Interleukin-6/glycoprotein 130-dependent pathways are protective during liver regeneration

After tissue loss the liver has the unique capacity to restore its mass by hepatocyte proliferation. Interleukin-6 (IL6)-deficient mice show a lack in DNA synthesis after partial hepatectomy (PH). To define better the role of IL6 and its family members for liver regeneration after PH, we used conditional knockout mice for glycoprotein 130 (gp130), the common signal transducer of all IL6 family members. We show that gp130-dependent pathways control Stat3 activation after PH. By using gene array analysis, we demonstrate that c-jun, NF-kappa B, c-myc, and tumor necrosis factor receptor expression is gp130-dependent. However, in gp130-deleted mice only minor effects on cell cycle and on the maximum of DNA synthesis after PH were found compared with controls. As in conditional gp130 animals, the acute phase response was completely abolished, we considered that other means are essential to define the role of gp130-dependent pathways for liver regeneration. LPS stimulation in gp130-deleted and also IL6 -/- animals after PH leads to a significant reduction in survival and DNA synthesis, which was associated with decreased Bcl-xL expression and higher apoptosis in the liver. These results indicate that the phenotype concerning the reduction in DNA synthesis might be linked to the degree of infection after PH. Thus our results suggest that the role of gp130-dependent signaling is not a direct influence on cell cycle progression after partial hepatectomy but is to activate protective pathways important to enable hepatocyte proliferation.     

IL-6 modulates hepatocyte proliferation via induction of HGF/p21cip1: regulation by SOCS3

The precise role of IL-6 in liver regeneration and hepatocyte proliferation is controversial and the role of SOCS3 in liver regeneration remains unknown. Here we show that in vitro treatment with IL-6 inhibited primary mouse hepatocyte proliferation. IL-6 induced p21cip1 protein expression in primary mouse hepatocytes. Disruption of the p21cip1 gene abolished the inhibitory effect of IL-6 on cell proliferation. Co-culture with nonparenchymal liver cells diminished IL-6 inhibition of hepatocyte proliferation, which was likely due to IL-6 stimulation of nonparenchymal cells to produce HGF. Finally, IL-6 induced higher levels of p21cip1 protein expression and a slightly stronger inhibition of cell proliferation in SOCS3+/- mouse hepatocytes compared to wild-type hepatocytes, while liver regeneration was enhanced and prolonged in SOCS3+/- mice. Our findings suggest that IL-6 directly inhibits hepatocyte proliferation via a p21cip1-dependent mechanism and indirectly enhances hepatocyte proliferation via stimulating nonparenchymal cells to produce HGF. SOCS3 negatively regulates liver regeneration.     

TNF-alpha induces tyrosine phosphorylation and recruitment of the Src homology protein-tyrosine phosphatase 2 to the gp130 signal-transducing subunit of the IL-6 receptor complex

Recently, it has been demonstrated that TNF-alpha and LPS induce the expression of suppressor of cytokine signaling 3 (SOCS3) and inhibit IL-6-induced STAT3 activation in macrophages. Inhibitor studies suggested that both induction of SOCS3 and inhibition of IL-6-induced STAT3 activation depend on the activation of p38 mitogen-activated protein kinase. Since recruitment of the tyrosine phosphatase Src homology protein tyrosine phosphatase 2 (SHP2) to the signal-transducing receptor subunit gp130 attenuates IL-6-mediated STAT-activation, we were interested in whether TNF-alpha also induces the association of SHP2 to the gp130 receptor subunit. In this study we demonstrate that stimulation of macrophages and fibroblast cell lines with TNF-alpha causes the recruitment of SHP2 to the gp130 signal-transducing subunit and leads to tyrosine phosphorylation of SHP2 and gp130. In this context the cytoplasmic SHP2/SOCS3 recruitment site of gp130 tyrosine 759 is shown to be important for the inhibitory effects of TNF-alpha, since mutation of this residue completely restores IL-6-stimulated activation of STAT3 and, consequently, of a STAT3-dependent promoter. In this respect murine fibroblasts lacking exon 3 of SHP2 are not sensitive to TNF-alpha, indicating that functional SHP2 and its recruitment to gp130 are key events in inhibition of IL-6-dependent STAT activation by TNF-alpha. Furthermore, activation of p38 mitogen-activated protein kinase is shown to be essential for the inhibitory effect of TNF-alpha on IL-6 signaling and TNF-alpha-dependent recruitment of SHP2 to gp130.     

SHP2 and SOCS3 contribute to Tyr-759-dependent attenuation of interleukin-6 signaling through gp130

Interleukin-6 (IL-6) activates the Jak/STAT pathway as well as the mitogen-activated protein kinase cascade. Tyrosine 759 of the IL-6 signal-transducing receptor subunit gp130 has been identified as being involved in negative regulation of IL-6-induced gene induction and activation of the Jak/STAT pathway. Because this site is known to be a recruitment motif for the protein-tyrosine phosphatase SHP2, it has been suggested that SHP2 is the mediator of tyrosine 759-dependent signal attenuation. We recently observed that the suppressor of cytokine-signaling SOCS3 also acts through the tyrosine motif 759 of gp130. However, the relative contributions of SHP2 and SOCS3 to the repression of IL-6 signaling are not understood. Therefore, we designed experiments allowing the independent recruitment of each of these proteins to the IL-6-receptor complex. We show that receptor- and membrane-targeted SHP2 counteracts IL-6 signaling independent of SOCS3 binding to gp130. On the other hand, SOCS3 inhibits signaling in cells expressing a truncated SHP2 protein, which is not recruited to gp130. These data suggest, that there are two, largely distinct modes of negative regulation of gp130 activity, despite the fact that both SOCS3 and SHP2 are recruited to the same site within gp130.     

IL-22-mediated liver cell regeneration is abrogated by SOCS-1/3 overexpression in vitro

The IL-10-like cytokine IL-22 is produced by activated T cells. In this study, we analyzed the role of this cytokine system in hepatic cells. Expression studies were performed by RT-PCR and quantitative PCR. Signal transduction was analyzed by Western blot experiments and ELISA. Cell proliferation was measured by MTS and [(3)H]thymidine incorporation assays. Hepatocyte regeneration was studied in in vitro restitution assays. Binding of IL-22 to its receptor complex expressed on human hepatic cells and primary human hepatocytes resulted in the activation of MAPKs, Akt, and STAT proteins. IL-22 stimulated cell proliferation and migration, which were both significantly inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin. IL-22 increased the mRNA expression of suppressor of cytokine signaling (SOCS)-3 and the proinflammatory cytokines IL-6, IL-8, and TNF-alpha. SOCS-1/3 overexpression abrogated IL-22-induced STAT activation and decreased IL-22-mediated liver cell regeneration. Hepatic IL-22 mRNA expression was detectable in different forms of human hepatitis, and hepatic IL-22 mRNA levels were increased in murine T cell-mediated hepatitis in vivo following cytomegalovirus infection, whereas no significant differences were seen in an in vivo model of ischemia-reperfusion injury. In conclusion, IL-22 promotes liver cell regeneration by increasing hepatic cell proliferation and hepatocyte migration through the activation of Akt and STAT signaling, which is abrogated by SOCS-1/3 overexpression.     

STAT3 contributes to the mitogenic response of hepatocytes during liver regeneration

STAT3 is rapidly induced during liver regeneration in an interleukin 6 (IL-6)-dependent fashion, and IL-6 is required for normal liver regeneration. We wanted to know whether STAT3 was also required for liver regeneration but disruption of the STAT3 gene during embryonic stages causes lethality. Therefore, an albumin promoter-driven Cre-loxP recombination system was used to create a STAT3 deletion in the adult mouse liver to study the role of STAT3 in liver regeneration. After partial hepatectomy, there was virtually no STAT3 RNA or protein induction in Alb(+) STAT3(fl/fl) livers. STAT3 DNA binding activity was also absent in Alb(+) STAT3(fl/fl) livers. Unlike in control livers, STAT1 was activated in STAT3 conditional-mutant livers posthepatectomy. Hepatocyte DNA synthesis at 40 h posthepatectomy in Alb(+) STAT3(fl/fl) livers was reduced to approximately one-third of the control. Alb(+) STAT3(fl/fl) livers had abnormalities in immediate-early gene activation that largely correlated with but were not identical to those seen in IL-6-/- livers. G(1) phase cyclins including cyclins D1 and E had lower expression levels in Alb(+) STAT3(fl/fl) livers, indicating an abnormal G(1) to S phase transition. Therefore, STAT3 accounts for part of the DNA synthetic response of the hepatocytes during liver regeneration, which cannot be compensated for by induction of STAT1. Normal activation of the MAPK pathway in Alb(+) STAT3(fl/fl) livers reinforces the fact that at least part of the effect of IL-6 on hepatocyte proliferation is not mediated by STAT3. This study provides the first in vivo evidence that STAT3 promotes cell cycle progression and cell proliferation under physiological growth conditions.     

Study on the activity of the signaling pathways regulating hepatocytes from G0 phase into G1 phase during rat liver regeneration

Under normal physiological conditions, the majority of hepatocytes are in the functional state (G0 phase). After injury or liver partial hepatectomy (PH), hepatocytes are rapidly activated to divide. To understand the mechanism underlying hepatocyte G0/G1 transition during rat liver regeneration, we used the Rat Genome 230 2.0 Array to determine the expression changes of genes, then searched the GO and NCBI databases for genes associated with the G0/G1 transition, and QIAGEN and KEGG databases for the G0/G1 transition signaling pathways. We used expression profile function (E _t ) to calculate the activity level of the known G0/G1 transition signal pathways, and Ingenuity Pathway Analysis 9.0 (IPA) to determine the interactions among these signaling pathways. The results of our study show that the activity of the signaling pathways of HGF, IL-10 mediated by p38MAPK, IL-6 mediated by STAT3, and JAK/STAT mediated by Ras/ERK and STAT3 are significantly increased during the priming phase (2–6 h after PH) of rat liver regeneration. This leads us to conclude that during rat liver regeneration, the HGF, IL-10, IL-6 and JAK/STAT signaling pathways play a major role in promoting hepatocyte G0/G1 transition in the regenerating liver.     

IL-6 trans-signaling modulates TLR4-dependent inflammatory responses via STAT3

Innate immune responses triggered by the prototypical inflammatory stimulus LPS are mediated by TLR4 and involve the coordinated production of a multitude of inflammatory mediators, especially IL-6, which signals via the shared IL-6 cytokine family receptor subunit gp130. However, the exact role of IL-6, which can elicit either proinflammatory or anti-inflammatory responses, in the pathogenesis of TLR4-driven inflammatory disorders, as well as the identity of signaling pathways activated by IL-6 in a proinflammatory state, remain unclear. To define the contribution of gp130 signaling events to TLR4-driven inflammatory responses, we combined genetic and therapeutic approaches based on a series of gp130(F/F) knock-in mutant mice displaying hyperactivated IL-6-dependent JAK/STAT signaling in an experimental model of LPS/TLR4-mediated septic shock. The gp130(F/F) mice were markedly hypersensitive to LPS, which was associated with the specific upregulated production of IL-6, but not TNF-α. In gp130(F/F) mice, either genetic ablation of IL-6, Ab-mediated inhibition of IL-6R signaling or therapeutic blockade of IL-6 trans-signaling completely protected mice from LPS hypersensitivity. Furthermore, genetic reduction of STAT3 activity in gp130(F/F):Stat3(+/-) mice alleviated LPS hypersensitivity and reduced LPS-induced IL-6 production. Additional genetic approaches demonstrated that the TLR4/Mal pathway contributed to LPS hypersensitivity and increased IL-6 production in gp130(F/F) mice. Collectively, these data demonstrate for the first time, to our knowledge, that IL-6 trans-signaling via STAT3 is a critical modulator of LPS-driven proinflammatory responses through cross-talk regulation of the TLR4/Mal signaling pathway, and potentially implicate cross-talk between JAK/STAT and TLR pathways as a broader mechanism that regulates the severity of the host inflammatory response.     

Proinflammatory cytokine production in liver regeneration is Myd88-dependent, but independent of Cd14, Tlr2, and Tlr4

TNF and IL-6 are considered to be important to the initiation or priming phase of liver regeneration. However, the signaling pathways that lead to the production of these cytokines after partial hepatectomy (PH) have not been identified. Enteric-derived LPS appears to be important to liver regeneration, possibly by stimulating proinflammatory cytokine production after surgery. To determine whether LPS signaling pathways are involved in the regulation of the proinflammatory cytokines TNF and IL-6 during the priming phase of liver regeneration, we performed PH on mice lacking the TLRs Tlr4 and Tlr2, the LPS coreceptor, Cd14, and Myd88, an adapter protein involved in most TLR and IL-1R pathways. In MyD88 knockout (KO) mice after PH, both liver Tnf mRNA and circulating IL-6 levels were severely depressed compared with heterozygous or wild-type mice. Activation of STAT-3 and three STAT-3 responsive genes, Socs3, Cd14, and serum amyloid A2 were also blocked. In contrast, Tlr4, Tlr2, and Cd14 KO mice showed no deficits in the production of IL-6. Surprisingly, none of these KO mice showed any delay in hepatocyte replication. These data indicate that the LPS receptor TLR4, as well as TLR2 and CD14, do not play roles in regulating cytokine production or DNA replication after PH. In contrast, MyD88-dependent pathways appear to be responsible for TNF, IL-6, and their downstream signaling pathways.     

Constitutive expression of cyclo-oxygenase 2 transgene in hepatocytes protects against liver injury

The effect of COX (cyclo-oxygenase)-2-dependent PGs (prostaglandins) in acute liver injury has been investigated in transgenic mice that express human COX-2 in hepatocytes. We have used three well-established models of liver injury: in LPS (lipopolysaccharide) injury in D-GalN (D-galactosamine)-preconditioned mice; in the hepatitis induced by ConA (concanavalin A); and in the proliferation of hepatocytes in regenerating liver after PH (partial hepatectomy). The results from the present study demonstrate that PG synthesis in hepatocytes decreases the susceptibility to LPS/D-GalN or ConA-induced liver injury as deduced by significantly lower levels of the pro-inflammatory profile and plasmatic aminotransferases in transgenic mice, an effect suppressed by COX-2-selective inhibitors. These Tg (transgenic) animals express higher levels of anti-apoptotic proteins and exhibit activation of proteins implicated in cell survival, such as Akt and AMP kinase after injury. The resistance to LPS/D-GalN-induced liver apoptosis involves an impairment of procaspase 3 and 8 activation. Protection against ConA-induced injury implies a significant reduction in necrosis. Moreover, hepatocyte commitment to start replication is anticipated in Tg mice after PH, due to the expression of PCNA (proliferating cell nuclear antigen), cyclin D1 and E. These results show, in a genetic model, that tissue-specific COX-2-dependent PGs exert an efficient protection against acute liver injury by an antiapoptotic/antinecrotic effect and by accelerated early hepatocyte proliferation.     

CCAAT enhancer-binding protein alpha is required for interleukin-6 receptor alpha signaling in newborn hepatocytes

The acute phase response is an evolutionarily conserved response of the liver to inflammatory stimuli, which aids the body in host defense and homeostasis. We have previously reported that CCAAT enhancer-binding protein alpha (C/EBPalpha) is required for the induction of acute phase protein (APP) genes in newborn mice in response to lipopolysaccharide. In this paper, we describe a mechanism by which C/EBPalpha knock-out mice are unable to induce APP gene expression in response to inflammatory stimuli. We demonstrate that the lack of acute phase response in C/EBPalpha knock-out mice is because of a hepatocyte autonomous defect. C/EBPalpha knock-out hepatocytes do not activate STAT3 in response to recombinant interleukin (IL)-6, indicating a defect in the IL-6 pathway. C/EBPalpha knock-out hepatocytes also do not show activation of other IL-6 receptor (IL-6R)-mediated Janus kinase substrates, gp130, SHP-2, and Tyk2. Further examination of the IL-6 pathway demonstrated that C/EBPalpha knock-out hepatocytes have decreased IL-6Ralpha protein levels caused, in part, by reduced protein stability. However, other components of the IL-6 pathway are intact, as demonstrated by rescue of STAT3 activation and APP gene induction with recombinant-soluble IL-6R linked to IL-6 cytokine (Hyper-IL-6) or with another gp130 signaling cytokine, Oncostatin M. In conclusion, C/EBPalpha is required for the proper regulation of IL-6Ralpha protein in hepatocytes resulting in a lack of acute phase protein gene induction in newborn C/EBPalpha null mice in response to lipopolysaccharide or cytokines.     

Overexpression of suppressor of cytokine signaling-3 in T cells exacerbates acetaminophen-induced hepatotoxicity

Cytokines have been implicated in the progression of acetaminophen (APAP)-induced acute liver injury. Suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine signaling by inhibiting the JAK-STAT pathway, but their role in APAP hepatotoxicity is unknown. In this present study, we attempted to explore the role of SOCS3 in T cells in APAP-induced liver injury. Mice with a cell-specific overexpression of SOCS3 in T cells (SOCS3Tg, in which Tg is transgenic) exhibited exaggerated hepatic injury after APAP challenge, as evidenced by increased serum alanine aminotransferase levels, augmented hepatic necrosis, and decreased survival relative to the wild-type mice. Adaptive transfer of SOCS3Tg-CD4(+) T cells into T and B cell-deficient RAG-2(-/-) mice resulted in an exacerbated liver injury relative to the control. In SOCS3Tg mice, hepatocyte apoptosis was enhanced with decreased expression of antiapoptotic protein bcl-2, whereas hepatocyte proliferation was reduced with altered cell cycle-regulatory proteins. Levels of IFN-gamma and TNF-alpha in the circulation were augmented in SOCS3Tg mice relative to the control. Studies using neutralizing Abs indicated that elevated IFN-gamma and TNF-alpha were responsible for the exacerbated hepatotoxicity in SOCS3Tg mice. Activation of STAT1 that is harmful in liver injury was augmented in SOCS3Tg hepatocytes. Alternatively, hepatoprotective STAT3 activation was decreased in SOCS3Tg hepatocytes, an event that was associated with augmented SOCS3 expression in the hepatocytes. Altogether, these results suggest that forced expression of SOCS3 in T cells is deleterious in APAP hepatotoxicity by increasing STAT1 activation while decreasing STAT3 activation in hepatocytes, possibly through elevated IFN-gamma and TNF-alpha.     

Cell proliferation and STAT6 pathways are negatively regulated in T cells by STAT1 and suppressors of cytokine signaling

Suppressor of cytokine signaling (SOCS) proteins have emerged as important regulators of cytokine signals in lymphocytes. In this study, we have investigated regulation of SOCS expression and their role in Th cell growth and differentiation. We show that SOCS genes are constitutively expressed in naive Th cells, albeit at low levels, and are differentially induced by Ag and Th-polarizing cytokines. Whereas cytokines up-regulate expression of SOCS1, SOCS2, SOCS3, and cytokine-induced Src homology 2 protein, Ags induce down-regulation of SOCS3 within 48 h of Th cell activation and concomitantly up-regulate SOCS1, SOCS2, and cytokine-induced Src homology 2 protein expression. We further show that STAT1 signals play major roles in inducing SOCS expression in Th cells and that induction of SOCS expression by IL-4, IL-12, or IFN-gamma is compromised in STAT1-deficient primary Th cells. Surprisingly, IL-4 is a potent inducer of STAT1 activation in Th2 but not Th1 cells, and SOCS1 or SOCS3 expression is dramatically reduced in STAT1(-/-) Th2 cells. To our knowledge, this is the first report of IL-4-induced STAT1 activation in Th cells, and suggests that its induction of SOCS, may in part, regulate IL-4 functions in Th2 cells. In fact, overexpression of SOCS1 in Th2 cells represses STAT6 activation and profoundly inhibits IL-4-induced proliferation, while depletion of SOCS1 by an anti-sense SOCS1 cDNA construct enhances cell proliferation and induces constitutive activation of STAT6 in Th2 cells. These results are consistent with a model where IL-4 has dual effects on differentiating T cells: it simulates proliferation/differentiation through STAT6 and autoregulates its effects on Th2 growth and effector functions via STAT1-dependent up-regulation of SOCS proteins.     

Interleukin-1 receptor antagonist modulates the early phase of liver regeneration after partial hepatectomy in mice

Background

Cytokine administration is a potential therapy for acute liver failure by reducing inflammatory responses and favour hepatocyte regeneration. The aim of this study was to evaluate the role of interleukin-1 receptor antagonist (IL-1ra) during liver regeneration and to study the effect of a recombinant human IL-1ra on liver regeneration.

Methods

We performed 70%-hepatectomy in wild type (WT) mice, IL-1ra knock-out (KO) mice and in WT mice treated by anakinra. We analyzed liver regeneration at regular intervals by measuring the blood levels of cytokines, the hepatocyte proliferation by bromodeoxyuridin (BrdU) incorporation, proliferating cell nuclear antigen (PCNA) and Cyclin D1 expression. The effect of anakinra on hepatocyte proliferation was also tested in vitro using human hepatocytes.

Results

At 24h and at 48h after hepatectomy, IL-1ra KO mice had significantly higher levels of pro-inflammatory cytokines (IL-6, IL-1β and MCP-1) and a reduced and delayed hepatocyte proliferation measured by BrdU incorporation, PCNA and Cyclin D1 protein levels, when compared to WT mice. IGFBP-1 and C/EBPβ expression was significantly decreased in IL-1ra KO compared to WT mice. WT mice treated with anakinra showed significantly decreased levels of IL-6 and significantly higher hepatocyte proliferation at 24h compared to untreated WT mice. In vitro, primary human hepatocytes treated with anakinra showed significantly higher proliferation at 24h compared to hepatocytes without treatment.

Conclusion

IL1ra modulates the early phase of liver regeneration by decreasing the inflammatory stress and accelerating the entry of hepatocytes in proliferation. IL1ra might be a therapeutic target to improve hepatocyte proliferation.