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• W2034307611 abstract "CpG oligodeoxynucleotides (CpG-ODNs), mimicking bacterial DNA, stimulate osteoclastogenesis via Toll-like receptor 9 (TLR9) in receptor activator of NF-κB ligand (RANKL)-primed osteoclast precursors. This activity is mediated via tumor necrosis factor (TNF)-α induction by CpG-ODN. To further reveal the role of the cytokine in TLR9-mediated osteoclastogenesis, we compared the ability of CpG-ODN to induce osteoclastogenesis in two murine strains, BALB/c and C57BL/6, expressing different TNF-α alleles. The induction of osteoclastogenesis and TNF-α release by CpG-ODN was by far more noticeable in BALB/c-derived than in C57BL/6-derived osteoclast precursors. Unexpectedly, as revealed by Northern analysis, CpG-ODN induction of TNF-α mRNA increase was more efficient in C57BL/6-derived cells. The cytokine transcript abundance was increased due to both increased message stability and rate of transcription. The difference between the two cell types was the result of a higher transcription rate in CpG-ODN-induced C57BL/6-derived cells caused by a single nucleotide polymorphism in κB2a site within the TNF-α promoter sequence. CpG-ODN enhanced the rate of the cytokine translation in BALB/c-derived cells. Thus, CpG-ODN modulated both transcription and translation of TNF-α. The induction of transcription was more evident in C57BL/6-derived cells, while the induction of translation took place only in BALB/c-derived osteoclast precursors. Altogether the cytokine was induced to a larger extent in BALB/c-derived osteoclast precursors, consistent with the increased CpG-ODN osteoclastogenic effect in these cells. CpG oligodeoxynucleotides (CpG-ODNs), mimicking bacterial DNA, stimulate osteoclastogenesis via Toll-like receptor 9 (TLR9) in receptor activator of NF-κB ligand (RANKL)-primed osteoclast precursors. This activity is mediated via tumor necrosis factor (TNF)-α induction by CpG-ODN. To further reveal the role of the cytokine in TLR9-mediated osteoclastogenesis, we compared the ability of CpG-ODN to induce osteoclastogenesis in two murine strains, BALB/c and C57BL/6, expressing different TNF-α alleles. The induction of osteoclastogenesis and TNF-α release by CpG-ODN was by far more noticeable in BALB/c-derived than in C57BL/6-derived osteoclast precursors. Unexpectedly, as revealed by Northern analysis, CpG-ODN induction of TNF-α mRNA increase was more efficient in C57BL/6-derived cells. The cytokine transcript abundance was increased due to both increased message stability and rate of transcription. The difference between the two cell types was the result of a higher transcription rate in CpG-ODN-induced C57BL/6-derived cells caused by a single nucleotide polymorphism in κB2a site within the TNF-α promoter sequence. CpG-ODN enhanced the rate of the cytokine translation in BALB/c-derived cells. Thus, CpG-ODN modulated both transcription and translation of TNF-α. The induction of transcription was more evident in C57BL/6-derived cells, while the induction of translation took place only in BALB/c-derived osteoclast precursors. Altogether the cytokine was induced to a larger extent in BALB/c-derived osteoclast precursors, consistent with the increased CpG-ODN osteoclastogenic effect in these cells. Bacterial products are the cause of pathological bone loss in a variety of diseases including periodontitis, osteomyelitis, bacterial arthritis, and infected metal orthopedic implant failure (1Nair S.P. Meghji S. Wilson M. Reddi K. White P. Henderson B. Infect. Immun. 1996; 64: 2371-2380Crossref PubMed Google Scholar, 2Orcel P. Feuga M. Bielakoff J. De Vernejoul M.C. Am. J. Physiol. 1993; 264: E391-E397PubMed Google Scholar, 3Millar S.J. Goldstein E.G. Levine M.J. Hausmann E. Infect. Immun. 1986; 51: 302-306Crossref PubMed Google Scholar, 4Ito H.O. Shuto T. Takada H. Koga T. Aida Y. Hirata M. Koga T. Arch. Oral Biol. 1996; 41: 439-444Crossref PubMed Scopus (37) Google Scholar, 5Bi Y. Seabold J.M. Kaar S.G. Ragab A.A. Goldberg V.M. Anderson J.M. Greenfield E.M. J. Bone Miner. Res. 2001; 16: 2082-2091Crossref PubMed Scopus (193) Google Scholar). Bacterial factors have been studied extensively for their ability to activate the innate immune system via Toll-like receptors (TLRs) 1The abbreviations used are: TLR, Toll-like receptor; ODN, oligodeoxynucleotide; RANKL, receptor activator of NF-κB ligand; ERK, extracellular signal-regulated kinase; BMM, bone marrow macrophage; BFA, brefeldin A; TNF, tumor necrosis factor; M-CSF, macrophage colony-stimulating factor; FCS, fetal calf serum; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay. 1The abbreviations used are: TLR, Toll-like receptor; ODN, oligodeoxynucleotide; RANKL, receptor activator of NF-κB ligand; ERK, extracellular signal-regulated kinase; BMM, bone marrow macrophage; BFA, brefeldin A; TNF, tumor necrosis factor; M-CSF, macrophage colony-stimulating factor; FCS, fetal calf serum; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay. present on the host immune cells (6Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4379) Google Scholar, 7Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1439) Google Scholar). Bone cells also express TLRs enabling the bacteria-derived factors to exert their effects on the skeleton (8Kikuchi T. Matsuguchi T. Tsuboi N. Mitani A. Tanaka S. Matsuoka M. Yamamoto G. Hishikawa T. Noguchi T. Yoshikai Y. J. Immunol. 2001; 166: 3574-3579Crossref PubMed Scopus (201) Google Scholar, 9Zou W. Amcheslavsky A. Bar-Shavit Z. J. Biol. Chem. 2003; 278: 16732-16740Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The most studied bacterial factor in this regard is lipopolysaccharide, recognized by both osteoclast and osteoblast lineage cells via TLR4 (8Kikuchi T. Matsuguchi T. Tsuboi N. Mitani A. Tanaka S. Matsuoka M. Yamamoto G. Hishikawa T. Noguchi T. Yoshikai Y. J. Immunol. 2001; 166: 3574-3579Crossref PubMed Scopus (201) Google Scholar, 9Zou W. Amcheslavsky A. Bar-Shavit Z. J. Biol. Chem. 2003; 278: 16732-16740Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Bacterial DNA has been shown to be a pathogen-derived structure activating the innate immune system via TLR9 (10Hemmi H. Takeuchi O. Kawai T. Kaisho T. Sato S. Sanjo H. Matsumoto M. Hoshino K. Wagner H. Takeda K. Akira S. Nature. 2000; 408: 740-745Crossref PubMed Scopus (5313) Google Scholar, 11Akira S. Takeda K. Kaisho T. Nat. Immunol. 2001; 2: 675-680Crossref PubMed Scopus (3898) Google Scholar, 12Klinman D.M. Yi A.K. Beaucage S.L. Conover J. Krieg A.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2879-2883Crossref PubMed Scopus (1353) Google Scholar, 13Krieg A.M. Curr. Opin. Immunol. 2000; 12: 35-43Crossref PubMed Scopus (311) Google Scholar, 14Krieg A.M. Annu. Rev. Immunol. 2002; 20: 709-760Crossref PubMed Scopus (2203) Google Scholar). This activity depends on unmethylated CpG dinucleotides in particular base contexts (“CpG motif”) (12Klinman D.M. Yi A.K. Beaucage S.L. Conover J. Krieg A.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2879-2883Crossref PubMed Scopus (1353) Google Scholar, 14Krieg A.M. Annu. Rev. Immunol. 2002; 20: 709-760Crossref PubMed Scopus (2203) Google Scholar). Synthetic oligodeoxynucleotides containing CpG motifs (CpG oligodeoxynucleotides (CpG-ODNs)) mimic the bacterial DNA immunostimulatory activity (15Yi A.K. Peckham D.W. Ashman R.F. Krieg A.M. Int. Immunol. 1999; 11: 2015-2024Crossref PubMed Scopus (80) Google Scholar).We have shown that CpG-ODNs inhibit the activity of the physiological osteoclast differentiation factor, receptor activator of NF-κB ligand (RANKL). Interestingly CpG-ODNs strongly increase osteoclastogenesis in RANKL-pretreated osteoclast precursors (16Zou W. Schwartz H. Endres S. Hartmann G. Bar-Shavit Z. FASEB J. 2002; 16: 274-282Crossref PubMed Scopus (51) Google Scholar). Thus, CpG-ODNs exert a dual effect on osteoclast differentiation: inhibition of osteoclastogenesis in early precursors but enhancement of osteoclastogenesis in precursors exposed to an osteoclastogenic signal. TNF-α mediates the enhanced osteoclastogenic effect of CpG-ODN by an autocrine mechanism (16Zou W. Schwartz H. Endres S. Hartmann G. Bar-Shavit Z. FASEB J. 2002; 16: 274-282Crossref PubMed Scopus (51) Google Scholar).BALB/c and C57BL/6 express different TNF-α alleles and differ in the response of their innate immune system (17Takao S. Mykytyn K. Jacob C.O. Immunogenetics. 1993; 37: 199-203Crossref PubMed Scopus (13) Google Scholar, 18Karupiah G. Vet. Immunol. Immunopathol. 1998; 63: 105-109Crossref PubMed Scopus (55) Google Scholar, 19Sapru K. Stotland P.K. Stevenson M.M. Clin. Exp. Immunol. 1999; 115: 103-109Crossref PubMed Scopus (36) Google Scholar, 20Uzonna J.E. Kaushik R.S. Gordon J.R. Tabel H. Parasite Immunol. 1999; 21: 57-71Crossref PubMed Scopus (62) Google Scholar, 21Iraqi F. Teale A. Immunogenetics. 1999; 49: 242-245Crossref PubMed Scopus (10) Google Scholar, 22Horai R. Saijo S. Tanioka H. Nakae S. Sudo K. Okahara A. Ikuse T. Asano M. Iwakura Y. J. Exp. Med. 2000; 191: 313-320Crossref PubMed Scopus (612) Google Scholar, 23Nicklin M.J. Hughes D.E. Barton J.L. Ure J.M. Duff G.W. J. Exp. Med. 2000; 191: 303-312Crossref PubMed Scopus (267) Google Scholar, 24Ulett G.C. Ketheesan N. Hirst R.G. Infect. Immunol. 2000; 68: 2034-2042Crossref PubMed Scopus (149) Google Scholar). In light of the pivotal role of TNF-α in mediating the osteoclastogenic effect of CpG-ODN, we studied the induction of osteoclastogenesis and TNF-α expression in cells derived from mouse strains expressing different alleles of the cytokine. We found that both osteoclastogenesis and TNF-α release induced by TLR9 activation were by far more evident in BALB/c-than in C57BL/6-derived cells, and we examined the mechanistic basis for the differential cytokine regulation.EXPERIMENTAL PROCEDURESMice—Seven- to 9-week-old male BALB/c and C57BL/6 mice were obtained from Harlan Laboratories Ltd. (Jerusalem, Israel).Reagents—Glutathione S-transferase-RANKL (residues 158–316) was prepared as described previously (16Zou W. Schwartz H. Endres S. Hartmann G. Bar-Shavit Z. FASEB J. 2002; 16: 274-282Crossref PubMed Scopus (51) Google Scholar). M-CSF and neutralizing anti-TNF-α antibodies (rat IgG) were purchased from R&D Systems, Inc. (Minneapolis, MN). Nuclease-resistant phosphorothioate oligodeoxynucleotide (5′-TCCATGACGTTCCTGACGTT-3′ (ODN 1826)) (15Yi A.K. Peckham D.W. Ashman R.F. Krieg A.M. Int. Immunol. 1999; 11: 2015-2024Crossref PubMed Scopus (80) Google Scholar) was purchased from BTG (Rehovot, Israel) and had undetectable endotoxin according to a limulus amoebocyte lysate assay (BioWhittaker, Walkersville, MD). 5,6-Dichloro-1-β-d-ribofuranosylbenzimidazole was purchased from Sigma. Mouse monoclonal anti-β-actin antibody was purchased from Sigma. Mouse monoclonal anti-phospho-extracellular signal-regulated kinase (ERK), anti-phospho-p38, and rabbit polyclonal anti-ERK antibodies were purchased from Cell Signaling Technology Inc. (Beverly MA). Mouse monoclonal anti-phospho-c-Jun N-terminal kinase and rabbit polyclonal anti-TNF-α antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-mouse TLR9 antibody was purchased from IMGENEX (San Diego, CA). Protein G-agarose was purchased from Oncogene Research Products (San Diego, CA). Brefeldin A (BFA) was purchased from Calbiochem. Media and sera were purchased from Biological Industries (Beth Haemek, Israel).In Vitro Osteoclast Formation Assay—Primary bone marrow macrophages (BMMs) were isolated as described previously (25Kurihara N. Tatsumi J. Arai F. Iwama A. Suda T. Exp. Hematol. 1998; 26: 1080-1085PubMed Google Scholar) and were plated in 96-well plates (1.3 × 105/well) in α-minimum Eagle's medium containing 10% FCS, M-CSF (50 ng/ml), and RANKL (10 ng/ml). After 3 days medium was changed to medium devoid of RANKL with or without CpG-ODN (100 nm), and osteoclast formation (tartrate-resistant acid phosphatase-positive cells with three or more nuclei) was evaluated 30 h later.Northern Blot Analysis—BMMs were plated in 35-mm tissue culture plates (2 × 106/plate) as described above. Total cellular RNA was extracted, fractionated, and transferred to nylon membranes as described previously (26Zou W. Bar-Shavit Z. J. Bone Miner. Res. 2002; 17: 1211-1218Crossref PubMed Scopus (179) Google Scholar). 32P-Labeled mouse TNF-α or mouse ribosomal protein L32 cDNA probes were used for hybridization. The hybridized membranes were then subjected to autoradiography, and the density of each of the mRNA bands was quantified by densitometry.Western Blot Analysis—Western analysis was performed as described previously (26Zou W. Bar-Shavit Z. J. Bone Miner. Res. 2002; 17: 1211-1218Crossref PubMed Scopus (179) Google Scholar). Bands were quantified by densitometry.Electrophoretic Mobility Shift Assay—Oligonucleotides corresponding to the κB2a site of the TNF-α promoter of BALB/c or C57BL/6 and to the κB consensus sequence (5′-CATGCCCTCTGGGGCCCCATA-3′, 5′-CATGCCCTCTCGGGCCCCATA-3′, and 5′-AGTTGAGGGGACTTTCCCAGGC-3′, respectively) were labeled, and nuclear extracts (5 μg) were incubated with labeled probes as described previously (9Zou W. Amcheslavsky A. Bar-Shavit Z. J. Biol. Chem. 2003; 278: 16732-16740Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Samples were then fractionated on a 7% polyacrylamide gel and visualized by exposing the dried gel to film.Nuclear Run-on Assay—BMMs were incubated in 150-mm tissue culture plates (2 × 107/plate). Cells were washed with PBS and then incubated for 1 h with or without CpG-ODN. Nuclei were harvested, resuspended in storage buffer (50 mm Tris-HCl, pH 8.3, 40% (v/v) glycerol, 5 mm MgCl2, 0.1 mm EDTA), and frozen at –80 °C until labeling. Transcripts that were initiated in the cells were allowed to continue in the presence of [α-32P]UTP (PerkinElmer Life Sciences) at 30 °C for 30 min. Labeled RNA was extracted with TRI Reagent and hybridized to nylon membrane cross-linked plasmid DNA for 48 h at 42 °C. The membranes were washed and exposed to x-ray film.ELISA—BMMs were incubated in 48-well plates (3.0 × 105 cells/well) for 3 days as above. Cell monolayers were washed with PBS and incubated in 0.4 ml of α-minimum Eagle's medium containing 10% FCS and M-CSF in the absence or presence of CpG-ODN (100 nm) for 4 or 6 h. TNF-α release was measured by ELISA using a DuoSet ELISA kit (R&D Systems, Inc.) according to the manufacturer's instructions. The relative cell number was estimated by the methylene blue uptake assay (27Goldman R. Bar-Shavit Z. J. Natl. Cancer Inst. 1979; 63: 1009-1016PubMed Google Scholar) using a Dynatech plate reader (Dynatech Laboratories, Inc., Chantilly, VA).Metabolic Labeling and Pulse-Chase Experiments—BMMs were incubated in 35-mm tissue culture plates (2 × 106/plate) as above. On day 3 cells were washed with PBS and incubated in methionine- and cysteine-free medium. After 30 min the medium was changed, and cells were incubated in methionine- and cysteine-free medium in the presence of 10% dialyzed FCS and 5 μg/ml BFA for 10 min. Then M-CSF was added to the cultures together with 100 μCi/ml 35S-labeled methionine and cysteine mixture (Redivue Pro-Mix l-[35S]methionine and l-[35S]cysteine in vitro cell labeling mixture, Amersham Biosciences). In pulse-chase experiments (28Mohagheghpour N. Waleh N. Garger S.J. Dousman L. Grill L.K. Tuse D. Cell. Immunol. 2000; 199: 25-36Crossref PubMed Scopus (76) Google Scholar, 29Takayanagi H. Kim S. Matsuo K. Suzuki H. Suzuki T. Sato K. Yokochi T. Oda H. Nakamura K. Ida N. Wagner E.F. Taniguchi T. Nature. 2002; 416: 744-749Crossref PubMed Scopus (584) Google Scholar), after a 10-min incubation with BFA, 50 ng/ml lipopolysaccharide was added to the cultures together with 100 μCi/ml 35S-labeled methionine and cysteine mixture. After 2 h cells were washed twice with ice-cold PBS. Cells were incubated in α-minimum Eagle's medium containing 10% dialyzed FCS with or without of 100 nm CpG-ODN for the indicated time and then washed extensively with PBS and lysed in RIPA lysis buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1.0% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS). We carried out immunoprecipitation with either polyclonal anti-TNF-α or anti-β-actin antibodies followed by SDS-PAGE and autoradiography.Protein Synthesis—BMMs were incubated in 48-well plates (3.0 × 105 cells/well). On day 3 cells were washed with PBS and incubated in 0.4 ml of leucine-free medium containing 10% dialyzed FCS. [3H]Leucine (5 μCi/well) was added to the cultures, and cells were incubated for 4 h in the absence or presence of CpG-ODN. Total protein was precipitated and dissolved (28Mohagheghpour N. Waleh N. Garger S.J. Dousman L. Grill L.K. Tuse D. Cell. Immunol. 2000; 199: 25-36Crossref PubMed Scopus (76) Google Scholar). The amount of 3H incorporated into cell-associated macromolecules was determined by scintillation counting.RESULTSModulation of Osteoclastogenesis and TNF-α Release by CpG-ODN—Since TNF-α mediates the osteoclastogenic effect of CpG-ODNs we compared this activity and CpG-ODN-induced TNF-α production in cells derived from BALB/c and C57BL/6 expressing two different alleles of the cytokine. It can be seen in Fig. 1A, as shown by us previously (16Zou W. Schwartz H. Endres S. Hartmann G. Bar-Shavit Z. FASEB J. 2002; 16: 274-282Crossref PubMed Scopus (51) Google Scholar), that CpG-ODN markedly increased osteoclast differentiation in RANKL-primed cells. In this experiment, cells were incubated for 72 h with M-CSF (50 ng/ml) and RANKL (10 ng/ml). Medium was then changed, and cells were incubated for an additional 30 h in the absence or presence of CpG-ODN (100 nm). As seen in the figure, the increase in osteoclast differentiation by CpG-ODN was more evident in BALB/c-than in C57BL/6-derived cells (∼6.6-versus ∼2.2-fold induction, respectively). To examine whether this difference is correlated with a differential TNF-α release from cells derived from the two strains in response to CpG-ODN we used ELISA. We found (Fig. 1B) that the release of TNF-α from BALB/c-derived cells was greater by ∼3-fold than the release from C57BL/6-derived cells. We did not detect any release of the cytokine in the absence of CpG-ODN up to 6 h of incubation.We next examined whether the correlation between CpG-ODN-induced osteoclastogenesis and TNF-α release represents cause and effect. To this end, we performed an osteoclastogenesis assay in the presence of anti-TNF-α neutralizing antibodies. As we showed previously for BALB/c-derived cells (16Zou W. Schwartz H. Endres S. Hartmann G. Bar-Shavit Z. FASEB J. 2002; 16: 274-282Crossref PubMed Scopus (51) Google Scholar), these antibodies markedly inhibited CpG-ODN-induced osteoclastogenesis also in C57BL/6-derived cells. At a dilution of 1:100 of the anti-TNF-α neutralizing antibodies, 60 and 80% inhibition were obtained with BALB/c and C57BL/6-derived cells, respectively (not shown). In parallel, we examined whether the anti-TNF-α neutralizing antibodies reduce TNF-α levels in medium conditioned by the CpG-ODN-treated cells. In this experiment, BALB/c- and C57BL/6-derived cells were plated in tissue culture plates (60-mm diameter, 5 × 106 cells/ plate in 5 ml), and after RANKL priming (3 days, 10 ng/ml) cells were incubated with CpG-ODN (100 nm) in 3 ml. Media were collected after 6 h, and aliquots were incubated without or with neutralizing anti-TNF-α antibodies overnight at 4 °C. Immunocomplexes were removed by immunoprecipitation. Aliquots were then subjected to ELISA. As seen in Table I, the cytokine levels were reduced. Already at a 1:1000 dilution of anti-TNF-α neutralizing antibody a dramatic decrease of TNF-α levels in serum was observed. Replacing the neutralizing antibodies with rat IgG did not reduce the cytokine levels (not shown). The possibility that a differential TNF receptor responsiveness in cells derived from the two strains contributes to the difference in CpG-ODN-induced osteoclastogenesis was ruled out by experiments showing no difference in TNF-α induction of c-Jun N-terminal kinase, ERK, and p38 phosphorylation in these cells (not shown).Table IAnti-TNF-α neutralizing antibodies reduce TNF-α levelsAntibody dilutionBALB/cC57BL/6No antibody424.0 ± 50.2aNumbers are pg of TNF-α/ml of methylene blue, average of triplicates ± S.E.134 ± 13.61:100047.9 ± 16.7NDbND, not detectable.1:50014.4 ± 8.1ND1:2001.9 ± 0.7ND1:100NDNDa Numbers are pg of TNF-α/ml of methylene blue, average of triplicates ± S.E.b ND, not detectable. Open table in a new tab Modulation of TNF-α mRNA Abundance by CpG-ODN in BALB/c- and C57BL/6-derived Cells—We then attempted to identify the mechanism(s) responsible for the higher TNF-α release from BALB/c-derived cells in response to CpG-ODN. To this end, we used Northern analysis to test the modulation of TNF-α mRNA steady-state levels by CpG-ODN. Addition of CpG-ODN to RANKL-primed bone marrow osteoclast precursors increased the cytokine mRNA levels in cells derived from the two strains (Fig. 2). Unexpectedly TNF-α transcript induction was more evident in C57BL/6-derived cells despite the lower cytokine release in response to CpG-ODN in these cells. Densitometric analysis showed that basal TNF-α transcript abundance was low and similar in the cells derived from the two strains. CpG-ODN addition increased TNF-α mRNA levels up to ∼5- and ∼15-fold in cells harvested from BALB/c and C57BL/6, respectively.Fig. 2Modulation of TNF-α mRNA abundance in BALB/c- and C57BL/6-derived cells by CpG-ODN. BMMs were plated as described under “Experimental Procedures.” Cells were then washed and treated with CpG-ODN as indicated. RNA was prepared and examined for transcript abundance of TNF-α. L32 was the loading control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To understand the differential modulation of TNF-α mRNA we first test whether CpG-ODN inhibits the cytokine transcript degradation and whether this activity differs in cells derived from the different strains. BALB/c- or C57BL/6-derived cells were grown for 72 h in medium containing M-CSF (50 ng/ml) and RANKL (10 ng/ml). Monolayers were then washed and incubated in the presence or absence of CpG-ODN (100 nm). After 50 min the transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole was added, and at the indicated time points RNA was collected for Northern analysis. Fig. 3A shows a representative autoradiogram showing TNF-α and L32 mRNA abundance. The TNF-α/L32 ratio was calculated from densitometric analyses (average from five independent experiments). As seen in Fig. 3B the rate of TNF-α mRNA degradation did not differ between cells derived from the two strains (half-life of 8.0 ± 1.0 and 8.0 ± 1.2 min in BALB/c- and C57BL/6-derived cells, respectively). CpG-ODN addition stabilized the cytokine transcript to a half-life of 17.1 ± 2.07 and 15.5 ± 0.84 min in BALB/c- and C57BL/6-derived cells, respectively. Thus, while message stabilization by CpG-ODN contributes to the increased TNF-α transcript abundance, it cannot be the reason for the difference in mRNA induction in the two strains.Fig. 3Modulation of TNF-α mRNA stability by CpG-ODN in BALB/c- and C57BL/6-derived cells. BMMs were plated as described under “Experimental Procedures.” Cells were then washed and incubated with or without CpG-ODN for 50 min. Then 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (2.5 μg/ml) was added at the indicated time. TNF-α and L32 mRNA abundance was examined using Northern blot analysis. A representative autoradiogram (A) and a densitometric analysis (B) of an average of five experiments are shown. The TNF/L32 ratio at time 0 was normalized to 100 on both control and CpG-ODN-treated cells in each experiment. Dashed and solid lines represent cells in the absence or presence of CpG-ODN, respectively; closed and open squares represent C57BL/6 and BALB/c-derived cells, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To examine directly the rate of transcription we performed a nuclear run-on assay. Cells were incubated for 60 min in the presence or absence of CpG-ODN. Nuclei were prepared and incubated with [32P]UTP for 30 min. RNA was collected and hybridized to filters containing plasmids carrying TNF-α and L32 cDNAs as well as to the corresponding empty plasmids. Filters were then subjected to autoradiography. The autoradiogram shown in Fig. 4 reveals that CpG-ODN increased the transcription rate in cells derived from the two strains, and consistent with a more noticeable increase in TNF-α mRNA abundance in C57BL/6 derived cells, the cytokine transcription rate in these cells was ∼3.3-fold greater than that observed in BALB/c cells.Fig. 4Nuclear run-on analysis of BALB/c- and C57BL/6-derived cells in the presence or absence of CpG-ODN. BMMs were plated as described under “Experimental Procedures.” Monolayers were then washed, and cells were incubated for 60 min in the presence or absence of CpG-ODN. Nuclei were subjected to nuclear run-on assay. Labeled RNA transcripts were prepared and hybridized to the target DNA as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We examined whether the difference in CpG-ODN-induced TNF-α transcription rate in cells derived from the two strains is due to differences in TLR9 expression levels and/or intracellular signaling pathways known to mediate CpG-ODN cellular activities. Cells derived from BALB/c and from C57BL/6 express similar levels of TLR9, and addition of CpG-ODN did not change these levels as determined by Western analysis (Fig. 5A). CpG-ODN induced the phosphorylation of p38, ERK, and c-Jun N-terminal kinase in cells derived from the two strains (Fig. 5B). Although the kinetics was not identical in the different cells, this slight variability does not seem to be responsible for the marked difference in TNF-α transcription.Fig. 5TLR9 expression and CpG-ODN-induced signaling molecule phosphorylation in BALB/c- and C57BL/6-derived cells. BMMs were plated as described under “Experimental Procedures.” Cells were incubated with CpG-ODN as indicated, and extracts were prepared for Western analyses. A, TLR9 levels were estimated using antibodies to this protein. Anti-actin antibody serves as the loading control. B, phosphorylation of ERK, p38, and c-Jun N-terminal kinase (JNK) was estimated using the corresponding antibodies. Anti-ERK antibody serves as the loading control. p-, phospho.View Large Image Figure ViewerDownload Hi-res image Download (PPT)NF-κB activation plays a role in CpG-ODN-induced transcriptional modulation of TNF-α (30Kwon H.J. Lee K.W. Yu S.H. Han J.H. Kim D.S. Biochem. Biophys. Res. Commun. 2003; 311: 129-138Crossref PubMed Scopus (31) Google Scholar). Three of the four κB sites in the TNF-α promoter are identical in the two strains. However, there is a single nucleotide difference between one of the κB sites (κB2a) in C57BL/6 and in BALB/c (ctcggctgcccc versus ctgggctgcccc, respectively) (21Iraqi F. Teale A. Immunogenetics. 1999; 49: 242-245Crossref PubMed Scopus (10) Google Scholar, 31Iraqi F. Teale A. Immunogenetics. 1997; 45: 459-461Crossref PubMed Scopus (9) Google Scholar). We used electrophoretic mobility shift assay to examine the possibility that nuclear extracts derived from CpG-ODN-stimulated cells bind to the BALB/c-derived κB2α less efficiently than to the C57BL/6-derived κB2α. To this end, BALB/c- and C57BL/6-derived κB2α oligodeoxynucleotide sequences were labeled, and their interactions with nuclear extracts derived from the two strains were analyzed. A shift was observed when the C57BL/6-derived κB2α was used but not with BALB/c-derived κB2α (Fig. 6A). Moreover κB2a derived from C57BL/6, but not from BALB/c, inhibited the shift when labeled κB consensus site was used (Fig. 6B). In both experiments similar results were obtained with BALB/c- and C57BL/6-derived nuclear extracts. Thus, we conclude that the single nucleotide polymorphism in κB2a is responsible for the difference in CpG-ODN-induced TNF-α transcription between cells derived from the two strains.Fig. 6BALB/c- and C57BL/6-derived κB2a interactions with nuclear proteins. BMMs were plated as described under “Experimental Procedures.” A, cells were incubated with CpG-ODN for the indicated times, and nuclear extracts (NE) from either BALB/c-derived cells or C57BL/6-derived cells (left and right 10 lanes, respectively) were prepared and subjected to electrophoretic mobility shift assay using κB2a derived from either BALB/c or C57BL/6 (B or C, respectively). B, cells were treated with CpG-ODN for 60 min (lanes 2–11; untreated control, lane 1), nuclear extracts were prepared from BALB/c- and C57BL/6-derived cells (upper and lower panels, respectively), and the ability of κB2a derived from either BALB/c (lanes 3–5) or C57BL/c (lanes 6–8) as well as unlabeled κB consensus sequence (lanes 9–11) to compete with the κB consensus sequence was measured using electrophoretic mobility shift assay.View Large Image" @default.
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• W2034307611 date "2004-12-01" @default.
• W2034307611 modified "2023-10-06" @default.
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• W2034307611 cites W1483162601 @default.
• W2034307611 cites W1514540900 @default.
• W2034307611 cites W1596485921 @default.
• W2034307611 cites W1655860314 @default.
• W2034307611 cites W1802262832 @default.
• W2034307611 cites W1905655085 @default.
• W2034307611 cites W1964464847 @default.
• W2034307611 cites W1967350690 @default.
• W2034307611 cites W1970120112 @default.
• W2034307611 cites W1977486425 @default.
• W2034307611 cites W1980095186 @default.
• W2034307611 cites W1981323152 @default.
• W2034307611 cites W1998574307 @default.
• W2034307611 cites W2003965263 @default.
• W2034307611 cites W2013203343 @default.
• W2034307611 cites W2014368997 @default.
• W2034307611 cites W2014770452 @default.
• W2034307611 cites W2016776106 @default.
• W2034307611 cites W2019146941 @default.
• W2034307611 cites W2027838401 @default.
• W2034307611 cites W2031504111 @default.
• W2034307611 cites W2033365840 @default.
• W2034307611 cites W2033441782 @default.
• W2034307611 cites W2041462231 @default.
• W2034307611 cites W2041712515 @default.
• W2034307611 cites W2045125789 @default.
• W2034307611 cites W2047110135 @default.
• W2034307611 cites W2050436579 @default.
• W2034307611 cites W2050802901 @default.
• W2034307611 cites W2058341787 @default.
• W2034307611 cites W2068515672 @default.
• W2034307611 cites W2068682899 @default.
• W2034307611 cites W2072038768 @default.
• W2034307611 cites W2073409518 @default.
• W2034307611 cites W2076145955 @default.
• W2034307611 cites W2078844869 @default.
• W2034307611 cites W2080677899 @default.
• W2034307611 cites W2091771822 @default.
• W2034307611 cites W2098387574 @default.
• W2034307611 cites W2102948981 @default.
• W2034307611 cites W2103581491 @default.
• W2034307611 cites W2106986504 @default.
• W2034307611 cites W2107462315 @default.
• W2034307611 cites W2108769690 @default.
• W2034307611 cites W2109703128 @default.
• W2034307611 cites W2110456176 @default.
• W2034307611 cites W2117439999 @default.
• W2034307611 cites W2117447669 @default.
• W2034307611 cites W2118011548 @default.
• W2034307611 cites W2131357215 @default.
• W2034307611 cites W2132933661 @default.
• W2034307611 cites W2150765396 @default.
• W2034307611 cites W2158089569 @default.
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• W2034307611 cites W2161697663 @default.
• W2034307611 cites W2163247322 @default.
• W2034307611 cites W2164909689 @default.
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