Yoichi Mizukami

Hepatitis C Virus–Induced Reactive Oxygen Species Raise Hepatic Iron Level in Mice by Reducing Hepcidin Transcription

BACKGROUND & AIMS Despite abundant clinical evidence, the mechanisms by which hepatic iron overload develops in patients with hepatitis C virus (HCV)-associated chronic liver disease remain unknown. The aim of this study was to investigate how hepatic iron overload develops in the presence of HCV proteins. METHODS Male transgenic mice expressing the HCV polyprotein and nontransgenic control mice (C57BL/6) were assessed for iron concentrations in the liver, spleen, and serum and iron regulatory molecules in vivo and ex vivo. RESULTS Transgenic mice had increased hepatic and serum iron concentrations, decreased splenic iron concentration, and lower hepcidin expression in the liver accompanied by higher expression of ferroportin in the duodenum, spleen, and liver. In response to hepatocellular iron excess, transferrin receptor 1 expression decreased and ferritin expression increased in the transgenic liver. Transgenic mice showed no inflammation in the liver but preserved the ability to induce hepcidin in response to proinflammatory cytokines induced by lipopolysaccharide. Hepcidin promoter activity and the DNA binding activity of CCAAT/enhancer-binding protein alpha (C/EBP) were down-regulated concomitant with increased expression of C/EBP homology protein, an inhibitor of C/EBP DNA binding activity, and with increased levels of reactive oxygen species in transgenic mice at the ages of 8 and 14 months. CONCLUSIONS HCV-induced reactive oxygen species may down-regulate hepcidin transcription through inhibition of C/EBPalpha DNA binding activity by C/EBP homology protein, which in turn leads to increased duodenal iron transport and macrophage iron release, causing hepatic iron accumulation.

A Novel Mechanism of JNK1 Activation NUCLEAR TRANSLOCATION AND ACTIVATION OF JNK1 DURING ISCHEMIA AND REPERFUSION

Cytokines and various cellular stresses are known to activate c-Jun NH2-terminal kinase (JNK), which plays a role in conveying signals from the cytosol to the nucleus. Here we investigate the translocation and activation of JNK1 during ischemia and reperfusion in perfused rat heart. Ischemia induces the translocation of JNK1 from the cytosol fraction to the nuclear fraction in a time-dependent manner. Immunohistochemical observation also shows that JNK1 staining in the nucleus is enhanced after ischemia. During reperfusion after ischemia, further nuclear translocation of JNK1 is apparently inhibited. In contrast, JNK1 activity in the nuclear fraction does not increased during ischemia but increases significantly during reperfusion with a peak at 10 min of reperfusion. The activation of JNK1 is confirmed by the phosphorylation of endogenous c-Jun (Ser-73) with similar kinetics. The level of c-jun mRNA also increases during reperfusion but not during ischemia. Based on fractionation and immunohistochemical analyses, an upstream kinase for JNK1, SAPK/ERK kinase 1 (SEK1), is constantly present in both the nucleus and cytoplasm throughout ischemia and reperfusion, whereas an upstream kinase for mitogen-activated protein kinase, MAPK/ERK kinase 1, remains in the cytosol. Furthermore, phosphorylation at Thr-223 of SEK1, necessary for its activation, rapidly increases in the nuclear fraction during postischemic reperfusion. These findings demonstrate that JNK1 translocates to the nucleus during ischemia without activation and is then activated during reperfusion, probably by SEK1 in the nucleus.

Ischemic preconditioning translocates PKC-δ and -ε, which mediate functional protection in isolated rat heart

Protein kinase C (PKC) plays an important role in mediating ischemic preconditioning (PC). However, the relationship between PKC isoforms and PC is still uncertain. We analyzed subcellular localization of PKC isoforms by Western blot analysis in isolated rat heart and demonstrate that PKC-α, -δ, and -ε were translocated to the membrane fraction associated with the improvement of cardiac function. Translocation of PKC-δ and -ε persisted after a 30-min period following PC, but the translocation of PKC-α was transient. Under low Ca2+ perfusion (0.2 mmol/l), PC improved the cardiac function associated with the translocation of PKC-δ. Chelerythrine (1.0 μmol/l) suppressed the translocation of all PKC isoforms associated with the loss of improvement of the cardiac function. On the other hand, bisindolylmaleimide (0.1 μmol/l) did not inhibit the improvement of cardiac function induced by PC, which was associated with the translocation of PKC-ε. These results indicate that the effect of PC on cardiac function is mediated by the translocation of either PKC-δ or -ε independently in rat hearts.

Eicosapentaenoic acid (EPA) induces Ca2+-independent activation and translocation of endothelial nitric oxide synthase and endothelium-dependent vasorelaxation

Eicosapentaenoic acid (EPA), but not its metabolites (docosapentaenoic acid and docosahexaenoic acid), stimulated nitric oxide (NO) production in endothelial cells in situ and induced endothelium‐dependent relaxation of bovine coronary arteries precontracted with U46619. EPA induced a greater production of NO, but a much smaller and more transient elevation of intracellular Ca2+ concentration ([Ca2+]i), than did a Ca2+ ionophore (ionomycin). EPA stimulated NO production even in endothelial cells in situ loaded with a cytosolic Ca2+ chelator 1,2‐bis‐o‐aminophenoxythamine‐N′,N′,N′‐tetraacetic acid, which abolished the [Ca2+]i elevations induced by ATP and EPA. The EPA‐induced vasorelaxation was inhibited by N ω ‐nitro‐L‐arginine methyl ester. Immunostaining analysis of endothelial NO synthase (eNOS) and caveolin‐1 in cultured endothelial cells revealed eNOS to be colocalized with caveolin in the cell membrane at a resting state, while EPA stimulated the translocation of eNOS to the cytosol and its dissociation from caveolin, to an extent comparable to that of the eNOS translocation induced by a [Ca2+]i‐elevating agonist (10 μM bradykinin). Thus, EPA induces Ca2+‐independent activation and translocation of eNOS and endothelium‐dependent vasorelaxation.

Sphingosylphosphorylcholine Is a Novel Messenger for Rho-Kinase–Mediated Ca2+ Sensitization in the Bovine Cerebral Artery: Unimportant Role for Protein Kinase C

Although recent investigations have suggested that a Rho-kinase–mediated Ca2+ sensitization of vascular smooth muscle contraction plays a critical role in the pathogenesis of cerebral and coronary vasospasm, the upstream of this signal transduction has not been elucidated. In addition, the involvement of protein kinase C (PKC) may also be related to cerebral vasospasm. We recently reported that sphingosylphosphorylcholine (SPC), a sphingolipid, induces Rho-kinase–mediated Ca2+ sensitization in pig coronary arteries. The purpose of this present study was to examine the possible mediation of SPC in Ca2+ sensitization of the bovine middle cerebral artery (MCA) and the relation to signal transduction pathways mediated by Rho-kinase and PKC. In intact MCA, SPC induced a concentration-dependent (EC50=3.0 &mgr;mol/L) contraction, without [Ca2+]i elevation. In membrane-permeabilized MCA, SPC induced Ca2+ sensitization even in the absence of added GTP, which is required for activation of G-proteins coupled to membrane receptors. The SPC-induced Ca2+ sensitization was blocked by a Rho-kinase inhibitor (Y-27632) and a dominant-negative Rho-kinase, but not by a pseudosubstrate peptide for conventional PKC, which abolished the Ca2+-independent contraction induced by phorbol ester. In contrast, phorbol ester–induced Ca2+ sensitization was resistant to a Rho-kinase inhibitor and a dominant-negative Rho-kinase. In primary cultured vascular smooth muscle cells, SPC induced the translocation of cytosolic Rho-kinase to the cell membrane. We propose that SPC is a novel messenger for Rho-kinase–mediated Ca2+ sensitization of cerebral arterial smooth muscle and, therefore, may play a pivotal role in the pathogenesis of abnormal contraction of the cerebral artery such as vasospasm. The SPC/Rho-kinase pathway functions independently of the PKC pathway.

Phosphoinositide 3-kinase accelerates autophagic cell death during glucose deprivation in the rat cardiomyocyte-derived cell line H9c2

We investigated cell death during glucose deprivation in rat cardiomyocyte-derived H9c2 cells. Electron microscopic analysis revealed accumulation of autophagic vacuoles during glucose deprivation. The addition of 3-methyladenine or LY294002, which are known to inhibit autophagosome formation, reduced cell death while Z-VAD-FMK, a caspase inhibitor, slightly affected cell death. Thus, cell death during glucose deprivation is not type I programmed cell death (apoptotic cell death) but type II programmed cell death (autophagic cell death). Moreover, we found that both insulin-like growth factor-I and the adenovirus-mediated overexpression of wild-type class I PI 3-kinase accelerated cell death as well as accumulation of autophagic vacuoles during glucose deprivation while dominant-negative PI 3-kinase reduced these phenomena. The results indicate that IGF-I/PI 3-kinase accelerates the accumulation of autophagic vacuoles and subsequent autophagic cell death during glucose deprivation, revealing the opposing role of IGF-I/PI 3-kinase in two distinct types of programmed cell death (apoptotic and autophagic cell death).

Involvement of Src Family Protein Tyrosine Kinases in Ca2+ Sensitization of Coronary Artery Contraction Mediated by a Sphingosylphosphorylcholine-Rho-Kinase Pathway

Abstract— We recently reported that sphingosylphosphorylcholine (SPC) is a novel messenger for Rho-kinase–mediated Ca2+ sensitization of vascular smooth muscle (VSM) contraction. Subcellular localization and kinase activity of Src family protein kinases (SrcPTKs), except for c-Src, is controlled by a reversible S-palmitoylation, an event inhibited by eicosapentaenoic acid (EPA). We examined the possible involvement of SrcPTKs in SPC-induced Ca2+ sensitization and effects of EPA. We used porcine coronary VSM and rat aortic VSM cells (VSMCs) in primary culture. An SrcPTKs inhibitor, PP1, and EPA inhibited SPC-induced contraction, concentration-dependently, without affecting [Ca2+]i levels and the Ca2+-dependent contraction induced by high K+ depolarization. A digitized immunocytochemical analysis in VSMCs revealed that SPC induced translocation of Fyn, but not of c-Src, from the cytosol to the cell membrane, an event abolished by EPA. Translocation of Rho-kinase from the cytosol to the cell membrane by SPC was also inhibited by EPA and PP1. The SPC-induced activation of SrcPTKs was blocked by EPA and PP1, but not by Y27632, an Rho-kinase inhibitor. Rho-kinase–dependent phosphorylation of myosin phosphatase induced by SPC was inhibited by EPA, PP1, and Y27632. Translocation and activation of SrcPTKs, including Fyn, play an important role in Ca2+ sensitization of VSM contractions mediated by a SPC-Rho-kinase pathway.

ERK1/2 regulates intracellular ATP levels through alpha-enolase expression in cardiomyocytes exposed to ischemic hypoxia and reoxygenation.

Extracellular signal-regulated kinase 1/2 (ERK1/2) is known to function in cell survival in response to various stresses; however, the mechanism of cell survival by ERK1/2 remains poorly elucidated in ischemic heart. Here we applied functional proteomics by two-dimensional electrophoresis to identify a cellular target of ERK1/2 in response to ischemic hypoxia. Approximately 1500 spots were detected by Coomassie Brilliant Blue staining of a sample from unstimulated cells. The staining intensities of at least 50 spots increased at 6-h reoxygenation after 2-h ischemic hypoxia. Of the 50 spots that increased, at least 4 spots were inhibited in the presence of PD98059, a MEK inhibitor. A protein with a molecular mass of 52 kDa that is strongly induced by ERK1/2 activation in response to ischemic hypoxia and reoxygenation was identified as α-enolase, a rate-limiting enzyme in the glycolytic pathway, by liquid chromatography-mass spectrometry and amino acid sequencing. The expressions of the α-enolase mRNA and protein are inhibited during reoxygenation after ischemic hypoxia in the cells containing a dominant negative mutant of MEK1 and treated with a MEK inhibitor, PD98059, leading to a decrease in ATP levels. α-Enolase expression is also observed in rat heart subjected to ischemia-reperfusion. The induction of α-enolase by ERK1/2 appears to be mediated by c-Myc. The introduction of the α-enolase protein into the cells restores ATP levels and prevents cell death during ischemic hypoxia and reoxygenation in these cells. These results show that α-enolase expression by ERK1/2 participates in the production of ATP during reoxygenation after ischemic hypoxia, and a decrease in ATP induces apoptotic cell death. Furthermore, α-enolase improves the contractility of cardiomyocytes impaired by ischemic hypoxia. Our results reveal that ERK1/2 plays a role in the contractility of cardiomyocytes and cell survival through α-enolase expression during ischemic hypoxia and reoxygenation.

Translocation of protein kinase C-α, δ and ϵ isoforms in ischemic rat heart

To explore the spatial and temporal localization of PKC isoforms during ischemia, we quantified PKC isoforms in the subcellular fractions in perfused rat heart by immunoblotting using specific antibodies against PKC isoforms. PKCs-alpha and epsilon translocated from the 100000 x g supernatant (S, cytosolic) fraction to the 1000 x g pellet (PI, nucleus-myofibril) and the 1000-100000 x g pellet (P2, membrane) fractions during 5-40 min of ischemia. PKC-delta redistributed from the P2 to the S fraction. A 50-kDa fragment of PKC-alpha appeared during ischemia possibly through calpain action. Immunohistochemical observations showed the different localizations of PKC-alpha, delta, and epsilon in the myocytes. The PKC assay displayed high basal levels of Ca(2+)-independent PKC, the activation of Ca(2+)-dependent PKC in the P1 and P2 fractions, and the activation of Ca(2+)-independent PKC in the P1 fraction after 20 min of ischemia. These observations show that ischemia induces different patterns of translocation of the three PKC isoforms, suggesting differences in their roles.

Nuclear translocation of PKCζ during ischemia and its inhibition by wortmannin, an inhibitor of phosphatidylinositol 3-kinase

Protein kinase Cζ (PKCζ), a member of the atypical PKC subgroup, is insensitive to Ca2+, diacylglycerol, and phorbol esters, but is activated by phospholipids such as phosphatidylinositol‐3,4,5‐triphosphate, a product of phosphatidylinositol 3‐kinase (PI3‐kinase). Here we show that PKCζ translocates from the cytosol to the 1000×g pellet (nuclear‐myofibrillar) fraction during ischemia for 40 min in Langendorff‐perfused rat hearts. In addition, immunohistochemical observation shows that ischemia induces the translocation of PKCζ to the nucleus. The nuclear translocation during ischemia is inhibited in a dose‐dependent manner by wortmannin (10−9–10−7 M), an inhibitor of PI3‐kinase.