Species-specific Regulation of Toll-like Receptor 3 Genes in Men and Mice
2003; Elsevier BV; Volume: 278; Issue: 24 Linguagem: Inglês
10.1074/jbc.m301476200
ISSN1083-351X
AutoresSven Heinz, Viola Haehnel, Marina Karaghiosoff, Lucia Schwarzfischer, Mathias Müller, Stefan W. Krause, Michael Rehli,
Tópico(s)Estrogen and related hormone effects
ResumoToll-like receptor 3 (TLR3) belongs to a family of evolutionary conserved innate immune recognition molecules and recognizes double-stranded RNA, a molecular pattern associated with viral infections. Earlier studies suggested a differential expression pattern in men and mice; the molecular basis for this observation, however, was unknown. Here we demonstrate that species-specific differences in tissue expression and responses to lipopolysaccaride (LPS) coincide with the presence of different, evolutionary non-conserved promoter sequences in both species. Despite the overall unrelatedness of TLR3 promoter sequences, mRNA expression of both TLR3 orthologues was induced by interferons, particularly by interferon (IFN)-β. The basal and IFN-β-induced activation of promoters from both species largely depended on similar interferon regulatory factor (IRF) elements, which constitutively bound IRF-2 and recruited IRF-1 after stimulation. In murine macrophages, IFN-β-induced TLR3 up-regulation required IFNAR1, STAT1, and in part IRF-1, but not the Janus kinase (Jak) family member Tyk2. We also show that LPS specifically up-regulates TLR3 expression in murine cells through the induction of autocrine/paracrine IFN-β. In humans, however, IFN-β-induced up-regulation of TLR3 was blocked by pretreatment with LPS, despite the efficient induction of IRF-1. Our findings reveal a mechanistic basis for the observed differences as well as similarities in TLR3 expression in men and mice. The IFN-β-TLR3 link further suggests a role of TLR3 in innate and adaptive immune responses to viral infections. It will be interesting and important to clarify whether the observed differences in the transcriptional regulation of TLR3 influence innate immune responses in a species-specific manner. Toll-like receptor 3 (TLR3) belongs to a family of evolutionary conserved innate immune recognition molecules and recognizes double-stranded RNA, a molecular pattern associated with viral infections. Earlier studies suggested a differential expression pattern in men and mice; the molecular basis for this observation, however, was unknown. Here we demonstrate that species-specific differences in tissue expression and responses to lipopolysaccaride (LPS) coincide with the presence of different, evolutionary non-conserved promoter sequences in both species. Despite the overall unrelatedness of TLR3 promoter sequences, mRNA expression of both TLR3 orthologues was induced by interferons, particularly by interferon (IFN)-β. The basal and IFN-β-induced activation of promoters from both species largely depended on similar interferon regulatory factor (IRF) elements, which constitutively bound IRF-2 and recruited IRF-1 after stimulation. In murine macrophages, IFN-β-induced TLR3 up-regulation required IFNAR1, STAT1, and in part IRF-1, but not the Janus kinase (Jak) family member Tyk2. We also show that LPS specifically up-regulates TLR3 expression in murine cells through the induction of autocrine/paracrine IFN-β. In humans, however, IFN-β-induced up-regulation of TLR3 was blocked by pretreatment with LPS, despite the efficient induction of IRF-1. Our findings reveal a mechanistic basis for the observed differences as well as similarities in TLR3 expression in men and mice. The IFN-β-TLR3 link further suggests a role of TLR3 in innate and adaptive immune responses to viral infections. It will be interesting and important to clarify whether the observed differences in the transcriptional regulation of TLR3 influence innate immune responses in a species-specific manner. Vertebrate Toll-like receptors (TLRs) 1The abbreviations used are: TLR, Toll-like receptor; IFN, interferon; IRF, interferon regulatory factor; ISRE, IFN-stimulated response element; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; RLM-RACE, RNA ligase-mediated rapid amplification of cDNA ends; Jak, Janus kinase; STAT, signal transducers and activators of transcription. recognize conserved microbial structures (pathogen-associated molecular patterns). Upon ligation they activate conserved intracellular signaling pathways, leading to the up-regulation of co-stimulatory molecules or the secretion of cytokines, responses that are necessary to combat infection in vertebrates (as reviewed in Refs. 1Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Crossref PubMed Scopus (2632) Google Scholar and 2Akira S. Curr. Opin. Immunol. 2003; 15: 5-11Crossref PubMed Scopus (478) Google Scholar). Two recent studies have implicated Toll-like receptor 3 (TLR3) in the recognition of double-stranded RNA, a molecular pattern associated with viral infections (3Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4959) Google Scholar, 4Matsumoto M. Kikkawa S. Kohase M. Miyake K. Seya T. Biochem. Biophys. Res. Commun. 2002; 293: 1364-1369Crossref PubMed Scopus (382) Google Scholar). Activation of the receptor with a chemical analog of double-stranded RNA, polyriboinosine-polyribocytidylic acid (poly(I:C)), was shown to induce the activation of NF-κB and the production of type I interferons (IFNs) in human and murine TLR3 expressing cell types (3Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4959) Google Scholar, 4Matsumoto M. Kikkawa S. Kohase M. Miyake K. Seya T. Biochem. Biophys. Res. Commun. 2002; 293: 1364-1369Crossref PubMed Scopus (382) Google Scholar). TLR3-deficient (TLR3–/–) mice exhibited reduced responses to poly(I:C) and reduced production of the inflammatory cytokines interleukin-6, interleukin-12, and tumor necrosis factor (3Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4959) Google Scholar). A contribution of TLR3 to antiviral immunity, however, remains to be demonstrated. Interestingly, different TLR3 expression patterns have been reported in mice and humans, a phenomenon also observed for at least three other TLR family members: TLR2, TLR4, and TLR9 (for a review, see Ref. 5Rehli M. Trends Immunol. 2002; 23: 375-378Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Within the human hematopoietic compartment, TLR3 mRNA expression has been shown to be restricted to dendritic cells (6Muzio M. Bosisio D. Polentarutti N. D'amico G. Stoppacciaro A. Mancinelli R. van't Veer C. Penton-Rol G. Ruco L.P. Allavena P. Mantovani A. J. Immunol. 2000; 164: 5998-6004Crossref PubMed Scopus (905) Google Scholar), as was the cytokine response to poly(I:C) stimulation (7Kadowaki N. Ho S. Antonenko S. Malefyt R.W. Kastelein R.A. Bazan F. Liu Y.J. J. Exp. Med. 2001; 194: 863-869Crossref PubMed Scopus (1691) Google Scholar). In mice, TLR3 is also strongly expressed in macrophages and its expression is markedly induced upon LPS stimulation, a feature that has not been observed in human cells (3Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4959) Google Scholar). Since the differential expression pattern of TLR3 could have a significant impact on TLR3 function in humans and mice, we have further characterized TLR3 expression and analyzed gene regulatory mechanisms acting in both species. We found that the non-coding regions, including 5′-exons and proximal promoter regions of TLR3 genes are different in both species, as is the cell type specificity and the regulated expression upon stimulation with LPS. However, despite the overall unrelatedness of promoter sequences, both species share the up-regulation of TLR3 by IFN-β. Evolutionary conservation of TLR3 induction by interferons indicate that this regulatory feature may be important during viral infections in both species. Chemicals—All chemical reagents used were purchased from Sigma (Berlin, Germany) unless otherwise noted. Protease inhibitors are from Roche Molecular Biochemicals. Purified lipopolysaccharide (LPS) from Salmonella abortus equi (LPSSAE) was a gift from C. Galanos (Max-Planck-Institut fuer Immunologie, Freiburg, Germany). LPS from Salmonella minnesota (LPSSM) and Escherichia coli (serotype 055:B5; LPSEC) were purchased from Sigma. Oligonucleotides were synthesized by TIB Molbiol (Berlin, Germany). Antisera for supershift analyses were purchased from Santa Cruz. Mice—The mutant mouse strain deficient in Tyk2 was generated by gene targeting, as described previously (8Karaghiosoff M. Neubauer H. Lassnig C. Kovarik P. Schindler H. Pircher H. McCoy B. Bogdan C. Decker T. Brem G. Pfeffer K. Muller M. Immunity. 2000; 13: 549-560Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). To obtain a pure C57BL6 background Tyk2-deficient mice were backcrossed with C57BL6 mice for eight generations. IFN-β-, IFNAR1-, STAT1-, and IRF-1-deficient mice (C57BL6 background) have been described previously (9Erlandsson L. Blumenthal R. Eloranta M.L. Engel H. Alm G. Weiss S. Leanderson T. Curr. Biol. 1998; 8: 223-226Abstract Full Text Full Text PDF PubMed Google Scholar, 10Matsuyama T. Kimura T. Kitagawa M. Pfeffer K. Kawakami T. Watanabe N. Kundig T.M. Amakawa R. Kishihara K. Wakeham A. et al.Cell. 1993; 75: 83-97Abstract Full Text PDF PubMed Scopus (558) Google Scholar, 11Durbin J.E. Hackenmiller R. Simon M.C. Levy D.E. Cell. 1996; 84: 443-450Abstract Full Text Full Text PDF PubMed Scopus (1304) Google Scholar, 12Muller U. Steinhoff U. Reis L.F. Hemmi S. Pavlovic J. Zinkernagel R.M. Aguet M. Science. 1994; 264: 1918-1921Crossref PubMed Scopus (2021) Google Scholar). Wild-type C57BL6 and Balb/c mice were obtained from Charles River. Cells—Monocytes were isolated and cultured to generate macrophages or immature dendritic cells as described earlier (13Heinz S. Krause S.W. Gabrielli F. Wagner H.M. Andreesen R. Rehli M. Genomics. 2002; 79: 608-615Crossref PubMed Scopus (20) Google Scholar). The human monocytic cell line THP-1 and the murine macrophage cell line RAW264.7 were maintained in RPMI 1640 medium plus 10% fetal calf serum and supplements. THP-1 cells were differentiated for 48–72h by adding PMA (10–8m) to the culture medium. To prepare peritoneal macrophages, mice (12 weeks of age) were injected intraperitoneally with 2 ml of 4% thioglycollate medium (Difco). Three days later, peritoneal exudate cells were isolated from the peritoneal cavity by washing with phosphate-buffered saline solution (Invitrogen). Cells were cultured in endotoxin-free Dulbecco's modified Eagle's medium, 5% fetal calf serum (Invitrogen) and after 1 h medium was changed to remove non-adherent cells. Adherent monolayer cells were used as peritoneal macrophages. Peritoneal macrophages were cultured in Dulbecco's modified Eagle's medium medium (Invitrogen) supplemented with 5% fetal calf serum (Invitrogen). Murine bone marrow-derived macrophages were cultured as described previously (14Ross I.L. Yue X. Ostrowski M.C. Hume D.A. J. Biol. Chem. 1998; 273: 6662-6669Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). RNA Preparation, Real-time PCR—Total RNA was isolated from different cell types by the guanidine thiocyanate/acid phenol method (15Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). RNA (1 μg) was reverse transcribed using Superscript II MMLV-RT (Invitrogen). Real-time PCR was performed on a Lightcycler (Roche Molecular Biochemicals) using the Quantitect kit (Qiagen) according to the manufacturer's instructions. Primers used were: TLR3 (specific for human and mouse): sense, 5′-TCA CTT GCT CAT TCT CCC TT-3′; antisense, 5′-GAC CTC TCC ATT CCT GGC-3′. TLR2 (mouse): sense, 5′-TTC TGA GTG TAG GGG CTT C-3′; antisense, 5′-CCC AGA AGC ATC ACA TGA C-3′. β-Actin: sense, 5′-TGA CGG GGT TCA CCC ACA CTG TGC CCA TCT A-3′; antisense, 5′-CTA GAA GCA TTT GTG GTG GAC GAT GGA GGG-3′. Cycling parameters were: denaturation 95 °C, 15 min, amplification 95 °C, 15 s, 56 °C, 20 s, 72 °C, 25 s, for 45 cycles. The product size was initially controlled by agarose gel electrophoresis, and melting curves were analyzed to control for specificity of the PCR reactions. TLR data were normalized for expression of the housekeeping gene β-actin. The relative units were calculated from a standard curve plotting three different concentrations of log dilutions against the PCR cycle number (CP) at which the measured fluorescence intensity reaches a fixed value. The amplification efficiency E was calculated from the slope of the standard curve by the formula: E = 10–1/slope. ETLR3 was in the range of 1.78 to 2.14, and ETLR2 ranged from 1.76 to 1.84. For each sample, data from three independent analyses were averaged. RNA Ligase-mediated RACE-PCR—Ten μg of total RNA from monocyte-derived dendritic cell or LPS-stimulated murine bone marrow-derived macrophages were used for cDNA synthesis with the First-Choice™ RLM-RACE kit (Ambion). The following TLR3-specific primers were used to amplify full-length 5′-cDNA fragments of human or murine TLR3, respectively: hTLR3-OUT (5′-TGT GAA GTT GGC GGC TGG-3′) and hTLR3-IN (5′-CAG GTG GCT GCA GTC AGC AAC-3′), mTLR3-OUT (5′-GTC AGC TAC GTT GTA TCT CAC AGT G-3′) and mTLR3-IN1 (5′-ACA CCC TTT CAT GAT TCA GCC-3′) or mTLR3-IN2 (5′-ACA CCA GAA TCC ATA GGG AC-3′). PCR products from both species were cloned into pCR2.1-TOPO (TOPO Cloning kit, Invitrogen), and inserts from at least 10 individual plasmid-containing bacterial colonies derived from each cell type were re-amplified by PCR and directly sequenced (performed by Geneart, Regensburg, Germany). Plasmid Construction and Purification—A 588-bp genomic fragment of the human TLR3-promoter was amplified from human genomic DNA using the Expand High Fidelity PCR system (Roche Molecular Biochemicals) and the primers hTLR3p_S (5′-GAT CAG ATC TCA GCT TTG CCA TGT TTG G-3′) and hTLR3p_AS (5′-ACG TGA ATT CTG TTG GAT GAC TGC TAG CCT TTC C-3′). Primer sequences were derived from a BAC clone containing the TLR3 sequence deposited in the GenBank™ data base (GenBank™ accession number AC104070). The obtained PCR fragment was subcloned into pGL3-B (Promega) and sequenced. Deletions of the hTLR3(–588) construct were generated by PCR using primers hTLR3(–400) _S (5′-GTC AAG ATC TTC GCA TGA GTC TAG CAG-3′) or hTLR3(–200) _S (5′-GAC TAG ATC TGG TTT GAA ACG CCT CTC TG-3′) together with the vector-specific primer GL2 (Invitrogen). Two fragments of the proximal murine TLR3 promoter (including intron 1 and exon 2) were similarly amplified from mouse genomic DNA using the primers mtlr3-F1_S (5′-TGC AAG ATC TGA GTG TAG CCA TGA GCC AGG-3′) and mtlr3-F2_AS (5′-CAT CAA GCT TCT ATC TTC TTT TGG TGC GCG-3′). Deletions of the resulting mtlr3(–1368) construct were generated using the internal SacI (–966) or EcoR I (–429) restriction sites. Mutations of putative transcription factor binding sites were carried out by PCR-mediated mutagenesis using the following primers: human ISRE/IRF element: htlr3IRF-M_S (5′-TTT TCA AGC TTT ACA CGC ACT TTC GAG AGT G-3′) and htlr3IRF-M_AS (5′-CAC TCT CGA AAG TGC GTG TAA AGC TTG AAA A-3′); human STAT element: htlr3STAT-M_S (5′-CCT TTG CCC TTC TTA TGA TGC ACC AAA CAT AA-3′) and htlr3STAT-M_AS (5′-TTA TGT TTG GTG CAT CAT AAG AAG GGC AAA GG-3′); murine exon 2 ISRE/IRF element: mtlr3IRFi-M_S (5′-CTC TCT CAA CTT AAG ACG CAC TTT CAG GCT GA-3′) and mtlr3IRFi-M_AS (5′-TCA GCC TGA AAG TGC GTC TTA AGT TGA GAG AG-3′); murine intron 1 ISRE/IRF element: mtlr3IRFo-M_S (5′-GGT AAG TGA ATG GCA CGC ACT TTG TTT AGA CA-3′) and mtlr3IRFo-M_AS (5′-TGT CTA AAC AAA GTG CGT GCC ATT CAC TTA CC-3′). DNA sequence analysis was performed by Geneart. For transfections, plasmids were isolated and purified using the Endofree Plasmid Kit from Qiagen. Transient and Stable DNA Transfections—THP-1 cells were transfected in duplicates using DEAE-dextran as described previously (16Rehli M. Poltorak A. Schwarzfischer L. Krause S.W. Andreesen R. Beutler B. J. Biol. Chem. 2000; 275: 9773-9781Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Undifferentiated THP-1 cells were cultivated for 48 h before harvesting. In stimulation experiments, THP-1 cells were treated with PMA the day after transfection and harvested after 72 h. Cell lysates were assayed for firefly and renilla luciferase activity using the dual-luciferase reporter assay system (Promega) on a Sirius luminometer (Berthold). Firefly luciferase activity of individual transfections was normalized against renilla luciferase activity. RAW264.7 cells were transfected using SuperFect reagent (Qiagen) according to the manufacturer's instructions as described previously (17Means T.K. Wang S. Lien E. Yoshimura A. Golenbock D.T. Fenton M.J. J. Immunol. 1999; 163: 3920-3927Crossref PubMed Google Scholar). Duplicate transfections were harvested after 24 h and cell lysates assayed for firefly luciferase activity using the luciferase reporter assay system (Promega). Firefly luciferase activity of individual transfections was normalized against protein concentration measured using a BCA assay (Sigma). For stimulation experiments, RAW264.7 cells were transfected in 10 cm tissue culture dishes as above using linearized reporter constructs (10 μg) as well as a plasmid (pcDNA3) carrying the neomycin resistance gene (5 μg). Cells were selected for stable integration of plasmid DNA by culturing cells in RPMI 1640 medium supplemented with 350 μg/ml G418 for 2–3 weeks. Stably transfected cells were pooled, expanded, and 7.5 × 105 cells/ml were seeded into six-well plates in duplicates the day before stimulation. Cells were harvested at the indicated timepoints, and cell lysates were assayed as described above. Nuclear Extracts and Electrophoretic Mobility Shift Assay—Nuclear extracts were prepared as described previously (16Rehli M. Poltorak A. Schwarzfischer L. Krause S.W. Andreesen R. Beutler B. J. Biol. Chem. 2000; 275: 9773-9781Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Double-stranded oligonucleotides corresponding to the STAT or IRF elements were labeled with [α-32P]dGTP using Klenow DNA polymerase. Sequences of IRF motifs are indicated in the figures; sequences of other oligonucleotides were: human STAT/GAS element, human STAT motif (5′-CTT TGC CCT TCT TGG AAT GCA CCA-3′) and mutated human STAT motif (5′-CTT TGC CCT TCT Tat gAT GCA CCA-3′); consensus GAS-element: 5′-CTT TGC ATT TCC CCG AAA TCA CCA-3′. The binding reaction contained 2.5 μg of nuclear extract protein, 0.5 μg of poly-(dI-dC), 20 mm HEPES, pH 7.9, 20 mm KCl, 1 mm dithiothreitol, 1 mm EDTA, pH 8.0, 5% glycerol, and 20 nmol of probe DNA in a final volume of 10 μl. Antisera used in supershift analyses were added after 15 min, and samples were loaded onto polyacrylamide gels after incubating at room temperature for a total of 30 min. Buffers and running conditions used have been described previously (16Rehli M. Poltorak A. Schwarzfischer L. Krause S.W. Andreesen R. Beutler B. J. Biol. Chem. 2000; 275: 9773-9781Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Gels were fixed in 5% acetic acid, dried, and autoradiographed. TLR3 mRNA Expression in Murine and Human Mononuclear Phagocytes—Recently published data suggest a differential expression pattern of TLR3 in human and murine mononuclear cells (5Rehli M. Trends Immunol. 2002; 23: 375-378Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). To directly compare the mRNA expression of TLR3 in human and murine cells, we designed PCR primers complementary to TLR3 sequences, which are identical in both species. Real-time PCR using various mononuclear cell types from both species confirmed the previously observed predominant expression of human TLR3 in "immature" myeloid dendritic cells (see Fig. 1). Highest TLR3 expression levels in mice, however, were detectable in macrophages, which expressed the highest TLR3 mRNA levels of all tested cell types. These results indicated that cell type-specific regulation of TLR3 might be different between the two species. Species-specific TLR3 Regulation in Response to LPS or IFNs—Previously published data indicated a differential, species-specific response in LPS-stimulated cells regarding TLR3 expression (3Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4959) Google Scholar, 6Muzio M. Bosisio D. Polentarutti N. D'amico G. Stoppacciaro A. Mancinelli R. van't Veer C. Penton-Rol G. Ruco L.P. Allavena P. Mantovani A. J. Immunol. 2000; 164: 5998-6004Crossref PubMed Scopus (905) Google Scholar, 18Visintin A. Mazzoni A. Spitzer J.H. Wyllie D.H. Dower S.K. Segal D.M. J. Immunol. 2001; 166: 249-255Crossref PubMed Scopus (515) Google Scholar). Additionally, TLR3 expression in human macrophages had been shown to be markedly induced upon stimulation with IFN-α (19Miettinen M. Sareneva T. Julkunen I. Matikainen S. Genes Immun. 2001; 2: 349-355Crossref PubMed Scopus (240) Google Scholar). To systematically compare the inducible expression patterns of TLR3 in both species, TLR3-expressing monocytic cells from both species were analyzed for TLR3 expression after stimulation with LPS, as well as type I or II interferons (IFN-β and IFN-γ, respectively), the latter being important mediators during viral infections. As shown in Fig. 2, all three stimuli induced TLR3 mRNA in murine bone marrow-derived macrophages after 4 h, with TLR3 being down-regulated to basal levels after 24 h. In the murine RAW264.7 macrophage cell line, again, all three stimuli up-regulated TLR3 mRNA at 4 h. In contrast to bone marrow-derived macrophages, IFN-γ led to a further increase in TLR3 expression after 24 h in RAW264.7 cells. In human monocyte-derived cell types, a consistent up-regulation of TLR3 expression was only observed in the presence of IFN-β. Shown are two representative examples (out of four) for monocyte-derived dendritic cells from different donors. While expressing different basal levels of TLR3 mRNA, primary blood monocytes and monocyte-derived macrophages up-regulated TLR3 in a comparable fashion in response to IFN-β (data not shown). The response to IFN-γ in human cells was variable depending on the donor. While the human monocytic cell lines MonoMac6 and THP-1 proved unresponsive under normal culture conditions, TLR3 mRNA was IFN-β-inducible in PMA-differentiated macrophage-like THP-1 cells (data not shown). In contrast to murine TLR3, we never observed a significant up-regulation of human TLR3 in response to LPS. Determination of Transcriptional Start Sites and Proximal Promoters—To further analyze the regulatory mechanisms of TLR3 expression in both species, we determined TLR3 transcriptional start sites by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) PCR using total RNA derived from LPS-stimulated murine BM-derived macrophages or human monocyte-derived dendritic cells as described under "Experimental Procedures." Comparison of the obtained 5′-cDNA sequences and publicly available genomic sequences revealed the complete structures of the human and murine TLR3 genes (see Fig. 3). The translation start codon of human TLR3 is located in exon II, whereas the murine coding sequence begins in exon IV. Murine TLR3 mRNA can be initiated from two alternative promoter regions preceding exon I or exon II, respectively, the latter being predominantly utilized in LPS-stimulated BM-derived macrophages. While the nucleotide sequences around coding regions of human and murine TLR3 share a high degree of homology (75% identity), sequence comparison using the ClustalW algorithm did not reveal a significant level of homology (3–16% identical nucleotides) between the proximal promoter regions and 5′-untranslated regions of murine and human TLR3 genes. Computational analysis revealed a differential organization of the proximal promoter sequences from both species. Whereas the human gene contains a TATA-like element, the murine promoters are TATA-less and instead contain several putative binding sites for the myeloid and B-cell-specific transcription factor PU.1 (sequences and structures of the proximal promoter regions are shown in Fig. 3). To facilitate further analysis of mechanisms regulating the differential expression pattern of TLR3 in humans and mice, we cloned fragments of the 5′-proximal promoter regions of both TLR3 genes into a luciferase reporter plasmid. Transient transfection analysis was performed in the human monocytic cell line THP-1 and the murine macrophage cell line RAW 264.7 to determine the basal activity of proximal TLR3 promoter regions. As shown in Supplemental Fig. 8, human TLR3 promoter constructs were weakly active in both cell lines. Constructs containing either both alternative murine promoters or the downstream promoter alone were strongly active in murine macrophages, but only weakly in the human monocytic cell line, suggesting that the proximal promoters display different activities in human and murine cell lines. TLR3 Promoter Elements and Signaling Molecules Involved in IFN Signaling—Initial transfection experiments indicated that the proximal TLR3 promoter regions from both species are responsive to IFN-β treatment in both human and murine cells. To determine cis-elements required for IFN-β-mediated induction of promoter activity, we mutated putative binding sites that could be involved in interferon-regulated gene responses, including two putative ISRE/IRF elements in the mouse promoter and an ISRE/IRF as well as a STAT element in the human promoter (see Fig. 4A). In human PMA-differentiated THP-1 cells, wild-type TLR3 constructs of both species significantly responded to IFN-β after 24 h, while only a marginal induction of promoter activity was observed after 4 h. Mutation of the putative ISRE/IRF element in the human promoter abolished both basal and induced activity. Mutation of the nearby human STAT site reduced basal activity to ∼50% and also abrogated the IFN-β-induced activation (Fig. 4B). In murine RAW264.7 cells, promoters from both species were activated by IFN-β after 4 h. Induction of both promoters by IFN-γ was delayed as compared with IFN-β. Promoters were only weakly induced by LPS in transfection experiments performed in RAW264.7 cells (Fig. 4C). These results were similar to those obtained for endogenous TLR3 expression in RAW264.7 cells upon stimulation (see Fig. 2B). Mutation of the inner ISRE/IRF element drastically reduced the basal activity of the murine TLR3 promoter in RAW264.7 macrophages (Fig. 4D) and resulted in complete loss of IFN-β-induced up-regulation of promoter activity (Fig. 4E). Mutation of an upstream ISRE/IRF element had no effect on the basal and the IFN-β induction of the TLR3 promoter (Fig. 3, D and E). Above results suggest that despite the overall unrelatedness of TLR3 promoter structures in mouse and man, promoters of both species contain functionally important ISRE/IRF elements, which are similar in sequence and located close to the transcription start sites. To identify the nuclear factors binding these elements under basal and induced conditions, gel shift experiments were performed using nuclear extracts of IFN-β-treated and untreated human dendritic cells and murine RAW264.7 macrophages. As demonstrated by competition and supershift assays (shown in Fig. 5), IRF-2 constitutively bound the IRF motif of human and murine origin, whereas IRF-1 was recruited after stimulation in both human and murine cells. Antibodies against other IRF family members (IRF-3, -4, -7, -8, and -9) or STAT proteins (STAT1–6) did not change the observed band pattern in gel shift assays (data not shown), indicating that IRF-1 and IRF-2 are the major factors binding IRF sites in both species. In initial co-transfection studies, both IRF-1 and IRF-2 were able to transactivate the human promoter in HT-29 cells (data not shown). We also performed gel shift assays to identify the nuclear proteins binding to the human STAT motif. The STAT motif specifically competed with the binding of STAT1 to a known STAT1 (GAS) binding site, indicating that STAT1 may be able to bind this site. However, we were unable to detect binding of STAT1 directly to the human STAT motif in gel shift assays (data not shown). For the murine gene, the role of several interferon signaling components in IFN-β induced TLR3 up-regulation, including the IFN-α/β receptor (IFNAR1), STAT1, Tyk2, as well as IRF-1, was analyzed in either peritoneal macrophages or bone marrow-derived macrophages from knock-out mice lacking the respective genes. As shown in Fig. 6, up-regulation of TLR3 by IFN-β in peritoneal macrophages depends on IFNARI, but not on the Jak family member Tyk2. The induction of TLR3 in bone marrow-derived macrophages also depended on STAT1 as well as IRF-1, albeit to a lesser degree, indicating that other family members may compensate for IRF-1 deficiency.Fig. 5Binding of nuclear proteins to the proximal human and murine IRF sites.A, sequence alignment of consensus (cons.) ISRE and IRF sites and murine and human TLR3 promoter sequences. B and C, labeled human IRF (hIRF) or murine IRF (mIRF) oligonucleotide was used in electrophoretic mobility shift assay with nuclear proteins from human dendritic cell (B) or RAW264.7 macrophages (C) untreated (0 h) or treated with IFN-β for 2 h. Addition of unlabeled oligonucleotides for competition analysis (lanes 2–5 and 7–10 in C) or antisera against IRF family transcription factors (lanes 3–5 and 7–10 in B and lanes 13–15 and 17–19 in C) is indicated above each lane. IRF-1- and IRF-2-containing complexes are marked with arrows, antibody supershifts with "SS," and unspecific complexes with an asterisks. NE, nuclear extract; DC, dendritic cell.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Analysis of signaling molecules involved in TLR3 mRNA
Referência(s)