CpG stimulation of primary mouse B cells is blocked by inhibitory oligodeoxyribonucleotides at a site proximal to NF-kappaB activation.
Bacterial DNA and CpG-oligodeoxyribonucleotides (ODN) are powerful B cell activators, inducing apoptosis protection, cell cycle entry, proliferation, costimulatory molecule expression, immunoglobulin (Ig) and interleukin-6 (IL-6) secretion. However, proximal events in B cell activation by ODN are only partially characterized, including the translocation of NF-kappaB to the nucleus. In this paper, we provide evidence that CpG-ODN-induced cell cycle entry and apoptosis protection are blocked by SN50 or gliotoxin and thus require NF-kappaB activation. NF-kappaB activation occurred within 30 minutes of stimulation of murine B cells with a phosphorothioate (S) CpG-ODN and persisted for up to 40 hours, with p50, p65, and c-Rel as the major components. Similar to other NF-kappaB inducers, CpG-ODN caused an early IkappaBalpha and IkappaBbeta degradation plus cleavage of the p50 precursor and subsequent NF-kappaB nuclear translocation. A group of closely related S-ODN, which specifically blocked CpG-induced B cell activation at submicromolar concentrations, also prevented NF-kappaB DNA binding and transcriptional activation. These inhibitory S-ODN differed from stimulatory S-ODN by having 2-3 G substitutions in the central motif. As inhibitory S-ODN did not directly interfere with the NF-kappaB DNA binding but prevented CpG-induced NF-kappaB nuclear translocation of p50, p65, and c-Rel and blocked p105, IkappaBalpha, and IkappaBbeta degradation, we concluded that their putative target must lie upstream of inhibitory kinase (IKK) activation.
Lenert
P
P
Department of Internal Medicine, University of Iowa, College of Medicine, Iowa City 52242, USA.
Stunz
L
L
YI
A K
AK
Krieg
A M
AM
Ashman
R F
RF
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001402:B-Lymphocytes / Q000187:drug effects* / Q000276:immunology; D001665:Binding Sites; D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D018899:CpG Islands / Q000502:physiology*; D003546:Cysteine Endopeptidases; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003864:Depression, Chemical; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D005912:Gliotoxin / Q000494:pharmacology; D020672:I-kappa B Proteins; D008213:Lymphocyte Activation / Q000187:drug effects; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D010455:Peptides / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013047:Specific Pathogen-Free Organisms; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D004268:DNA-Binding Proteins; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D009097:Multienzyme Complexes; D016328:NF-kappa B; C000606205:Nfkbia protein, mouse; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; C118258:SN50 peptide; D000072000:NF-KappaB Inhibitor alpha; D005912:Gliotoxin; D004247:DNA; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex