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Myeloid Differentiation Factor 88-dependent Post-transcriptional Regulation of Cyclooxygenase-2 Expression by CpG DNA

2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês

10.1074/jbc.m306280200

ISSN

1083-351X

Autores

Seon-Ju Yeo, Jae-Geun Yoon, Ae‐Kyung Yi,

Tópico(s)

NF-κB Signaling Pathways

Resumo

The immune stimulatory unmethylated CpG motifs present in bacterial DNA (CpG DNA) induce expression of cyclooxygenase-2 (cox-2). The present study demonstrates that CpG DNA can up-regulate cox-2 expression by post-transcriptional mechanisms in RAW264.7 cells. To determine the CpG DNA-mediated signaling pathway that post-transcriptionally regulates cox-2 expression, a cox-2 translational reporter (COX2–3′-UTR-luciferase) was generated by inserting sequences within the 3′-untranslated region (UTR) of cox-2 to the 3′ end of the luciferase gene under control of the SV40 promoter. CpG DNA-induced COX2–3′-UTR-luciferase activity was completely inhibited by an endosomal acidification inhibitor chloroquine, a Toll-like receptor 9 antagonist inhibitory CpG DNA, or overexpression of a dominant negative (DN) form of MyD88. However, overexpression of DN-IRAK-1 or DN-TRAF6 resulted in substantial, but not complete, inhibition of the CpG DNA-induced COX2–3′-UTR-luciferase activity. Activation of all three MAPKs (ERK, p38, and JNK) was required for optimal COX2–3′-UTR-luciferase activity induced by CpG DNA. Overexpression of DN-TRAF6 suppressed CpG DNA-mediated activation of p38 and JNK, but not ERK, explaining the partial inhibitory effects of DN-TRAF6 on CpG DNA-induced COX2–3′-UTR-luciferase activity. Co-expression of DN-TRAF6 and N17Ras completely inhibited CpG DNA-induced COX2–3′-UTR-luciferase activity, indicating the involvement of Ras in CpG DNA-mediated ERK and COX2–3′-UTR regulation. Collectively, our results suggest that MyD88 and MAPKs play a key regulatory role in CpG DNA-mediated cox-2 expression at the post-transcriptional level and that TRAF6 is a diverging point in the Toll-like receptor 9-signaling pathway for CpG DNA-mediated MAPK activation. The immune stimulatory unmethylated CpG motifs present in bacterial DNA (CpG DNA) induce expression of cyclooxygenase-2 (cox-2). The present study demonstrates that CpG DNA can up-regulate cox-2 expression by post-transcriptional mechanisms in RAW264.7 cells. To determine the CpG DNA-mediated signaling pathway that post-transcriptionally regulates cox-2 expression, a cox-2 translational reporter (COX2–3′-UTR-luciferase) was generated by inserting sequences within the 3′-untranslated region (UTR) of cox-2 to the 3′ end of the luciferase gene under control of the SV40 promoter. CpG DNA-induced COX2–3′-UTR-luciferase activity was completely inhibited by an endosomal acidification inhibitor chloroquine, a Toll-like receptor 9 antagonist inhibitory CpG DNA, or overexpression of a dominant negative (DN) form of MyD88. However, overexpression of DN-IRAK-1 or DN-TRAF6 resulted in substantial, but not complete, inhibition of the CpG DNA-induced COX2–3′-UTR-luciferase activity. Activation of all three MAPKs (ERK, p38, and JNK) was required for optimal COX2–3′-UTR-luciferase activity induced by CpG DNA. Overexpression of DN-TRAF6 suppressed CpG DNA-mediated activation of p38 and JNK, but not ERK, explaining the partial inhibitory effects of DN-TRAF6 on CpG DNA-induced COX2–3′-UTR-luciferase activity. Co-expression of DN-TRAF6 and N17Ras completely inhibited CpG DNA-induced COX2–3′-UTR-luciferase activity, indicating the involvement of Ras in CpG DNA-mediated ERK and COX2–3′-UTR regulation. Collectively, our results suggest that MyD88 and MAPKs play a key regulatory role in CpG DNA-mediated cox-2 expression at the post-transcriptional level and that TRAF6 is a diverging point in the Toll-like receptor 9-signaling pathway for CpG DNA-mediated MAPK activation. A motif of unmethylated CpG dinucleotides in a particular nucleotide context (CpG motif, GACGTT for murine and GTCGTT for human) in bacterial DNA is one of the newly identified pathogen-associated molecular patterns and is capable of activating cells of the immune system, including B lymphocytes, monocytes/macrophages, and dendritic cells that play a critical role in directing the host immune response to infection (reviewed in Ref. 1Krieg A.M. Annu. Rev. Immunol. 2002; 20: 709-760Crossref PubMed Scopus (2215) Google Scholar). The ability of CpG motifs in bacterial DNA to activate innate immunity can be mimicked by synthetic oligodeoxynucleotides containing the CpG motif (CpG DNA). CpG DNA is endocytosed by leukocytes, acidified, and then recognized by a pattern recognition receptor, Toll-like receptor 9 (TLR9), 1The abbreviations used are: TLR9, Toll-like receptor 9; COX, cyclooxygenase; UTR, untranslated region; DN, dominant negative; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IRAKs, IL-1R-associated kinases; TRAF6, tumor necrosis factor-α receptor-associated factor 6; TNF-α, tumor necrosis factor-α; IL, interleukin; GST, glutathione S-transferase; LPS, lipopolysaccharide; IFN, interferon; PG, prostaglandin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ARE, AU-rich element; ActD, actinomycin D; MEK, MAPK/ERK kinase.1The abbreviations used are: TLR9, Toll-like receptor 9; COX, cyclooxygenase; UTR, untranslated region; DN, dominant negative; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IRAKs, IL-1R-associated kinases; TRAF6, tumor necrosis factor-α receptor-associated factor 6; TNF-α, tumor necrosis factor-α; IL, interleukin; GST, glutathione S-transferase; LPS, lipopolysaccharide; IFN, interferon; PG, prostaglandin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ARE, AU-rich element; ActD, actinomycin D; MEK, MAPK/ERK kinase. in an endosomal compartment (2Yi A.-K. Tuetken R. Redford T. Waldschmidt M. Kirsch J. Krieg A.M. J. Immunol. 1998; 160: 4755-4761PubMed Google Scholar, 3Hemmi 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 (5359) Google Scholar, 4Ahmad-Nejad P. Hacker H. Rutz M. Bauer S. Vabulas R.M. Wagner H. Eur. J. Immunol. 2002; 32: 1958-1968Crossref PubMed Scopus (631) Google Scholar). Upon recognition of CpG DNA, TLR9 recruits the adaptor molecule myeloid differentiation factor 88 (MyD88) through an interaction between their C-terminal Toll/IL-1 receptor (TIR) domains. This recruitment of MyD88 to the TIR domain of TLR9 initiates a signaling pathway that sequentially involves IL-1R-associated kinases (IRAKs) and tumor necrosis factor-α receptor-associated factor 6 (TRAF6) (3Hemmi H. Takeuchi O. Kawai T. Kaisho T. Sato S. Sanjo H. Matsumoto M. Hoshino K. Wagner H. Takeda K. Akira S. 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Studies using gene-deficient mice and RAW264.7 cells transiently transfected with the dominant negative (DN) forms of these molecules indicated that this MyD88-mediated signaling pathway is essential for the CpG DNA-induced activation of NF-κB and c-Jun N-terminal kinase (JNK) (3Hemmi 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 (5359) Google Scholar, 5Hacker H. Vabulas R.M. Takeuchi O. Hoshino K. Akira S. Wagner H. J. Exp. Med. 2000; 192: 595-600Crossref PubMed Scopus (418) Google Scholar, 8Chuang T.H. Lee J. Kline L. Mathison J.C. Ulevitch R.J. J. Leukocyte Biol. 2002; 71: 538-544PubMed Google Scholar). In addition to MyD88, DNA-activated protein kinase and intracellular reactive oxygen species have also been demonstrated to be required for the CpG DNA-mediated activation of NF-κB and JNK, which in turn lead to the expression of various oncogenes and proinflammatory cytokines and mediators (2Yi A.-K. Tuetken R. Redford T. Waldschmidt M. Kirsch J. Krieg A.M. J. Immunol. 1998; 160: 4755-4761PubMed Google Scholar, 9Chu W. Gong X. Li Z. Takabayashi K. Ouyang H. Chen Y. Lois A. Chen D.J. Li G.C. Karin M. Raz E. Cell. 2000; 103: 909-918Abstract Full Text PDF PubMed Scopus (159) Google Scholar). The effects of CpG DNA on the immune system have been studied extensively. CpG DNA rapidly activates B cells to proliferate, to produce TNF-α, IL-6, IL-10, and immunoglobulin (Ig), and to express increased levels of co-stimulatory molecules (10Krieg A.M. Yi A.K. Matson S. Waldscmidt T. Bishop G. Teasdale R. Koretzky G.A. Klinman D. 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Saklatvala J. Clark A.R. Mol. Cell. Biol. 2000; 20: 4265-4274Crossref PubMed Scopus (370) Google Scholar). ARE/poly(U) binding factor 1 (AUF1) and CArG box-binding factor-A (CBF-A) have been shown to interact specifically with the AU-rich RNA stability determinants in cox-2 transcripts (48Lasa M. Mahtani K.R. Finch A. Brewer G. Saklatvala J. Clark A.R. Mol. Cell. Biol. 2000; 20: 4265-4274Crossref PubMed Scopus (370) Google Scholar, 49Dean J.L. Sully G. Wait R. Rawlinson L. Clark A.R. Saklatvala J. Biochem. J. 2002; 15: 709-719Crossref Google Scholar). Receptor-mediated signaling pathways that lead to the regulation of cox-2 mRNA stability have not been extensively studied. Three mitogen-activated protein kinase (MAPK) family members (extracellular signal-regulated kinase (ERK), JNK, and p38) have been reported to play an important regulatory role in mRNA stability of various cytokines and oncogenes (48Lasa M. Mahtani K.R. Finch A. Brewer G. Saklatvala J. Clark A.R. Mol. Cell. 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Blood. 2002; 99: 4048-4052Crossref PubMed Scopus (40) Google Scholar). In particular, it has been demonstrated that activation of p38 leads to the activation of MAPKAPK-2, which in turn activates heat shock protein 27 (HSP27). HSP27 has been shown to be involved in cox-2 mRNA stability via AUF1 (48Lasa M. Mahtani K.R. Finch A. Brewer G. Saklatvala J. Clark A.R. Mol. Cell. Biol. 2000; 20: 4265-4274Crossref PubMed Scopus (370) Google Scholar). Through the TLR9/MyD88-signaling pathway, CpG DNA induces activation of all three MAPKs in macrophages (3Hemmi 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 (5359) Google Scholar, 5Hacker H. Vabulas R.M. Takeuchi O. Hoshino K. Akira S. Wagner H. J. Exp. Med. 2000; 192: 595-600Crossref PubMed Scopus (418) Google Scholar). Of the three MAPKs activated by CpG DNA, p38, by leading to activation of the transcription factor cAMP-responsive element-binding protein, plays a critical regulatory role in the CpG DNA-mediated transcriptional regulation of cox-2 expression (28Yeo S.J. Gravis D. Yoon J.G. Yi A.K. J. Biol. Chem. 2003; 278: 22563-22573Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). However, previous studies have not addressed whether CpG DNA induces cox-2 mRNA stabilization nor have they determined what are the relative contributions of different TLR9/MyD88-signaling mediators to CpG DNA-induced cox-2 post-transcriptional regulation in macrophages. In the present study, we have demonstrated that CpG DNA up-regulates cox-2 expression, at least in part, through post-transcriptional regulation mechanisms that include mRNA stabilization. CpG DNA-mediated post-transcriptional regulation of cox-2 expression is dependent on the endosomal processing of CpG DNA and the TLR9/MyD88-dependent signaling pathway. In addition, our results demonstrated that all three MAPKs (ERK, p38, and JNK) are required for the CpG DNA-mediated post-transcriptional regulation of cox-2 expression and that TRAF6 only partially contributes to this post-transcriptional regulation process, due to its differential regulation of the different MAPKs. Furthermore, our results demonstrated that CpG DNA induces activation of the small GTP-binding protein Ras, and this CpG DNA-activated Ras, presumably through activation of ERK, positively regulates CpG DNA-mediated cox-2 expression at the post-transcriptional level. Oligodeoxynucleotides—Nuclease-resistant phosphorothioate oligodeoxynucleotides (S-ODN) were purchased from Operon (Alameda, CA) and further purified by ethanol precipitation. S-ODN had no detectable endotoxins by Limulus assay. The sequences of S-ODN used are 5′-TCCATGACGTTCCTGACGTT-3′ (CpG DNA, 1826), 5′-TCCAGGACTTCTCTCAGGTT-3′ (non-CpG DNA, 1982), and 5′-TCCTGGCGGGGAAGT-3′ (inhibitory CpG DNA and iCpG DNA, 2088). Cell Lines, Culture Conditions, and Reagents—RAW264.7 cells (ATCC, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 1.5 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin and cultured at 37 °C in a 5% CO2 humidified incubator. All culture reagents were purchased from Invitrogen. LPS (Salmonella minnesota Re 595) was purchased from Sigma. LPS purity was confirmed by its inability to induce IL-6 production in macrophages isolated from the LPS-unresponsive C3H/HeJ mouse strain. SB202190, U0126, and SP600125 were purchased from Calbiochem. Recombinant murine IFN-γ was purchased from R & D Systems (Minneapolis, MN). Preparation of RNA and Real Time PCR—RAW264.7 cells (2 × 106) were plated in 15-mm diameter dishes and then incubated for 24 h. Cells were stimulated with medium, CpG DNA (3 μg/ml), or non-CpG DNA (3 μg/ml) for the designated times. In some experiments, RAW264.7 cells pretreated with CpG DNA (3 μg/ml) for 12 h were stimulated with medium, CpG DNA (3 μg/ml), or non-CpG DNA (3 μg/ml) for the designated times in the presence of actinomycin D (ActD; 10 μg/ml) to inhibit new mRNA transcription. Cells were harvested, and total RNA was isolated by using RNeasy mini kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. Ten μg of total RNA served as the template for single strand cDNA synthesis in a reaction using oligo(dT) primers and Moloney murine leukemia virus-reverse transcriptase (Invitrogen) according to the manufacturer's protocol. To measure the relative amount of cox-2 gene transcripts, amplification of cDNA was monitored with the fluorescent DNA binding dye SYBR Green (PerkinElmer Applied Biosystems, Foster City, CA) in combination with the ABI 7900 sequence detection system (Perkin-Elmer Applied Biosystems) according to the manufacturer's instructions. Forward and reverse primers were designed using primer Express software (PerkinElmer Applied Biosystems). GAPDH was used for an internal control. The sequences of real time PCR primers used are as follows: cox-2 sense, 5′-CAGAACCGCATTGCCTCTG-3′ and cox-2 antisense, 5′-TTGAAGGTGTCGGGCAGC-3′; GAPDH sense, 5′-TTCACCACCATGGAGAAGGC-3′ and GAPDH antisense, 5′-GGCATGGACTGTGGTCATGA. ActD was purchased from Sigma. All real time PCR primers used were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). Plasmids—The 3′-UTR of cox-2 containing 763 bp was amplified by PCR using 5′-TCTAGAGTTCAACTGAGCTGTAAAAGTCT-3′ as a forward primer and 5′-TCTAGAACCAGCATTTTGACATACAAG-3′ as a reverse primer. The resulting 3′-UTR of cox-2 was ligated into pGEM-T easy vector (Promega, Madison, WI) and then subcloned into the 3′ end of the luciferase gene in pGL3-control vector (Promega, Madison, WI) to generate the cox-2 translational reporter (COX2–3′-UTR-luciferase). All PCR primers used for cloning were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). Generation of expression constructs encoding a dominant negative form (DN) of IRAK-1 (amino acids 1–215), DN-TRAF6 (amino acids 305–531), DN-p38, DN-JNK1, or DN-MEK1 were described previously (28Yeo S.J. Gravis D. Yoon J.G. Yi A.K. J. Biol. Chem. 2003; 278: 22563-22573Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). DN-MyD88 expression construct pIRES2-EGFP-DN-MyD88 was provided by Dr. S.-C. Hong (Indiana University, Indianapolis, IN). The AP-1-β-galactosidase construct, pCDNA3-JNK1 expression construct, and pCDNA3-N17Ras expression construct were provided by Dr. G. Koretzky (University of Pennsylvania, Philadelphia). Transient Transfection and Reporter Gene Assays—RAW264.7 cells (1.5 × 106 cells/plate) were plated into 60-mm cell culture plates and then incubated for 48 h to reach ∼80% confluence. Cells were transfected with pRL-TK-luciferase (0.5 μg) and COX2–3′-UTR-luciferase (1 μg) or pGL3-luciferase (1 μg), using LipofectAMINE Plus (Invitrogen). In some experiments, cells were co-transfected with an equal amount (2 μg) of control empty vector (pIRES-EGFP, pCDNA3, or pUSE), DNMyD88, DN-IRAK-1, DN-TRAF6, DN-MEK1, DN-p38, or DN-JNK1 and COX2–3′-UTR-luciferase (1 μg) plus pRL-TK-luciferase (0.5 μg) or AP-1-β-galactosidase (2 μg) and then incubated for 6 h. Transfected cells were pooled and washed 3 times with culture media. Cells (1 × 106 cells/15-mm cell culture plate) were stimulated with medium, CpG DNA (6 μg/ml), non-CpG DNA (6 μg/ml), IFN-γ (166 ng/ml), or LPS (50 ng/ml) for 12 h. In some experiments, transfected cells were pretreated with medium, iCpG DNA (6 μg/ml), chloroquine (2.5 μg/ml), Me2SO, U0126 (1.25 μm), SB202190 (2.5 μm), or SP600125 (5 μm) for 15 min before the stimulation. β-Galactosidase and luciferase activities in cell extracts were analyzed according to manufacturer's protocol using Galacto-Light Plus Reporter gene assay (Tropix, Bedford, MA) and Dual-Luciferase Reporter Assay System (Promega, Madison, WI), respectively. Luciferase activity was normalized using pRL-TK-luciferase activity (Renilla luciferase activity) in each sample. For AP1-β-galactosidase assay, equal concentrations of cell lysates were used. Generation of DN-TRAF6-expressing RAW264.7 Stable Transfectants (TRAF6DN-RAW264.7)—To generate RAW264.7 stable transfectants constitutively expressing DN-TRAF6, RAW264.7 cells (∼80% confluent in a 100-mm tissue culture dish) were transfected with pOPTRAF6DN constructs (30 μg) using LipofectAMINE PLUS (Invitrogen). To generate vector control cell line, RAW264.7 cells (∼80% confluent in a 100-mm tissue culture dish) were transfected with pOPRSVI.mcs1 cloning vector (30 μg) using LipofectAMINE PLUS (Invitrogen). Transfected cells (TRAF6DN-RAW264.7 cells and vector control cells) were selected and maintained in complete Dulbecco's modified Eagle's medium plus 500 μg/ml of geneticin (G418; Calbiochem). pOPTRAF6DN construct and pOPRSVI.mcs1 cloning vector were provided by Dr. G. Bishop at the University of Iowa (Iowa City, IA) (57Jalukar S.V. Hostager B.S. Bishop G.A. J. Immunol. 2000; 164: 623-630Crossref PubMed Scopus (79) Google Scholar). Preparation of Whole Cell Lysates and Western Blot Analysis— RAW264.7 cells (2 × 106 cells/ml) were pretreated with Me2SO or SP600125 (5 μm) for 15 min and then stimulated with medium or CpG DNA (6 μg/ml) for 45 min. In some experiments, TRAF6DN-RAW264.7 (2 × 106 cells/ml) were stimulated with medium, CpG DNA (6 μg/ml), or phorbol 12-myristate 13-acetate (50 ng/ml) for 45 min. Whole cell lysates were prepared as described previously (58Yi A.K. Yoon J.G. Yeo S.J. Hong S.C. English B.K. Krieg A.M. J. Immunol.

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