Abstract
CpG DNA induces macrophage cyclooxgenase-2 (Cox-2) production. In this study, we have investigated a biochemical signaling pathway and transcription factors responsible for transcriptional regulation of the Cox-2 gene expression induced by CpG DNA. CpG DNA-induced Cox-2 promoter activity was completely inhibited by an endosomal acidification inhibitor (chloroquine), a TLR9 antagonist inhibitory CpG DNA, or overexpression of a dominant negative (DN) form of MyD88. In contrast, overexpression of DN-IRAK1 or DN-TRAF6 only partially inhibited CpG DNA-induced Cox-2 promoter activity and NF-kappaB activation, indicating the presence of additional signaling modulators downstream of MyD88. CpG DNA-induced Cox-2 promoter activity was substantially suppressed in cells overexpressing super-suppressive IkappaB (IkappaB-arachidonic acid), DN-p38, or DN-CREB. In addition, Cox-2 promoter-luciferase reporters with alterations in predicted cis-acting transcriptional regulatory elements revealed that C/EBP, Ets-1, NF-kappaB, and CREB-binding sites are essential for optimal Cox-2 expression in response to CpG DNA. Conclusively, these results demonstrate that endosomal DNA processing and TLR9/MyD88-dependent activation of NF-kappaB and p38 are required for transcriptional regulation of Cox-2 expression induced by CpG DNA, and suggest that interleukin-1 receptor-associated kinase and/or TRAF6 may be a diverging point for NF-kappaB activation in response to CpG DNA in RAW264.7 cells.
Highlights
CpG DNA induces macrophage cyclooxgenase-2 (Cox-2) production
These results demonstrate that endosomal DNA processing and Toll-like receptor 9 (TLR9)/myeloid differentiation factor 88 (MyD88)-dependent activation of NF-B and p38 are required for transcriptional regulation of Cox-2 expression induced by CpG DNA, and suggest that interleukin-1 receptor-associated kinase and/or tumor necrosis factor-␣ receptor-associated factor 6 (TRAF6) may be a diverging point for NF-B activation in response to CpG DNA in RAW264.7 cells
We have investigated the molecular mechanisms by which CpG DNA induces Cox-2 expression at the transcriptional level in RAW264.7 cells
Summary
Oligodeoxynucleotides—Nuclease-resistant phosphorothioate oligodeoxynucleotides (S-ODN) were purchased from Operon (Alameda, CA) and further purified by ethanol precipitation. Site-directed mutagenesis was performed to modify each cis-acting element response site in the Cox-2 promoter region of the wild type Cox-2 promoter-luciferase reporter using the QuickChange mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. Cells were co-transfected with an equal amount (2 g) of control empty vector (pIRES-EGFP, pCDNA3, or pUSE), DNMyD88, DN-IRAK1, DN-TRAF6, DN-MEK1, DN-p38, DN-JNK1, IBAA, or DN-CREB and reporter gene Cox-2 promoter-luciferase (1 g) plus pRL-TK-luciferase (1 g), NF-B-luciferase (1 g) plus pRL-TKluciferase (1 g), CREB-luciferase (1 g) plus pRL-TK-luciferase (1 g), or AP-1--galactosidase (2 g) and incubated for 6 h. Cells (1 ϫ 106 cells/1.5-mm cell culture plate) were stimulated with medium, CpG
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
