Two new competing pathways establish the threshold for cyclin-B-Cdk1 activation at the meiotic G2/M transition.

Extracellular ligands control biological phenomena. Cells distinguish physiological stimuli from weak noise stimuli by establishing a ligand-concentration threshold. Hormonal control of the meiotic G2/M transition in oocytes is essential for reproduction. However, the mechanism for threshold establishment is unclear. In starfish oocytes, maturation-inducing hormones activate the PI3K-Akt pathway through the Gßγ complex of heterotrimeric G-proteins. Akt directly phosphorylates both Cdc25 phosphatase and Myt1 kinase, resulting in activation of cyclin-B-Cdk1, which then induces meiotic G2/M transition. Here, we show that cyclin-B-Cdk1 is partially activated after subthreshold hormonal stimuli, but this triggers negative feedback, resulting in dephosphorylation of Akt sites on Cdc25 and Myt1, thereby canceling the signal. We also identified phosphatase activity towards Akt substrates that exists independent of stimuli. In contrast to these negative regulatory activities, an atypical Gßγ-dependent pathway enhances PI3K-Akt-dependent phosphorylation. Based on these findings, we propose a model for threshold establishment in which hormonal dose-dependent competition between these new pathways establishes a threshold; the atypical Gßγ-pathway becomes predominant over Cdk-dependent negative feedback when the stimulus exceeds this threshold. Our findings provide a regulatory connection between cell cycle and signal transduction machineries.

Aging modulates susceptibility to mouse liver Mallory-Denk body formation.

Mallory-Denk bodies (MDBs) are hepatocyte cytoplasmic inclusions found in several liver diseases and consist primarily of the cytoskeletal proteins, keratins 8 and 18 (K8/K18). Recent evidence indicates that the extent of stress-induced protein misfolding, a K8>K18 overexpression state, and transglutaminase-2 activation promote MDB formation. In addition, the genetic background and gender play an important role in mouse MDB formation, but the effect of aging on this process is unknown. Given that oxidative stress increases with aging, the authors hypothesized that aging predisposes to MDB formation. They used an established mouse MDB model-namely, feeding non-transgenic male FVB/N mice (1, 3, and 8 months old) with 3,5 diethoxycarbonyl-1,4-dihydrocollidine for 2 months. MDB formation was assessed using immunofluorescence staining and biochemically by demonstrating keratin and ubiquitin-containing crosslinks generated by transglutaminase-2. Immunofluorescence staining showed that old mice had a significant increase in MDB formation compared with young mice. MDB formation paralleled the generation of high molecular weight ubiquitinated keratin-containing complexes and induction of p62. Old mouse livers had increased oxidative stress. In addition, 20S proteasome activity and autophagy were decreased, and endoplasmic reticulum stress was increased in older livers. Therefore, aging predisposes to experimental MDB formation, possibly by decreased activity of protein degradation machinery.

Keratin 8 phosphorylation regulates its transamidation and hepatocyte Mallory-Denk body formation.

Mallory-Denk bodies (MDBs) are hepatocyte inclusions that are associated with poor liver disease prognosis. The intermediate filament protein keratin 8 (K8) and its cross-linking by transglutaminase-2 (TG2) are essential for MDB formation. K8 hyperphosphorylation occurs in association with liver injury and MDB formation, but the link between keratin phosphorylation and MDB formation is unknown. We used a mutational approach to identify K8 Q70 as a residue that is important for K8 cross-linking to itself and other liver proteins. K8 cross-linking is markedly enhanced on treating cells with a phosphatase inhibitor and decreases dramatically on K8 S74A or Q70N mutation in the presence of phosphatase inhibition. K8 Q70 cross-linking, in the context of synthetic peptides or intact proteins transfected into cells, is promoted by phosphorylation at K8 S74 or by an S74D substitution and is inhibited by S74A mutation. Transgenic mice that express K8 S74A or a K8 G62C liver disease variant that inhibits K8 S74 phosphorylation have a markedly reduced ability to form MDBs. Our findings support a model in which the stress-triggered phosphorylation of K8 S74 induces K8 cross-linking by TG2, leading to MDB formation. These findings may extend to neuropathies and myopathies that are characterized by intermediate filament-containing inclusions.

Hepatitis C virus core protein upregulates the expression of vascular endothelial growth factor via the nuclear factor-κB/hypoxia-inducible factor-1α axis under hypoxic conditions.

AIM: Hepatitis C virus (HCV) core protein critically contributes to hepatocarcinogenesis, which is often observed in liver cirrhosis. Since the liver cirrhosis microenvironment is affected by hypoxia, we focused on the possible driving force of HCV core protein on signal relay from hypoxia-inducible factor (HIF)-1α to vascular endothelial growth factor (VEGF). METHODS: Human hepatocellular carcinoma cells stably overexpressing HCV core (Core cells) and NS5A (NS5A cells) were established; empty vector-transfected (EV) cells were used as controls. Hypoxia was induced by oxygen deprivation or by using cobalt chloride (CoCl(2) ). YC-1 was used to inhibit HIF-1α expression. Protein analyses for cultured cells and liver tissues obtained from CoCl(2) -treated HCV core-transgenic (Core-Tg) mice were performed by western blot and/or immunocytochemistry. Cellular mRNA levels were evaluated by quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: Under hypoxia, the sustained expression of HIF-1α, but not HIF-2α, was profoundly observed in Core cells but, was faint in EV and NS5A cells. Immunocytochemistry revealed increased HIF-1α in the nucleus. HIF-1α mRNA levels were significantly higher in Core cells than in EV cells under both normoxia and hypoxia. The HIF-1α-targeted VEGF and Bcl-xL expressions were increased in Core cells under hypoxia and abolished by YC-1 treatment. Hypoxic liver samples of Core-Tg mice indicated significant increases in both HIF-1α and VEGF expression compared with the wild type. CONCLUSIONS: Hepatitis C virus core protein has the distinct potential to transcriptionally upregulate and sustain HIF-1α expression under hypoxia, thereby contributing to increased VEGF expression, a key regulator in the hypoxic milieu of liver cirrhosis.

Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation.

Genetic factors impact liver injury susceptibility and disease progression. Prominent histological features of some chronic human liver diseases are hepatocyte ballooning and Mallory-Denk bodies. In mice, these features are induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in a strain-dependent manner, with the C57BL and C3H strains showing high and low susceptibility, respectively. To identify modifiers of DDC-induced liver injury, we compared C57BL and C3H mice using proteomic, biochemical, and cell biological tools. DDC elevated reactive oxygen species (ROS) and oxidative stress enzymes preferentially in C57BL livers and isolated hepatocytes. C57BL livers and hepatocytes also manifested significant down-regulation, aggregation, and nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH knockdown depleted bioenergetic and antioxidant enzymes and elevated hepatocyte ROS, whereas GAPDH overexpression decreased hepatocyte ROS. On the other hand, C3H livers had higher expression and activity of the energy-generating nucleoside-diphosphate kinase (NDPK), and knockdown of hepatocyte NDPK augmented DDC-induced ROS formation. Consistent with these findings, cirrhotic, but not normal, human livers contained GAPDH aggregates and NDPK complexes. We propose that GAPDH and NDPK are genetic modifiers of murine DDC-induced liver injury and potentially human liver disease.

Copper incorporation into ceruloplasmin is regulated by Niemann-Pick C1 protein.

AIM: Wilson disease is a genetic disorder of copper metabolism characterized by impaired biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. Our previous study showed the late endosome localization of ATP7B and described the copper transport pathway from the late endosome to trans-Golgi network (TGN). However, the cellular localization of ATP7B and copper metabolism in hepatocytes remains controversial. The present study was performed to evaluate the role of Niemann-Pick type C (NPC) gene product NPC1 on intracellular copper transport in hepatocytes. METHODS: We induced the NPC phenotype using U18666A to modulate the vesicle traffic from the late endosome to TGN. Then, we examined the effect of NPC1 overexpression on the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. RESULTS: Overexpression of NPC1 increased holo-Cp secretion to culture medium of U18666A-treated cells, but did not affect the secretion of albumin. Manipulation of NPC1 function affected the localization of ATP7B and late endosome markers, but did not change the localization of a TGN marker. ATP7B co-localized with the late endosome markers, but not with the TGN marker. CONCLUSION: These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via an NPC1-dependent pathway and incorporated into Cp.

Hepatocyte-derived Snail1 propagates liver fibrosis progression.

Chronic exposure of the liver to hepatotoxic agents initiates an aberrant wound healing response marked by proinflammatory, as well as fibrotic, changes, leading to compromised organ structure and function. In a variety of pathological states, correlative links have been established between tissue fibrosis and the expression of transcription factors associated with the induction of epithelial-mesenchymal cell transition (EMT) programs similar to those engaged during development. However, the role played by endogenously derived, EMT-associated transcription factors in fibrotic states in vivo remains undefined. Using a mouse model of acute liver fibrosis, we demonstrate that hepatocytes upregulate the expression of the zinc finger transcriptional repressor, Snail1, during tissue remodeling. Hepatocyte-specific ablation of Snail1 demonstrates that this transcription factor plays a key role in liver fibrosis progression in vivo by triggering the proximal genetic programs that control multiple aspects of fibrogenesis, ranging from growth factor expression and extracellular matrix biosynthesis to the ensuing chronic inflammatory responses that characterize this class of pathological disorders.

Keratin variants predispose to acute liver failure and adverse outcome: race and ethnic associations.

BACKGROUND & AIMS: Keratins 8 and 18 (K8/K18) provide anti-apoptotic functions upon liver injury. The cytoprotective function of keratins explains the overrepresentation of K8/K18 variants in patients with cirrhosis. However, K8/K18 variant-associated susceptibility to acute liver injury, which is well-described in animal models, has not been studied in humans. METHODS: We analyzed the entire coding regions of KRT8 and KRT18 genes (15 total exons and their exon-intron boundaries) to determine the frequency of K8/K18 variants in 344 acute liver failure (ALF) patients (49% acetaminophen-related) and 2 control groups (African-American [n = 245] and previously analyzed white [n = 727] subjects). RESULTS: Forty-five ALF patients had significant amino-acid-altering K8/K18 variants, including 23 with K8 R341H and 11 with K8 G434S. K8 variants were significantly more common (total of 42 patients) than K18 variants (3 patients) (P < .001). We found increased frequency of variants in white ALF patients (9.1%) versus controls (3.7%) (P = .01). K8 R341H was more common in white (P = .01) and G434S was more common in African-American (P = .02) ALF patients versus controls. White patients with K8/K18 variants were less likely to survive ALF without transplantation (P = .02). K8 A333A and G434S variants associated exclusively with African Americans (23% combined frequency in African American but none in white controls; P < .0001), while overall, K18 variants were more common in non-white liver-disease subjects compared to whites (2.8% vs 0.6%, respectively; P = .008). CONCLUSIONS: KRT8 and KRT18 are important susceptibility genes for ALF development. Presence of K8/K18 variants predisposes to adverse ALF outcome, and some variants segregate with unique ethnic and race backgrounds.

Gender dimorphic formation of mouse Mallory-Denk bodies and the role of xenobiotic metabolism and oxidative stress.

BACKGROUND & AIMS: Mallory-Denk bodies (MDBs) are keratin (K)-rich cytoplasmic hepatocyte inclusions commonly associated with alcoholic steatohepatitis. Given the significant gender differences in predisposition to human alcohol-related liver injury, and the strain difference in mouse MDB formation, we hypothesized that sex affects MDB formation. METHODS: MDBs were induced in male and female mice overexpressing K8, which are predisposed to MDB formation, and in nontransgenic mice by feeding the porphyrinogenic compound 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDB presence was determined by histologic, immunofluorescence, and biochemical analyses and correlated to liver injury using serologic and pathologic markers. Cytoskeletal and metabolic liver protein analysis, in vitro metabolism studies, and measurement of oxidative stress markers and protoporphyrin-IX were performed. RESULTS: Male mice formed significantly more MDBs, which was attenuated modestly by estradiol. MDB formation was accompanied by increased oxidative stress. Female mice had significantly fewer MDBs and oxidative stress-related changes, but had increased ductular reaction protoporphyrin-IX accumulation, and MDB-preventive K18 induction. Evaluation of the microsomal cytochrome-P450 (CYP) enzymes revealed significant gender differences in protein expression and activity in untreated and DDC-fed mice, and showed that DDC is metabolized by CYP3A. The changes in CYPs account for the gender differences in porphyria and DDC metabolism. DDC metabolite formation and oxidative injury accumulate on chronic DDC exposure in males, despite more efficient acute metabolism in females. CONCLUSIONS: Gender dimorphic formation of MDBs and porphyria associate with differences in CYPs, oxidative injury, and selective keratin induction. These findings may extend to human MDBs and other neuropathy- and myopathy-related inclusions.

Toward unraveling the complexity of simple epithelial keratins in human disease.

Simple epithelial keratins (SEKs) are found primarily in single-layered simple epithelia and include keratin 7 (K7), K8, K18-K20, and K23. Genetically engineered mice that lack SEKs or overexpress mutant SEKs have helped illuminate several keratin functions and served as important disease models. Insight into the contribution of SEKs to human disease has indicated that K8 and K18 are the major constituents of Mallory-Denk bodies, hepatic inclusions associated with several liver diseases, and are essential for inclusion formation. Furthermore, mutations in the genes encoding K8, K18, and K19 predispose individuals to a variety of liver diseases. Hence, as we discuss here, the SEK cytoskeleton is involved in the orchestration of several important cellular functions and contributes to the pathogenesis of human liver disease.

Niemann-Pick C1 protein transports copper to the secretory compartment from late endosomes where ATP7B resides.

Wilson disease is a genetic disorder characterized by the accumulation of copper in the body by defective biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. However, copper metabolism in hepatocytes has been still unclear. Niemann-Pick disease type C (NPC) is a lipid storage disorder and the most commonly mutated gene is NPC1 and its gene product NPC1 is a late endosome protein and regulates intracellular vesicle traffic. In the present study, we induced NPC phenotype and examined the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. The vesicle traffic was modulated using U18666A, which induces NPC phenotype, and knock down of NPC1 by RNA interference. ATP7B colocalized with the late endosome markers, but not with the trans-Golgi network markers. U18666A and NPC1 knock down decreased holo-Cp secretion to culture medium, but did not affect the secretion of other secretory proteins. Copper accumulated in the cells after the treatment with U18666A. These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via NPC1-dependent pathway and incorporated into apo-Cp to form holo-Cp.

Switching in discoid domain receptor expressions in SLUG-induced epithelial-mesenchymal transition.

BACKGROUND: Acquired features of cells under epithelial-mesenchymal transition (EMT) have not yet been fully identified. The current study was conducted to assess alterations in both the proliferative potential and the responsiveness to extracellular matrices (ECMs) in EMT. METHODS: MDCK cells and SLUG-transfected MDCK clones (SLUG-MDCK) were used in this study. The cell cycle was analyzed by using flow cytometry and Western blotting. ECM-stimulated cell proliferation was examined by using the following ECMs, type I collagen, type IV collagen, fibronectin, and laminin. Protein phosphorylation was detected by immunoprecipitation-Western by using the 4G10 antibody. RESULTS: Both G1 and G2/M arrest were found in the SLUG-MDCK cells, and the responsible molecules for the cell-cycle arrests were, at least in part, p21WAF1/Cip1 and Wee1. Once in contact with type I collagen, the SLUG-MDCK cells, showing the Wee1 degradation, dramatically started to proliferate up to 6-fold in cell number at Day 5, in contrast to only a 2-fold increase in the control. The analysis of the collagen receptors in the SLUG-MDCK cells disclosed a striking increase in the discoid domain receptor (DDR) 2 expression and a clear decrease in the DDR1 expression. The immunoprecipitated DDR2 protein extracted from SLUG-MDCK cells, which were cultured on collagen for 30 minutes, was tyrosine-phosphorylated, indicating valid functionality of the up-regulated receptor. The altered expression from DDR1 to DDR2 was also found in the naturally dedifferentiated sister cell lines of human liver cancer. CONCLUSIONS: Collectively, SLUG-induced EMT may alter the expression profile of receptor tyrosine kinases, including DDRs.

The genetic background modulates susceptibility to mouse liver Mallory-Denk body formation and liver injury.

UNLABELLED: Mallory-Denk bodies (MDBs) are hepatocyte inclusions found in several liver diseases and consist primarily of keratins 8 and 18 (K8/K18) and ubiquitin that are cross-linked by transglutaminase-2. We hypothesized that genetic variables contribute to the extent of MDB formation, because not all patients with an MDB-associated liver disease develop inclusions. We tested this hypothesis using five strains of mice (FVB/N, C3H/He, Balb/cAnN, C57BL/6, 129X1/Sv) fed for three months (eight mice per strain) the established MDB-inducing agent 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDB formation was compared using hematoxylin-and-eosin staining, or immunofluorescence staining with antibodies to K8/K18/ubiquitin, or biochemically by blotting with antibodies to transglutaminase-2/p62 proteins and to K8/K18/ubiquitin to detect keratin cross-linking. DDC feeding induced MDBs in all mouse strains, but there were dramatic strain differences that quantitatively varied 2.5-fold (P < 0.05). MDB formation correlated with hepatocyte ballooning, and most ballooned hepatocytes had MDBs. Immunofluorescence assessment was far more sensitive than hematoxylin-and-eosin staining in detecting small MDBs, which out-numbered (by approximately 30-fold to 90-fold) but did not parallel their large counterparts. MDB scores partially reflected the biochemical presence of cross-linked keratin-ubiquitin species but not the changes in liver size or injury in response to DDC. The extent of steatosis correlated with the total (large+small) number of MDBs, and there was a limited correlation between large MDBs and acidophil bodies. CONCLUSION: Mouse MDB formation has important genetic contributions that do not correlate with the extent of DDC-induced liver injury. If extrapolated to humans, the genetic contributions help explain why some patients develop MDBs whereas others are less likely to do so. Detection and classification of MDBs using MDB-marker-selective staining may offer unique links to specific histological features of DDC-induced liver injury.

Autophagy activation by rapamycin eliminates mouse Mallory-Denk bodies and blocks their proteasome inhibitor-mediated formation.

UNLABELLED: The proteasomal and lysosomal/autophagy pathways in the liver and other tissues are involved in several biological processes including the degradation of misfolded proteins. Exposure of hepatocyte cell lines to proteasome inhibitors (PIs) results in the formation of inclusions that resemble Mallory-Denk bodies (MDBs). Keratins are essential for MDB formation and keratin 8 (K8)-overexpressing transgenic mice are predisposed to MDB formation. We tested the hypothesis that PIs induce MDBs in vivo and that autophagy participates in MDB turnover. The effect of the PI bortezomib (which is used to treat some malignancies) on MDB formation was tested in K8-overexpressing mice and in cultured cells. Inclusion formation was examined using immune and conventional electron microscopy (EM). Bortezomib induced MDB-like inclusions composed of keratins, ubiquitin, and p62 in cultured cells. Short-term exposure to bortezomib induced similar inclusions in K8-overexpressing but not in nontransgenic mice, without causing liver injury. In bortezomib-treated mice, autophagy was activated in hepatocytes as determined by EM and biochemical analysis. Further activation of autophagy by rapamycin (Rap) decreased the number of inclusions in bortezomib-treated K8 transgenic mice significantly. Rap also led to resorption of spontaneously formed MDBs in aging K8-overexpressing mice. Immune EM demonstrated K8-positive and ubiquitin-positive structures in autophagic vacuoles in the mouse liver. CONCLUSION: PIs alone are sufficient to induce MDBs in susceptible animals, while Rap-mediated activation of autophagy prevents MDB formation and causes MDB resorption. These findings suggest that some patients treated with PIs may become predisposed to MDB formation. Autophagy provides a potential cellular mechanism for the resorption of cytoplasmic inclusions.

Mutation in keratin 18 induces mitochondrial fragmentation in liver-derived epithelial cells.

Microtubules (MTs) and microfilaments (MFs) are known to modulate mitochondrial morphology, distribution and function. However, little is known evidence about the role of intermediate filaments (IFs) in modulating mitochondria except desmin. To investigate whether or not the IFs regulate mitochondrial morphology, distribution, and function, we manipulated the IFs of cultured epithelial cells to express a mutant keratin 18 (K18). In contrast to the filamentous expression of wild K18, mutant K18 induced aggregation of K8/18, showing no fine IF network in the cells. In mutant K18-transfected cells, the mitochondria were fragmented into small spheroids, although they were observed as mitochondrial fibers in un-transfected or wild K18-transfected cells. Fluorescence recovery after photobleaching of fluorescence-labeled mitochondria was markedly less in the mutant K18-transfected cells, although a significant recovery was confirmed in wild K18-transfected cells. These findings suggest that the IFs are important for the maintenance of normal mitochondrial structures.

Keratin inclusions alter cytosolic protein localization in hepatocytes.

AIM: Mallory bodies have been observed in various liver diseases, however, the precise mechanism and significance of these structures have yet to be determined. METHODS: Previously we reported on the redistribution of cytosolic proteins to keratin inclusions in mutant keratin 18-transfected cells. In this study, we treated green fluorescent protein-tagged wild-type keratin 18-transfected cells with several proteasome inhibitors and performed immunofluorescent analyses. RESULTS: Proteasome inhibitors induced intracellular keratin inclusions, and desmoplakin, zonula occludens-1 and beta-catenin were relocated to keratin inclusions, while theintegral membrane proteins were intact. The cytosolic proteins, 14-3-3 zeta protein and glucose-6-phosphate dehydrogenase were also relocated to inclusions. Moreover, E-cadherin, a basolateral membrane protein, was present on both the apical and basolateral domains in inclusion-containing cells. CONCLUSION: These data are identical to those in the mutant keratin 18 transfection study and suggest that keratin inclusions induced by different treatments affect localization of various cytosolic components, which may influence cellular functions performed by these proteins.

Oxidative stress induces the endoplasmic reticulum stress and facilitates inclusion formation in cultured cells.

BACKGROUND/AIMS: The precise mechanism of formation and significance of Mallory bodies (MBs) are poorly understood. The endoplasmic reticulum (ER) is the organelle responsible for proper folding and elimination of unfolded proteins. Therefore, failure of this function increases defective proteins in the cell. METHODS: We examined the effects of oxidative stress on induction of ER stress and keratin 8 and 18 (K8/18)-containing inclusion formation in cultured human hepatoma cells and hepatocytes by immunofluorescence and immunoblot analyses. RESULTS: Generation of H(2)O(2) was detected in glucose oxidase (GO)-treated cells by 2',7'-dichlorodihydrofluorescein diacetate and co-treatment with GO and acetyl-leucyl-leucyl-norleucinal (ALLN), a proteasome inhibitor, induced formation of extensive keratin inclusions that were inhibited by pre-treatment with N-acetyl-cysteine. These inclusions shared similar features with MBs by immunofluorescence analysis. Electron microscopy showed that these structures appeared near the nuclei, surrounded by filamentous structures. GO and ALLN upregulated the expression of ER stress markers, however, 4-phenylbutyrate, a chemical chaperone, reduced formation of inclusions and expression of the ER stress markers. CONCLUSIONS: The oxidative stress coupled with limited inhibition of the proteasome induces dysfunction of the ER and results in inclusion formation in cultured cells. This suggests that ER stress plays a role in MB formation in liver disease.

Clearance of HCV improves insulin resistance, beta-cell function, and hepatic expression of insulin receptor substrate 1 and 2.

OBJECTIVES: Hepatitis C virus (HCV) infection is linked to greater insulin resistance. Although HCV itself is a candidate for the development of insulin resistance, the effects of antiviral treatment on impaired glucose metabolism remain unclear. The aim of this study is to examine the effects of clearance of HCV on insulin resistance, beta-cell function, and hepatic expression of insulin receptor substrate (IRS)1/2, central molecules for insulin signaling. METHODS: We analyzed 89 biopsy-proven patients with chronic HCV infection. Patients received interferon-alpha or interferon-alpha plus ribavirin for 6 months and were classified into three groups at 6 months after the conclusion of antiviral therapy according to their response to antiviral therapy: sustained responders (N = 29), relapsers (N = 12), and nonresponders (N = 48). Insulin resistance and beta-cell function were assessed by the homeostasis model assessment method (HOMA-IR and HOMA-%B, respectively). Hepatic expression of IRS1/2 was evaluated by immunoblotting and immunostaining in 14 sustained responders. RESULTS: In nonresponders and relapsers, there were no significant changes in HOMA-IR and HOMA-%B values after antiviral therapy. On the other hand, in sustained responders, HOMA-IR values significantly decreased to 1.7 +/- 0.8 from 3.1 +/- 1.1 (P < 0.05) after antiviral therapy. Similarly, HOMA-%B values significantly decreased to 90.6 +/- 10.0 from 113.7 +/- 15.3 (P < 0.05). Immunoblotting showed a threefold increase in IRS1/2 expression after clearance of HCV. Immunostaining revealed that greater IRS1/2 expression was seen in hepatocytes. CONCLUSIONS: We showed that clearance of HCV improves insulin resistance, beta-cell function, and hepatic IRS1/2 expression.

Endothelial progenitor cell transplantation improves the survival following liver injury in mice.

BACKGROUND & AIMS: Neovascularization, which is vital to the healing of injured tissues, recently has been found to include both angiogenesis, which involves in mature endothelial cells, and vasculogenesis, involving endothelial progenitor cells. The aim of this study was to clarify the possible roles of endothelial progenitor cells during postnatal liver regeneration. METHODS: To determine how endothelial progenitor cells participate in liver regeneration, human or mouse endothelial progenitor cells were transplanted into the mice with carbon tetrachloride-induced acute liver injury. Survival rate of the mice in endothelial progenitor cell-transplanted and control groups was calculated. Separately, livers removed temporally from both groups were examined. RESULTS: At an early stage, transplanted human endothelial progenitor cells were seen mainly surrounding hepatic central veins where hepatocytes showed extensive necrosis; later, the transplanted cells formed tubular structures. More of these cells were observed along hepatic sinusoids. Transplantation of human or mouse endothelial progenitor cells improved survival of the mice following liver injury (from 28.6% to 85.7%, P < .0005 and from 33.3% to 80.0%, P < .001, respectively), accompanied by greater proliferation of hepatocytes. Human endothelial progenitor cells produced several growth factors, such as hepatocyte growth factor, transforming growth factor-alpha, heparin-binding epidermal growth factor-like growth factor, and vascular endothelial growth factor, and also elicited endogenous growth factors. CONCLUSIONS: Endogenous and exogenous growth factors and direct neovascularization after endothelial progenitor cell transplantation promoted liver regeneration, thus improving survival after liver injury. Transplantation of endothelial progenitor cells could represent a new therapeutic strategy for promoting liver regeneration.

Keratin-containing inclusions affect cell morphology and distribution of cytosolic cellular components.

Many neurodegenerative diseases are characterized by the presence of protein aggregates bundled with intermediate filaments (IFs) and similar structures, known as Mallory bodies (MBs), are observed in various liver diseases. IFs are anchored at desmosomes and hemidesmosomes, however, interactions with other intercellular junctions have not been determined. We investigated the effect of IF inclusions on junction-associated and cytosolic proteins in various cultured cells. We performed gene transfection of the green fluorescent protein (GFP)-tagged cytokeratin (CK) 18 mutant arg89cys (GFP-CK18 R89C) in cultured cells and observed CK aggregations as well as loss of IF networks. Among various junction-associated proteins, zonula occludens-1 and beta-catenin were colocalized with CK aggregates on immunofluorescent analyses. Similar results were obtained on immunostaining for cytosolic proteins, 14-3-3 zeta protein, glucose-6-phosphate dehydrogenase and DsRed. E-cadherin, a basolateral membrane protein in polarized epithelia, was present on both the apical and basolateral domains in GFP-CK18 R89C-transfected cells. Furthermore, cells containing CK aggregates were significantly larger than GFP-tagged wild type CK18 (GFP-WT CK18)-transfected or non-transfected cells (P < 0.01) and sometimes their morphology was significantly altered. Our data indicate that CK aggregates affect not only cell morphology but also the localization of various cytosolic components, which may affect the cellular function.