Targeting a novel inducible GPX4 alternative isoform to alleviate ferroptosis and treat metabolic-associated fatty liver disease

Abstract

Metabolic-associated fatty liver disease (MAFLD), which is previously known as non-alcoholic fatty liver disease (NAFLD), represents a major health concern worldwide with limited therapy. Here, we provide evidence that ferroptosis, a novel form of regulated cell death characterized by iron-driven lipid peroxidation, was comprehensively activated in liver tissues from MAFLD patients. The canonical-GPX4 (cGPX4), which is the most important negative controller of ferroptosis, is downregulated at protein but not mRNA level. Interestingly, a non-canonical GPX4 transcript-variant is induced (inducible-GPX4, iGPX4) in MAFLD condition. The high fat-fructose/sucrose diet (HFFD) and methionine/choline-deficient diet (MCD)-induced MAFLD pathologies, including hepatocellular ballooning, steatohepatitis and fibrosis, were attenuated and aggravated, respectively, in cGPX4-and iGPX4-knockin mice. cGPX4 and iGPX4 isoforms also displayed opposing effects on oxidative stress and ferroptosis in hepatocytes. Knockdown of iGPX4 by siRNA alleviated lipid stress, ferroptosis and cell injury. Mechanistically, the triggered iGPX4 interacts with cGPX4 to facilitate the transformation of cGPX4 from enzymatic-active monomer to enzymatic-inactive oligomers upon lipid stress, and thus promotes ferroptosis. Co-immunoprecipitation and nano LC-MS/MS analyses confirmed the interaction between iGPX4 and cGPX4. Our results reveal a detrimental role of non-canonical GPX4 isoform in ferroptosis, and indicate selectively targeting iGPX4 may be a promising therapeutic strategy for MAFLD.

Keywords: Alternative isoform; Fatty liver; Ferroptosis; GPX4; High fat-fructose/sucrose diet; Methionine/choline-deficient diet; Oligomerization; Protein interaction.

Graphical abstract

Figure 1

Ferroptosis is triggered in liver tissue of MAFLD patients. (A) Representative H&E histological analysis showing the MAFLD pathology in liver tissue from individuals with or without MAFLD (n > 4 images per individual). Scale bar, 100 μm. (B) The levels of MDA, total-SOD and total-TAC in liver from individuals with or without MAFLD. (C) Immunohistochemistry analysis of 3-nitrotyrosine in liver from individuals with or without MAFLD. Scale bar, 100 μm. (D, E) Immunoblotting (D) and immunohistochemistry analysis (E) of 4-HNE-protein adducts in liver from individuals with or without MAFLD. Quantitative analyses were performed. Scale bar, 100 μm. (F) The mRNA level of NOX1 and NOX4 in liver from individuals with or without MAFLD. (G) The mRNA level of transferrin, FTH and FTL in liver from individuals with or without MAFLD. (H) Immunoblotting analysis of ferritin and transferrin in liver from individuals with or without MAFLD. Transferrin dimer was also detected by the antibody against transferrin in immunoblotting. Tubulin was used as a loading control in immunoblotting. (I) Relative mRNA levels of GPX4 isoform A and C in liver tissue from human with or without MAFLD. (J) Detection of GPX4 isoform A and GPX4 isoform C in liver tissues from human with or without MAFLD. Data are presented as mean ± SEM and analyzed by unpaired t-test, n = 7 per group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. Control.

Figure 2

Identification of a novel inducible GPX4 alternative isoform triggered upon lipid peroxidation stress. (A) The change mRNA levels of cGPX4 and nGPX4 in liver tissues from MAFLD mice induced by HFFD and MCD (n = 6 per group). (B) The change protein levels of cGPX4 and nGPX4 in liver tissues from MAFLD mice induced by HFFD and MCD (n = 3 per group). (C) Protein expression of nGPX4 in various mouse tissues was determined by immunoblotting with a polyclonal antibody against a specific amino acid sequence (SPRKRPGPRRRKARC) within the N-terminal of mouse nGPX4. (D) The distribution of nGPX4 in AML12 hepatocytes was determined by immunofluorescence with a polyclonal antibody against mouse nGPX4. DAPI was used to stain nuclei (n = 6 per group). Scale bar, 20 μm. (E) The nGPX4 was determined by immunofluorescence with a polyclonal antibody against mouse nGPX4 in liver tissue from Chow- or HFFD-treated mice (n = 6 per group). DAPI was used to stain nuclei. Scale bar, 100 μm. (F) Flag-tagged cGPX4 and nGPX4 were transfected into AML12 hepatocytes, and the subcellular localization of cGPX4 and nGPX4 was detected with a mouse monoclonal antibody recognizing Flag followed by an Alexa Fluor 568 goat-anti-mouse secondary antibody (n = 3 per group). DAPI was used to stain nuclei (blue). Scale bar, 20 μm. (G) Flag-tagged cGPX4 and nGPX4 were transfected into AML12 hepatocytes, and the cytosol and nuclear fractions were extracted to assess subcellular localization of cGPX4 and nGPX4 by immunoblotting with a mouse monoclonal antibody recognizing Flag (n = 3 per group). Lamin A/C was used a loading control for nuclear extract while GAPDH was used a loading control for cytosol extract. Data are presented as mean ± SEM and analyzed by one-way ANOVA, followed by Tukey's HSD test; ∗∗P < 0.01 vs. Chow. NS, no significance.

Figure 3

Knockin of cGPX4 inhibits ferroptosis and protects against MAFLD in mice. (A) Scheme illustrating experimental design for comparing MCD-induced MAFLD pathologies between R26^WT/WT and R26^cGPX4/cGPX4 mice. (B) The body weight curves of R26^WT/WT and R26^cGPX4/cGPX4 mice fed a MCD or a normal Chow diet (n = 6 per group). (C) The Fe²⁺ levels in liver tissues of R26^WT/WT and R26^cGPX4/cGPX4 mice fed a MCD or a normal Chow diet (n = 5 per group). (D) Immunoblotting of ACSL4, ALOX15, ferritin, transferrin, 3-nitrotyrosine, 4-HNE, TFR1 and ZIP14 in liver tissues of R26^WT/WT and R26^cGPX4/cGPX4 mice fed a MCD or a normal Chow diet (n = 3 per group). (E) PI staining showing the cell death in liver tissue of R26^WT/WT and R26^cGPX4/cGPX4 mice fed MCD or normal chow (n = 10 per group). DAPI was used to label nuclei. Scale bars, 100 μm. (F, G) The levels of MDA (F), GSH and SOD (G) in liver tissues from R26^WT/WT and R26^cGPX4/cGPX4 mice fed with Chow or MCD (n = 3 per group). (H) Serum ALT and AST levels in R26^WT/WT and R26^cGPX4/cGPX4 mice fed a MCD or a normal Chow diet (n = 4 per group). (I) MAFLD activity score calculated based on H&E staining in liver from R26^WT/WT and R26^cGPX4/cGPX4 mice fed a MCD or a normal Chow diet (n = 4 per group). Scale bars, 100 μm. (J) Immunohistochemistry staining of F4/80 in liver from R26^WT/WT and R26^cGPX4/cGPX4 mice fed a MCD or a normal Chow diet (n = 4 per group). Scale bar, 100 μm. (K, L) Liver fibrosis was determined by Masson's trichrome staining (K) and Sirius Red staining (L). Quantitative analyses were performed (n = 4 per group). Scale bars, 100 μm. Data are presented as mean ± SEM. Data were analyzed by two-way ANOVA followed by Tukey's HSD test; ∗P < 0.05, ∗∗P < 0.01 vs. Chow; ^#P < 0.05, ^##P < 0.01 vs. R26^WT/WT mice + MCD. NS, no significance.

Figure 4

Knockin of iGPX4 promotes ferroptosis in MAFLD murine model. (A) Scheme illustrating experimental design for comparing MCD-induced MAFLD pathologies between R26^WT/WT and R26^iGPX4/iGPX4 mice. (B) The Fe²⁺ level in liver tissues from R26^WT/WT and R26^iGPX4/iGPX4 mice fed MCD or normal chow (n = 5 per group). (C) The levels of MDA, GSH and SOD in liver tissues from R26^WT/WT and R26^iGPX4/iGPX4 mice fed MCD or normal chow (n = 4–5 per group). (D) Immunoblotting analyses of 3-nitrotyrosine and 4-HNE protein adducts in liver from R26^WT/WT and R26^iGPX4/iGPX4 mice under Chow or MCD conditions (n = 3 per group). (E) ROS production in liver from R26^WT/WT and R26^iGPX4/iGPX4 mice fed MCD or normal chow (n = 6 per group). Scale bar, 100 μm. (F) PI staining showing the cell death in liver tissue of R26^WT/WT and R26^iGPX4/iGPX4 mice fed MCD or normal chow. DAPI was used to label nuclei (n = 8 per group). Scale bars, 100 μm. (G) Immunoblotting analyses of ferroptosis-associated proteins Ferritin, Transferrin, TFR1, ZIP14, ACSL4 and ALOX15 in liver from R26^WT/WT and R26^iGPX4/iGPX4 mice fed MCD or normal chow. n = 3 per group. Data are presented as mean ± SEM and analyzed by two-way ANOVA followed by Tukey's post hoc test; ∗P < 0.05, ∗∗P < 0.01 vs. Chow; ^#P < 0.05, ^##P < 0.01 vs. R26^WT/WT mice + MCD. NS, no significance.

Figure 5

Knockin of iGPX4 exacerbates liver damage in MAFLD model. (A, B) Immunofluorescence (A) and immunohistochemistry (B) TUNEL assays in liver tissues from R26^WT/WT and R26^iGPX4/iGPX4 mice under Chow or MCD conditions (n = 3 per group). Scale bars, 100 μm. (C) H&E staining and MAFLD activity score in liver tissues from R26^WT/WT and R26^iGPX4/iGPX4 mice under Chow or MCD conditions (n = 3 per group). Scale bar, 100 μm. (D) Immunohistochemistry staining of F4/80 in liver tissues from R26^WT/WT and R26^iGPX4/iGPX4 mice under Chow or MCD conditions (n = 3 per group). Scale bar, 100 μm. (E) Liver fibrosis was determined by Masson's trichrome staining, α-SMA immunohistochemistry staining and Sirius Red staining in liver tissues from R26^WT/WT and R26^iGPX4/iGPX4 mice under Chow or MCD conditions (n = 3 per group). Scale bar, 100 μm. (F) Serum ALT and AST levels in R26^WT/WT and R26^iGPX4/iGPX4 mice under Chow or MCD conditions (n = 5 per group). (G–J) Heatmap derived from RNA-sequencing assay indicating the altered genes (red, upregulated; blue, downregulated) involved in glutathione and glutamine metabolism, cell survival a prolifendration, fibrosis and inflammation between R26^iGPX4/iGPX4 mice and R26^WT/WT mice liver tissues under MCD condition (n = 4 per group). Criteria: a fold change >1.5 and a corresponding adjusted P value < 0.05. Data are presented as mean ± SEM. Data was analyzed two-way ANOVA followed by Tukey's HSD test or unpaired t-test; ∗∗P < 0.01 vs. Chow; ^#P < 0.05, ^##P < 0.01 vs. R26^WT/WT mice + MCD. NS, no significance.

Figure 6

cGPX4 and iGPX4 isoforms have opposing effects on ROS production and ferroptosis in hepatocytes upon lipid stress. (A) Immunoblotting confirms the overexpression of cGPX4 and iGPX4 in AML12 hepatocytes transfected with pcDNA3.1-cGPX4 and pcDNA3.1-iGPX4 respectively. The anti-Pan-GPX4 antibody was used in left panel and the specific antibody against iGPX4 generated by us was used in right panel (n = 3 per group). (B, C) Fe²⁺ pool in AML12 hepatocytes was determined by immunofluorence analysis (B) and flow cytometry analysis (C). AML12 hepatocytes were transfected with pcDNA3.1-cGPX4 and pcDNA3.1-iGPX4 and then stimulated under methionine- and choline-deficient medium (MCDM). Calcein-AM probe was added into the medium (final concentration: 10 mol/L) for 30 min to monitorμ the Fe²⁺ pool under confocal microscopy or evaluated with flow cytometry (n = 6–8 per group). Scale bar, 100 μm. (D, E) ROS level in AML12 hepatocytes was determined by flow cytometry analysis (D) and immunofluorence analysis (E). AML12 hepatocytes were transfected with pcDNA3.1-cGPX4 and pcDNA3.1-iGPX4 and then stimulated under MCDM. In flow cytometry analysis, DCFH-DA fluorescence was analyzed. In immunofluorence analysis, DCFH-DA (5 μmol/L) and Mitotracker-Red-CMXRos dyes (200 nmol/L) were added into the medium for 30 min (n = 6–8 per group). The DCFH-DA fluorescence (green) and mitochondrial fluorescence (red) were visualized under a FV1000 confocal microscopy. Scale bar, 100 μm. (F) The levels of GSH and MDA in AML12 hepatocytes transfected with pcDNA3.1-cGPX4 and pcDNA3.1-iGPX4 plasmids respectively under normal culture medium or MCDM (n = 4 per group). (G, H) Immunoblotting analyses of 3-nitrotyrosine, 4-HNE, ACSL4, ALOX15 and Transferrin in AML12 hepatocytes transfected with plasmid of pcDNA3.1-cGPX4 (G) or pcDNA3.1-iGPX4 (H) under normal culture medium or MCDM (n = 3 per group). (I) The levels of GSH activity and MDA in AML12 hepatocytes transfected with pcDNA3.1-iGPX4 plasmids with or without ferroptosis inhibitor Fer-1 (100 nmol/L) under normal culture medium or MCDM (n = 4 per group). Data are presented as mean ± SEM and analyzed by two-way ANOVA followed by Tukey's post hoc test; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 7

Knockdown of iGPX4 isoform alleviates ferroptosis and lipid oxidation in an in vitro MAFLD model. (A) Oligonucleotide sequence of siRNAs targeting to iGPX4. (B) Efficiency of knockdown of iGPX4 by siRNA was confirmed by immunoblotting. GAPDH was used as a loading control (n = 3 per group). (C) Oil Red O staining showing the lipid content (red) induced by MCDM was largely prevented by knocking down of iGPX4 with siRNA (n = 6 per group). Scale bar, 100 μm. (D) Staining of with C11 BODIPY 581/591 probe showing the lipid oxidation (green) induced by MCDM was largely attenuated by knocking down of iGPX4 with siRNA (n = 6 per group). Scale bar, 100 μm. (E) Staining of with Calcein-AM probe, a fluorescein-derived dye with green fluorescence that is quenched upon binding to ferrous ion (Fe²⁺) showing the intracellular labile iron pool (LIP) induced by MCDM was prevented by knocking down of iGPX4 with siRNA (n = 6 per group). Scale bar, 100 μm. (F) Immunoblotting showing the levels of MDA, TFR and ACSL4 induced by MCDM were attenuated by knocking down of iGPX4 with siRNA (n = 3 per group). Data are presented as mean ± SEM, one way-ANOVA was performed; ∗∗P < 0.01 vs. MCDM.

Figure 8

Isoform iGPX4 interacts with cGPX4 and promotes cGPX4 oligomerization to facilitate ferroptosis upon lipid stress. (A) Immunoblotting analysis with an anti-Pan-GPX4 antibody showing GPX4 oligomerization by comparing the cGPX4 monomer (∼19 kDa) and oligomers (>50 kDa) in the liver tissues of mice fed normal chow diet, HFFD and MCD diet (left panel), as well as in AML12 hepatocytes stimulated under lipid stress by 4-HNE (20 μmol/L), PA (0.3 mmol/L) and MCDM (right panel). N-Acetyl-l-cysteine (NAC) was added into the culture medium to block the effect of 4-HNE. For detecting the GPX4 oligomerization, sample were incubated at 30 °C with EGS (300 μmol/L, 15 min) and lyzed with RIPA buffer with 5 mol/L guanidine-HCl. (B) Flag-tagged cGPX4 was transfected into AML12 hepatocyte cell line and the cells were stimulated with PA to evaluate the influence of lipid stress on cGPX4 oligomerization. The cGPX4 was detected with an antibody against Flag. The band in ∼19 kDa (monomer cGPX4) was designated with an arrow. pcDNA3.1 was used as a vector. (C) Flag-tagged cGPX4 and HA-tagged iGPX4 were transfected into AML12 hepatocyte cell line to evaluate the influence of iGPX4 on cGPX4 oligomerization. The cGPX4 was detected with an antibody against Flag. The band in ∼19 kDa (monomer cGPX4) was designated with an arrow. (D) Immunoblotting analysis showing overexpression of iGPX4 (pcDNA3.1-iGPX4) antagonized the cGPX4 overexpression (pcDNA3.1-cGPX4)-induced decrease of MDA and 4-HNE protein adducts in AML12 hepatocytes under PA stimuli (0.3 mmol/L). (E) Overexpression of iGPX4 abolished the cGPX4 overexpression-induced restoration of GSH activity in AML12 hepatocytes under PA stimuli (0.3 mmol/L). Data are presented as mean ± SEM and analyzed by one-way ANOVA followed by Tukey's post hoc test; ∗∗P < 0.01. (F) Flag-tagged cGPX4 and HA-tagged iGPX4 were co-transfected into AML12 hepatocyte cell line to evaluate the interaction between iGPX4 and cGPX4 with co-immunoprecipitation. WCL, whole cell lysate. Heavy chain and light chain were labeled. (G) A working model for the functional regulation of GPX4 alternative isoforms in ferroptosis control and hepatic damage in MAFLD. Lipid stress induces iGPX4, which interacts with cGPX4 and facilitates the transformation of cGPX4 from monomer (active) to oligomers (inactive). The isoform iGPX4 promotes ferroptosis and deteriorate MFALD.