Inhibition of 7-dehydrocholesterol reductase prevents hepatic ferroptosis under an active state of sterol synthesis

Abstract

Recent evidence indicates ferroptosis is implicated in the pathophysiology of various liver diseases; however, the organ-specific regulation mechanism is poorly understood. Here, we demonstrate 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme of cholesterol biosynthesis, as a regulator of ferroptosis in hepatocytes. Genetic and pharmacological inhibition (with AY9944) of DHCR7 suppress ferroptosis in human hepatocellular carcinoma Huh-7 cells. DHCR7 inhibition increases its substrate, 7-dehydrocholesterol (7-DHC). Furthermore, exogenous 7-DHC supplementation using hydroxypropyl β-cyclodextrin suppresses ferroptosis. A 7-DHC-derived oxysterol metabolite, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), is increased by the ferroptosis-inducer RSL-3 in DHCR7-deficient cells, suggesting that the ferroptosis-suppressive effect of DHCR7 inhibition is associated with the oxidation of 7-DHC. Electron spin resonance analysis reveals that 7-DHC functions as a radical trapping agent, thus protecting cells from ferroptosis. We further show that AY9944 inhibits hepatic ischemia-reperfusion injury, and genetic ablation of Dhcr7 prevents acetaminophen-induced acute liver failure in mice. These findings provide new insights into the regulatory mechanism of liver ferroptosis and suggest a potential therapeutic option for ferroptosis-related liver diseases.

Fig. 1. DHCR7 inhibition suppresses ferroptosis in Huh-7 cells.

a Strategy of CRISPR/Cas9-mediated whole-genome screening. Huh-7 cells were treated with RSL-3 (0.1 µM) for 24 h. The ferroptosis-resistant cells were selected 4 times, and the genomic DNA was purified and analyzed by NGS. (b) sgRNAs targeting DHCR7 were enriched in ferroptosis-resistant cells. (c, d) Control (sgGFP) and DHCR7-ablated (sgDHCR7) Huh-7 cells were treated with or without RSL-3 (0.03 and 0.1 µM) for 24 h. Cytotoxicity and cell death were assessed by an LDH release assay and SYTOX Green, respectively. Control (sgGFP) and DHCR7-ablated (sgDHCR7) Huh-7 cells were treated with or without (e) RSL-3 or (f) artesunate for 24 h, and (g) cysteine deprivation for 18 h, respectively. Cell viability was assessed by the MTT assay. h, i Huh-7 cells were treated with RSL3 for 24 h with or without AY9944 1 h prior to RSL-3 administration. Cytotoxicity and cell death were assessed by an LDH release assay and SYTOX Green, respectively. (j) Control (sgGFP) and GPX4-ablated (sgGPX4) cells were maintained with Fer-1 (0.1 µM), and then pretreated with Fer-1 (0.5 µM) or AY9944 (100 nM) for 1 h, followed by Fer-1 withdrawal. Cytotoxicity at 48 h was assessed by an LDH release assay. (d h) Data are representative of three independent experiments (c, e–g i, j). Data are means of (g and j) three or (c, e, f, and i) four independent experiments and expressed as dot plots and means ± SEM. Statistical significance was calculated using two-way ANOVA with Tukey’s post hoc test or Student’s t-test.

Fig. 2. DHCR7 inhibition suppresses lipid peroxidation.

Control (sgGFP) and DHCR7-ablated (sgDHCR7) Huh-7 cells were treated with RSL-3 (0.1 µM) for 6 h. The fluorescence intensity of C11-BODIPY^581/591 was analyzed by flow cytometry. a Representative plot of C11-BODIPY^581/591-stained cells. b The number of oxidized C11-BODIPY^581/591-positive cells and the mean fluorescence intensity of oxidized C11-BODIPY^581/591 were analyzed. (c, d) Control (sgGFP) and DHCR7-ablated (sgDHCR7) Huh-7 cells or A9944-treated cells were treated with RSL3 (0.1 µM) for 16 h, and lipid peroxides were measured by LC-MS/MS. a Data are representative and b means of three independent experiments and expressed as dot plots and means ± SEM. c, d Data are from four replicates. Statistical significance was calculated using two-way ANOVA with Tukey’s post hoc test.

Fig. 3. Accumulation of 7-DHC by DHCR7 inhibition, but not cholesterol deprivation, suppresses ferroptosis.

a Control (sgGFP) and DHCR7-ablated (sgDHCR7) Huh-7 cells were treated with or without RSL-3 (0.1 µM) for 16 h. b Huh-7 cells were pretreated with AY9944 (100 nM) for 1 h, and then treated with RSL-3 for 16 h. a, b Intracellular 7-DHC levels were assessed using LC-MS/MS analysis. n = 3, respectively. c Schematic diagram of a cholesterol synthesis pathway via lathosterol. The introduced or reduced double bond is shown by a red arrow. d Huh-7 cells transduced with sgDHCR7 and sgSC5D. Intracellular 7-DHC levels were assessed using LC-MS/MS analysis. Huh-7 cells transduced with sgDHCR7 and sgSC5D were treated with (e) cysteine deprivation for 18 h or (f) RSL-3 for 24 h. Cell viability was assessed by the MTT assay. g Cells were pretreated with MβCD (0.1 mM) for 1 h, and then treated with RSL-3 for 24 h. Cytotoxicity was assessed by an LDH release assay. (a, b, and d) Data are from (a and b) three or (d) four replicates. e–g Data are means of three independent experiments. Statistical significance was calculated using two-way ANOVA with Tukey’s post hoc test. Data are expressed as dot plots and means ± SEM.

Fig. 4. 7-DHC acts as a radical trapping agent to prevent ferroptosis.

a Huh-7 cells were treated with a 7-DHC-d7-HPβCD inclusion complex for 16 h, and the intracellular 7-DHC-d7 levels were measured by LC-MS/MS. b Huh-7 cells were pretreated with 7-DHC-HPβCD for 1 h and then treated with RSL-3 for 24 h. Cell viability was assessed by the MTT assay. c GPX4-ablated cells were pretreated with 7-DHC-HPβCD for 1 h, followed by Fer-1 withdrawal. Cytotoxicity at 48 h was assessed by an LDH release assay. d ESR signal intensity of TEMPOL (50 μM) after the addition of LOX (15 μg/ml) and AA (250 μM) with 7-DHC-HPβCD (30 µM or 100 µM), cholesterol-HPβCD (100 µM) or Lip-1 (100 µM). e Fluorescence intensities of NBD-Pen (5 μM) 5 min after the addition of LOX (5 μg/ml) and AA (500 μM) with 7-DHC-HPβCD (10 µM, 30 µM or 100 µM), cholesterol-HPβCD (100 µM) or Lip-1 (100 µM). f, g Control (sgGFP) and DHCR7-ablated (sgDHCR7) Huh-7 cells were treated with or without RSL-3 (0.1 µM) for 16 h. Intracellular DHCEO levels were assessed using LC-MS-MS analysis. Data are from (d and e) three or (a and g) four replicates. b, c) Data are means of three independent experiments. Statistical significance was calculated using one-way or two-way ANOVA with Tukey’s post hoc test. Data are expressed as dot plots and means ± SEM. *p < 0.05, **p < 0.01.

Fig. 5. Ferroptosis suppression by DHCR7 inhibition depends on the activity of sterol synthesis and DHCR7 expression.

a HT1080 cells and OVISE cells were treated with Fer-1 (1 µM), AY9944 (30 nM), Cholesterol-HPβCD inclusion complex, or 7-DHC-HPβCD inclusion complex for 1 h, followed by RSL-3 (0.1 µM) for 24 h. Cytotoxicity was assessed by an LDH release assay. b HT1080, OVISE, and Huh-7 cells were pretreated with Fer-1 (0.5 µM) or AY9944 (30 and 100 nM) for 1 h, and then treated with RSL-3 (0.1 µM) for 24 h. Cell viability was assessed by the MTT assay. c, d Expression of genes involved in cholesterol biosynthesis in human cancer cell lines in the CCLE dataset. c Scatter plot showing expression levels (log2 normalized TPM) of DHCR7 and either HMGCR or SC5D. d Expression score calculated using genes listed in the Cholesterol biosynthesis/Reactome. e Heatmap showing the expression levels of Cholesterol biosynthesis. f TRE-nSREBP-1a–HT1080 cells were treated with DOX (10 ng/mL or 30 ng/mL) for 6 h. The mRNA levels of DHCR7, SC5D, and HMGCR were assessed by real-time RT-PCR analysis. g and h TRE-nSREBP-1a–HT1080 cells were treated with or without RSL3 for 24 h after 6 h of treatment with DOX (30 ng/mL) and AY9944 (100 nM). Cytotoxicity and cell death were assessed by g an LDH release assay and h SYTOX Green. a, b, f, and g Data are means or h representative of (a, b, f, and h) three or g four independent experiments. Statistical significance was calculated using one-way or two-way ANOVA with Tukey’s post hoc test. Data are expressed as dot plots and means ± SEM.

Fig. 6. DHCR7 inhibition prevents ferroptosis-related liver diseases.

a DHCR7 expression in normal tissues and organs was analyzed using the RefEx database. b–e Liver samples and serum were obtained from hepatic ischemia-reperfusion or sham-operated C57BL/6J male mice treated with AY9944 (25 mg/kg) 1 h prior to open laparotomy. HE staining (b) and serum AST and ALT levels (c) were assessed. d Lipid peroxides in the liver and e 7-DHC in the liver and serum were assessed by LC-MS/MS. f, g C57BL/6J male mice were injected with 10 µg of px330 carrying a pair of Dhcr7-targeting gRNA or empty gRNA scaffold (NC) using a hydrodynamics-based procedure. PBS or acetaminophen (300 mg/kg) was intraperitoneally injected 7 days after gene transduction. Serum AST and ALT levels and HE staining were assessed. Data are collected from c 3–9 mice per group (n = 3 in vehicle/sham and AY9944/sham, n = 8 in vehicle/IRI, n = 9 in AY9944/IRI), d 6 mice per group, and e 3 mice per group. Statistical significance was calculated using two-way ANOVA with Tukey’s post hoc test. f–g Data are from 11 mice per group and statistical significance was calculated using two-tailed Mann-Whitney’s U test. Data are expressed as dot plots and/or means ± SEM.