RESUMO
<p><b>OBJECTIVE</b>To investigate the role of TNF receptor-associated factor 2 (TRAF-2) and TRAF6 in CD40-induced nuclear factor-kappaB (NF-kappaB) signaling pathway and whether CD40 signaling requires TRAF2.</p><p><b>METHODS</b>Human B cell lines were transfected with plasmids expressing wild type TRAF2 or dominant negative TRAF2, TRAF2-shRNA, or TRAF6-shRNA. The activation of NF-kappaB was detected by Western blot, kinase assay, transfactor enzyme-linked immunosorbent assay (ELISA), and fluorescence resonance energy transfer (FRET). Analysis of the role of TRAF-2 and TRAF-6 in CD40-mediated NF-kappaB activity was examined following stimulation with recombinant CD154.</p><p><b>RESULTS</b>TRAF2 induced activity of IkappaB-kinases (IKKalpha, IKKi/epsilon), phosphorylation of IkappaBalpha, as well as nuclear translocation and phosphorylation of p65/RelA. In contrast, TRAF6 strongly induced NF-kappaB activation and nuclear translocation of p65 as well as p50 and c-Rel. Engagement of CD154-induced nuclear translocation of p65 was inhibited by a TRAF6-shRNA, but conversely was enhanced by a TRAF2-shRNA. Examination of direct interactions between CD40 and TRAFs by FRET documented that both TRAF2 and TRAF6 directly interacted with CD40. However, the two TRAFs competed for CD40 binding.</p><p><b>CONCLUSIONS</b>These results indicate that TRAF2 can signal in human B cells, but it is not essential for CD40-mediated NF-kappaB activation. Moreover, TRAF2 can compete with TRAF6 for CD40 binding, and thereby limit the capacity of CD40 engagement to induce NF-kappaB activation.</p>
Assuntos
Animais , Humanos , Linfócitos B , Biologia Celular , Fisiologia , Antígenos CD40 , Metabolismo , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular , Metabolismo , Transferência Ressonante de Energia de Fluorescência , Quinase I-kappa B , Metabolismo , NF-kappa B , Genética , Metabolismo , Proteínas Proto-Oncogênicas c-fos , Metabolismo , Transdução de Sinais , Fisiologia , Fator 2 Associado a Receptor de TNF , Genética , Metabolismo , Fator 6 Associado a Receptor de TNF , Genética , Metabolismo , Fator de Transcrição RelA , Metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno , MetabolismoRESUMO
BACKGROUND: In contrast to evidences of Ig H chain receptor editing in transformed cell lines and transgenic mouse models, there has been no direct evidence that this phenomenon occurs in human developing B cells. METHODS: V(H)DJ(H) rearrangements were obtained from genomic DNA of individual IgM- B cells from liver and IgM+B cells from bone marrow of 18 wk of gestation human fetus by PCR amplification and direct sequencing. RESULTS: We found three examples of H chain receptor editing from IgM+ and IgM+human fetal B cells. Two types of V(H) replacements were identified. The first involved V(H) hybrid formation, in which part of a V(H) gene from the initial VDJ rearrangement is replaced by part of an upstream V(H) gene at the site of cryptic RSS. The second involved a gene conversion like replacement of CDR2, in which another V(H) gene donated a portion of its CDR2 sequence to the initial VDJ rearrangement. CONCLUSION: These data provide evidence of receptor editing at the H chain loci in developing human B cells, and also the first evidence of a gene conversion event in human Ig genes.