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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Acute liver failure is associated with significant mortality. However, the underlying pathophysiological mechanism is not yet fully understood. Suppressor of cytokine signaling-1 (SOCS1), which is a negative-feedback molecule for cytokine signaling, has been shown to be rapidly induced during liver injury. Here, using liver-specific SOCS1-conditional-knockout mice, we demonstrated that SOCS1 deletion in hepatocytes enhanced concanavalin A (ConA)–induced hepatitis, which has been shown to be dependent on activated T and natural killer T (NKT) cells. Although serum cytokine level and NKT cell activation were similar in wild-type (WT) and SOCS1-deficient mice after ConA treatment, proapoptotic signals, including signal transducers and activators of transcription 1 (STAT1) and Jun-terminal kinase (JNK) activation, were enhanced in SOCS1-deficient livers compared with those in WT livers. SOCS1-deficient hepatocytes had higher expression of Fas antigen and were more sensitive to anti-Fas antibody–induced apoptosis than were WT hepatocytes. Furthermore, SOCS1-deficient hepatocytes were more sensitive to tumor necrosis factor (TNF)-α-induced JNK activation and apoptosis. These data indicate that SOCS1 is important to the prevention of hepatocyte apoptosis induced by Fas and TNF-α. In contrast, SOCS1 overexpression in the liver by adenoviral gene transfer prevented ConA-induced liver injury. Conclusion: These findings indicate that SOCS1 plays important negative roles in fulminant hepatitis and that forced expression of SOCS1 is therapeutic in preventing hepatitis. (HEPATOLOGY 2008.)

Acute liver failure is a devastating liver disease associated with significant mortality worldwide (40%–80%).1 Acute liver failure is a clinical syndrome characterized by the sudden onset in a patient of severe acute hepatitis with associated symptoms, including jaundice and hepatic encephalopathy. Autoimmune hepatitis, alcohol consumption, viral hepatitis, and hepatotoxins have been identified as trigger factors of acute liver failure. However, the underlying pathophysiological mechanisms are not fully understood. Therefore, a therapeutic approach is difficult.

The lectin concanavalin A (ConA) is known to induce liver injury in mice. ConA-induced fulminant hepatitis mimics many aspects of human acute liver failure, including severe acute hepatitis, Fas-mediated hepatocyte cell death,2 systemic immune activation, and liver damage by activated T and natural killer T (NKT) cells.3 ConA-induced hepatitis is largely dependent on interferon (IFN)-γ/STAT1 as IFN-γ- and STAT1-deficient mice are resistant to ConA-induced hepatitis.4 On the other hand, IL-6 and cytokines that activate STAT3 protect mice from liver damage. In addition, JNK activation and reactive oxygen species (ROS) production have been shown to be involved in liver damage.5

These cytokines up-regulate the expression of the suppressor of cytokine signaling (SOCS) family proteins through the activation of the STAT- and nuclear factor–κB-mediated pathways. Among these, SOCS1 is relatively specific to IFN-γ/STAT1 and interleukin (IL)-4/STAT6, whereas SOCS3 is specific to STAT3.6 Both SOCS1 and SOCS3 have been shown to be rapidly induced in hepatocytes during liver injury.4 We have shown that lack of the SOCS3 gene in liver parenchymal cells protected mice from liver injury because of higher STAT3 activation. In contrast, SOCS1-deficient mice are born healthy but develop fulminant hepatitis as they grow.7, 8 Hyperactivation of SOCS1−/− NKT cells has been implicated in this mechanism.9 We have also shown that SOCS1-heterozygous mice were sensitive to chemical carcinogen–induced hepatitis.10 SOCS1 is a suppressor of liver fibrosis and hepatitis-induced carcinogenesis. However, the role of SOCS1 in hepatocytes has not been studied. To this end, we used hepatocyte-specific SOCS1-conditional-knockout (L-SOCS1 cKO) mice and overexpression of SOCS1 in the liver by adenovirus to clarify the relationship between hepatic SOCS1 expression and fulminant hepatitis. L-SOCS1 cKO mice were more sensitive to ConA-induced lethal effects and liver injury than wild-type (WT) mice. SOCS1 in hepatocytes protected the liver from various mechanisms of liver injury by conferring all cells with resistance to Fas and TNF-α. We also demonstrated that SOCS1 overexpression in the liver by adenoviral gene transfer prevented ConA-induced hepatitis. We propose that SOCS1 plays an important role in preventing liver damages induced by activated lymphocytes and that forced expression of SOCS1 in the liver is therapeutic for preventing hepatitis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals.

AlbCre mice on a C57BL/6 background expressing Cre recombinase under the control of the mouse albumin gene regulatory region were purchased from Jackson Laboratory (Wallenius). Construction of the SOCS1-flox vector and generation of SOCS1flox/flox mice will be described in another article (Tanaka et al., manuscript in preparation). AlbCre mice were crossed with SOCS1flox/flox mice, and the resulting AlbCre-SOCS1flox/flox mice (L-SOCS1 cKO mice) intercrossed with SOCS1flox/flox mice and their littermates were compared. Mice were given a single intravenous injection of ConA (Sigma) at 15 μg/g of body weight as described.19 All experiments on these mice were approved by and conducted in accordance with the Guidelines of the Animal Ethics Committee of Kyushu University, Fukuoka, Japan. Mice were housed in a temperature-controlled environment with a 12-hour light/12-hour dark cycle.

Histological Analysis.

The isolated livers were fixed in 10% formalin. Tissues were embedded in paraffin, and consecutive 5-μm sections were mounted on slides. Sections were stained with hematoxylin/eosin or terminal deoxynucleotidyl transferas–mediated deoxyuridine triphosphate nick-end labeling (TUNEL; Chemicon) staining according to the manufacturer's instructions.

Immunoblot Analysis and ELISA.

Livers were removed and frozen in liquid nitrogen 6 or 20 hours after the ConA injection. Western blotting was performed as described.26 Antibodies to phosphorylated STAT1 (Tyr701), phosphorylated STAT3 (Tyr705), phosphorylated STAT6 (Tyr641), phosphorylated Akt (Ser 473), Akt, phosphorylated IkB (Ser32/36), phosphorylated JNK (Thr183/Tyr185), phosphorylated ERK1/2 (Thr202/Tyr204), IRF-1, and caspase-3 were obtained from Cell Signaling Technology. The antibody to p53 was obtained from Novocastra. The antibody to SOCS3 was from IBL. Antibodies to STAT1, STAT3, STAT6, and IkB were obtained from Santa Cruz Biotechnology. Serum IL-6, IFN-γ, and TNF-α were measured by an enzyme-linked immunosorbent assay using an ELISA Ready-SET-GO kit (eBioscience) according to the manufacturer's instructions.

RNA Extraction and RT-PCR.

Total RNA was extracted from mouse livers or MEFs using TRIzol reagent (Invitrogen). First-strand cDNA was synthesized from 1 μg of total RNA with Mul V. reverse transcriptase using random hexamers. The cDNA was used as a template for polymerase chain reaction (PCR) using KOD-plus DNA polymerase (TOYOBO) according to the manufacturer's instructions. Quantitative real-time reverse transcription PCR (RT-PCR) was performed using an ABI-Prism 7000 (Applied Biosystems) as described.19 The oligonucleotides used for PCR were 5′-AAG TGC TGG AAA AGG AGA CAG G3′ (forward) and 5′-GAT TTG AGG CAT TCA TTG GTA TGG-3′ (reverse) for Fas and 5′-CTT CCG TTG TGC CAT GAA CTC3′ (forward) and 5′-TGC TGT GGT CAT CAG GTA GGG-3′ (reverse) for IRF-1. The oligonucleotides of SOCS1, SOCS3, Bcl2, BclxL, and G3PDH were described previously.24

Isolation of Hepatic Lymphocytes and Flow-Cytometric Analysis.

Hepatic lymphocytes were prepared from liver as described.24 The cells were incubated with antimouse CD16/CD32 mAb (2.4G2) to block the Fc receptor. They were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD3 antibody and phycoerythrin (PE)-conjugated NK1.1 antibody or FITC-conjugated CD4 antibody and PE-conjugated CD69 antibody (BD Pharmingen) in PBS containing 0.1% NaN3 and 2% fetal bovine serum. Stained cells were analyzed using a BD LSR system (BD Biosciences) and Flow Jo software (Tree Star).

Isolation of Hepatocytes and Flow Cytometric Analysis.

A liver was perfused via the portal vein with Hanks' balanced salt solution with 0.5 mM EGTA and then with a 0.05% collagenase type IV solution. Isolated hepatocytes were washed and purified in Hanks' balanced salt using low-speed centrifugation at 40g. Hepatocytes were stained with PE-conjugated anti-CD95 antibody (BD Pharmingen) in PBS containing 0.1% NaN3 and 2% fetal bovine serum. Stained cells were analyzed using a BD LSR system (BD Biosciences).

Detection of Cell Death.

Primary cultured hepatocytes were placed in a 96-well dish at 1 × 104 cells/well. To test for Fas antibody and TNF-α, primary cultured hepatocytes were stimulated by Jo2 (5 μg/mL) or TNF-α (10 ng/mL) for 18 hours, and living cells were analyzed with the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay using Cell Count Reagent SF (Nacalai tesque). MEFs were placed in 24-well plates at 5 × 104 cells/well, and were stimulated by TNF-α (10 ng/mL) for 8 hours. SP600125 (10 μM) was added 1 hour after TNF-α treatment. These cells were trypsinized and collected with the supernatants, and cell viability was determined by propidium iodide (Sigma) and Anenexin V (Sigma) staining using a BD LSR system (BD Biosciences) and Flow Jo software (Tree Star).

Recombinant Adenovirus.

Adenoviral vectors containing the genes for LacZ (AdLacZ), Myc-tagged SOCS1 (Ad-SOCS1), and Cre (AdCre) were prepared by homologous recombination in HEK293 cells as described previously.25, 27 For the in vivo gene transfer for the overexpression study, 200 μL of 1 × 109 plaque-forming units (PFU)/mL recombinant virus in PBS was intravenously injected into C57BL/6 mice. Three days later, ConA was injected into mice.

Statistical Analysis.

Data are expressed as the mean ± SEM. Statistical significance was tested with an unpaired 2-tailed Student t test.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Generation of Liver-Specific SOCS1-Conditional KO Mice.

SOCS1-deficient mice are born healthy but develop fulminant hepatitis as they grow. It is known that SOCS1-deficient NKT cells cause hepatitis9; however, the effect of SOCS1 in hepatocytes has not been studied. Therefore, we used a tissue-specific SOCS1-knockout (cKO) approach to investigate the relationship between hepatic SOCS1 expression and fulminant hepatitis. We generated mice homozygous for loxP-flanked (floxed) socs1 genes. A targeting vector was constructed so that the entire SOCS1 coding region could be deleted by expression of the Cre protein. We generated hepatocyte-specific SOCS1-deficient (L-SOCS1 cKO) mice by crossing SOCS1-flox mice and albumin-Cre transgenic mice (AlbCre), in which Cre recombinase is specifically expressed in liver parenchymal cells under the albumin promoter/enhancer.11

Genomic DNA extracted from the liver and other tissues was analyzed with PCR to verify the deletion of the socs1 gene. A 1.5-kb PCR product of with primers a and c corresponding to the deleted socs1 allele (socs1del; Fig. 1A,B) was detected in the liver from L-SOCS1 cKO mice but not in that from control SOCS1flox/flox (WT) mice. A 3-kb PCR product was detected in other tissues, including the thymus, spleen, pancreas, hypothalamus, and kidney, in L-SOCS1 cKO mice (Fig. 1B), indicating that liver-specific socs1 deletion occurred in these mice. RT-PCR analysis confirmed that socs1 gene expression was almost completely absent in parenchymal cells but was normal in nonparenchymal cells (Fig. 1C). The weight, gross appearance of the liver, and histological features of the liver were normal in L-SOCS1 cKO mice (data not shown).

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Figure 1. Generation of hepatocyte-specific SOCS1-deficient mice. (A) Schematic presentation of the wild-type locus (socs1WT), the targeted locus (socs1flox), and the deleted locus (socs1del). Arrows indicate the positions of the PCR primers. (B) PCR analysis of genomic DNA from liver (Li), thymus gland (Th), spleen (Sp), pancreas (Pa), hypothalamus (Hy), kidney (Ki), and tail (Co). WT, socs1flox, and socs1del alleles are indicated. (C) RT-PCR analysis. Parenchymal and non-parenchymal cells were isolated from liver by collagenase perfusion methods, and total RNA was extracted. Expressionof SOCS1 and control G3PDH mRNA was measured by RT-PCR.

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Enhanced ConA-Induced Hepatitis in L-SOCS1 cKO Mice.

We examined the effect of SOCS1 deletion on T cell-mediated and NKT cell-mediated hepatitis using the ConA-mediated model. At a high dose of ConA, L-SOCS1 cKO mice showed higher lethality than did WT control mice (Fig. 2A). Serum ALT levels induced by ConA injection were markedly higher in L-SOCS1 cKO mice than in WT mice (Fig. 2B), indicating more severe liver damage in L-SOCS1 cKO mice than in WT mice. This was confirmed by HE and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining, which showed more severe apoptosis and necrosis in L-SOCS1 cKO mice than in WT mice (Fig. 2C,D). The number of TUNEL-positive cells even in non-necrotic area was higher in L-SOCS1 cKO liver than in WT liver (Fig. 2D,E). Enhanced apoptosis of SOCS1-deficient liver was confirmed by processing the 32-kDa pro-caspase-3 to the 18-kDa active form. These findings indicate that L-SOCS1 cKO mice were more sensitive to ConA-induced hepatitis than were WT mice (Fig. 2F).

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Figure 2. Liver injury after the administration of ConA. (A) Survival after ConA injection. Mice were injected intravenously with ConA (15 μg/g body weight) and followed for 20 hours (n = 8). (B) Serum ALT level was measured at the indicated times (n = 6); *P < 0.05 (C, E). After 20 hours of ConA administration, livers from WT and L-SOCS1 cKO mice were stained with (C) hematoxylin and eosin or (D) TUNEL. Scale bar = 100 μm. (E) Percentage of TUNEL-positive cells in the nonnecrotic area (*P < 0.05). (F) Immunoblot for caspase-3. WT and L-SOCS1 cKO mice were injected with ConA, and the livers were collected after 6 or 20 hours. β-actin was used as the control. Positions of intact and cleaved caspase-3 are indicated. Anti-β-actin antibody was used as an internal control. Two representative samples from 2 independent animals are shown for each treatment.

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SOCS1 Deletion Did Not Affect Lymphocyte Activation and Cytokine Level.

After the injection of ConA, serum levels of cytokines were compared. Serum IL-6, IFN-γ, and TNF-α levels dramatically increased 6 hours after a single ConA injection, and there was no significant difference between WT mice and L-SOCS1 cKO mice (Fig. 3A-C). Sctivation of CD4+T cells and NKT cells, which has been shown to play an important role in ConA-induced hepatitis,3, 12 was not significantly different between WT and L-SOCS1 cKO mice (Fig. 3D). The decrease in the number of NKT cells after ConA injection in WT mice was equally that in L-SOCS1 cKO mice (Fig. 3E). These findings suggest that both types of mice showed similar activation of liver lymphocytes, including CD4+ T and NKT cells, and the cytokine production from these activated lymphocytes was similar. These data suggest that the difference between WT and L-SOCS1 cKO mice regarding liver injury was a result of differences in the cytokine sensitivity of hepatic cells.

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Figure 3. Activation of cytokine and lymphocytes after ConA administration. Serum levels of (A) IL-6, (B) IFN-γ, and (C) TNF-α were measured at the indicated times (n = 3). (D) Hepatic lymphocytes were isolated and stained with anti-CD4 and anti-CD69 monoclonal antibodies. Upper-right panel shows the activated CD4 T cells. Two similar experiments were performed. (E) Hepatic lymphocytes were stained with anti-NK1.1 and anti-CD3 antibodies. Upper-right panel shows the NKT cells.

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Enhanced Apoptosis Signals in Livers of L-SOCS1 cKO Mice.

To investigate the mechanism of severe liver injury in L-SOCS1 cKO mice, we analyzed the signals related to apoptosis. As expected, the phosphorylation of STAT1 and STAT6, but not of STAT3, was stronger in L-SOCS1 cKO mice than in WT mice (Fig. 4A). Consistent with the hyperactivation of STAT1, expression of the apoptosis-related IRF-1 gene was up-regulated in SOCS1 cKO mice, whereas the antiapoptotic Bcl2 was down-regulated in L-SOCS1 cKO mice (Fig. 4A,B). On the other hand, p53 expression and phosphorylation of the inhibitor of nuclear factor κB were not significantly different between WT and L-SOCS1 cKO mice (Fig. 4A). In particular, proapoptotic kinase JNK was more strongly activated in L-SOCS1 cKO mice than in WT mice (Fig. 4A).

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Figure 4. Enhanced apoptotic signals in L-SOCS1 cKO mice. (A) WT and L-SOCS1 cKO mice were injected with ConA, and after 6 or 20 hours the livers were collected, and immunoblot analysis was done with the indicated antibodies. Two representative samples from two independent animals are shown for each treatment. (B) Expression of SOCS1, Fas, IRF-1, and Bcl2 mRNA was analyzed by real-time RT-PCR. Each group was normalized with G3PDH. (n = 4–6). (C) Fas expression in hepatocytes of WT or L-SOCS1 cKO mice injected without (left) or with (right) ConA. Mean fluorescence intensity (MFI) of Fas staining is shown. (D, E) Effect of Fas-induced apoptosis of primary hepatocytes showing percentage of viable primary cultured hepatocytes after treatment with anti-Fas antibody. (D) Primary cultured hepatocytes from WT and L-SOCS1 cKO mice were treated with anti-Fas antibody for 18 hours after which viable cells were measured by MTT assay and compared with the number of viable cells without Fas treatment (*P < 0.05). (E) Immunoblot assays with anti-caspase-3 and anti–cleaved caspase-3 antibodies. β-Actin is shown as the input control.

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Fas-mediated hepatocyte apoptosis has been shown to be important in ConA-induced liver injury and in human liver disease.2 Fas mRNA was rapidly induced by ConA treatment in SOCS1-deficient liver but slowly induced in WT liver (Fig. 4B). Flow-cytometric analysis of the mean fluorescence intensity (MFI) revealed that surface Fas expression was about 5 times higher in SOCS1-deficient hepatocytes than in WT hepatocytes 6 hours after ConA treatment (Fig. 4C). However, unexpectedly, Fas expression on the cell surface was much higher in L-SOCS1 cKO mice than in WT mice even without ConA treatment (Fig. 4C, left). This may have been a result of accumulation of Fas protein because low expression of Fas mRNA was detected in SOCS1-deficient hepatocytes at basal levels (Fig. 4B).

To demonstrate enhanced sensitivity to Fas-mediated apoptosis, we isolated hepatocytes from L-SOCS1 cKO mice and treated them with anti-Fas antibody in vitro. Fas-mediated cell death was apparently enhanced in SOCS1-deficient hepatocytes (Fig. 4D). Consistently, the cleaved form of caspase-3 was increased in SOCS1-deficient hepatocytes compared with that in WT hepatocytes (Fig. 4E). These data indicate that SOCS1-deficient hepatocytes were more sensitive to Fas-induced apoptosis than were WT hepatocytes, probably because of higher Fas expression on the cell surface.

Enhanced TNFα-Mediated JNK Activation and Apoptosis in SOCS1-Deficient Hepatocytes.

As shown in Fig. 4A, JNK was more strongly activated in L-SOCS1 cKO mice than in WT mice after ConA treatment. Hyperactivation of JNK has been shown to be involved in apoptosis in many cases.5 To investigate the role of SOCS1 in JNK activation, we used primary cultured hepatocytes from L-SOCS1cKO and WT mice (Fig. 5A,B). WT hepatocytes did not show any sign of apoptosis in response to TNF-α, whereas a significant fraction of SOCS1-deficient hepatocytes were dead 18 hours after TNF-α treatment (Fig. 5A). This decrease in the number of viable cells in SOCS1-deficient hepatocytes was probably a result of apoptosis because cleaved caspase-3 was detected in SOCS1-deficient hepatocytes but not in WT hepatocytes (Fig. 5B). The JNK inhibitor SP600125 prevented both TNF-α-mediated cell death and cleavage of caspase-3 in SOCS1-deficient hepatocytes (JNKi+; Fig. 5A,B). Similar results were obtained in mouse embryonic fibroblasts (MEFs). Flow-cytometric analysis revealed that a significant number of SOCS1 KO MEFs underwent apoptosis by TNF-α treatment, which was prevented by a JNK inhibitor (Fig. 5C). TNF-α-mediated phosphorylation of JNK was apparently enhanced in SOCS1 KO MEFs compared with WT MEFs, which was blocked by the JNK inhibitor (Fig. 5D). These data indicate that SOCS1 negatively regulates TNF-α-induced apoptosis, which is dependent on JNK activation.

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Figure 5. Hyperactivation of JNK was involved in TNF-α-induced apoptosis of SOCS1 KO hepatocytes and MEFs. (A, B) Primary cultured hepatocytes were treated with TNF-α (10 ng/mL) with or without SP600125 (JNKi, 10 μM) for 18 hours. (A) The number of viable cells was assessed by the MTT assay and compared with the number of viable cells without TNF-α treatment (*P < 0.05). (B) Samples were analyzed with immunoblotting with anti-caspase-3 and anti-cleaved caspase-3 antibodies. (C, D) WT and SOCS1−/− MEFs were cultured without and with TNFα or with a combination of TNF-α (10 ng/mL) and SP600125 (JNKi, 10 μM). (C) After 8 hours, cells were stained with PI and annexin-V and analyzed with a flow cytometer. (D) Samples were also analyzed with imunoblotting with anti-phosphorylated JNK and anti-JNK antibodies.

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Prevention of Liver Injury by Overexpression of SOCS1.

The results described earlier led us to investigate whether overexpression of SOCS1 in the liver could potentially play a therapeutic role in cases of ConA-induced hepatitis. Mice were treated by intravenous injection of 1 × 109 PFU of the adenovirus-carrying socs1 gene (Ad-SOCS1) or the control adenovirus (Ad-LacZ) 3 days before ConA administration. Twelve hours after challenge with ConA, hepatic injury was assessed. We found that treatment with Ad-SOCS1 was able to strongly prevent increased serum aminotransferase level in mice challenged with ConA (Fig. 6A,B). This was confirmed by HE staining, which showed markedly reduced apoptosis and necrosis in mice treated with Ad-SOCS1 rather than with Ad-LacZ (Fig. 6C). Induction of proinflammatory cytokines such as IFN-γ and IL-6 were strongly suppressed in mice treated with Ad-SOCS1 rather than with Ad-LacZ 12 hours after ConA challenge (Fig. 6D,E). These data suggested that overexpression of SOCS1 or prevention of IFN-γ signal transduction is therapeutic for acute hepatitis induced by a hepatitis-virus infection.

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Figure 6. Prevention of liver injury by overexpression of SOCS1. (A, B) Mice were treated intravenously with Ad-SOCS1 (1 × 109) or Ad-LacZ (1 × 109) 3 days before the administration of ConA. After 12 hours of ConA administration, serum and liver from Ad-infected mice were collected. Serum levels of (A) ALT and (B) AST were measured (n = 4). (C) Liver samples from mice treated with Ad-SOCS1 or Ad-LacZ were stained with HE. Scale bar = 100 μm. (D, E) Serum levels of (D) IFN-γ and (E) IL-6 were measured (n = 5).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study is a report of the protective role of hepatic SOCS1 against ConA-mediated liver injury. We generated hepatocyte-specific SOCS1-conditional-knockout mice and showed that these mice were hypersensitive to ConA-induced hepatitis. However, we noticed that WT and L-SOCS1 cKO mice were similarly sensitive to chemical compound (acetaminophen)–induced hepatitis (M. Nakaya, unpublished data). Therefore, hepatic SOCS1 is very important for cytokine-mediated liver injury but not for toxic compound–induced necrosis in ConA-induced hepatitis. This is consistent with SOCS1/IFN-γ-DKO mice being resistant to ConA-induced hepatitis, similarly to IFN-γ-KO mice.9 Because SOCS1 is induced rapidly by liver injury models such as ConA-induced hepatitis and hepatectomy (Fig. 4B),4, 13 we propose that SOCS1 is important for preventing parenchymal cell death induced by activated lymphocytes during hepatitis.

A number of mechanisms seem to be involved in hypersensitivity to ConA-induced liver injury in L-SOCS1 cKO mice. We noticed strong activation of STAT1 in SOCS1-deficient liver, which is consistent with our previous observations that SOCS1 negatively regulates IFN-γ/STAT1.14 SOCS1 inhibited JAK-mediated STAT1 activation through its N-terminal kinase inhibitory region by direct binding to the activation loop of JAK.15 STAT1 up-regulates apoptosis by inducing apoptosis-related genes, such as IRF1, JNK, and Fas. JNK and IRF-1 have been shown to be involved in ConA-induced hepatitis.16 Although STAT1 reduces expression of antiapoptotic genes, such as Bcl-2 and Bcl-XL, Fas expression on cell surfaces was much higher in SOCS1-deficient liver than in WT liver even before ConA treatment. Expression of Fas mRNA in L-SOCS1 cKO liver (Fig. 4B) did not correlate with Fas antigen levels on the cell surfaces of SOCS1-deficient hepatocytes (Fig. 4C). This may be explained by the accumulation of Fas protein on cell surfaces because low expression of Fas mRNA was detected in SOCS1-deficient hepatocytes at basal levels (Fig. 4B). Alternatively, SOCS1/STAT1 may modulate the efficiency of the expression of Fas in SOCS1-deficient hepatocytes. It has been reported that STAT1 accelerates Fas trafficking to cell surfaces.17 However, the precise mechanism remains to be investigated. Several other mechanisms had been proposed to explain how STAT1 regulated apoptosis. STAT1 can promote apoptosis through the up-regulation of caspases, p53, and TRAIL.18, 19 However, we did not observe such changes in the L-SOCS1 cKO mice.

We noticed that JNK was strongly activated in SOCS1-deficient liver after ConA injection. We found that SOCS1-deficient hepatocytes and MEFs were more sensitive to TNF-α-induced apoptosis than were WT cells, which is consistent with a previous report.20 Recently, it was reported that SOCS1 functions as a negative regulator of TNF-α-induced apoptosis in endothelial cells, in part, by inducing ASK1 degradation.21 We also previously reported that JNK was hyperactivated in SOCS1-deficient mice.14, 22 Because SOCS1-deficient cells were resistant to TNF-α-induced apoptosis when a dominant-negative form of STAT1 was expressed (data not shown), hyperactivation of STAT1 may be involved in this higher JNK activation and stronger apoptosis in SOCS1-deficient cells.

In contrast to STAT1, STAT3 has been shown to protect against ConA-induced hepatitis. It was reported that a constitutively activated form of STAT3 (STAT3C) dramatically suppressed Fas-mediated liver injury.23 We previously reported that hepatocyte-specific SOCS3-deficient (L-SOCS3 cKO) mice were resistant to ConA-induced hepatitis because of increased activation of STAT3 and reduced activation of STAT1 in SOCS3-deficient liver. In SOCS3-deficient liver, up-regulation of Bcl-XL and reduced expression of IRF-1 were observed.24 Therefore, opposing effects of SOCS1 and SOCS3 were observed regarding hepatocyte apoptosis and STAT1/STAT3 activation. However, reduced STAT3 activation was not observed in L-SOCS1 cKO mice. This may have been a result of similar induction of SOCS3 in the livers of WT and L-SOCS1 cKO mice.

We found that overexpression of SOCS1 in the liver strongly suppressed ConA-induced hepatitis. We noticed that, in contrast to L-SOCS1 cKO mice, Ad-SOCS1-treated mice showed strong suppression of cytokine production. Similar liver protection by Ad-SOCS3 or membrane-permeable SOCS3 protein has been reported.25 Therefore, Ad-SOCS1 may prevent hepatitis not only because of the suppression of cytokine signaling in hepatocytes but also because of the suppression of cytokine production in lymphoid and myeloid cells. In any case, forced expression of SOCS1 or intracellular delivery of recombinant SOCS1 protein in liver cells could be therapeutic for acute liver injury induced by hepatitis-virus infection.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Y. Kawabata, T. Yoshioka, S. Sasaki, N. Kinoshita, and M. Ohstu for technical assistance and Y. Nishi for manuscript preparation.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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