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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

The Activator Protein 1 (AP-1) transcription factor subunit Fos-related antigen 1 (Fra-1) has been implicated in liver fibrosis. Here we used loss-of-function as well as switchable, cell type-specific, gain-of-function alleles for Fra-1 to investigate the relevance of Fra-1 expression in cholestatic liver injury and fibrosis. Our results indicate that Fra-1 is dispensable in three well-established, complementary models of liver fibrosis. However, broad Fra-1 expression in adult mice results in liver fibrosis, which is reversible, when ectopic Fra-1 is switched off. Interestingly, hepatocyte-specific Fra-1 expression is not sufficient to trigger the disease, although Fra-1 expression leads to dysregulation of fibrosis-associated genes. Both opn and cxcl9 are controlled by Fra-1 in gain-of-function and loss-of-function experiments. Importantly, Fra-1 attenuates liver damage in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine-feeding cholestatic liver injury model. Strikingly, manipulating Fra-1 expression affects genes involved in hepatic transport and detoxification, in particular glutathione S-transferases. Molecular analyses indicate that Fra-1 binds to the promoters of cxcl9 and gstp1 in vivo. Furthermore, loss of Fra-1 sensitizes, while hepatic Fra-1 expression protects from acetaminophen-induced liver damage, a paradigm for glutathione-mediated acute liver failure. Conclusion: These data define a novel function of Fra-1/AP-1 in modulating the expression of detoxification genes and the adaptive response of the liver to bile acids/xenobiotic overload. (Hepatology 2014;58:261–273)

Abbreviations
ALP

alkaline phosphatase

ALT

alanine aminotransferase

AP-1

Activator Protein 1

APAP

acetaminophen

BDL

bile duct ligation

CCl4

carbon tetrachloride

cDNA

complementary DNA

ChIP

chromatin immunoprecipitation

CK19

cytokeratin 19

Cxcl9

chemokine (C-X-C motif) ligand 9

DDC

3,5-diethoxycarbonyl-1,4-dihydrocollidine

Dox

doxycycline

ERK

extracellular signal-regulated kinases

ES

embryonic stem

Fra-1

Fos-related antigen 1

GGT

gamma glutamyl transferase

GSH

reduced glutathione

IHC

immunohistochemistry

JNK

Jun N-terminal kinases

OPN

osteopontin

The liver performs a wide range of functions including nutrient synthesis, transformation and storage, as well as endogenous and exogenous substance detoxification. Studies using genetically modified mice revealed essential functions of the dimeric transcription factor Activator Protein 1 (AP-1) in controlling liver development, homeostasis, and disease. For example, c-Jun is critical for hepatocyte proliferation and survival during liver development, regeneration, inflammation, and cancer.[1-4] Although the close homologs junb and jund are dispensable for liver homeostasis,[4, 5] JunD-deficient mice are sensitive to tumor necrosis factor alpha (TNF-α)-mediated hepatitis[6] and protected from carbon tetrachloride (CCl4)-induced liver fibrosis.[7] Furthermore, junb and jund can substitute for c-jun during fetal liver development.[8, 9]

In contrast, the functions of Fos proteins in liver physiology are less well defined. c-Fos, FosB, Fra-1, and Fra-2 form AP-1 complexes by association with Jun proteins. Genetic inactivation of single fos genes has no obvious effect on liver homeostasis (reviewed[10]) and the relevance of Fos proteins to liver disease in loss-of-function mouse models has not been reported. Interestingly, broad ectopic expression of Fra-1 or Fra-2 in transgenic mice resulted in increased bone mass, but also generalized fibrosis with predominant manifestation in the liver and lung.[11-14] In addition, Fra-1 transgenic mice (Fra-1Tg) developed an inflammation-associated ductular reaction preceding liver fibrosis, suggesting an involvement of Fra-1 in cholestatic liver disease.[14]

Hepatic fibrosis is the final common endpoint of most chronic liver diseases. We set out to define the contribution of Fra-1/AP-1 to the pathogenesis of cholestasis and hepatic fibrosis using Fra-1 loss-of-function mice and novel mouse models with switchable ectopic Fra-1 alleles. Our results indicate that Fra-1 is dispensable for liver fibrosis in three independent experimental models. However, broad Fra-1 expression results in reversible periportal liver fibrosis affecting opn and cxcl9 expression in hepatocytes. Strikingly, these experiments revealed that Fra-1 modulates the expression of genes involved in xenobiotic detoxification and that Fra-1/AP-1 is relevant for acetaminophen (APAP)-induced liver failure. Thus, we identify Fra-1/AP-1 as a novel regulator of the detoxification function of the liver.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

For more details, see the Supporting Information.

Mouse Lines and Treatment

All mouse experiments were performed in accordance with local and institutional regulations. The tet-switchable Fosl1 or Fosl2 alleles were generated according to a previous study.[15] The Fra-1Δembryo and the c-JunΔli* mouse lines are described elsewhere.[3, 16] doxycycline (Dox)- or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-chow was supplied ad libitum and CCl4 or APAP injected intraperitoneally.

Blood and Histological Analyses

Blood parameters and serum cytokines were measured using commercially available kits. Histology and immunohistochemistry (IHC) were performed on paraffin sections. Antibodies are listed in the Supporting Information.

Molecular and Cellular Analyses

Total RNA was extracted and complementary DNA (cDNA) synthesized using commercially available kits. Quantitative polymerase chain reaction (PCR) was performed using a SyberGreer-based kit on a Lightcycler. Immunoblots were performed according to a previous study.[17] Antibodies are described in the Supporting Information. Chromatin immunoprecipitation (ChIP) was performed as described.[4] Primary hepatocytes and embryonic fibroblasts were isolated according to a previous study.[3, 18] HuH-7 cells were cotransfected with a gstp1 reporter and Fra-1-expressing constructs and luciferase was quantified using a luminometer.[19, 20]

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information
Fra-1 Appears Dispensable in Experimental Mouse Models of Liver Fibrosis

Fra-1Δembryo mice are viable, overall healthy, and lack Fra-1, encoded by fosl1, in all tissues.[16] Adult mice were subjected to three models of liver injury, bile duct ligation (BDL) and DDC,[21] two paradigms of obstructive cholestasis and secondary biliary fibrosis, and CCl4, a model of postnecrotic parenchymal fibrosis.[22] In wild-type livers, Fra-1 was increased in all three models (Supporting Figs. 1A, 2A, 3A-C), although Fra-1Δembryo mice displayed an overall response comparable to control littermates. Liver histology revealed similar grades of fibrosis and/or ductular reaction, assessed by Sirius red staining (Fig. 1A; Supporting Fig. 1B) or cytokeratin 19 (CK19) IHC (Supporting Figs. 1C, 2B). No notable difference was observed in serum alanine aminotransferase (ALT) (Fig. 1B), organ weight, or other liver-related serum parameters (Supporting Figs. 1D, 2C). Quantitative reverse-transcription PCR (qRT-PCR) confirmed that most fibrosis and bile duct marker genes were similarly induced (Fig. 1C). c-Jun and all Fos proteins were induced in the liver upon BDL, DDC, and CCl4 (Fig. 1D), indicating that Fos proteins might compensate for the absence of Fra-1. Furthermore, Fra-2 was increased in untreated, BDL-, and to a lesser extent CCl4-treated, Fra-1Δembryo mutants, while c-Fos appeared higher upon DDC treatment (Fig. 1D). When analyzing fibrogenic genes in BDL-treated mice, decreased opn messenger RNA (mRNA) was observed in Fra-1-deficient livers (Supporting Fig. 1E). In addition, the anti-fibrogenic chemokine Cxcl9[23] was increased in the livers of mutant mice subjected to BDL or DDC (Fig. 1E) and circulating Cxcl9 was higher in Fra-1Δembryo mutants upon BDL (Fig. 1F).

image

Figure 1. fra-1 is dispensable in three experimental mouse models of liver fibrosis. (A) Sirius red staining of liver sections from Fra-1Δembryo mutants (fosl1Δ/Δ) and littermate controls (fosl1f/Δ) after BDL, DDC, or CCl4. Sections from sham-operated mice are shown for comparison. Scale bar = 500 μm. Quantification of fibrotic areas is shown on the right. n ≥ 5 per genotype, sham animals for each treatment were pooled. (B) Serum ALT in controls and Fra-1Δembryo mice after BDL, DDC, or CCl4. n ≥ 5 per genotype, sham animals were pooled. (C) qRT-PCR analysis of col1a1, col1a2, and ck19 expression in the liver of controls and Fra-1Δembryo mice after BDL, DDC, or CCl4. n ≥ 5 per genotype, sham animals were pooled. (D) Immunoblot analysis of Fra-2, c-Fos, FosB, and c-Jun in sham (sh), BDL, DDC, or CCl4-treated controls and Fra-1Δembryo livers. Tubulin and glyceraldehyde phosphate dehydrogenase (GAPDH) were used to control loading. (E) qRT-PCR analysis of cxcl9 expression in the liver of controls and Fra-1Δembryo mice after BDL, DDC, or CCl4. n ≥ 5 per genotype, sham animals were pooled. (F) Serum Cxcl9 in controls and Fra-1Δembryo mice determined by enzyme-linked immunosorbent assay (ELISA) after BDL, DDC, or CCl4. In the entire figure: BDL: 13 days, DDC: 17 days, and CCl4: 8 weeks.

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These data demonstrate that fosl1 inactivation does not significantly affect the extent of fibrotic reactions in three independent experimental models, possibly due to compensation by other Fos proteins, such as Fra-2. However, loss of Fra-1 impacts the hepatic expression of some fibrosis-associated genes upon BDL, such as opn and cxcl9.

Inducible Fra-1 Expression in Several Organs of Adult Mice Leads to Reversible Periportal Liver Fibrosis

We next assessed the consequences of ectopic Fra-1 expression using a novel switchable allele. A tetracycline-responsive element controlling a Flag-tagged fosl1 cDNA was targeted to the col1a1 locus in embryonic stem (ES) cells expressing a reverse tetracycline transactivator from the ROSA26 locus.[15] Correct transgene integration and Dox-inducible expression was confirmed in ES cells and double transgenic ROSA26::rtTA; Col1a1::TetOP-Fosl1 mice (here referred to as Fra-1tetON) by Southern blot and qRT-PCR (Supporting Fig. 4A-D). High levels of fra-1 overexpression were measured in several tissues upon Dox treatment (Fig. 2A). Ectopic Fra-1 was also detected by immunohistochemistry and western blots (Fig. 2C; Supporting Fig. 4E-F). In the liver, Fra-1 was expressed higher than in the previously generated Fra-1Tg animals (Supporting Fig. 4E) and similarly detectable in hepatocytes, cholangiocytes, and nonparenchymal liver cells (Fig. 2C; Supporting Fig. 5A). In addition, ectopic Fra-1 was efficiently shut down as early as 5 days after Dox removal (Fig. 2B,C; Supporting Fig. 5B) and mice that were not given Dox did not express the transgene (Supporting Fig. 4F), demonstrating a tight, Dox-dependent regulation of the ectopic Fosl1 allele.

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Figure 2. Fra-1tetON mice develop reversible liver fibrosis which is not observed in Fra-1hep-tetOFF mice. (A) qRT-PCR analysis of total (endogenous + ectopic) fra-1 mRNA expression in different organs of Fra-1tetON mice and control littermates after 9 days of Dox treatment. Control is set to 1. n = 2 per genotype. (B) Effect of Dox withdrawal on total fra-1 mRNA expression in Fra-1tetON and control mice. n = 2/group. Controls are set to 1. On: Dox supplied during 6 weeks. On-off: Dox supplied during 6 weeks then removed for 6 weeks. (C) Histological analysis of liver sections from Fra-1tetON mice and control littermates. On: Dox supplied during 6 weeks. On-off: Dox supplied during 6 weeks then removed for 6 weeks. Top: Fra-1 is detected by α-Fra-1 IHC in hepatocytes (black arrow) and cholangiocytes (red arrow). Middle: Bile ducts are visualized by α-cytokeratin-19 IHC (CK19). Bottom: Sirius red staining. Scale bar = 50 μM. (D) Serum ALP, albumin, cholesterol, and bile acids in controls and Fra-1tetON mice. On: Dox supplied during 6-8 weeks. n ≥ 11 per genotype. On-off: Dox supplied during 7 weeks then removed for ≥6 weeks. n = 6 per genotype. nd: values below detection limit (1 μMol). (E) Comparison of transgene expression in the liver between (Dox-treated 2-6 weeks) Fra-1tetON, and (1-month-old) Fra-1hep-tetOFF mice by qRT-PCR (Top, n ≥ 4 per genotype, controls set to 1 and pooled) and α-Flag Immunoblot (Bottom, actin used to control loading). (F) Histological analysis of liver sections from of 7 to 8-month-old Fra-1hep-tetOFF mice and control littermates. Top: α-Fra-1 IHC: Fra-1 is expressed in hepatocytes (black arrow) but not in cholangiocytes (red arrow) of Fra-1hep-tetOFF mice. Middle: Bile ducts (CK19). Bottom: collagen deposition (Sirius red). Scale bars = 50 μM. Representative pictures of n ≥ 3 per genotype are shown.

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Fra-1tetON mice lost weight and became lethargic after 6-8 weeks of Dox treatment and died or had to be euthanized (Supporting Fig. 5C,D). Splenomegaly, skeletal malformations, and sometime ascites were observed at necropsy (Supporting Fig. 5D and data no shown), while histological examination revealed periportal liver fibrosis, with collagen deposition and CK19, Fsp1, and Opn immunoreactivity (Fig. 2C; Supporting Fig. 6A). Increased Ki67-positive nuclei and immune infiltrates, in particular T and B cells, were observed around the portal tracts (Supporting Fig. 6A,B). These alterations were accompanied by higher serum bile acids and alkaline phosphatase (ALP) and decreased albumin and cholesterol (Fig. 2D). qRT-PCR revealed a significant increase in bile duct and fibrosis marker expression, in particular tgfb1 and pdgfc (Supporting Fig. 6C), consistent with previous observations.[14]

Importantly, the phenotype was largely reverted when mice were allowed to recover under a Dox-free diet. Mutant animals regained weight, serum parameters were normalized, and collagen deposition, CK19 immunoreactivity, and bile duct and fibrosis marker expression reverted to control and uninduced levels (Fig. 2C,D; Supporting Fig. 6D,E). This implies that continuous Fra-1 expression is necessary for the maintenance of the phenotype.

No Major Alterations in Liver Homeostasis Upon Ectopic Fra-1 Expression in Hepatocytes

To define the contribution of hepatocytes, we combined the tet-switchable Fosl1 allele with an LAP-tTA allele, where a tetracycline transactivator is controlled by the hepatocyte-specific cebpb promoter.[24] In double transgenic LAP::tTA; Col1a1::TetOP-Fosl1 mice, here referred to as Fra-1hep-tetOFF, transgene expression occurs when Dox is absent (tet-OFF).

High fra-1 RNA and protein expression was measured in mutant livers, while no ectopic fra-1 was detected in any other tissue tested (Fig. 2E; Supporting Fig. 7A,B). Fra-1 levels were consistently higher than in Fra-1tetON livers (Fig. 2E). Fra-1 expression was restricted to hepatocytes and notably absent in cholangiocytes (Fig. 2F). Interestingly, expression of the transgene since embryogenesis did not affect the viability of Fra-1hep-tetOFF mutants (Supporting Fig. 7C). Moreover, and irrespective of the timing of transgene induction, Fra-1hep-tetOFF mutants appeared healthy, with no consistent changes in body or organ weights (Supporting Fig. 7D). Histology revealed no signs of liver fibrosis (Fig. 2F), serum parameters were not altered, and bile duct and fibrosis marker genes, including tgfb1 and pdgfc, were overall unaffected (Supporting Fig. 7D,E). The only change was increased hepatic opn (Supporting Fig. 7E), albeit modest compared to Fra-1tetON mutants, indicating that Fra-1 likely modulates opn expression in hepatocytes. On the other hand, as pdgfc and tgfb1 are increased in Fra-1Tg bile ducts[14] and fibroblasts (Supporting Fig. 7F), ectopic Fra-1 expression in cholangiocytes and mesenchymal cells of the liver might contribute to increased fibrogenic cytokines in Fra-1Tg and Fra-1tetON livers.

Fra-1 Represses cxcl9 Expression in Hepatocytes

Hepatic cxcl9 mRNA and serum Cxcl9 were decreased in Fra-1tetON and Fra-1hep-tetOFF mutants (Fig. 3A,B), mirroring our observation in the Fra-1-deficient mice. AP-1 binding sites have been described in the human[25] and murine[26] cxcl9 promoters. ChIP analyses performed using whole liver extracts demonstrated that Fra-1 and c-Jun bind a cxcl9 promoter region containing the AP-1 sites (Fig. 3C,D). Furthermore, Fra-1 efficiently bound the same region in primary hepatocytes isolated from Fra-1tetON mice (Fig. 3E). Therefore, while mice overexpressing Fra-1 specifically in hepatocytes do not spontaneously develop liver fibrosis, Fra-1 affects the expression of a subset of fibrosis-associated genes, such as opn and cxcl9, and Fra-1/AP-1 regulate cxcl9 transcription by direct promoter binding.

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Figure 3. Fra-1 modulates cxcl9 expression in the liver. qRT-PCR analysis of cxcl9 expression in the liver (left) and serum Cxcl9 (right) in Fra-1tetON mice (A, Dox-treated 6-8 weeks) and Fra-1hep-tetOFF (B, 7-8 months old) mice compared to their respective control littermates. mRNA expression in controls was set to 1. n ≥ 6 per genotype. (C) Scheme of the murine cxcl9 promoter. The position of the putative AP-1 binding TRE elements is indicated relative to the transcription start site and the PCR amplicon is depicted in red. (D) ChIP analysis of the cxcl9 promoter in livers from untreated controls, Fra-1Δembryo, and c-JunΔli* mice using Fra-1, c-Jun antibody, or IgG. Input chromatin samples were run in parallel. Endpoint PCR for the fragment sketched in C is shown. (E) ChIP analysis of the cxcl9 promoter in primary hepatocytes from untreated (Off) or Dox-treated (On: 48 hours) controls and Fra-1tetON mice using Flag antibody or IgG. Input chromatin samples were run in parallel and percentage of amplification related to input for the fragment sketched in C was quantified by qPCR and plotted. n = 2. Note that due to the size of the chromatin fragments (around 500 bp) tested and the PCR strategy, ChIP analysis cannot discriminate between the four putative binding sites.

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Fra-1 Modulates the Adaptive Response to Bile Acid/Xenobiotic Overload

Fra-1hep-tetOFF mice were next subjected to the DDC model. After 12 days of treatment, control and mutant livers displayed histological signs of ductular reaction and periportal fibrosis (Fig. 4A). Interestingly, while body weight was comparable between the two genotypes, liver to body weight ratio was higher in Fra-1hep-tetOFF mutant mice and serum ALT was lower (Fig. 4B), indicating decreased liver damage. Nevertheless, fibrosis (Sirius red) and ductular reaction (CK19) appeared comparable to controls (Fig. 4A,C) and this was confirmed by qRT-PCR: the only notable changes were decreased cxcl9 and a striking increase in ggt expression (Fig. 4D).

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Figure 4. Response of Fra-1hep-tetOFF mice to DDC-induced liver injury. (A) Liver histology in Fra-1hep-tetOFF and control mice after 12 days of DDC treatment. Top: hematoxylin and eosin (H&E): arrows indicate characteristic porphyrin pigment plugs. Middle: Sirius red staining Bottom: CK19-IHC. Scale bar = 100 μM. (B) Body, relative liver weight, serum ALT and ALP in Fra-1hep-tetOFF and control mice after 12 days of DDC treatment. (C) Quantification of fibrotic areas in A (Sirius red). (D) qRT-PCR analysis of fibrosis and bile duct-associated genes in the livers of Fra-1hep-tetOFF and controls after 12 days of DDC treatment. Controls are set to 1. (E) Serum cholesterol and bile acids in Fra-1hep-tetOFF and control mice after 12 days of DDC treatment. n = 7 per genotype in the entire figure.

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Interestingly, decreased serum cholesterol and increased bile acids were observed in DDC-treated Fra-1hep-tetOFF mutants (Fig. 4E). Cholestatic liver injury is counteracted by hepatoprotective mechanisms including alterations in bile acid and xenobiotic transport and detoxification and gamma glutamyl transferase (GGT) is an enzyme of the gamma-glutamyl cycle, a pathway connected to xenobiotic detoxification. We therefore analyzed additional genes involved in xenobiotic metabolism. When wild-type mice were fed DDC, expression of hepatic bile acid uptake transporters decreased, while several efflux transporters and xenobiotic detoxification genes were up-regulated (Supporting Fig. 8A).[21] Similar alterations were observed upon BDL (Supporting Fig. 8B). In DDC-treated Fra-1hep-tetOFF mutants, uptake transporters were decreased compared to littermate controls, with slco1a4, slco1b2, and slc10a1 being the most significant (Fig. 5A). The most notably affected efflux transporter was abcb11, which was decreased, while abcc2 and abcg2 were increased (Supporting Fig. 9A). Detoxification enzymes were also affected, although no changes were observed at the protein level (Fig. 5A,B). Interestingly, untreated Fra-1hep-tetOFF mutants displayed lower slco1a1, slco1a4, and cyp2b10, unchanged efflux transporters and higher gsta1, gsta2, and gstp1 expression (Fig. 5C; Supporting Fig. 9B). Slco1a1, Cyp2b10, Gsta1/2, and Gstp1 protein levels were also affected (Fig. 5D). This indicates that ectopic expression of Fra-1 in hepatocytes affects the steady-state and DDC-regulated expression of genes involved in xenobiotic detoxification.

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Figure 5. Fra-1 affects the expression of a subset of hepatic transporters and detoxification genes. (A) mRNA expression of genes involved in various aspects of bile acid and xenobiotic metabolism in control and Fra-1hep-tetOFF mice after 12 days of DDC feeding. Controls are set to 1. n = 7 per genotype. (B) Slco1a1, Cyp2b10, Cyp2e1, Gsta1/2, and Gstp1 immunoblot in livers extracts of DDC-treated Fra-1hep-tetOFF mice. GAPDH was used to control loading. (C) Basal hepatic mRNA expression of genes involved in bile acid and xenobiotic metabolism in 6 to 8-month-old controls and Fra-1hep-tetOFF mice. Controls are set to 1. n ≥ 6/genotype. (D) Slco1a1, Cyp2b10, Cyp2e1, Gsta1/2, and Gstp1 immunoblot in livers extracts of 8-month-old Fra-1hep-tetOFF mice. GAPDH was used to control loading.

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Fra-1 Affects gsta1, gsta2, and gstp1 Expression and APAP-Induced Acute Liver Damage

We next assessed the relevance of our findings in the loss-of-function model. Transport and detoxification genes were comparable between untreated controls and mutant Fra-1Δembryo mice (Supporting Fig. 10A,B). However, three bile acid uptake transporters slco1a4, slco1b2, and slc10a1 and three canalicular efflux transporters abcb11, abcc2, and abcg2 were down-regulated in BDL-treated Fra-1Δembryo mutants (Fig. 6A; Supporting Fig. 10C). Gsta1, gata2, and gstp1 were also decreased (Fig. 6A) and this was further apparent at the protein level (Fig. 6B).

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Figure 6. Fra-1 affects xenobiotic metabolism under biliary stress conditions and binds the gstp1 promoter. (A) qRT-PCR analysis of hepatic transporters and detoxification enzymes in the livers of BDL-treated controls and mutant Fra-1Δembryo mice (13 days). Controls were set to 1. n ≥ 6/genotype. (B) Cyp2b10, Cyp2e1, Gsta1/2, and Gstp1 immunoblot in liver extracts from BDL-treated (13 days) control and mutant Fra-1Δembryo mice. GAPDH was used to control loading. (C) Scheme of the murine gstp1 promoter. The position of the putative AP-1 binding TRE element is indicated relative to the transcription start site and the PCR amplicon is depicted in red. (D) ChIP analysis of the gstp1 promoter in livers from untreated controls, Fra-1Δembryo, and c-JunΔli* mice using Fra-1, c-Jun antibody, or IgG. Input chromatin samples were run in parallel. Endpoint PCR for the fragment sketched in C is shown. (E) ChIP analysis of the gstp1 promoter in primary hepatocytes from untreated (Off) or Dox-treated (On: 48 hours) controls and Fra-1tetON mice using Flag antibody or IgG. Input chromatin samples were run in parallel and percentage of amplification related to input for the fragment sketched in C was quantified by qPCR and plotted. n = 2. (F) Reporter assay for a mouse gstp1 promoter fragment (−343/+1) in the presence of Fra-1, c-Jun∼Fra-1, JunB∼Fra-1, or JunD∼Fra-1 expression vectors in Huh-7 cells. Relative luminescence units (luciferase/renilla, Left). Empty expression vector was set to 1. Data represent mean ± SD of three independent experiments with technical triplicates for each. Comparable expression of Jun∼Fra-1 forced dimers was controlled by Fra-1 immunoblot. GAPDH was used to control loading (Right).

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The AP-1 binding site in the human GSTP1 promoter[27] is conserved in the mouse sequence (Fig. 6C). ChIP analyses performed with liver extracts demonstrated that Fra-1 and c-Jun efficiently bound chromatin fragments containing the putative AP-1 site (Fig. 6D). Furthermore, Fra-1 bound the same region in primary hepatocytes isolated from Fra-1tetON mice (Fig. 6E). Finally, reporter assays using Fra-1 forced dimers[19, 20] and HuH-7 hepatoma cells indicated that c-Jun is the dimerizing partner of Fra-1 to activate gstp1 transcription (Fig. 6F). Collectively, these data strongly support a contribution of Fra-1/AP-1 to the regulation of Gstp1 expression in vivo.

Glutathione S-transferases (GSTs) have been implicated in the liver's response to APAP overdose, a common cause of acute liver failure. Fra-1, c-Fos, and Fra-2 were strongly increased and extracellular signal-regulated kinases (ERK1/2), the main kinases responsible for Fra-1 induction,[28, 29] were found activated in the liver of APAP-treated mice (Fig. 7A,B). We therefore investigated APAP-induced hepatotoxicity in Fra-1 gain- and loss-of-function mice. Fra-1hep-tetOFF mutants exhibited a marked decrease in liver damage, as assessed by histology, cleaved caspase-3 IHC, serum ALT, and liver reduced glutathione (GSH) content (Fig. 7C,D). Conversely, increased APAP hepatotoxicity was observed in Fra-1Δembryo mice (Fig. 7E,F). This effect seemed specific to Fra-1, since xenobiotic transporter and detoxification enzyme were largely unaffected in the liver of Fra-2hep-tetOFF mutants, generated using a similar strategy. Despite high Fra-2 expression in hepatocytes, only Gstp1 was slightly increased (Supporting Fig. 11A-C). Consistently, Fra-2hep-tetOFF mutants displayed a comparable APAP response to control littermates (Supporting Fig. 11D).

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Figure 7. Fra-1 affects the liver's response to APAP-induced injury. (A) Immunoblot analysis of Phospho-ERK-1/2, ERK-1/2, c-Fos, Fra-1, and Fra-2 in sham- and APAP-injected wild-type mice. GAPDH was used to control loading. (B) IHC analysis Fra-1 in sham- and APAP-injected wild-type mice. Scale bar = 50 μM. (C) Liver histology in control and Fra-1hep-tetOFF mice after APAP injection. Top: H&E. Bottom: Cleaved Caspase-3 IHC. Scale bar = 200 μM. (D) Blood ALT levels and liver GSH contents in control and mutant Fra-1hep-tetOFF mice after APAP injection. (E) Liver histology in control and mutant Fra-1Δembryo mice after APAP injection. Top: H&E. Bottom: cleaved Caspase-3 IHC. Scale bar = 200 μM. (F) Blood ALT levels and liver GSH contents in control and mutant Fra-1Δembryo mice after APAP injection. In the entire figure n ≥ 4 per genotype, mice were analyzed 24 hours after APAP injection and controls for the fosl1Δ/Δ mice are fosl1f/Δ and fosl1+/Δ littermates.

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Collectively, while Fos proteins likely display redundant functions during the fibrotic response of the liver, our data point to an altered response of the Fra-1 mutant mice to xenobiotic overload, likely due to a specific Fra-1-dependent modulation of xenobiotic transporters and detoxification enzymes expression (Fig. 8).

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Figure 8. Model depicting the functions of Fra-1 in liver fibrosis and xenobiotic detoxification. In hepatocytes, Fra-1 modulates the expression of the Cxcl9 chemokine, as well as genes involved in xenobiotic detoxification. The small arrows indicate the changes induced by increased Fra-1 expression. Genes for which we demonstrated a direct promoter binding by Fra-1 in the whole liver and in solated hepatocytes are highlighted in red. Fra-1 expression in other cell types, such as stellate cells or cholangiocytes, is required to trigger liver fibrosis. Note that in the loss-of-function setting, Fra-2 likely compensates Fra-1 functions during liver fibrosis but not during APAP-injury.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

Mouse models provide alternative means to study human disease and evaluate therapeutic approaches.[30] Fra-1Tg mice have been proposed as a model for chronic biliary disease,[14] although this transgenic mouse presents a complex phenotype with osteosclerosis, generalized fibrosis, lipodystrophy, and lung alterations.[13, 31, 32] These facts limit its usefulness to dissect organ-specific functions of Fra-1. Here we generated novel mouse models harboring switchable, broad, or cell-specific Fra-1 alleles and investigated for the first time the physiological relevance of Fra-1 in liver disease using loss-of-function animals.

Broad Fra-1 expression in adult Fra-1tetON mice largely recapitulated the phenotypes observed in Fra-1Tg mice with a randomly integrated H2Kb-fosl1-LTR transgene.[13, 14] Specifically, mutant mice developed periportal liver fibrosis, ductular reaction, cytokine dysregulation, and immune infiltrates. This finding has two implications. First, we unambiguously rule out a contribution from the transgene insertion site, fosl1 intronic sequences or the FBJ-sarcoma virus LTR sequence included in the H2Kb-fosl1-LTR transgene to the phenotype. Second, we establish that broad Fra-1 expression in adult mice is sufficient to trigger the disease.

An advantage of this new model is the possibility to switch off transgene expression. Dox withdrawal in Fra-1tetON mutant mice led to a striking improvement of the liver phenotype. Such “transgene addiction” demonstrates the requirement for Fra-1 for the maintenance of the disease and provides a rationale for experimentally addressing the functional relevance of Fra-1 in experimental liver fibrosis and dissecting its transcriptional targets.

Depending on the site of injury, fibrosis may develop in the hepatic parenchyma, as seen in chronic hepatitis and modeled in mice by CCl4 administration, or can be restricted to the portal areas in biliary diseases and in mice upon BDL or DDC feeding. Surprisingly, Fra-1 is largely dispensable in all three models, as indicated by the extent of liver injury, ductular reaction, and liver fibrosis in Fra-1Δembryo mice. We propose that Fra-1 can trigger fibrosis when ectopically expressed, but is dispensable in cholestasis and fibrosis models, because of compensation by other AP-1 members. Indeed, all Fos proteins are induced upon injury and can compensate for the absence of Fra-1. Such functional overlap has already been documented, e.g., Fra-1 can substitute for most of c-Fos functions.[33, 34] The best candidate for compensation is the closest homolog Fra-2, encoded by fosl2 as Fra-2 transgenic mice develop systemic fibrosis highly reminiscent of Fra-1 transgenics.[12] In addition, Fra-2 is higher in untreated and in DDC- and CCL4-treated Fra-1Δembryo mutants. Analyzing mice with broad simultaneous fos11 and fosl2 inactivation would certainly be informative, but difficult to achieve, as fosl2 knockouts die within the first week after birth.[35] A tissue-specific gene inactivation strategy is required and determining the cell type(s) where ectopic Fra-1 expression triggers the liver phenotype is essential to define the adequate gene inactivation strategy.

We investigated the contribution of hepatocytes using a hepatocyte-specific mouse line. Surprisingly, Fra-1hep-tetOFF mutants appeared healthy and displayed unaltered fibrosis upon DDC. This suggests that Fra-1 expression in other liver cell types independently or in addition to hepatocytes is required to trigger the fibrotic phenotype. Crossing the Fra-1Tg to a Rag2-deficient background attenuated the disease and the contribution of cholangiocytes was also suggested, on the basis of increased tgfb1 and pdgf expression.[14] As Fra-1Tg fibroblasts express high amounts of tgfb1 and pdgfc, the contribution of hepatic stellate cells but also macrophages and endothelial cells deserves future investigation, using cell-specific tet-transactivator alleles. Nevertheless, some fibrosis-associated markers were found changed in Fra-1hep-tetOFF mutants and two genes: opn and cxcl9 were also deregulated in Fra-1 loss-of-function mutants, pointing to a specific and physiologically relevant regulation by Fra-1. Osteopontin, encoded by opn, is an extracellular matrix component and fibrotic marker. AP-1 regulates opn in vascular smooth muscle cells and macrophages[12, 36, 37] and opn was increased in Fra-1tetON and Fra-1hep-tetOFF mutants, while reduced in BDL-treated Fra-1Δembryo livers. Ectopic opn expression in hepatocytes triggers liver injury,[38, 39] but opn inactivation does not affect DDC-induced cholestasis.[40] Thus, the Fra-1-dependent changes in opn expression are likely not sufficient to impact fibrosis development in Fra-1 mutant mice.

Cxcl9 is a chemokine implicated in T, endothelial, and stellate cell chemotaxis, associated with human hepatic fibrosis and antifibrosis in mice.[23, 41] Cxcl9 was consistently down-regulated in Fra-1tetON and Fra-1hep-tetOFF mutant livers, while increased in BDL-treated Fra-1-deficient animals. Furthermore, Fra-1 binds to the mouse cxcl9 promoter in liver samples and primary hepatocytes, likely with c-Jun as a dimerizing partner. Cxcl9 is induced by interferon-gamma (IFNγ)/Stat1 and can be potentiated by numerous transcription factors.[42] Further studies will define whether Fra-1 actively represses cxcl9 transcription or interacts with cis-acting regulators at the cxcl9 promoter.

Bile acid levels are mainly regulated by transporters in the liver and intestine, while detoxification and excretion of harmful substances is an important hepatic function. Our analysis revealed an intriguing interaction between Fra-1 and genes involved in the liver's adaptive response to bile acids and xenobiotic overload. We observed an overall more pronounced adaptive response in Fra-1hep-tetOFF and Fra-1Δembryo mutant livers, with lower expression of bile acid uptake transporters. Consistently, circulating bile acids were increased in DDC-fed Fra-1hep-tetOFF mutants. The reason for unchanged bile acids in Fra-1Δembryo mutants upon BDL or DDC is not clear, but as these mice lack Fra-1 in all organs, a contribution from the adaptive response in the intestine or kidneys cannot be excluded. In addition, Fra-1 mutants exhibited altered basal or BDL/DDC-inducible expression of enzymes metabolizing xenochemicals and toxic endogenous compounds, such as cytochrome P450 members as well as gsta1, gsta2, and gstp1. Furthermore, Fra-1 and c-Jun bound the gstp1 promoter and c-Jun∼Fra-1 forced dimers activated transcription from a gstp1 reporter, indicating that Fra-1/AP-1 is a relevant transcriptional modulator of gstp1. GSTA1 induction has been associated with increased AP-1 activity in human hepatocyte cultures[43] and the mouse gsta1 and gsta2 promoters harbor potential AP-1 binding sites. Thus, these genes might also be direct transcriptional targets of Fra-1/AP-1.

GSTs catalyze the conjugation of toxic compounds with GSH, thus facilitating their excretion. Besides gsta1, gsta2, and gstp1, several genes deregulated in Fra-1 mutants are connected to glutathione metabolism. For instance, slco1a1, slco1a4 can mediate GSH export from hepatocytes, while abcc2 mediate biliary excretion of GSH-conjugates.[44] GSH attenuates hepatic oxidative stress, liver injury, and necrosis. Interestingly, Fra-1hep-tetOFF mutants had decreased liver injury upon DDC and were protected from APAP-induced liver damage, a paradigm for GSH-dependent acute liver failure, while Fra-1Δembryo mutants were more sensitive. Interestingly, cxcl9, opn, xenobiotic transporter, and detoxification enzymes were largely unaffected in Fra-2-overexpressing mutants and these mice were not protected from APAP-mediated liver injury. Our findings therefore imply a specific and novel role for Fra-1/AP-1 in the metabolic response of the liver to toxic compounds, likely through modulating GSH and xenobiotic handling. Preliminary data suggest that Fra-1 is increased in patients with APAP-induced liver injury (not shown), indicating that our findings are relevant for human disease.

ERK are the main kinases responsible for Fra-1 induction and stabilization.[28, 45] Consistent with increased Fra-1, increased ERK1/2 phosphorylation was observed in APAP-treated livers. While Jun N-terminal kinases (JNKs) have been extensively studied in APAP-induced liver injury,[46] the function of ERK and their targets has not been reported. JNK inhibition is hepatoprotective in APAP-treated mice. However, the function of JNK has mostly been ascribed to its role in maintaining mitochondrial integrity,[46] rather than affecting Jun proteins or APAP/GSH metabolism.[47, 48] Given that Fra-1 expression is not affected by JNK, including upon APAP,[48] and because our genetic data indicate that Fra-1 inhibition produces the opposite outcome as JNK inhibition, we propose that the ERK/Fra-1 pathway is hepatoprotective in APAP-mediated liver injury by regulating GSH metabolism, thus counteracting the deleterious function of JNK in the mitochondria.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

We thank Drs. A. Bozec, P. Hasselblatt, K. Matsuo, C. Oesterreicher, and M. Petruzzelli for valuable suggestions; the CNIO Transgenic Core Unit for ES cell injection; and G. Luque and G. Medrano for technical help with mouse procedures.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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hep26518-sup-0001-suppFig1.eps3361KSupporting Information Figure 1
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hep26518-sup-0012-SuppInfo.doc84KSupporting Information

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