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  • Activation of ferritinophagy is required for the RNA-binding protein ELAVL1/HuR to regulate ferroptosis in hepatic stellate cells.

Activation of ferritinophagy is required for the RNA-binding protein ELAVL1/HuR to regulate ferroptosis in hepatic stellate cells.

Autophagy (2018-08-08)
Zili Zhang, Zhen Yao, Ling Wang, Hai Ding, Jiangjuan Shao, Anping Chen, Feng Zhang, Shizhong Zheng
RESUMEN

Ferroptosis is a recently recognized form of regulated cell death that is characterized by lipid peroxidation. However, the molecular mechanisms regulating ferroptosis are largely unknown. In this study, we report that the RNA-binding protein ELAVL1/HuR plays a crucial role in regulating ferroptosis in liver fibrosis. Upon exposure to ferroptosis-inducing compounds, ELAVL1 protein expression was remarkably increased through the inhibition of the ubiquitin-proteasome pathway. ELAVL1 siRNA led to ferroptosis resistance, whereas ELAVL1 plasmid contributed to classical ferroptotic events. Interestingly, upregulated ELAVL1 expression also appeared to increase autophagosome generation and macroautophagic/autophagic flux, which was the underlying mechanism for ELAVL1-enhanced ferroptosis. Autophagy depletion completely impaired ELAVL1-mediated ferroptotic events, whereas autophagy induction showed a synergistic effect with ELAVL1. Importantly, ELAVL1 promoted autophagy activation via binding to the AU-rich elements within the F3 of the 3'-untranslated region of BECN1/Beclin1 mRNA. The internal deletion of the F3 region abrogated the ELAVL1-mediated BECN1 mRNA stability, and, in turn, prevented ELAVL1-enhanced ferroptosis. In mice, treatment with sorafenib alleviated murine liver fibrosis by inducing hepatic stellate cell (HSC) ferroptosis. HSC-specific knockdown of ELAVL1 impaired sorafenib-induced HSC ferroptosis in murine liver fibrosis. Noteworthy, we retrospectively analyzed the effect of sorafenib on HSC ferroptosis in advanced fibrotic patients with hepatocellular carcinoma receiving sorafenib monotherapy. Attractively, ELAVL1 upregulation, ferritinophagy activation, and ferroptosis induction occurred in primary human HSCs from the collected human liver tissue. Overall, these results reveal novel molecular mechanisms and signaling pathways of ferroptosis, and also identify ELAVL1-autophagy-dependent ferroptosis as a potential target for the treatment of liver fibrosis. Abbreviations: ACTA2/alpha-SMA: actin, alpha 2, smooth muscle, aorta; ACTB/beta-actin: actin beta; ARE: AU-rich element; ATG: autophagy related; BDL: bile duct ligation; BECN1: beclin 1; BSO: buthionine sulfoximine; COL1A1: collagen type I alpha 1 chain; ELAVL1/HuR: ELAV like RNA binding protein 1; FDA: fluorescein diacetate; FTH1: ferritin heavy chain 1; GOT1/AST: glutamic-oxaloacetic transaminase 1; GPT/ALT: glutamic-pyruvic transaminase; GPX4: glutathione peroxidase 4; GSH: glutathione; HCC: hepatocellular carcinoma; HSC: hepatic stellate cell; LCM: laser capture microdissection; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MDA: malondialdehydep; NCOA4: nuclear receptor coactivator 4; PTGS2: prostaglandin-endoperoxide synthase 2; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TBIL: total bilirubin; TEM: transmission electron microscopy; TGFB1: trasforming growth factor beta 1; UTR: untranslated region; VA-Lip-ELAVL1-siRNA: vitamin A-coupled liposomes carrying ELAVL1-siRNA.

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Cicloheximida, from microbial, ≥94% (TLC)
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Urea, powder, BioReagent, for molecular biology, suitable for cell culture
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Histodenz, nonionic density gradient medium
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Disolución salina tamponada con fosfato, BioPerformance Certified, pH 7.4
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Sodium orthovanadate, ≥90% (titration)
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Ethylenediaminetetraacetic acid, anhydrous, crystalline, BioReagent, suitable for cell culture
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Fluorescein diacetate, used as cell viability stain
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Live/Dead Cell Double Staining Kit, suitable for fluorescence
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L-Buthionine-sulfoximine, ≥97% (TLC)
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Glutathione Assay Kit, sufficient for 700 assays
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Mammalian Cell Lysis Kit
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Carbon-13C tetrachloride, 99 atom % 13C
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Normal Goat IgG, This Normal Goat IgG is validated for use in ELISA Flow Cytometry Immunoblotting Immunofluorescence Immunohistochemistry Immunoprecipitation for the detection of Goat IgG, Non-immune.