 mM EDTA-PBS, and lysed in lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.2 mM EDTA, 1.5 mM MgCl2, 0.5% Nonidet P-40, 1 mM DTT, 20 mM NaF) containing 1/25 v/v Complete™ (Roche Applied Science) on ice for 10 min. Nuclei were collected by centrifugation at 1000 ×g for 5 min at 4 °C and washed in the same buffer except Nonidet P-40. Nuclear pellets were resuspended in extraction buffer (10 mM HEPES pH 7.9, 420 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 25% glycerol, 1 mM DTT, 20 mM NaF) containing 1/25 v/v Complete™. 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, and the resultant supernatants were recovered and stored at −80 °C. Protein concentrations were measured with a Bradford method (Protein assay, Bio-Rad laboratories, Hercules, CA, USA). Gel shift assay Nuclear extracts (5 or 2 μg) were mixed with 32P-labeled or 5′-FITClabeled double-stranded oligonucleotide probe (0.1 or 5 pmol, respectively) in 20 μl of binding buffer (20 mM HEPES pH 7.9, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 250 μg/ml BSA, 6% glycerol, 2 μg poly dI-dC, 20 mM NaF) containing 1/25 v/v Complete™ for 20 min on ice. DNA probe/protein complexes were electrophoresed on a native 6% polyacrylamid gel at 2 mA for 4 h at 4 °C. For detecting 32P-labeled DNA probe/protein complexes, gels were dried, exposed to the imaging plate, and then evaluated using a BAS2500 bio-imaging analyzer (FUJIFILM Corp., Tokyo, Japan). Alternatively, fluorescence of DNA probe/protein complexes in the gels was detected with a LAS3000 image analyzer (FUJIFILM Corp.). For competition assays, 0.5–200-fold molar excesses of

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Table 1 Oligonucleotide primers used for the PCR-based site directed mutagenesis. Putative motifs

Primers sequences

NF-κB-1

Sense primer Antisense primer

NF-κB-2

3′- GGAATGTCAAGGTAGAGTAGGTTTTT-5′ −614 −594 −234 −217 5′-AAAAATGGGGTGCGGATTCTACC-3′ 3′-GAGTCCTCGATTTACCCGTTTTT-5′ −262 −245 −897 −874 5′-AAAAAGGTTTCTATTCCTGCACAAACATC-3′ 3′-CCCTTACACAGACTCCATCACTTTTT-5′ −933 −908 −256 −240 5′-AAAAAAGCTAAATGGGCTGGG-3′

Sense primer Antisense primer

CRE

−583 −562 5′-AAAAATCAACTGAAGCTCCTTTTTCTGTG-3′

Sense primer Antisense primer

AP-1-2

3′-CTCTACCAGGGTGGATGTTTTTTTT-5′ −669 −652

Sense primer Antisense primer

AP-1-1

−637 −615 5′-AAAAAACTTGATCACTAATTGAGAAAATG-3′

Sense primer Antisense primer

SP1-2

3′-GACTACGTCTCCGGTACCTTTTTT-5′ −761 −744

Sense primer Antisense primer

NF-κB-3

−729 −713 5′-AAAAAAAACTGGCTTGCTTCCCCTGG-3′

3′-GTTGTCCGGAGACAGAGTTTTT-5′ −284 −267 −91 −75 5′-AAAAGCCCGCAGGGTGGCCAG-3′

Sense primer Antisense primer

3′-CAGGGACTTGAAGGAATGTTTT-5′ −118 −100

Underlines indicate the nucleotides for the adenine substitutions of putative motif sequences in Fig. 1; NF-κB-1 (−743 to −730), NF-| B-2 (−651 to −638) and NF-| B-3 (−593 to −584); SP1-2 (−244 to −235); AP-1-1 (−907 to −898) and AP-1-2 (−266 to −257); CRE (−99 to −92).

unlabeled double-stranded oligonucleotides were incubated with nuclear extracts for 10 min on ice before the addition of probe. For antibody supershift experiments, DNA probe/protein complexes were incubated with 1 μg of antibodies (anti-CREB antibody, anti-C/EBPα antibody, antiNF-κB p65 antibody, anti-c-fos antibody, anti-c-jun antibody and normal rabbit IgG) or 20 μg of sera (anti-NF-κB p50 antiserum and normal rabbit serum) for 20 min on ice. Oligonucleotide probes used were as follows NF-κB-1, 5′-CCATGGAGGGAAGTTCCTTACTGGC-3′ (−749 to −724); CRE, 5′-CCTTACTGATG TCAGCCCGC-3′ (−105 to −88); AP-1-1, 5′-AGTGTCTGAGTCAGGGTTTC3′ (−913 to −892): consensus probe for NF-κB, 5′-AGTTGAGGGGACTTT CCCAGG-3′; consensus probe for CREB, 5′-GATTCGTGACGTCAGCACAG3′; consensus probe for C/EBPα, 5′-CCCTGATTGCGCAATAGGCT-3′; consensus probe for AP-1, 5′-CGCTTGATGACTCAGCCGGAA-3′ (putative or consensus sequences for transcription factors were indicated by underlines). For 32P labeling, oligonucleotide probes (50 pmol) were endlabeled by polynucleotide kinase and [γ-32P] ATP (6000 Ci/mmol; GE Healthcare, UK) for 60 min 37 °C, and unincorporated isotope was removed using Nick™ Column (GE Healthcare). Specific activities of labeled oligonucleotides were approximately 2×106 cpm/pmol DNA. Statistical analysis The data are expressed as the mean ± SD. Statistical analyses were performed using the unpaired Student's t-test (Statview 5.0, SAS Institute Inc., Cary, NC). A p-value b0.05 was considered to be statistically significant. Results Sequence of the 5′ upstream flanking region of mouse TREM-1 gene To clarify the expression mechanisms for the basal and LPS-induced expression of mouse TREM-1 gene, we first analyzed the cis-acting

motifs in the TREM-1 promoter (from −1200 to +54) using TFSERCH version 1.3 program; the transcription initiation site (+ 1) was estimated using EST database (NCBI reference sequence numbers: NT_039649.6 and BY192784). As shown in Fig. 1, the mouse TREM-1 promoter contained multiple potential binding motifs for AP-1 family (AP-1-1 and −2), NF-κB (NF-κB-1, -2 and -3), SP1 (SP1-1 and −2), GATA-1 (GATA-1-1, -2, -3 and −4), C/EBP (C/EBP-1 and -2) and CRE, although TATA-box sequence cannot be detected in the promoter. Luciferase activities of mouse TREM-1 promoter-containing plasmids with 5′ deletions or adenine substitution mutants To elucidate the potential role of cis-acting elements in the basal and LPS-induced transcription of mouse TREM-1 gene, the luciferase vector containing a series of 5′ truncated promoter was transfected into RAW264.7 macrophage-like cells and incubated in the absence (Resting) or presence of 250 ng/ml LPS. As shown in Fig. 2, a plasmid containing −1200 upstream region exhibited the substantial luciferase activity, which is consistent with the finding that TREM-1 gene is constitutively transcribed in resting macrophages/monocytes (Gingras et al., 2002; Ingersoll et al., 2010). Successive deletion of the upstream region to position −400 did not essentially affect, or moderately increased or reduced (pb 0.05) the basal activity. However, the deletion to position −50 resulted in the almost complete loss of the basal activity (p b 0.001). In accordance with the finding that the transcription of TREM-1 gene is upregulated by LPS-stimulation in macrophages/monocytes, the luciferase activity of a −1200 plasmid was enhanced by the addition of LPS (pb 0.01). The deletion to position −50 similarly resulted in the complete loss of the promoter activity even in the presence of LPS (pb 0.001) (Resting vs. LPS-stimulated, p N 0.05). Interestingly, the deletion to position −800 resulted in a significant loss of the LPS responsiveness (Resting vs. LPS-stimulated, p N 0.05), although other deletions to −1000, -600, -400, -200 and 100 did not essentially affect the LPS responsiveness (Resting vs. LPS-stimulated, p b 0.05).

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Fig. 1. Sequence of the 5′ upstream region of mouse TREM-1 gene. Transcription initiation site was estimated using EST database (NCBI reference sequence numbers: NT_039649.6 and BY192784). Analysis with TFSERCH version 1.3 program revealed that the 5′ upstream region of mouse TREM-1 gene contains putative cis-acting motifs for NF-κB, SP-1, AP-1, GATA-1, C/EBP and CREB; however, a TATA-box sequence can not be detected in the promoter. AP-1 sites are termed AP-1-1 (− 907 to − 898) and AP-1-2 (− 266 to − 257); NF-κB sites are termed NF-κB-1 (− 743 to − 730), NF-κB-2 (− 651 to − 638) and NF-κB-3 (− 593 to − 584); SP1 sites are termed SP1-1 (− 1159 to − 1150) and SP1-2 (− 244 to − 235); GATA-1 sites are termed GATA-1-1 (− 762 to − 753), GATA-1-2 (− 670 to − 661), GATA-1-3 (− 484 to − 476) and GATA-1-4 (−−464 to − 455); C/EBP are termed C/EBP-1 (− 566 to − 552) and C/EBP-2 (− 27 to − 17), respectively.

SP1-2 (−244 to −235), AP-1-1 (−907 to −898), AP-1-2 (−266 to −257), and CRE (−99 to −92) sequences in the constructs were substituted by adenine nucleotides (Fig. 3). Consistent with the results

To further determine the role of putative cis-acting elements in the basal and LPS-induced transcription of mouse TREM-1 gene, NF-κB-1 (−743 to −730), NF-κB-2 (−651 to −638), NF-κB-3 (−593 to −584), NF- B- 2 NF- B-1 NF- B-3 SP1-2 CRE AP-1-2 SP1-1 AP-1-1

-1200 GATA C/EBP GATA

Luc

**

C/EBP

*

-1000 -800

*

-600

**

***

***

-400

* ***

-200

**

*** *

-100

***

-50 0

Resting LPS-stimulated

1

2

3

Relative luciferase activity Fig. 2. Basal and LPS-induced luciferase activities of mouse TREM-1 promoter containing 5′ deletion constructs. Positions of putative cis-acting motifs (NF-κB, GATA, C/EBP, AP-1, SP1 and CRE) were indicated on the TREM-1 promoter flanking luciferase gene (Luc). RAW264.7 cells were transfected with a series of 5′ deletions reporter constructs (500 ng) and renilla luciferase expression plasmid phRL-TK (1 ng, an internal control), and incubated for 20 h. Thereafter, cells were incubated in the absence (Resting) or presence (LPSstimulated) of LPS (250 ng/ml) for 8 h, and then firefly and renilla luciferase activities were measured. Promoter activities were normalized with renilla luciferase activity, and expressed as a ratio relative to the firefly luciferase activity of − 1200 plasmid-transfected cells incubated without LPS. Values are mean ± SD of at least four independent experiments. Values are compared between − 1200 plasmid and 5′ deletion constructs in Resting cells, and between Resting and LPS-stimulated cells of each construct. *p b 0.05; **p b 0.01; ***p b 0.001.

H. Hosoda et al. / Life Sciences 89 (2011) 115–122 NF- BNF- 2B-1 NF- B-3 SP1-2 AP-1-2 CRE SP1-1 AP-1-1

119

Luc

-1200 GATA C/EBP GATA

AP-1-1 NF- B-1 NF- B-2 NF- B-3 AP-1-2 SP-1-2 CRE

***

C/EBP

A

**

A

A

*

A

* A

*** A

* ***

A

0

Resting LPS-stimulated

1

2

3

4

Relative luciferase activity Fig. 3. Basal and LPS-induced luciferase activities of mouse TREM-1 promoter containing adenine substitution constructs. Positions of adenine substitution mutation of putative cisacting motif (AP-1-1, NF-κB-1, NF-κB-2, NF-κB-3, AP-1-2, SP1-2 and CRE) were indicated on the TREM-1 promoter flanking luciferase gene (Luc). RAW264.7 cells were transfected with a series of reporter constructs with adenine substitutions (A) (500 ng) and renilla luciferase expression plasmid phRL-TK (1 ng, an internal control) and incubated for 20 h. Thereafter, cells were incubated in the absence (Resting) or presence (LPS-stimulated) of LPS (250 ng/ml) for 8 h, and then firefly and renilla luciferase activities were measured. Promoter activities were normalized with renilla luciferase activity, and expressed as a ratio relative to the firefly luciferase activity of − 1200 plasmid-transfected cells incubated without LPS. Values are mean ± SD of at least four independent experiments. Values are compared between − 1200 plasmid and adenine substitution constructs in Resting cells, and between Resting and LPS-stimulated cells of each construct. *p b 0.05; **p b 0.01; ***p b 0.001.

with a 5′ deletion construct to position −50 (deletion of CRE), the adenine substitution of CRE resulted in the significant decrease in the basal and LPS-induced TREM-1 promoter activity (pb 0.001). Moreover, similar to the results with a 5′ deletion construct to position −800 (deletion of AP-1-1), the substitution mutation in AP-1-1 substantially reduced the LPS responsiveness (Resting vs. LPS-stimulated, p N 0.05). Unexpectedly, the substitution mutation in NF-κB-1 enhanced the basal promoter activity (p b 0.01), but reduced the LPS responsiveness (Resting vs. LPS-stimulated, p N 0.05). These observations indicate that CRE and NF-κB-1 sites are involved in the positive and negative regulation of the basal promoter activity of mouse TREM-1 gene, respectively, whereas AP-1-1 site participates in the LPS-induced promoter activity of mouse TREM-1 gene.

the consensus C/EBPα (CCAAT box) and CREB (CRE) oligonucleotides as competitors. Interestingly, the DNA-protein complexes (nuclear proteins plus labeled-CRE sequence of the mouse TREM-1 promoter)

B

A

x0.5 x5 x50 x0.5 x5 x50 x0.5 x5 x50

*

Gel shift analyses of the CRE, NF-κB and AP-1 sites in the mouse TREM-1 promoter using RAW264.7 nuclear extracts We performed gel shift/supershift analyses to identify the transcription factor(s) that bind to the CRE, NF-κB-1 and AP-1-1 sites in the mouse TREM-1 promoter. When nuclear extracts from unstimulated RAW264.7 cells were incubated with a 32P-labeled CRE oligonucleotide (spanning −105 to − 88 of the TREM-1 promoter), the two specific bands of DNA-protein complexes were detected, which can be abolished with the excess of unlabeled CRE consensus oligonucleotide as well as unlabeled CRE sequence in the mouse TREM-1 promoter (Fig. 4A and B). It has been reported that C/EBP and CREB families of transcription factors such as C/EBPα and CREB can bind to the CRE, and upregulate the transcription (Montminy et al., 1990; Miller et al., 2003). To examine which transcription factors could bind to the CRE in the mouse TREM-1 promoter, we performed antibody supershift experiments. Of note, one strong supershifted band was observed by the addition of anti-C/EBPα Ab. In contrast, addition of anti-CREB Ab slightly reduced the two complex bands, although control IgG had no effect on the bands. These observations suggest that C/EBPα and possibly CREB can bind to the CRE sequence in the mouse TREM-1 promoter. To address this possibility, we used

Fig. 4. Gel shift assay of the CRE site in the mouse TREM-1 promoter. Gel shift assay was performed using the CRE sequence in the mouse TREM-1 promoter and nuclear extracts from unstimulated RAW264.7 cells. (A) Nuclear extracts (5 μg) were incubated with a 32 P-labeled CRE oligonucleotide probe in the mouse TREM-1 promoter. For competition assays, 200-fold molar excess of unlabeled CRE consensus oligonucleotide was incubated with nuclear extracts as a competitor. For antibody supershift experiments, DNA probe/protein complexes were incubated with 1 μg of anti-CREB antibody (+ αCREB), anti-C/EBPα antibody (+ α-C/EBP) or normal rabbit IgG as control (IgG). Arrowheads indicate the specific bands of DNA-protein complexes, which can be abolished by excess of unlabeled CRE consensus oligonucleotide (+ CRE consensus). Asterisks indicate a band of DNA-protein complexes supershifted by the addition of antibody. (B) Nuclear extracts (5 μg) incubated with a FITC-labeled CRE oligonucleotide probe in the mouse TREM-1 promoter. For competition assays, 0.5–50-fold molar excess (× 0.5–50) of unlabeled CRE in the mouse TREM-1 promoter (+ CRE TREM-1), CRE consensus (+ CRE consensus) or C/EBPα consensus (+ CCAATT consensus) oligonucleotides were incubated with nuclear extract as competitors. Arrowheads indicate the specific bands of DNA-protein complexes, which can be abolished by excess of unlabeled competitors. Data are from one of 3 separate experiments.

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can be more strongly abrogated by the consensus C/EBPα oligonucleotide compared with the consensus CRE oligonucleotide (Fig. 4B). Thus, it is likely that transcription factors, possibly C/EBPα rather than CREB, can strongly bind to the CRE sequence of mouse TREM-1 promoter in unstimulated RAW264.7 cells. Next, we performed the gel shift/supershift analysis of the NF-κB-1 site in mouse TREM-1 promoter. When nuclear extracts from unstimulated cells were incubated with a FITC-labeled NF-κB-1 oligonucleotide (spanning −749 to −724 of the TREM-1 promoter), the two specific bands of DNA-protein complexes were detected, which can be abolished with the excess of unlabeled NF-κB consensus oligonucleotide (Fig. 5). Of note, addition of p50 antiserum supershifted the lower complex. Moreover, addition of anti-p65 antibody moderately suppressed the upper complex. These observations suggest that the upper complex contains both p65 and p50 (p65/p50 heterodimer), whereas the lower complex is made of p50 (p50/p50 homodimer), as previously reported for the DNA-protein complex observed using NF-κB oligonucleotides and nuclear extracts from macrophage-like cells (Gerondakis et al., 1999; Kastenbauer and Ziegler-Heitbrock, 1999). Thus, NF-κB p50/p50 homodimer and p65/p50 heterodimer are likely to bind to the NF-κB-1 site of the mouse TREM-1 promoter in unstimulated RAW264.7 cells. Lastly, we performed the gel shift/supershift analysis of AP-1-1 site using nuclear extracts from unstimulated/LPS-stimulated cells and a 32 P-labeled AP-1-1 oligonucleotide (spanning −913 to −892 of the TREM-1 promoter). When nuclear extracts from LPS-stimulated RAW264.7 cells were used, a single band was detected, and was abolished with excess amounts of unlabeled AP-1 consensus sequence (Fig. 6) as well as unlabeled AP-1-1 sequence of mouse TREM-1 promoter (data not shown). In contrast, essentially no band of DNAprotein complex was detected using nuclear extracts from unstimulated RAW264.7 cells (Fig. 6). Notably, the addition of anti-c-fos antibody or anti-c-jun antibody obviously supershifted the complex. These observations suggest that the AP-1-1-recognizing transcription factors are upregulated by LPS stimulation, and possibly c-fos and c-jun families of transcription factor(s) bind to the AP-1-1 site of mouse TREM-1 promoter in LPS-stimulated RAW264.7 cells.

*

Fig. 5. Gel shift assay of the NF-κB-1 site in the mouse TREM-1 promoter. Gel shift assay was performed using the NF-κB-1 sequence in the mouse TREM-1 promoter and nuclear extracts from unstimulated RAW264.7 cells. Nuclear extracts (2 μg) were incubated with a FITC-labeled NF-κB-1 oligonucleotide probe. For competition assays, 30-fold molar excess of unlabeled NF-κB consensus oligonucleotide (+ NF-κB consensus) was incubated with nuclear extract as a competitor. For antibody supershift experiments, DNA probe/protein complexes were incubated with 1 μg of anti-NF-κB p65 antibody (+ α-p65) or normal rabbit IgG (+ IgG), or 20 μg of anti-NF-κB p50 antiserum (+ α-p50) or normal rabbit serum (+serum). Arrowheads indicate the specific bands of DNA-protein complexes, which can be abolished by excess of unlabeled competitors. Asterisk indicates the band of DNA-protein complexes supershifted by the addition of antiserum. Data are from one of 3 separate experiments.

Resting

LPS-stimulated

* *

Fig. 6. Gel shift assay of the AP-1-1 site in the mouse TREM-1 promoter. Gel shift assay was performed using the AP-1-1 sequence in the mouse TREM-1 promoter and nuclear extracts from unstimulated and LPS-stimulated RAW264.7 cells. Nuclear extracts (5 μg) from unstimulated or LPS-stimulated cells were incubated with a 32P-labeled AP-1-1 oligonucleotide probe. For competition assays, 200-fold molar excesses of unlabeled AP-1 consensus oligonucleotide (+ AP-1 consensus) were incubated with nuclear extracts as a competitor. For antibody supershift experiments, DNA probe/protein complexes were incubated with 1 μg of anti-c-fos antibody (+α-c-fos), anti-c-jun antibody (+ α-c-jun) or normal rabbit IgG (+IgG). An arrowhead indicates the specific band of DNA-protein complexes, which can be abolished by excess of unlabeled competitors. Asterisk indicates a band of DNA-protein complexes supershifted by the addition of antibody. Data are from one of 3 separate experiments.

Discussion In the present study, we elucidated the regulatory mechanism for the basal and LPS-induced transcription of mouse TREM-1 gene in mononuclear cells by investigating the TREM-1 promoter activities (by luciferase reporter assay) and the transcription factors associated with the TREM-1 promoter sequences (by gel shift assay) in resting and LPS-stimulated RAW264.7 murine macrophage-like cells. Reporter analyses using 5′ deletion and adenine substitution mutants revealed that the CRE site is involved in the basal and LPSinduced transcriptional activity of the TREM-1 gene (Figs. 2 and 3). Moreover, gel shift/supershift analysis indicated that C/EBPα rather than CREB interacts with the CRE site of the promoter in resting cells. In addition, we confirmed that C/EBPα and CREB are expressed in both resting and LPS-stimulated RAW264.7 cells (Supplementary Fig. 1). Interestingly, it has been reported that C/EBPα can bind to the promoter sequence regardless of its phosphorylation level (Ross et al., 2004), whereas CREB can bind to the promoter sequence only in its phosphorylation form (Wang et al., 1999; Usukura et al., 2000). Reportedly, phosphorylated CREB is not detected in resting RWA264.7 cells, but detected in LPS-stimulated RAW264.7 cells (Park et al., 2005). Furthermore, LPS stimulation increases the level of cAMP in macrophages and activates protein kinase A, which phosphorylates CREB (Murakami et al., 2007). Thus, it is interesting to speculate that C/EBPα interacts with the CRE site to regulate the basal transcription of mouse TREM-1 gene in resting cells, whereas CREB is possibly phosphorylated by protein kinase A and binds to the CRE site to upregulate the TREM-1 transcription in response to LPS-stimulation. Reporter analysis using adenine substitution mutants revealed that the NF-κB-1 site negatively and positively regulates the basal and LPS-induced TREM-1 promoter activities, respectively (Fig. 3). Furthermore, the DNA-protein complexes containing NF-κB p50/p50 homodimer and p65/p50 heterodimer can be detected by gel shift/ supershift analysis using NF-κB-1 sequence and nuclear extracts from resting (Fig. 5) and LPS-stimulated RAW264.7 cells (data not shown). Importantly, it is reported that NF-κB p50/p50 homodimer negatively regulates the gene transcription by binding with the NF-κB sequence in the promoter, whereas NF-κB p65/p50 heterodimer positively modulates the transcription (Kastenbauer and Ziegler-Heitbrock, 1999; Zhong et al., 2002). Of note, NF-κB p50 is abundantly expressed in both resting and LPS-stimulated RAW264.7 cell, and the expression of NF-κB p65 is markedly increased by LPS stimulation

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(Supplementary Fig. 1). Together, these observations possibly suggest that NF-κB p50/p50 homodimer mostly interacts with the NF-κB-1 site to downregulate the basal TREM-1 transcription in resting cells, whereas NF-κB p65/p50 heterodimer binds to the NF-κB-1 site to upregulate the TREM-1 transcription in response to LPS-stimulation. This is supported by the previous findings that NF-κB p65 can bind with the NF-κB site of mouse TREM-1 promoter (by ChiP assay) in LPS-stimulated RWA264.7 cells, and that the deletion of NF-κB-1 site reduced the LPS responsiveness in the cells (by reporter assay) (Zeng et al., 2007). Reporter analyses using 5′ deletion and adenine substitution mutants revealed that the AP-1-1 site is involved in the LPS-induced transcriptional activity of the mouse TREM-1 gene (Figs. 2 and 3). Moreover, gel shift/supershift analysis indicated that c-fos and c-jun interact with the AP-1-1 site of the promoter in LPS-stimulated RAW264.7 cells (Fig. 6). Importantly, it has been reported that c-fos and c-jun are phosphorylated by ERK and JNK, respectively, via the stimulation with LPS, and that phosphorylated c-fos and c-jun can bind with the AP-1 sequence in the promoter to upregulate the gene transcription (Hibi et al., 1993; Deng and Karin, 1994). Notably, LPS stimulation strikingly increased the expression as well as the phosphorylation of c-fos and c-jun in RAW264.7 cells (Supplementary Fig. 1). Together, these observations likely indicate that c-fos and c-jun phosphorylated by ERK and JNK interacts with the AP-1-1 site to upregulate the transcription of mouse TREM-1 gene in LPS-stimulated RAW264.7 cells. In contrast to our conclusion, Zeng et al. reported that AP-1 is not involved in the LPS-induced transcription of TREM-1, based on the results obtained with luciferase assay using mouse TREM-1 promoter and TLR4/MD-2-overexpressing human HEK293 cells (2007). The HEK293 cell originates from the human embryonic kidney cell, and does not express CD14, TLR4 and MD-2, the components of LPS receptor (Da Silva Correia et al., 2003). In the present study, we investigated the transcriptional regulation of TREM-1 using mouse TREM-1 promoter and mouse macrophage-like RAW 264.7 cells that express all the components of LPS receptor (CD14, TLR4 and MD-2) (Nagaoka et al., 2001; Shimura et al., 2004). Thus, it is easily expected that the LPS-medicated transcriptional response is different between CD14/TLR4/MD-2-expressing mouse RAW264.7 and TLR-4/MD-2expressing but CD14-unexpressing human HEK293 cells to mouse TREM-1 promoter. It is interesting to note that silencing or blocking of TREM-1 represses the cytokine production, thereby prolonging the survival of animal models with bacterial sepsis (Bouchon et al., 2001; Gibot et al., 2006a; Gibot et al., 2007). Thus, the present findings on the regulatory mechanisms for the TREM-1 gene transcription further raise a possibility that inflammatory disorders including sepsis could be regulated by the modulation of the expression of TREM-1, an amplifying molecule in inflammation. Conclusion In the present study, we elucidated the regulatory mechanism for the basal and LPS-induced transcription of mouse TREM-1 gene in mononuclear cells using RAW264.7 cells. In summary, it is suggested that C/EBPα and NF-κB p50/p50 homodimer are positively and negatively regulating the basal TREM-1 transcription via the interaction with the CRE and NF-κB-1, respectively. In addition, phosphorylated CREB and NF-κB p65/p50 heterodimer may participate in the LPSinduced upregulation of TREM-1 promoter activity possibly via the interaction with the CRE and NF-κB-1. Furthermore, phosphorylated c-fos/c-jun are likely to be involved in the LPS-induced TREM-1 transcription via the interaction with the AP-1-1. The present study has demonstrated for the first time the detailed mechanism for the basal and LPS-induced expression of TREM-1, an amplifying molecule in inflammation. This finding provides a novel

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insight into the role of TREM-1 in inflammation and raises a possibility that inflammatory disorders could be regulated by the modulation of the transcription/expression of TREM-1gene, one of the therapeutic targets in inflammation. Supplementary materials related to this article can be found online at doi:10.1016/j.lfs.2011.05.007. Conflict of interest statement The authors declare that they have no competing interests.

Acknowledgments This work was supported in part by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science, and a Grants-in-Aid for 21st Century COE Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, Seikagaku Biobusiness Corporation, Japan and Dainippon Sumitomo Pharma Co., Ltd., Japan.

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