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.

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.

A human T-cell lymphotropic virus type-1 (HTLV-1) carrier complicated with various autoimmune diseases including primary biliary cirrhosis.

A 47-year-old woman with primary biliary cirrhosis and scleroderma was examined at our hospital for a 1-week history of non-resolving fever, arthralgia, myalgia, muscle weakness and fatigue. A diagnosis of systemic lupus erythematosus was made based on arthralgia, low leukocyte count, low lymphocyte count, low serum concentration of complements, positive anti-nuclear antibody and positive anti-double-strand-DNA antibody. She was negative for anti-U1RNP antibody, but positive for anti-Jo1 antibody, and her initial serum concentration of creatine phosphokinase was elevated. We diagnosed her as having overlap syndrome with scleroderma, systemic lupus erythematosus and possible polymyositis associated with primary biliary cirrhosis. Prednisolone rapidly improved her symptoms. Lobulated leukocytes were observed in her peripheral blood specimen. She was positive for anti-HTLV-1 antibody, but Southern blot hybridization did not confirm monoclonal integration of HTLV-I proviral DNA in her peripheral blood. This suggests the possibility of a relationship between HTLV-1 infection and various autoimmune disorders including primary biliary cirrhosis.

Vascular endothelial growth factor is involved in angioedema associated with eosinophilia.

Angioedema associated with eosinophilia is a disorder characterized by angioedema and eosinophilia. However, the pathogenesis of this disorder has not been fully understood. We experienced 4 patients with angioedema associated with eosinophilia. All patients were young, 3 were female and 1 was male. All patients revealed edema of the limbs and eosinophilia. The symptom was rapidly improved after the initiation of low or medium dose of prednisolone. We evaluated the serum concentration of interleukin 5 (IL-5) and the plasma concentration of vascular endothelial growth factor (VEGF) in 1 patient. Both cytokines were markedly elevated before the treatment and decreased after the treatment. Angioedema associated with eosinophilia is not so rare, and IL-5 and VEGF are involved in the pathogenesis of this 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.

Redox state of albumin is not associated with colloid osmotic pressure.

Serum albumin exists in oxidized and reduced forms. Although the oxidation of albumin affects some of its functions, the relationship between oxidized albumin and colloid osmotic pressure (COP) remains unclear. The aim of this study was to determine whether there is an association between oxidized albumin and COP. Blood samples from 20 healthy volunteers were divided into two aliquots in order to prepare reduced (n=20) and oxidized albumin samples (n=20). This was achieved by treatment with L-cysteine and a redox-stabilizing agent before and after incubation at 37°C for 24 h. The percentage of oxidized albumin was determined by high-performance liquid chromatography. COP was measured using a colloid osmometer. Reduced and oxidized albumin samples showed 100% of reduced and 100% of oxidized albumin, respectively. There were no significant differences in albumin level and total protein level between the reduced and the oxidized albumin samples. No significant change was seen in COP between the reduced and the oxidized albumin samples (reduced albumin, 17.4±0.2 mmHg; oxidized albumin, 17.3±0.2 mmHg; P=0.465). Therefore, there is no significant difference in COP between reduced and oxidized albumin samples.

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.

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.

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.