lipid raft-dependent endocytosis of metallothionein in hepg2 cells

JOURNAL OF CELLULAR PHYSIOLOGY 210:428–435 (2007)

Lipid Raft-Dependent Endocytosis of Metallothioneinin HepG2 Cells

QIANG HAO,1 SUNG-HYE HONG,2,3 AND WOLFGANG MARET1,3,4*1Department of Preventive Medicine & Community Health,The University of Texas Medical Branch, Galveston, Texas

2Department of Biochemistry and Molecular Biology,University of British Columbia, Vancouver, British Columbia, Canada3Department of Pathology, Center for Biochemical and Biophysical

Sciences and Medicine, Harvard Medical School, Cambridge, Massachusetts4Department of Anesthesiology, The University of Texas Medical Branch,

Galveston, Texas

Human hepatocellular carcinoma (HepG2) cells take up metallothionein (MT) by endocytosis. MT co-localizes with albumin butnot with transferrin, indicating uptake via a non-classical pathway rather than via clathrin-mediated endocytosis. A lipid raft-dependent uptake is indicated by pravastatin inhibition of cholesterol synthesis andmethyl-b-cyclodextrin inhibition of cholesteroltranslocation to the plasma membrane, reducing MT uptake by 29% and 69%, respectively. Subcellular fractionation after MTuptake reveals significant amounts ofMT in vesicular fractions including lysosomes but virtually noMT in the cytosol.Metals boundtoMTare released into the cytosol, however. The findings define apathway for cellularmetal acquisition. Togetherwith results fromother studies demonstrating secretion of MT from different cells and the presence of MT in extracellular fluids, the results suggest afunction of MT in intercellular communication. J. Cell. Physiol. 210: 428–435, 2007. � 2006 Wiley-Liss, Inc.

Mammalian metallothioneins (MTs) bind seven zincions in two zinc/thiolate clusters (Arseniev et al., 1988;Robbins et al., 1991). Over a dozen genes encode humanMTs. MT-1, -2, -3, and -4 are the major isoforms.Originally considered intracellular, cytoplasmic pro-teins, MTs are now known to have extracellular actionsand to re-distribute from the cytosplasm to the inter-membrane space of mitochondria as well as to thenucleus (Apostolova and Cherian, 2000; Ye et al., 2001;Chung and West, 2004). Nuclear translocation dependson ATP, signaling kinases, the state of the cell, andoxidation of a cytosolic factor (Schmidt and Beyers-mann, 1999; Nagano et al., 2000; Woo et al., 2000;Takahashi et al., 2005). Mitochondrial uptake occursthrough an alternative (non-classical) pathway, andinvolves interactions of the lysine side chains of MT withthe outer mitochondrial membrane and release of zinc inthe intermembrane space (Ye et al., 2001). In addition,different cell lines secrete MT (Moltedo et al., 2000;Trayhurn et al., 2000a,b), and extracellular actions ofMTs have been observed. Intraperitoneally injected MT-1/-2 protects the injured mammalian brain and sup-presses collagen-induced arthritis (Youn et al., 2002;Penkowa, 2006). Extracellular MT-3 is a growthinhibitory factor (GIF) for neurons (Uchida et al.,1991). MT-3 is released from cultured astrocytes(Uchida et al., 2002), functions in a pathway that loadsneuronal vesicles with zinc (Cole et al., 2000), andinteracts with the small GTPase Rab3a (Knipp et al.,2005). Cadmium MT is transported from the liver to thekidney, where it is taken up and where the releasedcadmium damages proximal tubule cells (Nordberg,1972; Dudley et al., 1985; Chan et al., 1993). Uptakeof MT in kidney cell lines is an endocytotic processthat involves the scavenger receptors megalin/cubilin(Erfurt et al., 2003; Klassen et al., 2004).

Interactions of MTs with cells raise the question ofwhether or not MTs are taken up by cells other thankidney cells. New strategies for specific labeling of MTprovided fluorescent probes to address this question

(Hong and Maret, 2003; Hong et al., 2005). Employingthese probes, uptake of MT by lipid raft-dependentendocytosis in cultured hepatocytes is observed. Duringuptake, the metal but not the protein is released into thecytosol. This pathway of MT uptake might be physiolo-gically significant for functions of MT as an intercellu-lar effector and for its zinc-dependent cytoprotectivepotential.

MATERIALS AND METHODSMaterials

Sephadex G-25 and G-50 were purchased from GE Health-care (Piscataway, NJ); Cleland’s reagent (dithiothreitol, DTT)and pravastatin from Calbiochem (San Diego, CA); Alexa Fluor488 carboxylic acid succinimidyl ester, Alexa Fluor 546 C5

maleimide, Alexa Fluor 488 bovine serum albumin (F-albumin), and concanavalin A Alexa Fluor 627 conjugate fromMolecular Probes/Invitrogen (Eugene, OR); IMPACT proteinexpression kit from New England Biolabs (Beverly, MA); andcell culture products from Gibco (Carlsbad, CA). All otherchemicals were from Sigma (St. Louis, MO).

� 2006 WILEY-LISS, INC.

Abbreviations: CD, cyclodextrin; Cd7-MT2, metallothionein 2 with7 cadmium; DTT, dithiothreitol; F-albumin, FITC labeled bovineserum albumin; MT, metallothionein; R-transferrin, Alexa Fluor546 transferrin; T, thionein; R-MT, MT labeled with Alexa Fluor546 C5 maleimide at the S32C residue; Zn7-MT2, metallothionein2 with 7 zinc.

Contract grant sponsor: National Institutes of Health (to WM);Contract grant number: GM 065388.

*Correspondence to: Wolfgang Maret, Division of Human Nutri-tion, Preventive Medicine and Community Health, The Univer-sity of Texas Medical Branch, 700 Harborside Drive, Galveston,TX 77555. E-mail: [email protected]

Received 18 May 2006; Accepted 17 August 2006

DOI: 10.1002/jcp.20874

Expression of metallothionein andreconstitution with zinc or cadmium

Recombinant human MT2 reconstituted with either zinc orcadmium was prepared by using the IMPACT protein expres-sion system (Hong et al., 2001). The three-step purificationremoves any contaminating protein, including any bacterialendotoxin that could affect uptake of MT.

Labeling of MT with Alexa 546fluorophore

Site-specific labeling of MT2 at a cysteine side chainintroduced in the linker region between the two domains(S32C MT2 mutant) provides fluorescent tracers (Hong andMaret, 2003; Hong et al., 2005). Labeled MTs were preparedfrom reconstituted Cd7-MT (1 mg, 154 nmol) by adding 0.18 mg(223 nmol, i.e., a final molar ratio of 1.5:1) of the thiol-reactiveprobe Alexa 546 maleimide (a rhodamine derivative referred toas R) dissolved in 100 ml DMSO, and incubating the mixturefor 2 h at 258C with stirring under nitrogen to prevent MToxidation. Labeled Zn7-MT was prepared from labeled Cd7-MT(Hong and Maret, 2003). Labeled proteins were separated fromthe free probes on a Sephadex G-50 column (1� 50 cm). Thestoichiometries of labeling were 1:1 as determined from amolar absorptivity of E554¼ 111,100 M�1 � cm�1 for Alexa 546and both thiol and metal determinations for MT (Hong andMaret, 2003).

Tissue culture

Human hepatocellular carcinoma (HepG2) cells wereobtained from the American Type Culture Collection (Mana-ssas, VA). They were grown in DMEM containing 4.5 g/Lglucose, supplemented with 10% FBS (Hyclone, Salt Lake City,UT), 0.12 mg/ml streptomycin sulfate, and 0.1 mg/ml genta-micin sulfate. Cells were maintained at 5% CO2 and 378C in ahumidified atmosphere.

Cellular uptake of MT, albumin,and transferrin

Cells (1� 106 cells/400 ml or 1 ml) were seeded in either 24- or12-well culture plates on ethanol-treated microscope coverglasses, 12 mm (Fisher Scientific, Pittsburgh, PA) or in a Lab-TekII1 Chamber Slide system (Nalge Nunc International,Naperville, IL), and grown with DMEM/10% fetal bovineserum for 24 h. Cells were washed to remove the serum andincubated in serum-free medium for 2 h before addingfluorescent proteins. Uptake experiments were performedwith (i) free Alexa 546 fluorophore as control; (ii) Alexa 546labeled MT (R-MT), or (c) endocytosis markers, F-albumin, orR-transferrin. After incubation with these probes, DMEMmedium was decanted, and cells were washed with phosphate-buffered saline (PBS) three times to remove free probe. Cellswere also incubated with concanavalin A Alexa Fluor 647conjugate (20 mg/ml) to visualize the contour of the cells thatgrow in clusters. Cells were fixed with methanol or 4%formaldehyde at �208C for 10 min. Coverslides were mountedwith 50% glycerol or p-phenylene diamine and sealed withclear nailpolish.

Confocal laser microscopy

For imaging, cells on glass coverslides were placed inside achamber on the stage of an LSM 510 META confocal system,configured with an Axiovert 200 M inverted microscope (Zeiss,Jena, Germany). For detection of Alexa 546 dye, cells wereviewed with a rhodamine filter for images at 573 nm emission;for detection of Alexa 488 with an FITC filter for images at 494nm emission; for detection of Alexa 647 with a Cy5 filter forimages at 668 nm emission. Each image was corrected forbackground fluorescence. Data were acquired and processedwith a Zeiss LSM 510 workstation (version 3.0) and ZeissImage Browser (version 3.1) software. Exposure times wereidentical within each series of images and were chosen so thatall pixel intensities were within the linear response range ofthe camera.

Cellular uptake of fluorescent MT

Cells (6� 106) were incubated with 0.25mM R-MT for 30 min.Medium was decanted and saved for further analysis. Cellswere scraped into PBS, pelleted at 500g for 5 min, washed threetimes with 10 ml PBS to remove the free MT probe, and re-suspended in 1 ml PBS. A 500-ml aliquot of the cell suspensionwas removed, homogenized with a Potter-Elvehjem homoge-nizer (20 strokes), spun at 500g, and the protein concentrationof the supernatant determined with a Micro-BCA kit fromPierce (Rockford, IL). Fluorescence at 570 nm was determinedwith 530 nm excitation using an SLM-8000 spectrofluorimeterwith ISS (Champaign, IL) data acquisition and Vinci Multi-dimensional Fluorescence Spectroscopy software.

Membrane cholesterol depletion

Cellular cholesterol was depleted from the cytoplasmicmembrane by incubation of 1� 106 cells with differentconcentrations of methyl-b-cyclodextrin (CD) for 30 min inserum-free medium. Cells were incubated for 30 min witheither 0.25 mM R-MT or unlabeled Cd7-MT for fluorescence andcadmium measurements, respectively. MT that was not takenup was removed with three washes of 10 ml PBS. The cells werefixed with 10 mM methanol for fluorescence microscopy afterremoving them from Petri dishes with a cell scraper.

Inhibition of cholesterol synthesis

Cellular cholesterol synthesis was inhibited by incubating1� 106 cells with different concentrations of pravastatin (froma 10 mM stock solution in DMSO) for 24 h in serum-freemedium. Cells were incubated with 0.25 mM R-MT for 30 minand processed as described in the cholesterol depletionexperiment.

Subcellular fractionation

Cells (1� 107) were incubated with R-MT (0.25 mM) for30 min with or without prior treatment with methyl-b-cyclo-dextrin (5 mM) for 30 min. R-MT-loaded cells were scraped into5 ml PBS, pelleted by centrifugation, washed three times with10 ml PBS, and re-suspended in 2 ml of the same buffer. Cellswere homogenized in a Potter-Elvehjem homogenizer with atleast 20 strokes. Observation under a microscope verified thatmost of the cells were broken. Differential velocity sedimenta-tion resulted in five fractions: (1) P1 (pellet from homogenatespun at 1,000g, 10 min), containing nuclei, plasma membranesheets, and heavy mitochondria; (2) P2 (pellet from the P1supernatant spun at 3,000g, 10 min), containing plasmamembrane fragments and heavy mitochondria; (3) P3 (pelletfrom the P2 supernatant spun at 10,000g, 20 min), containingmitochondria, lysosomes, peroxisomes, and Golgi membranes;(4) P4 (pellet from the P3 supernatant spun at 100,000g,60 min), containing smaller intracellular membranes andvesicles; and (5) cytosol (the supernatant from the 100,000gcentrifugation, 60 min) (Rickwood, 1985). The first threecentrifugations were performed with a Beckman J2-21centrifuge, while a Beckman OPTIMATM XL PreparativeUltracentrifuge was used for the last step. For fluorescencemeasurements, the different pellets were re-suspended in 1 mlPBS. For determination of total cadmium by atomic absorptionspectrophotometry, washed cells were dried overnight at 658C,digested with 150 ml concentrated nitric acid at 808C for 12 h,and diluted to 4 ml with Milli-Q water (Millipore, Medfield,MA). Media from the uptake experiments and the PBS washeswere also analyzed for cadmium and MT by fluorimetry andatomic absorption spectrophotometry. Total cadmium contentof each fraction was analyzed accordingly. Fluorescence andcadmium contents of cytosolic fractions were determined fromaliquots of the sample.

Translocation of MT to the cytosol

To introduce MT into the cytosol of cells, a peptidetransporter (Endo-Porter) was used according to instructionsfrom the manufacturer (Gene Tools LLC, Philomath, OR).Briefly, 1� 106 cells grown on coverslides in 1 ml DMEMmedium were mixed with 10 ml Endo-Porter, loaded with R-MT(0.25mM final concentration) for 30 min, and washed with PBS.

CELLULAR UPTAKE OF METALLOTHIONEIN 429

Journal of Cellular Physiology DOI 10.1002/jcp

The coverslides with attached cells were then removed andimaged by confocal fluorescence microscopy.

Statistical analyses

Values are mean�SD and were analyzed by Student’st-test. Significance was assessed at the P< 0.05 level.

RESULTSNovel fluorescent probes for cellular

traffic of metallothionein

Fluorescence resonance energy transfer (FRET) sen-sors of MT employ a labeling strategy that introducesfluorophores at defined positions in the protein withoutinterference of the labeling with the zinc/thiolateclusters (Hong and Maret, 2003; Hong et al., 2005).Recombinant human MT2 with an S32C mutationbetween the two protein domains and a free N-terminusallows attachment of maleimide derivatives of fluoro-phores at the free cysteine and succinimidyl esterderivatives of fluorophores at the free N-terminus.These fluorescence-labeled MTs are novel probes forstudies of cellular uptake and traffic of MT.

Cellular uptake of MT by endocytosis

Cellular uptake of R-MT and R-transferrin was studiedin HepG2 cells. Labeled transferrin was employed todetermine whether or not cells take up MT by receptor-mediated, clathrin-dependent endocytosis. R-transferrinendocytosis occurs immediately after addition to themedium (1 min) (Fig. 1). The staining is more diffusethan that of R-MT (Fig. 1). While no significant changeof R-transferrin fluorescence is observed up to 30 min,cellular fluorescence of R-MT increases significantlyduring this time period (Fig. 1). No uptake of the proteinsis observed at 48C, demonstrating endocytosis. The freefluorophore is neither taken up nor diffuses into the cellbecause incubation of cells with the Alexa 546 probe doesnot result in any cellular fluorescence.

MT co-localizes with albumin but nottransferrin in the endocytotic pathway

For further characterization of the uptake pathway,possible co-localization of albumin and MT during

cellular uptake was investigated. Albumin enters thecell via a caveolae-dependent endocytosis pathway(Franke et al., 1987; Schubert et al., 2001). R-MT wasmixed with F-albumin, and the uptake of the proteinmixture was imaged in a single cell (Fig. 2, upper part)or in a cell cluster (Fig. 2, lower part). In both cases,F-albumin co-localizes with R-MT (arrows). In the cellclusters, some of the non-peripheral cells do not showMT or albumin uptake, suggesting contact inhibition asan attenuation mechanism for the uptake pathway. Todemonstrate the localization of the proteins relative tothe cells, cell contours were visualized with Alexa 647-concanavalin A (Fig. 2, lower part). Taken together, thedata demonstrate MT uptake by a pathway similar toalbumin but dissimilar to transferrin uptake.

Albumin has no effect on cellularuptake of MT

Co-localization of MT and albumin could indicate thatalbumin affects MT uptake. Albumin binds MT with abinding constant of 11 mM (Churchich et al., 1989).Kinetics of R-MT uptake in the absence or presence of F-albumin demonstrate that MT uptake is completewithin 60 min and that co-treatment with F-albuminhas very little effect on the rate of MT uptake (Fig. 3).Although confocal microscopy detects uptake at MTconcentrations of 25 nM, the fluorescence signal at thisconcentration is too weak when using a spectrofluori-meter for quantitative studies. Accordingly, concentra-tions of 0.25 mM R-MT were used in these studies. About5% of the MT probes are taken up at an MT concentra-tion of 0.25 mM. At this concentration of MT, uptake issaturated because longer incubations with MT do notincrease uptake (Fig. 3).

Cyclodextrin inhibits MT uptake

Endothelial cells take up albumin by caveolae-dependent endocytosis (Schubert et al., 2001). Thispathway is one of several non-classical pathways thatinvolve lipid rafts and are linked to cellular cholesterollevels. Cholesterol is required to maintain the structuralintegrity of caveolae (Hailstones et al., 1998). Because

Fig. 1. MT uptake by HepG2 cells: HepG2 cells were cultured in a Lab-Tek II1 Chamber SlideTM Systemin DMEM supplemented with 10% fetal bovine serum. Cells were incubated in serum-free medium for 2 h,and then incubated with 0.25 mM R-transferrin or R-MT at 378C for the times indicated. After washingthoroughly with PBS, cells were fixed with methanol, dried, and mounted with p-phenylene diamine.Cells were imaged using a Zeiss-510 confocal microscope (Rhodamine filter).

430 HAO ET AL.


MT and albumin co-localize the effect of CD, an inhibitorof caveolae and lipid raft formation (Tiruppathi et al.,2004), on MT uptake was investigated. Exposing thecells to CD removes cholesterol from the plasmamembrane and causes the disappearance of caveolae(Christian et al., 1997). Membrane cholesterol levels incells are affected only when CD concentrations are 5 mM

or higher (Mouat et al., 2003). Treating HepG2 cells with5 mM methyl-b-cyclodextrin reduces the cellular fluor-escence of R-MT significantly (Fig. 4A), inhibiting R-MTuptake by 69�4.5% (Fig. 4B). Incubating HepG2 cellswith 0.5 mM methyl-b-cyclodextrin, which has no effecton membrane cholesterol, has virtually no effect on MTuptake.

Pravastatin inhibits MT uptake

The cholesterol synthesis inhibitor pravastatin wasemployed for further investigation of the relationshipbetween cholesterol levels and MT uptake. Pravastatindecreases cellular cholesterol by inhibiting HMG-CoAreductase (Pan, 1991). When R-MT is added to cells thatwere incubated with 5–50 mM pravastatin for 24 h,uptake is reduced by 14�4% and 29�4%, respectively(Fig. 5).

Subcellular distribution of MT andcytosolic cadmium uptake

In order to determine the fate of MT after cellularuptake, R-MT-loaded cells were subjected to subcellularfractionation. Cd7-MT was used in this study becausethere is no cadmium in the cells. Thus, if cadmium isreleased from MT, it is detected readily. When the fivefractions obtained by differential velocity centrifugationare examined for their Alexa 546 fluorescence and totalcadmium content (Fig. 6), fluorescent MT is detected inthe P1, P2, P3, and P4 fractions but is virtually absentin the cytoplasmic fraction (Fig. 6A, black bar). The P3(10,000g) fraction shows the highest fluorescence(after subtracting the background fluorescence inuntreated cells). Since the P3 fraction contains mainlylysosomes, peroxisomes, and Golgi membranes, thisresult indicates that endocytosed MT does not enter thecytosol but accumulates in other cellular compartments,

Fig. 2. MT co-localization with albumin: HepG2 cells were cultured and treated as described in thelegend of Figure 1. Cells were incubated with 0.25 mM R-MT with the same amount of F-albumin eitherwithout (upper part) or with (lower part) Alexa 647-concanavalin A for 30 min at 378C. After washingthoroughly with PBS, cells were fixed with methanol, dried, and mounted with p-phenylene diamine.Filters: Cy5 for Alexa 647-concanavalin A, FITC for F-albumin, and Rhodamine for R-MT. Arrowsindicate co-localization.

Fig. 3. Effect of albumin on MT uptake: HepG2 cells were incubatedwith 0.25 mM R-MT alone (open squares) or with F-albumin (closedsquares) for different time periods (1, 5, 40, 60, 120, and 160 min),washed three times each with PBS, PBSþ 1% BSA, and PBS for 5 minat 48C, incubated with 0.1% pronase for 1 h in medium (DMEMþ10%FBS), harvested by centrifugation (500g for 5 min), rinsed with PBS,and lysed with 100 ml of 0.01% (v/v) Triton X-100 in PBS. Proteinconcentrations in the supernatant were determined with a bicincho-ninic acid (BCA) assay kit. Amounts of R-MT taken up were quantifiedby measuring fluorescence at 573 nm with 530 nm excitation.



mostly in lysosomes. In contrast, a significant amount ofcadmium is in the cytosolic fraction (Fig. 6B, black bar),demonstrating dissociation of cadmium from MT andtransport into the cytosol. To exclude the possibility

that the additional cysteine residue introduced into MTaffects cadmium binding and uptake, experiments wererepeated with wild-type Cd7-MT2. The same amounts ofcadmium are released into cytosol wild-type Cd7-MT2and S32C mutant Cd7-MT2.

Incubating cells with 5 mM methyl-b-cyclodextrinsignificantly reduces the fluorescence and cadmiumcontent of all five fractions (Fig. 6A, B, dark gray bar). Incells incubated with 5 mM methyl-b-cyclodextrin, thecytosolic cadmium concentration decreases from 19 to5 mg/L (Fig. 6B, dark gray bar).

Translocation of MT to the cytosol

Since endogenous MT is in the cytosol but extra-cellular MT probes remain in the vesicular system afteruptake, a method was sought for introducing probes intothe cytosol so that functions of MT in the cytosol could beexplored by fluorescence techniques. The cellular pep-tide transporter Endo-Porter was designed to deliversubstances into the cytosol of cells by an endocytosis-mediated process. Endo-Porter does not compromisemembrane integrity but renders endosomes ‘‘leaky,’’thus releasing endocytosed proteins into cytosol (Sum-merton, 2005). Endo-Porter alone does not have anyfluorescence (Fig. 7A). With the assistance of Endo-Porter, R-MT can be introduced into the cytosol ofHepG2 cells (Fig. 7B).

DISCUSSION

A fundamental issue for the functions of mammalianMTs is their localization. The finding of a cholesterol-dependent uptake pathway with subsequent transloca-tion of the metal ions but not the protein to the cytosol

Fig. 4. Cyclodextrin inhibition of MT uptake: HepG2 cells were incubated with different concentrationsof methyl-b-cyclodextrin (CD) (0, 0.5, and 5 mM) for 30 min. R-MT (0.25 mM final concentration) wasadded, and incubation continued for 30 min. A: Cells were observed under a confocal fluorescencemicroscope. B: Cells (1�106) were detached from the well plate, washed three times with PBS, andresuspended in 1 ml PBS. Fluorescence at 573 nm was determined with 530 nm excitation. Data aremean�SD of triplicate determinations. Asterisk indicates significance at P<0.05. No significantdifference was found for 0.5 mM CD treatment.

Fig. 5. Pravastatin inhibition of MT uptake: HepG2 cells wereincubated with pravastatin (5 and 50 mM) for 24 h. R-MT (0.25 mMfinal concentration) was added, and incubation continued for 30 min.Cells (1� 106) were detached from the well plate, washed three timeswith PBS, and resuspended in 1 ml PBS. Fluorescence at 573 nm wasdetermined with 530 nm excitation. Data are mean�SD of triplicatedeterminations. Asterisk indicates significance at P<0.05. Nosignificant difference was found for 5 mM pravastatin treatment.

432 HAO ET AL.


provides a new perspective on the functions of MT inmetal traffic and intercellular communication. Oncein the cell, the translocated metal ions bind to theapoprotein thionein, which is present in amounts

commensurate with those of MT in the hepatic cytosol(Yang et al., 2001). In a tightly controlled process, theresulting MT then redistributes metal ions in the cell,for example, from the cytoplasm to the nucleus or tomitochondria (Apostolova and Cherian, 2000; Ye et al.,2001).

HepG2 cells are epithelial cells that serve as a modelfor the secretion and uptake of biomolecules by hepato-cytes (Pohl et al., 2002). MT uptake via lipid raft-mediated endocytosis is supported by co-localizationwith albumin and the effects of inhibitors of thecholesterol pathway. However, the effects of theseinhibitors should not be taken as the only criterion fora lipid raft mechanism as cholesterol depletion alsoaffects the formation of endocytotic vesicles fromclathrin-coated pits (Rodal et al., 1999; Subtil et al.,1999). Uptake might involve caveolae that are mainlylocalized on the sinusoidal blood-facing membrane of thehepatocyte (Pol et al., 1999; Calvo and Enrich, 2000). Forthe MT uptake pathway to be significant for physiology,there must be extracellular MT. Indeed, MT is secretedfrom cells and can be detected in the blood, bile, andurine, although MT does not have a sequence motif thatwould identify it as a secreted protein. The liver releasescadmium MT, which subsequently is taken up by theproximal tubule cells in the kidney through interactionswith the scavenger receptors megalin/cubulin (Nord-berg, 1972; Dudley et al., 1985; Klassen et al., 2004). Inthis process, MT localizes to late endosomal/lysosomalmembrane vesicles (Erfurt et al., 2003). The results ofthe differential centrifugation study (Fig. 6) suggestthat the endocytosed MT also localizes to lysosomes inliver cells. Metal release from MT in lysosomes couldoccur by direct metal transfer to an acceptor or by thelower pH and/or the more oxidizing redox environmentof endosomes/lysosomes (Austin et al., 2005). Zincbinding to MT is both redox- and pH-dependent (Jianget al., 2000; Maret, 2004). Accounting for the effect of pHonly, it was estimated that about 60% of zinc and 20% ofcadmium are displaced from MT at a lysosomal pH of 4.7,and that the resulting apoprotein thionein is 1,500-foldmore sensitive towards degradation by lysosomalproteases, among which cathepsin B is the most active(McKim et al., 1992). However, this enzyme is verytightly inhibited by zinc ions with a KI value of about160 nM (pH 7.4) (unpublished results), raising thepossibility that thionein is not degraded by lysosomes

Fig. 6. Subcellular localization of MT and cadmium: R-MT (0.25 mMfinal concentration) was incubated with HepG2 cells (1�107) in 3 mlmedium at 378C for 30 min. Cells were also incubated with 5 mMmethyl-b-cyclodextrin for 30 min, and R-MT added to the medium.Cells were detached from the well plate, washed three times with PBS,homogenized, and subjected to differential centrifugation. A: Theamount of MT in each fraction was determined by fluorescence ofAlexa 546. B: The amount of cadmium in each fraction wasdetermined by atomic absorption spectrophotometry. The three barsrefer to: Light gray: cells without incubation, control; Black: cellsincubated with MT2; Dark gray: cells incubated with 5 mM methyl-b-cyclodextrin and then with MT2.

Fig. 7. Translocation of MT2 to the cytosol: HepG2 cells were incubated with 1% (v/v) Endo-Porter, andthen R-MT added to a final concentration of 0.25 mM. A: Cells loaded with Endo-Porter only; (B) Cellsloaded with Endo-Porter and R-MT.



but recycled to the plasma membrane. Endogenous MTis degraded in lysosomes, in particular when the cellularzinc content decreases (Chen and Failla, 1989). Incontrast, copper MT is resistant to lysosomal proteoly-sis, accumulates under conditions of copper overload,and is excreted from the liver into both plasma and bile(Bremner et al., 1989).

Relatively small amounts of MT exist in extracellularfluids (Bremner and Mehra, 1991). A role of extracel-lular MT in the acute phase response has beendiscussed. Thionein is induced by an IL-6-dependentmechanism (Schroeder and Cousins, 1990). It is by nomeans a foregone conclusion that MT is formedconcomitantly from newly synthesized thionein becausethe cellular availability of zinc is tightly controlled(Maret, 2004). The plasma concentration of MTincreases during various forms of stress (Sato et al.,1993). In the plasma of newborn rat pups, MT concen-trations are up to 1,000-fold higher than in the plasma ofadult rats (Morrison and Bremner, 1987). Since acuteimmobilization stress increases serum MT concentra-tions in rats, a function of MT in the physiologicaladaptation to stress has been suggested (Hidalgo et al.,1988). During surgery, MT increases sevenfold in theblood plasma of humans from a normal reference valueof 32� 16 ng/ml (Akintola et al., 1995). The antibodiesused to detect plasma MT do not provide informationabout the redox state and metal load of MT (Maret,1995). Therefore, it is not clear in which form the proteinis secreted as an acute phase reactant. If MT partici-pates in the liver uptake of zinc during the acute phaseresponse (Falchuk, 1977), then it must be the apoproteinthionein that is secreted.

In blood, MT is expected to bind to albumin based on adissociation constant of 11 mM for the MT/albumincomplex (Churchich et al., 1989). Whether or not MTdelivers zinc to a specific zinc-binding site in albumin isunknown (Stewart et al., 2003). Albumin enhances zincuptake of endothelial cells, and endocytotic co-transportof zinc and albumin has been postulated (Rowe andBobilya, 2000). Since albumin participates in transcy-tosis of myeloperoxidase in endothelial cells (Tiruppathiet al., 2004), albumin could be the carrier of MT throughthe endothelial cells that face the space of Disse and thesinusoids of hepatocytes. MT- and albumin-dependentzinc uptake could represent two independent processesthat relate to either endothelial or epithelial cells, andthe interaction of these systems might be important forcellular zinc uptake in endothelial and epithelial cells inthe liver. In primary cultures of rat hepatocytes,cadmium bound to MT but not to albumin is availablefor the cells (Pham et al., 2004). In the human breastepithelial cell line MCF-10A, depletion of plasmamembrane cholesterol content inhibits zinc uptake(Mouat et al., 2003). It might also be significant for zincuptake that cholesterol increases significantly in theVLDL and HDL fractions in zinc-deficient mice and thatzinc supplementation decreases cholesterol (Reitereret al., 2005).

The intestinal cell line Caco-2 (Moltedo et al., 2000)and primary white adipocytes secrete MT (Trayhurnet al., 2000a,b). As a putative adipokine, MT may havean endocrine role in signaling between adipocytes andthe liver. A role of MT in energy metabolism is indicatedby the fact that MT-1/2 knock-out mice become obese(Beattie et al., 1998). The secretion of MT-3 fromastrocytes, growth inhibition of neuronal cells, and afunction of loading synaptic vesicles with zinc suggestthat MT also has a paracrine function. Thus, in addition

to its role in cellular translocations, MT appears toparticipate in signaling between organs and cells.

In summary, a decisive advance for studying thelocalization of MT is the preparation of site-specificallylabeled MTs (Hong and Maret, 2003; Hong et al., 2005),which in their single-labeled form serve as tracers forthe protein and in their double-labeled form are FRETsensors. Because MT probes also can be brought into thecytosol with protein transduction systems such as Endo-Porter, it is now possible to study metal release in livingcells. These tools should greatly aid in elucidating theprotective functions of extracellular MT and details ofthe endosomal/lysosomal traffic.

ACKNOWLEDGMENTS

We thank Dr. Bo Xu and Xiaolian Liang in the ProteinExpression Core Facility of UTMB for help withfermentation of bacteria for protein expression;Dr. Leoncio Vergara in the Optical Imaging Laboratoryof UTMB for help with confocal laser microscopy;Dr. V.M. Sadagopa Ramanujam, Associate Professor,Department for Preventive Medicine and CommunityHealth, The University of Texas Medical Branch, forzinc and cadmium analyses by atomic absorptionspectrophotometry (supported by the Human NutritionResearch Facility); Nicki Watson at the WhiteheadInstitute, W. K. Keck Biological Imaging Facility(Massachusetts Institute of Technology) for trainingand help with confocal laser microscopy.

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lipid raft-dependent endocytosis of metallothionein in hepg2 cells

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