TNF receptor-associated factor-2 is involved in both IL-1 beta and TNF-a signaling cascades leading to NF-kappa B activation and IL-8 expression in human intestinal epithelial cells

Cytokine signaling involves the participation of many adaptor proteins, inc luding the docking protein TNF receptor-associated factor-2 (TRAF-2), which is believed to transmit the TNF-alpha signal through both the I kappa B/NF -kappa B and c-Jun N-terminal kinase (JNK)/stress-related protein kinase (S APK) pathways. The physiological role of TRAF proteins in cytokine signalin g in intestinal epithelial cells (IEC) is unknown. We characterized the eff ect of a dominant-negative TRAF-2 delivered by an adenoviral vector (Ad5dnT RAF-2) on the cytokine signaling cascade in several LEC and also investigat ed whether inhibiting the TRAF-2-transmitting signal blocked TNF-alpha-indu ced TNF-kappa B and IL-8 gene expression. A high efficacy and level of Ad5d nTRAF-2 gene transfer were obtained in IEC using a multiplicity of infectio n of 50. Ad5dnTRAF-2 expression prevented TNF-cu-induced, but not IL-1 beta -induced, I kappa B alpha degradation and NF-kappa B activation in NIH-3T3 and IEC-6 cells. TNF-alpha-induced JNK activation was also inhibited in Ad5 dnTRAF-2-infected HT-29 cells. Induction of IL-8 gene expression by TNF-alp ha was partially inhibited in Ad5dnTRAF-2-transfected HT-29, but not in con trol Ad5LacZ-infected, cells. Surprisingly, IL-1 beta-mediated IL-8 gene ex pression was also inhibited in HT 29 cells as measured by Northern blot and ELISA, We concluded that TRAF-3 is partially involved in TNF-alpha-mediate d signaling through I kappa B/NF-kappa B in LEG. In addition, our data sugg est that TRAF-2 is involved in IL-1 beta signaling in HT-29 cells. Manipula tion of cytokine signaling pathways represents a new approach for inhibitin g proinflammatory gene expression in IEC.