A20 promotes liver regeneration by decreasing SOCS3 expression to enhance IL-6/STAT3 proliferative signals

Authors

  • Cleide G. da Silva,

    1. Division of Vascular and Endovascular Surgery, Center for Vascular Biology Research and the Transplant Institute, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
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  • Peter Studer,

    1. Division of Vascular and Endovascular Surgery, Center for Vascular Biology Research and the Transplant Institute, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
    Current affiliation:
    1. Department of Visceral Surgery and Medicine, University Hospital Bern, Bern, Switzerland
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  • Marco Skroch,

    1. Division of Vascular and Endovascular Surgery, Center for Vascular Biology Research and the Transplant Institute, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
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  • Jerome Mahiou,

    1. Division of Vascular and Endovascular Surgery, Center for Vascular Biology Research and the Transplant Institute, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
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  • Darlan C. Minussi,

    1. Division of Vascular and Endovascular Surgery, Center for Vascular Biology Research and the Transplant Institute, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
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  • Clayton R. Peterson,

    1. Division of Vascular and Endovascular Surgery, Center for Vascular Biology Research and the Transplant Institute, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
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  • Suzhuei W. Wilson,

    1. Division of Vascular and Endovascular Surgery, Center for Vascular Biology Research and the Transplant Institute, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
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  • Virendra I. Patel,

    1. Division of Vascular and Endovascular Surgery, Center for Vascular Biology Research and the Transplant Institute, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
    Current affiliation:
    1. Division of Vascular Surgery, Massachusetts General Hospital, Boston, MA
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  • Averil Ma,

    1. Division of Gastroenterology, Department of Medicine, University of California San Francisco, San Francisco, CA
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  • Eva Csizmadia,

    1. Division of Vascular and Endovascular Surgery, Center for Vascular Biology Research and the Transplant Institute, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
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  • Christiane Ferran

    Corresponding author
    1. Division of Vascular and Endovascular Surgery, Center for Vascular Biology Research and the Transplant Institute, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
    2. Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
    • M.D., Ph.D., Beth Israel Deaconess Medical Center, Research North #370F/G, 99 Brookline Ave., Boston MA 02215
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    • fax: 617-667-0445


  • Potential conflict of interest: Nothing to report.

Abstract

Liver regeneration is of major clinical importance in the setting of liver injury, resection, and transplantation. A20, a potent antiinflammatory and nuclear factor kappa B (NF-κB) inhibitory protein, has established pro-proliferative properties in hepatocytes, in part through decreasing expression of the cyclin dependent kinase inhibitor, p21. Both C-terminal (7-zinc fingers; 7Zn) and N-terminal (Nter) domains of A20 were required to decrease p21 and inhibit NF-κB. However, both independently increased hepatocyte proliferation, suggesting that additional mechanisms contributed to the pro-proliferative function of A20 in hepatocytes. We ascribed one of A20′s pro-proliferative mechanisms to increased and sustained interleukin (IL)-6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation, as a result of decreased hepatocyte expression of the negative regulator of IL-6 signaling, suppressor of cytokine signaling 3 (SOCS3). This novel A20 function segregates with its 7Zn not Nter domain. Conversely, total and partial loss of A20 in hepatocytes increased SOCS3 expression, hampering IL-6-induced STAT3 phosphorylation. Following liver resection in mice pro-proliferative targets downstream of IL-6/STAT3 signaling were increased by A20 overexpression and decreased by A20 knockdown. In contrast, IL-6/STAT3 proinflammatory targets were increased in A20-deficient livers, and decreased or unchanged in A20 overexpressing livers. Upstream of SOCS3, levels of its microRNA regulator miR203 were significantly decreased in A20-deficient livers. Conclusion: A20 enhances IL-6/STAT3 pro-proliferative signals in hepatocytes by down-regulating SOCS3, likely through a miR203-dependent manner. This finding together with A20 reducing the levels of the potent cell cycle brake p21 establishes its pro-proliferative properties in hepatocytes and prompts the pursuit of A20-based therapies to promote liver regeneration and repair. (HEPATOLOGY 2013)

The liver has a unique regenerative capacity, restoring liver mass after surgical resection or toxic/viral hepatocyte damage.1 Liver regeneration (LR) is characterized by hepatocytes' rapid, synchronized transition from quiescence into cell cycle. Rodent models of partial hepatectomy (PH) unveiled molecular drivers of successful LR, such as the priming cytokines tumor necrosis factor (TNF) and interleukin (IL)-6, that drive hepatocyte entry into cell cycle.1, 2 Shortly following hepatectomy, Kupffer cells release TNF, activating nuclear factor kappa B (NF-κB) in neighboring hepatocytes, thus increasing IL-6 secretion. In turn, IL-6 binds to its receptor, activating the JAK/STAT3 (signal transducer and activator of transcription 3) pathway to promote hepatocyte proliferation.3 Accordingly, the superagonistic IL-6/soluble IL-6R fusion protein enhances LR.4 In contrast, mice with targeted disruption of IL-6 demonstrate impaired regeneration that could be reversed by overexpression or induction of STAT3.5-8 Levels of IL-6 after PH determine its hepatotrophic effect. Indeed, inappropriately high IL-6 levels rather inhibit LR by increasing expression of the cyclin dependent kinase inhibitor (CDKI), p21.9 IL-6 signaling in hepatocytes is finely regulated by a negative feedback loop provided by IL-6/STAT3-mediated induction of suppressor of cytokine signal-3 (SOCS3).10 Accordingly, hepatocyte-specific SOCS3 knockout (hKO SOCS3) mice show improved LR following hepatectomy.11 In sum, the IL-6/STAT3/SOCS3 pathway is a promising target for proregenerative strategies.

We previously demonstrated that A20/tnfaip3, a key element of the cellular response to injury and inflammation,12, 13 exerts multiple hepatoprotective functions through combined anti-inflammatory, anti-apoptotic, and pro-proliferative effects in hepatocytes.14-16 A20 restrains inflammation by inhibiting NF-κB activation,17 blocks apoptosis by disrupting caspase-8 activation,18 limits ischemic damage by increasing peroxisome proliferator-activated receptor alpha (PPARα) expression,16 optimizes energy production through improved lipid/fatty acid metabolism, and promotes proliferation by decreasing p21.15, 19 A20′s hepatoprotective functions improve LR and survival in mouse models of extreme liver injury, including extended (78%, EH) and radical/lethal (83%) hepatectomy, acute toxic hepatitis and lethal ischemia/reperfusion injury.14-16 A20 is an NF-κB-dependent gene14 that is part of the regenerative response of the liver, and accordingly, its expression levels increase in deceased and living donor liver grafts hours following reperfusion.20 A20 KO mice are born cachectic and die within 4-5 weeks of birth, mainly from unfettered liver inflammation, which shows A20′s high rank in the hierarchy of the liver physiologic anti-inflammatory armamentarium.21

Since transcription of key priming cytokine IL-6 is in part NF-κB-dependent,22 we questioned whether the NF-κB inhibitory protein A20 would decrease IL-6 levels, thereby attenuating its pro-proliferative advantage in hepatocytes. This work examines this question.

Abbreviations

7Zn, A20′s 7 zinc C-terminal domain; ANOVA, analysis of variance; β-gal; β-galactosidase; CCNA, cyclin A; CCND1, cyclin D1; CDKI, cyclin dependent kinase inhibitor; EDTA, ethylenediaminetetraacetic acid; EH, extended hepatectomy; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; FGG, fibrinogen gamma chain; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HA, hemagglutinin; HT, heterozygous; IHC, immunohistochemistry; IL-6, interleukin-6; LR, liver regeneration; KO, knockout; LPS, lipopolysaccharide; miR, microRNA; MPH, mouse primary hepatocytes; NF-κB, nuclear factor kappa B; Nter, A20′s N-terminal domain; PCNA, proliferating cell nuclear antigen; PH, partial hepatectomy; qPCR, quantitative real time polymerase chain reaction; rAd, recombinant adenovirus; SAA1, serum amyloid A 1; SOCS3, suppressor of cytokine signaling 3; STAT3, signal transducer and activator of transcription 3; TBP, TATA box binding protein; TNF, tumor necrosis factor alpha.

Materials and Methods

Reagents.

Human recombinant TNF and IL-6 were from R&D Systems (Minneapolis, MN). Lipopolysaccharide (LPS), insulin, fetal bovine serum (FBS), gelatin, and collagenase type IV were from Sigma-Aldrich (St. Louis, MO).

Mice.

Eight to 12-week-old BALB/c mice (Taconic Farms, Germantown, NY) and A20 heterozygous (HT) and wildtype littermate (WT) mice were used in models of hepatectomy.21 Four to 5-week-old A20 WT, HT, and KO mice were used for hepatocyte isolation. All procedures were performed in accordance with the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals, and approved by the Institutional Committee for Use and Care of Laboratory Animals.

Cell Culture.

Mouse normal liver epithelial cell line (NMuLi, CRL-1638), human hepatocellular carcinoma cell line (HepG2, HB-8065), and human kidney embryonic cell line (HEK-293) were purchased from the American Type Culture Collection (Manassas, VA).16 Mouse primary hepatocytes (MPH) were isolated using a modified two-step EDTA/collagenase protocol.23

Hepatocyte Proliferation.

NMuLi and HepG2 hepatocytes were synchronized in G0/G1 phase of the cell cycle by 24-hour serum starvation. Cell proliferation was determined by cell count using Trypan blue exclusion before and 24 hours after addition of 10% FBS.

Western Blot Analysis.

HepG2 and MPH whole cell lysates were recovered before and following IL-6, TNF, and/or LPS treatment, and protein concentration determined.16 Samples were analyzed by western blot (WB) using the following primary antibodies: rabbit anti-STAT3, rabbit anti-IκBα, mouse anti-β-actin, (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho-STAT3 (P-STAT3 Tyr705) (Cell Signaling Technology, Danvers, MA), chicken anti-TNFAIP3 (A20) (Abcam, Cambridge, MA), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Calbiochem/EMD Biosciences, San Diego, CA), anti-hemagglutinin (HA) (Roche Applied Science, Indianapolis, IN); and secondary antibodies (Thermo Scientific, Rockford, IL). Immunoblots were scanned and band intensity quantified by densitometry using ImageJ 1.41 (NIH, Bethesda, MD).

IL-6 Enzyme-Linked Immunosorbent Assay (ELISA).

Supernatants from nontreated and LPS/TNF-treated HepG2 and MPH cultures were assayed for IL-6 levels using human and mouse IL-6 ELISA Ready-SET-Go! (eBioscience, San Diego, CA). Results were normalized to total protein concentration.

Quantitative Reverse Transcriptase Polymerase Chain Reaction (qPCR).

RNA was extracted using the Qiagen RNeasy Mini Kit (Valencia, CA) or Trizol reagent (Sigma-Aldrich), and cDNA was synthesized using iScript cDNA synthesis kit (Bio-Rad). Gene expression was quantified using iTaq Fast SYBR Green Supermix with ROX (Bio-Rad) and gene-specific primers (Invitrogen, Coralville, IA) listed in Supporting Table 1, or TaqMan Mm00627280_m1 (tnfaip3), Mm00607939_s1 (β-actin). Expression of target genes was normalized to that of the housekeeping genes β-actin, TATA box binding protein (TBP), or 28S. MicroRNA (miRNA) was extracted using the mirVana kit (Life Technologies, Grand Island, NY), and assayed for miR203, and the housekeeping miRNA, snoRNA202, using TaqMan (Applied Biosystems, Foster City, CA). qPCR were performed on a 7500 Fast Real-Time PCR System (Applied Biosystems).

Recombinant Adenoviruses.

We generated recombinant adenovirus (rAd).A20 using a plasmid provided by Dr. V. Dixit (Genentech, San Francisco, CA).24 The rAd.βgal was a gift of Dr. Robert Gerard (University of Texas SW, Dallas, TX). By RT-PCR, we generated HA-tagged deletion mutants comprising the N-terminus (Nter) and seven Zinc (7Zn) domains of A20 and cloned them in pAC CMVpLpA SR(+) expression plasmid to generate rAd. (Supporting Methods). We used HEK293 cells to generate, produce, and titer rAd. that were purified by cesium chloride density gradient centrifugation for in vivo,24 or the AdenoPure LS Kit (Puresyn, Malvern, PA) for in vitro experiments. Hepatocyte cultures (60% confluent) were transduced with rAd. at a multiplicity of infection (MOI) of 50-200 plaque-forming units per cell (pfu/cell), leading to transgene expression in >95% of cells without toxicity14, 15 (Supporting Fig. S1). In vivo, we injected 1 × 109 pfu of rAd. in 100 μL saline into the mouse penile vein. This dose and route of administration achieves maximal transgene expression in 30% of hepatocytes, 5 days after injection.15 Transgene expression was analyzed by WB (A20) and X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) staining (β-gal).

Hepatectomy Model.

A 78% hepatectomy (EH) was performed as described.15 Livers harvested before and after surgery were either frozen in liquid nitrogen for protein and RNA extraction, or fixed in 10% formalin for immunohistochemistry (IHC) and immunofluorescence (IF) analysis.

Immunochemistry and Immunofluorescence.

For IHC and IF staining we used the following primary antibodies: goat anti-SOCS3, rabbit anti-P-STAT3 (Cell Signaling), rat anti-Ki67 (Dako), chicken anti-albumin (Novus Biologicals, Littleton, CO), and goat anti-HNF4α (Santa Cruz), followed by horseradish peroxidase (HRP) or Alexa Fluor 488 (green) and 594 (red) conjugated secondary antibodies (Invitrogen, Carlsbad, CA). Ki67, P-STAT3, and SOCS3-positive cells per high-power field (HPF) were counted using ImageJ automated or manual cell counting.

Statistical Analysis.

Data were analyzed by one-way or two-way analysis of variance (ANOVA) followed by post-hoc Bonferroni multiple range test when F was significant, using Prism 5 (GraphPad Software, La Jolla, CA). Differences between groups were rated significant at a probability error (P) of less than 0.05.

Results

N-Terminal and 7Zn C-Terminal Domains of A20 Independently Promote Hepatocyte Proliferation in a p21-Independent Fashion.

We evaluated cell proliferation in nontransduced (C), rAd.A20, rAd.Nter, rAd.7n, and rAd.βgal-transduced NMuLi cells. This cell line responds in a physiologic manner to growth factor-induced cell cycle progression.15 Overexpression of A20 increased by 1.6-fold cell counts/well when compared to C and rAd.βgal transduced cells, 24 hours after addition of 10% FBS, (Fig. 1A, n = 4-6; P < 0.05). Similarly, rAd.Nter and rAd.7Zn-transduced cells showed a 1.8- and 1.9-fold increase, respectively, in cell counts/well (Fig. 1A; n = 3-4; P < 0.05 versus C and P < 0.01 versus rAd.βgal). This indicated that independent overexpression of Nter or 7Zn increases proliferation in NMuLi cells. We reproduced these results in HepG2 cells, validating their use in subsequent experiments (Fig. S2A; n = 4; P < 0.001).

Figure 1.

C-terminal and N-terminal domains of A20 independently promote hepatocyte proliferation but neither can independently decrease p21 expression or inhibit IκBα degradation. (A) NMuLi cells were transduced with rAd.A20, rAd.7Zn, and rAd.Nter, serum-starved for 24 hours to synchronize their cell cycle, then supplemented with 10% FBS-enriched medium to drive cell proliferation. Cell count/well was evaluated 24 hours later by Trypan blue exclusion and plotted as mean ± SEM of 3-6 independent experiments. (B) Relative p21 mRNA levels measured by qPCR in HepG2 cells transduced with rAd.A20, rAd.7Zn, and rAd.Nter for 48 hours. Histograms represent mean ± SEM of relative mRNA levels after normalization by β-actin mRNA (n = 3-5 independent experiments). (C) Representative IκBα western blot of cell lysates from rAd.A20, rAd.7Zn, and rAd.Nter HepG2 cells treated with TNF (200 U/mL) for 15 minutes. β-Actin was used for loading control (n = 3 independent experiments). Nontransduced (C) and rAd.βgal transduced cells were used as controls. *P < 0.05, **P < 0.01.

We previously reported that A20′s pro-proliferative effect in hepatocytes related, at least in part, to decreased p21 expression.15 We confirmed in HepG2 that overexpression of full-length A20, but not Nter or 7Zn, significantly decreased p21 messenger RNA (mRNA) levels as compared to β-gal-expressing cells (Fig 1B; n = 3-5; P < 0.05). As for NF-κB inhibition17 (Fig. 1C; n = 3), cooperation between Nter and 7Zn domains was required to decrease p21, signifying that other mechanism(s) must account for their independent pro-proliferative effect in hepatocytes. Given potential discrepancies between cell lines and primary cells, we validated these results in MPH: full-length A20 but neither 7Zn nor Nter decreased p21 mRNA levels (Fig. S2B; n = 2; P < 0.05), or inhibited TNF-induced IκBα degradation (Fig. S2C; n = 3).

A20 Enhances IL-6/STAT3 Signaling Despite Overall Decreasing IL-6 Production in Hepatocytes.

Since IL-6 is central to hepatocyte proliferation, we measured IL-6 levels in supernatants of C, rAd.A20, rAd.Nter, rAd.7Zn, and rAd.βgal transduced HepG2 stimulated with TNF (200 U/mL) and LPS (10 μg/mL) for 6 hours to mimic the physiologic triggers of IL-6 secretion after hepatectomy.1 IL-6 levels significantly increased in all groups following TNF/LPS, as compared to corresponding nonstimulated cells (6.5- to 9.9-fold, Fig. 2A). However, IL-6 levels were significantly lower in supernatants of A20 overexpressing HepG2 compared to all other groups (Fig. 2A; n = 4-7; P < 0.01 versus C; and P < 0.001 versus rAd.Nter, rAd.7Zn, and rAd.βgal). This result indicates that IL-6 transcription partially relies on NF-κB.22 Notably, neither Nter nor 7Zn decreased TNF/LPS-induced IL-6 secretion, with 7Zn overexpression moderately, yet significantly increasing it (Fig. 2A; P < 0.01 versus C).

Figure 2.

Overexpression of A20 increases IL-6-induced STAT3 phosphorylation through down-regulation of SOCS3 expression, despite lower IL-6 production. HepG2 were nontransduced (C), or transduced with rAd.A20, rAd.7Zn, rAd.Nter, and control rAd.βgal (A) IL-6 levels were determined by ELISA in cell culture supernatants 6 hours following TNF (200 U/mL) and LPS (10 ng/mL). Results are expressed as mean ± SEM of 4-7 independent experiments. Representative phospho (P-STAT3) and total STAT-3 western blots following (B) TNF (200 U/mL) and LPS (10 ng/mL) for 6 to 24 hours or (C) IL-6 (50 ng/mL) for 15 minutes to 6 hours. β-Actin was used as loading control. Corrected densitometry results, presented as percentage of control nonstimulated cells, are expressed as mean ± SEM of 3-5 independent experiments. (D) Relative SOCS3 mRNA levels were measured by qPCR 1 to 3 hours following IL-6 (50 ng/mL). Histograms represent mean ± SEM of relative mRNA levels after normalization with 28S (n = 4-5 independent experiments). *P < 0.05, **P < 0.01 and ***P < 0.001 versus C, and #P < 0.05, ##P < 0.01, and ###P < 0.001 versus rAd.βgal within each treatment group.

Despite lower IL-6 levels in supernatants of A20-overexpressing cells, STAT3 phosphorylation, downstream of IL-6, was enhanced at baseline (∼150-fold; P < 0.001), 6 hours (P < 0.001) and 24 hours (P < 0.05) after LPS/TNF treatment, when compared to C and rAd.βgal-transduced cells (Fig. 2B; n = 3-4). Like A20, overexpression of 7Zn but not Nter also increased STAT3 phosphorylation, reflecting either higher production of IL-6 by 7Zn-expressing cells, or that A20′s 7Zn domain accounts for its ability to increase STAT3 phosphorylation.

To clarify this issue, we washed out the basal medium of C and rAd. transduced (A20, Nter, 7Zn, βgal) HepG2 cell cultures, then treated them with exogenous IL-6 (50 ng/mL) and checked for STAT3 phosphorylation 15 minutes to 6 hours later. Control, rAd.Nter, and rAd.βgal transduced HepG2 showed low basal P-STAT3 levels, that transiently increased (peaking 15 minutes) after IL-6 stimulation. A20 and 7Zn overexpressing HepG2 had significantly higher baseline levels of P-STAT3 (comparable to IL-6 induced peak levels) that were slightly enhanced and sustained for at least 6 hours after IL-6 addition (Fig. 2C; n = 4-5). These results indicate that this novel effect of A20 indeed maps to its 7Zn domain.

To investigate the molecular basis for the A20-mediated increase in STAT3 phosphorylation, we assessed STAT3-dependent expression of the negative regulator of IL-6 signaling, SOCS3. Our results showed that both A20 and 7Zn, but not Nter, significantly decreased basal and IL-6-induced up-regulation of SOCS3 mRNA in HepG2 cells, compared to controls (Fig. 2D; n = 4-5; P < 0.05 versus C and P < 0.01 versus rAd.βgal). Altogether, these results uncover a novel mechanism by which A20 (7Zn domain) promotes hepatocyte proliferation through decreasing SOCS3 expression.

A20 Knockdown Increases SOCS3 Expression and Attenuates IL-6/STAT3 Signaling, Despite Increasing IL-6 Production in Hepatocytes.

To investigate the physiologic role of A20 in regulating IL-6/STAT3/SOCS3 signaling, we performed loss of function experiments, using MPH isolated from A20 KO, A20 HT, and WT littermate mice. We confirmed by qPCR that A20 mRNA was absent in A20 KO, and reduced by 50% in A20 HT MPH, as compared to WT (Fig. 3A; n = 3; P < 0.001). Total loss of A20 significantly increased basal (P < 0.05) and TNF-induced (P < 0.001) IL-6 production by MPH, when compared to HT and WT (Fig. 3B; n = 4). Heterozygous MPH showed an intermediate result. Increased basal IL-6 levels in A20 KO and HT hepatocytes were paralleled by higher basal P-STAT3 levels, indicating a chronic state of IL-6-mediated activation of these hepatocytes (Fig. S3; n = 2). However, when we washed away endogenously produced IL-6 prior to adding exogenous IL-6 (50 ng/mL), STAT3 phosphorylation was almost abolished in A20 KO, and attenuated (but with similar kinetics) in A20 HT MPH, as compared to WT (Fig. 3C; n = 3). Decreased STAT3 phosphorylation in A20 KO MPH correlated with significantly higher basal (P < 0.05) and IL-6-induced (P < 0.01 at 1 hour, P < 0.05 at 3 hours) SOCS3 mRNA levels, compared to WT (Fig. 3D; n = 4-5). We obtained similar results in whole livers, when hepatocytes where still in their physiologic multicellular environment; SOCS3 mRNA levels were significantly higher in KO versus HT (P < 0.001) and WT (P < 0.01), and slightly greater in HT versus WT livers (Fig. 4A; n = 7-9). Consequently, we noted significantly more SOCS3/albumin positive hepatocytes around the portal and central veins of A20 KO livers, as compared to HT and WT livers (Fig. 4B,C; n = 3-4; P < 0.01). In line with in vitro results, the number of P-STAT3/HNF4α-positive hepatocytes, at baseline, was higher in A20 KO than in WT and HT livers (Fig. 4D,E; n = 3-4; P < 0.01, and <0.05, respectively). Despite increased P-STAT3 levels in A20 KO livers, the number of Ki67 proliferating hepatocytes was not significantly different among groups (Fig. S4C,D; n = 3-4). We attribute this result to very high IL-6 levels in A20 KO mice, creating an IL-6 “hyperstimulation” outcome, i.e., impaired hepatocyte proliferation despite increased P-STAT3 levels.9 Indeed, intrahepatic IL-6 levels were significantly higher in A20 KO versus HT and WT livers (Fig. S4A; n = 5; P < 0.001), with a corresponding increase in mRNA levels of STAT3-dependent cell cycle inhibitor, p21 (Fig. S4B; n = 5; P < 0.05). We verified by qPCR that A20 mRNA levels were absent or reduced by 50% in A20 KO and HT livers, respectively, as compared to WT (Fig. S4E; n = 4-6). Since SOCS3 mRNA levels are also epigenetically regulated by miR203,26 we checked by qPCR for miR203 levels and found them significantly lower in KO (P < 0.01) and HT (P < 0.05) versus WT livers (Fig. 4F; n = 5-7).

Figure 3.

A20 knockdown increases IL-6 production but decreases IL-6 induced STAT3 phosphorylation by up-regulating SOCS3. (A) A20 mRNA levels in MPH isolated from WT, A20 HT and A20 KO livers, as measured by qPCR. Histograms represent mean ± SEM of relative A20 mRNA levels after normalization with β-actin (n = 3 mice per group). (B) IL-6 levels determined by ELISA in cell culture supernatants of MPH 6 hours following TNF (200 U/mL). Results are expressed as mean ± SEM of 3-6 independent experiments. (C) Representative phospho (P-STAT3) and total STAT-3 western blots of MPH treated with IL-6 (50 ng/mL) for 30 minutes to 6 hours. GAPDH was used as loading control. Corrected densitometry results, presented as percentage of WT nonstimulated cells are expressed as mean ± SEM of three independent experiments. (D) Relative SOCS3 mRNA levels measured by qPCR in MPH cultures stimulated for 1 to 3 hours with IL-6 (50 ng/mL). Histograms represent mean ± SEM of relative mRNA levels after normalization with β-actin (n = 4-5 independent experiments). *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4.

A20 knockdown increases SOCS3 expression, which inversely correlates with lower miR-203 levels. Livers of 4 to 5-weeks old WT, A20 HT, and A20 KO mice were analyzed for (A) SOCS3 mRNA levels by qPCR. Histograms represent mean ± SEM of relative SOCS3 mRNA levels after normalization with β-actin (n = 7-9 mice per group). (B) Graph representing mean ± SEM of SOCS3-positive hepatocytes per high-power field (HPF), as analyzed (C) by immunohistochemistry (brown). Double immunofluorescence staining, using fluorescence labeled SOCS3 and hepatocyte-specific albumin antibodies (SOCS3: green; albumin: red; DAPI: blue), confirmed that SOCS3-positive cells were hepatocytes. (D) Graph representing mean ± SEM of P-STAT3-positive hepatocytes per HPF, as analyzed by (E) immunohistochemistry (brown). Double immunofluorescence staining, using fluorescence labeled P-STAT3 and hepatocyte-specific hepatocyte nuclear factor 4α (HNF) (P-STAT3: red; HNF: green; DAPI: blue). Yellow arrows indicate P-STAT3/HNF/DAPI-positive hepatocytes; pink arrows indicate P-STAT3/DAPI-positive but HNF-negative cells. Photomicrographs are representative of 3-4 mice per group. Original magnification ×200 (light microscopy) and ×400 (fluorescence microscopy). (F) Histograms representing mean ± SEM of relative miR-203 mRNA levels measured by qPCR after normalization with the housekeeping miRNA gene sno202 (n = 5-7 mice per group). *P < 0.05 **P < 0.01 and ***P < 0.001.

A20 Overexpression in Mouse Livers Enhances and Accelerates Hepatocyte Proliferation Following Extended Hepatectomy by Inhibiting SOCS3 Up-regulation.

We chose to confirm the effect of A20 on IL-6/STAT3/SOCS3 in the mouse model of EH, characterized by delayed LR and high lethality rate (50%), to magnify A20′s pro-proliferative mechanism(s).15 We performed EH in BALB/C mice that did not receive any rAd. (C) or were intravenously injected with rAd.A20 or the control rAd.βgal. Overexpression of A20 inhibited EH-induced hepatic up-regulation of SOCS3 mRNA when compared to C or rAd.βgal treated livers 24 hours following surgery (Fig. 5A; n = 3-6). We confirmed these results by showing decreased SOCS3 immunostaining in A20-overexpressing livers, 36 hours following EH (Fig. 5B; n = 5-6).

Figure 5.

Overexpression of A20 in livers down-regulates SOCS3 to promote hepatocyte proliferation following extended hepatectomy. Livers from mice that did not receive recombinant adenovirus (C) or were injected with rAd.A20 or rAd.βgal were recovered before (pre) and 24 hours (mRNA) or 36 hours (immunostaining) after extended hepatectomy (post) and evaluated for (A) SOCS3 mRNA levels by qPCR. Histograms represent mean ± SEM relative SOCS3 mRNA levels (n = 3-6 mice per group). (B) SOCS3 protein expression by immunohistochemistry (brown) (n = 5-6 mice per group). (C) Cyclin D1 (CCND1) and (D) cyclin A (CCNA) mRNA levels by qPCR. Histograms represent mean ± SEM relative CCND1 and CCNA mRNA levels (n = 3-6 mice per group). (E) Hepatocyte proliferation was evaluated by Ki67 immunostaining (brown). Double immunofluorescence, using fluorescence-labeled Ki67 and hepatocyte-specific HNF (Ki67: red; HNF: green; DAPI: blue), confirm that ki67-positive cells are hepatocytes. Histograms represents mean ± SE of Ki67-positive hepatocytes per HPF. Photomicrographs in (B,E) are representative of 5-6 (light microscopy) or 3-6 (fluorescence microscopy) mice per group. Original magnification ×200 (light microscopy) and ×400 (fluorescence microscopy). mRNA levels in (A,C,D) were normalized using TATA box binding protein (TBP) mRNA. *P < 0.05, **P < 0.01, ***P < 0.001

EH resulted in a moderate increase in cyclin D1 (CCND1) and cyclin A (CCNA) mRNA in C livers. This is expected, given impaired LR in this EH model. On the other hand, rAd-treated livers had higher basal mRNA levels of CCND1 and CCNA when compared to C, possibly reflecting adenoviral toxicity. Whereas rAd. toxicity repressed further EH-induced up-regulation of CCND1 and CCNA mRNA in rAd.βgal-treated livers, overexpression of A20 rescued this outcome, allowing for a significant increase of both cyclins, 24 hours following EH (Fig. 5C,D; n = 3-6). We ascribe this positive outcome in A20-treated livers to lower SOCS3 mRNA levels, hence enhanced IL-6/STAT3 signals, culminating in increased mRNA levels of downstream targets, CCND1 and CCNA. Consequently, hepatocyte proliferation was significantly enhanced, as shown by increased numbers of Ki67/HNF4α-positive hepatocytes, in rAd.A20 versus C and rAd.βgal-treated livers (Fig. 5E; n = 5-6; P < 0.001). Interestingly, greater hepatocyte proliferation in rAd.A20 versus rAd.βgal livers occurred despite significantly higher basal hepatic IL-6 levels in the latter (Fig. S5; n = 4-5; P < 0.001). Higher IL-6 in rAd.βgal-treated livers is caused by the inflammatory response to rAd. that is contained in rAd.A20-treated livers by the anti-inflammatory function of this protein.

Increased SOCS3 Expression in A20 Heterozygous Livers Correlates with Impaired Hepatocyte Proliferation During Liver Regeneration.

We evaluated SOCS3 expression in livers of A20 HT and WT mice before and 24 hours following EH. A20 KO mice die within 3-6 weeks of age, and hence cannot be used for these experiments.21 HT livers showed a tendency towards higher SOCS3 mRNA and protein (IHC) (Fig 6A; n = 5-6) and lower miR203 levels than WT, 24 hours after EH (Fig. 6B; n = 5-6). Increased SOCS3 levels in HT livers associated with impaired hepatocyte proliferation and delayed LR, as evidenced by lower number of Ki67/HNF4α-positive hepatocytes in HT versus WT livers, 36 hours after EH (Fig. 6C; n = 2 (WT) and 5 (HT); P < 0.001). This also correlated with lower P-STAT3 levels in HT versus WT livers 4 hours following EH (Fig. 6D; n = 2), and with inadequate up-regulation of CCND1 (P < 0.05) and CCNA (P < 0.05) mRNA, 24 hours after EH (Fig. 6E,F; n = 5-6).

Figure 6.

A20 knockdown increases SOCS3 expression, impairing hepatocyte proliferation following extended hepatectomy. Mice livers from A20 WT and A20 HT mice were recovered and evaluated for (A) SOCS3 mRNA levels by qPCR and protein expression by immunohistochemistry (brown), before (pre) and 24 hours after (post) EH. Histograms represent mean ± SEM relative SOCS3 mRNA levels (n = 5-6 mice per group). Photomicrographs are representative of 5-6 mice per group. (B) miR-203 mRNA levels by qPCR, pre- and 24 hours post-EH. Histograms represent mean ± SEM relative miR203 mRNA levels after normalization with the housekeeping miRNA gene sno202 (n = 5-6 mice per group). (C) Hepatocyte proliferation by Ki67 immunostaining (brown) and double immunofluorescence (Ki67: red; hepatocyte nuclear factor 4α (HNF): green; DAPI: blue), pre- and 36 hours post-EH. Histograms represent mean ± SEM of Ki67-positive cells per HPF (n = 2 (WT) and 5 (HT) mice). (D) Phospho (P-STAT3) and total STAT-3 by western blot analysis, pre- and 4 hours post-EH. Immunoblots for GAPDH were used as loading control. Histograms represent mean ± SEM of corrected densitometry results, calculated as percentage of P-STAT3/STAT3 ratio in pre WT livers. (E) Cyclin D1 (CCND1) and (F) cyclin A (CCNA) mRNA levels, by qPCR pre- and 24 hours post-EH. Histograms represent mean ± SEM of relative CCND1 and CCNA mRNA levels (n = 5-6 mice per group). Original magnification in (A,C) ×200 (light microscopy) and ×400 (fluorescence microscopy). In (A,E,F) mRNA levels were normalized using TATA box binding protein (TBP) mRNA. *P < 0.05, ***P < 0.001.

A20 Overexpression Attenuates While A20 Knockdown Enhances Expression of STAT3-Dependent Acute Phase Response Genes Following Liver Resection.

IL-6/STAT3 signaling also triggers an acute inflammatory response in hepatocytes that, if uncontrolled, causes liver damage and counterproductively stuns cell cycle progression.9 Accordingly, we measured mRNA levels of STAT3-dependent liver-derived acute phase response proteins serum amyloid A 1 (SAA1) and fibrinogen (FGG) in A20-overexpressing and knockdown livers, at baseline and following EH. Overexpression of A20 significantly decreased EH-induced up-regulation of FGG (Fig. 7A,B; n = 6; P < 0.01), and reduced baseline and EH-induced up-regulation of SAA1. Conversely, A20 HT livers expressed higher levels of SAA1 (Fig 7C; n = 5-6; P < 0.01) and FGG (Fig. 7D; n = 6) following EH. These findings suggest that A20 does not aggravate and may even moderately decrease IL-6-induced pro-inflammatory signals.

Figure 7.

A20 expression regulates acute phase response gene expression following liver resection. Serum amyloid A 1 (SAA1) and fibrinogen gamma chain (FGG) mRNA levels were evaluated by qPCR in livers from: (A,B) rAd.A20 and rAd.βgal transduced livers, and (C,D) WT and A20 HT livers before (pre) and 24 hours after (post) EH (n = 5-6 mice per group). Histograms represent mean ± SEM of relative target gene mRNA levels after normalization with TATA box binding protein (TBP). **P < 0.01.

Discussion

A20, a newly identified player in LR, is induced in mice and humans as part of the hepatocyte's regenerative response following hepatectomy.15, 20 Overexpression of A20 dramatically improves LR and survival in mouse models of extended (78%) and lethal (87%) hepatectomy in part through decreasing the expression of CDKI, p21.15 In contrast, A20 knockdown significantly impairs and delays LR (Studer et al., in prep.). We initiated this work to determine which A20 domain supports this function in hepatocytes. Our results showed that both Nter and 7Zn mutants independently promote hepatocyte proliferation, but neither recapitulates A20′s effect on p21. Akin to A20′s NF-κB inhibitory function, both domains were required to decrease p21. Inhibition of NF-κB by A20 has been ascribed to its sequential N-terminus-mediated deubiquitination of signaling mediators such as RIP and NEMO, followed by C-terminus-mediated degradative polyubiquitination of the same substrates.17 Whether ubiquitin editing or other cooperative mechanisms account for A20 decreasing p21 mRNA is being explored. Regardless, these results suggested that mechanisms other than those involving p21 account for the pro-proliferative function of A20 in hepatocytes. In this work, we identified one of these mechanisms by demonstrating that A20 decreases expression of the negative regulator of IL-6 signaling, SOCS3.

IL-6, produced following hepatectomy by Kupffer cells and hepatocytes,25 is the central trigger of LR by way of phosphorylation/activation of STAT3.27 Successful LR depends on an intact TNF-NFκB-IL-6-STAT3 pathway. TNF-R1 KO, and hepatocyte-specific IL-6 or STAT3 KO mice have impaired regeneration, sometimes causing lethality following PH.5, 27-29 IL-6 administration decreases lethality rates post-PH in TNF-R1 KO mice, indicating that TNF promotes LR mostly by inducing IL-6.28 The impact of A20 on IL-6/STAT3 signaling was unknown. We surmised that overexpression of A20, by blocking LPS/TNF-mediated NF-κB activation in hepatocytes, could reduce IL-6 production and hence limit its own pro-proliferative advantage. We confirmed that overexpression of A20 (but neither Nter nor 7Zn, which do not inhibit NF-κB) significantly decreased LPS/TNF-induced up-regulation of IL-6 in HepG2, without eliminating it, which indicated that IL-6 expression was not exclusively NF-κB-dependent in hepatocytes.22 However, despite lower IL-6 levels, there was stronger baseline and LPS/TNF-induced phosphorylation of STAT3 in A20-overexpressing hepatocytes. This effect was mimicked by 7Zn mutant. In fact, mere overexpression of A20 or 7Zn in HepG2 significantly increased STAT3 phosphorylation at baseline, and these levels were moderately increased or unchanged by IL-6 treatment, indicating that exogenous IL-6 was not necessary to produce this effect. We believe that high and sustained STAT3 phosphorylation in A20/7Zn-overexpressing hepatocytes is key to their pro-proliferative advantage, regardless of whether these cells are treated with IL-6.

Loss of function experiments supported A20′s physiologic impact on IL-6/STAT3 signaling, as they showed significantly higher TNF-induced IL-6 secretion, with paradoxically lower STAT3 phosphorylation in A20 KO and HT hepatocytes. We attribute this paradox to A20 knockdown increasing SOCS3 expression. STAT3 inducible SOCS3 is part of a negative feedback loop that inhibits IL-6 signaling, i.e., STAT3 phosphorylation.30 Gain of function studies confirmed that SOCS3 was the prime target of A20 in modulating IL-6 signaling. Overexpression of A20 or 7Zn in hepatocytes significantly decreased SOCS3 expression, thereby increasing STAT3 phosphorylation. Increased and sustained STAT3 phosphorylation in A20 overexpressing hepatocytes is similar to that seen in IL-6 treated hKO SOCS3 hepatocytes.11, 30 Furthermore, hKO SOCS3 livers, similar to A20-overexpressing livers, demonstrate enhanced and accelerated regeneration following PH.11, 30

In vivo, basal SOCS3 mRNA and protein levels were also significantly enhanced in A20 KO versus WT livers, with A20 HT showing an intermediate phenotype. Basal immunostaining for P-STAT3 was higher in A20 KO versus WT livers. We believe this result represents enhanced inflammation, as indicated by significantly higher IL-6 levels in A20 KO livers causing increased, but still inadequate, STAT3 phosphorylation. This is consistent with impaired hepatocyte proliferation following PH in mice either chronically exposed to high IL-6 levels (like A20 KO), or overexpressing the soluble IL-6-receptor gp80 and concomitantly treated with IL-6.9 Impaired proliferation in these conditions results, at least in part, from IL-6-dependent up-regulation of p21,9, 31 as in A20 KO livers.

IL-6 levels were increased in A20 HT livers at baseline, yet these livers still showed a trend towards higher SOCS3 levels. We discovered that A20 knockdown (KO and HT) significantly decreased hepatic levels of miR-203. Since SOCS3 is an evolutionarily conserved target of miR-203,26 A20-mediated modulation of SOCS3 expression in hepatocytes is, at least in part, epigenetically regulated by A20′s effect on miR-203.

We validated these findings in mouse models of EH. A20 overexpression significantly decreased SOCS3 mRNA and protein levels in mice livers following EH, while A20 knockdown had the opposite effect. Accordingly, STAT3-dependent CCNA and CCND1 levels increased in A20 overexpressing livers, enhancing hepatocyte proliferation following EH. These results are consistent with increased expression of cyclins (D, E, A) and improved LR in SOCS3 heterozygous and hKO SOCS3 mice after PH.11, 31 In contrast, A20 HT livers failed to adequately up-regulate CCND1 and CCNA, hence showed decreased hepatocyte proliferation following EH. We plan to overexpress A20 in IL-6 and SOCS3 KO mice undergoing EH in order to evaluate the contribution of A20′s impact on the IL-6/STAT3/SOCS3 pathway to its overall pro-proliferative function in hepatocytes. We recognize that SOCS3 knockdown / STAT3 activation are linked to hepatocarcinogenesis,11, 32 a potential concern for A20-overexpressing livers. Our previous studies, however, indicate that short-term overexpression of A20 does not carry a significant carcinogenic risk. Indeed, no rAd.A20-treated mice developed liver carcinomas during the 6 months monitoring period.14-16 Longer follow-up periods may be required to completely rule out this risk.

In contrast to cell cycle targets of STAT3, overexpression of A20 slightly decreased and A20 knockdown increased STAT3-induced proinflammatory acute phase response genes, SAA1 and FGG, following EH.33 These data agree with NF-κB (inhibited by A20 overexpression) synergizing with STAT3 to induce acute phase response proteins,34 and with data demonstrating that increased SOCS3 (as in A20 KO) enhances NF-κB activation.35

In summary, this is the first report demonstrating that A20 enhances IL-6 proliferative signals in hepatocytes through down-regulation of SOCS3 while decreasing IL-6 levels and reducing its proinflammatory signals, all of which optimize liver regeneration (Fig. 8). This key finding along with A20 reducing p21 levels underscore A20′s pro-proliferative properties in hepatocytes, and support pursuit of A20-based therapies to promote LR following extensive liver resections for living donation or large tumors.

Figure 8.

A20 promotes proliferative and reduces inflammatory IL-6/STAT3 signals. In response to decreased liver mFfigass and relative increase in hepatic LPS concentration, activated Kupffer cells secrete TNF. When bound to TNF, the TNF receptor transduces mitogenic but also proinflammatory signals in neighboring hepatocytes, which are mediated by the transcription factor NF-κB. Activation of NF-κB occurs by way of phosphorylation of the NF-κB inhibitor IκBα, resulting in the dissociation and subsequent nuclear localization of phosphorylated NF-κB (p65/p50), initiating transcription of NF-κB-dependent genes such as IL-6 and the NF-κB regulatory protein A20/tnfaip3. Upon IL-6 binding, the IL-6R/gp130 dimer induces phosphorylation of JAK1 and 3, which in turn phosphorylates STAT3. Phosphorylated STAT3 dimerizes and translocates to the nucleus, where it binds STAT3 binding element (SBE) and initiates transcription of cyclins, acute phase proteins, and the negative regulator of IL-6 signaling, socs3. A20 regulates IL-6/STAT3 signaling by increasing miR203 levels, which down-regulates SOCS3 mRNA, thus unleashes STAT3 signaling and transcription of STAT3-dependent mitogenic genes such as cyclin d1 and cyclin a. On the other hand, A20 blocks NF-κB signaling, thus reducing transcription of STAT3-dependent proinflammatory genes, such as saa1 and fgg, whose transcription relies on synergy between STAT3 and NF-κB. In sum, overexpression of A20 in hepatocytes promotes their proliferation by favoring IL-6 proliferative over inflammatory signals.

Acknowledgements

We thank Dr. Vishva Dixit and Robert Gerard for providing the A20 plasmid and the recombinant β-galactosidase adenovirus. We also thank Mr. Alon Neidich for help in editing the article.

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