title: Myeloid differentiation factor 88-dependent post-transcriptional regulation of cyclooxygenase-2 expression by CpG DNA: tumor necrosis factor-alpha receptor-associated factor 6, a diverging point in the Toll-like receptor 9-signaling.
abstract: 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.
authors.affiliation: Children's Foundation Research Center at Le Bonheur Children's Medical Center, Department of Pediatrics, University of Tennessee Health Science Center, Memphis, Tennessee 38103, USA.
authors.forename: Seon-Ju
authors.initials: SJ
authors.lastname: Yeo
authors.forename: Jae-Geun
authors.initials: JG
authors.lastname: Yoon
authors.forename: Ae-Kyung
authors.initials: AK
authors.lastname: Yi
mesh_terms: D020413:3' Untranslated Regions; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D002460:Cell Line; D018899:CpG Islands; D051546:Cyclooxygenase 2; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D008156:Luciferases / Q000378:metabolism; D020935:MAP Kinase Signaling System; D051379:Mice; D053594:Myeloid Differentiation Factor 88; D010957:Plasmids / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis* / Q000235:genetics; D011506:Proteins / Q000378:metabolism / Q000502:physiology*; D012323:RNA Processing, Post-Transcriptional; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D048029:TNF Receptor-Associated Factor 6; D051217:Toll-Like Receptor 9; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; D048868:Adaptor Proteins, Signal Transducing; D000943:Antigens, Differentiation; D004268:DNA-Binding Proteins; D007527:Isoenzymes; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D011506:Proteins; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D048029:TNF Receptor-Associated Factor 6; C495370:Tlr9 protein, mouse; D051217:Toll-Like Receptor 9; D004247:DNA; D008156:Luciferases; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases