INHIBITION OF PROINFLAMMATORY MOLECULE PRODUCTION BY ADENOVIRUS-MEDIATED EXPRESSION OF A NUCLEAR FACTOR KAPPA-B SUPER-REPRESSOR IN HUMAN INTESTINAL EPITHELIAL-CELLS

NF-kappa B plays a major role in the transcriptional regulation of man y proinflammatory genes in multiple cell lineages, including intestina l epithelial cells (IEC), Activation of NF-kappa B requires both phosp horylation and degradation of its natural cytoplasmic inhibitor, I kap pa B. We tested whether a super-repressor of NF-kappa B activity, whic h is a mutated nondegradable I kappa B alpha resistant to phosphorylat ion and degradation, could be delivered into IEC using an adenoviral v ector (Ad5I kappa B) and determined the antiinflammatory potential of this inhibitor following different stimuli. We showed for the first ti me that recombinant adenovirus efficiently infected (>80%) transformed as well as primary IEC, Cytoplasmic levels of the NF-kappa B super-re pressor protein were more than 50-fold higher than those of endogenous I kappa B, and this mutated I kappa B was resistant to IL-1 beta-indu ced degradation. Immunofluorescent RelA nuclear staining was strongly inhibited in Ad5I kappa B-infected IEC compared with control Ad5LacZ, and NF-kappa B, but not AP-1 binding activity, was reduced by more tha n 70% as measured by electrophoretic mobility shift assay (EMSA), Indu ction of inducible nitric-oxide synthase (iNOS), IL-1 beta, and IL-8 g enes by IL-1 beta, TNF-alpha, or PMA was blocked in Ad5I kappa B-infec ted cells but not in Ad5LacZ controls as assayed by RT-PCR and ELISA. In addition, IL-1 beta-induced IL-8 secretion was totally inhibited by Ad5I kappa B in primary colonic IEC, We conclude that an adenoviral v ector efficiently transfers a nondegradable I kappa B in both transfor med and native IEC. The strong inhibition of NF-kappa B activity and t he resulting down-regulation of multiple proinflammatory molecules by Ad5I kappa B suggests an exciting approach for in vivo intestinal gene therapy and illustrates the key role of NF-kappa B in transcriptional regulation of the inflammatory phenotype of IEC.