Cellular Signalling 26 (2014) 260–267

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Cellular Signalling

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β-Catenin-dependent pathway activation by both promiscuous“canonical” WNT3a–, and specific “noncanonical” WNT4– andWNT5a–FZD receptor combinations with strong differences in LRP5and LRP6 dependency

Larisa Ring a, Peter Neth a, Christian Weber a,b, Sabine Steffens a,b, Alexander Faussner a,⁎a Institute for Cardiovascular Prevention, Ludwig-Maximilians-University Munich, Pettenkoferstraße 9, 80336 Munich, Germanyb DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, 80336 Munich, Germany

⁎ Corresponding author. Tel.: +49 89 5160 2602.E-mail addresses: larisa.ring@med.uni-muenchen.de (

peter.neth@med.uni-muenchen.de (P. Neth), christian.we(C. Weber), sabine.steffens@med.uni-muenchen.de (S. Stealexander.faussner@med.uni-muenchen.de (A. Faussner).

0898-6568/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.cellsig.2013.11.021

a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 September 2013Received in revised form 4 November 2013Accepted 18 November 2013Available online 21 November 2013

Keywords:WNTFZDLRP5/6WNT/β-catenin signalling

TheWNT/β-catenin signalling cascade is the best-investigated frizzled receptor (FZD)pathway, however,wheth-er and how specific combinations ofWNT/FZD and co-receptors LRP5 and LRP6 differentially affect this pathwayare not well understood. This is mostly due to the fact that there are 19 WNTs, 10 FZDs and at least two co-receptors. In our attempt to identify the signalling capabilities of specific WNT/FZD/LRP combinations wemade use of our previously reported TCF/LEF Gaussia luciferase reporter gene HEK293 cell line (Ring et al.,2011). Generation of WNT/FZD fusion constructs – but not their separate transfection – without or withadditional isogenic overexpression of LRP5 and LRP6 in our reporter cells permitted the investigation of specificWNT/FZD/LRP combinations. The canonical WNT3a in fusion to almost all FZDs was able to induce β-catenin-dependent signalling with strong dependency on LRP6 but not LRP5. Interestingly, noncanonical WNT ligands,WNT4 andWNT5a,were also able to act “canonically” but only in fusionwith specific FZDs andwith selective de-pendence on LRP5 or LRP6. These data and extension of this experimental setup to the poorly characterized otherWNTs should facilitate deeper insight into the complex WNT/FZD signalling system and its function.

© 2013 Elsevier Inc. All rights reserved.

1. Introduction

TheWNT/FZD signalling pathways play pivotal roles in developmen-tal processes such as cell proliferation, differentiation andmigration [1].The importance of this signalling becomes obvious when it is dysregu-lated, leading to many different, mostly severe pathologies, like osteo-porosis, Alzheimer's disease or cancer [2–4]. In humans, there are 19WNT ligands, ten frizzled receptors (FZDs) and at least two co-receptors, LRP5 and LRP6 (low-density lipoprotein receptor-relatedproteins 5 and 6), making the WNT/FZD signalling system highly com-plex. The so far best investigated WNT/FZD pathway depends onthe regulation of the β-catenin level, whereby stabilized β-cateninaccumulates in the cytosol, then translocates to the nucleus and bindsto the T cell factor and lymphoid enhancer factor (TCF/LEF) family oftranscriptional cofactors, thus activating the transcription of targetgenes, e.g. c-myc, cyclin D1, and axin2 [5–8]. According to their biolog-ical activity WNTs have been historically classified into two differentgroups: the canonical WNTs, such as WNT1, WNT3a and WNT8, that

L. Ring),ber@med.uni-muenchen.deffens),

ghts reserved.

are able to induce theβ-catenin-dependent pathway and aremostly es-sential for embryonic development, and the noncanonical, i.e. suppos-edly β-catenin independently signalling WNTs, such as WNT4, WNT5aandWNT11, that participate in developmental processes such as planarcell polarity and convergent extension movements during gastrulation[9–11]. The most accepted current model is that canonical WNT ligandsnucleate the formation of a physical complex with FZDs and LRPs thatactivates the β-catenin-dependent pathway, and that WNTs are thecomponent most important for the signalling outcome [12,13]. Theten FZDs form their own separate family of G protein-coupled receptors[14] although there have been only a few reports on their competenceto activation of G proteins [15–17]. They share a conserved extracellularcysteine-rich domain (CRD), which has been identified as the bindingsite for WNT ligands [18,19]. The most sequential diversity amongFZDs is located in their intracellular C-termini that harbour, however,a KTxxxWdomain, which presumably is themost important transducerof the WNT/β-catenin pathway [20,21]. But to which extent and howthe secreted WNT glycoproteins that also contain a highly conservedcysteine-rich domain [22] discriminate between these receptors arestill notwell understood. Furthermore the relatively unknown functionsand specificities of LRP5 and LRP6 or of ROR1 and ROR2 as co-receptorscontribute to the challenge to identify the role of specific WNT/FZD/co-receptor interactions in the induction of the β-catenin signalling cas-cade [23,24]. Considerable work has been done that resulted in reports

261L. Ring et al. / Cellular Signalling 26 (2014) 260–267

on the function of certain canonical or noncanonical WNTs with select-ed FZDs with regard to β-catenin-dependent and independent signal-ling, investigating in part also the dependency on the co-receptorsLRP5 and LRP6 [25–28]. Still, as these results were often obtainedwith completely different experimental background, little directly com-paring information about the specificities of WNT/FZD/co-receptorcombinations is available, although, for example the prototype of non-canonical WNTs, WNT5a is suggested to transduce not only β-catenin-independent but also β-catenin-dependent pathways relying on the(co-)receptor context [29,30]. A systematic study that might also betransferred to the many less well-characterized members of the WNTfamily should therefore be of great general interest. In order to performsuch systematic investigations we have generated a TCF/LEF Gaussia lu-ciferase reporter gene HEK293 cell line that also allowed the regulatableisogenic overexpression of the co-receptors LRP5 and LRP6 through theuse of the Flp-In TRex-system [31]. In the present study we determinedthe effects of the combinations of all FZDs (with exception of FZD3)with three prototypically canonical or noncanonical WNTs (WNT3a;WNT4 & WNT5a) – either expressed separately or as fusion proteins –on β-catenin-dependent signalling. We also investigated the effect ofan overexpression of the co-receptors LRP5 and LRP6 on the signallingof these combinations.

This experimental setup allowed us to identify specific WNT/FZDcombinations with strong differences in their dependency on the co-receptor LRP5 and LRP6 in activating the WNT/β-catenin-dependentpathway. The generation of WNT/FZD fusion proteins as compared tothe separate transfection of respective WNTs and FZDs, proved thebest approach to characterize the properties of specific combinations.With efficient tools like the available reporter gene cell lines andWNT/FZD fusion proteins it is possible to study further specific WNT/FZD combinations, especially by extending this approach also to theless-well characterized WNT ligands, for a better understanding oftheir role and therapeutic potential in the complex WNT/FZD/co-receptor system.

2. Materials and methods

2.1. Reagents

Flp-In T-Rex HEK-293 cells were obtained from Life Technologies(Carlsbad, CA, USA). Cell culture media and additions were from PAALaboratories (Coelbe, Germany). EcoTransfect was purchased from OZBiosciences (Marseille, France). Poly-D-lysine and EZview™ Red anti-myc affinity matrix were purchased from Sigma-Aldrich (Taufkirchen,Germany). Fugene HD reagent and EDTA-free protease inhibitor cock-tail were fromRoche (Mannheim,Germany). G418-BC sulphatewas ob-tained from Biochrom AG (Berlin, Germany). Oligonucleotides weresynthesized from MWG-Biotech (Ebersberg, Germany).

2.2. Expression constructs

Plasmids harbouring the human coding sequences of FZD1, FZD2,FZD5, FZD9 FZD10,WNT4 andWNT5awere delivered by OriGene Tech-nologies (Rockville, MD, USA). The plasmid with the coding sequencefor FZD4 was purchased from Missouri S&T cDNA Resource Center(Rolla, MO, USA). The cDNA for FZD6 was obtained by RT/PCR fromHEK293 cells. Plasmids containing human FZD7, FZD8 and WNT3acDNAs have been previously described [32,33]. Using the signal peptidesequence of FZD4 for all FZDs to simplify further cloning procedures, theFZD coding sequences without their specific signal peptide sequenceswere ligated into the pcDNA5/FRT/TO vector (Life Technologies), thusall constructs consisted of the signal peptide sequence of FZD4, followedby a NotI cleavage site, a hemagglutinin (HA)/myc-double tag and thesequence of the respective FZD with an XhoI restriction site after thestop codon as described earlier [31]. As XhoI and NotI cleavage siteswere to be used for all subcloning, the internally existing XhoI cleavage

site of FZD7 and NotI cleavage site of FZD9 were changed by silent mu-tation. All the primers used with standard PCR techniques are depictedin Table S1.

For the introduction of FZD8 into the described standard vector thesingle internal NotI cleavage site at a suitable position after the FZD8 sig-nal peptidewas used in combination with an XhoI cleavage site after itsstop codon. The coding regions of all constructs were sequenced by au-tomated DNA sequencing (MWG-Biotech, Ebersberg, Germany) to ver-ify their correctness.

For the generation ofWNT/FZD fusion proteins, the FZD4 signal pep-tide sequence of the FZD constructs described above was substituted bythe respectiveWNT sequence, followed by a short spacer and amyc-tagas previously described for the fusion construct WNT3a/FZD4 [31]. TheWNT primer pairs used for the introduction of HindIII/KpnI restrictionsites via standard PCR techniques are given in Table S1. The spacermyc-tag sequence (harbouring also a BamHI restriction site) wasinserted accordingly via KpnI/NotI cleavage sites. For C-terminally2xFLAG-tagged WNT constructs the FZD4 sequence of the respectiveWNT/FZD4 fusion construct was exchanged for a 2xFLAG epitope se-quence with a stop codon and NotI/XhoI restriction sites. The plasmidwith the human coding sequence for LRP5 was previously described[34]. The N-terminally 2xFLAG-tagged LRP5 pcDNA5/FRT/TO plasmidwas generated by firstly introducing the 2xFLAG-tag into the pcDNA5/FRT/TO via HindIII/NotI restriction sites followed by insertion of theLRP5 sequence via NotI/NotI digestion and determination of the rightorientation. The cloning strategy for introducing the sequence of LRP6into a pcDNA5/FRT/TO plasmid with a C-terminal HA-tagwas describedpreviously [31].

2.3. Cell culture and stable transfection

Flp-In T-RexHEK-293 cells were cultured inDMEMhigh glucose sup-plemented with 10% foetal calf serum and 1% penicillin/streptomycin. ApcDNA3 Gaussia luciferase reporter plasmid with 10xTCF/LEF responseelementswas transfected into the Flp-In T-RexHEK-293 cells and severalclones with stable random integration selected with G418 (1 mg/ml) asdescribed [31]. For additional directed stable transfection with pcDNA5/FRT/TO expression vectors harbouring FZD1, FZD5, FZD8, LRP5 or LRP6cDNAs the Flp-In site of the cells was used and clones were selectedwith hygromycin B (0.5 mg/ml) as previously reported [31].

2.4. Reporter gene assay

Transient transfection of the 10xTCF/LEF reporter cells without orwith LRP5 and LRP6 overexpression was performed with EcoTransfectreagent according tomanufacturer's instructions as described previous-ly [31]. 72 h post-transfection the supernatant containing the secretedGaussia luciferase was collected and bioluminescence was measuredupon addition of coelenterazine, as previously reported [31]. The activ-ity was monitored within 1 min in a Mithras LB940 Multimode Micro-plate Reader (Berthold Technologies, Bad Wildbad, Germany). Relativelight units were normalized to empty vector- (mock-) transfectedcells and are presented as x-fold over mock.

2.5. Immunoblot analysis

For functional analysis of the transiently transfected constructs, cellsin 6-well plates were transfected with 1 μg DNA using EcoTransfect fol-lowing the manufacturer's instructions. After induction of gene expres-sionwith tetracycline (0.5 μg/ml) and incubation for 24 h the cells werelysed for 10 min on ice in 200 μl RIPA lysis buffer (50 mMTris, 150 mMNaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, pH 8.0)containing a protease inhibitor cocktail. Thereafter, lysates were collect-ed and vortexed for 30 min every 5 min on ice. After 15 min centrifuga-tion at 13,000 xg at 4 °C, lysates were mixed 5:1 with 5× Laemmlibuffer (0.625 M Tris–HCl, 10% SDS (w/v), 50% glycerine, under reducing

Table 1Correlation score of surface expression and total protein expression level of transiently transfected FZD1–10 (except FZD3).

FZD1 FZD2 FZD4 FZD5 FZD6 FZD7 FZD8 FZD9 FZD10

Surface expression ++ ++ ++ +++ + ++ +/− + +++Protein expression +++ +++ +++ +++ + ++ ++ + ++

Correlation scorewas estimatedwith:+/− b10-fold,+ N20-fold,++ N30-fold and+++ N50-fold over untransfected HEK293 cells for surface expression of FZD receptors (see Fig. 1B),and in ascending order for the band intensity (+, ++, +++) for protein expression levels of FZD receptors (see Fig. 1C).

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conditions with 25% DTT, bromophenol blue, pH 6.8) and incubated for5 min at 70 °C. For analysing myc-tagged fusion constructs, lysateswere incubated with 15 μl EZview™ Red anti-myc affinity matrix over-night at 4 °C. After intensive washing with RIPA lysis buffer withoutprotease inhibitor cocktail the matrix was incubated at 70 °C for5 min with 2:1 2×-Laemmli buffer and analysed by Western blotting.The membrane was blocked for 1 h at room temperature (RT) withblocking buffer consisting of 1% (w/v) non-fat dry milk in TBST(50 mM Tris-buffered saline, 150 mM NaCl, 0.05% Tween 20, pH 7.5).Myc-tagged proteins were detected with anti-myc (9E10) antibody(1:1000) (sc-40, Santa Cruz, CA, USA) and FLAG-tagged proteins withanti-FLAG antibodies (1:3000) (F921, Sigma), both followed by anti-mouse-HRP (1:5000) (7726, Cell Signalling, Danvers, MA, USA) assecondary antibody. HA-tagged constructs were detected with anti-HA-HRP (1:3000) (11867423001, Roche). Incubation with all antibod-ieswas carried out in TBST for 1 h at RT. Visualizationwas accomplishedusing Chemiluminescence Reagent Plus (Roche) and X-ray films(Hyperfilms ECL, GE Healthcare, Munich, Germany).

2.6. Flow cytometry analysis

HEK293 cells were seeded in 12-wells at about 70% confluency.After 24 h cells were transiently transfected with 1 μg DNA/well withEcoTransfect according to the manufacturer's instructions and gene ex-pression was induced with tetracycline (0.5 μg/ml) after another 24 hincubation. 48 h post-transfection cells were washed with PBS and de-tached by incubation with Accutase at 37 °C for 5 min. After additionof 1 ml PBS the cells were centrifuged for 5 min (400 xg, 4 °C), dyedwith anti-c-myc FITC conjugated antibody (3 μg/μl, MA1-19617, thermoscientific) in 100 μl PBS for 30 min at 4 °C in the dark, intensivelywashedand resuspended in PBS. The mean fluorescence intensity of cells ex-pressing FZD receptors with or withoutWNT ligand fusion was analysedwith Attune Acoustic Focusing Cytometer (Life Technologies™,Darmstadt, Germany).

2.7. Immunostaining

HEK293 cells stably overexpressed the cDNA of FZD1, FZD5 or FZD8were seeded in a poly-D-lysine coated 8-well chambered coverglass(Thermo Scientific, Germany)with about 60% confluency. After tetracy-cline induction (0.5 μg/ml) and further incubation for 24 h the cellswere prepared for the immunostaining. Firstly, cellswere permeabilisedwith 3.7% PFA (paraformaldehyd)/PBS for 15 min at RT. After washingthe cells with ice cold PBS, cells were directly blocked with 10% FCS/PBS at 4 °C. For intracellular staining cells were incubated with 0.25%Triton X-100/PBS for 10 min on ice, washed three times with ice coldPBS and then blocked as described before. Anti-cmyc-FITC conjugatedantibody (3 μg/μl, MA1-1967, Thermo Scientific) was used for dyingfor 30 min at 4 °C in the dark. After washing the cells three times withice cold PBS the immunofluorescence images were captured with 10 sof exposure time with an Olympus inverted fluorescence microscope(IX70) using Image ProPlus 5.0.

2.8. Data analysis

The relative light units of three different reporter cell clones inat least four independent experiments were measured for Gaussia

luciferase activity and are presented asmean ± SEM. One-way analysisof variance (ANOVA) with the Tukey's Multiple Comparison test wasperformed with *p b 0.05, **p b 0.01, ***p b 0.001 as statistical signifi-cant. Flow cytometry data were measured as mean fluorescence inten-sity in at least four independent experiments and are presented asmean ± SEM. One-way ANOVA with Dunnett's multiple comparisontestwas performedwith *p b 0.05, **p b 0.01, ***p b 0.001 as statisticalsignificant. Data analyses were performed with GraphPad Prism forMacintosh, version 5.0d (GraphPad Software, Inc., San Diego, Ca, USA).

3. Results

WNT-induced inhibition of constitutive β-catenin degradation re-sults in its cytosolic accumulation followed by translocation into thenucleus where it acts as a transcription factor promoting TCF/LEF de-pendent transcription of respective target genes [2,5]. Recently, wedemonstrated the functionality of TCF/LEF reporter HEK293 cells (lateraddressed as reporter cells) in combination with the Flp-In-TRex ex-pression system (Life Technologies) for stable, but regulatable proteinexpression as an excellent tool for studying the WNT/β-catenin path-way [31]. In order to investigate differences of definedWNT/FZD combi-nations with regard to the WNT/β-catenin signalling pathway, wecoexpressed FZD1–10 (except for FZD3, because it was not available)with three selected WNTs: i) WNT3a, known as the “activator” ofWNT/β-catenin signalling [35], ii) WNT4, considered to be a “non-canonical” FZD agonist [36], and iii) WNT5a, discussed of activating dif-ferent signalling pathways in a receptor context dependent manner[29,30]. Moreover, we took advantage of the Flp-In sites of the reportercells for the isogenic overexpression of LRP5 and LRP6 (see Fig. 1D, E). Asa first result, we observed that reporter cells with a stable overexpres-sion of LRP6 displayed a twice as high basal luciferase activity as com-pared to cells without tetracycline induction, whereas LRP5 expressionhad no such effect (see Fig. S1).

3.1. Basal signalling and localisation of FZD receptors in HEK293 cells

Before starting to investigate the effects of WNT-induced stimula-tion in the reporter cells, we analysed the basal behaviour of FZD recep-tors alone or in combination with LRP5 or LRP6 overexpression.Plasmids harbouring the FZD receptors were transiently transfectedinto the reporter cells without or with stable overexpression of LRP5or LRP6. Compared tomock-transfected cells none of the FZD constructsby itself significantly induced luciferase activity (Fig. 1A, white bars).Analysis by flow cytometry was performed to ensure that all FZDswere expressed properly at the cell surface. The presence of all FZDs atthe cell surface could be demonstrated, but although all under the con-trol of the same CMV promoter and their sequence starting with thesame signal peptide (taken from FZD4), they displayed very differentlevels (Fig. 1B, see also Fig. S2A–I). The range varied from about 10-fold for FZD8 to 70-fold for FZD10 over untransfected cells. Their differ-ential expression pattern correlated mostly with their overall proteinlevels (Fig. 1C, see also Table 1), only FZD8 displayed relatively strongerbands in the immunoblot as compared to its surface expression deter-mined by flow cytometry.

Of all FZD constructs only transfection of FZD9 and FZD10 into re-porter cells with overexpression of LRP6 resulted in a significant induc-tion (about 4-fold) of luciferase activity (Fig. 1A, black bars). This

Fig. 1. Characteristics of FZD receptors (FZD1–10, except FZD3). (A) Gaussia luciferase activity of transiently transfected FZD receptors (FZDs) of three independent clones of TCF/LEF HEK293cells (white bars), with stably overexpressed LRP5 (grey bars) or LRP6 (black bars) presented as fold increase over mock-transfected cells. Values represent means ± SEM of 9 independentexperiments (n = 3 of each independent clone), performed in triplicates. One-way ANOVAwith Tukey's test: ***p b 0.001, **p b 0.01. (B) Flow cytometry analysis of transiently transfectedFZDs.Meanfluorescent intensity represented as fold overHEK293 cells in triplicates of at least four independent experiments (means ± SEM). One-wayANOVAwithDunnett'smultiple com-parison test: ***p b 0.001, **p b 0.01, *p b 0.05. (C) Representative immunoblot from cell lysates of three independent experiments of transient transfected hemagglutinin (HA)-myc-taggedFZDs, inducedwith tetracycline (tet) anddetectedwith anti-HA antibody and the corresponding loading controlwith anti-actin. Black arrowheads indicateHA-detected FZDs and correspond-ing actin as loading control. Asterisks signify the protein band for the corresponding FZD. (D, E) Representative anti-HA and anti-FLAG immunoblot of cell lysates of TCF/LEF reporter HEK293cells stably overexpressing LRP5 and LRP6, respectively without and with tet induction. Black arrowhead indicates predicted size of LRP5 and LRP6 (approx. 20 μg protein/lane).

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demonstrates that there is no direct proportionality between basal ac-tivity –whether in the presence of co-receptors or not – and expressionlevels of the receptors, and of course proves that despite its low proteinlevel FZD9 is functional.

In order to determine whether the weak surface expression of FZD8in comparison to its strong band in the immunoblot is caused by apronounced specific intracellular localisation of FZD8 we performed acomparative immunostaining with HEK293 cells stably overexpressingFZD1, FZD5 or FZD8. For both, for FZD5 with about 50-fold and forFZD1 with about 40-fold expression over untransfected cells (seeFig. 1B), immunostaining demonstrated strong cell surface expression(Fig. 2A and C), and only a very weak one for FZD8 (Fig. 2E). Afterpermeabilisation of the cellswith TritonX-100 to be able to label also in-tracellularly located receptors, for FZD5 still most of the fluorescencewas associated with the cell membrane with only a few punctae visibleintracellularly (Fig. 2B). In contrast, after Triton X-100 treatment ofFZD1 expressing cells, high intracellular staining was detected in addi-tion to themembrane associated fluorescence (Fig. 2D), indicating con-siderable intracellular localisation of FZD1 under normal cell cultureconditions. Most intriguingly, after permeabilisation the cells expressingFZD8 displayed strong intracellular staining with hardly any staining ofthe cell membrane (Fig. 2F). This strong basal intracellular localisationexplains nicely the observed discrepancy between the weak signal inthe flow cytometry analysis (see Fig. 1B) and the fairly strong band inthe immuno blot (see Fig. 1C).

3.2. Signalling of co-transfected WNT3a and FZD1-10 (except FZD3)

After having determined the basal activity of the FZD constructs weanalysed the transfection of WNT3a, WNT4 and WNT5a in our reporter

cells, without or with stably overexpressed LRP5 or LRP6. Transfectionof WNT3a alone increased the luciferase activity about 3- to 5-foldover mock-transfected cells (Fig. 3A, white bars), representing the pos-sible WNT3a-induced TCF/LEF reporter activation without any potenti-ation of the signalling through LRP5 (grey bars) or LRP6 (black bars) asco-receptors. WNT4 andWNT5a did not activate the luciferase activity,neither in reporter cells alone nor with LRP5 or LRP6 overexpression.

The next stepwas to co-transfect the FZDswithWNT3a and identifythe luciferase activity in reporter cells (white bars), with stablyoverexpressed LRP5 (grey bars) or LRP6 (black bars). Fig. 3B clearlydemonstrates that almost none of the combinations, without or withstably overexpressed LRP5 or LRP6 did significantly increase the lucifer-ase activity as compared to mock-transfected cells. The question arosehow well these results actually reflect the capabilities of the variousWNT/FZD/LRP combinations to induce β-catenin-dependent signalling.An obvious disadvantage of this experimental approach is that an inter-action of the overexpressed proteins with endogenously expressedWNTs and FZDs (and LRPs) is rather likely and might generate unclearresults with regard to the contribution of the overexpressed proteins.In an attempt to widely exclude an interaction of the overexpressedWNTs and FZDs with their respective endogenous counterparts, wetherefore continued with expression of fusion proteins of WNT ligandsand FZDs.

3.3. Signalling of WNT3a/FZD fusion proteins and the effects of LRP5 andLRP6 overexpression

Tofirstly determine the ability of theWNT3a/FZD constructs to inducea β-catenin-dependent TCF/LEF transcription response, the chimericWNT3a/FZD1–10 (except FZD3) plasmids were transiently transfected

Fig. 2. Anti-cmyc-FITC staining of naïve and permeabilised HEK293 cells stably overex-pressing FZD1, FZD5 or FZD8. Representative fluorescence microscopy images ofHEK293 cells with stably overexpressed FZD5 (A, B), FZD1 (C, D), and FZD8 (E, F) fixedwith 3.7% PFA for staining of the HA-myc-tagged FZD receptors on the cell surfacewithout (A, C, E) or after permeabilisation with 0.25% Triton X-100 for intracellular stain-ing (B, D, F) with anti-cmyc-FITC antibody (3 μg/μl).

Fig. 3. Gaussia luciferase activation induced by WNT alone and with transiently co-transfected FZD proteins. (A)WNT3a-, WNT4- andWNT5a-induced Gaussia luciferase ac-tivity of three independent clones each of reporter cells (white bars), or with stablyoverexpressed LRP5 (grey bars) or LRP6 (black bars). (B) Gaussia luciferase activity ofco-transfected WNT3a and FZD1–10 (except FZD3) of three independent clones each ofreporter cells (white bars), or with stably overexpressed LRP5 (grey bars) or LRP6 (blackbars). Luciferase activity is presented as fold over mock-transfected cells. Representeddata are means ± SEM of at least 4 independent experiments performed in triplicates.One-way ANOVA with Tukey's test: ***p b 0.001, **p b 0.01, *p b 0.05.

264 L. Ring et al. / Cellular Signalling 26 (2014) 260–267

into reporter cells without or with stable overexpression of the co-receptors LRP5 or LRP6.

Of all constructs, only transfection withWNT3a/FZD1 and –FZD7 re-sulted per se in a luciferase activity that was significantly increased ascompared to mock-transfected cells (Fig. 4A, white bars). Some otherconstructs, however, showed at least a trend to increased activity. Asthe WNT3a/FZD fusion proteins can be considered as constitutively ac-tivated receptors, their signalling strength or their lack thereof mightbe caused by desensitisation/internalisation processes and be due tothe inherent trafficking patterns of the respective FZD after activationresulting in different surface expressions. Thus, we compared their sur-face expression (Fig. 4B) relative to unfused receptors (see also Fig. 1B).Although as expected for activated seven transmembrane receptorsmost of the fusion constructs displayed reduced surface expression ascompared to the naïve wild types, a low signal or lack thereof couldnot be explained by a low surface expression of the respectiveconstructs as there was no direct correlation (e.g. WNT3a/FZD8 withno signalling activity but high surface expression, Fig. 4A and B). Over-expression of the co-receptor LRP5 had no significant effect on the lucif-erase activities observed with the different WNT3a/FZD constructs(Fig. 4A, grey bars). In contrast, stable overexpression of LRP6 in the re-porter cells had dramatically different effects on their signalling.Where-as several constructs still displayed no significant luciferase activity ortrends at best (WNT3a/FZD6, –FZD8, –FZD9), four other constructs(WNT3a/FZD4, –FZD5, –FZD7, –FZD10) in the presence of LRP6 stronglyinduced the signal between 25- to 40-fold over mock-transfected cells(Fig. 4A, black bars). Intriguingly, there was no direct relation betweenthe activity exhibited in the absence or presence of overexpressed

LRP6. WNT3a/FZD1 and –FZD7 signalled very similarly in the absenceof LRP6, but only WNT3a/FZD7 displayed a strong increase whencoexpressed with LRP6 whereas WNT3a/FZD1 did not at all (Fig. 4A).

Taken together, these results demonstrate that the effect of WNT3aon the WNT/β-catenin signalling pathway in HEK293 cells strongly de-pends on the FZD receptor it is fused to. The response pattern covers awide range: There are FZDs that in fusion with WNT3a transduce thesignal activity in a good fashion even without overexpression of LRP5or LRP6 (FZD1 and FZD7, with only signalling of FZD7 being strongly in-creased by LRP6 overexpression), others that display (strong) signallingonlywith overexpression of LRP6 (FZD2, FZD4, FZD5, FZD7, FZD10), andfinally those that hardly induce any luciferase activity even with LRP6overexpression (FZD6, FZD8, FZD9).

3.4. Noncanonical WNT4- and WNT5a-induced signalling is stronglyreceptor specific

For further investigation under what conditions specific FZD recep-tors can stimulate β-catenin-dependent signalling the historicallytermed noncanonical WNT ligands, WNT4 and WNT5a, in fusion withFZD receptors were transfected into the reporter cells without or withoverexpression of the co-receptors LRP5 and LRP6. The first noticeableobservation was that no WNT4/FZD construct was able to activate theGaussia luciferase expression (Fig. 5A, white bars) by itself (as also

Fig. 4.WNT3a/FZD1–10 (except FZD3) induced Gaussia luciferase activity and surface ex-pression in the presence or absence of LRP5 and LRP6. (A) Gaussia luciferase activity ofthree independent clones each of TCF/LEF reporter HEK293 cells (white bars), stably over-expressing LRP5 (grey bars) or LRP6 (black bars), transiently transfected with WNT3a/FZD1–10 (except FZD3) chimeric constructs. Luciferase activity is presented as fold in-crease over mock-transfected cells. One-way ANOVA with Tukey's test: ***p b 0.001,**p b 0.01, *p b 0.05. (B) Cell surface expression of transiently transfected WNT3a/FZD1–10 (except FZD3) chimera presented as mean fluorescent intensity relative to tran-sient expression of corresponding FZD alone (=100%) and was analysed via flow cytom-etry. Values represent means ± SEM of at least four independent experiments. One-wayANOVA with Dunnett's multiple comparison test: ***p b 0.001, **p b 0.01.

Fig. 5.WNT4–, andWNT5a/FZD1–10 (except FZD3) Gaussia luciferase activity dependingon LRP5 or LRP6. (A–B) Gaussia luciferase expression of three independent clones each ofTCF/LEF reporterHEK293 cells (white bars), orwith stably overexpressed LRP5 (grey bars)or LRP6 (black bars) all transiently transfectedwithWNT4– (A), andWNT5a (B)/FZD1–10(except FZD3) and presented as fold increase over mock-transfected cells. Values repre-sent means ± SEM of 9 independent experiments (n = 3 of each independent clone),performed in triplicates. One-way ANOVA with Tukey's test: ***p b 0.001, *p b 0.05.

265L. Ring et al. / Cellular Signalling 26 (2014) 260–267

was shown with transfection of WNT4 alone in the reporter cells (seeFig. 3A)). However, when the WNT4/FZD10 chimera was transfectedinto reporter cells with overexpression of LRP5 a weak (about 4-fold)but significant increase in luciferase activity was present (Fig. 5A, greybars). The enormous luciferase activities, which were achieved throughtransfection with the four different WNT3a/FZD chimeric proteins (seeFig. 4A), in LRP6 overexpressing reporter cells could not be observedwith the four corresponding WNT4/FZD fusions (Fig. 5A, black bars). Asignificant but only moderate (about 5-fold) increase was seen withWNT4/FZD4 chimeric protein. Transfection with WNT4/FZD5 revealedonly a weak activation. The strongest induction of luciferase activationwas achievedwithWNT4/FZD10 fusion protein and LRP6 (Fig. 5A) com-parable to that ofWNT3a/FZD10 in combinationwith LRP6 (see Fig. 4A).In contrast, the combination of WNT4/FZD7 showed no effect at all, al-though WNT3a/FZD7 fusion with LRP6 overexpression had been oneof the strongest stimulators (see Fig. 4A).

A similarly highly specific signalling pattern was obtained when re-porter cells were transfected with WNT5a/FZD chimeric proteins (seealso Fig. 3A for WNT5a transfection alone in the reporter cells). OnlyWNT5a in combinationwith FZD4was able to stimulate Gaussia lucifer-ase activity by itself (Fig. 5B, white bars). As observed forWNT4,WNT5a

when fused to FZD10was able to increase luciferase activity in amoder-ate fashion (about 4-fold over mock) when LRP5 was overexpressed(Fig. 5B, grey bars), making these the two investigated WNT/FZD com-binations that suggest some positive role for LRP5 in β-catenin signal-ling. Intriguingly, whereas WNT4/FZD10 induced luciferase activitycould be strongly amplified by LRP6 overexpression (Fig. 5A) therewas only a weak effect of LRP6 on WNT5a/FZD10 signalling (Fig. 5B,black bars). On the other hand, whereas WNT4/FZD4 displayed onlymoderate signalling (about 4-fold) in LRP6 overexpressing cells(Fig. 5A), the signalling of WNT5a/FZD4 was strongly increased (about25-fold) by LRP6 overexpression (Fig. 5B).

Taken together, these data demonstrate that depending on the con-text of FZD and LRP co-receptors not only canonical WNT proteins likeWNT3a can activate the WNT/β-catenin signalling but also noncanoni-cal ligands, like WNT4 and WNT5a. These results, however, also showthat the requirements for WNT4 and WNT5a signalling with regard toFZDs and LRPs are distinctly more specific than for WNT3a, which ap-pears to be more promiscuous.

4. Discussion

In this study we set out to systematically investigate the specificityof the interaction of WNT ligands with FZD receptors with regard toβ-catenin-dependent signalling. In addition, we wanted to assess in di-rect comparison the contributions of the co-receptors LRP5 and LRP6

266 L. Ring et al. / Cellular Signalling 26 (2014) 260–267

that in general are not dealt with separately and therefore often onlyone or the other has been considered in respective studies [37–39].We took all 10 human FZDs (with exception of FZD3, that unfortunatelywas not available to us) and selected three representative ligands fromthe 19 existing human WNTs. According to the literature these threeWNTs should behave quite differently with regard to β-catenin signal-ling, and therefore also have been historically classified as canonical(WNT3a) or noncanonical (WNT4, WNT5a), i.e. acting or not via theβ-catenin-dependent pathway, respectively [29,30,35,36].

Our first results indicated that the experimental setup in HEK293cells might be too complex for WNTs and FZDs to be co-transfectedand studied as separate entities: Although the transient transfection ofFZDs or the selected WNTs alone produced well-defined data with re-gard toβ-catenin-dependent signalling (see Figs. 1A and 3A), their tran-sient co-transfection generated results that were difficult to interpret,displaying mostly only trends as even apparent differences hardlyturned significant. Several of the FZDs [13,40] as well as LRP5 andLRP6 and WNT binding ROR2 [30] are expressed endogenously inHEK293 cells. Consequently, transiently coexpressed WNTs could bindin principal to all available endogenous and overexpressed FZDs andco-receptors making it difficult to relate the resulting luciferase activityto the respective overexpressed FZDs. Moreover, FZD6 that is endoge-nously expressed in HEK293 cells [40] has been reported to have evennegative effects on β-catenin-dependent signalling [41] making the in-terpretation of the results even more complex. As a side note, thisinhibiting effect of FZD6 would be in good agreement with our datashowing that FZD6 overexpression in combination with expression offree WNT3a results in a lower signal than WNT3a alone (see Fig. 3B).

In contrast to the separate transfection, fusion of the selected WNTsto the N-terminus of FZD receptors allowed us to determine the effectsof the selectedWNT/FZD combinations on β-catenin-dependent signal-ling and to clearly assess the contributions of the co-receptors LRP5 andLRP6. Apparently, the FZD-fused WNT ligands do not activate the en-dogenous FZDs and thus do not confuse the results by doing so. This isindicated by the fact that almost all WNT3a-fusion constructs displayedactivities that were below that observed for unfused WNT3a (seeFigs. 3A and 4A, white bars). Thus, it is also unlikely that the fusedWNTs interact significantly with other WNT-binding receptors such asROR2 or RYK [42,43], although they obviously can interact sufficientlywith the co-receptors LRP5 and LRP6.

Our data suggest so far that for induction of β-catenin-dependentsignalling the respectiveWNT ligand ismore critical than the FZD recep-tor and in some way justify the historical classification of WNTs ascanonical and noncanonical [22]. Almost all FZD-fusions with the ca-nonical WNT3a (strongly) induced luciferase activity, in particularwith overexpression of LRP6 (see Fig. 4A). Thus, fused with WNT3aonly FZD6 – which actually might act inhibiting as mention before –

and FZD8 showed no significant effect or trend to induce β-catenin-dependent signalling (FZD3 was not tested). WNT3a therefore, can beconsidered as highly promiscuous und should be able to induce β-catenin signalling in almost all cellular settings. In contrast, the nonca-nonical WNT4 and WNT5a induced luciferase activity only with threeout of nine tested FZDs, and that onlywith LRP5 or LRP6 overexpression.Intriguingly, for both ligands it were the same three FZDs (FZD4, FZD5,and FZD10), although the effect of an LRP6 overexpression was verystrongonWNT4/FZD10 but onlymoderate onWNT4/FZD4, and precise-ly the other way around for WNT5a/FZD4 and –FZD10, pointing to ahighly WNT/FZD specific interaction with LRP6.

The effects of an overexpression of the co-receptors LRP5 and LRP6were highly diverse. Signalling of almost all WNT3a fusions was in-creased by LRP6 but none by LRP5 overexpression, as also has been re-ported in earlier studies [23,44]. LRP5 had at least some moderateeffects on three of the noncanonical WNT/FZD fusions. Our data there-fore, suggest that noncanonicalWNTs asWNT4 orWNT5a can in princi-pal act “canonically”, i.e. activate β-catenin-dependent signalling, that,however, a much more specific cellular environment is needed for

them than for the canonical WNT3a to do so. The strong contributionof LRP6 to effective β-catenin-dependent signalling of all three WNTsis a good indicationwhy LRP6 plays such an important role in the devel-opmental embryogenesis of mice [45].

Genetic fusion of an agonistic acting WNT ligand to a FZD receptorshould result in the synthesis of a permanently activated FZD that ac-cordingly should constitutively evoke desensitising cellular responses.As several FZDs have been reported to be internalised afterWNT stimu-lation [46–49] similar results have to be expected for at least someof theactive WNT/FZD fusion proteins. As a fact we saw less surface expres-sion and distinctly less total protein for the fusion constructs ascompared to the respective unfused FZD (see Fig. S3). At this point,though, it is not clear whether this is a consequence of ongoinginternalisation and degradation or just of an overall reduced expressionof the fusion proteins. However, clearly no correlation between the sig-nalling response and the surface expression level of the correspondingWNT/FZD fusion proteins was observed (see also Figs. 4A, 5A and B)as all WNT/FZD fusion constructs, also the inactive ones, displayedlower surface expression levels (see Fig. S3). In this regard one has tokeep in mind that the data on surface expression and total protein re-flect the state after 48 h being the result of several processes such asneosynthesis, internalisation, and degradation or recycling of the con-structs. In contrast, the amount of luciferase activity in the cell superna-tant should theoretically depend only on the rate of synthesis andsecretion of Gaussia luciferase over 72 h.

For most unfused FZDs their surface expression determined by flowcytometry was in good correlation with their total protein shown in theimmunoblot, indicating that most of the receptors are located at the cellsurface (see Fig. 1B and C, Table 1). One distinct exception was FZD8where we saw very low surface expression although there was a strongband in the immunoblot (see Fig. 1C, lane 7). Immunostaining ofpermeabilised cells indeed indicated that FZD8 is highly localisedintracellularly (see Fig. 2F) as compared to other FZDs, like FZD1 andFZD5, which are located preferably at the cell surface, also afterpermeabilisation (see Fig. 2B and D) that is also suggested in previouswork [50].

5. Conclusion

To summarize, the results of our study using three different WNT li-gands and nine FZD receptors suggest that canonical WNT ligands aremuch more promiscuous in their ability to activate the β-catenin-dependent pathway via different FZD receptors, an action in whichthey are strongly supported by LRP6 but not LRP5 as co-receptor. Non-canonical WNTs can also induce β-catenin-dependent signalling, re-quire, however, a much more selected setting with regard to FZDsubtype and LRP5 or LRP6. Our results also demonstrate that the useof WNT/FZD fusion in combination with the Flp-In system that allowsthe stable isogenic overexpression of large proteins such as the LRPsshould be an excellent approach to further unravel the specificity ofWNT/FZD interactions and thus help to shed light also on the functionof the less well-characterized WNT ligands. Especially using the ap-proach to fuseWNT ligandswith FZDs one should be able to investigatespecific WNT/FZD combinations, which are associated with aberrantdisorders, like cancer, where some WNTs or FZDs are highly over-expressed [3,51–53] and therefore may induce abnormal activation ofβ-catenin signalling.

Acknowledgements

The study was supported by the European Research Council(ERC AdG °249929 to C.W.) and Deutsche Forschungsgemeinschaft(FOR809 to C.W.).

The authors thank C. Seidl for excellent technical assistance.

267L. Ring et al. / Cellular Signalling 26 (2014) 260–267

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.cellsig.2013.11.021.

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