Kyoko Okumura

Death receptor 5 mediated-apoptosis contributes to cholestatic liver disease

Chronic cholestasis often results in premature death from liver failure with fibrosis; however, the molecular mechanisms contributing to biliary cirrhosis are not demonstrated. In this article, we show that the death signal mediated by TNF-related apoptosis-inducing ligand (TRAIL) receptor 2/death receptor 5 (DR5) may be a key regulator of cholestatic liver injury. Agonistic anti-DR5 monoclonal antibody treatment triggered cholangiocyte apoptosis, and subsequently induced cholangitis and cholestatic liver injury in a mouse strain-specific manner. TRAIL- or DR5-deficient mice were relatively resistant to common bile duct ligation-induced cholestasis, and common bile duct ligation augmented DR5 expression on cholangiocytes, sensitizing mice to DR5-mediated cholangitis. Notably, anti-DR5 monoclonal antibody-induced cholangitis exhibited the typical histological appearance, reminiscent of human primary sclerosing cholangitis. Human cholangiocytes constitutively expressed DR5, and TRAIL expression and apoptosis were significantly elevated in cholangiocytes of human primary sclerosing cholangitis and primary biliary cirrhosis patients. Thus, TRAIL/DR5-mediated apoptosis may substantially contribute to chronic cholestatic disease, particularly primary sclerosing cholangitis.

Role of adipocytokines in hepatic fibrogenesis

Obesity and insulin resistance are the key factors for progression of hepatic fibrosis in various chronic liver diseases including non‐alcoholic steatohepatitis (NASH). Recently it has been shown that leptin plays a pivotal role in development of hepatic fibrosis. Leptin promotes hepatic fibrogenesis through upregulation of transforming growth factor‐β in Kupffer cells and sinusoidal endothelial cells. Further, leptin facilitates proliferation and prevents apoptosis of hepatic stellate cells. There is a paradox, however, in that ob/ob mice and Zucker rats, which are the obese and diabetic strains, had minimal profibrogenic responses in the liver, most likely because they lack leptin and its receptors. To establish a more clinically relevant model to study the mechanism of fibrogenesis under steatohepatitis, fatty changes and profibrogenic responses in the liver caused by methionine–choline deficiency (MCD) were investigated in the KK‐Ay mouse, which is an obese and diabetic strain. KK‐Ay mice developed more severe hepatic steatosis, inflammation and fibrosis induced by an MCD diet as compared to C57Bl/6 controls. Importantly, KK‐Ay mice lack physiological upregulation of adiponectin levels, suggesting that adiponectin plays a pivotal role not only in regulation of insulin sensitivity but also in modulation of inflammatory and profibrogenic responses in dietary steatohepatitis. Collectively, these findings support the hypothesis that the balance of adipocytokine expression is a key regulator for the progression of hepatic fibrosis in the setting of steatohepatitis.

Role of apoptosis in acetaminophen hepatotoxicity

Acetaminophen overdose causes liver injury by mechanisms involving glutathione depletion, oxidative stress and mitochondrial dysfunction. The role of apoptosis in acetaminophen‐induced cell killing is still controversial. Here, our aim was to evaluate the mitochondrial permeability transition (MPT) as a key factor in acetaminophen‐induced necrotic and apoptotic killing of primary cultured mouse hepatocytes. Acetaminophen (10 μmol/L) induced necrotic killing in approximately 50% of hepatocytes after 6 h and cyclosporin A (CsA), MPT inhibitor, temporarily decreased necrotic killing after 6 h, but cytoprotection was lost after 16 h. Confocal microscopy revealed mitochondrial depolarization and inner membrane permeabilization at approximately 4.5 h after acetaminophen. CsA delayed these changes indicative of the MPT to about 11 h after acetaminophen. TUNEL labeling and caspase 3 activation also increased after acetaminophen. Fructose (20 mmol/L, an ATP‐generating glycolytic substrate) plus glycine (5 mmol/L, a membrane stabilizing amino acid) prevented nearly all necrotic cell killing but paradoxically increased apoptosis. In conclusion, acetaminophen induces the MPT and ATP‐depletion‐dependent necrosis or caspase‐dependent apoptosis as determined, in part, by ATP availability from glycolysis.

Involvement of tumor necrosis factor α and very late activation antigen 4/vascular cell adhesion molecule 1 interaction in surgical-stress-enhanced experimental metastasis

Abstract We examined the influence of surgical stress on hematogenous metastasis of malignant tumor cells. The study was performed by focusing on the involvement of inflammatory cytokines in the serum, raised acutely after surgery, and endothelial adhesion molecules in the metastatic process. Surgical stress, given to C57BL/6 mice before B16-BL6 melanoma inoculation, significantly enhanced the pulmonary metastasis. This enhancement was seen when the surgery lasted for more than 2 h. After the 2-h surgery, the enhancement of pulmonary metastasis was seen most remarkably when B16-BL6 was inoculated 24 h after surgery. The serum level of tumor necrosis factor α (TNFα) in the mice that underwent the 2-h surgery peaked 12 h after the surgery. In contrast, serum interferon γ was not detectable. Administration of an anti-TNFα mAb before the surgery inhibited the enhanced metastasis by inhibiting the increased expression of vascular cell adhesion molecule 1 (VCAM-1) on lung vascular endothelium after the surgery. Pretreatment of B16-BL6 cells with an anti-very late activation antigen 4 (anti-VLA-4) mAb completely inhibited the enhanced metastasis after surgery. Administration of an anti-VCAM-1 mAb before surgery also inhibited the enhancement. These results indicate that serum TNFα , raised by surgical stress, is critically involved in the enhanced pulmonary metastasis of mouse melanoma by inducing VCAM-1 expression on lung vascular endothelium.

Pioglitazone promotes survival and prevents hepatic regeneration failure after partial hepatectomy in obese and diabetic KK‐Ay mice

Pathogenesis of metabolic syndrome–related nonalcoholic steatohepatitis (NASH) involves abnormal tissue‐repairing responses in the liver. We investigated the effect of pioglitazone, a thiazolidinedione derivative (TZD), on hepatic regenerative responses in obese, diabetic KK‐Ay mice. Male KK‐Ay mice 9 weeks after birth underwent two‐thirds partial hepatectomy (PH) after repeated intragastric injections of pioglitazone (25 mg/kg) for 5 days. Almost half of the KK‐Ay mice died within 48 hours of PH;however, mortality was completely prevented in mice pretreated with pioglitazone. In KK‐Ay mice, bromodeoxyuridine (BrdU) incorporation to hepatocyte nuclei 48 hours after PH reached only 1%; however, pioglitazone pretreatment significantly increased BrdU‐positive cells to 8%. Cyclin D1 was barely detectable in KK‐Ay mice within 48 hours after PH. In contrast, overt expression of cyclin D1 was observed 24 hours after PH in KK‐Ay mice pretreated with pioglitazone. Hepatic tumor necrosis factor alpha (TNF‐α) messenger RNA (mRNA) was tremendously increased 1 hour after PH in KK‐Ay mice, the levels reaching ninefold over C57Bl/6 given PH, whereas pioglitazone blunted this increase by almost three‐fourths. Pioglitazone normalized hypoadiponectinemia in KK‐Ay mice almost completely. Serum interleukin (IL)‐6 and leptin levels were elevated extensively 24 hours after PH in KK‐Ay mice, whereas the levels were largely decreased in KK‐Ay mice given pioglitazone. Indeed, pioglitazone prevented aberrant increases in signal transducers and activators of transcription (STAT)3 phosphorylation and suppressor of cytokine signaling (SOCS)‐3 mRNA in the liver in KK‐Ay mice. Conclusion: These findings indicated that pioglitazone improved hepatic regeneration failure in KK‐Ay mice. The mechanism underlying the effect of pioglitazone on regeneration failure most likely involves normalization of expression pattern of adipokines and subsequent cytokine responses during the early stage of PH. (HEPATOLOGY 2009.)

Ursolic acid ameliorates hepatic fibrosis in the rat by specific induction of apoptosis in hepatic stellate cells.

BACKGROUND & AIMS Specific induction of cell death in activated hepatic stellate cells (HSCs) is a promising therapeutic strategy for hepatic fibrosis. In this study, we evaluated the cell-killing effect of ursolic acid (UA), a pentacyclic triterpenoid, in activated HSCs both in vitro and in vivo. METHODS Culture-activated rat HSCs were treated with UA (0-40μM), and the mechanisms of cell death were evaluated. The cell killing effect of UA on activated HSCs in rats chronically treated with thioacetamide (TAA) was detected by dual staining of TdT-mediated dUTP nick-end labeling (TUNEL) and smooth muscle α-actin (αSMA) immunohistochemistry, and resolution of hepatic fibrosis was evaluated. Further, the protective effects of UA on progression of hepatic fibrosis caused by TAA and bile duct ligation (BDL) were evaluated. RESULTS UA induced apoptotic cell death in culture-activated HSCs, but not in isolated hepatocytes and quiescent HSCs. Mitochodrial permeability transition (MPT) preceded the cleavage of caspase-3 and -9 following UA treatment. UA also decreased phosphorylation levels of Akt, and diminished nuclear localization of NFκB in these cells. In rats pretreated with TAA for 6weeks, a single injection of UA induced remarkable increases in TUNEL- and αSMA-dual-positive cells in 24h, and significant regression of hepatic fibrosis within 48h. Moreover, UA ameliorated hepatic fibrogenesis caused by both chronic TAA administration and BDL. CONCLUSIONS UA ameliorated experimental hepatic fibrosis most likely through specific induction of apoptosis in activated HSCs. It is therefore postulated that UA is a potential therapeutic reagent for resolution of hepatic fibrosis.

Adoptive transfer of cytotoxic T lymphocytes induced by CD86-transfected tumor cells suppresses multi-organ metastases of C1300 neuroblastoma in mice

Abstract In this study, we examined the therapeutic antitumor effect of cytotoxic T lymphocytes (CTL) generated against CD86-transfected mouse neuroblastoma C1300. We first generated the transfectant, CD86+C1300, expressing a high level of mouse CD86 on the cell surface. While CD86+C1300 cells were rejected in syngeneic A/J mice when inoculated subcutaneously, neither vaccination nor any therapeutic antitumor effect was obtained, implying that C1300 may be a poorly immunogenic tumor. However, in vitro stimulation of splenocytes from either C1300-bearing or CD86+C1300-rejecting mice with CD86+C1300 cells resulted in remarkable CTL activity against C1300 cells. The CTL activity induced by CD86+C1300 was mediated by T cell receptor/CD3 and CD8 and was further enhanced by the addition of interleukin-2. Intravenous inoculation of C1300 cells led to multiple organ metastases including the liver, lung, kidney, ovary, lymph node and bone marrow. To examine the therapeutic effect of CTL in this metastasis model, CTL induced by parental or CD86+C1300 cells were administrated into C1300-bearing mice. Adoptive transfer of CD86+C1300-induced CTL resulted in marked elimination of multi-organ metastases and prolonged survival in almost all mice, 70% of which survived indefinitely. These results indicate that adoptive transfer of CTL induced by CD86-transfected tumor cells in vitro would be effective and useful for tumor immunotherapy against poorly immunogenic tumors.

High Fat Diet Increases Susceptibility to Hypoxia-Induced Zone 3-Dominant Hepatocyte Apoptosis in Mice

PCR), real time PCR, chromatin immunoprecipitation (ChIP) assay, and immunoblot analysis. Results: Cellular TG content was increased when HepG2 were cultured in the presence of oleic acid, and this increase was prevented by guggulsterone treatment. Guggulsterone prevented LXRα agonist (T0901317) induced activation of SREBP-1c, inhibited its mRNA and protein induction, thereby resulting in the down-regulation of SREBP-1c target genes. This inhibitory effect of guggulsterone was blocked by the protein kinase C (PKC) inhibitor. Moreover, guggulsterone enhanced LXRα phosphorylation at threonine residue and decreased phosphorylation at serine residue, which caused impaired DNA binding activity, leading to inhibition of LXRα transactivation and expression. Conclusion: These results show that guggulsterone prevents LXRα mediated SREBP-1c dependent hepatic steatosis through PKC dependent pathway, and suggests that guggulsterone can therapeutically be used to treat nonalcoholic fatty liver disease.

169 Trail Receptor Dr5-Mediated Apoptosis Signals Induce Screrosing Cholangiopathy in Mice

culture markedly decreased nuclear LXR protein levels, suggesting that 25HC3S may complex with LXR to prevent its activation. 25HC3S administration was followed by doseand timedependent decreases in SREBP-1 protein and mRNA, consistent with down regulation of SREBP-1 transcription. 25HC3S administration also led to a decrease in mature SREBP-1 accompanied by an increase in SREBP-1 precursor, consistent with post-translational downregulation of SREBP-1 activity. 25HC3S decreased the expression of a number of SREBP1-responsive genes including acetyl CoA carboxylase-1 (ACC-1) and fatty acid synthase (FAS), key enzymes involved in fatty acid biosynthesis, as well as Cyp7A1, HMGR, and LDLR, which are key proteins involved in cholesterol metabolism. 25HC, the precursor of 25HC3S, had opposite effects, increasing SREBP-1 and FAS mRNA levels in the hepatocytes. Conclusion: 25HC3S, initially produced in mitochondria in response to excess cholesterol, is a potent down-regulator of the cholesterol and fatty acid biosynthetic pathways. The effects of 25HC3S appear to be mediated via inhibition of LXR/SREBP-1 signaling.