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Potential conflict of interest: Nothing to report.
Gp130-mediated IL-6 signaling may play a role in oval cell proliferation in vivo. Levels of IL-6 are elevated in livers of mice treated with a choline-deficient ethionine-supplemented (CDE) diet that induces oval cells, and there is a reduction of oval cells in IL-6 knockout mice. The CDE diet recapitulates characteristics of chronic liver injury in humans. In this study, we determined the impact of IL-6 signaling on oval cell-mediated liver regeneration in vivo. Signaling pathways downstream of gp130 activation were also dissected. Numbers of A6+ve liver progenitor oval cells (LPCs) in CDE-treated murine liver were detected by immunohistochemistry and quantified. Levels of oval cell migration and proliferation were compared in CDE-treated mouse strains that depict models of gp130-mediated hyperactive ERK-1/2 signaling (gp130ΔSTAT), hyperactive STAT-3 signaling (gp130Y757F and Socs-3−/ΔAlb) or active ERK-1/2 as well as active STAT-3 signaling (wild-type). The A6+ve LPC numbers were increased with IL-6 treatment in vivo. The gp130Y757F mice displayed increased A6+ve LPCs numbers compared with wild-type and gp130ΔSTAT mice. Numbers of A6+ve LPCs were also increased in the livers of CDE treated Socs-3−/ΔAlbmice compared with their control counterparts. Lastly, inhibition of ERK-1/2 activation in cultured oval cells increased hyper IL-6-induced cell growth. For the first time, we have dissected the gp130-mediated signaling pathways, which influence liver progenitor oval cell proliferation. Conclusion: Hyperactive STAT-3 signaling results in enhanced oval cell numbers, whereas ERK-1/2 activation suppresses oval cell proliferation. (HEPATOLOGY 2007;45:486–494.)
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Preexisting hepatocytes and cholangiocytes may replace liver tissue lost as a consequence of liver injury.1 If this pathway is impaired during chronic liver injury, then survival depends on recruitment of either a stem cell or a progenitor cell. Liver progenitor cells (LPCs) migrate into the liver, proliferate, and then differentiate to give rise to both hepatocytes and cholangiocytes. The LPC expresses markers in common with cholangiocytes and embryonic hepatocytes and in rodents it is called an “oval cell.” One of the most commonly used markers to detect liver progenitor oval cells in murine liver is A6.2 Tumor necrosis factor and IL-6 may stimulate hepatocyte division.1, 3 In contrast, the function of cytokines in chronic liver injury is not as well characterized.
The signaling pathways that control oval cell proliferation and differentiation remain poorly understood. The role of glycoprotein 130 (gp130)-mediated IL-6 signaling and the gp130-mediated signaling pathways in oval cells in vivo are currently not known. When IL-6 binds to the membrane-bound IL-6 receptor (IL-6R), a gp130 homodimer is recruited. The gp130 molecule is the signal transducing chain and interacts with the tyrosine kinases known as the Janus kinases. These Janus kinase proteins phosphorylate tyrosine residues on gp130, which provides docking bays for the signal transducers and activators of transcription (STAT) proteins. These STAT proteins dock and after phosphorylation translocate to the nucleus, where they regulate transcription of genes such as the suppressor of cytokine signaling 3 (SOCS-3).4 Dimerization of gp130 also leads to activation of the extracellular signal-related kinases (ERK-1/2), which are mitogen-activated protein kinases. SOCS-3 is considered to be the physiological negative regulator of IL-6 signaling.5 It is induced by 40-fold after acute liver injury because of partial hepatectomy.6 The status and role of SOCS-3 expression in chronic liver injury has not been defined. The absence of SOCS-3 in mouse liver (Socs3−/Δ Alb) results in greater IL-6-induced STAT-3 phosphorylation and therefore it mimics a mouse model of hyperactive STAT-3 signaling in the liver.5, 7 Another hyperactive STAT-3 signaling mouse model (gp130Y757F) was recently derived when the tyrosine 757 residue of gp130 was mutated to a phenylalanine that prevented SOCS-3 binding and SHP2-Ras-ERK signaling upon gp130 activation.8 Interestingly, multiple organs including the liver reliably demonstrated high-level STAT-3 phosphorylation both under basal and IL-6 in vivo stimulated conditions.9
In this study, we determine the impact of IL-6 signaling on LPC migration and proliferation in vivo. The signaling pathways downstream of gp130 activation were also dissected. SOCS-3 was shown to be up-regulated in chronically injured mouse and human liver. Interleukin-6 administration resulted in increased migration and proliferation of oval cells in vivo. Using knock-in mouse models, we demonstrate for the first time that hyperactive STAT-3 signaling enhances LPC numbers, whereas ERK-1/2 activation suppressed LPC proliferation.
Pathogen-free 4-week-old male C57BL/6J, (gp130Δ STAT and gp130Y757F8, 10) and liver conditional SOCS-3 knockout mice ([Socs3−/ΔAlb]5) were housed in accordance with the guidelines of the Animal Ethics Committee of the University of Western Australia. The Socs3−/ΔAlb mice possessed a SOCS-3 loxP-flanked allele that was excised in the liver when crossed with mice expressing Cre recombinase under the control of the albumin promoter. The control mice for the Socs3−/Δlb animals possessed the SOCS-3 loxP-flanked allele that was not deleted because of absence of Cre. All genotypes were confirmed by PCR. Mice were fed normal chow and drinking water (Chow) or choline-deficient chow (ICN) and drinking water containing 0.165% ethionine (Sigma) (CDE). Mice were injected with Hanks balanced salt solution (HBSS), human IL-6 (4 μg), or hyper IL-6 (human IL-6/sIL-6R fusion protein; 4 μg)11 every 2 days as indicated in Peters et al.12
Cell Lines and Reagents.
The isolation and characterization of p53 null immortalized liver (PIL)-2 and PIL-2.1 cells has been documented.13–15 The bipotential mouse oval liver (BMOL) cell line was isolated from the livers of wild-type C57BL6/J mice placed on the CDE diet for 2 weeks. The BMOL cell line was derived by using Percoll gradient centrifugation,15 followed by the “plate and wait” method, which enabled spontaneous immortalization.16 Oval cell lines were grown as previously described.14 The anti-ERK 2 (C-14), phospho-ERK (E-4), and phospho-STAT-3 antibodies were purchased from Santa Cruz Biotechnology, the anti-Flag horseradish peroxidase antibody was bought from Sigma (Sydney, Australia), and polyclonal horseradish peroxidase–coupled goat anti-rabbit and anti-mouse antibodies were obtained from Biorad (Hercules, CA). Recombinant human IL-6 and hyper IL-6 were expressed in stably transfected CHO cells and purified from the supernantant as previously described. The ERK inhibitor U0126 was purchased from Chemicon (Melbourne, Australia).
Tissue and Serum Preparation.
Healthy and chronically injured human liver biopsies were kindly provided by Associate Professor Lim Seng Gee (Division of Gastroenterology, Department of Medicine, National University Hospital, Singapore). Sample collection was approved by the Ethics Committee of Gleneagles Hospital and National University Hospital, Singapore.
Mice were anesthetized by injection of 2.5% Avertin, and blood was taken via cardiac puncture. Liver was perfused with PBS, and liver tissue was either snap frozen, embedded in Cryomatrix (Shandon, Pittsburgh, PA) and snap frozen, or fixed in Carnoy's fixative and embedded in paraffin.
Oval Cell Isolation.
Collagenase digestion of intact liver and fractionation by centrifugal elutriation of the isolated cells was performed as described.17 This was followed by affinity purification of the oval cell-containing fraction using a MiniMACS column containing an anti-CD45 antibody (Miltenyl Biotec, Auburn, CA).17 The cells in the CD45−ve column fraction were tested for oval cell markers (such as A6) by immunocytochemistry and determined to contain oval cells at approximately 95% purity. The CD45+ve fraction was considered to contain inflammatory cells.
RNA Isolation and Reverse Transcription Polymerase Chain Reaction.
Total RNA from cell lines or liver was isolated using Trizol (Gibco BRL) and reverse transcription (RT)-PCR conducted as described.14 The following primers were used in RT-PCR analyses:
β-Actin: 5′-GCCAACACAGTGCTGTCTGG-3′ (Forward) and
GAPDH: 5′-TGCCCCCTCTGCTGATGCC-3′ (Forward) and
SOCS-3 (mouse): 5′-GGAGTGGTGGCTCCTGGCTCT-3′ (Forward) and
SOCS-3 (human): 5′-GTCACCCACAGCAAGTTTCC–3′ (Forward) and
Cyclin D1: 5′-CTGACACCAATCTCCTCAACGAC-3′ (Forward) and
Frozen sections were stained for A6 whereas paraffin-embedded sections were stained for proliferating cell nuclear antigen (PCNA), muscle pyruvate kinase, and SOCS-3 as described previously.18–20 The number of oval cells was determined in each mouse liver by counting 10 adjacent periportal nonoverlapping fields at 400× magnification. The mean of the 10 field counts was then calculated. The number of positive cells for A6 or PCNA that were approximately 10 μm in diameter with ovoid nucleus and scant cytoplasm was expressed as a percentage of total cells.
Membrane Gene Array.
The Atlas mouse 1.2 cDNA expression membrane array (Clontech) was conducted according to the manufacturer's instructions.
Serum AST concentrations were determined in serum using the GO-transaminase kit (Sigma), following the manufacturer's instructions.
Cells were stimulated, lysates prepared, and Western blotting performed as described.14
Cell migration was carried out according to Vuillermoz et al.21
MTT Proliferation Assay.
The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assay was conducted as described.14 PIL-2.1 cells were grown in 0.5% fetal bovine serum (FBS) containing media with cytokine or without cytokine (control). The ERK inhibitor U0126 was used during a 30-minute pretreatment and then for the duration of the experiment at a concentration of 10 μM.
The BMOL cell line was transiently transfected with either the pEF BOS empty Flag vector or the pEF BOS SOCS-3 Flag vector using Lipofectamine and Lipofectamine Plus reagent (Invitrogen) and assayed 24 hours and 48 hours after transfection.
Mean values were calculated for all parameters, whereas mean values and standard errors were calculated for migration/proliferation assay data and SOCS-3 messenger RNA (mRNA) expression levels. Mann-Whitney tests were applied to the data, with P ≤ 0.05 indicating a significant difference.
SOCS-3 Is Increased in Injured Mouse and Human Liver, and LPCs Are a Major Source of SOCS-3.
To ascertain the signaling pathways involved in LPC activation, a gene array was conducted on RNA obtained from CDE-injured mouse liver. This revealed that the expression of numerous STAT-3 target genes including Bcl-xL, Akt, and SOCS-3 were up-regulated during the CDE diet (Supplementary Table 1). Both Bcl-xL and Akt are antiapoptotic genes whereas SOCS-3 is the physiological negative regulator of IL-6 signaling.5, 22, 23 Livers from wild-type mice on a CDE and normal chow diet for 2 and 4 weeks were also used to measure SOCS-3 mRNA expression by RT-PCR. The SOCS-3 mRNA levels were increased in mice on a CDE diet (Fig. 1A). The levels of SOCS-3 mRNA in chronically injured human liver showing (1) mild inflammatory cell infiltration, (2) cirrhosis, or (3) HBV positivity were also increased (Fig. 1B). The SOCS-3 mRNA levels were independent of the degree of cirrhosis, HBV infection, or inflammation.
Our next aim was to determine the cellular source of SOCS-3. Primary mouse oval cells, inflammatory cells and hepatocytes all contributed to the increase in SOCS-3 (Fig. 1C). After stimulation with IL-6, high levels of SOCS-3 mRNA were observed in the PIL-2 oval cell line, after 30 minutes, and remained at this level after 60 minutes of stimulation (Fig. 1D). Because SOCS-3 was elevated in chronically injured mouse and human liver and in response to IL-6 stimulation of LPCs, this suggests that IL-6 signaling may play a role during LPC migration and proliferation.
Gp130-Mediated Interleukin-6 Signaling Induces Migration and Proliferation of A6+ve LPCs In Vivo.
IL-6 signaling via the membrane-bound and soluble IL-6R may promote proliferation of oval cells in vivo. We have created a fusion protein that consists of IL-6 covalently fused to the soluble IL-6R, which is called Hyper IL-6.11 Mice were administered the CDE diet for 2 weeks with concurrent stimulations with IL-6 and hyper IL-6. The SOCS-3 mRNA levels were elevated with IL-6 and hyper IL-6 treatment of mice on normal chow compared with HBSS-treated counterparts, which confirms that the cytokines were bioactive in vivo (Fig. 2A).
Serum AST, a marker of liver damage, was measured to determine the extent of liver damage engendered by the CDE diet. Serum AST rose in the HBSS, IL-6, and hyper IL-6-treated mice in response to the CDE diet. There was no consistent trend in serum AST levels in the IL-6 and hyper IL-6-treated CDE mice that would indicate that the degree of liver damage in these mice was different from the HBSS-treated CDE mice (data not shown).
The CDE diet resulted in elevated A6+ve oval cell numbers in the HBSS (Fig. 2B), IL-6 (4 μg; Fig. 2C) and hyper IL-6 (4 μg; Fig. 2D)-treated mice. The A6+ve oval cell numbers were enhanced in CDE IL-6-treated mice (Fig. 2E) and CDE hyper IL-6-treated mice (Fig. 2E) compared with CDE HBSS-treated mice. We then determined whether IL-6 and hyper IL-6 could influence the ability of oval cells to home or migrate to the liver (number of clusters) and then proliferate (number of cells per cluster and PCNA staining) to regenerate the livers of CDE-fed mice. The increased number of A6+ve clusters in IL-6 and hyper IL-6-treated mice suggests that oval cell migration was also increased (Fig. 2F). Oval cell proliferation was also enhanced as IL-6 and hyper IL-6 treatment increased the numbers of A6+ve cells in each cluster (Fig. 2G) and the number of proliferating cell nuclear antigen+ve oval cells (Figure 2H). When CDE-treated mice were injected with 1 μg IL-6 every 2 days for 14 days, an increase in oval cell numbers was not evident (Supplementary Fig. 2).
Gp130 Signaling Promotes Migration and Proliferation of LPCs In Vitro.
The direct biological consequence of hyper IL-6 stimulation on liver progenitor cells was investigated using a migration assay and MTT proliferation assay. Hyper IL-6 significantly increased migration (P = 0.029; Figure 2I) of the PIL2.1 oval cell line. Hyper IL-6 resulted in marked LPC proliferation after 5 and 7 days of treatment (P ≤ 0.0079; Fig. 2J).
Hyperactive STAT-3 Signaling Correlates with Increased Oval Cell-mediated Liver Regeneration.
As IL-6 signaling was shown to influence oval cell migration and proliferation in vivo, it was important to dissect the gp130-mediated signaling pathways (SHP2-Ras-ERK and STAT-3), which are critical. We used the gp130ΔSTAT and gp130Y757F mice, which have been demonstrated to be reliable mouse models of hyperactive SHP2-Ras-ERK signaling and STAT-3 signaling respectively.8–10, 24
The gp130Y757F mice have increased numbers of A6+ve LPCs compared with wild-type and gp130ΔSTAT mice under normal chow conditions (Fig. 3A; P = 0.0082) and after CDE-mediated liver injury (Fig. 3A and Supplementary Fig. 4; P = 0.0031). Migration of A6+ve oval cells was increased in the livers of gp130Y757F mice on the CDE diet as evidenced by the increased number of A6+ve clusters compared with wild-type and gp130ΔSTAT mice (Fig. 3B). Proliferation of A6+ve oval cells was also increased in the livers of gp130Y757F mice on the CDE diet, as the number of A6+ve cells in each cluster was increased compared with wild-type and gp130ΔSTAT mice (Fig. 3C; P = 0.0357). The increased oval cells in the livers of gp130Y757F mice on the normal chow and CDE diet is unlikely to be a consequence of increased injury because they did not display increased serum AST levels compared with their wild-type and gp130ΔSTAT counterparts on their corresponding diets (data not shown). Because SOCS-3 is a STAT-3-regulated gene, the livers of wild-type, gp130Y757F and gp130ΔSTAT mice on the CDE diet were examined for expression of SOCS-3. The SOCS-3 levels were increased 2-fold in the livers of gp130Y757F mice compared with wild-type mice (Fig. 3D). Conversely, SOCS-3 expression in the livers of gp130ΔSTAT mice was reduced to 25% of wild-type counterpart levels (P = 0.0061; Fig. 3D). The expression of another STAT-3-regulated gene, cyclin D1, was also specifically up-regulated in a majority of the livers of gp130Y757F mice on the CDE diet (Fig. 3E).
Oval Cell Proliferation Is Negatively Regulated by ERK-1/2 Signaling.
To determine which signaling pathways are critical for hyper IL-6-induced oval cell proliferation, we inhibited ERK-1/2 phosphorylation 2-fold both before and after hyper IL-6 stimulation using the inhibitor U0126 (Fig. 4A). The levels of STAT-3 phosphorylation were not affected by U0126 treatment. Inhibition of ERK-1/2 activation increased hyper IL-6-induced cell growth (P = 0.0079; Fig. 4B), strongly suggesting that the ERK-1/2 signaling pathway is critical for suppression of proliferation.
To further support the view that decreased STAT-3 signaling dampens proliferation of oval cells, we determined the effect that SOCS-3 overexpression would have on proliferation. When the BMOL cell line was transfected with the SOCS-3 expression construct, the oval cells overexpressing SOCS-3 (Fig. 5A) showed decreased cell growth compared with their empty-vector transfected control counterparts (Fig. 5B; P = 0.0047). To further support that hyperactive STAT-3 activity increases LPC migration and proliferation, we used Socs3−/ΔAlb mice [SOCS-3 knockout (KO)]. These mice are genetically engineered to lack SOCS-3 in albumin-expressing liver cells such as hepatocytes and oval cells (Supplementary Fig. 6D). These mice possess hyper-active STAT-3 activity in response to gp130 activation (Supplementary Fig. 3A;5). The livers of control and Socs3−/ΔAlb (SOCS-3 KO) mice on normal chow possessed less than 0.8% A6+ve oval cells (Fig. 5C). In mice administered the CDE diet, the number of A6+ve LPCs were increased in the livers of control and Socs3−/ΔAlb mice but to a greater extent in the latter (Fig. 5C and Supplementary Fig. 5; P = 0.0401). The increased LPC numbers in the livers of Socs3−/ΔAlb mice on the CDE diet is unlikely to be a consequence of increased injury because they did not display increased serum AST levels compared with their control counterparts on the CDE diet (data not shown). Both migration and proliferation of A6+ve oval cells were increased in the livers of Socs3−/ΔAlb mice as evidenced by the increased number of A6+ve clusters (Fig. 5D; P = 0.0330) and an increased number of A6+ve cells in each cluster (Fig. 5E) compared with control mice, respectively.
In this study we sought to investigate the impact of IL-6 administration in vivo on oval cell migration and proliferation and the responsible gp130-mediated signaling pathways.
We established that gp130Y757F and Socs3−/ΔAlb mice may display enhanced STAT-1/3 signaling in the liver after IL-6 stimulation compared with wild-type counterparts.5, 8, 9 We have demonstrated in gp130Y757F and Socs3−/ΔAlb mice on the CDE diet that hyper-active STAT-3 signaling (Supplementary Fig. 3A) results in increased oval cell proliferation compared with wild-type or control counterparts. Aside from the knock-in mouse setting, the phenomenon of hyperactive STAT-3 signaling may occur in vivo because of numerous cytokines converging on the STAT-3 pathway. Numerous studies have demonstrated a marked increase in STAT-3 phosphorylation in tissue or cells deriving from the liver when SOCS-3 is inactivated.5, 7, 25 Although STAT-1 phosphorylation may also be increased in the liver in the absence of SOCS-3, the extent is less compared with STAT-3.7 Activated STAT-3 and not STAT-1 efficiently translocates to the nucleus after IL-6 stimulation,26 and STAT-3 has the primary role in controlling the acute phase response in the liver after systemic IL-6 treatment.27 Also, it is STAT-3 and not STAT-1 that up-regulates SOCS-3 levels in an effort to dampen STAT-3 signaling. When Western blots for STAT-1(P) and STAT-3(P) were conducted on the livers of gp130Y757F and Socs3−/ΔAlb mice on the CDE diet for 2 weeks, only STAT-3(P) levels were found to be increased compared with wild-type or control counterparts (Supplementary Fig. 3A). In light of the abovementioned studies and our results, we may conclude that hyperactive STAT-3 and not STAT-1 signaling appears to be the promoter of oval cell proliferation. In support of our study, Sanchez and colleagues28 demonstrated that only the highly proliferative oval cell compartment activated in rats after treatment with 2-acetylaminofluorene/partial hepatectomy displays STAT-3 phosphorylation. Because STAT-3 is not activated in CDE-treated gp130ΔSTAT mice, but it is in wild-type mice to some degree, it may have been expected that the gp130ΔSTAT mice may have a blunted oval cell response compared with wild-type mice. The reason the CDE-treated gp130ΔSTAT mice possessed similar oval cell levels to wild-type mice is attributable to compensatory pathways such as tumor necrosis factor signaling29 still being active in the gp130ΔSTAT mice.
Here, we show that SOCS-3 acts to control the oval cell numbers in the liver. Thus, a model for the role of SOCS-3 in chronic liver injury can now be proposed. Because SOCS-3 is increased in oval cells residing in chronically damaged liver, it dampens the proliferative response of oval cells to IL-6 signaling. Our experiments in Socs3−/ΔAlb mice demonstrated that the absence of SOCS-3 enhances oval cell numbers, whereas SOCS-3 overexpression in oval cell lines decreased oval cell proliferation. There is experimental evidence to implicate oval cells as the source of liver cancer.13, 17 Liver cancer may be a consequence of hyper-active proliferation of oval cells attributable to elevated gp130-mediated STAT-3 signaling. By controlling proliferation, SOCS-3 may enhance the regenerative activity of the oval cells, while limiting the detrimental role of oval cells in generating hepatocellular carcinoma. Interestingly, wild-type mice treated with or without IL-6 during the CDE diet possess equivalent SOCS-3 mRNA levels in the liver (Supplementary Fig. 1). Likely, the levels of SOCS-3 in CDE-fed mice are close to maximum expression and further increases due to IL-6 administration may not occur. We may speculate that the increased oval cell migration and proliferation due to IL-6 treatment is a direct result of SOCS-3 not being greater compared with mice fed the CDE diet only. Hence, the increased STAT-3 signaling in IL-6-treated CDE mice is not negatively regulated as efficiently as in mice fed the CDE diet only.
Liver progenitor oval cells are clinically significant, because they are potentially useful for cell and gene therapy to treat metabolic liver diseases.30 Knowledge of the critical cytokine signaling pathways that affect oval cells will underpin strategies to expand and convert these liver progenitor cells in vitro. Our current study highlights that modulation of gp130-mediated signaling in the liver may be beneficial to enhance oval cell–mediated liver regeneration or inhibit oval cell-mediated tumorigenesis.
We thank Elizabeth Klinken and Ross McCulloch for excellent technical assistance. We are grateful to Janina Tirnitz-Parker for use of the BMOL cell line and thankful to Jian-Guo Zhang for providing the rabbit anti-mouse SOCS-3 antibody.