Evidence for altered regulation of I kappa B alpha degradation in human colonic epithelial cells.
The nuclear factor kappaB (NF-kappaB) regulates the transcription of genes bearing the kappaB consensus motif. Transmigration of NF-kappaB from the cytoplasm to the nucleus is regulated by the IkappaB family of inhibitory NF-kappaB-binding proteins. Dissociation of the NF-kappaB-IkappaB complex requires both phosphorylation and degradation of IkappaBs. We demonstrate that IL-1beta induces complete IkappaB alpha degradation in Caco-2 cell lines but not in HT-29, T84, SW-480 transformed cell lines, or native colonic epithelial cells. A similar lack of IkappaB alpha degradation in HT-29 cells followed TNF-alpha and bacterial polymer stimulation. IL-1beta stimulated NF-kappaB nuclear translocation and NF-kappaB-dependent IL-1beta and IL-8 expression in both Caco-2 and HT-29 cells as assayed by electrophoretic mobility shift assay, immunofluorescence, kappaB-luciferase transfection, reverse transcriptase-PCR analysis and ELISA. In HT-29 cells stimulated with IL-1beta, IkappaB alpha was phosphorylated and when cycloheximide blocked new protein synthesis, IkappaB alpha was partially degraded. NF-kappaB cytoplasmic to nuclear transmigration was incomplete and preceded IkappaB alpha degradation in 9T-29 cells, in contrast to complete coordinated NF-kappaB nuclear translocation and IkappaB alpha degradation in Caco-2 cells. Greater sensitivity of HT-29 cells to a calpain inhibitor, as measured by IL-8 secretion, suggested enhanced resistance to IkappaB alpha proteolysis. These data show that IL-1beta induces NF-kappaB activity and expression of NF-kappaB-dependent genes in colonic epithelial cells and suggest altered regulation of IkappaB alpha degradation compared with other cell lineages, which may result in their increased responsiveness to therapeutic blockade.
Jobin
C
C
Department of Medicine, University of North Carolina at Chapel Hill, 27599, USA.
Haskill
S
S
Mayer
L
L
Panja
A
A
Sartor
R B
RB
D002461:Cell Line, Transformed; D003106:Colon / Q000166:cytology* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D020672:I-kappa B Proteins; D007375:Interleukin-1 / Q000096:biosynthesis / Q000494:pharmacology; D016209:Interleukin-8 / Q000096:biosynthesis; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D010766:Phosphorylation
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D007375:Interleukin-1; D016209:Interleukin-8; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha