Interferon-α stimulates DExH-box helicase 58 to prevent hepatocyte ferroptosis

Abstract

Background: Liver ischemia/reperfusion (I/R) injury is usually caused by hepatic inflow occlusion during liver surgery, and is frequently observed during war wounds and trauma. Hepatocyte ferroptosis plays a critical role in liver I/R injury, however, it remains unclear whether this process is controlled or regulated by members of the DEAD/DExH-box helicase (DDX/DHX) family.

Methods: The expression of DDX/DHX family members during liver I/R injury was screened using transcriptome analysis. Hepatocyte-specific Dhx58 knockout mice were constructed, and a partial liver I/R operation was performed. Single-cell RNA sequencing (scRNA-seq) in the liver post I/R suggested enhanced ferroptosis by Dhx58^hep-/-. The mRNAs and proteins associated with DExH-box helicase 58 (DHX58) were screened using RNA immunoprecipitation-sequencing (RIP-seq) and IP-mass spectrometry (IP-MS).

Results: Excessive production of reactive oxygen species (ROS) decreased the expression of the IFN-stimulated gene Dhx58 in hepatocytes and promoted hepatic ferroptosis, while treatment using IFN-α increased DHX58 expression and prevented ferroptosis during liver I/R injury. Mechanistically, DHX58 with RNA-binding activity constitutively associates with the mRNA of glutathione peroxidase 4 (GPX4), a central ferroptosis suppressor, and recruits the m⁶A reader YT521-B homology domain containing 2 (YTHDC2) to promote the translation of Gpx4 mRNA in an m⁶A-dependent manner, thus enhancing GPX4 protein levels and preventing hepatic ferroptosis.

Conclusions: This study provides mechanistic evidence that IFN-α stimulates DHX58 to promote the translation of m⁶A-modified Gpx4 mRNA, suggesting the potential clinical application of IFN-α in the prevention of hepatic ferroptosis during liver I/R injury.

Keywords: DExH-box helicase 58 (DHX58); Glutathione peroxidase 4 (GPX4); Ischemia/reperfusion (I/R); YT521-B homology domain containing 2 (YTHDC2); m6A modification.

Fig. 1

Hepatic DHX58 expression is markedly decreased during liver I/R injury. a Expression of hepatic DDX/DHX family members following liver I/R injury. b Immunofluorescence staining images of DHX58, hepatocyte and DAPI in liver sections analyzed by confocal microscopy. Scale bar = 50 µm. c DHX58 protein level in the isolated hepatocytes and non-parenchymal cells (NPCs) of the liver was examined by Western blotting. d DHX58 protein level in liver tissues after I/R injury was examined by Western blotting. e Dhx58 mRNA level in liver tissues after I/R injury was examined by qRT-PCR. f DHX58 protein level in primary hepatocytes following H/R injury was examined by Western blotting. g Dhx58 mRNA level in primary hepatocytes following H/R injury was examined by qRT-PCR. h DHX58 and COX2 in human normal liver tissues, obtained from distal normal liver tissues of liver hemangioma patients, and human liver tissues post I/R, obtained from biopsy approximately 3 h post reperfusion in donors during liver transplantation, were examined by Western blotting. The donor livers for transplantation underwent 5 to 10 min of warm ischemia followed by 5 to 7 h of cold ischemia. Quantified DHX58 expression was shown. i The correlation between DHX58 and COX2 in (h) was analyzed by Pearson’s correlation coefficient assay. Data are shown as mean ± SD (n = 3 or indicated) or photographs from one representative of three independent experiments. ^**P < 0.01 compared with the sham/control group by Student’s two-tailed t-test. DHX58 DExH-box helicase 58, I/R ischemia/reperfusion, DAPI 4’,6-diamidino-2-phenylindole, H/R hypoxia/re-oxygenation, COX2 cyclooxygenase-2, SD standard deviation

Fig. 2

Excessive ROS production during liver I/R injury decreases DHX58 expression. a DHX58 protein level in primary hepatocytes treated with H₂O₂ was examined by Western blotting. b Dhx58 mRNA level in primary hepatocytes treated with H₂O₂ was examined by qRT-PCR. c DHX58 protein level in liver tissues with NAC or BHA pre-treatment and then I/R was examined by Western blotting. d Dhx58 mRNA level in liver tissues with NAC or BHA pre-treatment and then I/R was examined by qRT-PCR. e DHX58 protein level in primary hepatocytes with NAC or BHA pre-treatment and then H/R was examined by Western blotting. f Dhx58 mRNA level in primary hepatocytes with NAC or BHA pre-treatment and then H/R was examined by qRT-PCR. g DHX58 protein level in primary hepatocytes with NAC or BHA pre-treatment and then H₂O₂ (2 mmol/L) administration for 3 h was examined by Western blotting. h Dhx58 mRNA level in primary hepatocytes with NAC or BHA pre-treatment and then H₂O₂ (2 mmol/L) administration for 3 h was examined by qRT-PCR. Data are shown as mean ± SD (n = 3) or photographs from one representative of three independent experiments. ^*P < 0.05, ^**P < 0.01. ROS reactive oxygen species, I/R ischemia/reperfusion, DHX58 DExH-box helicase 58, NAC N-acetylcysteine, BHA butylated hydroxyanisole, H/R hypoxia/re-oxygenation, SD standard deviation

Fig. 3

Decreased DHX58 aggravates liver I/R injury. a DHX58 protein level in liver tissues and isolated hepatocytes from Dhx58^f/f and Dhx58^hep−/− mice was confirmed by Western blotting. Liver I/R injury was administrated in Dhx58^f/f and Dhx58^hep/− mice, liver injury was shown by gross appearances of representative livers, and white arrow indicated injured area (b), liver pathology was analyzed by HE staining (c), necrotic area and Suzuki’s score were analyzed (n = 4) (d), serum ALT and AST were examined (n = 4) (e), IL-6, IL-1β, and MCP1 mRNA levels in liver tissues were examined by qRT-PCR (n = 4) (f), infiltration of neutrophils and macrophages were analyzed by flow cytometry (g). Scale bar = 20 μm. Data are shown as mean ± SD or photographs from one representative of three independent experiments. ^*P < 0.05, ^**P < 0.01. DHX58 DExH-box helicase 58, I/R ischemia/reperfusion, HE hematoxylin-eosin, ALT alanine aminotransferase, AST aspartate aminotransferase, IL-6 interleukin-6, IL-1β interleukin-1β, MCP1 monocyte chemoattractant protein 1

Fig. 4

The downregulation of DHX58 expression following I/R injury promotes ferroptosis in hepatocyte. a UMAP visualization of cells in the livers of Dhx58^f/f and Dhx58^hep−/− mice underwent sham or I/R, and each dot corresponded to one single cell colored according to the cell cluster. b The bubble diagram of signature genes for each cell type is displayed. UMAP visualization of hepatocytes in I/R-treated Dhx58^f/f and Dhx58^hep−/− mice was shown as indicated (c), cell counts of Dhx58^f/f and Dhx58^hep−/− mice in each hepatocyte cluster were shown (d), and functional gene enrichment in each hepatocyte cluster by QuSAGE analysis was shown (e). Liver I/R injury was administrated in Dhx58^f/f and Dhx58^hep/− mice, hepatic iron (f) and LPO (g) levels were analyzed accordingly (n = 4), representative transmission electron microscope images show the morphology of ferroptosis in hepatocyte with black arrows indicated mitochondrion and lipid droplets (h), GSH and GSSG were analyzed and the ratio was calculated (n = 4) (i). Scale bar = 20 μm (f) and 2 μm (h). j Serum ALT and AST of Dhx58^f/f and Dhx58^hep−/− mice treated with ferroptosis inducer erastin (n = 3). Data are shown as mean ± SD or photographs from one representative of three independent experiments. ^*P < 0.05, ^**P < 0.01. DHX58 DExH-box helicase 58, I/R ischemia/reperfusion, UMAP uniform manifold approximation and projection, QuSAGE quantitative set analysis of gene expression, LPO lipid peroxide, GSH glutathione, GSSG oxidized glutathione, ALT alanine aminotransferase, AST aspartate aminotransferase, SD standard deviation, HSEC hepatic sinusoid endothelium cell, GMP granulocyte-monocyte progenitor, NK natural killer, AIM2 absent in melanoma 2, NLRs nucleotide-binding leucine-rich repeat receptors, NLRP1 nucleotide-binding oligomerization domain-like receptor protein 1, IPAF interleukin-1β-converting enzyme-protease activating factor

Fig. 5

DHX58 associates Gpx4 mRNA and promotes its translation. a Schematic workflow of DHX58 downstream targets analysis. b The association between DHX58 and Gpx4 mRNA in primary hepatocytes were determined by RIP-qRT-PCR. ^*P < 0.05. c The top motif identified by HOMER of DHX58-bound peaks in Gpx4 mRNA. d The association between the CTD domain of DHX58 and endogenous Gpx4 mRNA in primary hepatocytes was determined by RIP-qRT-PCR. ^*P < 0.05, ^**P < 0.01, ns non-significant. e DHX58, ACSL4, COX2, SLC7A11, and GPX4 protein levels in liver tissues of Dhx58^f/f and Dhx58^hep−/− mice after I/R injury were examined by Western blotting. f DHX58, ACSL4, COX2, SLC7A11, and GPX4 protein levels in primary hepatocytes from Dhx58^f/f and Dhx58^hep−/− mice following H/R injury were examined by Western blotting. g GPX4 protein level in DHX58-overexpressed primary hepatocytes was examined by Western blotting. h DHX58, ACSL4, COX2, SLC7A11, and GPX4 protein levels in primary hepatocytes from Dhx58^f/f or Dhx58^hep−/− mice with GPX4 overexpression and treatment of H/R or erastin were examined by Western blotting. i In primary hepatocytes from Dhx58^f/f and Dhx58^hep−/− mice, relative Gpx4 mRNA distribution in each ribosome fractions was analyzed by qRT-PCR. Data are shown as mean ± SD (n = 3) or photographs from one representative of three independent experiments. ^#P < 0.05, ^##P < 0.01 vs. Dhx58^f/f. ▲CTD C-terminal domain deleted, DHX58 DExH-box helicase 58, GPX4 glutathione peroxidase 4, RIP RNA immunoprecipitation, HOMER hypergeometric optimization of motif enrichment, CTD C-terminal domain, ACSL4 acyl-CoA synthetase long chain family member 4, COX2 cyclooxygenase-2, SLC7A11 solute carrier family 7 member 11, I/R ischemia/reperfusion, H/R hypoxia/re-oxygenation, LMW low molecular weight, HMW high molecular weight, SD standard deviation

Fig. 6

DHX58 recruits YTHDC2 to promote the translation of m⁶A-modified Gpx4 mRNA. a The endogenous association between DHX58 and YTHDC2 in liver tissues was examined by Co-IP. b GPX4 protein level in primary hepatocytes with Ythdc2 overexpression or knockdown was examined by Western blotting. c In primary hepatocytes with Ythdc2 overexpression or knockdown, the relative distribution of Gpx4 mRNA in each ribosome fraction was analyzed by qRT-PCR. ^##P < 0.01 vs. Empty vector. d In primary hepatocytes from Dhx58^f/f and Dhx58^hep−/− mice with knockdown of Ythdc2, DHX58, YTHDC2, and GPX4 protein levels were examined by Western blotting. e Sequencing read clusters from MeRIP-seq analysis of Gpx4 mRNA in primary hepatocytes and top consensus motif identified by HOMER with MeRIP-seq peaks. f m⁶A modification of Gpx4 and Acsl4 mRNAs in primary hepatocytes were examined by RIP-qRT-PCR. g GPX4 protein level in primary hepatocytes with knockdown of Mettl3 was examined by Western blotting. h In primary hepatocytes transfected with Flag-tagged DHX58 or YTHDC2, together with Mettl3 knockdown, Flag-tag, METTL3, and GPX4 protein levels were examined by Western blotting. Data are shown as mean ± SD (n = 3) or photographs from one representative of three independent experiments. ^**P < 0.01. ns non-significant, Si-1 No.1 siRNA targeting YTHDC2, Si-2 No.2 siRNA targeting YTHDC2, DHX58 DExH-box helicase 58, YTHDC2 YT521-B homology domain containing 2, m⁶A N⁶-methyladenosine, Gpx4 glutathione peroxidase 4, Co-IP co-immunoprecipitation, MeRIP-seq methylated RNA immunoprecipitation sequencing, HOMER hypergeometric optimization of motif enrichment, Acsl4 acyl-CoA synthetase long chain family member 4, RIP RNA immunoprecipitation, METTL3 methyltransferase complex methyltransferase-like 3, CTD C-terminal domain, SD standard deviation, LMW low molecular weight, HMW high molecular weight

Fig. 7

Pretreatment with IFN-α can inhibit hepatic ferroptosis by stimulating DHX58. a DHX58 and GPX4 protein levels in the liver with IFN-α pretreatment and then I/R. Wild-type (WT) and Dhx58^hep/− mice were pretreated with IFN-α, and then underwent I/R, liver damage was examined by serum ALT and AST (b), liver pathology was analyzed by HE staining (c), necrotic area and Suzuki’s score were examined (d), ROS production was analyzed by DHE staining (e), hepatic iron (f) and LPO (g) levels were analyzed accordingly. Scale bar = 20 μm. Data are shown as mean ± SD (n = 4) or photographs from one representative of three independent experiments. ^*P < 0.05, ^**P < 0.01. ns non-significant, IFN-α interferon-α, DHX58 DExH-box helicase 58, GPX4 glutathione peroxidase 4, I/R ischemia/reperfusion, ALT alanine aminotransferase, AST aspartate aminotransferase, HE hematoxylin-eosin, ROS reactive oxygen species, DHE dihydroethidium, LPO lipid peroxide, SD standard deviation

Fig. 8

The pretreatment with IFN-α induces the activation of DHX58, which recruits YTHDC2 to recognize and enhance the translation of m⁶A-modified Gpx4 mRNA, thus preventing hepatic ferroptosis. IFN-α interferon-α, DHX58 DExH-box helicase 58, GPX4 glutathione peroxidase 4, ROS reactive oxygen species, YTHDC2 YT521-B homology domain containing 2, m⁶A N⁶-methyladenosine