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Specific Inhibition of Ikkβ in Hepatocytes Induces Proliferation of LiverProgenitors in Injured LiversYoungmi Jung, Rafal P. Witek, Wing-Kin Syn, Steve S. Choi, Alessia Omenetti, Anna MaeDiehl

Background and Aims: The transcription factor NF-κB induces expression of hepatocyteviability factors. The IKK complex, consisting of 3 subunits, IKKα, IKKβ and IKKγ regulatesnuclear localization of NF-κB. Hepatocyte-specific deletion of IKKβ (ΔHEP) increases suscept-ibility to apoptotic stimuli, worsening certain liver injuries. HowΔHEP influences regenerativeresponses in injury is unclear. If ΔHEP compromises hepatocyte replicative capacity, progen-itors are expected to undergo compensatory proliferation to regenerate injured livers. Hedge-hog (Hh) pathway activation is known to regulate this process and promote epithelial-mesenchymal transition (EMT) of progenitors. Therefore, we evaluated the hypothesis thathepatocyte proliferation is reduced, and Hh signaling, EMT, and progenitor proliferationincrease after injury in ΔHEP mice. Methods: To induce liver injury, ΔHEP and F(floxed)/F mice were fed methionine choline-deficient, ethionine supplemented (MCDE) diets. Activecaspase 3 (apoptotic marker), Ki67 (proliferation marker), AE1/AE3 and A6 (progenitormarkers), EMT markers, and Hh signaling were compared in ΔHEP and F/F mice beforeand after 1 week of feeding MCDE diets. Results: Although the livers of ΔHEP mice appearedgrossly normal at baseline, they had significantly higher levels of activated caspase 3 onWestern blot. FACS of primary hepatocytes that were double-stained for annexin V andalbumin, confirmed that ΔHEPmice had more apoptotic hepatocytes than F/F mice (p<0.05).Immunohistochemistry (IHC) demonstrated significantly more progenitors in ΔHEP thanF/F mice. These cells were uniformly negative for annexin V by FACS. MCDE feedinginduced liver injury and comparably increased liver weight in both groups. Most hepatocytenuclei in MCDE+F/F mice contained NF-κB p65, whereas almost none of the MCDE+ΔHEPhepatocyte nuclei were NF-κB p65(+). MCDE+F/F mice showed greater hepatocyte prolifera-tion by Ki67 IHC than MCDE+ΔHEP mice (p<0.005). In contrast, ductular cell proliferationwas greater in MCDE+ΔHEP mice (p<0.05), with dramatic expansions of AE1/AE3 and A6-expressing progenitors (p<0.005). Increased Hh signaling in ΔHEP mice was shown byQRT-PCR. The number of Gli2(+) hepatocytes were higher inMCDE+ΔHEP than inMCDE+F/F mice (p<0.05), and induction of TGFβ and EMT-related genes was greater in MCDE+ΔHEPthan MCDE+F/F mice. Increased fibrosis in ΔHEP mice was confirmed by morphometryand hydroxyproline assay. Conclusion: Specific deletion of IKKβ in hepatocytes promotestheir apoptosis and inhibits their proliferative activity, forcing expansion of Hh-responsiveprogenitor populations and induction of EMT to repair liver injury.

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NIM811 Inhibits Mitochondrial Dysfunction, Prevents Liver Injury andStimulates Liver Regeneration After Massive HepatectomyZhi Zhong, Junjiang Sun, Robert Currin, John J. Lemasters

Background. Massive hepatectomy (PHX) leads to failure of remnant livers, but the underly-ing mechanisms remain unclear. Mitochondrial function is critical for cell survival and liverregeneration. A hypermetabolic burden in remnant liver tissue may lead to mitochondrialdysfunction and hepatic failure. Aim. Accordingly, this study investigated whether blockingthe mitochondrial permeability transition (MPT) with N-methyl-4-isoleucine cyclosporin(NIM811), a nonimmunosuppressive analog of cyclosporin A, could decrease injury andimprove regeneration of remnant livers after massive hepatectomy. Methods. C57BL/6 micewere gavaged with NIM811 at a dose of 10 mg/kg or vehicle 2 h before surgery and then5 mg/kg daily afterwards. Mice underwent sham operation or 90% PHX. Results. Serumalanine aminotransferase (ALT) increased to ~1200 U/L 24 h after PHX and then decreasedgradually. NIM811 decreased peak ALT release by ~65%. Apoptotic cells detected by TUNELwere 197 cells/high power field (hpf) after 90% PHX, which decreased to 124 cells/hpf withNIM811. Caspase-3 activity was also 50% lower inNIM811-treatedmice compared to vehicle-treated mice after 90% PHX, confirming decreased apoptosis. 5-Bromo-2'-deoxyuridineincorporation was 30-fold higher in livers of NIM811-treated mice after PHX compared tovehicle-treated mice, indicating improved liver regeneration by NIM811. Hepatic mitochon-drial depolarization and cell death were detected by intravital confocalmicroscopy of rhodam-ine 123 (Rh123) and propidium iodide (PI) in living mice under pentobarbital anesthesia.In sham-operated mice, green Rh123 fluorescence was punctate in virtually all hepatocytes,indicating mitochondrial polarization. By contrast, mitochondria in many hepatocytes didnot take up Rh123 at 3 h after 90% PHX (13 cells/hpf). Importantly, NIM811 decreasedthe number of hepatocytes with depolarized mitochondria by 66%. Non-viable cells wereundetectable in sham-operated mice and increased slightly to 1.4 cells/hpf 3 h after PHX.NIM811 decreased cell death at this early stage after PHX by ~60%. Immunohistochemicalstaining showed clear punctuate localization of cytochome c in mitochondria of sham-operated livers, which became diffuse after PHX, indicating release of cytochrome c frommitochondria. Cytochrome c release was partially blocked by NIM811. Conclusion. NIM811minimizes liver injury and restores liver regeneration after 90% PHX, at least in part, bypreventing MPT onset, subsequent compromised energy supply and proapoptotic cytochomec release (NIDDK).

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The MicroRNA miR-181b Is Involved in Extracellular Matrix RemodelingDuring Hepatic Regeneration Following Partial HepatectomyLyudmyla I. Khrapenko, Nianyuan Huang, Erica Swenson, Chiara Braconi, Tushar Patel

Background: Liver regeneration involves a coordinated program of altered gene expression.Matrix remodeling, an essential component of hepatic regeneration, involves alterated expres-sion of matrix metalloproteases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs).miRNA are potent regulators of gene expression but their role in hepatic regeneration isunknown. Our aim was to identify regulatory miRNA that may contribute to matrixremodeling during hepatic regeneration. Methods: Hepatic regeneration was experimentallyinduced by 2/3 partial hepatectomy in mice. miRNA expression was assessed using a custom

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miRNA microarray. miRNA expression was quantitated by real-time PCR. Protein expressionwas assessed by immunoblot and immunofluorescence assays. Luciferase reporter assayswith downstream regulatory native and mutated miRNA complementary regions were usedto validate miRNA target interactions. HepSV40 immortalized mouse hepatocytes weretransfected with miRNA precursors or anti-miR oligonucleotides to modulate miRNA expres-sion. MMP activity was assessed by in situ zymography in liver tissues and by gel zymographyin cells. Results: The expression of 35 of 318 miRNA (11%) was altered by >1.5-fold (withp<0.05) during the first 240 minutes after partial hepatectomy. Of these only one miRNA,miR-181b, exhibited a prominent and sustained suppression. By real-time PCR analysis, miR-181b expression was reduced to 25±13%, 18±6%, 31±12% and 56±8% of basal expression at15, 45, 90 and 240 minutes respectively following hepatectomy. TIMP-3 was identified asa putative target of miR-181b using target prediction databases. The miR-181b complement-ary sites on the 3'-UTR of TIMP-3 were verified as a direct target for miR-181b using luciferaseassays. Moreover, hepatic TIMP-3 expression was rapidly increased after hepatectomy to126±9% of basal after 45 minutes. In situ zymography identified a transient increase inenzymatic activity following hepatectomy with a peak of ~3-fold of basal by 90 minutes.Over-expression of miR-181b reduced TIMP-3 levels by 36±7% and increased metalloprote-ase activity by 52%±6%, whereas inhibition of miR-181b increased TIMP-3 expression by30±4%. Summary and Conclusions: We report for the first time that: (a) altered expressionof miR-181b occurs immediately following hepatic regeneration, and (b) TIMP-3, a criticalmediator of hepatic regeneration, can be targeted by miR-181b. These studies identify anovel role for miRNA in the regulation of hepatic regeneration and implicate miR-181b asa critical modulator of matrix remodeling and architectural rearrangement following hepatec-tomy.

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The Interferon Stimulated Gene 15 Promotes Hepatitis C Virus Replication,Attenuates the IFN Response in Hepatocytes and Predicts Outcome ofAntiviral TherapyRuth Broering, Martin Trippler, Min Jiang, Mengji Lu, Guido Gerken, Joerg F. Schlaak

Background: The molecular basis for non-response to antiviral therapy of patients withchronic hepatitis C (HCV) that are treated with pegylated interferon-α and ribavirin is notclear. However, non-response to combination therapy has previously been associated witha strong hepatic up-regulation of the interferon stimulated gene 15 (ISG15). Therefore, theaim of this study was to further elucidate the functional role of ISG15 in HCV infection.Methods: ISG15 gene expression was suppressed in human (con1) and murine (MH1)hepatoma cells harboring the HCV con1 replicon I377/NS3-3 using specific siRNAs. Effectson HCV replication and ISG expression were determined by quantitative rtPCR and westernblot analysis. In addition, ISG15 expression was analyzed in liver samples of HCV patientsprior to antiviral therapy. Results: Knockdown of ISG15 expression suppressed HCV replica-tion to levels comparable to IFNs. This was sustained during long-term treatment for morethan 3 months without evidence for the selection of resistant mutations. The antiviral effectof ISG15 knockdown was not mediated by the release of IFN or induction of ISGs. Tripletherapy consisting of ISG15 knockdown, IFN-α and ribavirin led to complete suppressionof NS5a protein expression which corresponded to 99% suppression of HCV-RNA comparedto 75% suppression by IFN-α and ribavirin. The combination of ISG15 knockdown andIFN resulted in enhanced and prolonged expression of selected ISGs. Consistent with theIn Vitro data, significantly elevated hepatic ISG15 levels were found in HCV genotype 1 (vs.genotype 2/3) and in patients with a low In Vivo antiviral response to IFN. Conclusions:These data indicate that ISG15 plays an important role in the replication cycle of HCV andas an attenuator of the IFN-response. Therefore, therapies that are based upon the suppressionof ISG15 may provide a promising strategy to overcome non-response to standard combina-tion treatment in the future.

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Rab7 Dependent DR5 Trafficking to Lysosomes Is Required for the LysosomalPathway of TRAIL Mediated ApoptosisYuko Akazawa, Justin L. Mott, Steven F. Bronk, Nathan W. Werneburg, M. EugeniaGuicciardi, Xue Meng, Mark A. McNiven, Gregory J. Gores

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively triggers celldeath in malignant liver cells, hepatocellular and cholangiocarcinoma cell lines, via deathreceptor 5 (DR5). This TRAIL cytotoxicity is mediated by lysosomal permeabilization (Guicci-ardi ME et al, Am J Physiol Gastrointest Liver Physiol, 2007, Werneburg NW et al, J BiolChem, 2007). However, the cellular processes mediating lysosomal permeabilization byTRAIL:DR5 remain unclear. Our AIM was to explore the concept that TRAIL:DR5 internaliz-ation and trafficking to the lysosomes is required for the lysosomal pathway of apoptosis.METHODS: Apoptosis was induced by treating the hepatocellular carcinoma cell line, Huh-7 cells with human recombinant TRAIL. Internalization of TRAIL:DR5 complex was analyzedby total internal reflection fluorescence (TIRF) microscopy using DR5-EGFP. Co-localizationof DR5-EGFP with lysosomes was examined by confocal microscopy. Lysosomal permeabiliz-ation was evaluated by examining the subcellular localization of cathepsin D a lysosomalprotease, by immunofluorescence. RESULTS: The TRAIL:DR5 complex underwent rapidcellular internalization within 30 minutes upon TRAIL treatment. Within 60 minutes, DR5-EGFP co-localized with Lysotracker red as observed by confocal microscopy (17% co-localization without TRAIL treatment vs. 80% co-localization with TRAIL treatment, p<0.05).Immunoblot analysis also demonstrated DR5 in lysosomal fractions from TRAIL-treated butnot vehicle treated cells. This DR5 trafficking to the lysosomes was explored by using smallinterfering RNA knock down of Rab7, a small GTPase which regulate late endosomaltrafficking of receptors to lysosomes. We first confirmed Rab7 siRNA did not reduce eitherexpression of lysosomal cathepsins nor co-localization of cathepsin B with lysosomes. Rab7siRNA significantly reduced TRAIL-mediated trafficking of DR5 to lysosomes, (80% co-localization in Control cells vs. 42% co-localization in Rab7 siRNA transfected cells, p<0.05),lysosomal disruption (63% diffuse fluorescence cells in control vs. 32% diffuse fluorescencecells in Rab7 siRNA, p<0.05) and apoptosis (48% in control cells vs. 23% in Rab7 siRNAtransfected cells, p<0.05). CONCLUSIONS: Our results indicate that trafficking of

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