Sanae Haga

Role of STAT-3 in regulation of hepatic gluconeogenic genes and carbohydrate metabolism in vivo.

The transcription factor, signal transducer and activator of transcription-3 (STAT-3) contributes to various physiological processes. Here we show that mice with liver-specific deficiency in STAT-3, achieved using the Cre-loxP system, show insulin resistance associated with increased hepatic expression of gluconeogenic genes. Restoration of hepatic STAT-3 expression in these mice, using adenovirus-mediated gene transfer, corrected the metabolic abnormalities and the alterations in hepatic expression of gluconeogenic genes. Overexpression of STAT-3 in cultured hepatocytes inhibited gluconeogenic gene expression independently of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), an upstream regulator of gluconeogenic genes. Liver-specific expression of a constitutively active form of STAT-3, achieved by infection with an adenovirus vector, markedly reduced blood glucose, plasma insulin concentrations and hepatic gluconeogenic gene expression in diabetic mice. Hepatic STAT-3 signaling is thus essential for normal glucose homeostasis and may provide new therapeutic targets for diabetes mellitus.

Stat3 protects against Fas-induced liver injury by redox-dependent and -independent mechanisms

Signal transducer and activator of transcription-3 (Stat3) is one of the most important molecules involved in the initiation of liver development and regeneration. In order to investigate the hepatoprotective effects of Stat3, we examined whether Stat3 protects against Fas-mediated liver injury in the mouse. A constitutively activated form of Stat3 (Stat3-C) was adenovirally overexpressed in mouse liver by intravenous injection, and then a nonlethal dose of Fas agonist (Jo2) was injected intraperitoneally into the mouse (0.3 microg/g body wt). Stat3-C dramatically suppressed both apoptosis and necrosis induced by Jo2. In contrast, liver-specific Stat3-knockout mice failed to survive following Jo2 injection. Stat3-C upregulated expression of FLICE inhibitor protein (FLIP), Bcl-xL, and Bcl-2, and accordingly downregulated activities of FLICE and caspase-3 that were redox-independent. Interestingly, Stat3-C also upregulated the redox-associated protein redox factor-1 (Ref-1) and reduced apoptosis in liver following Jo2 injection by suppressing oxidative stress and redox-sensitive caspase-3 activity. These findings indicate that Stat3 activation protects against Fas-mediated liver injury by inhibiting caspase activities in redox-dependent and -independent mechanisms.

The survival pathways phosphatidylinositol-3 kinase (PI3-K)/phosphoinositide-dependent protein kinase 1 (PDK1)/Akt modulate liver regeneration through hepatocyte size rather than proliferation.

Liver regeneration comprises a series of complicated processes. The current study was designed to investigate the roles of phosphoinositide‐dependent protein kinase 1 (PDK1)‐associated pathways in liver regeneration after partial hepatectomy (PH) using liver‐specific Pdk1‐knockout (L‐Pdk1KO) and Pdk1/STAT3 double KO (L‐DKO) mice. There was no liver regeneration, and 70% PH was lethal in L‐Pdk1KO mice. Liver regeneration was severely impaired equally in L‐Pdk1KO and L‐DKO mice, even after nonlethal 30% PH. There was no cell growth (measured as increase of cell size) after hepatectomy in L‐Pdk1KO mice, although the post‐PH mitotic response was the same as in controls. As expected, hepatectomy did not induce hepatic Akt‐phosphorylation (Thr308) in L‐Pdk1KO mice, and post‐PH phosphorylation of Akt, mammalian target of rapamycin (mTOR), p70 ribosomal S6 kinase (p70S6K), and S6 were also reduced. To examine the specific role of PDK1‐associated signals, a “pif‐pocket” mutant of PDK1, which allows PDK1 only to phosphorylate Akt, was used. Liver regeneration was recovered in L‐Pdk1KO mice with a “pif‐pocket” mutant of PDK1. This re‐activated Akt in L‐Pdk1KO mice liver and induced post‐PH cell growth, without affecting cell proliferation. Further deletion of STAT3 (L‐DKO mice) did not further deteriorate liver regeneration, although this certainly reduced post‐PH mitotic response. These findings indicate that PDK1/Akt contribute to liver regeneration by regulating cell size. Regarding phosphatidylinositol‐3 kinase (PI3‐K), immediate upstream signal of PDK1, activation of PI3‐K induced cell proliferation via STAT3 activation in the liver of L‐Pdk1KO mice but did not improve impaired liver regeneration. This confirmed the pivotal role of PDK1 in liver regeneration and cell growth. Conclusion: PDK1/Akt‐mediated responsive cell growth is essential for normal liver regeneration after PH, especially when cell proliferation is impaired. (HEPATOLOGY 2009;49:204‐214.)

Preventing hypoxia/reoxygenation damage to hepatocytes by p66shc ablation : Up-regulation of anti-oxidant and anti-apoptotic proteins

BACKGROUND/AIMS Ischemia/reperfusion damage to the liver remains a serious concern in many clinical situations. Major mechanisms for this certainly include oxidative stress. METHODS The effects of ablating the p66 isoform of ShcA (p66(shc)) on hypoxia/reoxygenation (H/R)-induced oxidative stress and cell injury in hepatocytes were investigated. RESULTS Immediately after reoxygenation, AML12 cells were clearly under oxidative stress; many cells underwent apoptosis. However, knockdown of p66(shc) by specific RNAi markedly decreased cellular oxidative stress and H/R-induced apoptosis, as well as conferring resistance to H(2)O(2) insult. These data suggest that prevention of apoptosis conferred by ablation of p66(shc) results from changed ROS-scavenging, but not inhibition of ROS generation. These data were also confirmed in fibroblasts from p66(shc) knockout mice. Anti-oxidant molecules, such as MnSOD and Ref-1 and the anti-apoptotic molecule Bcl-xL were up-regulated, and pro-apoptotic FLICE was down-regulated, by ablation of p66(shc). Interestingly, catalase expression was not affected in p66(shc)-knockdown-AML12 cells although it is a major target in other cell types. CONCLUSIONS Our findings suggest that in hepatocytes, ablation of p66(shc) is cytoprotective against H/R-induced oxidative stress, with MnSOD and Ref-1 playing critical roles, and with up-regulation of Bcl-xL and down-regulation of FLICE contributing jointly to preventing cells from undergoing oxidant-induced apoptosis.

Hypoxia/re-oxygenation-induced, redox-dependent activation of STAT1 (signal transducer and activator of transcription 1) confers resistance to apoptotic cell death via hsp70 induction

STAT1 (signal transducer and activator of transcription 1) is potentially involved in cell survival, as well as cell death, in different types of cells. The present study was designed to examine the effects of STAT1 on hypoxia/re-oxygenation (H/R)-induced cell death and/or survival, and the underlying mechanisms of any such effects. H/R was shown to induce apoptotic cell death of rat hepatocytes. The addition of a STAT1-specific inhibitor, fludarabine, significantly increased the fraction of apoptotic cells after H/R. Following H/R, STAT1 was activated and sequential phosphorylation of Tyr701 and Ser727 was observed, which could be inhibited by the antioxidant N-acetyl-L-cysteine. Tyrosine and serine phosphorylation of STAT1 was mediated by Janus kinase 2 and phosphoinositide 3-kinase/Akt kinase respectively in a redox-dependent manner following H/R. STAT1-induced HSP70 (heat-shock protein 70) expression and the suppression of apoptosis occurred concomitantly. In conclusion, STAT1 activation, in a redox-dependent manner, following H/R may play crucial roles in cell survival, at least partly via HSP70 induction.

Obstructive jaundice increases sensitivity to lipopolysaccharide via TLR4 upregulation: Possible involvement in gut-derived hepatocyte growth factor-protection of hepatocytes

Background:  Patients with obstructive jaundice are prone to sepsis after biliary tract surgery. The present study was designed to determine the effect of biliary obstruction on cytokine responses to lipopolysaccharide (LPS).

Control of allograft rejection by applying a novel nuclear factor-κB inhibitor, dehydroxymethylepoxyquinomicin

Background. Nuclear factor (NF)-&kgr;B plays a crucial role in lymphocyte activation, proliferation, and survival. We examined the immunosuppressive effect of a newly developed NF-&kgr;B inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) in allotransplantation. Methods. Purified C57BL/6 (H-2b) T cells were used for in vitro studies examining activation, proliferation, cytokine production and nuclear NF-&kgr;B and nuclear factor of activated T cells (NFAT) protein levels. A fully major histocompatibility complex incompatible BALB/c (H-2d)-to-C57BL/6 mice cardiac transplantation model was utilized for in vivo studies. DHMEQ was given intraperitoneally to transplant recipients at a various dose starting from day 0. In some, DHMEQ was administered concomitantly with tacrolimus. Results. DHMEQ significantly suppressed &agr;CD3+&agr;CD28 monoclonal antibody–triggered T-cell proliferation, CD25/CD69 expressions, and both interleukin-2 and interferon (IFN)-&ggr; production in a dose-dependent fashion. DHMEQ blocked nuclear translocation of NF-&kgr;B but not NFAT in activated T cells. Combined treatment with DHMEQ and tacrolimus significantly suppressed T cell activation as compared to that of mono-therapy with either agent alone. Single DHMEQ treatment moderately prolonged cardiac allograft survival. Further, combination of DHMEQ plus tacrolimus markedly prolonged graft mean survival time (MST) to 59.5 days when compared to either DHMEQ (MST: 10 days) or tacrolimus (MST: 13 days) treatment alone. Such effect was associated with inhibition of mixed lymphocyte reaction against donor antigen, IFN-&ggr; producing splenocytes and graft cellular infiltration as examined at 5 and 12 days posttransplantation. Conclusion. DHMEQ inhibits nuclear translocation of NF-&kgr;B but not NFAT in activated T cells, and prolongs allograft survival. Blocking both NF-&kgr;B and NFAT by DHMEQ and tacrolimus induces potent immunosuppression, which may become a new modality in controlling allograft rejection.

Contribution of toll-like receptor 2 to the innate response against Staphylococcus aureus infection in mice.

Staphylococcus aureus is a common pathogen that causes a wide range of infectious diseases. The function of TLRs, specifically TLR2, during S. aureus infection is still debated. In this study, we investigated the extent to which TLR2 contributes to the host innate response against the bacterial infection using TLR2-deficient mice. Intravenous inoculation with S. aureus resulted in all TLR2-deficient mice dying within 4 d, along with a high bacterial burden in the livers. However, histological examination showed the same degree of macrophage and neutrophil accumulation in the livers of infected TLR2-deficient mice as that in infected wild-type (WT) mice. TLR2-deficient mouse macrophages also showed normal phagocytic activity, although they failed to express CD36 that appeared on the surface of WT mouse cells upon challenge with heat-killed S. aureus. These data indicate that TLR2, as well as CD36, does not directly affect S. aureus clearance and that CD36 expression on macrophages depends on the presence of TLR2. In vivo infection with S. aureus caused significantly elevated production of TNF-α and IL-6 in the livers and blood of TLR2-deficient mice compared with those in WT mice, while the hepatic and serum levels of IL-10 decreased in these mice. In contrast, lower expression of IL-6 and IL-10, but not of TNF-α, at both the gene and protein levels was found in TLR2-deficient mouse macrophages compared to that in WT mouse cells, in response to challenge with heat-killed S. aureus. These findings suggest that the S. aureus-induced pro-inflammatory cytokine response is not dependent on macrophages and that TLR2 deficiency results in decreased IL-10 release by macrophages, which contributes to dysregulated cytokine balance, impaired bacterial clearance, and mouse death. Therefore, TLR2 possesses a protective function during S. aureus infection by regulating pro- and anti-inflammatory cytokine responses.

Improved Hepatic Regeneration With Reduced Injury by Redox Factor-1 in a Rat Small-Sized Liver Transplant Model

Redox factor‐1 (Ref‐1) has been shown to function in a redox‐dependent manner in the cell. This study was designed to examine the effects of Ref‐1 on liver regeneration as well as protection against postischemic injury in a rat model of 20% partial liver transplantation. Adenovirus carrying the full length of Ref‐1 gene was introduced into liver grafts by ex vivo perfusion via the portal vein during preservation. Liver graft weights were assessed, as well as graft histology, serum levels of alanine aminotransferase (ALT)/bilirubin, DNA binding activities of AP‐1 and Stat3. Redox factor‐1 successfully expressed in the liver graft, improved regeneration by promoting cell proliferation. Overexpression of Ref‐1 protein also reduced post‐transplant injury and inflammatory reactions in the grafts. The increased serum levels of ALT and bilirubin observed after transplantation were significantly reduced by Ref‐1 overexpression. Furthermore, adenovirally overexpressed Ref‐1 in mouse liver successfully promoted liver regeneration after simple partial hepatectomy. Interestingly, Ref‐1 significantly increased DNA binding of Stat3, but not AP‐1. Overexpressed Ref‐1 effectively promoted graft regeneration and reduced postischemic injury in a small‐sized liver transplantation model. The results of the present study may open a new avenue to clinical transplantation of disproportionately sized grafts in living‐related liver transplantation.

Mechanism of impaired regeneration of fatty liver in mouse partial hepatectomy model

Background and Aim:  The mechanism of injury in steatotic liver under pathological conditions been extensively examined. However, the mechanism of an impaired regeneration is still not well understood. The aim of this study was to analyze the mechanism of impaired regeneration of steatotic liver after partial hepatectomy (PH).