Overexpression of DTX1 inhibits D-GalN/TNF-α-induced pyroptosis and inflammation in hepatocytes by regulating NLRP3 ubiquitination - PubMed (original) (raw)

Overexpression of DTX1 inhibits D-GalN/TNF-α-induced pyroptosis and inflammation in hepatocytes by regulating NLRP3 ubiquitination

Mingshui Liu et al. Toxicol Res (Camb). 2024.

Abstract

Background: Acute liver injury (ALI) is characterized by massive hepatocyte death and has high mortality and poor prognosis. Hepatocyte pyroptosis plays a key role in the pathophysiology of ALI and is involved in the inflammatory response mediated by NOD-like receptor protein 3 (NLRP3) inflammasome activation. Deltex 1 (DTX1) is a single transmembrane protein with ubiquitin E3 ligase activity and is closely involved in cell growth, differentiation, and apoptosis, as well as intracellular signal transduction. However, little is known about the influence of DTX1 on ALI. This study aimed to investigate the role of DTX1 in pyroptosis and inflammation induced by D-galactosamine (D-GalN) and tumor necrosis factoralpha (TNF-α) in human hepatocytes (LO2 cells) in vitro.

Methods: Cell pyroptosis was measured by flow cytometry. The levels of DTX1, pyroptosis-associated proteins, and inflammatory cytokines were detected by quantitative real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. Immunofluorescence staining, co-immunoprecipitation, ubiquitination, and luciferase reporter and chromatin immunoprecipitation assays were performed to detect the regulation between DTX1 and NLRP3 or hepatocyte nuclear factor 4 alpha (HNF4α). Analysis of variance was performed to compare groups.

Results: We found that DTX1 was decreased in D-GalN/TNF-α-induced LO2 cells. DTX1 overexpression significantly inhibited D-GalN/TNF-α-induced cell pyroptosis and inflammation. DTX1 interacted with NLRP3 and induced NLRP3 ubiquitination and degradation. Furthermore, by targeting NLRP3, DTX1 knockdown significantly induced cell pyroptosis and inflammation. In addition, HNF4α promoted DTX1 transcription by binding with its promoter.

Conclusion: Our study revealed that DTX1 suppressed D-GalN/TNF-α-induced hepatocyte pyroptosis and inflammation by regulating NLRP3 ubiquitination.

Keywords: DTX1; NRLP3; acute liver injury; inflammation; ubiquitination.

© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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Figures

Fig. 1

Fig. 1

DTX1 overexpression inhibits pyroptosis and inflammation in D-GalN/TNF-α-stimulated LO2 cells. After D-GalN and TNF-α treatment of LO2 cells for 0, 12, 24, and 48 h, the mRNA (A) and protein (B) levels of DTX1 were detected by qRT-PCR and western blotting. LO2 cells were treated with empty or DTX1-expressing vectors for 48 h to construct models that overexpressed DTX1 or with D-GalN and TNF-α to construct an acute liver injury model. C) The rate of pyroptosis was evaluated by flow cytometry. D) Protein levels of DTX1, caspsae-1 p20, and GSDMD-N were detected by western blotting. E) IL-1β and IL-18 concentrations were determined by ELISA. Values are presented as mean ± SD, n = 3 for the independent biological experiments. **P < 0.01, ***P < 0.001 vs. 0 h or control. ###P < 0.001 vs. D-GalN + TNF-α + vector.

Fig. 2

Fig. 2

DTX1 interacts with and induces ubiquitination of NLRP3. LO2 cells were treated with empty or DTX1-expressing vectors for 48 h to construct models that overexpressed DTX1 or with D-GalN and TNF-α to construct an acute liver injury model. A and B) The mRNA and protein levels of NLRP3 were measured by qRT-PCR and western blotting. C) Colocalization of DTX1 and NLRP3 was shown on laser confocal microscopy. D) Binding between DTX1 and NLRP3 was measured by Co-IP assay. E) Cells were treated with empty or DTX1-expressing vectors for 48 h to construct models overexpressing DTX1. LO2 cells were incubated with CHX for 0, 3, and 6 h. the NLRP3 level in LO2 cells is shown. F) Lysates of stable DTX1-overexpressing LO2 cells were immunoprecipitated with anti-NLRP3 antibody and examined using anti-Ub antibody. Values are presented as mean ± SD, n = 3 for the independent biological experiments. *P < 0.05, ***P < 0.001 vs. control or vector. ###P < 0.001 vs. D-GalN + TNF-α + vector.

Fig. 3

Fig. 3

DTX1 silencing promotes pyroptosis and inflammasome activation in LO2 cells. Cells were treated with small interfering RNA of DTX1 for 48 h to construct a cell model with low DTX1 expression. DTX1-silenced cells were treated with vehicle or NLRP3 inhibitor (MCC950). A) The rate of pyroptosis was assessed by flow cytometry. B) Protein levels of caspsae-1 p20 and GSDMD-N were measured by western blotting. C) The cell concentrations of IL-1β and IL-18 were analyzed by ELISA. Values are presented as mean ± SD, n = 3 for the independent biological experiments. *P < 0.05, ***P < 0.001 vs. siNC. ###P < 0.001 vs. siDTX1-1 + vehicle.

Fig. 4

Fig. 4

HNF4α promotes DTX1 expression through transcription regulation. LO2 cells were treated with D-GalN and TNF-α for 0, 12, 24, and 48 h. the mRNA (A) and protein (B) levels of HNF4α were detected by qRT-PCR and western blotting. Cells were treated with small interfering RNA of HNF4α for 48 h to construct a cell model with low HNF4α expression. Results of qRT-PCR and western blotting of the mRNA (C) and protein (D) levels of DTX1. (E) Luciferase activity assay of the DTX1 promoter in HNF4α-silenced LO2 cells. (F) ChIP analyses of HNF4α binding to the DTX1 promoter. Values are presented as mean ± SD, n = 3 for the independent biological experiments. *P < 0.05, ***P < 0.001 vs. 0 h, siNC, or IgG.

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