TRAF2 Promotes Liver Fibrosis via Regulation of the HIF-1α/GLUT1-Mediated Glycolysis in Hepatic Stellate Cells

Abstract

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is an intracellular aptamer protein with E3 ligase activity and has been reported to be involved in the pathogenesis of hepatitis and liver cancer. However, the specific mechanism for liver fibrosis mediated by TRAF2 is a still-unresolved issue. In this study, we uncovered high TRAF2 expression in activated hepatic stellate cells (HSCs) and fibrotic livers of both human and two mouse liver fibrosis models. TRAF2 in HSCs correlated positively with liver fibrosis and could directly prompt HSC activation, as evidenced by in vitro gain-of-function and loss-of-function models. In vivo, HSC-specific knockout of TRAF2 could alleviate liver injury and fibrosis in mice. Mechanistically, we demonstrated that TRAF2 in HSCs promoted the increase of hypoxia-inducible factor-1α (HIF-1α) levels by inhibiting von Hippel-Lindau (pVHL)-mediated HIF-1α degradation and inducing HIF-1α translation via activating mTORC1 pathway. Elevated HIF-1α expression predisposed to a rise in its transcriptional target glucose transporter 1 (GLUT1) expression and glycolytic activity in HSCs, eventually developing liver fibrosis. Thus, TRAF2 exerts a significant impact upon activating HSCs and may become a candidate molecule for anti-liver fibrosis therapy.

Keywords: GLUT1; Glycolysis; HIF-1α; Hepatic stellate cells (HSCs); Liver fibrosis; TRAF2.

Figure 1

High TRAF2 abundance was observed in fibrotic livers from human and two mouse liver fibrosis models. (A) Liver samples obtained from individuals with HBV-related (n = 9) or alcohol-related (n = 6) liver fibrosis underwent H&E, sirius red and immunohistochemistry staining for α-SMA and TRAF2 after paraffin embedding and section. Tissues adjacent to hepatic hemangioma were employed as controls (n = 10). Image Pro Plus software was applied for the measurement of positive staining regions. Scale bars = 50 µm. (B) Protein levels of TRAF2 and α-SMA in clinical samples from HBV or alcohol-related liver fibrosis were detected by immunoblot assay, and hepatic hemangioma-adjacent normal tissues were employed as controls. (C) Liver tissues from mice subjected to olive oil or CCl₄ after paraffin embedding and section underwent H&E, sirius red and immunohistochemical staining for α-SMA and TRAF2 and relative quantitative results of the regions of positive staining. Scale bars = 50 µm. (D) Protein levels of TRAF2 and α-SMA in the livers from vehicle or CCl₄ injected-mice were probed using immunoblotting. (E) Liver sections from mice subject to sham operation or BDL underwent H&E, sirius red and immunohistochemical staining for α-SMA and TRAF2 and quantification of the regions of positive staining. Scale bars = 50 µm. (F) Hepatic expression of TRAF2 and α-SMA proteins in mice suffering sham operation or BDL was detected using immunoblotting. All data are represented as the means ± SD. *, P<0.05; **, P<0.01; ****, P<0.0001. Abbreviations: H&E: Hematoxylin and eosin; α-SMA: α-smooth muscle actin; TRAF2: Tumor necrosis factor receptor-associated factor 2; HBV: Hepatitis B virus; CCl₄: Carbon tetrachloride; BDL: Bile duct ligation.

Figure 2

Increased TRAF2 expression was basically localized in HSCs and hepatocytes in fibrotic livers of humans and mice. (A) Normal and fibrotic liver tissues of humans were stained with an anti-HNF4α or anti-α-SMA antibody (green) for labeling hepatocytes and HSCs, respectively. Fluorescent TRAF2 was shown in red and the cell nucleus stained with DAPI was shown in blue. Scale bars = 10 µm. (B) Vehicle or CCl₄-induced liver tissues of mice were stained with an anti-HNF4α or anti-α-SMA antibody (green) for labeling hepatocytes and HSCs, respectively. Fluorescent TRAF2 was shown in red and the cell nucleus stained with DAPI was shown in blue. Scale bars = 10 µm. (C) Liver tissues from mice receiving olive oil or CCl₄ underwent perfusion and enzyme digestion for isolating primary HSCs and hepatocytes, followed by the confirmation of TRAF2 and α-SMA proteins in these two kinds of cells by immunoblotting analyses. (D) Double immunofluorescence was done on the primary HSCs isolated from mice receiving olive oil or CCl₄. Fluorescent α-SMA was displayed in green, fluorescent TRAF2 was shown in red and the cell nucleus stained with DAPI was shown in blue. Scale bars = 5 µm. Abbreviations: HNF4α: Hepatocyte nuclear factor 4α; α-SMA: α-smooth muscle actin; TRAF2: Tumor necrosis factor receptor-associated factor 2; DAPI: 4,6-diamidino-2-phenylindole; CCl₄: Carbon tetrachloride; HSC: Hepatic stellate cell; WT: Wild type.

Figure 3

HSC-specific ablation of TRAF2 protected against liver fibrosis in mice after CCl₄ treatment. (A) Schematic illustration of TRAF2^f/f and TRAF2^△HSC mice receiving twice-weekly intraperitoneal injections of olive oil or CCl₄ for 8 weeks. (B) Liver tissues from TRAF2^f/f and TRAF2^△HSC mice after paraffin embedding and section underwent H&E, sirius red as well as immunohistochemical staining for α-SMA and COL1A1. Scale bars = 50 µm. (C-E) Relative quantitative results of the regions of sirius red, α-SMA and COL1A1 positive staining. (F-G) ALT and AST were evaluated as indicators of liver function. (H) Statistic analysis showed the liver/body weight ratio. (I) Hepatic α-SMA expression in transgenic mice receiving vehicle or CCl₄ was detected using immunoblotting. All data are represented as the means ± SD. **, P<0.01; ***, P<0.001; ****, P<0.0001. Abbreviations: H&E: Hematoxylin and eosin; α-SMA: α-smooth muscle actin; COL1A1: Collagen type 1; TRAF2: Tumor necrosis factor receptor-associated factor 2; CCl₄: Carbon tetrachloride; HSC: Hepatic stellate cell; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase.

Figure 4

HSC-specific ablation of TRAF2 protected against liver fibrosis in mice after BDL treatment. (A) Schematic diagram of TRAF2^f/f and TRAF2^△HSC mice receiving sham operation or BDL. (B) Livers from transgenic mice after paraffin embedding and section underwent H&E, sirius red and immunohistochemical staining for α-SMA and COL1A1. Scale bars = 50 µm. (C-E) Relative quantitative results of the regions of sirius red, α-SMA and COL1A1 positive staining. (F-G) ALT and AST were evaluated as indicators of liver function. (H) Statistic analysis showed the liver/body weight ratio. (I) Hepatic α-SMA expression in transgenic mice receiving sham operation or BDL was detected using immunoblotting. All data are represented as the means ± SD. ***, P<0.001; ****, P<0.0001. Abbreviations: H&E: Hematoxylin and eosin; α-SMA: α-smooth muscle actin; COL1A1: Collagen type 1; TRAF2: Tumor necrosis factor receptor-associated factor 2; BDL: Bile duct ligation; HSC: Hepatic stellate cell; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase.

Figure 5

TRAF2 promoted HSC activation, profibrogenic phenotype and glycolysis in vitro. (A) Schematic illustration of primary mouse HSCs cultured at specified time intervals. (B) Livers from normal mice underwent perfusion and enzyme digestion for isolating primary HSCs and culturing these cells in vitro for 3, 7, 14 days, and then determination of the transcriptional levels of TRAF2 and ACTA2 was done on the cultured HSCs. (C) Comparison of double immunofluorescence results of primary mouse HSCs cultured for 6 h, 3, 7 and 14 days. Fluorescent α-SMA was displayed in green and fluorescent TRAF2 was shown in red. Scale bars = 10 µm. (D) TRAF2, COL1A1, PDGFRβ and α-SMA levels in LX-2 cells cultured on Matrigel were detected using immunoblotting. (E) Transcript levels of TRAF2 and indicated fibrosis-related markers in LX-2 cells cultured on Matrigel. (F) Immunoblotting analyses of TRAF2, COL1A1, PDGFRβ and α-SMA protein levels in LX-2 cells with TRAF2 inhibition or not. (G) Immunoblotting analyses of TRAF2, COL1A1, PDGFRβ and α-SMA expression in LX-2 cells with TRAF2 overexpression or not. (H) KEGG pathway enrichment analysis based on the identified proteins which were differentially expressed in LX-2 cells. (I) Flow cytometry analyses of the cell uptake of 2-NBDG. (J) The cell uptake of 2-NBDG was determined using immunofluorescence. (K) The cell supernatants collected were processed to assess glucose concentration. (L) The cell supernatants collected were processed to examine lactate concentration. All data are represented as the means ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Abbreviations: HSC: Hepatic stellate cell; TRAF2: Tumor necrosis factor receptor-associated factor 2; α-SMA: α-smooth muscle actin; COL1A1: Collagen type 1; PDGFRβ: Platelet-derived growth factor receptor β; KEGG: Kyoto Encyclopedia of Genes and Genomes; 2-NBDG: 2-(N-(7-nitrobenzen-2-oxa-1, 3- diazol-4-yl) amino)-2-deoxyglucose.

Figure 6

TRAF2 activated HIF-1α-GLUT1 axis to induce HSC activation and profibrogenic phenotype. (A) Determination of transcript levels of glycolytic genes. (B) Immunoblotting analyses of protein levels of TRAF2 and GLUT1 after silencing TRAF2 in LX-2 cells. (C) Immunoblotting analyses of TRAF2 and GLUT1 expression in LX-2 cells overexpressing TRAF2 or not. (D) GLUT1 siRNA blocked elevated expression of COL1A1 and α-SMA caused by TRAF2 overexpression. (E) Immunoblotting analyses showed protein levels of TRAF2 and HIF-1α when vehicle or TRAF2 siRNA was administered into LX-2 cells. (F) Immunoblotting analyses of TRAF2 and HIF-1α protein levels in LX-2 cells overexpressing TRAF2 or not. (G) Immunoblotting analyses of nuclear and cytoplasm fraction of HIF-1α after TRAF2 knockdown. (H) LX-2 cells transfected with vector or TRAF2 plasmid for 48 h were collected for determining the transcriptional activity of HIF-1α. (I) Elevated protein level of GLUT1 by TRAF2 overexpression was reversed by HIF-1α siRNA. All data are represented as the means ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Abbreviations: TRAF2: Tumor necrosis factor receptor-associated factor 2; SLC2A1: Solute carrier family 2 member 1; HK2: Hexokinase 2; PKM2: Phosphofructokinase muscle isoform; GPI: Glucose-6-phosphate isomerase; LDHA: Lactate dehydrogenase A; PFKM: Phosphofructokinase muscle isoform; PFKL: Phosphofructokinase liver isoform; ALDOA: Fructose-bisphosphate aldolase A; PGK1: Phosphoglycerate kinase 1; PGAM1: Phosphoglycerate mutase 1; ENO1: Enolase 1; ENO2: Enolase 2; GLUT1: Glucose transporter 1; α-SMA: α-smooth muscle actin; COL1A1: Collagen type 1; HIF-1α: Hypoxia-inducible factor-1α.

Figure 7

TRAF2 upregulated HIF-1α levels by obstructing pVHL-dependent proteasomal degradation of HIF-1α and activating mTORC1-mediated increased HIF-1α translation. (A) Immunoblotting analyses of protein levels of hydroxylated HIF-1α in LX-2 cells transfected with or without TRAF2 siRNA for 48 h, followed by a 6 h-treatment of MG132 (10 µM). (B) Immunoblotting analyses of pVHL protein level in LX-2 cells silencing TRAF2 or not. (C) Determination of the relative mRNA level of pVHL in LX-2 cells silencing TRAF2 or not. (D) After a 6 h-treatment of MG132 (10 µM), IP was performed on LX-2 cells overexpressing TRAF2 or not using antibody against HIF-1α and protein A/G agarose. Precipitated proteins and whole-cell lysates were analyzed using indicated antibodies. (E) Immunoblotting analyses of HIF-1α protein level in LX-2 cells. (F) Representative bands of p-S6K and S6K in LX-2 cells silencing TRAF2 or not were determined by immunoblotting. (G) Representative bands of p-4E-BP1 and 4E-BP1 in LX-2 cells silencing TRAF2 or not were measured by immunoblotting. (H) Elevated protein levels of HIF-1α, p-S6K and p-4E-BP1 induced by TRAF2 overexpression were blocked by rapamycin. All data are represented as the means ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Abbreviations: TRAF2: Tumor necrosis factor receptor-associated factor 2; HIF-1α: Hypoxia-inducible factor-1α; pVHL: von Hippel-Lindau; Ub: Ubiquitin; S6K: p70 ribosomal S6 kinase; 4E-BP1: eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1.

Figure 8

Graphical abstract explained the mechanism of TRAF2 promoting HSC activation and liver fibrosis development. TRAF2 promoted HIF-1α translation by mediating mTORC1 signaling and increased HIF-1α protein stability by inhibiting its pVHL-dependent degradation. HIF-1α recognized the HRE of DNA after nuclear translocation, which facilitated the transcriptional expression of several glycolytic proteins, such as glucose transporter GLUT1. Enhanced GLUT1 expression and translocation to the PM facilitated glucose uptake of HSCs, which in turn promoted glycolysis and aggravated the development of liver fibrosis. Abbreviations: HSC: Hepatic stellate cell; TRAF2: Tumor necrosis factor receptor-associated factor 2; AKT: Serine/threonine kinase; mTOR: Mammalian target of rapamycin; mTORC1: mTOR Complex 1; HIF-1α: Hypoxia-inducible factor-1α; pVHL: von Hippel-Lindau; HRE: Hypoxia response element; GLUT1: Glucose transporter 1; DNA: Deoxyribonucleic acid; PM: Plasma membrane.