Activation of ferritinophagy is required for the RNA-binding protein ELAVL1/HuR to regulate ferroptosis in hepatic stellate cells - PubMed (original) (raw)

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

Zili Zhang et al. Autophagy. 2018.

Abstract

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.

Keywords: Autophagy; ELAVL1; ferritinophagy; ferroptosis; hepatic stellate cell; liver fibrosis; therapeutic target.

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Figures

Figure 1.

Figure 1.

RNA-binding protein ELAVL1 expression is increased during HSC ferroptosis. HSC-LX2 and HSC-T6 cells were treated with erastin (10 μM), BSO (200 μM), and sorafenib (5 μM) with or without the indicated inhibitors (ZVAD-FMK, 10 μM; ferrostatin-1, 1 μM; necrosulfonamide, 0.5 μM) for 24 h. (a) Cell viability and (b) MDA levels were assayed (n = 3 in every group, **, p < 0.01). HSC-LX2 and HSC-T6 cells were treated with erastin (10 μM), BSO (200 μM), and sorafenib (10 μM) for 24 h. (c) ELAVL1 protein and (d) mRNA levels were determined (n = 3 in every group). (e) HSC-LX2 cells were treated with erastin (10 μM) with or without MG-132 (5 μM) or cycloheximide (CHX, 20 μg/ml) for 24 h, and ELAVL1 protein level was assayed (n = 3 in every group, **, p < 0.01, ***, p < 0.001, #, p < 0.05). (f) HSC-LX2 cells were treated with CHX (20 μg/ml) for 1 h or treated with erastin (10 μM) for 4 h followed by CHX (20 μg/ml) for 1 h. ELAVL1 protein level was determined at the indicated time points (n = 3 in every group).

Figure 2.

Figure 2.

Increased ELAVL1 expression contributes to HSC ferroptosis. The indicated HSC cells were stably transfected with ELAVL1 siRNA or ELAVL1 plasmid, and then were treated with erastin (0–10 μM) or sorafenib (0–10 μM) for 24 h. (a) The transfection efficiency was confirmed by western blot analysis (n = 3 in every group). (b) Cell viability was assayed (n = 3 in every group, *, p < 0.05, N.S., not significant). (c) GSH, ROS, MDA, and iron levels were assayed (n = 3 in every group, *, p < 0.05, N.S., not significant). (d) The indicated _ELAVL1_-overexpressing HSC cells were treated with erastin (10 μM) and sorafenib (10 μM) with or without the indicated inhibitors (ferrostatin-1, 1 μM; liproxstatin-1, 100 nM; ZVAD-FMK,10 μM; necrostatin-1, 10 μM; necrosulfonamide, 0.5 μM) for 24 h, and cell viability was assayed (n = 3 in every group, ***, p < 0.001).

Figure 3.

Figure 3.

Enhanced ferroptosis by ELAVL1 is associated with autophagy activation. The indicated HSC cells were stably transfected with ELAVL1 siRNA or ELAVL1 plasmid, and then were treated with erastin (10 μM) for 24 h. (a) LC3-I/II protein expression was determined by western blot analysis (n = 3 in every group, **, p < 0.01). (b) 5 autophagy-related genes (ATG12-ATG5, BECN1, ATG7, and ATG14) were assayed by western blot analysis (n = 3 in every group). (c) BECN1 and MAP1LC3B protein expression were determined by immunofluorescence analysis. Scale bars: 50 μm. Representative photographs were shown (n = 3 in every group, **, p < 0.01). (d) SQSTM1 protein expression was assayed by western blot analysis (n = 3 in every group, **, p < 0.01, ***, p < 0.001). (e) HSC-LX2 cells were stably transfected with ELAVL1 siRNA or ELAVL1 plasmid, and then were treated with erastin (10 μM) for 24 h. Autophagosomes and autolysosomes were determined by transmission electron microscopy analysis. Scale bars: 0.5 μm. Representative photographs were shown (n = 3 in every group, **, p < 0.01).

Figure 4.

Figure 4.

Disruption of autophagy by BECN1 siRNA impairs ELAVL1-enhanced ferroptosis. The indicated HSC cells were stably transfected with BECN1 siRNA or BECN1 plasmid, and then were treated with erastin (10 μM) for 24 h. (a) BECN1 and LC3-I/II protein expression was determined by western blot analysis. The indicated HSC cells were stably transfected with BECN1 siRNA, BECN1 plasmid, BECN1 siRNA+ ELAVL1 plasmid, or BECN1 plasmid+ ELAVL1 plasmid, and then were treated with erastin (0–10 μM) or sorafenib (0–10 μM) for 24 h. (b) Cell viability was assayed (n = 3 in every group, *, p < 0.05, N.S., not significant). (c) GSH, ROS, MDA, and iron levels were assayed (n = 3 in every group, *, p < 0.05, N.S., not significant).

Figure 5.

Figure 5.

ELAVL1 promotes autophagy activation and BECN1 mRNA stability via binding to the AU-rich elements. (a) The predicted hits of the ELAVL1 signature motif in human BECN1 and TNF mRNA 3ʹ-UTR were assayed. (b) Association of endogenous ELAVL1 with endogenous BECN1 mRNA was measured by real-time PCR after ribonucleoprotein immunoprecipitation (RNP IP) (n = 3 in every group, **, p < 0.01). (c) mRNA affinity isolation assay was performed with biotinylated transcripts of the BECN1 mRNA 5ʹ-UTR, coding region (CR), 3ʹ-UTR or the TNF mRNA 3ʹ-UTR. ACTB served as a negative control (n = 3 in every group). (d) ELAVL1 binding to different fractions of the 3ʹ-UTR of BECN1 mRNA was determined by mRNA affinity isolation assay (n = 3 in every group). (e) The constructs of reporter that expressed chimeric RNA containing luciferase and the BECN1 mRNA 3ʹ-UTR are illustrated schematically. Activities of various luciferase reporters treated as indicated was determined (n = 3 in every group, *, p < 0.05, **, p < 0.01). (f) The deletion of specific ELAVL1-binding sites in the BECN1 3ʹ-UTR is illustrated. The luciferase reporter activity after ELAVL1 overexpression was assayed (n = 3 in every group, *, p < 0.05).

Figure 6.

Figure 6.

Sorafenib treatment alleviates murine liver fibrosis by inducing HSC ferroptosis. Fifty mice were randomly divided into 5 groups of 10 animals each with comparable mean body weight. Mice of 5 groups were treated with Sham, BDL+ VA-Lip-Control-siRNA, BDL+ VA-Lip-Control-siRNA+ sorafenib, BDL+ VA- Lip-_ELAVL1-_siRNA, or BDL+ VA-Lip-_ELAVL1_-siRNA+ sorafenib. (a and b) The pathological changes of the livers were observed by macroscopic examination. Scale bars: 1 cm. Representative photographs were shown. Thin sections (4 μm) were stained with H&E, Sirius Red, and Masson for histopathological study. The liver fibrosis stage was assessed by Ishak scale. Liver hydroxyproline level was determined using the Hydroxyproline Assay Kit (n = 6 in every group, *, p < 0.05, **, p < 0.01). (c) Immunohistochemical staining of ACTA2 and COL1A1 was determined. Scale bars: 50 μm. Representative photographs were shown (n = 6 in every group, *, p < 0.05, **, p < 0.01, ***, p < 0.001).

Figure 7.

Figure 7.

HSC-specific knockdown of ELAVL1 impairs sorafenib-induced HSC ferroptosis in murine liver fibrosis. Fifty mice were randomly divided into 5 groups of 10 animals each with comparable mean body weight. Mice of 5 groups were treated with Sham, BDL+VA-Lip-Control-siRNA, BDL+VA-Lip-Control-siRNA+sorafenib, BDL+VA-Lip-_ELAVL1_-siRNA, or BDL+VA-Lip-_ELAVL1_-siRNA+sorafenib. Primary HSCs were further isolated from BDL+VA-Lip-Control-siRNA+sorafenib or BDL+VA-Lip-_ELAVL1_-siRNA+sorafenib treated mice, and total RNAs were extracted for RNA-Seq. (a) Microarray heat map demonstrates clustering of isolated primary HSCs. Hierarchical cluster analysis of significantly differentially expressed mRNAs: bright green, underexpression; gray, no change; bright red, overexpression (BDL+VA-Lip-Control-siRNA+sorafenib, n = 5; BDL+VA-Lip-_ELAVL1_-siRNA+sorafenib, n = 5). (b) Volcano plot demonstrates clustering of isolated primary HSCs. Hierarchical cluster analysis of significantly differentially expressed mRNAs: bright blue, underexpression; gray, no change; bright red, overexpression (BDL+VA-Lip-_ELAVL1_-siRNA, n = 5; BDL+VA-Lip-Control-siRNA+sorafenib, n = 5). (c) Fold changes of autophagy-related genes and ferroptosis-related genes were identified. (d) The mRNA expression of autophagy markers MAP1LC3B/LC3B, BECN1, and SQSTM1 was determined by real-time PCR in isolated primary HSCs (n = 6 in every group, *, p < 0.05, **, p < 0.01, ***, p < 0.001). (e) Ferroptosis markers FTH1 and PTGS2 mRNA expression and ferroptotic events including iron accumulation and lipid peroxidation were determined in isolated primary HSCs (n = 6 in every group, **, p < 0.01, ***, p < 0.001).

Figure 8.

Figure 8.

ELAVL1 upregulation, ferritinophagy activation, and ferroptosis induction occur in primary human HSCs from fibrotic patients with HCC receiving sorafenib monotherapy. Primary human HSCs were isolated from the collected liver tissue by laser capture microdissection (LCM), and total RNAs were extracted for RNA-Seq. (a) Microarray heat map demonstrates clustering of isolated primary HSCs before and after sorafenib treatment. Hierarchical cluster analysis of differentially expressed mRNAs: bright green, underexpression; gray, no change; bright red, overexpression (No treatment, n = 5; sorafenib treatment, n = 5). (b) Volcano plot demonstrates clustering of isolated primary HSCs before and after sorafenib treatment. Hierarchical cluster analysis of differentially expressed mRNAs: bright blue, underexpression; gray, no change; bright red, overexpression (No treatment, n = 5; sorafenib treatment, n = 5). (c) Fold changes of liver fibrosis-related genes, autophagy-related genes, and ferroptosis-related genes were identified. (d) Blood samples were collected for the analyses of liver functions including GPT/ALT, GOT1/AST, and TBIL (total bilirubin) (n = 28 in every group, *, p < 0.05, ***, p < 0.001). (e) The mRNA expression of liver fibrosis markers ACTA2 and COL1A1 was determined by real-time PCR in sorafenib-treated liver biopsies and untreated controls (n = 5 in every group, *, p < 0.05, **, p < 0.01). (f and g) The mRNA expression of ferritinophagy markers MAP1LC3B/LC3B, BECN1, SQSTM1, ATG3, FTH1, and NCOA4 was determined by real-time PCR in isolated primary HSCs before and after sorafenib treatment (n = 5 in every group, *, p < 0.05, **, p < 0.01, ***, p < 0.001, N.S., not significant). (h) Ferroptosis markers ELAVL1 and PTGS2 mRNA expression and ferroptotic events including iron accumulation, lipid ROS production, and lipid peroxidation were all determined in isolated primary HSCs before and after sorafenib treatment (No treatment, n = 5; sorafenib treatment, n = 5; *, p < 0.05, ***, p < 0.001).

Figure 9.

Figure 9.

The RNA-binding protein ELAVL1 regulates ferroptosis by inducing ferritinophagy activation in HSCs. Ferroptosis-inducing compounds increase ELAVL1 expression through the inhibition of the ubiquitin-proteasome pathway. Increased ELAVL1 can bind to the AREs of the BECN1 mRNA 3ʹ-UTR, trigger autophagy activation, promote autophagic ferritin degradation, and in turn, lead to HSC ferroptosis.

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