Effects of Vomitoxin (Deoxynivalenol) on Transcription Factor NF-κB/Rel Binding Activity in Murine EL-4 Thymoma and Primary CD4+T Cells

Abstract

The trichothecene mycotoxin vomitoxin (VT, deoxynivalenol) superinduces the gene expression of IL-2 and several other cytokines in both cellular and murine models. Because transcription factor NF-κB/Rel has been shown to play a crucial role in control of cytokine gene transcription, we assessed thein vitroeffects of VT on NF-κB/Rel binding activity by electrophoretic mobility shift assay using both cloned (EL-4) and primary (CD4+) murine T cells. When EL-4 thymoma cells were stimulated with phorbol 12-myristate 13-acetate plus ionomycin in the presence of 500 ng/ml VT, DNA binding activity by NF-κB/Rel in nuclear extracts was increased from 2 to 48 hr when compared to controls employing no VT. VT preferentially induced a slower migrating electrophoretic band of the NF-κB/Rel complex particularly in later time points (8–48 hr). The band was found to contain a c-Rel/p50 heterodimer by supershift assay using antibodies specific for c-Rel and p50. NF-κB/Rel binding activity was enhanced by VT in a dose-dependent fashion. As little as 50 ng/ml VT was sufficient to increase NF-κB/Rel binding in a 1-hr EL-4 culture. Using Western blot analysis, effects on EL-4 cells were further related to VT-mediated inhibition of resynthesis of IκBα, a cytoplasmic inhibitor of NF-κB/Rel. Decreased IκBα levels were observed with 250–1000 ng/ml VT from 4 to 48 hr. Using primary murine CD4+T cell cultures, elevated NF-κB/Rel and c-Rel/p50 binding activities were also observed at VT of 500 ng/ml from 1 to 72 hr concurrently with decreased IκBα levels. These data suggest that VT increased NF-κB/Rel binding activity and, in particular, the transactivating form, c-Rel. Increased NF-κB/Rel binding activity in the later (48 hr) stages of cell incubation may be explained, in part, by VT-mediated inhibition of resynthesis of its cytoplasmic inhibitor IκBα and by decreases in the inhibitory p50 homodimer. Elevated NF-κB/Rel binding activity may be involved mechanistically in VT-induced gene expression of the cytokines and resultant toxic and autoimmune effects.