Báo cáo khoa học: Membrane trafficking of CD98 and its ligand galectin 3 in BeWo cells ) implication for placental cell fusion pot

Results Expression of CD98 increases over time almost linearly after forskolin treatment Fusion of BeWo cells is enhanced by forskolin treat-ment, which, by activating adenylyl cyclase,

in BeWo cells ) implication for placental cell fusion

Paola Dalton1, Helen C Christian1, Christopher W G Redman2, Ian L Sargent2and C A R Boyd1

1 Department of Physiology, Anatomy and Genetics, University of Oxford, UK

2 Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Oxford, UK

CD98, a multifunctional membrane protein originally

discovered on the surface of activated T cells [1], is

now known to be present in many cell types and all

malignant cell lines [2] The CD98 antigen (also known

as FRP-1 and 4F2) is a dimeric structure consisting of

a type 2 heavily glycosylated integral membrane

pro-tein of around 80 kDa (heavy chain) covalently

attached to a nonglycosylated integral membrane

pro-tein of 40 kDa (light chain); there are six possible light

chains, which are expressed differentially according to

the tissue of origin [3,4] The heavy and light chains

are linked by a single extracellular disulfide bond In

this heterodimeric form, the CD98 protein is an amino

acid transporter transferring specific groups of amino acids across the plasma membrane, the group and the mechanism depending on the properties of the specific light chain Transfection studies in mammalian cells have indicated that whereas CD98hc can be expressed

on the plasma membrane on its own, trafficking of the light chain to the cell surface is possible only in the heterodimeric form and apparently independently of disulfide linkage [5]

Although roles for CD98 in cellular differentiation, adhesion, growth, apoptosis and amino acid transport have been reported, plausible mechanisms underlying most of these functions are only starting to emerge,

Keywords

brefeldin A; CD98; cell fusion; galectin 3;

trafficking

Correspondence

P Dalton, Department of Physiology,

Anatomy and Genetics, University of

Oxford, Oxford OX1 3QX, UK

Fax: +44 186 527 2420

Tel: +44 795 286 8502

E-mail: paoladalton@yahoo.co.uk

(Received 28 December 2006, revised 6

March 2007, accepted 23 March 2007)

doi:10.1111/j.1742-4658.2007.05806.x

CD98 heavy chain (CD98hc), expressed at high levels in developing human trophoblasts, is an integral membrane protein with multiple N-linked gly-cosylation sites and known to be important for cell fusion, adhesion, and amino acid transport Western blotting and flow cytometry were used to study the effect of brefeldin A, an inhibitor of protein translocation through the Golgi, on CD98hc in the human placental trophoblast cell line BeWo Although brefeldin A treatment caused increased cell surface expression of CD98hc, a novel partially glycosylated form of the protein was found and, concomitantly, cell fusion was reduced Western blotting showed that CD98 and galectin 3, a proposed ligand for the glycosylated extracellular domain of CD98hc, co-immunoprecipitated, and double-label immuno-electron microscopy confirmed that CD98hc associated with galec-tin 3 Furthermore, cell fusion was reduced (specifically) by the disacchar-ide lactose, a known ligand for the carbohydrate recognition domain of galectin 3, suggesting that the association was functional Taken together, the data suggest that N-glycosylation of CD98 and subsequent interaction with galectin 3 is critical for aspects of placental cell biology, and provides

a rationale for the observation that, in the mouse, truncation of the CD98hc extracellular domain leads to early embryonic lethality [Tsum-ura H, Suzuki N, Saito H, Kawano M, Otake S, Kozuka Y, Komada H, Tsurudome M & Ito Y (2003) Biochem Biophys Res Commun 308, 847–851]

Abbreviations

BFA, brefeldin A; CRD, carbohydrate recognition domain; EM, electron microscopy; ER, endoplasmic reticulum; FACS, fluorescence activated cell sorting; Lac, lactose; PFA, paraformaldehyde.

and formation of activated complexes with other

pro-teins, in particular b1-integrin, galectin 3 and CD147,

has been proposed by various investigators [6–9]

CD98 expression is also necessary for virus-induced

cell fusion and for osteoclast formation [10–12] and,

importantly, it is found in cytotrophoblasts and on the

plasma membrane of the syncytiotrophoblast of the

human placenta [13,14] Furthermore, manipulation of

CD98 expression by antisense oligoneucleotide and

small interfering RNA affects both amino acid

trans-port and cell fusion in BeWo cells [15–17] More

recently, we have shown that CD98 involvement in the

process of cell fusion that is necessary for

syncytiotro-phoblast formation is a distinct function from its role

in amino acid transport Indeed, by crosslinking

CD98hc with monoclonal antibodies to CD98, we have

shown increased surface expression of this molecule

and increased fusion of BeWo cells (a well-established

choriocarcinoma cell line that can undergo fusion and

morphologic differentiation similar to the formation of

syncytiotrophoblast by the cytotrophoblasts in the

pla-centa) In contrast, LAT1 (one of the six known light

chains) surface expression and amino acid transport

were disrupted [18]

The macrocyclic lactone brefeldin A (BFA) is a

metabolite of the fungus Eupenicillium brefeldianum

and has antiviral, antibacterial and antifungal

activit-ies Most importantly, though, it specifically and

reversibly blocks protein transport from the

endoplas-mic reticulum (ER) to the Golgi apparatus in many

cell types and species Distinct morphologic changes

accompany several specific and reversible effects on

cellular protein traffic; however, the precise effects of

BFA vary among cell types Because of its numerous

and reversible effects on protein transport and

process-ing, BFA has become an important tool for cell

biolo-gists [19,20] We decided to employ this drug to

perturb the protein trafficking and function of CD98

and galectin 3, which has been proposed as an

endog-enous crosslinker for CD98 [21–23] Galectin 3 was

originally found by Ho and Springer as a surface

mar-ker called Mac2, which is present on the cell surface of

inflammatory macrophages [24] Galectins belong to a

b-galactoside-binding family of proteins defined by

their conserved peptide sequence elements, which are

crucial for the carbohydrate-binding activity of those

lectins Fourteen galectins (galectin 1–14) have been

found in mammals so far, and are also known in birds,

amphibians, fish, nematodes, Drosophila, sponges, and

fungi A common feature of all galectins is the strong

modulation of their expression during development

Galectin 3 is expressed widely in epithelial and immune

cells, and its expression is correlated with cancer

aggressiveness and metastasis It is reported to be involved in various biological phenomena, including cell growth, adhesion, differentiation, angiogenesis, and apoptosis (indeed, it is the only antiapoptotic galectin family member) Galectin 3 is composed of one carbohydrate recognition domain (CRD), consist-ing of 130 amino acids, and of an additional non-CRD domain, which is involved in the oligomerization

of galectin 3 The oligomerization results in the forma-tion of a galectin 3 molecule that possesses multivalent CRDs Oligomerization enables galectin 3 to mediate crosslinking of its ligands In order to crosslink surface ligands to exert its activities, galectin 3, which is mainly intracellular, has to be released extracellularly; however, this protein contains no hydrophobic sequences that may function as signal sequences or transmembrane domains, and is secreted by unknown mechanisms [21,25] (although alternative spliced forms

of galectin 3 that contain transmembrane domains have been detected in chicken osteoblast-like cells and

in intestine [26]) Finally, there is evidence for galec-tin 3 as a factor in RNA splicing, based on the local-ization of the protein in the nucleus [27]

In this article, we further examine to what extent the functions of CD98, other than amino acid transport, are independent of dimerization with the light chain LAT1, and whether interaction with galectin 3 is necessary to facilitate fusion The properties of and putative relationship between these two molecules are discussed in the context of cellular distribution and cel-lular fusion

Results

Expression of CD98 increases over time almost linearly after forskolin treatment

Fusion of BeWo cells is enhanced by forskolin treat-ment, which, by activating adenylyl cyclase, results in

an increase in intracellular cAMP concentration We have previously shown, using single-color flow cytome-try [fluorescence activated cell sorting (FACS)], a signi-ficant increase of CD98 expression on intact BeWo cells after forskolin treatment for 24 h [18] Here, we determined the levels of expression of CD98 by west-ern blotting in cell extracts from BeWo cells cultured with or without forskolin for 12 h, 24 h, 36 h, or 48 h (Fig 1A,B): CD98 expression in cells cultured in the presence of the vehicle (dimethylsulfoxide, control cells) was not substantially different from that in cells treated with 100 lm forskolin after 12 h of culture After 24 h, whereas there was no increase in control cells, the addition of forskolin produced a 20%

increase in CD98 expression This stimulation

increased almost linearly up to 48 h, at which time

there was approximately 35% more CD98 in BeWo

cells cultured in forskolin-containing medium than in

control cells The mean CD98 expression of the two

types of culture was significantly different, with a

P-value of 0.032 (two-tailed paired t-test)

CD98 surface expression increases after cell

treatment with BFA

In this series of experiments, BeWo cells were cultured

in six-well plates in the presence of the vehicle or

100 lm forskolin for 24 h At 20 h, 22 h, 23 h, and

23.5 h (for a total of 4 h, 2 h, 1 h, and 30 min,

respect-ively), BFA was added to half of the wells to a final

concentration of 5 lgÆmL)1, and the cells were

returned to culture for the remaining period

Single-color FACS, while confirming that forskolin

stimula-tion significantly enhanced CD98 surface expression as

compared to control cells (dimethylsulfoxide), clearly

showed, contrary to expectation, a time-dependent

increase of CD98 surface expression on intact BeWo cells after BFA treatment for both control and forsko-lin-incubated cells (Fig 2A) However, this was not due to an increase in the amount of CD98, as total (surface plus cytoplasm) CD98 expression did not sig-nificantly change (Fig 2B), suggesting that BFA treat-ment had increased CD98 trafficking to the cell surface

Detection of partially glycosylated⁄ unglycosylated CD98 after cell treatment with BFA and tunicamycin

We then used SDS⁄ PAGE and western blotting to look at CD98 expression in cell lysates from BeWo cells cultured for 24 h as above but with or without BFA (5 lgÆmL)1) only for the last 4 h We speculated whether BFA, known to produce distinct morpho-logic⁄ structural effects at the ER–Golgi level, could cause alteration not only of CD98 trafficking to the plasma membrane but also of its structure

Interestingly, after incubation of the blots with rabbit anti-(human CD98), we observed an extra band that ran lower than the normal CD98 band of

 80 kDa (reduced blots) or  110–120 kDa (nonre-duced blots) and had an approximate molecular mass

of  64 kDa or  80 kDa, depending on whether the gels had been run under reducing or nonreducing conditions The extra band was present only in the samples treated with BFA in either control or forsk-olin-stimulated cells (Fig 3A,B), and presumably cor-responds to partially glycosylated CD98 proteins that failed to complete the complex process of N-glycosy-lation in the ER–Golgi apparatus This is consistent with the results obtained when we treated BeWo cells for 24 h with tunicamycin, an antibiotic that inhibits the first steps of N-linked glycosylation and blocks the formation of new N-glycosidic protein–carbo-hydrate linkages Under reducing conditions, in the absence of forskolin, an extra band of lower mole-cular mass ( 53 kDa) was detected in these lysates (Fig 3A2) After forskolin treatment, known to sti-mulate CD98 expression, in addition to the 53 kDa band, a tight band running at approximately 49 kDa was clearly identifiable This, we suggest, corresponds

to the fully unglycosylated CD98 molecule, and is compatible with the theoretical 30 kDa mobility shift that is predicted based on the four potential extracel-lular N-glycosylation sites The number of bands seen

in Fig 3A2 must reflect the total population of immunoreactive CD98 molecules after 24 h of tunica-mycin treatment; these molecules normally will only

be present transiently, and thus the duration of

A

B

12.5 Forskolin

dimethylsulfoxide

24 hrs

12 hrs - 36 hrs - 48 hrs

-+F

97

64 97

64

CD98hc

10.0

7.5

5.0

2.5

0.0

12 hours 24 hours 36 hours 48 hours

Fig 1 The expression of CD98 increases over time almost linearly

after forskolin treatment Western blotting on a 4–12% Bis⁄ Tris

NuPage gel run under reducing conditions with Mops running buffer:

BeWo cells at 50–60% confluence were treated with

dimethyl-sulfoxide (vehicle control, – F) or 100 l M forskolin (+ F) for the

indi-cated times at 37 C (A) Immunoblot after incubation with rabbit

anti-(human CD98) (Santa Cruz, 1 : 200), horseradish

peroxidase-conjucated goat anti-rabbit IgG and 3,3¢-diaminobenzidine (DAB);

the data shown are representative of two independent experiments

performed in triplicate (B) Absorbance of the 80 kDa band

quanti-fied by densitometry The data are the means of two independent

experiments performed in triplicate ± SEM The mean CD98

expression of the two types of culture was significantly different,

with a P-value of 0.032 (two-tailed paired t-test).

Fig 2 CD98 surface expression increases after BFA treatment BeWo cells at 50–60% confluence were incubated in medium containing di-methylsulfoxide (vehicle control) or 100 l M forskolin (Forsk) for 24 h at 37 C BFA was added for the indicated times before harvesting, and CD98 was detected by single-color flow cytometry Cells were labeled with goat anti-(human CD98) and rabbit anti-(goat IgG) conjugated with fluorescein isothiocyanate (A) Labeling of surface antigens on intact BeWo cells (B) Labeling of surface and intracellular antigens after cell permeabilization n ¼ number of cell samples.

A 2

Fig 3 Detection of partially glycosylated and unglycosylated CD98 after cell treatment with BFA and tunicamycin Western blotting under redu-cing (A) or non reduredu-cing (B) conditions on a 10% Bis ⁄ Tris NuPage gel: BeWo cells at 50–60% confluence were treated with dimethylsulfoxide (vehicle control, – Forskolin) or with 100 l M forskolin (+ Forskolin) for 24 h at 37 C Immunoblots were incubated with rabbit anti-(human CD98) (1 : 200), horseradish peroxidase-conjucated goat anti-rabbit IgG and DAB A novel band (arrow) of  64 kDa (A 1 ) or  80 kDa (B) was present in whole cell lysates from BFA-treated cells in both control and forskolin-stimulated cells, presumably a partially glycosylated form of CD98 A band

of lower molecular mass ( 53 kDa) was present in both control and forskolin-stimulated cell lysates after tunicamycin treatment (A 2 ), with an additional band of  49 kDa after forskolin treatment Single representative blots from two experiments run in duplicate.

glycosidase inhibition will determine the precise

pat-tern observed

Cell fusion decreases after pulse treatment

with BFA for 4 h

To investigate the relationship between the changes

observed in CD98 expression and structure after BFA

treatment of BeWo cells with functional alterations, we

used two-color FACS to quantify cellular fusion

We used 3,3¢-dioctadecyloxacarbocyanine

perchlo-rate (DiO) cell-labeling solution, a lipophilic tracer that

is weakly fluorescent in water but highly fluorescent

and quite photostable when incorporated into

mem-branes, and Mitotracker deep red 633, a cell-permeant

mitochondrion-selective dye, to uniformly label

suspen-ded BeWo cells as previously described [18] Briefly,

flow cytometry analysis of a 50 : 50 mixed cell

popula-tion from cells stained either with DiO or Mitotracker

red and then cultured together allows us to quantify

cellular fusion⁄ stable aggregation, which is represented

by double-positive cells To better evaluate the effects

of BFA treatment on cell fusion in this group of

experiments, BFA was added for 4 h in the middle of

a 24–26 h culture to cells incubated with or without

100 lm forskolin The medium was then changed back

to dimethylsulfoxide or forskolin alone for the

remain-ing culture time We note that, inevitably, the

magni-tude of the effect will be reduced by the diverse

cellular stages (proliferation, aggregation, fusion, etc.)

of the BeWo cell population in the window of the

pulse of BFA Two-color FACS analysis showed a

decrease in cellular fusion after pulse BFA treatment

as compared to both groups of BFA-untreated cells

(P¼ 0.048, one-way ANOVA) (Fig 4) However, when

we measured CD98 surface expression in control or

forskolin-stimulated cells, we found that this was still

increased in the presence of BFA (data not shown)

Galectin 3 and CD98 co-immunoprecipitate

We have previously postulated a role for galectin 3,

an S-type lectin containing a carbohydrate-binding

domain, as a physiological ligand of CD98 in vivo [18]

Figure 5A shows the primary intracellular

distribu-tion of galectin 3 in the cytoplasm and nucleus of

BeWo cells, determined by indirect

immunofluores-cence To investigate the possible ligand-binding role of

galectin 3 in relation to CD98, BeWo cell lysates were

incubated with a goat polyclonal antibody against

CD98 or a goat polyclonal or mouse monoclonal

anti-body against galectin 3 Original whole cell lysates and

immunoprecipitates were then subjected to SDS⁄ PAGE

and western blotting (Fig 5B), and cut into single strips as described in Experimental procedures Whole lysates of BeWo cells (lanes 2 and 7) were probed with rabbit (human CD98) (lane 2) or with goat anti-(human galectin 3) (lane 7) Goat anti-anti-(human CD98) immunoprecipitates (lanes 3–6) were probed with rab-bit anti-(human CD98) (lane 3), goat anti-(human galectin 3) (lane 4), mouse anti-(phosphatidylinositol 3-kinase) (lane 5, as irrelevant control antibody), and rabbit IgG (lane 6, as negative control) Goat anti-(human galectin 3) immunoprecipitates (lanes 8, 9 and 11) were probed with mouse anti-(human galectin 3) (lane 8), rabbit anti-(human CD98) (lane 9), and rabbit IgG (lane 11) Mouse anti-(human galectin 3) immuno-precipitate (lane 10) was probed with rabbit anti-(human CD98) The results clearly demonstrate galectin 3 and CD98 co-immunoprecipitation in BeWo cell extracts A band equivalent to the molecular mass

of galectin 3 monomers ( 28–30 kDa) was present in

Fig 4 Cell fusion decreases after pulse treatment with BFA for 4 h Double-color flow cytometry assays for detection of fused (double-positive) cells BeWo cells were prestained with DiO (maximum emission 501 nm) or Mitotracker deep red 633 (maximum emission

665 nm) dye Single-color cells or a 50 : 50 mixture of both cells were then cultured for 12–14 h in medium containing dimethylsulfoxide or 100 l M forskolin (Forsk) at 37 C, and BFA (final concentration 5 lgÆmL)1) was then added to the medium for 4 h After that, the medium was replaced with fresh medium containing dimethylsulfoxide or 100 l M forskolin, and cells were cultured for a further 8–10 h The graph shows data normalized to dimethylsulfox-ide control (dimethylsulfoxdimethylsulfox-ide ¼ 10%); n ¼ number of cell samples Statistical analysis: one-way ANOVA , P-value 0.048.

CD98 immunoprecipitates (with an additional band of

higher molecular mass, probably corresponding to

galectin 3 dimers) A band equivalent to the molecular

mass of CD98 ( 80 kDa) was present in the reverse

immunoprecipitation experiment, whether or not the

immunoprecipitates were prepared using the goat or

the mouse anti-(human galectin 3)

CD98 and galectin 3 co-localize in the plasma

membrane, cytoplasm and nucleus

We have previously shown CD98 expression and

distri-bution by immuno-electron microscopy (immuno-EM)

[18] In the current study, we performed immuno-EM

of galectin 3 We found that galectin 3 was uniformly

distributed in the cytoplasm and nucleus, even if it was

scarce on the cellular membrane, in both the

dimethyl-sulfoxide-treated and the forskolin-treated groups,

although in the latter, sporadic clustering of

immuno-reactivity was observed (Fig 6A) To further confirm

the close localization of the galectin 3 and CD98

mole-cules, we used immuno-EM and a standard double gold

technique: double immunoreactivity was determined

using an appropriate secondary antibody)10 nm gold complex to detect anti-CD98 (smaller-diameter parti-cles) and an appropriate secondary antibody)15 nm gold complex to detect anti-galectin 3 (larger-diameter particles) The electronmicrographs in Fig 6B,C clearly show co-localization of these two molecules in the plasma membrane, in the cytoplasm and in the nucleus

of forskolin-treated BeWo cells

Inhibition of galectin 3 binding to membrane glycoproteins affects cellular fusion

We then investigated whether the close proximity of CD98 and galectin 3 in several cellular locations was indicative of a functional association

Galectin 3, like most members of the galectin family, acts as a receptor for ligands containing poly(N-acetyl-lactosamine) sequences through the C-terminus CRD

We used the high affinity of galectin 3 for lactose (Lac) to inhibit binding between the glycosylated sites

of CD98 and galectin 3 CDR domains, and measured its effect on cellular fusion The cells were labeled either with DiO and Mitotracker Deep Red 633, or

A

B

Fig 5 Galectin 3 is detected in BeWo cells and co-immunoprecipitates with CD98 (A) Immunofluorescence: galectin 3 (fluorescein isothiocyanate) primary distribution in the cytoplasm and nucleus of BeWo cells; nuc-lei are stained with DAPI (B) Co-immuno-precipitation: western blotting of a 10% Bis ⁄ Tris NuPage gel run under reducing con-ditions with Mes running buffer BeWo cell original total lysate (lanes 2 and 7) was probed with rabbit anti-(human CD98) (lane 2) or with goat anti-(human galectin 3) (lane 7) Goat anti-(human CD98) immuno-precipitates (lanes 3–6) were probed with rabbit (human CD98) (lane 3), goat (human galectin 3) (lane 4), mouse anti-(human PI3Kinase) (lane 5, irrelevant control antibody) and rabbit IgG (lane 6) Goat anti-(human galectin 3) immunoprecipitates (lanes 8, 9 and 11) were probed with mouse (human galectin 3) (lane 8), rabbit anti-(human CD98) (lane 9) and rabbit IgG (lane 11) Mouse anti-(human galectin 3) im-munoprecipitate (lane 10) was probed with rabbit anti-(human CD98) Arrows indicate CD98 immunoreactivity (upper arrows) or galectin 3 immunoreactivity (lower arrows).

with DiO and

1,1¢-dioctadecyl-3,3,3¢,3¢-tetramethyl-indodicarbocyanine perchlorate (DiD), another

lipo-philic tracer with markedly red-shifted fluorescence

excitation and emission spectra in the same range as

Mitotracker Deep Red We followed the protocol

employed for two-color FACS after BFA treatment;

here, however, pulsed incubation with BFA for 4 h was substituted by an equal incubation time with

50 mm Lac or, in some experiments, with 50 mm malt-ose, which has a much lower affinity for galectin 3 Interestingly, we observed a small but significant reduction in cell fusion in the presence of Lac (Fig 7)

Discussion

Syncytial fusion is a rare event in cell biology

In humans, we typically find three syncytial tissues: syncytiotrophoblast, striated muscle fibers and

Fig 7 Inhibition of galectin 3 crosslinking membrane glycoproteins affects cellular fusion Double-color flow cytometry assays for detection of fused (double-positive) cells BeWo cells were pre-stained with DiO (em 501 nm) or DiD (em 665 nm) dye Single-color cells or a 50 : 50 mixture of both cells were then cultured for 12–14 h in medium containing dimethylsulfoxide or 100 l M forsko-lin (Forsk) at 37 C, when Lac (final concentration 50 m M ) or malt-ose (Malt, final concentration 50 m M ) was added to the medium for

4 h The medium was then replaced with fresh medium containing dimethylsulfoxide or 100 l M forskolin, and cells were cultured for a further 8–10 h The graph shows data normalized to dimethylsulfox-ide control (dimethylsulfoxdimethylsulfox-ide ¼ 10%); n ¼ number of cell samples Statistical analysis: one-way ANOVA , P-value 0.0148.

A

B

C

Fig 6 Galectin 3 co-localizes with CD98 (A) Immuno-EM: electron micrograph of forskolin-treated cell (· 25 000) showing galectin 3 (arrows); note occasional clustering of gold particles (B, C) Double labeling immuno-EM: electron micrographs of forskolin-treated cells showing co-localization of galectin 3 and CD98 at the plasma mem-brane (B,C), in the nucleus (B), and in the cytoplasm (C) (arrows) Sections were sequentially stained using as secondary antibodies anti-(goat IgG) )10 nm gold complex to detect anti-CD98 (smaller-diameter particles) and anti-(rabbit IgG) )15 nm gold complex to detect anti-galectin 3 (larger-diameter particles) Three representa-tive fields; scale bars 200 nm.

chondro-osteoclast Syncytiotrophoblast forms during

implantation, and is then maintained at the villous

maternal–fetal interface throughout pregnancy A useful

model of trophoblast syncytialization is the

choriocarci-noma cell line BeWo; these cells are able to fuse, and

fusion can be also enhanced by forskolin treatment

Recently, the use of the fungal metabolite BFA to

cause Golgi breakdown showed that part of Golgi

gly-cosylation enzymes recycle to the ER, whereas Golgi

matrix proteins are retained in a set of cytoplasmic

membranes; this has led to the suggestion that BFA

disrupts a dynamic membrane-recycling pathway

between the ER and cis⁄ medial Golgi, effectively

blocking membrane transport out of but not back to

the ER [28] However, both the dynamic interaction

between ER and Golgi and the mechanism of action

of BFA are still subjects of intense discussion

N-linked glycosylation of membrane proteins is

acquired as a post-translational modification in the

ER, and further processing takes place in the Golgi

before the proteins reach the cell surface CD98 has

been previously shown to be involved, among its many

other functions, in trophoblast fusion [17,18] As

gly-cosylation seems to be important for correct protein

folding and for ligand–receptor interactions, and

because CD98 is an N-glycosylated protein [29,30], in

this study we investigated the effect of BFA on the

expression and the function of CD98 and its direct

and indirect effect on galectin 3, which binds

glycosyl-ated proteins through its CDR site [25]

By analysing BeWo cells at different time points with

SDS⁄ PAGE, we showed that, as a consequence of

fors-kolin treatment, there is a time-dependent increase in

CD98 protein expression comparable to that of the

CD98 mRNA previously observed [31] Unexpectedly,

we found that CD98 surface expression was also

incre-ased in a time-dependent manner in BeWo cells treated

with BFA This result implied the existence, for CD98,

of an alternative route to the plasma membrane that is

independent of the classic secretory pathway through

the trans-Golgi apparatus, which is used by most

secre-tory and transmembrane proteins and can be inhibited

by BFA Furthermore, analysis of western blots probed

with CD98 antibody, under reducing and non reducing

conditions, showed the presence of an additional band

in the BFA-treated cell lysates; this band presumably

corresponded to a partially glycosylated form of CD98,

after breakdown of the Golgi apparatus, in the last 4 h

of culture Taken together, these two findings would

suggest that a partially glycosylated form of CD98 is

capable of reaching and inserting into the cell membrane

via an unknown mechanism of transport that is

inde-pendent of the ER–trans-Golgi pathway

We next investigated whether CD98 glycosylation was necessary for its role in cellular fusion For this purpose, it was important to add BFA to the culture when the cells were just starting to fuse; previous experiments had indicated that this occurs after 12–

14 h Moreover, we had found that BeWo cells undergo morphologic changes followed by detachment and death if cultured with BFA for over 6–8 h (data not shown) However, BFA effects are reversible if the drug is removed We decided, therefore, to add BFA for 4 h in the middle of the culture time, to remove it, and then to observe the number of cells that under-went fusion as compared to untreated cells This could

be quantified by two-color FACS of BeWo cells previ-ously labeled with one of two well-separated fluores-cent dyes and then calculation of the number of double-fluorescent cells [18] The results showed that glycosylation of CD98 is important for the fusion of BeWo cells as, although the molecule was still over-expressed on the surface of BFA-treated cells (data not shown), cellular fusion was decreased as compared to untreated cells However, as anticipated, the magnitude

of the observed effect was moderate

It has been suggested that galectin 3 is an endo-genous crosslinker for CD98 and may promote CD98 dimerization (and consequent integrin activation) [21]

We have shown in BeWo cells, both by immunofluo-rescence and by immuno-EM, that galectin 3 is expressed in all three cellular compartments Immuno-precipitation of BeWo cell total lysates with either goat anti-(human CD98) or goat or mouse anti-(human galectin 3) has also shown that CD98 and galectin 3 co-immunoprecipitate Consequently, we used imm-uno-EM to confirm the relative positions of galectin 3 and CD98 in the cells We showed unambiguous co-localization of the two molecules, both

intracellular-ly, in the nucleus and cytoplasm, and at the plasma membrane The relative abundance of CD98 molecules

as compared to that of galectin 3 molecules at the same location supports the hypothesis of dimerization

of CD98 molecules through linking with either mono-meric or oligomono-meric forms of galectin 3 In the latter case, galectin 3 would have several CDR sites and be able to interact with many CD98 molecules

If there is an association between CD98 and galec-tin 3, then disturbing it should disrupt cellular fusion Indeed, by blocking galectin 3 CDR sites with 50 mm Lac, we showed that we could reduce the fusion of BeWo cells

Getting proteins to the correct place at the right time is a logistical challenge for any cell Proteins des-tined for the classic secretory pathway, such as immu-noglobulins, typically contain N-terminal signal

peptides that mediate membrane translocation into the

lumen of the ER followed by ER–Golgi-dependent

transport to the cell surface On the other hand, a

growing number of proteins (angiogenic growth

fac-tors, galectins, inflammatory cytokines, viral proteins)

lack a signal peptide but are still secreted from the cell

These proteins do not contain modifications such as

glycosylation (which happen at the ER–Golgi level),

and their secretion is not inhibited by BFA or similar

inhibitors of the classic secretory pathway In recent

years, several distinct ‘nonclassic’ secretory pathways

have been demonstrated [32]

As any morphologic and functional modification of

the ER–Golgi–trans-Golgi complex would affect the

proteins using this pathway, both structurally

(incom-plete or null secondary modifications) and functionally

(as a result of the failure to reach correct cell

loca-tions), in this study we used BFA to investigate CD98

function and protein interactions

Our data suggest that CD98 can traffic to the

plasma membrane via at least two distinct transport

mechanisms in BeWo cells, one dependent upon the

classic secretory pathway (glycosylated protein), and

the other on an alternative route (nonglycosylated

pro-tein) Furthermore, we demonstrate that CD98

glyco-sylation is necessary for cell fusion and that this in

turn requires interaction between CD98 and galectin 3

This lectin, like CD98, is present both in the

cytotro-phoblasts and in the syncytiotrophoblast [33,34]

Hence, crosslinking of these two molecules in vivo

could be an essential molecular mechanism to enable

syncytiotrophoblast formation Our findings now need

to be investigated in the intact placenta, e.g by

look-ing for co-localization of these two molecules in

nor-mal placental tissue and in primary cell lines

The results reported in this article fit unexpectedly

with a recent study on CD98hc knockout mice that

suggested an essential role for CD98 in early mouse

development Embryonic lethality was found when the

transgene encoding the molecule was truncated at the

extracellular domain, leaving intact both the

intracellu-lar and the transmembrane parts of the molecule [35]

Our work emphasizes the way in which the external

domain of CD98 may play a critical role in

tropho-blast cell biology

Experimental procedures

Primary antibodies

Rabbit anti-(human galectin 3) was obtained from

Chem-icon Europe Ltd (Chandlers Ford, UK) Goat anti-(human

CD98) (C-20), rabbit anti-(human CD98) (H-300), normal

goat IgG and normal rabbit IgG (isotype-matched con-trols), goat anti-(human galectin 3) (D-20) and mouse anti-(human galectin 3) (B-2) were obtained from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA) Mouse anti-(human PI3Kinase p85a) was obtained from Serotec (Kidlington, UK)

Secondary antibodies

Horseradish peroxidase-conjugated goat anti-(rabbit IgG), horseradish peroxidase-conjugated rabbit anti-(mouse IgG) and fluorescein isothiocyanate-conjugated swine anti-(rabbit IgG) were obtained from Dako (Glostrup, Denmark) Horseradish peroxidase-conjugated donkey anti-(goat IgG) and protein A⁄ G plus agarose were obtained from Santa Cruz Biotechnology Inc Fluorescein isothiocyanate-conju-gated rabbit anti-(goat IgG) was obtained from Sigma (Gillingham, UK) Rabbit anti-(goat IgG))10 nm gold complex to detect anti-CD98 and goat anti-(rabbit IgG))15 nm gold complex to detect anti-galectin 3 were obtained from British Biocell (Cardiff, UK)

Cell culture

BeWo cells were cultured at 37C as monolayers in F-12K Nutrient Mixture (Kaighn’s modification) supple-mented with 10% fetal bovine serum, 2 mm l-glutamine (all Gibco, Paisley, UK), 100 UÆmL)1 penicillin and

100 UÆmL)1 streptomycin (Sigma) in a humidified atmo-sphere of 5% CO2 and 95% air Confluent cells were har-vested by trypsinization with trypsin⁄ EDTA in HBSS without Ca2+ and Mg2+ (Gibco), resuspended in fresh medium, and plated in six-well culture plates (BD Falcon, Oxford, UK) When the cells reached 65–70% confluence, forskolin (Sigma) or vehicle (dimethylsulfoxide) was added

in fresh medium at a final concentration of 100 lm for

24 h, unless otherwise indicated In some wells, BFA (Sigma) (final concentration 5 lgÆmL)1), tunicamycin (Sigma) (final concentration 10 lgÆmL)1), 50 mm Lac or

50 mm maltose were added as indicated in the different experiments For the two-color FACS experiments, before plating, viable cells were counted by the trypan blue (Sigma) method, resuspended in serum-free medium, and stained with either 10 lL of vybrant DiO or 5 lL of vybrant DiD cell labeling solutions (1 mm) (Molecular Probes, Invitrogen, Paisley, UK) per 106cellsÆmL)1 cells for 30 min, or with MitoTracker Deep Red633 (Molecular Probes) at a concentration of 25 nm per 106cellsÆmL)1 cells for 15 min; labeling was carried out at 37C in the dark with gentle shaking After extensive washing with warm serum-free medium, each group of stained cells was resupended in complete growth medium and plated either

on its own or in a 50 : 50 mixture (DiO-labeled and Mito-tracker Red-labeled or DiO-labeled and DiD-labeled cells)

in six-well culture plates

SDS⁄ PAGE and western blotting

Confluent cultures from six-well plates were washed with

ice-cold Ca2+-free and Mg2+-free Dulbecco’s

phosphate-buf-fered saline (D-NaCl⁄ Pi) (Gibco) and then lysed at 4C in

ice-cold modified RIPA buffer containing 50 mm Tris⁄ HCl

(pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mm

NaCl, 1 mm EDTA, and 10 lL of protease inhibitor mixture

(Sigma), for 15 min on a rocker Samples were sonicated

three times for 30 s, and clarified by centrifugation at

10 000 g for 15 min at 4C (Beckman GS15-R, rotor F402,

Beckman Coulter Ltd., High Wycombe, UK) Supernatants

(10 lg of protein) were retained, solubilized in NuPAGE

sample buffer (Invitrogen), with or without reducing agent,

warmed for 10 min at 75C, and then run on 10% Novex

Bis⁄ Tris NuPAGE gels (Invitrogen) The proteins were

trans-ferred to nitrocellulose membranes, blocked using 5% (w⁄ v)

nonfat dry milk in 0.01 m NaCl⁄ Pi(Sigma) with 0.05% (v⁄ v)

Tween 20 for 1 h at room temperature, and then incubated

with rabbit anti-(human CD98) (H-300, 1 : 200) overnight at

4C Horseradish peroxidase-conjugated goat anti-(rabbit

IgG) was used for secondary labeling Immunoreactive bands

were identified by SIGMAFAST 3,3¢-diaminobenzidine

tab-lets (Sigma) according to the manufacturer’s instructions

Co-immunoprecipitation

Whole cell lysates were prepared as described above

Aliqu-ots (10 lg of protein) were retained and solubilized in

Nu-PAGE sample buffer (Invitrogen) for analysis by western

blotting The remaining lysates were precleared with 1 lg of

goat IgG and 10 lL of protein A⁄ G plus agarose [for goat

anti-(human CD98) and goat anti-(human galectin 3)

immu-noprecipitates] or with 10 lL of protein A⁄ G plus agarose

[for mouse anti-(human galectin 3)] for 10 min at 4C; the

agarose beads were removed by centrifugation at 4000 g

(Beckman GS15-R, rotor F4202), and the cleared lysates

were incubated overnight with 2 lg of goat anti-(human

CD98) or goat (human galectin 3) or mouse

anti-(human galectin 3) at 4C Immune complexes were

cap-tured using 20 lL of protein A⁄ G agarose beads for 2 h at

4C, and then washed three times with lysis buffer

Follow-ing elution with NuPAGE buffer, samples were boiled for

5 min to dissociate beads from the immunocomplexes, and

centrifuged at 100 000 g (Eppendorf 5415C, Eppendorf UK

Ltd., Cambridge, UK); associated proteins in the

superna-tants were resolved on a 10% Novex Bis⁄ Tris NuPAGE gel

(Invitrogen) under reducing condition with Mes running

buf-fer (Invitrogen) The proteins were transbuf-ferred to a

nitrocellu-lose membrane, and this was cut into strips corresponding to

single protein lanes (revealed after reversibly staining with

Ponceau red) Single strips were blocked using either 5%

(w⁄ v) nonfat dry milk in 0.01 m NaCl ⁄ Pi(Sigma) with 0.05%

(v⁄ v) Tween 20 (for strips to be probed with anti-CD98), or

0.01 m NaCl⁄ Pi (Sigma) with 0.05% (v⁄ v) Tween 20 plus

10% normal serum of the host species of the secondary anti-body for 1 h at room temperature, and then incubated with either rabbit (human CD98) (H-300, 1 : 200), goat anti-(human galectin 3) (1 : 100), mouse anti-anti-(human galectin 3) (1 : 100), mouse anti-(human PI3 kinase) (1 : 100) as irre-levant control antibody, normal rabbit IgG or normal goat IgG (1 : 100) overnight at 4C Horseradish dase-conjugated goat anti-(rabbit IgG), horseradish peroxi-dase-conjugated donkey anti-(goat IgG) or horseradish peroxidase-conjugated rabbit anti-(mouse IgG) were used for secondary labeling Immunoreactive bands were identified by SIGMAFAST 3,3¢-diaminobenzidine tablets (Sigma) accord-ing to the manufacturer’s instructions

Flow cytometry) surface staining on intact cells

Cells from six-well plates were detached with trypsin⁄ EDTA (Gibco) Aliquots of 1· 106cells were washed in NaCl⁄ Pi

and resuspended in 250 lL of FACS buffer (NaCl⁄ Pi, 1% fetal bovine serum, 0.1% NaN3) with goat anti-(human CD98) (C-20, 1 : 20) or isotype control IgG or no primary antibody Cells were incubated for 45 min on ice, and then washed three times with FACS buffer Samples were then incubated with fluorescein isothiocyanate-conjugated rabbit anti-(goat IgG) (1 : 50) for 45 min on ice and washed three times Samples were finally resuspended in FACS buffer and 2% paraformaldehyde (PFA), and the number of events was analyzed by flow cytometry using a FACSCalibur (BD Biosciences, Oxford, UK) flow cytometer and cell quest software and⁄ or an EPICS Altra (Beckman Coulter Ltd., High Wycombe, UK) flow cytometer and expo32 software

Flow cytometry) surface and intracellular staining

Cell suspensions were fixed in 2% PFA for 20 min at room temperature, washed once in NaCl⁄ Pi, permeabilized with 1% saponin in FACS buffer for 15 min at room tempera-ture, and then stained following the surface staining proto-col After the final wash, samples were fixed again in 2% PFA before analysis

Immunofluorescence

Cells (1· 103

) were plated onto chamber wells (Lab-Tek, Fisher Scientific UK Ltd., Loughborough, UK), grown for

24 h, washed with NaCl⁄ Pi, and fixed with 2% PFA and rin-sed Nonspecific binding sites were blocked with blocking buffer (NaCl⁄ Pi, 0.05% Tween 20, 10% fetal bovine serum, 10% goat serum) for 20 min at room temperature Cells were then incubated with rabbit anti-(human galectin 3), 1 : 1000

in diluting buffer (NaCl⁄ Pi, 0.05% Tween 20, 1% fetal bovine serum, 1% goat serum) for 1 h at room temperature, washed three times for 5 min, and then incubated with