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. 2020 Aug;16(8):1482-1505.
doi: 10.1080/15548627.2019.1687985. Epub 2019 Nov 11.

RNA-binding protein ZFP36/TTP protects against ferroptosis by regulating autophagy signaling pathway in hepatic stellate cells

Zili Zhang  1 , Mei Guo  2 , Yujia Li  1 , Min Shen  1 , Desong Kong  1 , Jiangjuan Shao  1 , Hai Ding  3 , Shanzhong Tan  3 , Anping Chen  4 , Feng Zhang  1   5 , Shizhong Zheng  1   5
Affiliations

RNA-binding protein ZFP36/TTP protects against ferroptosis by regulating autophagy signaling pathway in hepatic stellate cells

Zili Zhang et al. Autophagy. 2020 Aug.

Abstract

Ferroptosis is a recently discovered form of programmed cell death, but its regulatory mechanisms remain poorly understood. Here, we show that the RNA-binding protein ZFP36/TTP (ZFP36 ring finger protein) plays a crucial role in regulating ferroptosis in hepatic stellate cells (HSCs). Upon exposure to ferroptosis-inducing compounds, the ubiquitin ligase FBXW7/CDC4 (F-box and WD repeat domain containing 7) decreased ZFP36 protein expression by recognizing SFSGLPS motif. FBXW7 plasmid contributed to classical ferroptotic events, whereas ZFP36 plasmid impaired FBXW7 plasmid-induced HSC ferroptosis. Interestingly, ZFP36 plasmid inhibited macroautophagy/autophagy activation by destabilizing ATG16L1 (autophagy related 16 like 1) mRNA. ATG16L1 plasmid eliminated the inhibitory action of ZFP36 plasmid on ferroptosis, and FBXW7 plasmid enhanced the effect of ATG16L1 plasmid on autophagy. Importantly, ZFP36 plasmid promoted ATG16L1 mRNA decay via binding to the AU-rich elements (AREs) within the 3'-untranslated region. The internal mutation of the ARE region abrogated the ZFP36-mediated ATG16L1 mRNA instability, and prevented ZFP36 plasmid-mediated ferroptosis resistance. In mice, treatment with erastin and sorafenib alleviated murine liver fibrosis by inducing HSC ferroptosis. HSC-specific overexpression of Zfp36 impaired erastin- or sorafenib-induced HSC ferroptosis. Noteworthy, we analyzed the effect of sorafenib on HSC ferroptosis in fibrotic patients with hepatocellular carcinoma receiving sorafenib monotherapy. Attractively, sorafenib monotherapy led to ZFP36 downregulation, ferritinophagy activation, and ferroptosis induction in human HSCs. Overall, these results revealed novel molecular mechanisms and signaling pathways of ferroptosis, and also identified ZFP36-autophagy-dependent ferroptosis as a potential target for the treatment of liver fibrosis.

Abbreviations: ARE: AU-rich elements; ATG: autophagy related; BECN1: beclin 1; CHX: cycloheximide; COL1A1: collagen type I alpha 1 chain; ELAVL1/HuR: ELAV like RNA binding protein 1; FBXW7/CDC4: F-box and WD repeat domain containing 7; FN1: fibronectin 1; FTH1: ferritin heavy chain 1; GPX4/PHGPx: glutathione peroxidase 4; GSH: glutathione; HCC: hepatocellular carcinoma; HSC: hepatic stellate cell; LSEC: liver sinusoidal endothelial cell; MAP1LC3A: microtubule associated protein 1 light chain 3 alpha; MDA: malondialdehyde; NCOA4: nuclear receptor coactivator 4; PTGS2/COX2: prostaglandin-endoperoxide synthase 2; RBP: RNA-binding protein; ROS: reactive oxygen species; SLC7A11/xCT: solute carrier family 7 member 11; SQSTM1/p62: sequestosome 1; TNF: tumor necrosis factor; TP53/p53: tumor protein p53; UTR: untranslated region; ZFP36/TTP: ZFP36 ring finger protein.

Keywords: Autophagy; FBXW7; ZFP36; ferroptosis; hepatic stellate cell.

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Conflict of interest statement

No potential conflict of interest was reported by the authors.

Figures

Figure 1.
Figure 1.
RNA-binding protein ZFP36 expression is decreased during HSC ferroptosis. HSC-LX2 and HSC-T6 cells were treated with erastin (10 μM), sorafenib (10 μM), and RSL3 (2.5 μM), with or without the indicated inhibitors (liproxstatin-1, 100 nM; ZVAD-FMK, 10 μM; necrostatin-1, 10 μM) for 24 h. (A) Cell viability, MDA, and iron levels were assayed (n = 3 in every group, **, p < 0.01). (B and C) HSC-LX2 and HSC-T6 cells were treated with erastin (10 μM), sorafenib (10 μM), and RSL3 (2.5 μM) for 24 h. ZFP36 protein and mRNA levels were determined (n = 3 in every group, *, p < 0.05, **, p < 0.01, ***, p < 0.001). (D) HSC-LX2 and HSC-T6 cells were treated with sorafenib (10 μM) with or without MG-132 (10 μM) for 24 h. Cells were harvested for ubiquitination assay (n = 3 in every group). (E) HSC-LX2 cells were treated with erastin (10 μM) with or without cycloheximide (CHX, 20 μg/ml) or MG-132 (10 μM) for 24 h, and ZFP36 protein level was assayed (n = 3 in every group, *, p < 0.05, **, p < 0.01). (F) HSC-LX2 cells were treated with CHX (20 μg/ml) for 24 h or treated with erastin (10 μM) and CHX (20 μg/ml) for 24 h. ZFP36 protein levels were determined at the indicated time points (n = 3 in every group).
Figure 2.
Figure 2.
The ubiquitin ligase FBXW7 decreases ZFP36 protein expression by recognizing SFSGLPS motif. (A) Amino acid sequence alignment between human and rat ZFP36 protein was determined. (B) HSC-LX2 cells were treated with erastin (10 μM), sorafenib (10 μM), and RSL3 (2.5 μM) for 24 h. The binding of ZFP36 and FBXW7 was determined by immunoprecipitation assay (n = 3 in every group). (C and D) FBXW7-deficient HSC-LX2 and HSC-T6 cells were treated with erastin (10 μM) for 24 h. The ubiquitylation of ZFP36 and the protein levels of ZFP36 and FBXW7 were determined (n = 3 in every group). (E) FBXW7-deficient HSC-LX2 cells were treated with erastin (10 μM) for 24 h. the mRNA levels of ZFP36 and FBXW7 were determined (n = 3 in every group, *, p < 0.05, **, p < 0.01, N.S., not significant). (F-H) HSC-LX2 cells were stably transferred with ZFP36 Δ186-192 plasmid, ZFP36 plasmid, or FBXW7 plasmid, and then were treated with erastin (10 μM) for 24 h. The binding of ZFP36 and FBXW7, the ubiquitylation of ZFP36, and the protein levels of ZFP36 were determined (n = 3 in every group, *, p < 0.05, N.S., not significant). (I) HSC-LX2 cells were treated with erastin (10 μM) with or without autophagy inhibitors (3-MA, 10 mM; bafilomycin A1, 5 nM) for 24 h. The protein levels of ZFP36 were determined (n = 3 in every group, ***, p < 0.001, N.S., not significant).
Figure 3.
Figure 3.
ZFP36 overexpression confers resistance to HSC ferroptosis. (A) HSC-LX2 and HSC-T6 cells overexpressing ZFP36 were treated with erastin (10 μM) for 24 h. The ZFP36 protein levels were determined (n = 3 in every group, ***, p < 0.001). (B) HSC-LX2 and HSC-T6 cells overexpressing ZFP36 and FBXW7 were treated with erastin (0–10 μM) or sorafenib (0–10 μM) for 24 h. Cell viability was assayed (n = 3 in every group, *, p < 0.05). (C-F) HSC-LX2 and HSC-T6 cells overexpressing ZFP36 and FBXW7 were treated with erastin (10 μM) or sorafenib (10 μM) for 24 h. The levels of iron, ROS, GSH, and MDA were assayed (n = 3 in every group, *, p < 0.05). (G) ZFP36-deficient HSC-LX2 and HSC-T6 cells were treated with erastin (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 4.
Figure 4.
Reduced ferroptosis by ZFP36 plasmid is associated with autophagy inactivation. (A) HSC-LX2 cells overexpressing ZFP36 were treated with sorafenib (10 μM) for 24 h, and total RNAs were extracted for RNA-Seq. Microarray heat map demonstrates clustering of HSC-LX2 cells. Hierarchical cluster analyses of significantly differentially expressed mRNAs: bright blue, underexpression; gray, no change; bright red, overexpression (Control vector, n = 3; ZFP36 plasmid, n = 3). (B-D) HSC-LX2 cells overexpressing ZFP36 and FBXW7 were treated with erastin (10 μM), sorafenib (10 μM), and RSL3 (2.5 μM) for 24 h. The protein levels of ATG3, ATG4A, ATG12–ATG5, BECN1, ATG7, ATG9A, and ATG16L1 were determined (n = 3 in every group). (E) HSC-LX2 cells overexpressing ZFP36 and FBXW7 were treated with sorafenib (10 μM) for 24 h. The protein complex of ATG12–ATG5-ATG16L1 was assayed by immunocoprecipitation (n = 3 in every group). (F) HSC-LX2 and HSC-T6 cells overexpressing ZFP36 and FBXW7 were treated with sorafenib (10 μM) for 24 h. The protein levels of LC3-I/II were determined (n = 3 in every group).
Figure 5.
Figure 5.
ZFP36 plasmid inhibits HSC ferroptosis by regulating autophagy signaling. (A) HSC-LX2 cells overexpressing ZFP36 and FBXW7 were stably transferred with pGM-CMV-GFP-hLC3 plasmid, and then were treated with sorafenib (10 μM) for 24 h. The green fluorescence spots were detected. Scale bars: 50 μm. Representative photographs were shown (n = 3 in every group, **, p < 0.01, ***, p < 0.001). (B) HSC-LX2 cells overexpressing ZFP36 and FBXW7 were treated with sorafenib (10 μM) for 24 h. The endogenous LC3 levels were determined by immunofluorescence. Scale bars: 50 μm. Representative photographs were shown (n = 3 in every group, **, p < 0.01, ***, p < 0.001). (C) HSC-LX2 cells were stably transferred with CMV-TurboRFP-EGFP-LC3-PGK-Puro plasmid and ZFP36 plasmid, and then were treated with sorafenib (10 μM) with or without autophagy inhibitors (3-MA, 10 mM; bafilomycin A1, 5 nM) for 24 h. The yellow fluorescence and red fluorescence spots were detected. Scale bars: 50 μm. Representative photographs were shown (n = 3 in every group, *, p < 0.05, **, p < 0.01). (D) HSC-LX2 cells overexpressing ZFP36 and FBXW7 were treated with sorafenib (10 μM) for 24 h. Autophagosomes and autolysosomes were determined by transmission electron microscopy. Scale bars: 0.5 μm. Representative photographs were shown (n = 3 in every group, *, p < 0.05, **, p < 0.01, ##, p < 0.01).
Figure 6.
Figure 6.
Induction of autophagy by ATG16L1 plasmid impairs ZFP36-induced resistance to HSC ferroptosis. (A-D) HSC-LX2 and HSC-T6 cells overexpressing ATG16L1, ZFP36 and FBXW7 were treated with sorafenib (10 μM) for 24 h. The mRNA expression of ATG16L1, MAPILC3B, SQSTM1, and FTH1 were determined (n = 3 in every group, *, p < 0.05, **, p < 0.01, ***, p < 0.001). (E and F) HSC-LX2 cells overexpressing ATG16L1, ZFP36 and FBXW7 were stably transferred with pGM-CMV-GFP-hLC3 plasmid, and then were treated with sorafenib (10 μM) for 24 h. The green fluorescence spots were detected. Scale bars: 50 μm. Representative photographs were shown (n = 3 in every group, *, p < 0.05, **, p < 0.01). (G) HSC-LX2 and HSC-T6 cells overexpressing ATG16L1, ZFP36 and FBXW7 were treated with sorafenib (0–10 μM) or erastin (0–10 μM) for 24 h. Cell viability were examined (n = 3 in every group, *, p < 0.05). (H) HSC-LX2 and HSC-T6 cells overexpressing ATG16L1, ZFP36 and FBXW7 were treated with erastin (10 μM) or sorafenib (10 μM) for 24 h. The levels of iron, GSH, and MDA were assayed (n = 3 in every group, *, p < 0.05).
Figure 7.
Figure 7.
ZFP36 plasmid promotes autophagy inactivation and ATG16L1 mRNA decay via binding to the AU-rich elements. (A) The predicted hits of the ZFP36 signature motif in human ATG16L1 and TNF mRNA 3ʹ-UTR were assayed. (B) HSC-LX2 cells overexpressing ZFP36 were treated with sorafenib (10 μM), ActD (10 μg/ml) and DBR (5 μM) for indicated times. The remaining ATG16L1 mRNA levels were measured by real-time PCR and normalized to the results at 0 min after ActD/DRB treatment (n = 3 in every group, **, p < 0.01). (C) HSC-LX2 cells overexpressing ZFP36 were treated with sorafenib (10 μM) and CHX (20 μg/ml) for 150 min. ATG16L1 protein level was determined at the indicated time points (n = 3 in every group). (D) Association of endogenous ZFP36 with endogenous ATG16L1 mRNA was measured by real-time PCR after ribonucleoprotein immunoprecipitation (RNP IP) (n = 3 in every group, **, p < 0.01). (E) mRNA affinity isolation assay was performed with biotinylated transcripts of the ATG16L1 mRNA 5ʹ-UTR, coding region (CR), 3ʹ-UTR or the TNF mRNA 3ʹ-UTR (n = 3 in every group). (F) Luciferase constructs carrying the 3ʹ-UTRs of genes encoding ACTB (3ʹ-UTR-ACTB), TNF (3ʹ-UTR-TNF), ATG16L1 (3ʹ-UTR-ATG16L1) or empty vector (pGL3) was stably transfected into HSC-LX2 cells. Cells were treated with sorafenib (10 μM) for 24 h, and luciferase activities were measured and normalized to the activities obtained in pGL3-transfected cells without treatment (n = 3 in every group, ***, p < 0.001). (G) Luciferase constructs carrying the 3ʹ-UTRs of genes encoding ACTB (3ʹ-UTR-ACTB) and ATG16L1 (3ʹ-UTR-ATG16L1) were stably transfected into HSC-LX2 cells. Luciferase mRNA half-life was measured by real-time PCR after sorafenib (10 μM) and ActD/DRB treatment for indicated times (n = 3 in every group, ***, p < 0.001). (H) HSC-LX2 cells overexpressing ZFP36 were stably transfected with the luciferase constructs carrying the 3ʹ-UTRs of genes encoding ACTB (3ʹ-UTR-ACTB), TNF (3ʹ-UTR-TNF), ATG16L1 (3ʹ-UTR-ATG16L1) or empty vector (pGL3), and then were treated with sorafenib (10 μM) for 24 h. Luciferase activities were measured and normalized to the activities obtained in pGL3-transfected cells without treatment (n = 3 in every group, ***, p < 0.001). (I) ATG16L1 3ʹ-UTR luciferase construct was co-transfected with different amounts of ZFP36 plasmid into HSC-LX2 cells. Luciferase activity was detected after sorafenib (10 μM) treatment for 24 h and normalized to the cells transfected with empty plasmid (n = 3 in every group, *, p < 0.05, **, p < 0.01, ***, p < 0.001). (J) mRNA pull down assay was performed by mixing ATG16L1-AGE-Bio, TNF-AGE-Bio, and Mut-ATG16L1-AGE-Bio with total cell extracts from HSC-LX2 cells. Precipitates were prepared for western blot (n = 3 in every group). (K) Cold ATG16L1-AGE probes or Mut-ATG16L1-AGE probes with different concentrations were used to compete for the binding between ATG16L1-AGE-Bio and ZFP36 (n = 3 in every group).
Figure 8.
Figure 8.
HSC-specific knock-in of Zfp36 impairs erastin- or sorafenib-induced HSC ferroptosis in murine liver fibrosis. Mice of 7 groups were treated with sham, BDL+VA-Lip-control-vector, BDL+VA-Lip-control-vector+erastin, BDL+VA-Lip- control-vector+sorafenib, BDL+VA-Lip-Zfp36-plasmid, BDL+VA-Lip-Zfp36- plasmid+erastin or BDL+VA-Lip-Zfp36-plasmid+sorafenib. (A) The pathological changes of the livers were observed by macroscopic examination. Scale bars: 1 cm. Thin sections (4 μm) were stained with H&E, Sirius Red, and Masson for histopathological study. Scale bars: 50 μm. Representative photographs were shown (n = 6 in every group, *, p < 0.05, **, p < 0.01, ***, p < 0.001). (B and C) The mRNA expression of liver fibrosis markers (Acta2, Col1a1, Fn1, and Des) and autophagy markers (Atg16l1, Lc3, Sqstm1, and Fth1) was determined by real-time PCR in isolated primary HSCs (n = 6 in every group, *, p < 0.05, **, p < 0.01, ***, p < 0.001, N.S., not significant). (D) The Ptgs2 mRNA expression, iron level, ROS level, and MDA level were determined (n = 6 in every group, *, p < 0.05, **, p < 0.01, ***, p < 0.001, N.S., not significant).
Figure 9.
Figure 9.
Sorafenib monotherapy leads to ZFP36 downregulation, ferritinophagy activation, and ferroptosis induction in human HSCs from fibrotic patients. (A-C) Primary human HSCs were isolated from the collected liver tissue by laser capture microdissection (LCM). The mRNA expression of ZFP36, ACTA2, COL1A1, ATG16L1, LC3, SQSTM1, and FTH1 was determined by real-time PCR (No treatment, n = 28; Sorafenib treatment, n = 18, **, p < 0.01, ***, p < 0.001). (D) The PTGS2 mRNA expression, iron level, ROS level, and MDA level were determined (No treatment, n = 28; Sorafenib treatment, n = 18, ***, p < 0.001).
Figure 10.
Figure 10.
RNA-binding protein ZFP36 protects against ferroptosis by regulating autophagy signaling pathway in HSCs. ZFP36 overexpression can result in ATG16L1 mRNA decay via binding to the AREs in the 3ʹ-UTR, thus triggering autophagy inactivation, blocking autophagic ferritin degradation, and eventually conferring resistance to ferroptosis.
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References

    1. Zhang K, Han X, Zhang Z, et al. The liver-enriched lnc-LFAR1 promotes liver fibrosis by activating TGFβ and notch pathways. Nat Commun. 2017;8:144. - PMC - PubMed
    1. De Magalhaes Filho CD, Downes M, Evans R.. Bile acid analog intercepts liver fibrosis. Cell. 2016;166:789. - PubMed
    1. Ding BS, Cao Z, Lis R, et al. Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis. Nature. 2014;505:97–102. - PMC - PubMed
    1. Kostallari E, Hirsova P, Prasnicka A, et al. Hepatic stellate cell-derived platelet-derived growth factor receptor-alpha-enriched extracellular vesicles promote liver fibrosis in mice through SHP2. Hepatology. 2018;68:333–348. - PMC - PubMed
    1. Gandhi CR. Hepatic stellate cell activation and pro-fibrogenic signals. J Hepatol. 2017;67:1104–1105. - PMC - PubMed

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This work was supported by the National Natural Science Foundation of China [81270514, 31401210, 81600483, and 31571455], the Open Project Program of Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica [JKLPSE 201804], and the Project of the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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