24(S)-Hydroxycholesterol induces RIPK1-dependent but MLKL-independent cell death in the absence of caspase-8

Abstract 24(S)-Hydroxycholesterol (24S-OHC), which is enzymatically produced in the brain, is known to play an important role in maintaining brain cholesterol homeostasis. We have previously reported that 24S-OHC induces a type of non-apoptotic programmed necrosis in neuronal cells expressing little caspase-8. Necroptosis has been characterized as a type of programmed necrosis in which activation of receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) is involved in the signaling pathway. In the present study, we investigated the involvement of these three proteins in 24S-OHC-induced cell death. We found that RIPK1 but neither RIPK3 nor MLKL was expressed in human neuroblastoma SH-SY5Y cells, while all three proteins were expressed in human T lymphoma caspase-8-deficient Jurkat (Jurkat^Cas8-/-) cells. In Jurkat^Cas8-/- cells, tumor necrosis factor α (TNFα)-induced cell death was significantly suppressed by treatment with respective inhibitors of RIPK1, RIPK3, and MLKL. In contrast, only RIPK1 inhibitor showed significant suppression of 24S-OHC-induced cell death, and even this was less prominent than was observed in TNFα-induced cell death. In Jurkat^Cas8-/- cells, knockdown of either RIPK1 or RIPK3 caused moderate but significant suppression of 24S-OHC-induced cell death, but no such effect was observed as a result of knockdown of MLKL. Collectively, these results suggest that, for both SH-SY5Y cells and Jurkat^Cas8-/- cells, 24S-OHC-induced cell death is dependent on RIPK1 but not on MLKL. We therefore conclude that, in the absence of caspase-8 activity, 24S-OHC induces a necroptosis-like cell death which is RIPK1-dependent but MLKL-independent.