Effects of vomitoxin (deoxynivalenol) on transcription factor NF-kappa B/Rel binding activity in murine EL-4 thymoma and primary CD4+ T cells.
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-kappaB/Rel has been shown to play a crucial role in control of cytokine gene transcription, we assessed the in vitro effects of VT on NF-kappaB/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-kappaB/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-kappaB/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-kappaB/Rel binding activity was enhanced by VT in a dose-dependent fashion. As little as 50 ng/ml VT was sufficient to increase NF-kappaB/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 IkappaBalpha, a cytoplasmic inhibitor of NF-kappaB/Rel. Decreased IkappaBalpha levels were observed with 250-1000 ng/ml VT from 4 to 48 hr. Using primary murine CD4+ T cell cultures, elevated NF-kappaB/Rel and c-Rel/p50 binding activities were also observed at VT of 500 ng/ml from 1 to 72 hr concurrently with decreased IkappaBalpha levels. These data suggest that VT increased NF-kappaB/Rel binding activity and, in particular, the transactivating form, c-Rel. Increased NF-kappaB/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 IkappaBalpha and by decreases in the inhibitory p50 homodimer. Elevated NF-kappaB/Rel binding activity may be involved mechanistically in VT-induced gene expression of the cytokines and resultant toxic and autoimmune effects.
Department of Food Science and Human Nutrition, Michigan State University, East Lansing 48824-1224, USA.
Y L
YL
Ouyang
S
S
Li
J J
JJ
Pestka
D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism*; D002478:Cells, Cultured; D051379:Mice; D016328:NF-kappa B / Q000187:drug effects*; D011485:Protein Binding / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000187:drug effects*; D020569:Proto-Oncogene Proteins c-rel; D013945:Thymoma / Q000235:genetics* / Q000378:metabolism*; D014255:Trichothecenes / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D014255:Trichothecenes; C007262:deoxynivalenol