Effects of TNFα on expression of ICAM-1 in human airway epithelial cells in vitro: oxidant-mediated pathways and transcription factors

We demonstrate that two different cell-permeable antioxidants, pyrrolidine dithiocarbamate (PDTC) and dimethylthiourea (DMTU), inhibit TNFα-induced ICAM-1 surface and gene expression in primary cultures of differentiated normal human bronchial epithelial (NHBE) cells. In addition, TNFα stimulates binding of nuclear proteins to the nuclear factor kappa beta (NFκB) and the CAAT/enhancer binding protein (C/EBP) consensus sites in the ICAM-1 promoter in these cells. Because these transcription factors have been suggested to be oxidant-sensitive and important in ICAM-1 expression, the potential involvement of reactive oxygen species (ROS) in the response to TNFα was investigated. Interestingly, neither PDTC nor DMTU altered binding of NFκB complexes. In contrast, either the proteasome inhibitor carbobenzoxy-L-leucy-L-leucy-L-leucinal (MG 132) or the IκBα inhibitor BAY 11-7082 ablated TNFα-induced ICAM-1 gene expression and MG132 inhibited TNFα-induced NFκB complexes. Surprisingly, either PDTC or DMTU inhibited the binding of TNFα-enhanced C/EBP complexes to the consensus site directly adjacent to the NFκB site. These results suggest that although TNFα enhances binding of C/EBP and NFκB complexes in NHBE cells, C/EBP binding seems to involve an oxidant-dependent mechanism, whereas activation of NFκB complexes utilizes the ubiquitin-proteasome pathway, a mechanism that seems to be unaltered by the presence of antioxidants. Because interference with either signaling pathway abrogates TNFα-induced ICAM-1 expression, activation of both complexes seems to be involved in this response to TNFα, but this activation occurs via different intracellular pathways.