Increased susceptibility of renal epithelial cells to TNFα-induced apoptosis following treatment with fumonisin B₁

Previous studies have shown that tumor necrosis factor α (TNFα) is involved in the pathogenic events following exposure to fumonisin B₁ (FB₁), a potent inhibitor of ceramide synthase and sphingolipid biosynthesis. The intimate role of sphingolipid mediators in TNFα signaling and cellular death suggests that FB₁ may alter the sensitivity of cells to TNFα-induced apoptosis. We tested the hypothesis that FB₁ treatment will increase the sensitivity of porcine renal epithelial cells to TNFα. Porcine renal epithelial cells (LLC-PK₁) were treated with FB₁ for 48 h prior to treatment with TNFα. A dose-dependent increase in TNFα-induced apoptosis was observed in cells pretreated with FB₁. Cells treated with FB₁ showed increased DNA fragmentation and terminal uridine nucleotide end labeling in response to TNFα treatment. FB₁ increased DNA synthesis and resulted in cell cycle arrest in the G₂/M phase of the cell cycle. Flow cytometric analysis of the cell cycle indicated that TNFα predominantly killed cells in the G₂/M phase. The activation of JNK, a mitogen-activated protein kinase (MAPK), was increased following 48 h exposure to FB₁. Phosphorylation of p38 and ERK remained unchanged following treatment with FB₁. FB₁ also increased free sphingoid base levels under identical treatment conditions. Results suggest that FB₁ increased free sphingoid base levels and the population of cells in the G₂/M phase. This population was shown to be most susceptible to TNFα-induced apoptosis. Phosphorylation of pro-apoptotic JNK may play an important role in these effects.