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Cited by 112Liver Disease Diagnosis and TreatmentLiver physiology and pathologyLiver Diseases and Immunity

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The transcription factor c-Jun/AP-1 promotes liver fibrosis during non-alcoholic steatohepatitis by regulating Osteopontin expression

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Schulien, Isabel, Hockenjos, Birgit, Schmitt-Graeff, Annette, Perdekamp, Markus Große, Follo, Marie, Thimme, Robert, Hasselblatt, Peter·2019·Cell Death & Differentiation
DOI 10.1038/s41418-018-0239-8PMC PMC6748141
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Abstract

Progression of non-alcoholic fatty liver disease (NAFLD) from steatosis to non-alcoholic steatohepatitis (NASH) is a key step of NASH pathogenesis. The AP-1 transcription factor c-Jun is an important regulator of hepatic stress responses, but its contribution to NASH pathogenesis remains poorly defined. We therefore addressed c-Jun expression in liver biopsies of patients with steatosis and NASH. The role of c-Jun during NASH pathogenesis was analyzed mechanistically in c-Jun mutant mice fed with a methionine- and choline-deficient diet (MCDD). Disease progression from steatosis to NASH in patients correlated with increased c-Jun expression in hepatocytes, while its expression in non-parenchymal liver cells (NPLCs) particularly correlated with fibrosis. Analysis of untreated and MCDD-fed mice lacking c-Jun in hepatocytes ( c-Jun ∆li ) revealed that c-Jun promotes hepatocyte survival, thereby protecting against the regenerative ductular reaction (DR) of Sox9/Osteopontin (Opn) co-expressing NPLCs, expression of the Opn receptor CD44 and fibrosis, which were all exacerbated in c-Jun ∆li mice. Since Opn and c-Jun were co-expressed by NPLCs in mice and patients with NASH, we wondered whether the increased fibrosis observed in c-Jun ∆li mice could be rescued by additional c-Jun deletion in NPLCs ( c-Jun ∆li* ). c-Jun ∆li* mice with NASH indeed exhibited reduced expression of Opn and CD44 in NPLCs, impaired DR and reduced fibrosis. A similar phenotype was observed in Opn knockout mice, suggesting that the observed functions of c-Jun were indeed Opn-dependent. In conclusion, c-Jun expression correlates with disease progression from steatosis to NASH in patients and exerts cell-type-specific functions in mice: In hepatocytes, it promotes cell survival thereby limiting the DR and fibrogenesis. In NPLCs, it rather promotes the DR and fibrogenesis by regulating expression of Opn and CD44.

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Figures

Fig. 1 c-Jun expression correlates with disease progression from NAFL to NASH in humans and mice. a Representative immunohistochemistry for c-Jun of human liver biopsies from healthy controls, patients with NAFL or NASH. b c-Jun expression in hepatocytes and NPLCs of NAFL (n = 9) and NASH patients (n = 27) was quantified. c Hepatocellular c-Jun expression was correlated with the NAS (c, left panel) and fibrosis stage (c, right panel). d c-Jun expression in NPLCs was correlated with the NAS (d, left panel) and fibrosis stage (d, right panel). e Representative immunohistochemistry for c-Jun of livers from mice treated with a CD, HFD or MCDD. f c-Jun expression in hepatocytes and NPLCs of HFD- (n = 4) and MCDD-fed mice (n = 5) was quantified. Significance was tested by Mann–Whitney test and correlation was tested by Pearson correlation coefficient. p values are indicated if significant. Scale bar = 50 µm. Hepatocytes and NPLCs are depicted by blue and red arrows, respectively
Fig. 1 c-Jun expression correlates with disease progression from NAFL to NASH in humans and mice. a Representative immunohistochemistry for c-Jun of human liver biopsies from healthy controls, patients with NAFL or NASH. b c-Jun expression in hepatocytes and NPLCs of NAFL (n = 9) and NASH patients (n = 27) was quantified. c Hepatocellular c-Jun expression was correlated with the NAS (c, left panel) and fibrosis stage (c, right panel). d c-Jun expression in NPLCs was correlated with the NAS (d, left panel) and fibrosis stage (d, right panel). e Representative immunohistochemistry for c-Jun of livers from mice treated with a CD, HFD or MCDD. f c-Jun expression in hepatocytes and NPLCs of HFD- (n = 4) and MCDD-fed mice (n = 5) was quantified. Significance was tested by Mann–Whitney test and correlation was tested by Pearson correlation coefficient. p values are indicated if significant. Scale bar = 50 µm. Hepatocytes and NPLCs are depicted by blue and red arrows, respectivelyCC BY · PubMed Central
Fig. 2 Increased fibrosis in MCDD-fed c-Jun∆li mice. a, b Representative immunohistochemistry for c-Jun following 49 days of MCDD feeding (a) and hematoxylin and eosin (H&E) stainings following 0 and 49 days of MCDD feeding (b) of livers from mice with the indicated genotypes. Nuclei of hepatocytes and NPLCs are depicted by blue and red arrows, respectively. c Cells with caspase 3 cleavage were assessed by immunofluorescence and quantified (n = 5-10/genotype and timepoint). d Hepatic mRNA expression of the indicated genes following 49 days of MCDD (n = 5-8/genotype). mRNA expression is shown relative to untreated controls. e Quantification and representative Sirius Red stainings following 49 days of MCDD (left, n = 7/genotype). Significance was tested by Mann–Whitney test. P-values are indicated if significant. Scale bar = 50 µm
Fig. 2 Increased fibrosis in MCDD-fed c-Jun∆li mice. a, b Representative immunohistochemistry for c-Jun following 49 days of MCDD feeding (a) and hematoxylin and eosin (H&E) stainings following 0 and 49 days of MCDD feeding (b) of livers from mice with the indicated genotypes. Nuclei of hepatocytes and NPLCs are depicted by blue and red arrows, respectively. c Cells with caspase 3 cleavage were assessed by immunofluorescence and quantified (n = 5-10/genotype and timepoint). d Hepatic mRNA expression of the indicated genes following 49 days of MCDD (n = 5-8/genotype). mRNA expression is shown relative to untreated controls. e Quantification and representative Sirius Red stainings following 49 days of MCDD (left, n = 7/genotype). Significance was tested by Mann–Whitney test. P-values are indicated if significant. Scale bar = 50 µmCC BY · PubMed Central
Fig. 3 Increased DR in MCDD-fed c-Jun∆li mice. a The number of CD3-, F4/80-, Ly6G-, and NKp46-positive cells was assessed by immunohistochemistry (n = 4-16/genotype and timepoint). b Quantification of Ki67-positive NPLCs by immunohistochemistry (n = 4–9/genotype and timepoint). c Sox9-positive cells were assessed by immunohistochemistry (n = 5–15/genotype and timepoint). d–f Hepatic expression of Opn and of its receptor CD44 was determined by qPCR (d and e, n = 4–8/genotype and timepoint) and immunohistochemistry (f, representative stainings following 49 days of MCDD). mRNA expression is shown relative to untreated controls. Significance was tested by Mann–Whitney test. P-values are indicated if significant. Scale bar = 50 µm
Fig. 3 Increased DR in MCDD-fed c-Jun∆li mice. a The number of CD3-, F4/80-, Ly6G-, and NKp46-positive cells was assessed by immunohistochemistry (n = 4-16/genotype and timepoint). b Quantification of Ki67-positive NPLCs by immunohistochemistry (n = 4–9/genotype and timepoint). c Sox9-positive cells were assessed by immunohistochemistry (n = 5–15/genotype and timepoint). d–f Hepatic expression of Opn and of its receptor CD44 was determined by qPCR (d and e, n = 4–8/genotype and timepoint) and immunohistochemistry (f, representative stainings following 49 days of MCDD). mRNA expression is shown relative to untreated controls. Significance was tested by Mann–Whitney test. P-values are indicated if significant. Scale bar = 50 µmCC BY · PubMed Central
Fig. 4 Co-expression of c-Jun with the HPC markers Sox9, CK-19 and Opn. a Representative double-immunofluorescence stainings for Opn (green) and Sox9 (red) (upper panel) or αSMA (red) (lower panel) of livers from mice with the indicated genotypes following 49 days of MCDD feeding. Double-positive cells are indicated by red arrows. b, c Representative immunohistochemistry of OPN (brown) and c-Jun (red) double-positive cells of mice of the indicated genotypes following 49 days of MCDD feeding (b) or liver sections of patients with NASH (c). d, e Representative immunohistochemistry of c-Jun (d = brown, e = red) and Sox9 (d = red) or CK-19 (e = brown) double-positive cells of mice of the indicated genotypes following 49 days of MCDD feeding. Double-positive hepatocytes and NPLCs are indicated by blue and red arrows, respectively. Single positive cells are indicated by black arrows. Scale bar (immunofluorescence) = 25 µm. Scale bar (immunohistochemistry) = 10 µm
Fig. 4 Co-expression of c-Jun with the HPC markers Sox9, CK-19 and Opn. a Representative double-immunofluorescence stainings for Opn (green) and Sox9 (red) (upper panel) or αSMA (red) (lower panel) of livers from mice with the indicated genotypes following 49 days of MCDD feeding. Double-positive cells are indicated by red arrows. b, c Representative immunohistochemistry of OPN (brown) and c-Jun (red) double-positive cells of mice of the indicated genotypes following 49 days of MCDD feeding (b) or liver sections of patients with NASH (c). d, e Representative immunohistochemistry of c-Jun (d = brown, e = red) and Sox9 (d = red) or CK-19 (e = brown) double-positive cells of mice of the indicated genotypes following 49 days of MCDD feeding. Double-positive hepatocytes and NPLCs are indicated by blue and red arrows, respectively. Single positive cells are indicated by black arrows. Scale bar (immunofluorescence) = 25 µm. Scale bar (immunohistochemistry) = 10 µmCC BY · PubMed Central
Fig. 5 Expression analysis of CD44-positive NPLCs. a Representative double-immunofluorescence stainings for CD44 (green) and Sox9 (red) (upper panel) or αSMA (red) (lower panel) of livers from mice with the indicated genotypes following 49 days of MCDD feeding. b, c Representative immunohistochemistry of CD44 (red) and CD3 (b = brown) or c-Jun (c = brown) double-positive cells of livers from mice with the indicated genotypes following 49 days of MCDD feeding. Double-positive cells are indicated by red arrows. Scale bar (immunofluorescence) = 25 µm. Scale bar (immunohistochemistry) = 10 µm
Fig. 5 Expression analysis of CD44-positive NPLCs. a Representative double-immunofluorescence stainings for CD44 (green) and Sox9 (red) (upper panel) or αSMA (red) (lower panel) of livers from mice with the indicated genotypes following 49 days of MCDD feeding. b, c Representative immunohistochemistry of CD44 (red) and CD3 (b = brown) or c-Jun (c = brown) double-positive cells of livers from mice with the indicated genotypes following 49 days of MCDD feeding. Double-positive cells are indicated by red arrows. Scale bar (immunofluorescence) = 25 µm. Scale bar (immunohistochemistry) = 10 µmCC BY · PubMed Central
Fig. 6 c-Jun promotes Opn expression in NPLCs and the subsequent DR. a Representative immunohistochemical stainings for c-Jun, OPN and CD44 of liver sections from mice with the indicated genotype following 49 days of MCDD. b Hepatic Opn and Cd44 mRNA expression (n = 5–13/genotype and timepoint). mRNA expression is shown relative to untreated controls. c Caspase 3 cleavage was assessed by immunofluorescence (n = 4-10/genotype and timepoint). d Serum ALT concentrations (n = 4–12/genotype and timepoint). e Quantification of Sox9-positive cells by immunohistochemistry (left, n = 5–13/genotype and timepoint). Representative double-immunofluorescence stainings for Sox9 (red) and Opn (green) of livers from mice with the indicated genotype following 49 days of MCDD feeding are shown (right). Double-positive cells are indicated by red arrows. Significance was tested by Mann–Whitney test. P-values are indicated if significant. Scale bar (immunofluorescence) = 25 µm. Scale bar (immunohistochemistry) = 50 µm
Fig. 6 c-Jun promotes Opn expression in NPLCs and the subsequent DR. a Representative immunohistochemical stainings for c-Jun, OPN and CD44 of liver sections from mice with the indicated genotype following 49 days of MCDD. b Hepatic Opn and Cd44 mRNA expression (n = 5–13/genotype and timepoint). mRNA expression is shown relative to untreated controls. c Caspase 3 cleavage was assessed by immunofluorescence (n = 4-10/genotype and timepoint). d Serum ALT concentrations (n = 4–12/genotype and timepoint). e Quantification of Sox9-positive cells by immunohistochemistry (left, n = 5–13/genotype and timepoint). Representative double-immunofluorescence stainings for Sox9 (red) and Opn (green) of livers from mice with the indicated genotype following 49 days of MCDD feeding are shown (right). Double-positive cells are indicated by red arrows. Significance was tested by Mann–Whitney test. P-values are indicated if significant. Scale bar (immunofluorescence) = 25 µm. Scale bar (immunohistochemistry) = 50 µmCC BY · PubMed Central
Fig. 7 c-Jun expression in NPLCs promotes fibrosis by regulating Opn expression. a Hepatic mRNA expression of the depicted genes following 49 days of MCDD (n = 5–13/genotype). mRNA expression is shown relative to untreated controls. b Quantification of Sirius red-stained area following 49 days of MCDD (left panel; n = 11–12/genotype) and representative stainings (right panel). c Serum ALT concentrations of mice with the indicated genotypes treated with CD or MCDD (n = 4–8/genotype and timepoint). d Quantification of Sox9-positive cells by immunohistochemistry (n = 4–10/genotype and timepoint). e Quantification of the Sirius red-positive area following 49 days of MCDD (n = 5–6/genotype). Significance was tested by Mann–Whitney test. P-values are indicated. Scale bar = 50 µm
Fig. 7 c-Jun expression in NPLCs promotes fibrosis by regulating Opn expression. a Hepatic mRNA expression of the depicted genes following 49 days of MCDD (n = 5–13/genotype). mRNA expression is shown relative to untreated controls. b Quantification of Sirius red-stained area following 49 days of MCDD (left panel; n = 11–12/genotype) and representative stainings (right panel). c Serum ALT concentrations of mice with the indicated genotypes treated with CD or MCDD (n = 4–8/genotype and timepoint). d Quantification of Sox9-positive cells by immunohistochemistry (n = 4–10/genotype and timepoint). e Quantification of the Sirius red-positive area following 49 days of MCDD (n = 5–6/genotype). Significance was tested by Mann–Whitney test. P-values are indicated. Scale bar = 50 µmCC BY · PubMed Central

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Metadata

Authors
Schulien, I., Hockenjos, B., Schmitt-Graeff, A., Perdekamp, M. G., Follo, M., Thimme, R., & Hasselblatt, P.
Affiliations
University of Freiburg (DE)
Corresponding author
Hasselblatt, Peter
ORCID
Venue
Cell Death & Differentiation
Publisher
Springer Science and Business Media LLC
Published
2019-02-18
Volume
26
Issue
9
Pages
1688-1699
ISSN
  • 1350-9047 (ISSN-L)
  • 1476-5403
DOI
10.1038/s41418-018-0239-8
PMC
PMC6748141
OpenAlex
W2913699817
Cited by
111
References
43
Citations by year
2026: 12 · 2025: 26 · 2024: 16 · 2023: 12 · 2022: 11 · 2021: 24 · 2020: 9 · 2019: 1
FWCI
8.56
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Death and Differentiation (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
Deutsche Forschungsgemeinschaft (Ha4314/2-3)
Topics
  • Health Sciences › Medicine › Hepatology › Liver Disease Diagnosis and Treatment
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
  • Health Sciences › Medicine › Hepatology › Liver Diseases and Immunity
Keywords
c-Jun, AP-1 transcription factor, nonalcoholic steatohepatitis, liver fibrosis, osteopontin, CD44, non-parenchymal liver cells, hepatocyte viability, steatosis, methionine-choline-deficient diet, SOX9
MeSH terms
Animals, Diet, Humans, Liver Cirrhosis, Male, Mice, Inbred C57BL, Regeneration, Proto-Oncogene Proteins c-jun, Mice, Knockout, Disease Progression, Hyaluronan Receptors, Hepatocytes, Cell Proliferation, Mice, Osteopontin, Non-alcoholic Fatty Liver Disease

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