Superinduction of IL-2 Gene Expression by Vomitoxin (Deoxynivalenol) Involves Increased mRNA Stability

Abstract

To better understand molecular mechanisms by which the trichothecene vomitoxin (VT) superinduces cytokine gene expression, we studied the posttranscriptional effects of this mycotoxin on interleukin-2 (IL-2) gene expression in murine EL-4 thymoma cells stimulated with phorbol 12-myristate 13-acetate and ionomycin (PMA + ION). Northern analysis revealed that doses of 50 to 500 ng/ml VT superinduced IL-2 mRNA expression in a dose- and time-dependent manner in a synchronous model where VT was added at onset of PMA + ION stimulation. In accordance with the mRNA levels, IL-2 production was significantly elevated in the presence of 50 to 250 ng/ml VT. Superinduction of IL-2 mRNA was also observed in a delayed synchronous model (VT added 20 hr after PMA + ION stimulation) and an asynchronous model (VT added 20 hr after PMA + ION stimulation and removal). To assess the effects of VT (500 ng/ml) on IL-2 mRNA half-life, three transcriptional inhibitors were used in the delayed synchronous model. Actinomycin D (ActD) had a pronounced stabilizing effect on IL-2 mRNA but not on mRNA for the housekeeping gene GAPDH. VT did not affect IL-2 mRNA levels in ActD-treated cells. Although 5,6-dichloro-β- d-ribofuranosyl-benzimidazole (DRB) also had a stabilizing effect on IL-2 mRNA, IL-2 mRNA half-life t 1/2in VT-treated cells was three times that of control. In contrast, inclusion of cyclosporin A (CsA) into the cultures specifically arrested IL-2 transcription in EL-4 cells without any stabilizing effect. VT exposure in the presence of CsA markedly prolonged the half-life of IL-2 mRNA in a dose-dependent manner. The t 1/2for IL-2 mRNA in the control culture was 2.1 hr, whereas t 1/2was 3.1, 3.4, 4.2, and 10.5 hr in cultures containing 50, 100, 250, and 500 ng/ml VT, respectively. These results suggest that VT can superinduce IL-2 at both the mRNA and the protein level and that this superinduction can be explained, in part, by posttranscriptional mechanisms such as enhanced mRNA stability.