RESUMO
Clostridium difficile causes an intense inflammatory colitis through the actions of two large exotoxins, toxin A and toxin B. IL-8 is believed to play an important role in the pathophysiology of C. difficile-mediated colitis, although the mechanism whereby the toxins up-regulate the release of IL-8 from target cells is not well understood. In this study, we investigated the mechanisms through which toxin A induces IL-8 secretion in human monocytes. We found that cellular uptake of toxin A is required for the up-regulation of IL-8, an effect that is not duplicated by a recombinant toxin fragment comprising the cell-binding domain alone. Toxin A induced IL-8 expression at the level of gene transcription and this effect occurred through a mechanism requiring intracellular calcium and calmodulin activation. Additionally, the effects of toxin A were inhibited by the protein tyrosine kinase inhibitor genistein, but were unaffected by inhibitors of protein kinase C and phosphatidylinositol-3 kinase. We determined that toxin A activates nuclear translocation of the transcription factors NF-kappa B and AP-1, but not NF-IL-6. NF-kappa B inhibitors blocked the ability of toxin A to induce IL-8 secretion, and supershift analysis indicated that the major isoform of NF-kappa B activated by the toxin is a p50-p65 heterodimer. This study is the first to identify intracellular signaling pathways and transcription factors involved in the C. difficile toxin-mediated up-regulation of IL-8 synthesis and release by target cells. This information should increase our understanding of the pathogenesis of C. difficile colitis and the nature of IL-8 gene regulation as well.
Assuntos
Toxinas Bacterianas , Cálcio/fisiologia , Clostridioides difficile/imunologia , Enterotoxinas/fisiologia , Interleucina-8/metabolismo , Monócitos/metabolismo , NF-kappa B/fisiologia , Regulação para Cima/imunologia , Transporte Biológico/genética , Proteínas Estimuladoras de Ligação a CCAAT , Cálcio/metabolismo , Sinalização do Cálcio/imunologia , Calmodulina/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Enterotoxinas/antagonistas & inibidores , Enterotoxinas/genética , Enterotoxinas/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Interleucina-8/biossíntese , Interleucina-8/genética , Líquido Intracelular/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/imunologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição RelA , Transcrição Gênica/imunologia , Regulação para Cima/efeitos dos fármacosRESUMO
A human tryptophan hydroxylase intron seven polymorphism previously associated with low CSF 5-HIAA and suicidal behavior was sequenced and characterized for its potential role in TPH pre-mRNA splicing. Two polymorphic sites were identified: A218C and A779C. The 779A allelic frequency in various populations ranged from 0.43 to 0.61 and was in strong linkage disequilibrium with the A218C site. A218C provides a site for restriction fragment length polymorphism analysis. TPH mRNA was reverse-transcribed and sequenced. No aberrant splice products from the 779A or 779G TPH genes were detected nor were any other polymorphic nucleotides found.
