Abstract
Tumor necrosis factor (TNF) has a critical role in diverse cellular events including inflammation, apoptosis and necroptosis through different signaling complexes. However, little is known about how the transition from inflammatory signaling to the engagement of death pathways is modulated. Here we report that the cytoplasmic retinoic acid receptor gamma (RARγ) controls receptor-interacting protein kinase 1 (RIP1)-initiated cell death when cellular inhibitor of apoptosis (cIAP) activity is blocked. Through screening a short hairpin RNA library, we found that RARγ was essential for TNF-induced RIP1-initiated apoptosis and necroptosis. Our data suggests that RARγ initiates the formation of death signaling complexes by mediating RIP1 dissociation from TNF receptor 1. We demonstrate that RARγ is released from the nucleus to orchestrate the formation of the cytosolic death complexes. In addition, we demonstrate that RARγ has a similar role in TNF-induced necroptosis in vivo. Thus, our study suggests that nuclear receptor RARγ provides a key checkpoint for the transition from life to death.
Highlights
Tumor necrosis factor (TNF) has a critical role in diverse cellular events including inflammation, apoptosis and necroptosis through different signaling complexes
The expression levels of RARγ, RARα, receptor-interacting protein kinase 1 (RIP1), RIP3, TNFR1-associated death domain protein (TRADD), mixed lineage kinase-domain-like (MLKL), CYLD and TNFR-associated factor 2 (TRAF2) in the three RARγ-short hairpin RNA (shRNA) clones and cont-shRNA cells were examined by western blotting (Fig. 1a) and the results indicated that the RARγ-shRNA knocked down the level of RARγ protein and did not affect the expression of other related proteins
TNF-induced apoptosis and necroptosis could be initiated by TRADD or RIP1 respectively when de novo protein synthesis is blocked or inhibitor of apoptosis (IAP) E3 ligases are inhibited[1, 3]
Summary
RARγ is required for cell death initiated by RIP1. To identify additional components of TNF-induced necroptosis, we used a retroviral short hairpin RNA (shRNA)-mediated genetic screen to identify genes whose knockdown resulting in resistance to necroptosis. To examine whether RARγ is required for necroptosis, we treated these cells with TNF-α and Smac mimetic (TS) to induce RIP1-initiated apoptosis. RARα does not have any redundant role with RARγ in the process since knockdown of RARα in RARγ-shRNA-A cells did not further increase the resistance of the cells to TNF-induced RIP1-initiated apoptosis and necroptosis (Supplementary Fig. 4b). Inhibition of TSZ-induced necroptosis by the specific RIP1 inhibitor, Necrostatin-1, had no effect on the dramatic increase of RARγ localization in the cytoplasm (Supplementary Fig. 8). These results indicated that RARγ was released to the cytoplasm from the nucleus during RIP1-initiated apoptosis and necroptosis. Smac-mimetic treatment alone was sufficient to trigger the release of RARγ from the nucleus
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