Abstract
Cadmium(Cd) induces cytotoxicity via autophagy-induced apoptosis in non-activated mouse monocytes; however, the molecular mechanism remains unclear. Here, we show that autophagy induces Fas (CD95/APO-1)-mediated apoptosis by promoting accumulation of p62/SQSTM1 in response to Cd. Cd produced tumor necrosis factor (TNF)-α, peaking at 6 h, and exhibiting a concentration-dependent increase. Immunoblot analysis revealed polyubiquitinated (polyUb) full-length Fas (antibody clone G-9) and reduced cytosolic Fas (antibody clone M-20) in Cd-exposed RAW264.7 cells. The accumulation of polyUb-Fas was transient and positively correlated with polyUb-p62 and polyUb-proteins. Autophagy inhibition via chemical and genetic modulation suppressed Cd-induced polyUb-p62, polyUb-Fas, and polyUb-protein levels, whereas the level of cytosolic Fas recovered to that of the control. Immunofluorescence (IF) staining for full-length Fas, p62, and ubiquitin revealed an aggregated pattern in Cd-induced apoptotic cells, which was inhibited by blocking autophagy. Fas colocalized with microtubule-associated protein 1 light chain (LC)-3B. IF staining and immunoprecipitation assays revealed colocalization and interaction among p62, Ub, and Fas. Knockdown of p62 reduced the binding of Ub and Fas. Together, these data suggest that polyUb-p62 targets Fas and recruits it to autophagosomes, where Fas transiently aggregates to promote apoptosis and is degraded with polyUb-p62. In conclusion, autophagy regulates C-terminal cytosolic Fas aggregation via p62 polyubiquitination, which is required for apoptosis and may play a critical role in the production of select cytokines.
Highlights
The cell death receptor, a member of the tumor necrosis factor receptor (TNFR) superfamily, consists of tumor necrosis factor receptor (TNFR)-1, Fas (CD95/APO-1), and the tumor necrosis factor-related apoptosis-inducing ligand death receptors DR4 and DR51
Damage to RAW264.7 cells caused by Cd exposure was examined by fluorescence-activated cell sorting (FACS) analysis
Western blot analysis revealed a decrease in C-terminal cytosolic Fas that was dependent on Cd concentration and exposure time used the half maximal inhibitory concentration (IC50) of Cd
Summary
The cell death receptor, a member of the tumor necrosis factor receptor (TNFR) superfamily, consists of tumor necrosis factor receptor (TNFR)-1, Fas (CD95/APO-1), and the tumor necrosis factor-related apoptosis-inducing ligand death receptors DR4 and DR51. Autophagy and apoptosis are programmed cell death pathways that are induced by different molecular mechanisms. FADD plays a role in the formation of DISC, which is involved in the extrinsic apoptotic pathway, as well as in autophagic cell death by interacting with Atg[527]. Since low doses of Cd is more effective in cell proliferation[30,31], our study focused on cell death by toxic doses of Cd. Cd toxicity is associated with different modes of cell death, including autophagy, apoptosis, and necrosis, which can impair the immune response[32]. The aim of this study was to clarify the relationship between Fas signaling and autophagy in Cd-induced toxicity. For this purpose, we used the monocytes/macrophage cell line Raw264.7, which plays a central role in the activation of the immune system[35]
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