Cross-Talk between the Pathways Leading to the INduction of Apoptosis and the Secretion of Tumor Necrosis Factor-α in Ricin-Treated RAW 264.7 Cells

Abstract

Ricin induced apoptotic nuclear morphological changes in mouse macrophage cell line RAW264.7 cells at concentrations sufficient to cause severe protein synthesis inhibition. Ricin also induced the release of tumor necrosis factor-α (TNF-α) from this cell line in a dose-dependent manner but the profile was bell-shaped. However, the isolated galactose-specific ricin B-chain had no such effects. These results suggest that the receptor-binding of ricin through the B-chain is not enough, and subsequent attack on the intracellular target, i.e., the 28 S ribosomal RNA (rRNA), by the A-chain of internalized ricin is required for the effects of ricin. Z-D-CH₂-DCB, a caspase family inhibitor, showed potent inhibition of the release of TNF-α from RAW264.7 cells as well as blockage of the induction of apoptosis by ricin. Furthermore, SB202190, a specific P 38 mitogen-activated protein (MAP) kinase inhibitor that strongly inhibits the release of TNF-α, also showed significant inhibition of ricin-induced apoptosis. These results suggest that there may be cross-talk between the pathways leading to the release of TNF-α and apoptosis. Time course analysis revealed that the activation of p 38 MAP kinase started prior to the induction of TNF-α release and apoptosis. Since the activation of p 38 MAP kinase in ricin-treated RAW264.7 cells was not prevented by Z-D-CH₂-DCB, the activation of p 38 MAP kinase may occur upstream of the caspase cascade. Among the other protein synthesis inhibitors examined, modeccin and anisomycin, which can trigger a ribotoxic stress response similar to ricin, induced the release of TNF-α, but emetine and cycloheximide did not. These results suggest that the specific attack on the 28 S ribosomal RNA and the resulting ribotoxic stress response may trigger the multiple signal transduction pathways through the activation of p 38 MAP kinase, which in turn leads to TNF-α release and apoptosis.