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

E X P E R I M E N TA L C E L L R E S E A RC H 315 ( 2 0 0 9 ) 119 – 12 6

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Niemann-Pick C1 protein transports copper to the secretory compartment from late endosomes where ATP7B resides Chikatoshi Yanagimoto a,⁎, Masaru Harada a,b , Hiroto Kumemura a , Hironori Koga a , Takumi Kawaguchi a , Kunihiko Terada c , Shinichiro Hanada a , Eitaro Taniguchi a , Yukio Koizumi d , Souichi Koyota d , Haruaki Ninomiya e , Takato Ueno a , Toshihiro Sugiyama d , Michio Sata a a

Division of Gastroenterology, Department of Medicine and Research Center for Innovative Cancer Therapy of the 21st Century COE Program for Medical Science, Kurume University School of Medicine, 67 Asahi-Machi, Kurume 830-0011, Japan b The Third Department of Internal Medicine, University of Occupational and Environmental Health, Japan c Department of Medicine, Onoba Hospital, Akita, Japan d Biochemistry, Akita University School of Medicine, Akita, Japan e Department of Neurobiology, Tottori University Faculty of Medicine, Japan

A R T I C L E I N F O R M AT I O N

AB ST R AC T

Article Chronology:

Wilson disease is a genetic disorder characterized by the accumulation of copper in the body by

Received 3 April 2008

defective biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper

Revised version received

incorporation to ceruloplasmin (Cp) and biliary copper excretion. However, copper metabolism in

28 August 2008

hepatocytes has been still unclear. Niemann-Pick disease type C (NPC) is a lipid storage disorder and

Accepted 15 October 2008

the most commonly mutated gene is NPC1 and its gene product NPC1 is a late endosome protein

Available online 31 October 2008

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.

Keywords:

The vesicle traffic was modulated using U18666A, which induces NPC phenotype, and knock down

Wilson disease

of NPC1 by RNA interference. ATP7B colocalized with the late endosome markers, but not with the

ATP7B

trans-Golgi network markers. U18666A and NPC1 knock down decreased holo-Cp secretion to

Niemann-Pick disease type C

culture medium, but did not affect the secretion of other secretory proteins. Copper accumulated in

NPC1

the cells after the treatment with U18666A. These findings suggest that ATP7B localizes in the late

Rab7

endosomes and that copper in the late endosomes is transported to the secretory compartment via

Ceruloplasmin

NPC1-dependent pathway and incorporated into apo-Cp to form holo-Cp. © 2008 Elsevier Inc. All rights reserved.

Introduction Wilson disease is a genetic disorder characterized by the excessive accumulation of copper in the body [1–5]. Wilson disease gene was

cloned and shown to encode 1465 amino-acid protein [6]. Wilson disease gene product, designated ATP7B, is a transmembrane protein and a copper-translocating P-type ATPase, which has 65% homology with ATP7A (Menkes disease protein) [7–12]. It

⁎ Corresponding author. Fax: +81 942 34 2623. E-mail address: [email protected] (C. Yanagimoto). Abbreviations: AFP, α-fetoprotein; Cp, ceruloplasmin; FITC, fluorescence isothiocyanate; GFP, green fluorescent protein; Lamp, lysosomeassociated membrane protein; MPR, mannose 6-phosphate receptor; NPC, Niemann-Pick disease type C; PBS, phosphate buffered saline; TGN, transGolgi network; TRITC, tetramethylrhodamine isothiocyanate 0014-4827/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2008.10.022

120

E X P E R I M E N TA L C E L L R E S E A RC H 315 ( 2 0 0 9 ) 119 – 12 6

functions in copper secretion into plasma coupled with ceruloplasmin (Cp) synthesis, incorporation of copper into apo-Cp to form holo-Cp, and biliary copper excretion [13–15]. Defective ATP7B causes progressive copper accumulation in the body. We have described the late endosomal localization of ATP7B in hepatocytes [16–20]. This localization of ATP7B is important for the copper excretion into the bile canaliculus via lysosomes [3,17,21–23]. Holo-Cp is generated in the trans-Golgi network (TGN) by incorporating copper into apo-Cp after copper reaches the TGN [5,24]. Copper transported into the lumen of the late endosomes may be transported from the late endosomes to the TGN by the mannose 6-phosphate receptor (MPR) recycling vesicles [3,25]. Others have described ATP7B localized in the TGN [15,26–35]. It has been reported that the localization of ATP7B changes from the TGN to pericanalicular vesicles when the intracellular copper concentration is increased [30,31,36,37]. However, we showed that the change of copper concentration did not affect the endosome localization of ATP7B [16,19,20]. Therefore, the precise physiological action of ATP7B in copper metabolism in hepatocytes has remained unclear. Elucidation of the accurate localization of ATP7B within hepatocytes is very important for understanding the biological mechanisms of copper homeostasis in general as well as the pathogenesis of Wilson disease. Rab proteins are small GTPases localized at the cytoplasmic face of specific intracellular membranous compartments in the endocytic and exocytic pathways, where they regulate vesicle traffic [38]. Rab7 is a representative small GTPase that is localized in the late endosomes and functions in vesicle transport in the late endocytic compartments [39–41]. Niemann-Pick disease type C (NPC) is an autosomal recessive disorder characterized by progressive neurological degeneration. The disease is one of the so-called lysosomal storage disorders and results in the accumulation of cholesterol and sphingolipids [42,43]. The most commonly mutated gene in this disorder, NPC1, has been cloned [44], and its gene product NPC1 is a membrane protein localized in the late endosomes [45,46], and regulates intracellular cholesterol trafficking [47–49]. In NPC fibroblasts, the transport of MPR from the late endosomes to the TGN was impaired [47–50]. This indicates that NPC1 contributes to the transport of MPR from the late endosomes to the TGN. In the present study, we induced NPC phenotype and examined the effect of the resulting disturbance of membrane traffic on the distribution of ATP7B and secretion of Cp in hepatoma cell lines. ATP7B was localized in the late endosomes in normal conditions and in late endosome–lysosome hybrid organelles in NPC-like conditions. The NPC phenotype decreased copper incorporation into apo-Cp and decreased the secretion of holo-Cp to the culture medium. These findings indicate that ATP7B functions in the late endosomes, and that copper in the late endosomes is transported to the secretory compartment in a NPC1-dependent manner and then incorporated into apo-Cp to form holo-Cp.

Materials and methods Cells Huh7 and Hep3B cells (hepatoma cell lines) were cultured in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO)

supplemented with 10% fetal calf serum (Wako Pure Chemical Industries, Ltd., Osaka, Japan), penicillin (100 U/ml, crystalline penicillin G Meiji, Meiji Seika Kaisya, Tokyo, Japan), and streptomycin (0.1 mg/ml, Meiji Seika Kaisya) at 37 °C in 5% CO2. Copper concentration in the culture medium as measured by direct colorimetric assay was below the detectable level (<5 μg/dl).

U18666A treatment After the culture medium was exchanged into new culture medium, cells were treated with U18666A (2 μg/ml, Biomol, Plymouth Meeting, PA) for 24 h to manipulate the vesicle traffic in the late endocytic structures and fixed or harvested [47–50].

Construction and transfection of cDNAs GFP-ATP7B and GFP-Rab7 were constructed as described previously [17,48]. GFP-Endo was purchased from Takara Bio. (Shiga, Japan). cDNAs were transiently transfected into cultured cells using Effecten Transfection Reagent (Qiagen GmbH, Hilden, Germany) according to the manufacturer's recommendations at 24 h after plating and cells were fixed 48 h after the transfection.

RNA interference siRNA for human NPC1 and scramble RNA were from Dharmacon Inc. (Lafayette, CO). Transfection was performed using HiPerFect Transfection Reagent (Qiagen) according to the manufacturer's protocol, and cells were harvested at 48 or 72 h after transfection. At 48 h after the transfection, the medium was changed to normal medium, then cells were further cultured for 24 h and the culture medium was collected.

Antibodies Antibodies against the following antigens were used: γ-adaptin (Sigma); ATP7B [20]; GM130 (BD Biosciences, San Jose, CA); galactosyltransferase (a kind gift from Dr. Suganuma); p230 (BD Biosciences); lysosome-associated membrane protein (Lamp) 1 and Lamp2 [51]; NPC1 (Novus Biologicals, Littleton, CO); Cp (Dako Japan, Kyoto, Japan); holo-Cp (NIPRO Incorporated, Osaka, Japan); albumin (ICN Biomedicals Incorporated, Costa Mesa, CA); α-fetoprotein (AFP) (Dako Japan); and actin (Sigma). The following were used as secondary antibodies: FITC-conjugated rabbit antimouse immunoglobulin (Dako Japan); TRITC-conjugated rabbit antimouse immunoglobulin (Dako Japan); and peroxidase-conjugated secondary antibody (GE Healthcare, Buckinghamshire, England).

Immunofluorescence At 48 h after the transfection, cells were fixed with freshly prepared 4% paraformaldehyde in PBS for 30 min and permeabilized with 0.1% Triton X-100 in PBS for 10 min or 0.1% saponin in PBS for 30 min. Nonspecific binding was blocked by incubation with Protein Block Serum Free (Dako Japan) for 30 min followed by incubation with the primary and secondary antibodies for 1 h respectively. A confocal laser scanning microscope (Fluoview FV 300, Olympus, Tokyo, Japan) equipped with an Argon/Krypton laser was used. For double-labeling analysis, images were acquired sequentially using separate excitation wavelengths of 488 nm for GFP or FITC and 568 nm for TRITC, and then merged.

E X P E R I M E N TA L C E L L R E S E A RC H 315 ( 2 0 0 9 ) 119 – 12 6

121

copper content of the same protein concentration of cell lysate was measured by an atomic absorption spectrometer Z-5000 (HITACHI, Tokyo, Japan).

Statistical analysis Statistical analysis of Cp secretion and Copper contents in cell lysates was performed by Mann Whitney U test and Wilcoxon Matched-Pair Signed-Rank test respectively. Results were expressed as mean ± SE and p value <0.05 was considered as significant.

Results Both GFP-ATP7B and endogenous ATP7B were localized in the late endosomes

Fig. 1 – Confocal laser scanning microscopic images of Huh7 cells transfected with GFP-ATP7B (green). Cells were treated with or without U18666A for 24 h, and labeled with anti-γ-adaptin or anti-Lamp2 antibody (red). GFP-ATP7B was colocalized with Lamp2 but not with γ-adaptin in all cells, with or without U18666A treatment. Bar, 10 μm.

GFP-ATP7B was not colocalized with γ-adaptin (Fig. 1A), GM130, galactosyltransferase and p230 (data not shown), which are markers of the TGN, in GFP-ATP7B-transfected Huh7 cells. We then examined the effect of U18666A. U18666A is a sterol derivative that induces the NPC phenotype by inhibiting the function of NPC1 or NPC1-related proteins and induces the formation of late endosome–lysosome hybrid organelles [20,45,46]. U18666A did not affect the cell viability. When cells were treated with U18666A, GFP-ATP7B was not colocalized with γ-adaptin (Fig. 1A), GM130, galactosyltransferase and p230 (data

Electrophoresis and immunoblotting Cell culture medium was collected and cell debris were cleared by centrifugation at 1200 g for 5 min at 4 °C. Cells were lysed in lysis buffer (50 mM HEPES at pH 7.5, 250 mM NaCl, 20 mM EDTA, and 0.1% Nonidet P-40 additionally containing 1 mM phenylmethylsulfonyl fluoride, a protease inhibitor cocktail (Sigma)), 10 nM NaF, and 1 mM Na3VO4. Lysates were incubated at 4 °C for 15 min and then cell debris were cleared by centrifugation at 14,500 g for 30 min at 4 °C. Supernatants from cell culture medium and lysate were subjected to protein determinations using a DC protein assay kit (Bio-RAD Laboratories, Hercules, CA). Equal volumes of protein were separated by 7.5% to 12.5% sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Samples for Cp were loaded onto sodium dodecyl sulfatepolyacrylamide gel electrophoresis under non-reducing conditions. Proteins were transferred to polyvinylidene difluoride membrane, and the membranes were processed by routine immunoblotting. Antibody reactivity was detected using Amersham ECL Advance Western Blotting Detection Kit (Amersham Pharmacia Biotech, Piscataway, NJ). Specific bands were visualized by allowing the membrane to expose blue-light-sensitive autoradiographic films. Densities of immunoreactive Cp bands were quantified by densitometer using NIH-Image J (developed at the National Institutes of Health). The density of holo-Cp was divided by that of apo-Cp to measure the ratio of holo-Cp to apo-Cp.

Measurement of copper content Cell lysates were collected as described above. We made 6 paired samples from cells with or without U18666A treatment. The

Fig. 2 – Confocal laser scanning microscopic images of Huh7 cells transfected with GFP-Rab7 (green). Cells were treated with or without U18666A for 24 h, and labeled with anti-γ-adaptin or anti-Lamp2 antibody (red). GFP-Rab7 was colocalized with Lamp2 but not with γ-adaptin in all cells, with or without U18666A treatment. Bar, 10 μm.

122

E X P E R I M E N TA L C E L L R E S E A RC H 315 ( 2 0 0 9 ) 119 – 12 6

not shown). Without the treatment of U18666A, GFP-ATP7B was colocalized in part but not all with Lamp1 (data not shown) and Lamp2 (Fig. 1B), which are markers of the late endosomes and lysosomes. After the treatment with U18666A, GFP-ATP7B was almost completely colocalized with Lamp1 (data not shown) and Lamp2 (Fig. 1B). We used Rab7 as a late endosome marker because Rab7 was shown to be localized in the late endosomes [39–41]. In GFPRab7-transfected Huh7 cells, GFP-Rab7 was not colocalized with γ-adaptin either with or without treatment of U18666A (Fig. 2A). GFP-Rab7 was colocalized in part but not all with Lamp2. After the treatment with U18666A, GFP-Rab7 was almost completely colocalized with Lamp2 (Fig. 2B). This distribution was identical with that of GFP-ATP7B. We examined the localization of endogenous ATP7B using GFP-Rab7, because the anti-ATP7B antibody and most of the antibodies for organelle markers used in the present study were mouse monoclonal antibodies. GFPRab7 was colocalized with endogenous ATP7B in cells with or without treatment of U18666A (Fig. 3A). Similar results were obtained using Hep3B cells (data not shown). Furthermore, another endosome marker, GFP-Endo, was also colocalized with endogenous ATP7B in cells with or without treatment of U18666A (Fig. 3B).

U18666A decreased the secretion of copper-binding holo-Cp to the culture medium U18666A impaired the transport of MPR from the late endosomes to the TGN [50]. Therefore, we hypothesized that U18666A inhibits the copper transport from the late endosomes to the TGN, affecting holo-Cp secretion, since copper seems to be transported by the MPR recycling vesicles from the late endosomes to the TGN [3,16,18–20]. We performed Western blotting of culture medium for Cp using two anti-Cp antibodies (Fig. 4Aa). The purified Cp was also subjected to Western blotting, and an anti-Cp antibody revealed two bands of about 135 and 85 kDa corresponding to apo-Cp and holo-Cp respectively [52,53]. The anti-holo-Cp antibody which recognized only holo-Cp revealed a band of about 85 kDa [54,55]. The secretion of holo-Cp to medium from U18666A-treated cells was significantly decreased in Western analysis using both the anti-Cp and the anti-holo-Cp antibodies compared with that of medium from untreated cells. The secretions of albumin and AFP to the medium were not influenced by the U18666A treatment (Fig. 4Aa). Coomassie brilliant blue staining of the culture medium revealed that the same amounts of proteins were loaded (Fig. 4Ab). The ratio of holo-Cp to apo-Cp of medium from the U18666A-treated cells significantly decreased compared with that of untreated cells (Fig. 4B). When the medium supplemented with 10% fetal calf serum without culture was examined by Western analysis, we could detect apo-Cp but not holo-Cp (data not shown). When the medium non-supplemented with 10% fetal calf serum without culture was used to Western analysis, we could not detect either apo-Cp or holo-Cp (data not shown). When we examined the secretion of holo-Cp to medium from U18666A-treated cells for 24 h using non-fetal calf serum after supplemented medium, the ratio of holo-Cp to apo-Cp significantly decreased compared with that of untreated cells (data not shown). The similar results were obtained using another hepatoma cell line, Hep3B (Fig. 4C).

Fig. 3 – Confocal laser scanning microscopic images of Huh7 cells. (A) Cells transfected with GFP-Rab7 (green) were treated with or without U18666A for 24 h, and labeled with anti-ATP7B antibody (red). (B) Cells transfected with GFP-Endo (green) were treated with or without U18666A for 24 h, and labeled with anti-ATP7B antibody (red). Endogenous ATP7B was colocalized with GFP-Rab7 and GFP-Endo in all cells, with or without U18666A treatment. Bar, 10 μm.

U18666A caused decrease of the intracellular holo-Cp and accumulation of copper in cultured cells We examined the protein expressions including Cp and copper content in cultured cells. The expression of holo-Cp of lysate of U18666A-treated Huh7 cells was decreased compared with that of untreated cells (Fig. 5A). The expressions of ATP7B and actin were not affected by the treatment with U18666A (Fig. 5A). Copper content in the U18666A-treated cells was significantly higher than that of untreated cells (Fig. 5B), indicating that the NPC phenotype induces the accumulation of copper in the cells.

NPC1 knock down reduced the holo-Cp secretion U18666A treatment, which induces the NPC phenotype, decreased holo-Cp content in the cells and secretion to the culture medium. Therefore, we further examined the effect of NPC1 knock down using siRNA on holo-Cp secretion. NPC1 siRNA successfully decreased the expression of NPC1 at 48 (data not shown) and 72 h after the transfection (Fig. 6A). Then we examined the secretion of holo-Cp to the culture medium by Western blotting. The secretion of holo-Cp to the medium of NPC1 siRNA-transfected cells was decreased compared with that of control siRNAtransfected cells (Fig. 6B). The secretion of albumin was not affected by NPC1 knock down (Fig. 6B).

E X P E R I M E N TA L C E L L R E S E A RC H 315 ( 2 0 0 9 ) 119 – 12 6

123

Discussion The novel findings of the present study are; (1) endogenous ATP7B is localized in the late endosomes in hepatoma cells, (2) U18666A and NPC1 knock down, which impair vesicular transport from the late endosomes to the TGN, decrease copper incorporation into Cp and induce accumulation of copper in the cells. These findings suggest that ATP7B is localized in the late endosomes and that the late endosomes play important roles in copper metabolism in a NPC1-dependent manner in hepatocytes. Proper intracellular localization of the ATP7B is very important for copper metabolism in hepatocytes. However, the precise intracellular localization of ATP7B has been controversial [5,15–20,26–35]. Determination of this issue is essential for the understanding of copper metabolism in the body and the mechanisms of Wilson disease. ATP7B transports copper from cytoplasm into the lumen of its residing organelles. Copper is exported from hepatocytes by two different pathways. One is secretion to the plasma via copper incorporation into Cp [13], and the other is biliary copper excretion, probably via lysosomes [14,22,23]. Various studies have reported that ATP7B is localized to the TGN in low-copper medium. After the addition of copper, some studies reported that ATP7B moved to unidentified vesicular structures [30,36], while others described ATP7B as being localized at the canalicular membrane itself [31,33,37].

Fig. 5 – (A) Immunoblot of lysates from Huh7 cells with or without U18666A treatment for 24 h. The expression of holo-Cp decreased after the treatment with U18666A, although the expression of ATP7B and actin was not affected by the U18666A treatment. (B) Copper content in cell lysates from Huh7 cells with or without U18666A treatment for 24 h. Copper content of cell lysates was significantly increased after the treatment with U18666A (n = 6). ⁎P < 0.05. Copper accumulated within the cells treated with U18666A.

We previously reported that ATP7B was localized in the late endosomes in various cells using GFP-ATP7B and ATP7B-DsRed [16–20]. In the present study, GFP-ATP7B was not colocalized with TGN markers (γ-adaptin, GM130, galactosyltransferase and p230), but was colocalized partly with Lamp1 and Lamp2, which are

Fig. 4 – (A) a: Immunoblot of culture medium from Huh7 cells with or without U18666A treatment for 24 h. Purified Cp was used as a positive marker for Cp. b: Coomassie brilliant blue staining of culture medium from Huh7 cells with or without U18666A treatment for 24 h. (B) Summary of densitometry analysis of holo-Cp level relative to apo-Cp level as in A (n = 6). ⁎P < 0.05. (C) Immunoblot of culture medium from Hep3B cells with or without U18666A treatment for 24 h. Treatment with U18666A decreased secretion of holo-Cp into the culture medium.

124

E X P E R I M E N TA L C E L L R E S E A RC H 315 ( 2 0 0 9 ) 119 – 12 6

Fig. 6 – Effect of NPC1siRNA in Huh7 cells. (A) Immunoblotting of cell lysate from NPC1 siRNA-transfected or control siRNA-transfected Huh7 cells. (B) Immunoblotting of culture medium from NPC1 siRNA-transfected or control siRNA-transfected Huh7 cells. (C) Densitometry analysis of relative protein level of holo-Cp in B. NPC1 knock down decreased the secretion of holo-Cp to the culture medium.

markers of late endosomes and lysosomes. These results verify our previous results using various cell types. We further examined the localization of endogenous ATP7B using two endosome markers, GFP-Rab7 and GFP-Endo. GFP-Rab7, a late endosome marker, was not colocalized with γ-adaptin but was colocalized with Lamp2. GFP-Rab7 was almost completely colocalized with endogenous ATP7B. Endosome localization of endogenous ATP7B was further demonstrated using GFP-Endo, another endosome marker. Therefore, both GFP-ATP7B and endogenous ATP7B are localized in the late endosomes. Furthermore, we manipulated the membrane transport in cultured cells and examined the localization of GFP-ATP7B and endogenous ATP7B. In U18666A-treated cells, GFP-ATP7B was almost completely colocalized with Lamp1 and Lamp2. Lamp1 and Lamp2 are localized in both late endosomes and lysosomes in ordinary conditions. This observation indicates the formation of late endosome–lysosome hybrid organelles as we previously described [20]. GFP-Rab7 was not colocalized with γ-adaptin but rather with Lamp2 even in these conditions. This distribution is identical to that of GFP-ATP7B. GFP-Rab7 was almost completely colocalized with Lamp2 and endogenous ATP7B in U18666A-treated cells. These results apparently indicate that ATP7B is localized in the late endosomes but not in the TGN. The reason why our results differ from those in previous reports is unknown. However, it is unlikely to be due to differences of the cells used, because we used various cell types including primary isolated cultured cells and obtained the same results in all cell types [20]. The effect of GFP tag or transfection is also unlikely considering the present results with endogenous ATP7B. The effect of copper concentration in the medium is unlikely, because our previous studies demonstrated that copper concentration did not affect the distribution of endogenous ATP7B and GFP-ATP7B [16,18,20].

Next we examined the effect of U18666A on copper incorporation into Cp. If ATP7B is localized in the late endosomes, ATP7B translocates copper into the late endosomes from the cytoplasm. Copper seems to be transported from the late endosomes to the TGN by the MPR recycling vesicles, where it is then incorporated into Cp. U18666A would be expected to decrease the holo-Cp synthesis and accumulate copper in the cells because retrograde vesicular transport from late endosomes to TGN was shown to be inhibited in NPC-deficient cells and U18666A-treated cells [49,50]. However, if ATP7B were localized in the TGN, U18666A would not affect the holo-Cp synthesis. In the present study, we examined the secretion of holo-Cp to the culture medium. The ratio of holo-Cp to apo-Cp in the culture medium of U18666A-treated Huh7 cells was significantly decreased compared with that of untreated cells. Similar results were obtained from Hep3B cells. Protein synthesis and secretion of U18666A-treated cells were unchanged, because the secretion of albumin and AFP was not affected by U18666A treatment. These results indicated that U18666A decreased copper incorporation into Cp. Although the difference between the ratio of holo-Cp to apo-Cp in the culture medium of non-treated Huh7 cells and that in U18666A-treated Huh7 cells is significant, it was modest. This may be due to copper pooling in the ER, because there is the cycling vesicle movement between the ER and Golgi apparatus [56]. Moreover, we examined the effects of NPC1 knock down on holo-Cp secretion. Holo-Cp in the culture medium of NPC1 siRNAtransfected cells decreased compared with that of control siRNAtransfected cells. Although the difference of holo-Cp secretion seemed to be modest, it can be explained by the same reason as the case of U18666A treatment. The secretion of albumin was not altered by NPC1 knock down. Therefore, the effect of U18666A is identical to NPC1 knock down. NPC1 is important for vesicle transport from the late endosomes to the TGN [49,50]. Thus, copper in the late endosomes translocated by ATP7B must be transported to the TGN in a NPC1-dependent manner. Recent study demonstrated that human copper transporter 2, a copper transporter, localizes in the late endosomes and lysosomes, and regulates intracellular copper level [57]. COMMD1/MURR1, gene product defective in canine copper toxicosis, also localizes in the endosomes and lysosomes [58]. And lysosomes are an important source of biliary copper excretion. Therefore, tight regulation of intracellular copper concentration in the late endosomes and lysosomes are very reasonable [22,23]. We examined the effects of U18666A on intracellular copper content and expression of ATP7B. Intracellular copper content was increased in U18666A-treated cells compared with that of untreated cells, although the expression of ATP7B was not affected. One explanation for this observation could be that U18666A decreases the copper incorporation into apo-Cp and decreases the secretion of holo-Cp, which induces copper accumulation in the cells. Another possibility is that NPC1 is important for vesicular transport between late endosomes and lysosomes, and U18666A inhibits biliary lysosomal copper excretion. This is because NPC1 is important for transport of various substances from the late endocytic structures, and cholesterol accumulates in lysosomes in patients with NPC [45]. This latter possibility should be further investigated in future. In the present study, we used U18666A to manipulate the membrane transport. U18666A did not seem to affect cell viability because it did not affect the cell shape, cell growth, and protein synthesis and secretion of other proteins.

E X P E R I M E N TA L C E L L R E S E A RC H 315 ( 2 0 0 9 ) 119 – 12 6

In conclusion, ATP7B was clearly confirmed to be localized in the late endosomes. This finding was confirmed not only by an immunocytochemical approach, but also by analysis of Cp secretion into the culture medium. This proper localization of ATP7B enables hepatocytes to appropriately export copper by means of Cp secretion and biliary copper excretion via lysosomes [13,14,17,22,23]. We suggest that some copper translocated into the late endosome lumen from cytoplasm is transported to the TGN by MPR recycling vesicles, and incorporated into apo-Cp in the TGN in a NPC1-dependent manner. Copper-incorporated holo-Cp is secreted to the sinusoidal space through the secretory pathway. Thus, various types of mutations of ATP7B may cause defective copper ATPase activity in hepatocyte late endosomes, and this must be the main defect in Wilson disease.

Acknowledgment We would like to thank Dr. David Marks for the kind gift of GFP-Rab7.

REFERENCES

[1] M.C. Linder, M. Hazegh-Azam, Copper biochemistry and molecular biology, Am. J. Clin. Nutr. 63 (1996) 797S–811S. [2] J. Camakaris, I. Voskoboinik, J.F. Mercer, Molecular mechanisms of copper homeostasis, Biochem. Biophys. Res. Commun. 261 (1999) 225–232. [3] M. Harada, Wilson disease, Med. Electron Microsc. 35 (2002) 61–66. [4] A. Ala, A.P. Walker, K. Ashkan, J.S. Dooley, M.L. Schilsky, Wilson's disease, Lancet 369 (2007) 397–408. [5] S. La Fontaine, J.F. Mercer, Trafficking of the copper-ATPases, ATP7A and ATP7B: role in copper homeostasis, Arch. Biochem. Biophys. 463 (2007) 149–167. [6] K. Petrukhin, S. Lutsenko, I. Chernov, B.M. Ross, J.H. Kaplan, T.C. Gilliam, Characterization of the Wilson disease gene encoding a P-type copper transporting ATPase: genomic organization, alternative splicing, and structure/function predictions, Hum. Mol. Genet. 3 (1994) 1647–1656. [7] P.C. Bull, G.R. Thomas, J.M. Rommens, J.R. Forbes, D.W. Cox, The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene, Nat. Genet. 5 (1993) 327–337. [8] K. Petrukhin, S.G. Fischer, M. Pirastu, R.E. Tanzi, I. Chernov, M. Devoto, L.M. Brzustowicz, E. Cayanis, E. Vitale, J.J. Russo, D. Matseoane, B. Boukhgalter, W. Wasco, A.L. Figus, J. Loudianos, A. Cao, I. Sternlieb, O. Evgrafov, E. Parano, L. Pavone, D. Warburton, J. Ott, G.K. Penchaszadeh, I.H. Scheinberg, T.C. Gilliam, Mapping, cloning and genetic characterization of the region containing the Wilson disease gene, Nat. Genet. 5 (1993) 338–343. [9] R.E. Tanzi, K. Petrukhin, I. Chernov, J.L. Pellequer, W. Wasco, B. Ross, D.M. Romano, E. Parano, L. Pavone, L.M. Brzustowicz, M. Devoto, J. Peppercorn, A.I. Bush, I. Sternlieb, M. Pirastu, J.F. Gusella, O. Evgrafov, G.K. Penchaszadeh, B. Honig, I.S. Edelman, M.B. Soares, I.H. Scheinberg, T.C. Gilliam, The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene, Nat. Genet. 5 (1993) 344–350. [10] Y. Yamaguchi, M.E. Heiny, J.D. Gitlin, Isolation and characterization of a human liver cDNA as a candidate gene for Wilson disease, Biochem. Biophys. Res. Commun. 197 (1993) 271–277. [11] G.A. Scarborough, Structure and function of the P-type ATPases, Curr. Opin. Cell Biol. 11 (1999) 517–522.

125

[12] S. Lutsenko, M.J. Petris, Function and regulation of the mammalian copper-transporting ATPases: insights from biochemical and cell biological approaches, J. Membr. Biol. 191 (2003) 1–12. [13] K. Terada, T. Nakako, X.L. Yang, M. Iida, N. Aiba, Y. Minamiya, M. Nakai, T. Sakaki, N. Miura, T. Sugiyama, Restoration of holoceruloplasmin synthesis in LEC rat after infusion of recombinant adenovirus bearing WND cDNA, J. Biol. Chem. 273 (1998) 1815–1820. [14] K. Terada, N. Aiba, X.L. Yang, M. Iida, M. Nakai, N. Miura, T. Sugiyama, Biliary excretion of copper in LEC rat after introduction of copper transporting P-type ATPase, ATP7B, FEBS Lett. 448 (1999) 53–56. [15] I.H. Hung, M. Suzuki, Y. Yamaguchi, D.S. Yuan, R.D. Klausner, J.D. Gitlin, Biochemical characterization of the Wilson disease protein and functional expression in the yeast Saccharomyces cerevisiae, J. Biol. Chem. 272 (1997) 21461–21466. [16] M. Harada, S. Sakisaka, T. Kawaguchi, R. Kimura, E. Taniguchi, H. Koga, S. Hanada, S. Baba, K. Furuta, R. Kumashiro, T. Sugiyama, M. Sata, Copper does not alter the intracellular distribution of ATP7B, a copper-transporting ATPase, Biochem. Biophys. Res. Commun. 275 (2000) 871–876. [17] M. Harada, S. Sakisaka, K. Terada, R. Kimura, T. Kawaguchi, H. Koga, E. Taniguchi, K. Sasatomi, N. Miura, T. Suganuma, H. Fujita, K. Furuta, K. Tanikawa, T. Sugiyama, M. Sata, Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes, Gastroenterology 118 (2000) 921–928. [18] M. Harada, S. Sakisaka, K. Terada, R. Kimura, T. Kawaguchi, H. Koga, M. Kim, E. Taniguchi, S. Hanada, T. Suganuma, K. Furuta, T. Sugiyama, M. Sata, A mutation of the Wilson disease protein, ATP7B, is degraded in the proteasomes and forms protein aggregates, Gastroenterology 120 (2001) 967–974. [19] M. Harada, H. Kumemura, S. Sakisaka, S. Shishido, E. Taniguchi, T. Kawaguchi, S. Hanada, H. Koga, R. Kumashiro, T. Ueno, T. Suganuma, K. Furuta, M. Namba, T. Sugiyama, M. Sata, Wilson disease protein ATP7B is localized in the late endosomes in a polarized human hepatocyte cell line, Int. J. Mol. Med. 11 (2003) 293–298. [20] M. Harada, T. Kawaguchi, H. Kumemura, K. Terada, H. Ninomiya, E. Taniguchi, S. Hanada, S. Baba, M. Maeyama, H. Koga, T. Ueno, K. Furuta, T. Suganuma, T. Sugiyama, M. Sata, The Wilson disease protein ATP7B resides in the late endosomes with Rab7 and the Niemann-Pick C1 protein, Am. J. Pathol. 166 (2005) 499–510. [21] M. Harada, T. Kawaguchi, H. Kumemura, M. Sata, Where is the site that ATP7B transports copper within hepatocytes? Gastroenterology 125 (2003) 1911–1912 1911; author reply. [22] J.B. Gross Jr., B.M. Myers, L.J. Kost, S.M. Kuntz, N.F. LaRusso, Biliary copper excretion by hepatocyte lysosomes in the rat. Major excretory pathway in experimental copper overload, J. Clin. Invest. 83 (1989) 30–39. [23] M. Harada, S. Sakisaka, M. Yoshitake, S. Shakadoh, K. Gondoh, M. Sata, K. Tanikawa, Biliary copper excretion in acutely and chronically copper-loaded rats, Hepatology 17 (1993) 111–117. [24] K. Terada, Y. Kawarada, N. Miura, O. Yasui, K. Koyama, T. Sugiyama, Copper incorporation into ceruloplasmin in rat livers, Biochim. Biophys. Acta 1270 (1995) 58–62. [25] G. Griffiths, B. Hoflack, K. Simons, I. Mellman, S. Kornfeld, The mannose 6-phosphate receptor and the biogenesis of lysosomes, Cell 52 (1988) 329–341. [26] X.L. Yang, N. Miura, Y. Kawarada, K. Terada, K. Petrukhin, T. Gilliam, T. Sugiyama, Two forms of Wilson disease protein produced by alternative splicing are localized in distinct cellular compartments, Biochem. J. 326 (Pt 3) (1997) 897–902. [27] S.L. La Fontaine, S.D. Firth, J. Camakaris, A. Englezou, M.B. Theophilos, M.J. Petris, M. Howie, P.J. Lockhart, M. Greenough, H. Brooks, R.R. Reddel, J.F. Mercer, Correction of the copper transport defect of Menkes patient fibroblasts by expression of

126

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38] [39]

[40]

[41] [42] [43]

[44]

E X P E R I M E N TA L C E L L R E S E A RC H 315 ( 2 0 0 9 ) 119 – 12 6

the Menkes and Wilson ATPases, J. Biol. Chem. 273 (1998) 31375–31380. A.S. Payne, E.J. Kelly, J.D. Gitlin, Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 10854–10859. K. Nagano, K. Nakamura, K.I. Urakami, K. Umeyama, H. Uchiyama, K. Koiwai, S. Hattori, T. Yamamoto, I. Matsuda, F. Endo, Intracellular distribution of the Wilson's disease gene product (ATPase7B) after in vitro and in vivo exogenous expression in hepatocytes from the LEC rat, an animal model of Wilson's disease, Hepatology 27 (1998) 799–807. M. Schaefer, R.G. Hopkins, M.L. Failla, J.D. Gitlin, Hepatocyte-specific localization and copper-dependent trafficking of the Wilson's disease protein in the liver, Am. J. Physiol. 276 (1999) G639–G646. M. Schaefer, H. Roelofsen, H. Wolters, W.J. Hofmann, M. Muller, F. Kuipers, W. Stremmel, R.J. Vonk, Localization of the Wilson's disease protein in human liver, Gastroenterology 117 (1999) 1380–1385. J.R. Forbes, D.W. Cox, Copper-dependent trafficking of Wilson disease mutant ATP7B proteins, Hum. Mol. Genet. 9 (2000) 1927–1935. H. Roelofsen, H. Wolters, M.J. Van Luyn, N. Miura, F. Kuipers, R.J. Vonk, Copper-induced apical trafficking of ATP7B in polarized hepatoma cells provides a mechanism for biliary copper excretion, Gastroenterology 119 (2000) 782–793. S. La Fontaine, M.B. Theophilos, S.D. Firth, R. Gould, R.G. Parton, J.F. Mercer, Effect of the toxic milk mutation (tx) on the function and intracellular localization of Wnd, the murine homologue of the Wilson copper ATPase, Hum. Mol. Genet. 10 (2001) 361–370. D. Huster, M. Hoppert, S. Lutsenko, J. Zinke, C. Lehmann, J. Mossner, F. Berr, K. Caca, Defective cellular localization of mutant ATP7B in Wilson's disease patients and hepatoma cell lines, Gastroenterology 124 (2003) 335–345. M.A. Cater, S. La Fontaine, K. Shield, Y. Deal, J.F. Mercer, ATP7B mediates vesicular sequestration of copper: insight into biliary copper excretion, Gastroenterology 130 (2006) 493–506. Y. Guo, L. Nyasae, L.T. Braiterman, A.L. Hubbard, NH2-terminal signals in ATP7B Cu-ATPase mediate its Cu-dependent anterograde traffic in polarized hepatic cells, Am. J. Physiol. Gasterointest. Liver Physiol. 289 (2005) G904–G916. J. Somsel Rodman, A. Wandinger-Ness, Rab GTPases coordinate endocytosis, J. Cell. Sci. 113 (Pt 2) (2000) 183–192. P. Chavrier, R.G. Parton, H.P. Hauri, K. Simons, M. Zerial, Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments, Cell 62 (1990) 317–329. M.J. Tuvim, R. Adachi, S. Hoffenberg, B.F. Dickey, Traffic control: Rab GTPases and the regulation of interorganellar transport, News Physiol. Sci. 16 (2001) 56–61. M.C. Seabra, E.H. Mules, A.N. Hume, Rab GTPases, intracellular traffic and disease, Trends Mol. Med. 8 (2002) 23–30. S. Mukherjee, F.R. Maxfield, Lipid and cholesterol trafficking in NPC, Biochim. Biophys. Acta 1685 (2004) 28–37. T.Y. Chang, P.C. Reid, S. Sugii, N. Ohgami, J.C. Cruz, C.C. Chang, Niemann-Pick type C disease and intracellular cholesterol trafficking, J. Biol. Chem. 280 (2005) 20917–20920. E.D. Carstea, J.A. Morris, K.G. Coleman, S.K. Loftus, D. Zhang, C. Cummings, J. Gu, M.A. Rosenfeld, W.J. Pavan, D.B. Krizman, J. Nagle, M.H. Polymeropoulos, S.L. Sturley, Y.A. Ioannou, M.E. Higgins, M. Comly, A. Cooney, A. Brown, C.R. Kaneski, E.J.

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55] [56]

[57]

[58]

Blanchette-Mackie, N.K. Dwyer, E.B. Neufeld, T.Y. Chang, L. Liscum, J.F. Strauss III, K. Ohno, M. Zeigler, R. Carmi, J. Sokol, D. Markie, R.R. O'Neill, O.P. van Diggelen, M. Elleder, M.C. Patterson, R.O. Brady, M.T. Vanier, P.G. Pentchev, D.A. Tagle, Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis, Science 277 (1997) 228–231. E.B. Neufeld, M. Wastney, S. Patel, S. Suresh, A.M. Cooney, N.K. Dwyer, C.F. Roff, K. Ohno, J.A. Morris, E.D. Carstea, J.P. Incardona, J.F. Strauss III, M.T. Vanier, M.C. Patterson, R.O. Brady, P.G. Pentchev, E.J. Blanchette-Mackie, The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo, J. Biol. Chem. 274 (1999) 9627–9635. M. Zhang, N.K. Dwyer, E.B. Neufeld, D.C. Love, A. Cooney, M. Comly, S. Patel, H. Watari, J.F. Strauss III, P.G. Pentchev, J.A. Hanover, E.J. Blanchette-Mackie, Sterol-modulated glycolipid sorting occurs in Niemann-Pick C1 late endosomes, J. Biol. Chem. 276 (2001) 3417–3425. M. Walter, J.P. Davies, Y.A. Ioannou, Telomerase immortalization upregulates Rab9 expression and restores LDL cholesterol egress from Niemann-Pick C1 late endosomes, J. Lipid Res. 44 (2003) 243–253. A. Choudhury, M. Dominguez, V. Puri, D.K. Sharma, K. Narita, C.L. Wheatley, D.L. Marks, R.E. Pagano, Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells, J. Clin. Invest. 109 (2002) 1541–1550. I.G. Ganley, S.R. Pfeffer, Cholesterol accumulation sequesters Rab9 and disrupts late endosome function in NPC1-deficient cells, J. Biol. Chem. 281 (2006) 17890–17899. T. Kobayashi, M.H. Beuchat, M. Lindsay, S. Frias, R.D. Palmiter, H. Sakuraba, R.G. Parton, J. Gruenberg, Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport, Nat. Cell Biol. 1 (1999) 113–118. J.W. Chen, T.L. Murphy, M.C. Willingham, I. Pastan, J.T. August, Identification of two lysosomal membrane glycoproteins, J. Cell Biol. 101 (1985) 85–95. T. Wang, S.A. Weinman, Involvement of chloride channels in hepatic copper metabolism: ClC-4 promotes copper incorporation into ceruloplasmin, Gastroenterology 126 (2004) 1157–1166. N.E. Hellman, S. Kono, G.M. Mancini, A.J. Hoogeboom, G.J. De Jong, J.D. Gitlin, Mechanisms of copper incorporation into human ceruloplasmin, J. Biol. Chem. 277 (2002) 46632–46638. S. Hiyamuta, K. Ito, Monoclonal antibody against the active site of caeruloplasmin and the ELISA system detecting active caeruloplasmin, Hybridoma 13 (1994) 139–141. S. Hiyamuta, K. Shimizu, T. Aoki, Early diagnosis of Wilson's disease, Lancet 342 (1993) 56–57. J. Lippincott-Schwartz, L.C. Yuan, J.S. Bonifacino, R.D. Klausner, Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER, Cell 56 (1989) 801–813. P.V. van den Berghe, D.E. Folmer, H.E. Malingre, E. van Beurden, A.E. Klomp, B. van de Sluis, M. Merkx, R. Berger, L.W. Klomp, Human copper transporter 2 is localized in late endosomes and lysosomes and facilitates cellular copper uptake, Biochem. J. 407 (2007) 49–59. A.E. Klomp, B. van de Sluis, L.W. Klomp, C. Wijmenga, The ubiquitously expressed MURR1 protein is absent in canine copper toxicosis, J. Hepatol. 39 (2003) 703–709.