Signalling via the osteopontin and high mobility group box-1 axis drives the fibrogenic response to liver injury

Abstract

Objective: Liver fibrosis is associated with significant collagen-I deposition largely produced by activated hepatic stellate cells (HSCs); yet, the link between hepatocyte damage and the HSC profibrogenic response remains unclear. Here we show significant induction of osteopontin (OPN) and high-mobility group box-1 (HMGB1) in liver fibrosis. Since OPN was identified as upstream of HMGB1, we hypothesised that OPN could participate in the pathogenesis of liver fibrosis by increasing HMGB1 to upregulate collagen-I expression.

Design and results: Patients with long-term hepatitis C virus (HCV) progressing in disease stage displayed enhanced hepatic OPN and HMGB1 immunostaining, which correlated with fibrosis stage, whereas it remained similar in non-progressors. Hepatocyte cytoplasmic OPN and HMGB1 expression was significant while loss of nuclear HMGB1 occurred in patients with HCV-induced fibrosis compared with healthy explants. Well-established liver fibrosis along with marked induction of HMGB1 occurred in CCl₄-injected Opn^Hep transgenic yet it was less in wild type and almost absent in Opn^-/- mice. Hmgb1 ablation in hepatocytes (Hmgb1^ΔHep) protected mice from CCl₄-induced liver fibrosis. Coculture with hepatocytes that secrete OPN plus HMGB1 and challenge with recombinant OPN (rOPN) or HMGB1 (rHMGB1) enhanced collagen-I expression in HSCs, which was blunted by neutralising antibodies (Abs) and by Opn or Hmgb1 ablation. rOPN induced acetylation of HMGB1 in HSCs due to increased NADPH oxidase activity and the associated decrease in histone deacetylases 1/2 leading to upregulation of collagen-I. Last, rHMGB1 signalled via receptor for advanced glycation end-products and activated the PI3K-pAkt1/2/3 pathway to upregulate collagen-I.

Conclusions: During liver fibrosis, the increase in OPN induces HMGB1, which acts as a downstream alarmin driving collagen-I synthesis in HSCs.

Keywords: BASIC SCIENCES; HEPATIC FIBROSIS; HEPATIC STELLATE CELL; HEPATOCYTE; SIGNALING.

Figure 1

Osteopontin (OPN) and high-mobility group box-1 (HMGB1) colocalise and their expression correlates with fibrosis progression in patients with chronic HCV-induced fibrosis. H&E staining, OPN and HMGB1 immunohistochemistry (IHC) in paraffin-embedded archived human liver biopsies from a deidentified control and from a patient with clinically proven hepatitis C virus (HCV)-induced fibrosis that did not progress in disease stage (stage 2) over 3 years show similar expression of OPN (yellow arrows, insets) and HMGB1 (green arrows, insets) (A). H&E staining, OPN and HMGB1 IHC from a patient with clinically proven HCV-induced fibrosis that progressed from stage 2 to 3 in 3 years show increased expression of OPN (yellow arrows, insets) and HMGB1 (green arrows, insets) (B). Total OPN and HMGB1 morphometry analysis according to fibrosis stage. Results are expressed as fold-change of the healthy liver explants, which are assigned a value of 1; n=10/group, ***p<0.001 for stages 1, 2, 3 or 4 vs 0 (C). Immunofluorescence shows colocalisation of OPN (red) and HMGB1 (green) in a patient with chronic HCV-induced fibrosis at stage 3 (D). DAPI, 4′,6-diamidino-2-phenylindole; IOD, integrated optical density.

Figure 2

Osteopontin (OPN) and high-mobility group box-1 (HMGB1) colocalise and their expression correlates in carbon tetrachloride (CCl₄)-induced liver injury in mice. Wild-type (WT), Opn^−/− and Opn^Hep Tg mice were injected with mineral oil (MO) or CCl₄ for 1 month. OPN (A) and HMGB1 (B, top) immunohistochemistry (IHC) and morphometry analysis in livers from CCl₄-injected mice show increased OPN (yellow arrows, insets) along with HMGB1 (green arrows, insets) expression, which is greater in CCl₄-injected Opn^Hep Tg than in WT and less in Opn^−/− mice. Western blot analysis for HMGB1 in livers from MO-injected WT and CCl₄-injected WT, Opn^−/− and Opn^Hep Tg mice. The results from the western blot analysis are corrected by calnexin (loading control) (B, middle). Quantification of nuclear, cytoplasmic, nuclear-to-total and cytoplasmic-to-total HMGB1 expression (B, bottom). Immunofluorescence shows colocalisation of OPN and HMGB1 as well as induction in CCl₄-injected WT mice (C, top), which was quantified by morphometry (C, bottom). Immunofluorescence demonstrates colocalisation of OPN and HMGB1 with HNF4α (hepatocyte marker, nuclear) along with induction by CCl₄ treatment (D, top and middle). There is also colocalisation of HMGB1 with desmin (hepatic stellate cell (HSC) marker, cytoplasmic) along with induction by CCl₄ treatment (D, bottom). Collagen-I IHC and morphometry assessment in livers from MO-injected or CCl₄-injected WT, Opn^−/− and Opn^Hep Tg mice (E). In all panels, the results are expressed as fold-change of the WT mice injected MO, which are assigned a value of 1 and are mean values±SEM; n=8/group. *p<0.05, **p<0.01 and ***p<0.001 for CCl₄-injected mice versus MO-injected mice; ^●p<0.05, ^●●p<0.01 and ^●●●p<0.001 for Opn^Hep Tg or Opn^−/− versus WT mice. CV, central vein; DAPI, 4′,6-diamidino-2-phenylindole; IOD, integrated optical density; PV, portal vein.

Figure 2

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Figure 3

Hmgb1 ablation in hepatocytes partially prevents carbon tetrachloride (CCl₄)-induced liver fibrosis in mice. Hmgb1^ΔHep and control littermates were injected mineral oil (MO) or CCl₄ for 1 month. H&E staining (A, top), the pathology scores (A, middle) and serum alanine aminotransferase (ALT) plus aspartate aminotransferase (AST) activities (A, bottom) show lower necrosis, inflammation, hepatocyte ballooning degeneration and fibrosis in Hmgb1^ΔHep compared with control littermates. High-mobility group box-1 (HMGB1); green arrows, insets and collagen-I immunohistochemistry (IHC) and morphometry analysis show reduced HMGB1 and collagen-I deposition in livers from CCl₄-injected Hmgb1^ΔHep compared with control littermates. IHC and western blot analysis demonstrate similar expression of osteopontin (OPN) and receptor for advanced glycation end-products (RAGE) in these mice (B). The results are expressed as fold-change of the MO-injected control littermates, which are assigned a value of 1 and are mean values±SEM; n=8/group. *p<0.05, **p<0.01 and ***p<0.001 for CCl₄-injected versus MO-injected mice; ^●p<0.05, ^●●p<0.01 and ^●●●p<0.001 for Hmgb1^ΔHep versus control littermates. CV, central vein; IOD, integrated optical density; PV, portal vein; WT, wild type.

Figure 3

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Figure 4

Osteopontin (OPN) is also upstream of high-mobility group box-1 (HMGB1) in hepatic stellate cells (HSCs) and they both regulate collagen-I expression in an autocrine fashion in vitro. Primary HSCs from wild-type (WT) and Opn^−/− mice were cultured for 5 days. Western blot analysis of intracellular OPN, HMGB1 and collagen-I plus extracellular collagen-I expression (A, left). Rat HSCs were infected with Ad-LacZ or Ad-OPN for 48 h. Western blot analysis of intracellular OPN and HMGB1 in rat HSCs infected with Ad-LacZ or Ad-OPN (A, right). Mouse embryonic skin fibroblasts (MEFs) from WT and Hmgb1^−/− mice were cultured for 1 day. Western blot analysis of intracellular OPN, HMGB1 and collagen-I plus extracellular collagen-I expression (B). Hepatocytes are a major source of OPN and HMGB1 signalling to HSCs to increase collagen-I production. Primary rat HSCs were cultured alone for 5 days and then cocultured with primary hepatocytes from mineral oil (MO)-treated or CCl₄-treated Opn^−/−, Hmgb1^ΔHep and their matching control littermates for 1 day in the presence or absence of non-immune IgG or a neutralising antibody (Ab) to HMGB1 or OPN, respectively. Western blot analysis of intracellular and extracellular collagen-I is shown (C). In all panels, the results are corrected by the specific loading control and are expressed as fold-change of the control, which are assigned a value of 1 and are mean values±SEM; n=3/group. Experiments were performed in triplicate four times. *p<0.05, **p<0.01 and ***p<0.001 for Ad-OPN or CCl₄ versus Ad-LacZ or MO; ^●p<0.05, ^●●p<0.01 and ^●●●p<0.001 for Opn^−/−, Hmgb1^−/−, HMGB1 Ab or OPN Ab versus WT or IgG; °p<0.05, °°p<0.01 and °°°p<0.001 for the Opn^−/− and Hmgb1^ΔHep coculture versus the WT and the control littermate cocultures.

Figure 5

Recombinant osteopontin (rOPN) induces high-mobility group box-1 (HMGB1) expression and translocation in hepatic stellate cells (HSCs) and drives the increase in collagen-I production. Primary rat HSCs cultured for 4 days (quiescent) or 7 days (activated) were treated with rOPN for 6 h. Western blot analysis for intracellular HMGB1 and for intracellular plus extracellular collagen-I (A, left). Western blot analysis for HMGB1 in primary rat HSCs treated with 50 nM rOPN for 6 h in the presence or absence of 100 µM cycloheximide (A, right). Primary rat HSCs cultured for 7 days were treated with rOPN for 6 h. Western blot analysis of nuclear plus cytoplasmic HMGB1 and intracellular collagen-I (B). In (A and B), the results are expressed as fold-change of the corresponding control, which are assigned a value of 1 if signal is present and are mean values±SEM; n=3/group in experiments performed in triplicate four times. *p<0.05, **p<0.01 and ***p<0.001 for rOPN versus control; ^●●●p<0.001 for cycloheximide cotreated versus rOPN. Primary mouse HSCs treated with 50 nM rOPN for 6 h. Immunofluorescence analysis for HMGB1 (green) and collagen-I (red) (C). HMGB1 structure and schematic representation of the lysine residues targeted in the HMGB1 constructs (D). Rat HSCs were transfected with a series of constructs driving HMGB1 localisation followed by 0–50 nM rOPN treatment for 6 h. The constructs were (1) pGFP, an empty vector as a negative control; (2) wild-type (WT).Hmgb1.GFP containing nuclear localisation signals 1 (NLS1) and NLS2 to overexpress HMGB1 and (3) Hmgb1.NLS1/2(8K→8A).GFP containing all eight lysines in the two NLS mutated to alanines, which cannot be acetylated and result in nuclear localisation. Immunofluorescence for collagen-I (yellow arrows) and GFP fluorescence (HMGB1 localization: white arrows point at nuclear HMGB1 and white arrowheads point at cytosolic HMGB1) were visualised by confocal microscopy (E) and quantified by morphometry assessment (F). In (F), the results are expressed as fold-change of the control WT.Hmgb1.GFP, which are assigned a value of 1 and are mean values±SEM; n=3/group in experiments performed in triplicate four times. *p<0.05, **p<0.01 and ***p<0.001 for rOPN versus control; ^●●p<0.01 and ^●●●p<0.001 for Hmgb1(8K→8A).GFP versus WT.Hmgb1.GFP. IOD, integrated optical density.

Figure 5

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Figure 6

Recombinant osteopontin (rOPN) activates NADPH oxidase (NOX) and inhibits histone deacetylases (HDACs) 1/2 promoting high-mobility group box-1 (HMGB1) acetylation and translocation along with collagen-I upregulation in hepatic stellate cells (HSCs). Rat HSCs were treated with rOPN for 6 h. Immunoprecipitation of intracellular HMGB1 and immunoblotting for acetylated lysines (A). Identification of the HMGB1 isoforms in HSCs lysates and in the cell culture medium. Spectra of whole protein electrospray ionisation–liquid chromatography–mass spectrometry of the HMGB1 isoforms. A schematic representation of each isoform is on each spectra (grey boxes); n=3/group (B). Rat HSCs were treated with rOPN for 2 h. Western blot analysis for PCAF and p300 (C). Rat HSCs were treated with rOPN for 1 and 2 h. Western blot analysis for HDACs1-6 (D). NOX activity in rat HSCs treated with rOPN for 6 h alone or pretreated for 0.5 h with apocynin or diphenyleneiodonium (DPI), the two NOX inhibitors. The percentage of dihydroethidium (DHE)-positive cells was measured by flow cytometry as an indirect measurement of O₂^.− production (E). Rat HSCs were treated with rOPN for 6 h in the presence or absence of apocynin or DPI. Western blot analysis of HDACs1/2 along with intracellular and extracellular collagen-I (F). The results from the western blot analysis are corrected by the specific loading control and are expressed as fold-change of the controls, which are assigned a value of 1 and are mean values±SEM; n=3/group in experiments performed in triplicate four times. *p<0.05, **p<0.01 and ***p<0.001 for rOPN versus control; •p<0.05 and ••p<0.01 for cotreated versus rOPN. HDACs, histone deacetylases.

Figure 6

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Figure 7

rHMGB1 signals via receptor for advanced glycation end-products (RAGE) to upregulate collagen-I expression through the PI3K–pAkt1/2/3 pathway in hepatic stellate cells (HSCs). Rat HSCs were treated with 50 nM rHMGB1 for 6 h. Western blot analysis for intracellular plus extracellular OPN and collagen-I (A). HSCs were challenged with rHMGB1 up to 6 h and western blot analysis was performed for PI3K, pAkt1/2/3 and Akt1/2/3 (B). Western blot for pAkt1/2/3, pAkt1/2/3 and intracellular plus extracellular collagen-I in HSCs treated with rHMGB1 in the presence or absence of wortmannin or LY294002 (two PI3K inhibitors) (C). Rage ablation was performed in HSCs by transduction with shRNA lentiviral particles and isolation of stable clones expressing the shRNA via puromycin dihydrochloride selection. Cells were treated with rHMGB1 for 6 h followed by western blot analysis for RAGE, PI3K, pAkt1/2/3, Akt1/2/3 intracellular and extracellular collagen-I (D). The results from the western blot analysis are corrected by the specific loading control and are expressed as fold-change of the controls, which are assigned a value of 1 and are given as mean values±SEM; n=3/group in experiments performed in triplicate four times. *p<0.05, **p<0.01 and ***p<0.001 for rHMGB1 versus control; •p<0.05, ••p<0.01 and •••p<0.001 for cotreated or Rage ablated versus rHMGB1 or Gfp. Wild-type (WT) mice were injected CCl₄ for 1 month along with non-immune IgG or RAGE neutralising Ab. H&E staining and collagen-I immunohistochemistry (IHC) and morphometry analysis showing that neutralisation of RAGE protects mice from liver fibrosis (E). The results are expressed as fold-change of the IgG group, which are assigned a value of 1 and are given as mean values±SEM; n=3/group. ••p<0.01 for RAGE Ab versus IgG. CV, central vein; PV, portal vein.