Tsuneo Kitamura

Senescence marker protein-30 knockout mouse liver is highly susceptible to tumor necrosis factor-α- and fas-mediated apoptosis

Senescence marker protein-30 (SMP30) is a calcium-binding protein that decreases in an androgen-independent manner with aging. To elucidate the physiological role of this protein, we introduced a null mutation of the SMP30 gene into the germ line of mice. Despite the complete lack of SMP30 (SMP30-/-), these mutant mice were indistinguishable from their wild-type (SMP30+/+) littermates in terms of development and fertilization capability. We then investigated the tissue susceptibility for apoptosis induced by cytokine using primary cultured hepatocytes, because SMP30 could rescue cells from cell death caused by calcium influx, using a calcium ionophore as previously described. SMP30-/- hepatocytes were found to be more susceptible to apoptosis induced by tumor necrosis factor-alpha (TNF-alpha) plus actinomycin D (ActD) than SMP30+/+ hepatocytes. In addition, the TNF-alpha/ActD-induced caspase-8 activity in SMP30-/- hepatocytes was twofold greater than that in SMP30+/+ hepatocytes. In contrast, no significant difference was observed in the TNF-alpha/ActD-induced nuclear factor-kappa B activation of SMP30+/+ versus SMP30-/- hepatocytes, indicating that SMP30 is not related to TNF-alpha/ActD-induced nuclear factor-kappa B activation itself. Moreover, deletion of the SMP30 gene enhanced liver injury after treatment in vivo with anti-Fas antibody and the SMP30+/- mice showed intermediate susceptibility to Fas-induced apoptosis. Collectively, these results demonstrate that SMP30 acts to protect cells from apoptosis.

Role of Kupffer cells and gut-derived endotoxins in alcoholic liver injury1

The hepatotoxic effects of alcohol have been described in detail, but factors responsible for its hepatotoxicity have only partially been characterized. For example, it is known that chronic ethanol ingestion increases hepatotoxicity and produces fatty liver, hepatitis and cirrhosis. However, acute ethanol consumption reduces endotoxin hepatotoxicity. It now appears that Kupffer cells participate in several aspects of these phenomena. Previously, most studies on the effects of alcohol on liver function have focused chiefly on the hepatocyte. Recently, attention has been directed towards the effect of ethanol ingestion on Kupffer cell function, which is stimulated by gut‐derived endotoxins (lipopolysaccharides) via mechanisms dependent on increased gut permeability and the possible relationship between Kupffer cells and alcohol‐induced liver injury. Here we will review new evidence for the proposal that Kupffer cells and endotoxins play a pivotal role in hepatotoxicity following alcohol exposure, based on studies using the continuous intragastric enteral feeding model developed by Tsukamoto and French and an acute model developed by us.

Inhibition of hepatitis C virus replication by chloroquine targeting virus-associated autophagy.

BackgroundAutophagy has been reported to play a pivotal role on the replication of various RNA viruses. In this study, we investigated the role of autophagy on hepatitis C virus (HCV) RNA replication and demonstrated anti-HCV effects of an autophagic proteolysis inhibitor, chloroquine.MethodsInduction of autophagy was evaluated following the transfection of HCV replicon to Huh-7 cells. Next, we investigated the replication of HCV subgenomic replicon in response to treatment with lysosomal protease inhibitors or pharmacological autophagy inhibitor. The effect on HCV replication was analyzed after transfection with siRNA of ATG5, ATG7 and light-chain (LC)-3 to replicon cells. The antiviral effect of chloroquine and/or interferon-α (IFNα) was evaluated.ResultsThe transfection of HCV replicon increased the number of autophagosomes to about twofold over untransfected cells. Pharmacological inhibition of autophagic proteolysis significantly suppressed expression level of HCV replicon. Silencing of autophagy-related genes by siRNA transfection significantly blunted the replication of HCV replicon. Treatment of replicon cells with chloroquine suppressed the replication of the HCV replicon in a dose-dependent manner. Furthermore, combination treatment of chloroquine to IFNα enhanced the antiviral effect of IFNα and prevented re-propagation of HCV replicon. Protein kinase R was activated in cells treated with IFNα but not with chloroquine. Incubation with chloroquine decreased degradation of long-lived protein leucine.ConclusionThe results of this study suggest that the replication of HCV replicon utilizes machinery involving cellular autophagic proteolysis. The therapy targeted to autophagic proteolysis by using chloroquine may provide a new therapeutic option against chronic hepatitis C.

Sphingosine 1‐phosphate protects rat liver sinusoidal endothelial cells from ethanol‐induced apoptosis: Role of intracellular calcium and nitric oxide

In alcoholic liver disease, ethanol‐induced damage to sinusoidal endothelial cells (SECs) appears to be important in the progression of liver damage. However, little is known about the mechanisms responsible for protection of SECs against ethanol‐induced injury. To elucidate the role of sphingosine 1‐phosphate (S1P), which is stored in platelets and may be released from them on their activation, we investigated the effect of S1P on rat liver SECs in primary culture. Pretreatment of cells with 1 μmol/L S1P attenuated ethanol‐induced apoptosis. Electron microscopy confirmed this protective effect of S1P on damaged SECs in liver tissues after perfusion of ethanol. In the absence of ethanol, S1P increased DNA synthesis as determined via incorporation of bromodeoxyuridine. S1P also ameliorated the decreased DNA synthesis of cells induced by ethanol. Addition of S1P to cells induced an increase in intracellular calcium concentrations and NO production in cells. Western blotting revealed that S1P significantly induced the activation of endothelial NO synthase (eNOS), but not Akt, and that S1P‐induced activation of eNOS was blocked by trifluoperazine, a calmodulin inhibitor. Furthermore, NG‐nitro‐L‐arginine methyl ester, a NO synthase inhibitor, cancelled the effect of S1P on DNA synthesis, apoptosis, and NO production in vitro as well as the protective effect of S1P on cell damage in situ. In conclusion, the biological effect of S1P is at least partially mediated by Ca2+‐sensitive eNOS activation and subsequent NO formation; extracellular S1P could contribute to sinusoidal protection and remodeling in alcoholic liver injury. (HEPATOLOGY 2006;44:1278–1287.)

Protective Effect of Thalidomide on Endotoxin-Induced Liver Injury

BACKGROUND Activation of Kupffer cells by lipopolysaccharide (LPS) plays a pivotal role in the onset of pathophysiological events that occur during endotoxemia, and intracellular calcium concentration ([Ca2+]i) is involved in LPS-stimulated cytokine production. Tumor necrosis factor (TNF)-alpha is produced exclusively by the monocyte-macrophage lineage, which is mostly made up of Kupffer cells, and thalidomide has been shown to reduce TNF-alpha production from macrophages. However, there is increasing evidence that TNF-alpha may play a role in the initiation or progression of multiple organ failure syndrome. Therefore, the purpose of this work was to determine whether thalidomide could prevent LPS-induced liver injury. METHODS Rats were given a single oral dose of thalidomide (5 mg/kg). To assess the sensitization of Kupffer cells, LPS (5 or 10 mg/kg) was administered intravenously, and mortality, liver histology, and transaminases were evaluated 24 hr later. Kupffer cells were isolated 2 hr after thalidomide treatment. After the addition of LPS, [Ca2+]i was measured by using a microspectrofluorometer with the fluorescent indicator fura-2, and TNF-alpha was measured by enzyme-linked immunosorbent assay. RESULTS LPS caused focal necrosis with neutrophil infiltration in the liver. Moreover, LPS dramatically increased transaminases. These pathologic parameters and increases of serum transaminases were diminished markedly by thalidomide. In isolated Kupffer cells, LPS-induced increases in [Ca2+]i and TNF-alpha production were suppressed by treatment with thalidomide. To further explore the mechanism by which thalidomide directly abrogated Kupffer cell sensitivity to LPS, we determined the effect of thalidomide (5 microM) in vitro on LPS-induced [Ca2+]i response and TNF-alpha production. With the addition of thalidomide (5 microM) in vitro to the culture media for 2 hr before LPS, these parameters were suppressed. CONCLUSIONS Thalidomide prevents LPS-induced liver injury via mechanisms dependent on the suppression of TNF-alpha production from Kupffer cells.

Senescence marker protein-30 (SMP30) enhances the calcium efflux from renal tubular epithelial cells

AbstractBackground. Senescence marker protein-30 (SMP30), a calcium binding protein, is preferentially expressed in the renal proximal tubules and hepatocytes and is presumed to play a role in Ca2+ homeostasis. Methods. To explore its physiological functions in the tubular cells, we investigated the effect of SMP30 on Ca2+ efflux via the ATP-dependent plasma membrane calcium pump. LLC-PK1 cells were stably transfected with a cDNA encoding SMP30, and the established transfectants were subjected to ATP responses. Results. Overexpression of SMP30 significantly increased Ca2+ efflux under both basal and ATP-stimulated conditions. Inhibition of calmodulin by trifluoperazine abrogated the enhanced Ca2+ efflux, suggesting that SMP30 activated the calmodulin-dependent Ca2+ pump. It is known that Ca2+ superfluous influx induces cellular injury. Compared with mock-transfected cells, LLC-PK1 cells expressing SMP30 showed resistance to cellular death triggered by Ca2+ superfluous influx. Conclusion. These results suggest the possibility that, in renal tubular cells, endogenous SMP30 participates in Ca2+ efflux via activating the calmodulin-dependent Ca2+ pump and thereby confers resistance of the cells against injury caused by high intracellular Ca2+ concentrations.

Dalteparin Sodium Prevents Liver Injury Due to Lipopolysaccharide in Rat Through Suppression of Tumor Necrosis Factor-α Production by Kupffer Cells

BACKGROUND Sensitization of Kupffer cells (KC) to lipopolysaccharide (LPS) and overproduction of tumor necrosis factor (TNF)-alpha play important roles in the pathogenesis of alcoholic liver damage and sepsis-associated organ injury. Therefore, suppression of TNF-alpha should prove useful for treatment of LPS-induced liver injury. Recently, heparin has been reported to diminish TNF-alpha production from macrophages in response to LPS. Dalteparin sodium (DS) is a low-molecular-weight heparin with a mean molecular weight of 5,000. DS elicits an antithrombotic effect through a mechanism depending on anti-factor Xa activity but not on the antithrombin activity. DS is thus suitable for treatment of disseminated intravascular coagulation because it has a much smaller prohemorrhagic property. In this study, we evaluated whether DS could prevent LPS-induced liver injury. METHODS Female Wistar rats were administered DS (50 IU/kg intraperitoneally) followed by challenge with LPS (5 mg/kg intravenously) 2 hr later. Livers and sera were collected 24 hr later. KC from rats were isolated and cultured in RPMI 1640 supplemented with 10% fetal bovine serum. After the addition of LPS (10 microg/ml) to the culture media, intracellular Ca2+ was measured by using a fluorescent indicator, fura-2. RESULTS LPS (5 mg/kg intravenously) caused focal necrosis and neutrophil infiltration in the control liver. The histological changes and increased alanine aminotransferase levels caused by LPS injection were diminished by treatment with DS. LPS increased intracellular Ca2+ of KC in control rats from the basal level (26 +/- 6 nmol/liter) to 280 +/- 18 nmol/liter. This increase was blunted by DS (126 +/- 28 nmol/liter). The DS treatment decreased the LPS-induced TNF-alpha production by KC from 911 +/- 78 pg/ml to 309 +/- 45 pg/ml (p < 0.05). CONCLUSIONS These results indicate that DS reduces the LPS-induced liver injury through suppression of TNF-alpha production.

Liver regeneration, liver cancers and cyclins

Accumulating evidence has revealed that malignant cell growth is regulated by complex mechanisms involved in genetic and epigenetic factors. Among human cancers, cancer in the liver (hepatocellular carcinoma (HCC)) is characterized by the evidence that it is usually based on chronic liver diseases such as liver cirrhosis or chronic hepatitis, in which the liver is persistently regenerating following hepatic injury. This raises the possibility that repeated hepatocyte proliferation may cause disorder of genes that are regulating the cell cycle in hepatocytes, thus causing HCC. In this article, recent studies focusing on liver regeneration and cancer are reviewed from the viewpoint of the cell cycle that is regulated by cyclin and the associated proteins.

Burn injury sensitizes rat Kupffer cells via mechanisms dependent on gut-derived endotoxin

BackgroundBacterial translocation occurs after thermal injury in association with intestinal barrier loss. Recently, we found that sensitization of Kupffer cells involved gut-derived endotoxin; therefore, the purpose of this work was to study the mechanisms of sensitization of Kupffer cells in burn injury.MethodsRats received a 30% body surface area full-thickness steam burn 24 h before experiments. Serum alanine aminotransferase (ALT) was measured to assess liver damage, and plasma endotoxin in the portal vein were measured. Kupffer cells were isolated 24 h after the burn. Intracellular calcium ([Ca2+]i) in Kupffer cells was measured using a microspectrofluorometer with the fluorescent indicator, fura-2, and tumor necrosis factor (TNF)-α was measured by enzyme-linked immunosorbent assay (ELISA).ResultsLipopolysaccharide (LPS)-induced mortality was increased by burn treatment. This increase was blocked by gadolinium chloride, a Kupffer-cell toxicant. Accordingly, Kupffer cells were involved in this system. The LPS-induced increase of ALT was upregulated by the burn injury. This increase was blocked by pretreatment with antibiotics. Endotoxin levels were increased to almost 300 pg/ml (normal, <20 pg/ml) in the portal veins of rats that received a burn. This increase was blunted by antibiotics. In Kupffer cells isolated from untreated control rats, [Ca2+]i increased to 82 ± 7 nM after the addition of LPS (100 ng/ml). Levels were elevated twofold over control levels in the cells from rats with burn (174 ± 15 nM). In addition, TNF-α production by Kupffer cells isolated from rats with burn was increased fourfold over the based level. Sterilization of the gut with antibiotics completely blocked all effects of the burn on [Ca2+]i and TNF-α release.ConclusionsKupffer cells isolated from rats with burn exhibited sensitization to LPS, involving gut-derived endotoxin. It is concluded that burns sensitize Kupffer cells to LPS via mechanisms that are dependent on gut-derived endotoxin.

Expression of leptin receptors in hepatic sinusoidal cells

Emerging evidence has suggested a critical role of leptin in hepatic inflammation and fibrogenesis, however, the precise mechanisms underlying the profibrogenic action of leptin in the liver has not been well elucidated. Therefore, the present study was designed to investigate the expression and functions of leptin receptors (Ob-R) in hepatic sinusoidal cells. Hepatic stellate cells (HSCs), Kupffer cells and sinusoidal endothelial cells (SECs) were isolated from rat livers by in situ collagenase perfusion followed by differential centrifugation technique, and expression of Ob-Ra and Ob-Rb, short and long Ob-R isoforms, respectively, were analyzed by RT-PCR. Ob-Ra mRNA was detected ubiquitously in HSCs and SECs. In contrast, Ob-Rb was detected clearly only in SECs and Kupffer cells, but not in 7-day cultured HSCs. Indeed, tyrosine-phosphorylation of STAT-3, a downstream event of Ob-Rb signaling, was observed in SECs, but not in HSCs, 1 hr after incubation with leptin. Further, leptin increased AP-1 DNA binding activity and TGF-beta 1 mRNA levels in Kupffer cells and SECs, whereas leptin failed to increase TGF-beta 1 mRNA in HSCs. These findings indicated that SECs and Kupffer cells, but not HSCs, express functional leptin receptors, through which leptin elicits production of TGF-beta 1. It is hypothesized therefore that leptin, produced systemically from adipocytes and locally from HSCs, up-regulates TGF-beta 1 thereby facilitate tissue repairing and fibrogenesis in the sinusoidal microenvironment.