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B Super-Repressor in Human Intestinal Epithelial Cells1
*
Departments of Medicine, Microbiology, Immunology and
the Center for Gastrointestinal Biology and Disease, University of North Carolina, Chapel Hill, NC 27599;
Division of Clinical Immunology, Mount Sinai Medical Center, New York, NY 10029; and
§
Department of Pharmacology, University of California, San Diego, CA 92093
NF-
B plays a major role in the transcriptional regulation of
many proinflammatory genes in multiple cell lineages, including
intestinal epithelial cells (IEC). Activation of NF-B requires both
phosphorylation and degradation of its natural cytoplasmic inhibitor,
IB. We tested whether a super-repressor of NF-B activity, which
is a mutated nondegradable IB
resistant to phosphorylation and
degradation, could be delivered into IEC using an adenoviral vector
(Ad5IB) and determined the anti-inflammatory potential of this
inhibitor following different stimuli. We showed for the first time
that recombinant adenovirus efficiently infected (>80%) transformed
as well as primary IEC. Cytoplasmic levels of the NF-B
super-repressor protein were more than 50-fold higher than those of
endogenous IB, and this mutated IB was resistant to
IL-1ß-induced degradation. Immunofluorescent RelA nuclear staining
was strongly inhibited in Ad5IB-infected IEC compared with control
Ad5LacZ, and NF-B, but not AP-1 binding activity, was reduced by
more than 70% as measured by electrophoretic mobility shift assay
(EMSA). Induction of inducible nitric-oxide synthase (iNOS), IL-1ß,
and IL-8 genes by IL-1ß, TNF-, or PMA was blocked in
Ad5IB-infected cells but not in Ad5LacZ controls as assayed by
RT-PCR and ELISA. In addition, IL-1ß-induced IL-8 secretion was
totally inhibited by Ad5IB in primary colonic IEC. We conclude that
an adenoviral vector efficiently transfers a nondegradable IB in
both transformed and native IEC. The strong inhibition of NF-B
activity and the resulting down-regulation of multiple proinflammatory
molecules by Ad5IB suggests an exciting approach for in vivo
intestinal gene therapy and illustrates the key role of NF-B in
transcriptional regulation of the inflammatory phenotype of IEC.
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N. Giannoukakis, W. A. Rudert, M. Trucco, and P. D. Robbins Protection of Human Islets from the Effects of Interleukin-1beta by Adenoviral Gene Transfer of an Ikappa B Repressor J. Biol. Chem., November 17, 2000; 275(47): 36509 - 36513. [Abstract] [Full Text] [PDF]
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E. G. A. Cafferata, A. M. G. Guerrico, O. H. Pivetta, and T. A. Santa-Coloma NF-kappa B Activation Is Involved in Regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Interleukin-1beta J. Biol. Chem., April 27, 2001; 276(18): 15441 - 15444. [Abstract] [Full Text] [PDF]
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R. LaMonica, S. S. Kocer, J. Nazarova, W. Dowling, E. Geimonen, R. D. Shaw, and E. R. Mackow VP4 Differentially Regulates TRAF2 Signaling, Disengaging JNK Activation while Directing NF-kappa B to Effect Rotavirus-specific Cellular Responses J. Biol. Chem., June 1, 2001; 276(23): 19889 - 19896. [Abstract] [Full Text] [PDF]
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A. K. Cardozo, H. Heimberg, Y. Heremans, R. Leeman, B. Kutlu, M. Kruhoffer, T. Orntoft, and D. L. Eizirik A Comprehensive Analysis of Cytokine-induced and Nuclear Factor-kappa B-dependent Genes in Primary Rat Pancreatic beta -Cells J. Biol. Chem., December 21, 2001; 276(52): 48879 - 48886. [Abstract] [Full Text] [PDF]
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