GABAergic signaling in primary lens epithelial and lentoid cells and its involvement in...

Please cite this article in press as: M. Schwirtlich, et al., GABAergic signaling in primary lens epithelial and lentoid cells and its involvement inintracellular Ca2+ modulation. Cell Calcium (2011), doi:10.1016/j.ceca.2011.07.002

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GABAergic signaling in primary lens epithelial and lentoid cells and itsinvolvement in intracellular Ca2+ modulation

Marija Schwirtlicha,1, Andrea Kwakowskya,d,1, Zsuzsa Emrib, Károly Antalb,Zsombor Laczac, Attila Cselenyákc, Zoya Katarovaa,2, Gábor Szabóa,!,2

a Laboratory of Molecular Biology and Genetics, Department of Gene Technology and Developmental Neurobiology, Institute of Experimental Medicine,Hungarian Academy of Sciences, Budapest, Hungaryb Department of Zoology, Eszterházy College, Eger, Hungaryc Department of Human Physiology and Clinical Experimental Research, Semmelweis University, Budapest, Hungaryd Centre for Neuroendocrinology and Department of Physiology, Otago School of Medical Sciences, University of Otago, Dunedin 9054, New Zealand

a r t i c l e i n f o

Article history:Received 7 January 2011Received in revised form 6 July 2011Accepted 7 July 2011Available online xxx

Keywords:GADGABA receptorsIntracellular calciumLensNeurotransmittersGABA signaling in developmentLECCell differentiation

a b s t r a c t

Primary lens epithelial cell (LEC) cultures derived from newborn (P0) and one-month-old (P30) mouselenses were used to study GABA (gamma-aminobutyric acid) signaling expression and its effect on theintracellular Ca2+ ([Ca2+]i) level. We have found that these cultures express specific cellular markers forlens epithelial and fiber cells, all components of the functional GABA signaling pathway and GABA, thusrecapitulating the developmental program of the ocular lens. Activation of both GABA-A and GABA-Breceptors (GABAAR and GABABR) with the specific agonists muscimol and baclofen, respectively induces[Ca2+]i transients that could be blocked by the specific antagonists bicuculline and CGP55845 and weredependent on extracellular Ca2+. Bicuculline did not change the GABA-evoked Ca2+ responses in Ca2-containing buffers, but suppressed them significantly in Ca2+-free buffers suggesting the two receptorscouple to convergent Ca2+ mobilization mechanisms with different extracellular Ca2+ sensitivity. Pro-longed activation of GABABR induced wave propagation of the Ca2+ signal and persistent oscillations.The number of cells reacting to GABA or GABA + bicuculline in P30 mouse LEC cultures expressing pre-dominantly the synaptic type GABAAR did not differ significantly from the number of reacting cellsin P0 mouse LEC cultures. The GABA-induced Ca2+ transients in P30 (but not P0) mouse LEC could beentirely suppressed by co-application of bicuculline and CGP55845. The GABA-mediated Ca2+ signalingmay be involved in a variety of Ca2+-dependent cellular processes during lens growth and epithelial celldifferentiation.

© 2011 Elsevier Ltd. All rights reserved.

1. Introduction

GABA (gamma-aminobutyric acid) is a versatile molecule serv-ing as an inhibitory neurotransmitter in the central nervous system

Abbreviations: CNS, central nervous system; GABA, gamma-aminobutyric acid;GAD65, glutamic acid decarboxylase, 65 kDa form; GAD67, glutamic acid decarboxy-lase, 67 kDa form; GAT, GABA transporter (membrane); GABAR, GABA receptor(s);GABAAR, GABA-A receptor(s); GABAA!3, GABA-A receptor !3 subunit (GABA-binding); GABABR, GABA-B receptor; GABABR2, GABA-B receptor R2 subunit; VGCC,voltage-gated calcium channel(s); VGAT, vesicular GABA transporter; [Ca2+]i, intra-cellular Ca2+ concentration; P0, postnatal day 0; P30, postnatal day 30; AAH, artificialaqueous humor; DAPI, 4" ,6-diamidino-2-phenylindole; LEC, lens epithelial cell(s);N-cad, N-cadherin; E-cad, E-cadherin; Cx43, connexin 43.

! Corresponding author at: Institute of Experimental Medicine, HungarianAcademy of Sciences, POB 67, H-1450 Budapest, Hungary. Tel.: +36 1 210 9980;fax: +36 1 323 3011.

E-mail address: [email protected] (G. Szabó).1 These authors have contributed equally to this work.2 These authors share senior authorship.

(CNS) of vertebrates, a paracrine/autocrine signaling molecule andmetabolite in numerous cells of the nervous system and periphery[1–5]. GABA is synthesized by GAD (glutamic acid decarboxylase)in GABAergic neurons and non-neuronal cells and is thereafterreleased by either a vesicular (VGAT) or membrane (GAT) GABAtransporters into the extracellular space activating two types ofGABA receptors: the ionotropic GABAAR and metabotropic GABABR.In contrast to the adult CNS where GABARs mediate fast synap-tic or tonic inhibitory neurotransmission, activation of GABAARin neuronal progenitors triggers a rise in the intracellular Ca2+

([Ca2+]i) which in turn regulates a variety of key cellular processes,including cell proliferation, migration and differentiation [6–8].The G protein-coupled metabotropic GABABR is generally thoughtto inhibit the [Ca2+]i elevation through presynaptic inhibition ofVGCCs (voltage-gated calcium channels) or post-synaptic stimu-lation of GIRK (G-protein coupled inwardly rectifying potassium)channels [9,10]. However, it has been revealed that GABABR canalso induce [Ca2+]i rise in hippocampal astroglia [11–13] as well asneurons [14,15].

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Recently we have shown that all components of the GABAsignaling machinery are expressed in two non-neuronal cellularsystems: mouse embryonic stem cells (mESC) and the developinglens [16–18] both affording valuable in vitro and in vivo models tostudy the GABA signaling and the effect of GABAR activation onthe [Ca2+]i level during embryonic development and cellular dif-ferentiation. We have demonstrated that undifferentiated mouseembryonic stem cells (mESC) express molecular components ofthe GABA and Ca2+ signaling indistinguishable from their nervoussystem counterparts, but in a contrasting way both GABARs evokesynergistic [Ca2+]i rise, albeit acting through different mechanisms[18].

Our studies in the mouse lens have revealed that the GABAsignaling machinery is present from the earliest stages of lensdevelopment in the epithelial sheet forming the lens placode[16,17]. Later, the placode cells give rise to the ocular lens, apolarized organ, composed of a monolayer of cubical epitheliumat the anterior pole, central differentiated primary lens fibersand differentiating secondary fiber cells. The latter arise fromcontinuously proliferating epithelial cells, which undergo termi-nal differentiation characterized by extensive fiber elongation,concomitant loss of all cellular organelles and upregulation ofthe lens crystallins [19,20]. During development multiple GABAreceptor subunits and other components of the GABA signal-ing show characteristic spatio-temporal patterns of expressionthat overlap in the lens epithelium, but segregate to primary orsecondary lens fibers along with the two GAD isoforms GAD65and GAD67, respectively [16,17]. The two GABARs exhibit a clearembryonic-to-postnatal shift in subunit composition characterizedby an upregulation of the fast synaptic type GABAAR and down-regulation of the GABABR2 receptor subunit during postnatal stages[16]. The functionality of the lens-specific GABAR was demon-strated by GABA administration to acutely isolated newborn mouselenses, which resulted in the elevation of [Ca2+]i level in elongat-ing fiber cells at the equatorial region. We have proposed thatthe responding cells may correspond to the population of chickenannular pad cells that also respond to mAchR (muscarinic acetyl-choline receptor) stimulation with a [Ca2+]i rise [16,21]. However,using the above approach only superficial fibers could be visu-alized with a poor resolution at the cellular level (discussed in[16]).

Here we have expanded these studies in primary mouse LECcultures, which represent a faithful in vitro model system of lensepithelial cell differentiation [22,23] and have been successfullyused before to study different aspects of the lens Ca2+ signaling[24–29]. We show that primary LEC cultures prepared from new-born and one-month-old mouse lenses express GAD, GABA, GABAreceptors and transporters. Activation of both GABARs induceda rise in [Ca2+]i that was dependent on the presence of extra-cellular Ca2+ and could be blocked by specific antagonists inboth P0 and P30 mouse LEC cultures. This synergistic elevationof [Ca2+]i following activation of GABAAR and GABABR may beinvolved in the maintenance of Ca2+ homeostasis, which is akey regulator of a variety of cellular processes involved in lensdevelopment.

2. Materials and methods

2.1. Animal use

Mice of strain FVB/Ant [30] housed in the Medical Gene Techno-logical Unit of the Institute of Experimental Medicine were used. Allexperiments with animals were conducted in compliance with NIH(NIH Publication #85-23, 1985) and EC (86/609/EEC/2) guidelinesand approved by in-house and national committees.

2.2. Primary lens epithelial cell (LEC) culture andimmunocytochemistry

Lenses dissected from newborn (P0) mice were cleaned of ciliaryepithelium and retina with fine forceps and collected in ice-coldcell-culture grade Tyrode’ salts (Sigma–Aldrich, St. Louis, MO, USA).Subsequently the lenses were incubated in 0.02% EDTA solution in0.5 mM Dulbecco’s Phosphate Buffered Saline (Sigma–Aldrich) at37 #C for 10 min, transferred to pre-warmed Medium 199 (Invit-rogen, Carlsbad, CA, USA) supplemented with 10% fetal bovineserum (FBS, EuroClone, Pero-Milano, Italy) triturated 10 times andcentrifuged at 200 $ g for 2 min. Cells were resuspended in freshMedium 199 + 10% FBS and plated at the equivalent of one lens/cm2

on glass coverslips pre-coated with BD MatrigelTM (BD Biosciences,NJ, USA; 0.4 mg/ml in PBS). In addition, primary LEC were alsocultured from lens capsules isolated from one-month-old (P30)mice and plated on matrigel-coated coverslips at 1 capsule/cm2

in Medium 199 + 10% FBS. Primary LEC cultures were grown at37 #C at 5%CO2/95%O2 with medium changed every two days.Two to three-week-old cultures containing single lens epithelialcells, epithelial monolayers and lentoids of different sizes wereused for immunocytochemistry and Ca2+ imaging. Cultures werefixed in 4% (w/v) paraformaldehyde (PFA) in PBS (phosphate-buffered saline) for 20 min at room temperature (RT) then washed3$ 5 min in PBS. For GABA detection fixation was done in 4%PFA–0.1% glutaraldehyde–PBS for 30 min at RT. Cells were per-meabilized in 0.2% Triton X-100 (Sigma–Aldrich)–PBS for 5 min atRT and blocked in 0.5% blocking reagent (Roche Applied Sciences,Penzberg, Germany)–PBS for 1 h at RT. Primary antibodies wereapplied in 1% BSA–PBT (PBS + 0.05% TWEEN® 20, Sigma–Aldrich)overnight at 4 #C as follows: goat anti-"A-crystallin (Santa CruzBiotechnology, Santa Cruz, CA, USA, 1:500), rabbit anti-GABAA!3(1:1000) and rabbit anti-"B-crystallin (1:500) both from NovusBiologicals, Littleton, CO, USA, mouse anti-uvomorulin (E-cadherin,Sigma–Aldrich, 1:1000), mouse anti-N-cadherin (1:1000), mouseanti-GABABR2 (1:500) and mouse anti-Cx43 (1:1000) all fromBD Biosciences; rabbit anti-GABA (Sigma–Aldrich, 1:5000); rab-bit anti-GAD serum #6799 [17] at 1:500 or mouse anti-GAD67(EMD Millipore-Chemicon, Billerica, MA, USA) mouse anti-GAD65developed by David Gottlieb and obtained through the Develop-mental Studies Hybridoma Bank (Iowa City, IA, USA, 1:250); guineapig anti-VGAT (Calbiochem, Merck KGaA, Darmstadt, Germany;1:5000), guinea pig anti-mouse GAT-1 (gift from N. Nelson, Tel AvivUniversity), rabbit anti-GAT-3 (Abcam, Cambridge, UK, 1:1000).Coverslips were washed 3$ 10 min in PBT and incubated withanti-rabbit, mouse, goat or guinea-pig secondary antibodies con-jugated either with Alexa Fluor® 594 or with Alexa Fluor® 488(Invitrogen, diluted 1:500 in 1% BSA-PBT) for 1 h at RT. The anti-GABA antibody was reacted first with biotinylated goat anti-rabbitIgG (Vector, Burlingame, CA, USA) for 90 min at RT in PBT–1%BSA, followed by Cy3-streptavidin (Jackson ImmunoResearch, WestGrove, PA, USA, 1:5000) in PBS for 1 h at RT. The actin cytoskele-ton was stained with rhodamin-phalloidin (Invitrogen, 1:100) for30 min at RT in PBS. Cells were washed 3$ 10 min in PBT, 1$ PBSand incubated for 5 min at RT with DAPI (Invitrogen) as recom-mended by the manufacturer. Coverslips were mounted in Mowiol(Calbiochem) containing DABCO (Sigma–Aldrich, 0.1 mg/ml) andexamined under Zeiss Axioscop-2 equipped with AxioCam HRc dig-ital camera using AxioVision 4.6 software or Zeiss LSM 510 META.

2.3. Ca2+ imaging studies

Primary LEC were loaded with 5 #M Fluo-4/AM in the pres-ence of 0.1% pluronic acid F-127 (both from Invitrogen) in growthmedium at 37 #C for 45 min, in a humidified atmosphere of 5%CO2/95%/O2. Subsequently the medium was changed and de-




M. Schwirtlich et al. / Cell Calcium xxx (2011) xxx– xxx 3

esterification of the dye was allowed to proceed for another15–30 min. The glass coverslips with attached cells were placed in aPOC-R type submerged chamber (PeCon GmbH, Erbach, Germany).In each experiment cells were perfused for 2 min at 2 ml/min withartificial aqueous humor (AAH) of the following composition (inmM): NaCl, 130; KCl, 5; CaCl2, 1; MgCl2, 0.5; d-glucose, 5; NaHCO3,5; HEPES, 10; pH 7.25 to obtain a steady baseline. GABA (1 mM),muscimol (100 #M) or baclofen (20 #M), all from Sigma–Aldrichwere applied for 2 min, followed by a 10 min perfusion with AAH. Ingeneral, the visual field selected under phase contrast and fluores-cence microscopy for recordings included single cells, monolayersand multilayered lentoid-like structures.

Whenever GABAR agonists in combination with antagonistswere used, cells were initially perfused with the antagonist: 20 #Mbicuculline (Sigma–Aldrich; a GABAAR antagonist) for 2 min or20 #M of CGP55845 (Tocris Bioscience, Bristol, UK; a GABABRantagonist) for 2–4 min. This was followed by the co-application ofagonist and antagonist: 100 #M muscimol + 20 #M bicuculline or20 #M baclofen + 20 #M CGP55845, respectively, for 10 min withAAH. Cells were then perfused for 2 min with AAH containingthe agonist alone (muscimol or baclofen, respectively). WheneverGABA was applied as agonist cells were initially perfused withAAH (with or without Ca2+) until steady baseline was achieved,then 50 mM GABA drop was added (to final concentration 1 mM)followed by AAH (4 min), AAH + bicuculline (2 min), GABA drop,AAH + bicuculline + CGP55845 (7 min), GABA drop, AAH (10 min).GABA (50 mM) and KCl (1 M) drops were added to verify theexcitability of the cells at the end of the recording. Cultures wereobserved under a Zeiss LSM 510 META (Carl Zeiss) laser scan-ning confocal microscope and scanned every second. All settingsof the laser, optical filter and microscope as well as data acquisi-tion were controlled by LSM 510 META software. Excitation was at488 nm and emitted light was read at 515 nm. Fluorescence inten-sity within regions of interest covering a single cell was followedover time and expressed in arbitrary units. Data are presented asrepresentative single experiments or the mean ± S.E.M. of pooleddata from a number of experiments, as indicated. Statistical signifi-cance was estimated by Mann–Whitney unpaired test, with p < 0.05

considered statistically significant compared to control if not statedotherwise.

3. Results

3.1. Primary LEC cultures – features of the in vitro differentiationof lens epithelial cells

Primary LEC cultures were derived primarily from newbornmouse lenses, as they express high levels of GABA signaling com-ponents [17] as well as provide an ample supply of easy-to-dissectepithelial and fiber cells. In some experiments primary LEC werederived from P21 to P30 (postnatal days 21–30) mouse lens cap-sules containing the attached lens epithelial cells. Under the cultureconditions used here, the lens epithelial cells continuously prolifer-ate, migrate and subsequently differentiate. After two weeks in vitrothe primary LEC cultures were comprised of single epithelial cellsof flattened irregular shape (Fig. 1A and E), epithelial monolayers(Fig. 1B and C) and randomly distributed lentoids–multilayered cellaggregates of various sizes and shapes that appear to bulge from thesurrounding epithelial cells (Fig. 1B–D and G). Based on morphol-ogy and expression profiling lentoids are considered an equivalentof aggregates of elongating fiber cells [23,31–33]. The transitionbetween epithelial cells, monolayers and lentoids involves a grad-ual change in cell compaction and nuclear morphology that couldbe traced by staining of the actin cytoskeleton and nuclear DAPIstaining (Fig. 1E–G). Single cells are clearly distinguished by theirlarge nuclei and the presence of stress fibers – bundles of actinfilaments (Fig. 1E). Cells with large nuclei initially form monolay-ers with strong staining of cortical actin fibers (Fig. 1F). Furthercompaction of cells with concomitant reduction of nuclear sizeand chromatin condensation produces compact monolayers andmultilayered bulging lentoids that could be distinguished morpho-logically (Fig. 1B, C and F) and by staining with cellular markers(see below). Lentoids may contain transluscent anuclear parts cor-responding to terminally differentiated fiber cells (Fig. 1D). Cellborders of cells forming lentoids could not be distinguished underphase contrast.

Fig. 1. Features of primary LEC cultures prepared from newborn mouse lenses. Images of LEC cultured grown for two weeks in vitro. These cultures comprise epithelial cellswith large intact nuclei and strong staining for actin stress fibers (arrowheads in A, E and G), flat cell monolayers with large nuclei and cortical actin filaments (arrows in Aand F), flat sheets of tightly packed cells with small pyknotic nuclei (empty asterisks in B, C and F) and bulging multilayered lentoids made of highly compacted cells withsmall pyknotic nuclei (B–D, F and G, solid asterisks). At later stages of lentoid differentiation cells around lentoids become sparse with some lentoid parts developing intoanuclear bulges (solid asterisks in D). Cells were visualized by phase contrast (C), DIC microscopy (A, B and D) or by fluorescence following staining of filamentous actin withrhodamin-phalloidin (E–G). Cell nuclei were counterstained with DAPI (blue color in A, B, D and E–G). Fluorescent images were superimposed on the DIC images in (A, B, Dand G) using AxioVersion 4.6 software. Scale bars: 50 #m (A, D and E); 100 #m (B–C, F, and G). (For interpretation of the references to color in this figure legend, the readeris referred to the web version of the article.)





In addition to morphological criteria, we also used cellularmarkers to distinguish between epithelial and differentiated fibercells (Fig. 2). The molecular chaperone "A-crystallin, one of themost abundant proteins of the lens epithelium and fibers andthe small "B-crystallin, more abundant in fiber cells [34] weredetected at high levels in multilayered lentoids and were onlyweakly expressed in epithelial cells and monolayers (Fig. 2A, D,G and K). Lentoids, but not epithelial cells stained strongly for the

adhesion protein N (neural) cadherin typically expressed on thebroad face of fiber cells (Fig. 2B, C and E), while E-cad (epithelial-cadherin) only stained flat, presumably epithelial, but not bulginglentoid regions (Fig. 2H and J and [35]) distinguished by the promi-nent "B-crystallin staining (Fig. 2G and J). Similarly, Cx43 (connexin43), a gap junction channel protein known to be expressed abun-dantly in lens epithelium [36] was localized to flat monolayersincluding those found at the periphery of "B-crystallin stained

Fig. 2. Newborn mouse primary LEC cultures express different markers for lens epithelial and differentiating fiber cells. Antibodies staining differentially epithelial or fibercells were used: "A- and "B-crystallin, N- and E-cadherin (N-cad and E-cad, respectively) and connexin-43 (Cx43, a gap juction protein channel specific for lens epithelium).Visualization was by indirect immunofluorescence using Alexa 488 (A, D, G and K) or Alexa 594 (B, C, E, H and L) conjugated secondary antibodies. Nuclei were counterstainedwith DAPI (A–C, F, I, J and M). (A) The fluorescent image is superimposed on the phase-contrast image. "A-crystallin is strongly expressed in small cellular aggregates (insetin A) as well as larger multilayered lentoids (A) staining also for N-cad (B). N-cad is detected in multicellular lentoids (C, arrows) while epithelial cells remain unstained(C, arrowhead). (D) "B-crystallin strongly stains lentoids (solid asterisk), also displaying a prominent membrane staining for N-cad (arrows in E). Epithelial cells are weaklylabeled with a "B-crystallin-specific antibody (arrowheads in D and F) and negative for N-cad (E and F). (K–L) Flat monolayer area at the periphery of the lentoid (emptyasterisks in G and H) stains weakly for "B-crystallin (G) and strongly for the epithelial marker E-cad (H), while the multilayered regions (solid asterisk in G and J) stainsin a complementary manner. (K–M) Flat region (K and L – empty asterisks) and bulging regions (solid asterisks in K and M) of a multicellular lentoid-like structure stainoppositely for Cx43 and "B-crystallin, respectively. Scale bars: 50 #m.





multilayered lentoid-like regions (Fig. 2K–M). DAPI staining ofnuclei clearly demonstrated that compact monolayers composedof closely packed cells expressing epithelial markers and bulginglentoids both possess similar sized “pyknotic” nuclei (Fig. 2I, J, Mand data not shown).

3.2. Expression of GABA signaling components in primary LECcultures

Similar to intact lenses [16,17] primary LEC cultures preparedfrom newborn mouse lenses expressed both forms of the GABA-synthesizing enzyme GAD (i.e. GAD65 and GAD67), as well asvesicular (VGAT) and membrane GABA transporters (Fig. 3A–I). Ingeneral, the bulging multilayered lentoids clearly identifiable underphase contrast (Fig. 3A, D and G) showed the strongest expres-sion for GABA signaling components, while epithelial cells wereless prominently stained (Fig. 3B, C, E, F, H and I). GAD65 and thevesicular GABA transporter VGAT showed a uniform distributionwithin multilayered lentoid-like aggregates (Fig. 3B and C), whilethe membrane GABA transporter GAT-1 and especially GAD67 wereenriched in the most differentiated lentoid thickenings devoid ofnuclear DAPI staining (Fig. 3D–F) that also stained strongly forGABA (see below). GAT-3 was found in virtually all types of cells:in both single or monolayer epithelial cells as well as multilayerlentoid-line structures (Fig. 3G–I and data not shown). GAD67was also expressed in a subpopulation of previously describedsmall spindle-like cells of epithelial origin (data not shown and[31]).

Prominent labeling of the GABA-binding GABAA!3 subunit wasdetected over the cell borders of flattened (putative monolayer)regions found at the periphery of bulging lentoid-like structuresand was stronger in the multilayer region (Fig. 4A and C). GABABR2

staining displayed a uniform staining over the entire lentoid sur-face (Fig. 4G and I). An almost identical staining pattern of thetwo receptors was observed in epithelial monolayers with typi-cal large nuclei (Fig. 4D–F and J–L). The staining product was of apunctate appearance outlining cell borders at the periphery of themuticellular assembly (Fig. 4D, F, J and L) and highly accumulatedat presumptive intercellular interfaces and underlying perinuclearspace of internally positioned cells, as demonstrated in confocalimages (Fig. 4D–F and J–L). Only a subpopulation of single cellsstained for GABAR (data not shown).

In conclusion, some single epithelial cells, most if not all cellsof epithelial monolayers and multilayered lentoid-like structuresexpressed all components required for a functional GABA signalingpathway.

3.3. Primary newborn mouse LEC cultures produce GABA

Using a highly specific antibody, we could detect strong GABAstaining in newborn mouse primary LEC cultures. Multilayeredlentoid structures showed high accumulation of GABA especiallyin their more differentiated anuclear regions, but also in theadjacent flatter regions (Fig. 5A and A"-boxed area in A), whilesingle cells remained unstained (Fig. 5C and C"-boxed area inC; fluorescence in C" is digitally enhanced to demonstrate thepresence of cells that are NOT stained). This expression gradientwas typically found in the vicinity of all lentoid-like structurescontaining epithelial cells at different stages of differentiationcharacterized by typical changes in nuclear shape and chromatincondensation normally accompanying fiber maturation (Fig. 5B,B"-boxed area in B and [37]). Rare neurons could be also identi-fied by their typical morphology and high content of GABA (Fig. 5Aand A").

Fig. 3. Primary LEC cultures from newborn mouse lenses express different members of the GABA signaling pathway. The following specific antibodies were used: GAD65–GAD-6 (C); GAD67-rabbit #6799 (F) or mouse GAD67 (I); VGAT (B); GAT-1 (E) and GAT-3 (H). (A, D and G) Phase contrast images corresponding to the fluorescent images shownin (B, C), (E, F) and (H, I), respectively. (B, F and H) Nuclei were counterstained for DAPI. (A–C) VGAT co-localizes with GAD65 in bulging lentoid-like structures. (D and E)Co-localization of GAT-1 and GAD67: The flat area of the lentoid-like structure (empty asterisks in D–F) exhibits a weak membrane staining for GAT-1 (E) and no staining forGAD67 (F), while the bulging multilayered regions (E, solid asterisks) are strongly labeled for both (E and F). GAT-3 (H) and GAD67 (I) antibodies both stain the multilayeredlentoid structure shown in (G); the surrounding flat monolayer is weakly labeled. Scale bar: 100 #m.





Fig. 4. Expression of GABAA and GABAB receptors in primary LEC cultures from newborn mice. Primary LEC cultures were stained with rabbit anti-GABAA!3 (A–F) or mouseanti-GABABR2 (G–L) antibodies and visualized by epifluorescence (A–C and G–I) or laser scanning microscopy (D–F and J–L). Nuclei were counterstained with DAPI (B, C,E, F, H, I, K and L). Staining shows patchy vesicular appearance outlining the presumed cellular membranes at the lentoid periphery (arrows in A, D, F and J). Considerableenrichment is observed at intercellular interfaces of LEC monolayers (arrowheads in D and J) and lentoid surfaces (A and G). Bar 20 #m in (D–F and J–L); 50 #m in (A–C andG–I).

3.4. GABA receptor-mediated Ca2+ responses

The functional assembly and activity of GABAA and GABABreceptors expressed by primary LEC was studied by measuring[Ca2+]i changes in cultures loaded with Fluo-4/AM upon applica-tion of selective agonists/antagonists. Most of the cells of the visualfield selected for recordings were successfully loaded, includingepithelial cells, either singly or in monolayers and multicellularlentoid-like aggregates distinguished under phase contrast andfluorescence (Supplementary Fig. 1; Supplementary movies 1–6and data not shown). Larger lentoids, likely to represent a laterstage of lens cell differentiation [31] remained unlabeled. Thelabeled cells did not show spontaneous Ca2+ transients in AAHsolution.

3.4.1. Modulation of [Ca 2+]i levels by selective GABAAR orGABABR ligands

The GABAAR agonist muscimol (100 #M) and GABABR agonistbaclofen (20 #M), applied in AAH induced a simultaneous increaseof fluorescence (Fig. 6A1–4 and B1–2) mostly in small lentoid-likestructures (Fig. 6A1 and B1; Supplementary movie 1), but also inepithelial cells in flat monolayers; Fig. 6A3), which were identifiedin initially obtained phase contrast images. The average number of

cells per observation field responding to muscimol was 4.2 ± 1.02,p < 0.05, n = 10 fields (Fig. 6C) with a mean amplitude of fluores-cence change !F = 46.5 ± 5, p < 0.05. In comparison, Ca2+ transientswere almost fully blocked in the presence of the GABAAR antago-nist bicuculline (cell number 0.5 ± 0.3, n = 6, p < 0.05; amplitude offluorescence change !F = 19.5 ± 5.84; Fig. 6C) demonstrating thespecificity of the response.

Baclofen induced relatively small Ca2+ transients in a por-tion of single cells (data not shown), which could be explainedby their low number of receptors or the relative insensitivityof the latter to baclofen. The response to baclofen (average cellnumber 3.4 ± 0.5, n = 8 fields, p < 0.05; mean amplitude change!F = 37.0 ± 14.5, n = 25, p < 0.05; Fig. 6C) was typically completelyblocked in the presence of the GABABR antagonist CGP55845(0.3 ± 0.3; n = 3 fields, p < 0.05; mean amplitude change !F = 93.6,n = 1; Fig. 6C). The large-amplitude transients exhibited by rarecells responding to baclofen in the presence of CGP55845 mayresult from release of Ca2+ from the intracellular Ca2+ stores [18,38].Continuous application of baclofen sometimes induced persistentoscillations and a secondary wave of signal propagation within themultilayered cell aggregates and adjacent epithelial monolayers(Fig. 6B1; Supplementary movie 2), which were sustained evenafter complete washout of baclofen (Fig. 6B2).





Fig. 5. GABA detection in primary LEC cultures derived from newborn mice with anti-GABA antibody. (A, A" , C and C") Visualization was with streptavidin-Cy3. (B, B") Nucleiwere revealed by DAPI staining. Boxed regions in A, B and C are enlarged in A" , B" and C" , respectively. A large multicellular structure (A–C) is highly positive for GABA inits bulging anucleated lentoid part (solid asterisk in A, A" and C) and less pronouncedly stained at the flat, presumably epithelial monolayer regions (empty asterisk in A").Rare neurons express similar levels of GABA (arrows in A, A"). Cells outside the multicellular structure with large (C" arrowheads) or partially disintegrated (numbered 1–3in B") nuclei remained unstained (B, B" , C and C"; boxed area in C devoid of staining was digitally enhanced in C" to reveal the presence of unstained cells in this area). Cellsbelonging to the lentoid structure with strongly stained small nuclei (#4 in B") are GABA+ (yellow asterisk in B and C). (B") Circled nuclei of different shapes and level ofchromatin compaction (numbered 1–4) correlating with different stages of lens epithelial-to-fiber cell differentiation proceeding from less differentiated (#1) [5] to moredifferentiated (# 4) stages [37]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 6. Changes of [Ca2+]i in primary newborn mouse LEC cultures in response to muscimol (a GABAAR agonist) and baclofen (a GABABR agonist). Cultures grown for twoweeks in vitro and loaded with Fluo-4/AM were used. (A1 and A2) Perfusion was with AAH buffer followed by AAH containing 100 #M muscimol. (A3 and A4) Perfusion wasdone initially with AAH containing 100 #M muscimol in the presence of 20 #M of the GABAAR antagonist bicuculline then by 100 #M muscimol alone, followed by 1 mMGABA and a drop addition of 1 mM KCl (A4). Note the complete blockade of muscimol action by bicuculline (A3 and A4). (B1 and B2) representative recordings of baclofen-evoked fluorescence changes. Cells were initially perfused with AAH containing 20 #M CGP55845 (CGP, a GABABR antagonist) then AAH containing 20 #M CGP55845 + 20 #Mbaclofen followed by baclofen alone. Baclofen induces a simultaneous rise of fluorescence (*) in closely apposed cell stacks followed by oscillations (**). Note that CGP55845 at20 #M completely blocks the baclofen-evoked Ca2+ responses (B1, B2). (A1, A3 and B1) Cells manifesting Ca2+ transients are circled on the confocal images taken at differenttime points of the recording using fluorescent images taken at the peak of fluorescence. (A2, A4, and B2): traces of fluorescence intensity changes in arbitrary units (F) overtime in cells circled on the corresponding images shown in (A1, A3 and B1). (C) Bar chart illustrating the average number of cells (upper panel) and amplitude of fluorescencechanges (!F, lower panel) responding to muscimol (Musc), muscimol in the presence of bicuculline (Musc + Bic), baclofen (Bacl) and baclofen in the presence of CGP55845(Bacl + CGP). Data represent the means ± S.E.M.





Fig. 7. GABA induces [Ca2+]i rise in primary LEC cultures from newborn mouse lenses through synergistic activation of GABAAR and GABABR. (A1–A6) Representativerecordings of GABA-evoked changes in fluorescence of Fluo-4/AM loaded LEC cultures grown in vitro for two weeks. Perfusion was initially with AAH followed by AAH withbicuculline (Bic; 20 #M) and finally with AAH containing 20 #M bicuculline + 20 #M CGP55845 (Bic + CGP). (A1–A6) Drop application of GABA (final concentration 1 mM)induced a similar Ca2+ rise in cells perfused with AAH or AAH + bicuculline, but not in AAH containing both GABAR antagonists bicuculline and CGP (Bic + CGP). (A1, A3 and A5):Cells manifesting [Ca2+]i transients are circled on the confocal images taken at different time points of the recording. (A2, A4 and A6) Traces of fluorescence intensity changes(F, in arbitrary units) measured over time in cells circled in the corresponding images in (A1, A3 and A5). (B) GABA-evoked responses in cells perfused in Ca2+-free AAH buffer(0 Ca2+), followed by AAH (0 Ca2+) containing either 20 #M bicuculline or 20 #M bicuculline (Bic) and 20 #M CGP55845 (CGP). (C1 and C2) Bar charts illustrating the averagenumber of cells (C1) and amplitude of fluorescence change !F in arbitrary units (C2) responding to GABA. Cultures were perfused with either AAH buffer containing 1 mMCaCl2 (closed bars) or AAH buffer without CaCl2 (0 Ca2+, open bars). Data represent the mean ± S.E.M.

3.4.2. Responses to GABA in newborn mouse LEC culturesIn general, both cells of larger lentoid surfaces and adjacent

epithelial cells of primary newborn mouse LEC responded to GABAwith a quick rise in fluorescence (Fig. 7A1–4; Supplementarymovies 3–5). The average cell number was 5.0 ± 1.4; p < 0.05;F = 58.9 ± 9.5, p < 0.05, n = 10 fields (Fig. 7C1–2). Addition ofGABA in the presence of the GABAAR antagonist bicucullineproduced similar responses compared to GABA added in AAH(Fig. 7A1–2 and A5–6; Supplementary movie 6) with regard toboth cell number and amplitude of reaction (average cell num-ber 5.6 ± 2.6; !F = 68.4 ± 10.6, p > 0.05 for differences comparedto GABA responses, n = 3 fields; Fig. 7C1–2). Some cells clearlyshowed a delayed response first at this stage, as shown in

Fig. 7A5–6. Blockade of both GABAR by applying bicuculline andCGP55845 generally suppressed all GABA-evoked Ca2+ responses(Fig. 7A1–6) and only occasional single Ca2+ transients wererecorded under these conditions (average cell number 0.8 ± 0.6,n = 6 fields, p < 0.05 for calculated differences compared to GABAresponses; !F = 97.3 ± 56.6, n = 6 fields; Fig. 7C1–2).

While wave propagation of the signal was clearly observed(Supplementary movies 3–5) it was rarely followed by oscilla-tions. Drop application of KCl induced a simultaneous Ca2+ risein most, if not all cells of a visual field (Supplementary movie3; Supplementary Fig. 1), which demonstrated that the late Ca2+

transients and waves do not result from a slow diffusion ofGABA.





Fig. 8. GABA induces Ca2+ transients through activation of both GABAA and GABAB receptors in LEC cultures derived from lens capsules of one-month-old (P30) mice:similarities with newborn (P0) mouse LEC cultures. LEC cultures grown for 2–3 weeks in vitro were used. (A1 and A2) Bar charts illustrating the average cell number (A2)and the amplitude of fluorescence change expressed in arbitrary units !F (A2) of primary LEC cultures derived from newborn (solid black bars) or P30 (open bars) micedisplaying Ca2+ transients in response to GABA. (B) Traces of fluorescence intensity (F) changes induced by GABA over time recorded in individual cells of P30 mouse LECculture. Initial perfusion was with AAH followed by AAH containing bicuculline (20 #M; Bic) and AAH containing both bicuculline and CGP55845 (20 #M each; Bic + CGP).GABA as added in a drop (1 mM final concentration) induced Ca2+ transients in LEC cultures derived from either newborn or one-month-old mouse lenses during perfusionwith AAH or AAH containing bicuculline, but only newborn cultures respond to GABA in the presence of both bicuculline and CGP55845 (A1, A2 and B).

Using Ca2+-free buffer we still observed Ca2+ transients inresponse to GABA (Fig. 7B), although with considerably reducedaverage cell number (2.8 ± 0.8, n = 4, p < 0.05) and mean amplitudeof the responses (!F = 33.5 ± 16.4, n = 4 fields, p > 0.05; Fig. 7C1–2)compared to GABA responses in the presence of Ca2+. The num-ber of cells reacting to GABA with Ca2+ transients in the presenceof bicuculline or bicuculline + CGP55845 in Ca2+-free buffer wasreduced even further (GABA + bicuculline: cell number: 1 ± 0, n = 3fields; !F = 27.0 ± 4.6, n = 3 fields; GABA + bicuculline + CGP55845:cell number 1 ± 0.6, n = 3 fields; !F = 30.4 ± 5.0, n = 3 fields).

3.4.3. Responses to GABA in one-month-old mouse LEC culturesWe also studied the GABA-induced Ca2+ signaling in LEC cul-

tures derived from one-month-old (P30) mouse lens capsuleswith attached epithelium (Fig. 8A1–2 and B). As described fornewborn mouse LEC, the P30 mouse LEC cultures contain sin-gle epithelial cells, epithelial sheets and lentoids that derive fromlens epithelial cells migrating out of the lens capsules (data notshown).

Similar to P0, the P30 mouse LEC responded to GABA with Ca2+

transients (4 and 8 cells, n = 2 fields compared to LEC from P0mouse lenses: average cell number 5.2 ± 0.9, n = 26 fields, respec-tively; Fig. 8A1). Furthermore, the responses observed for GABAadded in the presence of bicuculline did not differ significantlybetween the two types of culture (P0: GABA + bic: 5.7 ± 2.2, n = 7fields vs. P30: 8.0 ± 2.2 cells, n = 3 fields). However, the mean ampli-tude of Ca2+ transients in response to GABA in P30 mouse LECcultures differed compared to P0 cultures (P30: !F = 31.8 ± 5.7,n = 2 vs. P0: !F = 58.3 ± 3.9, n = 10 fields, respectively; Fig. 8A2).This difference between P0 and P30 mouse LEC cultures wasreduced when GABA was applied in the presence of bicuculline(Fig. 8A2; P0: !F = 69.4 ± 10.6, n = 3 fields; P30: !F = 68.4 ± 10.8,n = 2 fields, p > 0.05). Blockade of both GABAAR and GABABR withbicuculline + CGP55845 abolished all responses to GABA in P30mouse LEC cultures (0, n = 2). In comparison, low numbers of P0mouse LEC still produced Ca2+ transients (0.8 ± 0.6, p < 0.05 n = 6;Fig. 8A1) with higher amplitude (97 ± 56.0, n = 2; Fig. 8A2) underidentical conditions.

4. Discussion

4.1. Primary LEC cultures as a model system for studying GABAsignaling during development

GABA signaling has been drawing continuous attention due toits widespread expression and versatile role in variety of neuronal

and non-neuronal cell types. Its hyperpolarizing inhibitory effectmediated by the two classes of GABARs is indispensable for thefunctionality of neuronal circuitry and control of excitability ofthe vertebrate CNS. In recent years it has become apparent thatthe GABAARs exert a predominantly Ca2+-elevating activity in neu-ronal progenitors prior to synaptic engagement [5], whereas muchless is known about the effect of GABABR, the possible interactionsbetween the two receptors and downstream effects in differenti-ating neural precursors due to the complexity of the developingnervous system. We chose to use primary LEC cultures derivedfrom postnatal mouse lenses since they recapitulate the develop-ment of the mammalian lens [23], which expresses high levels ofGABA signaling pathway components [16,17,39,40]. In addition,primary LEC cultures have been extensively used before for highresolution Ca2+-imaging [24,29,41–43]. Two weeks after platingthe mouse LEC cultures from both newborn and one-month-oldlenses contain cells at virtually all stages of differentiation, whichcan be easily identified by size, nuclear shape, chromatin con-densation and staining for different cellular markers like "A- and"B-crystallin (epithelial cells and lentoids), Cx43 and E-cadherin(epithelial monolayers) or N-cadherin (lentoids). The transitionbetween different differentiated stages, however, is gradual andcells at intermediate stages may share common morphological andphysiological features.

The highest expression of GABA receptors and transporters wasdetected in multilayer lentoid structures composed of nucleatedcells corresponding to the elongating equatorial lens fibers [24],while epithelial monolayers and a population of single epithe-lial cells showed lower level of expression. Interestingly, GADand GABA were highly enriched in anucleated lentoid regions,corresponding to terminally differentiated lens fiber cells. Fur-thermore, GABA and VGAT were never detected in single cellsindicating GABA synthesis and VGAT-coupled release increasesin more differentiated cells. This expression pattern correspondswell to that described for the intact mouse lens [17] and clearlydemonstrates that primary LEC cultures faithfully recapitulatethe GABAergic signaling expression pattern during lens develop-ment.

4.2. Both GABAAR and GABABR induce synergistic elevation of[Ca2+]i in primary LEC cultures from newborn or one-month-oldmouse lenses

This is the first ever study dealing with the GABA-evoked Ca2+

signaling in primary lens epithelial cultures and one of few that hasaddressed the role of both GABARs in conveying the GABA signal





to downstream Ca2+ signaling. Therefore, here we have addressedbasic issues like the nature of responses of primary LEC culturesderived from newborn (P0) or P30 mice to specific GABA recep-tor agonists or GABA, and sensitivity of the responses to selectiveantagonists in the presence/absence of extracellular Ca2+. Since thevast majority if not all cells exhibiting Ca2+ transients throughoutthese experiments were nucleated cells belonging to monolayers orsmall lentoid-like structures that did not differ substantially in theirresponses, the recorded fluorescent intensity values were pooledfor statistical analysis. Anucleated lentoid cells presumably alsolacking intracellular Ca2+ stores [24] did not load under conditionsdescribed here and were excluded from this analysis.

4.2.1. Studies with GABAR-selective agonistsBoth muscimol (GABAAR agonist) and baclofen (GABABR ago-

nist) evoked Ca2+ transients in all responding cells that could beblocked by the selective antagonists bicuculline and CGP55845,respectively. While the GABAAR-mediated Ca2+ rise in primary LECis entirely consistent with the channel properties in immature neu-rons [1,5,44] and/or non-neuronal cells [3,45], the GABABR-evokedCa2+ transients were unusual, as GABABR is widely considered tobe inhibitory in both embryos and in the adult [3,44,46,47]. Suchtransients were also observed in the rare occasionally surviving(retinal) neurons in the LEC cultures indicating that this propertyof GABABR might be a much wider phenomenon during develop-ment. We have also found a greater variability in the P0 mouse LECresponses to baclofen compared to muscimol. This phenomenonhas been previously described for hippocampal astroglia and isthought to be an intrinsic feature of the GABABR [11]. This vari-ability may arise from the superimposed inhibitory and excitatoryeffects of GABABR existing in different cells as described for mEScells [18]. However, the inhibitory component is probably smallcompared to the excitatory one, since application of the antago-nist CGP55845 alone usually had no effect, but occasionally evokedlong-lasting small-amplitude oscillations, the mechanism of whichis obscure at present.

4.2.2. GABA responsesResponses to GABA alone or in the presence of GABAR antag-

onists revealed the nature of the interactions between the tworeceptors. The elevation of the intracellular Ca2+ level by the twoGABAR in Ca2+-containing buffers was not additive since GABAevoked similar responses alone or in the presence of bicuculline.This indicates that convergent intracellular Ca2+ mobilizationmechanisms are activated by the two receptors as has been previ-ously shown for histamine1R and histamine2R in splenocytes [48],or P2XR and P2YR in retinal cells [49]. Another explanation maybe a GABAAR-independent potentiation effect of bicuculline on theCa2+ transients described previously in rat cerebellar granule cells[50]. Finally, this effect may be caused by the increased GABABRactivity due an elevated level of ambient GABA [51] followingblockade of GABAAR. Even application of both GABAR antagonistscannot suppress all GABA-evoked Ca2+ transients, which may resultfrom a non-canonical GABA-evoked Ca2+ signaling, like the recentlydescribed GABA transporter (GAT)-mediated Ca2+ rise in olfactorybulb astrocytes [52]. A full blockade of the two GABAR was effec-tively achieved in P30 mouse LEC cultures (see below).

Compared to P0, Ca2+ responses to GABA in LEC cultures derivedfrom P30 lenses were characterized by smaller amplitudes andgreater sensitivity to CGP55845 and bicuculline. These changes arepartly attributable to the switch of expression to a predominantlyfast synaptic type GABAAR since GABABR or other GABA signalingcomponents do not change significantly between the two stages[16]. Interestingly, this switch is not paralleled by a reversal of thereceptor properties from excitatory to inhibitory as would be pre-

dicted from studies in the CNS. It remains to be seen whether anexcitatory-inhibitory shift of GABAAR-evoked responses occurs atlater developmental stages.

4.2.3. Cell-to-cell wave propagation and oscillationsFollowing prolonged applications of baclofen or GABA (but not

muscimol) we have also been able to record the synchronous reac-tion of cell groups followed by wave-propagation of the signal,delayed Ca2+ transients and, in the case of baclofen long-lastingoscillations. The cell-to-cell signal propagation is reminiscent ofresponses involving mobilization of intracellular Ca2+ in lens cul-tures mediated by ATP receptors and strongly suggests that it maybe propagated through intercellular gap junctions and/or puriner-gic receptors [25,28,29,41]. Whereas GABA, GABAA or GABABreceptor agonists evoked Ca2+ increases with similar amplitudes,only the activation of GABABR by baclofen lead to sustained [Ca2+]iresponses, similarly to cerebellar mGluRs where both mGluR1 andmGluR5 induced [Ca2+]i mobilization but only the activation ofmGluR5 results in intracellular Ca2+ oscillation [53].

4.2.4. Dependence of GABA-evoked Ca2+ responses onextracellular Ca2+

The primary mouse LEC did not elicit responses to baclofen inthe absence of extracellular Ca2+ (data not shown), but reactedto GABA under the same conditions, although the number andamplitudes of responses decreased compared to Ca2+-containingbuffers. The GABA responses observed in Ca2+-free buffers are prob-ably due to release of Ca2+ from the intracellular Ca2+ stores. Thedecreased responsiveness in Ca2+-free solution and the robust Ca2+

transients evoked by KCl application in AAH implicate VGCCs asmediators of Ca2+ influx. The presence of L-type VGCCs in primaryLEC has previously been documented [43]. Bicuculline reduced sig-nificantly the responses to GABA in Ca2+-free conditions, in contrastto Ca2+-containing buffers, where it did not exert a significanteffect (Section 4.2.2) highlighting the differential (extracellular)Ca2+ sensitivity of GABAAR and GABABR-activated Ca2+-mobilizingmechanisms.

Following extensive washes with Ca2+-free buffer lens epithe-lial and fiber cells reacted to addition of 1mM Ca2+ with a sustainedand robust capacitive Ca2+ rise (data not shown), which is usuallyattributed to Ca2+ influx through the store-operated channel (SOC)mediating the refilling of the internal stores [24,25,41,43]. There-fore, the GABA-induced changes in [Ca2+]i in lens epithelial andfiber cells of primary mouse LEC cultures are mediated throughmultiple mechanisms employing both extracellular Ca2+ influx andCa2+ efflux from (IP3-sensitive) internal stores, which is also con-sistent with expression data.

In comparison, dependence of the Ca2+ rise exclusively onintracellular (mainly IP3-dependent) Ca2+ store release coupled tostore-operated Ca2+ influx has been described for primary humanlens cells in culture after activation of G-protein coupled recep-tors [18,41] and also in astroglia and mouse ES cells after GABABRactivation [11,18]. In this respect, GABABR of primary LEC culturesderived from P0 or P30 mouse lenses show similar sensitivity toexternally added Ca2+ as CNS neurons [54].

4.3. Possible role of GABA signaling in the developing lens andimplications for developing CNS and lens pathologies

GABA is a novel neurotransmitter for the lens in addition toacetylcholine [55], which is known to increase [Ca2+]i rise in lensepithelial and fiber cells through muscarinic receptors [21,55]. Thelens expresses a fully functional GABA signaling machinery whichis remarkably similar to that of the developing nervous system asdemonstrated by expression profiling [16,17]. Hence, it affords anideal model system to study the mechanisms of GABA-induced Ca2+





signaling, the interplay between the two GABA receptors, possi-ble involvement of other GABA signaling components and couplingto downstream second messenger pathways. Finally, although thelens cells possess a remarkably high resting [Ca2+]i [24] a sustainedCa2+ overload is the prime reason for lens cataract [43], underliesmany pathological conditions and triggers apoptosis [56,57]. LECcultures of human origin could be potentially useful in studies onthe interplay between GABA, Ach and Ca2+ signaling in patholog-ical conditions associated with cataract, like IDDM or Alzheimer[51,58].

Conflict of interest

The authors of this paper declare no conflict of interest for thepast three years with regard to the content of this paper.

Acknowledgements

We acknowledged the excellent work of the members of theGene Technology Unit, Institute of Experimental Medicine. Thiswork was partially supported by Hungarian National Grant Agency(OTKA) grant # T038098 (to Z.K.).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ceca.2011.07.002.

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