Cellular differentiation causes a selective down-regulation of interleukin (IL)-1β-mediated NF-κB activation and IL-8 gene expression in intestinal epithelial cells

Interleukin (IL)-1 beta signals through various adapter proteins and kinases that lead to activation of numerous downstream targets, including the transcription factors including NF-kappa B. In this study, we analyzed and characterized the effect of the differentiation of intestinal epithelial cells on IL-1 beta-mediated NF-kappa B activation and IL-8 gene expression. We report that IL-8 mRNA accumulation and protein secretion were down-regulated in IL-1 beta- and lipopolysaccharide-stimulated differentiated HT-29 cells (HT-29/MTX, where MTX is methotrexate) compared with undifferentiated cells (HT-29/p), whereas no differential effects were found following tumor necrosis factor (TNF)-alpha or phorbol myristate acetate stimulation. Cross-linking and affinity binding studies reveal that IL-1 beta exclusively binds the type I receptor (IL-1RI) and not IL-1RII in both HT-29/p and HT-29/MTX cells. IL-1 beta-mediated I kappa B kinase and c-Jun N-terminal kinase (JNK) activity were both diminished in differentiated HT-29 cells. DNA binding activity in differentiated HT-29 cells relative to HT-29/p cells was strongly reduced following IL-1 beta exposure but not after TNF-alpha stimulation. The proximal IL-1 signaling molecule IL-1 receptor-associated kinase was not degraded in IL-1 beta-stimulated HT-29 cells, in contrast to Caco-2 cells, kappa B-luciferase reporter gene activity was 16-fold higher following TNF receptor-associated factor-6 transfection after IL-1 beta stimulation in HT-29/MTX cells. We conclude that cellular differentiation of HT-29 cells selectively impairs the IL-1 beta signaling pathway inhibiting both NF-kappa B and JNK activity in response to IL-1 beta. This relative unresponsiveness to IL-1 beta may represent an important regulatory mechanism of differentiated intestinal epithelial cells.