SIRT2-mediated deacetylation and deubiquitination of C/EBPβ prevents ethanol-induced liver injury - PubMed (original) (raw)

doi: 10.1038/s41421-021-00326-6.

Xidai Long # 2, Xin Ruan # 1, Qian Wei 1, Lin Zhang 3, Lulu Wo 1, Dongdong Huang 1, Longshuai Lin 4, Difei Wang 1, Li Xia 5, Qinghua Zhao 4, Junling Liu 3, Qian Zhao 6, Ming He 7

Affiliations

SIRT2-mediated deacetylation and deubiquitination of C/EBPβ prevents ethanol-induced liver injury

Yingting Zhang et al. Cell Discov. 2021.

Erratum in

Abstract

Protein acetylation has emerged to play pivotal roles in alcoholic liver disease (ALD). Sirutin 2 (SIRT2) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase involved in the regulation of aging, metabolism, and stress. However, the role of SIRT2 in ALD remains unclear. Here, we report that the SIRT2-mediated deacetylation-deubiquitination switch of CCAAT/enhancer-binding protein beta (C/EBPβ) prevents ALD. Our results showed that hepatic SIRT2 protein expression was negatively correlated with the severity of alcoholic liver injury in ALD patients. Liver-specific SIRT2 deficiency sensitized mice to ALD, whereas transgenic SIRT2 overexpression in hepatocytes significantly prevented ethanol-induced liver injury via normalization of hepatic steatosis, lipid peroxidation, and hepatocyte apoptosis. Mechanistically, we identified C/EBPβ as a critical substrate of SIRT2 implicated in ALD. SIRT2-mediated deacetylation at lysines 102 and 211 decreased C/EBPβ ubiquitination, resulting in enhanced protein stability and subsequently increased transcription of C/EBPβ-target gene LCN2. Importantly, hepatic deacetylated C/EBPβ and LCN2 compensation reversed SIRT2 deletion-induced ALD aggravation in mice. Furthermore, C/EBPβ protein expression was positively correlated with SIRT2 and LCN2 expression in the livers of ALD patients and was inversely correlated with ALD development. Therefore, activating SIRT2-C/EBPβ-LCN2 signaling pathway is a potential therapy for ALD.

© 2021. The Author(s).

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

Fig. 1. SIRT2 protein level negatively correlates with the severity of ALD in patients.

a Representative IHC images of liver tissues from ALD patients for the lower (SIRT2low, IRS ≤ 5, n = 47) and higher (SIRT2high, IRS > 5, n = 55) expressions of SIRT2. The scale bar represents 50 μm. b–f Plots of serum ALT (b) and AST (c), cl.Caspase-3 IRS scores (d), and the percentages of liver tissues with high and low necrosis (e) or fibrosis (f) in SIRT2low and SIRT2high groups. Statistical significance was determined by two-tailed Student’s _t-_test (b–d), Pearson’s _χ_2 test (e, f). Data are shown as means ± SD and were considered statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 2

Fig. 2. Liver-specific SIRT2 KO sensitizes mice to alcoholic liver injury.

SIRT2 f/f Alb-Cre – (LoxP) and SIRT2 f/f Alb-Cre + (_SIRT2_-KO) male mice were treated with pair (Pair) and ethanol diet (EtOH) according to NIAAA model (n = 8–10/group). a–h Liver injury, steatosis, lipid peroxidation, and cell apoptosis were assessed by images of the indicated livers (scale bar, 1 cm), mouse hepatic H&E staining (scale bar, 100 μm), IHC detection of 4-HNE and TUNEL (scale bar, 100 μm) (a), serum ALT (b) and AST (c), liver triglyceride (TG) (d), hepatic MDA content (e), and PTGS2 mRNA (f), quantitative analysis of TUNEL-positive hepatocytes (magnification, ×200) (g), Western blot analysis of cl.Caspase-3 in murine liver tissues (h). Student’s _t-_test was used for statistical evaluation. Data are shown as means ± SD and are considered statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 3

Fig. 3. Hepatocyte-specific SIRT2 overexpression prevents alcoholic liver injury in mice.

Male C57BL/6 mice tail injected with AAV8-Ctrl (Ctrl), AAV8-SIRT2 (SIRT2), or AAV8_-_SIRT2-H187A (H187A) were treated with pair (Pair) and ethanol diet (EtOH) according to NIAAA model (n = 7/group). a Images of the indicated livers (scale bar, 1 cm), murine liver staining by H&E (scale bar, 50 μm), IHC detection of 4-HNE and TUNEL assay (scale bar, 100 μm). b–d Serum ALT (b) and AST (c) measurements and TG content (d). e–f Hepatic MDA measurements (e) and PTGS2 mRNA analysis by qRT-PCR (f). g Quantitative analysis of TUNEL-positive hepatocytes (magnification, ×200) in murine livers. h Western blot analysis of cl.Caspase-3 and SIRT2 expression in murine liver tissues. Student’s _t-_test was used for statistical evaluation. Data are shown as means ± SD and are considered statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4

Fig. 4. Hepatic SIRT2 protects mice from ALD through upregulating LCN2.

a Heatmap of differentially expressed genes associated with acute-phase response (Gene Ontology: 0006953) identified by RNA-seq using liver tissues from pair- or EtOH-fed LoxP or _SIRT2_-KO mice (n = 3). b, c qRT-PCR (b) and Western blot (c) analyses of LCN2 mRNA and protein expression in murine livers. d, e qRT-PCR (d) and Western blot analysis (e) of LCN2 expression in SIRT2 knockdown (SIRT2 KD) or control AML12 hepatocytes. f, g qRT-PCR (f) and Western blot analysis (g) of LCN2 in SIRT2 overexpression hepatocytes. h–o LoxP and _SIRT2_-KO male mice tail-vein injected with AAV8-Ctrl (Ctrl) or AAV8-LCN2 (LCN2) were treated with pair (Pair) or ethanol diet (EtOH) according to NIAAA model construction (n = 3–5/group). h Images of the indicated livers (scale bar, 1 cm), H&E staining of murine livers (scale bar, 50 μm), IHC detection of 4-HNE and TUNEL (scale bar, 100 μm). i, j Liver TG and MDA content of indicated mice. k Hepatic PTGS2 mRNA by qRT-PCR analysis of indicated mice. l, m Serum ALT and AST of indicated mice. n Quantitative analysis of TUNEL-positive hepatocytes (magnification, ×200). o Western blot analysis of cl.Caspase-3 protein in murine liver tissues. Student’s _t-_test was used for statistical evaluation. Data are shown as means ± SD and are considered statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5

Fig. 5. C/EBPβ mediates the upregulation of LCN2 by SIRT2 under ethanol stress.

a Venn diagram shows common transcription factors predicted as the putative shared regulators on LCN2, Hp, Saa3, Saa1, Hpx, and Orm2. b Western blot analysis of hepatic LCN2, C/EBPβ, and SIRT2 expression in pair and EtOH-fed mice. c, d LCN2 mRNA expression after C/EBPβ knockdown (KD) or overexpression (C/EBPβ) in AML12 cells by qRT-PCR analysis. #P < 0.05 and ###P < 0.001 are used to indicate statistical significance compared between the group with EtOH treatment and the corresponding group without EtOH treatment. e Dual-luciferase reporter assay was performed in HK293T cells. Western blot analysis of C/EBPβ expression (top) and luciferase activities of the LCN2 promoter-reporter system (bottom) are shown. f UCSC Epigenome Browser tracks of the C/EBPβ ChIP-seq signal −3 kb before TSS of LCN2 from Cistrome DB ToolKit (top); information about predicted C/EBPβ binding sites and the primers targeting different sites on LCN2 promoter (middle); ChIP analysis showing C/EBPβ occupancy at the LCN2 proximal promoter in AML12 cells treated with EtOH and control (bottom). PPARγ and distant region primers were, respectively, used as a positive and negative control. g qRT-PCR analysis of LCN2 mRNA expression in SIRT2 knockdown AML12 cells transfected with C/EBPβ. Student’s _t_-test was used for s_t_atistical evaluation. Data are shown as means ± SD and are considered statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 6

Fig. 6. SIRT2 decreases C/EBPβ ubiquitination and stabilizes C/EBPβ protein by deacetylation under ethanol stress.

a Immunofluorescence analysis of SIRT2 and C/EBPβ in AML12 cells treated with EtOH (scale bar, 50 μm). b Endogenous SIRT2–C/EBPβ interaction was analyzed by the amount of C/EBPβ coimmunoprecipitated with the same loading amount of SIRT2 in AML12 cells. c Acetylation levels of the same loading amount of endogenous C/EBPβ purified by IP in ethanol-treated AML12 cells detected by pan-acetyllysine antibody are shown, which indicates SIRT2 deacetylates C/EBPβ. d, e Acetylation levels of IP-purified Flag-C/EBPβ in HEK293T cells coexpressing SIRT2, SIRT2-H187A (d) or with SIRT2 knockdown (e). f, g qRT-PCR (f) and Western blot analysis (g) of C/EBPβ expression in SIRT2 knockdown or control AML12 cells treated with or without ethanol. h, i HEK293T cells co-transfected Flag-tagged C/EBPβ with the indicated plasmids or siRNA were treated with 10 μM MG132 or DMSO for 6 h. Ubiquitination level of C/EBPβ was probed by anti-HA antibody. j Ubiquitination levels of the same loading amount endogenous C/EBPβ purified by IP were probed by pan-ubiquitin antibody. k Acetylation levels of IP-purified C/EBPβ and its mutants in HEK293T cells with SIRT2 knockdown. K98R is used as a negative control. l Acetylation levels of IP-purified Flag-C/EBPβ and its mutants in HEK293T cells. m Ubiquitination levels of IP-purified Flag-C/EBPβ and its mutants in HEK293T cells with SIRT2 knockdown. n Ubiquitination levels of IP-purified Flag-C/EBPβ and its mutants in HEK293T cells. The experiments were repeated at least for three times with the same results, and the results of one representative experiment are shown.

Fig. 7

Fig. 7. C/EBPβ deacetylation reverses hepatic SIRT2 deficiency-aggravated ALD in mice.

LoxP and _SIRT2_-KO mice tail injected with AAV8-Ctrl (Ctrl) or AAV8_-_C/EBPβ (C/EBPβ) or AAV8-C/EBPβ K102R (K102R) or AAV8-C/EBPβ K211R (K211R) were treated with NIAAA model (n = 7/group). a–h The effects of wild-type C/EBPβ or constitutively deacetylated C/EBPβ mutants on steatosis, lipid peroxidation, and cell apoptosis were assessed by images of the indicated livers (scale bar, 1 cm), hepatic H&E staining (scale bar, 50 μm), IHC detection of 4-HNE and TUNEL (scale bar, 100 μm) (a), liver/body weight ratios (b), liver TG (c), hepatic MDA content (d), and PTGS2 mRNA (e), serum ALT (f), and AST (g), quantitative analysis of TUNEL-positive hepatocytes (magnification, ×200) (h). i Hepatic LCN2 mRNA analysis by qRT-PCR. j Western blot analysis of cl.Caspase-3, LCN2, C/EBPβ, and SIRT2 protein expression in the indicated livers. Student’s _t_-test was used for statistical evaluation. Data are shown as means ± SD and are considered statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 8

Fig. 8. Hepatic C/EBPβ negatively correlates with alcoholic liver injury and positively correlates with SIRT2 and LCN2 expression in patient livers.

a–f Plots of serum ALT (a) and AST (b), cl.Caspase-3 IRS scores (c), the percentages of liver tissues with different degrees of necrosis (d), fibrosis (e), and proliferation (f) in ALD patients with high (C/EBPβhigh, n = 48) and low (C/EBPβlow, n = 54) C/EBPβ expressions. g Representative IHC images of patient liver samples for the low and high expressions of indicated proteins (scale bar, 100 μm). h–j Correlation analysis of relative protein expression of SIRT2, C/EBPβ, and LCN2 in liver tissues from ALD patients (n = 102). Statistical significance was determined by two-tailed Student’s t test (a–c), Pearson’s _χ_2 test (d–f), and linear correlation and regression (h–j). Data are shown as means ± SD and are considered statistically significant at **P < 0.01.

Fig. 9

Fig. 9. Model. Protective mechanism of hepatic SIRT2-C/EBPβ-LCN2 axis in ALD.

Ethanol upregulated SIRT2 in hepatocytes, which directly deacetylates C/EBPβ on lysines 102 and 211 and inhibits ubiquitination of C/EBPβ, and subsequently promotes the transcription of LCN2. This in turn prevents oxidative stress and lipid peroxidation induced by ethanol. SIRT2 KO increases ubiquitination and degradation of C/EBPβ and aggravates ethanol-induced liver injury.

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