Panoply™ Human RIPK1 Knockdown Stable Cell Line

As a form of programmed cell death, necroptosis has intrinsic initiators, including receptor-interacting serine/threonine protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like proteins (MLKL). These factors collectively constitute the necroptosis signaling pathway and mediate necroptosis induced by various necroptosis-stimulating factors, such as tumor necrosis factor (TNF). Although chemical inhibition of RIPK1 can block TNF-induced necroptosis, gene knockout of RIPK1 does not inhibit it but rather promotes TNF-induced necroptosis. Furthermore, RIPK3 has been reported to mediate RIPK1-independent necroptosis. Here, researchers found that TRADD is crucial for TNF-induced necroptosis in RIPK1-knockdown L929 and HT-22 cells. Mechanistic studies revealed that TRADD binds to RIPK3 to form a novel protein complex. This complex promotes RIPK3 oligomerization and subsequently phosphorylation, ultimately activating the RIPK3-MLKL signaling pathway. Therefore, TRADD, as a RIPK3 chaperone protein, initiates necroptosis in RIPK1-knockdown L929 and HT-22 cells upon TNF stimulation. Furthermore, TRADD is crucial for the accumulation of reactive oxygen species (ROS), a key factor in TNF-induced RIPK1-independent necroptosis. These results indicate that TRADD is a novel target protein for TNF-induced RIPK3 activation and subsequent RIPK1-independent necroptosis.

It has been reported that RIPK3 phosphorylation promotes the accumulation of intracellular ROS, which is crucial for TNF-induced necrotizing apoptosis in L929 cells. Since TRADD is essential for RIPK3 phosphorylation in RIPK1-independent necrotizing apoptosis, researchers then investigated the effect of TRADD on ROS accumulation. As shown in Figure 1A, ROS levels were significantly increased in both RIPK1 knockdown cells and negative control L929 cells under TNF stimulation. Furthermore, the antioxidant butylated hydroxyanisole (BHA) significantly inhibited both RIPK1-dependent and RIPK1-independent necrotizing apoptosis (Figure 1B). Therefore, these data indicate that ROS also accumulates in RIPK1-knockdown L929 cells and mediates RIPK1-independent necrotizing apoptosis. However, simultaneous knockdown of TRADD inhibited ROS accumulation in TNF-induced RIPK1-knockdown L929 cells (Figure 1C). Moreover, restoring TRADD expression in L929 cells with both TRADD and RIPK1 knockdown restored TNF plus Z-VAD-induced ROS accumulation (Figure 1D). Therefore, these data indicate that TRADD is crucial for TNF-induced ROS accumulation in the absence of RIPK1.

Figure 1. ROS accumulate in a TRADD-dependent manner and mediate necroptosis in RIPK1-knockdown L929 cells upon TNF stimulation. (Wang, et al., 2020)