Differential activation of the calcium/protein kinase C and thecanonical b-catenin pathway by Wnt1 and Wnt7a producesopposite effects on cell proliferation in PC12 cells

Paola Spinsanti,* Teresa De Vita,* Alessandra Caruso,* Daniela Melchiorri,* Roberta Misasi,�Andrea Caricasole� and Ferdinando Nicoletti*,§

*Department of Human Physiology and Pharmacology, University of Rome La Sapienza, Rome, Italy

�Department of Experimental Medicine, University of Rome La Sapienza, Rome, Italy

�Sienabiotech s.p.a., Siena, Italy

§I. N. M. Neuromed, Pozzilli, Italy

The Wnt proteins constitute a large family of secretedglycoproteins that bind to 7-transmembrane Frizzled (FZD)receptors and to type-5 or-6 low density lipoprotein receptor-related proteins (LRP5/6). The diverse biological effects ofWnts are mediated by at least three signaling pathways(Kohn and Moon 2005; Gordon and Nusse 2006). Althoughthe presence in mammalian genomes of 19 genes encodingdistinct Wnts, 10 genes encoding different FZD receptors and2 genes encoding LRP co-receptors potentially allows for alarge number of ligand–receptor combinations, little infor-mation is available on what ligand–receptor complex canactivate one or the other of the known Wnt pathways(Caricasole et al. 2003a). Another degree of complexity isadded by the finding that Wnts can signal via some receptor

tyrosine kinases (Lu et al. 2004), and individual Wnts canoften signal through more than one pathway (Mikels andNusse 2006). Interaction of Wnts with different FZD

Received August 1, 2007; revised manuscript received October 2, 2007;accepted October 22, 2007.Address correspondence and reprint requests to Ferdinando Nicoletti,

MD, Department of Human Physiology and Pharmacology, P.le AldoMoro, 5, Roma 00185, Italy. E-mail: ferdinandonicoletti@hotmail.comAbbreviations used: ChAT, choline acetyltransferase; Dkk-1, dick-

kopf-1; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetalbovine serum; FZD, frizzled; GSK, glycogen synthase kinase; JNK,Jun-N-terminal kinase; LEF, lymphoid enhancer-binding protein factor;LRP, low density lipoprotein receptor-related protein; NFAT, nuclearfactor of activated T cells; PKC, protein kinase C; TCF, T-cell factor.

Abstract

We examined the effect of Wnt1 and Wnt7a on cell proliferation

using undifferentiated PC12 cells, which originate from the

neural crest and are widely employed as a neuronal cell model.

Heterologous expression of Wnt1 enhanced [3H]thymidine

incorporation and expression of cyclin D1 and cylin E in PC12

cells. Opposite effects were observed in PC12 cells expressing

Wnt7a. Searching for the mechanisms underlying the opposite

effects of Wnt1 and Wnt7a on PC12 cell proliferation, we

examined the activation of the canonical b-catenin/T-cell–

lymphoid enhancer-binding protein transcription factor path-

way and the ‘calcium pathway’ by co-transfecting the cells with

a reporter gene controlled by either T-cell–lymphoid enhancer-

binding protein transcription factor or the calcium-activated

transcription factor, NFAT. Wnt1 and Wnt7a activated both

pathways, but to a different extent. While Wnt1 preferentially

activated the calcium pathway, Wnt7a mainly activated the

canonical pathway. Pharmacological inhibition of protein

kinase C, which is a component of the calcium pathway,

abrogated the increase in cell proliferation induced by Wnt1

without affecting the antiproliferative action of Wnt7a. The

action of Wnt7a was instead occluded by lithium ions, which

mimic the activation of the canonical pathway, and was largely

reduced by Dickkopf-1, which acts as an inhibitor of the

canonical pathway. In addition, expression of a constitutively

active mutant of b-catenin potently activated the canonical Wnt

pathway and reduced [3H]thymidine incorporation. These data

challenge the view that the canonical Wnt pathway invariably

supports cell growth and suggest that, at least in PC12 cells,

cell proliferation is regulated by the balance between the cal-

cium/protein kinase C pathway and the canonical pathway.

Keywords: calcium/protein kinase C pathway, PC12 cells,

proliferation, b-catenin pathway, Wnt1, Wnt7a.

J. Neurochem. (2008) 104, 1588–1598.

d JOURNAL OF NEUROCHEMISTRY | 2008 | 104 | 1588–1598 doi: 10.1111/j.1471-4159.2007.05111.x

1588 Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 1588–1598� 2007 The Authors

receptors and LRP5/6 activates a ‘canonical’ signalingpathway leading to a reduced degradation of b-catenin. Inthe absence of Wnt, b-catenin is recruited into a multiproteincomplex including adenomatous polyposis coli and axin,which facilitates phosphorylation of b-catenin by caseinkinase 1 and glycogen synthase kinase-3b (GSK3b). Thisleads to b-catenin degradation through the ubiquitin/protea-some pathway. Interaction of Wnt with FZD activates theprotein disheveled leading to the membrane recruitment ofaxin, which binds to the cytoplasmic tail of LRP5/6. As aconsequence, b-catenin is no longer degraded and translo-cates into the nucleus where it activates gene transcription byinteracting with T-cell factor (TCF) and lymphoid enhancer-binding protein (LEF) transcription factors (reviewed byMoon et al. 2004).

Wnt signaling coordinates a diverse array of processesduring embryonic development such as the organization ofthe body plan and organogenesis. A growing body ofevidence supports a role for Wnt signaling in neuronaldevelopment and homeostasis (reviewed by Caricasole et al.2005). Wnt abnormalities have been associated with neuro-developmental disorders including Williams syndrome (Zhaoet al. 2005), autism (Wassink et al. 2001; but see alsoMcCoy et al. 2002; and Li et al. 2004 for contrastingresults), and schizophrenia (Cotter et al. 1998; Miyaokaet al. 1999). Interestingly, however, a deregulation of Wntsignaling is also associated with neuronal degeneration inmodels of Alzheimer’s disease (De Ferrari and Inestrosa2000; Garrido et al. 2002; Inestrosa et al. 2002, 2004, 2005;Caricasole et al. 2003a, 2004; De Ferrari et al. 2003;Alvarez et al. 2004; Fuentealba et al. 2004; Scali et al.2006). A number of drugs, including activators of theperoxisome proliferator-activated receptor-c, antioxidants,bifunctional anti-inflammatory drugs, cholinesterase inhibi-tors, and muscarinic receptor agonists, protect neuronsagainst b-amyloid toxicity by modulating Wnt signaling(Farias et al. 2004, 2005; Inestrosa et al. 2005; Quintanillaet al. 2005). The established role of GSK3b in phosphory-lating tau protein (Busciglio et al. 1995; Takashima et al.1998; Jackson et al. 2002; Pierrot et al. 2006) suggests arole for the canonical Wnt pathway in the formation ofpathological aggregates of hyperphosphoylated tau (neurofi-brillay tangles) found in Alzheimer’s disease and otherneurodegenerative disorders. Another possible link betweenWnt signaling and Alzheimer’s disease is the associationbetween presenilins (which are mutated in most cases offamiliar Alzheimer’s disease) and b-catenin (Nishimura et al.1999; Parisiadou et al. 2004; Teo et al. 2005; Hwang et al.2006).

Recent evidence suggests that an unscheduled activationof cell cycle in differentiated neurons is a common determi-nant of cell death in a variety of neurodegenerative disorders,including Alzheimer’s disease (reviewed by Arendt et al.1998; Raina et al. 1999; Copani et al. 2001; Caricasole et al.

2003a; Herrup et al. 2004; Nagy 2000, 2005; Neve andMcPhie 2006). Cultured neurons challenged with b-amyloid(the main component of amyloid plaques of the Alzheimer’sbrain) re-enter the cell cycle and die during the S phase(Copani et al. 1999). Cell death has been related to anunscheduled DNA synthesis carried out by DNA pol-b(Copani et al. 2006) and perhaps to other mechanismsrelated to the cell cycle (reviewed by Herrup et al. 2004).The molecular events that drive the abnormal cell cycle inneurons destined to die are unknown. Wnt signaling wasconsidered as a potential candidate because Wnt stimulatesproliferation of a variety of cells, including CNS neuropro-genitors. Transgenic mice expressing constitutively activeb-catenin in neuroprogenitor cells develop enlarged brainsbecause a greater proportion of neuroprogenitors re-enter thecell cycle after mitosis (Chenn and Walsh 2002). However,the role of Wnt may not be univocal because Wnt7a inhibitsproliferation of lung cancer cells (Winn et al. 2005, 2006),and Wnt5a may act as tumor suppressor (Liang et al. 2003).We therefore decided to examine how different Wnts,presumably activating different signaling pathways, affectproliferation in cells of neural crest origin. As a first step, wecompared the action of Wnt1 and Wnt7a in PC12 cells,which derive from the neural crest, are easily transfected, andhave already been used for the molecular characterization ofWnt signaling (Caricasole et al. 2003a).

Materials and methods

MaterialsDulbecco’s modified Eagle’s medium (DMEM) and fetal bovine

serum (FBS) were purchased from Invitrogen (Milan, Italy).

Bisindolylmaleimide and all other chemicals were purchased from

Sigma Aldrich (Milan, Italy). [3H]Thymidine was purchased from

GE Healthcare Europe GmbH (Milan, Italy).

Cell culturePC12 cells were cultured in DMEM supplemented with 2 mmol/L

glutamine, 200 U/mL penicillin, 100 lg/mL streptomycin, and

10% FBS, and maintained at 37�C in a 5% CO2 humidified

atmosphere.

PlasmidsConstruction of plasmids was as follows: for the Wnt7a expression

construct, the Wnt7a cDNA was amplified from human adult brain

cDNA by using primers carrying EcoRVand XbaI restriction sites at

the flanking ends. The amplified cDNA was sequenced and

subcloned into the EcoRV and XbaI restriction enzyme sites of the

eukaryotic expression pCIN4 vector (Rees et al. 1996; Caricasoleet al. 2002). The expression plasmid for mouse Wnt1 was provided

by Marc van de Wetering (Hubrecht Laboratory, Utrecht, The

Netherlands). The expression plasmid for mouse Dickkopf-1 (Dkk-

1) was a kind gift from Drs C. Niehrs and M. Semenov, and was

reported previously (Tamai et al. 2000; Wu et al. 2000; Mao et al.2001). The expression plasmid for constitutively active b-catenin

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 1588–1598

Regulation of PC12 cell proliferation by Wnt1 and Wnt7a | 1589

was provided by Dr R. T. Moon (University of Washington, School

of Medicine, Seattle, WA, USA).

Transient transfectionTransient transfections of PC12 cells were carried out in triplicate

employing LipofectAMINE 2000 (Invitrogen) according to the

manufacturer’s instructions. In brief, PC12 cells were plated 1 day

before transfection and then incubated for 4 h with appropriate

amount of DNA-lipofectAMINE 2000 complexes in Opti-Mem I

serum-free medium. Four hours later, cells were added with half

volume of DMEM supplemented with 4 mmol/L glutamine, 200 U/

mL penicillin, 200 lg/mL streptomycin, and 20% FBS.

RT-PCR analysisTotal RNA was extracted from the cultures with Trizol reagent

(Invitrogen, Milan, Italy) and subjected to DnaseI treatment

(Promega, Italy) according to manufacturer’s instructions. Total

RNA (1 lg) was then employed for cDNA synthesis, using ImProm-

II reverse transcriptase (Promega) and random examers primers

according to manufacturer’s instructions. The RT product was

diluted to 100 lL with sterile, distilled water and 1 lL of cDNAwas

employed for amplification. The following primers were used:

Human Wnt7a: For, 5¢-TTCACCTACGCCATCATTGC-3¢ (621–640 of human Wnt7a) and Rev, 5¢-TTACAGTTGCACTGCCA-CAC-3¢ (1285–1266 of human Wnt7a); Rat Wnt7a: For, 5¢-GCCTGGGCCACCTCTTTC-3¢ (279–296 of rat Wnt7a) and Rev,

5¢-GCCACAACCGAAGAGAAGTCA-3¢ (348–328 of rat Wnt7a);

Mouse/rat Wnt1: For, 5¢-TCCTCCACGAACCTGCTGACA-3¢(445–464 of mouse Wnt1 and 450–470 of rat Wnt1) and Rev 5¢-GTCGCAGGTGCAGGATTCGAT-3¢ (783–763 of mouse Wnt1 and

788–768 of rat Wnt1); and Rat b-actin: For, 5¢-TGAACCC-TAAGGCCAACCGTG-3¢ and Rev, 5¢-GCTCATAGCTCTTCTC-CAGGG-3¢.

The primers used for the amplification of b-actin (Roelen et al.1994) span an intron and yield products of different size depending

on whether cDNA or genomic DNA is used as a template (400 and

600 bp, respectively).

Assessment of [3H]thymidine incorporation

PC12 cells were seeded in 96-well plates at a density of 50 000/

well. A total of 0.4 lg of DNA encoding for Wnt1, Wnt7a, Dkk-1,

or b-catenin was transfected into each well as described above.

Thirty-two hours later, the medium was replaced with fresh DMEM

supplemented 2 mmol/L glutamine, 200 U/mL penicillin, 100 lg/mL streptomycin, and 0.5% or 10% FBS; [3H]thymidine (1lCi/well) was added, and the incubation was continued for 16 h. At the

end of the incubation, cells were washed with phosphate-buffered

saline and incubated for 20 min with 5% trichloroacetic acid. The

precipitates were solubilized with 1 mol/L NaOH, and radioactivity

was counted by scintillation spectrometry. When needed, lithium

ions (added as LiCl) or bisindolylmaleimide were added since the

time of cell transfection and maintained in the medium for 48 h.

Fluorescent chromatin stainingApoptotic death was assessed by fluorescent chromatin staining with

Hoechst 33258, as described previously (Copani et al. 1995).

Apoptotic cells were considered as those showing chromatin

fragmentation or nuclear pyknosis.

Luciferase reporter assayTransfections and reporter assay were carried out essentially as

described (Caricasole et al. 2003b). PC12 cells were plated in 96-wellculture plates at a density of 90 000/well. A total of 0.65 lg of DNAwas transfected into each well, including luciferase reporter plasmid

(150 ng), expression constructs (200 ng), a carrier plasmid DNA

(pGEM4Z, to 650 ng; Promega) as appropriate, and the Renillaluciferase CMV-driven internal reporter (100 ng; Promega) for

normalization, in 100 lL of Opti-Mem Medium (Invitrogen) lacking

fetal calf serum. The luciferase reporter plasmid is the p4TCF,

comprising four copies of a TCF-responsive element upstream of a

TATA element luciferase coding sequence transcriptional unit (Bettini

et al. 2002). The luciferase nuclear factor of activated T cells (NFAT)

reporter plasmid is a commercial vector (pNFAT-TA-Luc, Vector

Mercury pathway profiling systems; Clontech, Milan, Itlay). Luci-

ferase activity wasmeasured at different times from transfection using

the Promega luciferase assay reagent and read using a Berthold

LUMAT LB3907 tube luminometer (Bad Wildbad, Germany).

Western blotWestern blot analysis was performed as described previously

(Iacovelli et al. 2002). For the analysis of b-tubulin and choline

acetyltransferase (ChAT), PC12 cells were lysed in Triton X lysis

buffer (10 mmol/L Tris–HCl, pH 7.4, 150 mmol/L NaCl, 1% Triton

X-100, 1 mmol/L EDTA, 10% glycerol, 1 mmol/L phenylmethyl-

sulfonyl fluoride, 10 lg/mL leupeptin, 10 lg/mL aprotinin, 1 mmol/

L sodium o-vanadate, 50 mmol/L sodium fluoride, and 10 mmol/L

b-glycerophosphate) for 15 min. Total cell lysates were clarified by

centrifugation (10 000 g for 10 min). For the analysis of b-catenine,cyclin D1, and cylin E, cell nuclear fractions were isolated by

differential centrifugations using a commercially available kit (Pierce,

Rockford, IL, USA) (Calderone et al. 2003). Protein cell lysates

(80 lg) were separated by sodium dodecyl sulfate–polyacrylamide

gel electrophoresis, blotted onto nitrocellulose membranes, and

probed anti-ChAT (1 : 250; Chemicon, Milan, Italy), monoclonal

antineuronal class III b-tubulin (Tuj1, 1 : 500; Covance, Berkeley,

CA, USA), polyclonal anti-b-catenin antibody (1 : 500; Cell Signal-

ing technology, Milan, Italy), monoclonal cyclin D1 (1 : 250; Santa

Cruz Biotechnology, Milan, Italy), polyclonal cyclin E (1 : 250;

Santa Cruz Biotechnology), and monoclonal anti-b-actin (1 : 1000;

SigmaAldrich) antibodies. Immunoreactive bands were visualized by

enhanced chemiluminescence (Amersham Biosciences, Milan, Italy),

using horseradish peroxidase-linked secondary antibodies.

Results

Wnt1 and Wnt7a differentially regulate proliferation ofPC12 cellsWe examined the regulation of cell proliferation by Wnt inundifferentiated PC12 cells, a model in which we havecharacterized the molecular determinants of the ‘canonical’pathway activated by Wnt7a (Caricasole et al. 2003a).Undifferentiated PC12 cells express FZD2, FZD5, FZD7,and LRP6 Wnt co-receptors, but lack LRP5 (Caricasole et al.2003a). We transfected PC12 cells with expression vectors

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 1588–1598� 2007 The Authors

1590 | P. Spinsanti et al.

encoding for mouse Wnt1 or human Wnt7a. Expression ofmouse Wnt1 and human Wnt7a after transfection wasconfirmed by RT-PCR (Fig. 1). Undifferentiated PC12 cellsare known to endogenously express Wnt3, Wnt3a, Wnt4,Wnt5a, Wnt6, Wnt10a, and Wnt11, but not Wnt1 and Wnt7a(Erdreich-Epstein and Shackleford (1998). We confirmed thelack of endogenous expression of Wnt1 and Wnt7a by usingthe same primers used for the detection of mouse Wnt1(which also detect rat Wnt1; see ‘control vector’ in Fig. 1),and specific primers for rat Wnt7a (not shown).

Transfections were carried out in serum-free medium, and10% serum was added 4 h later to allow the expression oftransfected proteins. The luciferase assay (see below)suggested that expression of both Wnt1 and Wnt7a wasmaximal 24 h after transfection and declined afterwards; incontrast, 48 h was an optimal time for the detection of

[3H]thymidine incorporation. The following conditions wereconsidered optimal for our experiments: 4 h of transfection inserum-free medium, followed by 28 h of incubation inmedium containing 10% serum, and additional 16 h ofincubation in fresh medium containing 0.5% serum and[3H]thymidine. Under these conditions, ectopically expressedWnt1 and Wnt7a had opposite effects on [3H]thymidineincorporation. Expression of Wnt1 increased [3H]thymidineincorporation, in agreement with previous findings (Issackand Ziff 1998; Chou et al. 2000), whereas expression ofWnt7a largely reduced [3H]thymidine incorporation. Thesame trend was observed when the whole incubation (44 h)after transfection was carried out in medium containing 10%serum (Table 1). We also examined the expression of themid-late G1 phase markers, cyclin D1 and cyclin E, 20 hafter cell transfection. Transfection was carried out after 24 hof cell starvation in serum-free medium. Under theseconditions, expression of Wnt1 and Wnt7a increased andreduced nuclear levels of cyclin D1 and cyclin E, respectively(Fig. 2a and b). Reduction of cell proliferation by Wnt7a wasnot an epiphenomenon of cell death and was not associatedwith cell differentiation. Accordingly, no difference in the

Table 1 [3H]thymidine incorporation in PC12 cells transfected with a

control vector or with a vector encoding for Wnt1 or Wnt7a (0.4 lg/

0.2 mL) and incubated with 0.5% or 10% FBS during the last 16 h of

incubation (see Results section)

cpm/well

0.5% FBS (%) 10% FBS (%)

Control vector 38 072 ± 3134 (100) 24 655 ± 2365 (100)

Wnt1 52 800 ± 4087 (139)* 29 266 ± 4085 (119)

Wnt7a 22 063 ± 1918 (58)* 15 455 ± 2232 (63)*

Values are means ± SEM of six determinations from two independent

experiments. *p < 0.05 (one-way ANOVA + Fisher’s PLSD) versus the

respective values obtained with the control vector. FBS, fetal bovine

serum.

(a)

(b)

(c)

Fig. 1 RT-PCR analysis of Wnt1 (a) and Wnt7a (b) mRNA in PC12

cells transfected with a control vector, or a vector encoding for Wnt1 or

Wnt7a (1 lg/0.5 mL) for 24 h. Note the absence of the two transcripts

in cells transfected with the control vector. b-Actin mRNA was de-

tected in (c) and appears as a single amplification product at 400 bp.

This excludes contamination by genomic DNA (see Materials and

methods).

(a)

(b)

Fig. 2 Levels of cyclin D1 (a) and cyclin E

(b) in nuclear extracts from PC12 cells

expressing either Wnt7a or Wnt1. Cultures

were transfected with plasmids encoding for

Wnt7a or Wnt1 and assessed for the

expression of cyclins 20 h later. Densito-

metric values are means ± SEM and were

calculated from three independent experi-

ments. *p < 0.05 (one-way ANOVA + Fisher’s

PLSD) versus the respective controls. Pro-

tein values in nuclear extracts were nor-

malized by nuclear b-actin (Cappuccio et al.

2005).

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Regulation of PC12 cell proliferation by Wnt1 and Wnt7a | 1591

number of apoptotic cells (defined as cells bearing chromatinfragmentation or nuclear piknosis after staining with Hoechst33258) was found between control cultures and culturesexpressing Wnt1 or Wnt7a (Table 2). In addition, no changesin cell morphology, b-tubulin expression or ChAT expressionwere found in Wnt7a-expressing cells after 48 h (Fig. 3a andb). No changes in cell morphology were also detected in cellsexpressing Wnt1 (Fig. 3a). Induction of a neuronal pheno-type associated with an increased expression of b-tubulin andChAT were instead observed following a 48-h exposure to10 ng/mL of nerve growth factor, as expected (Fig. 3a and b).

Wnt1 and Wnt7a differ in their ability to activate thecanonical pathway and the calcium pathway in PC12 cellsWe therefore examined whether Wnt1 and Wnt7a differed fortheir ability to stimulate the two main Wnt signalingpathways, i.e. the ‘canonical’ b-catenin/TCF/LEF pathway,and the ‘calcium’ pathway. We assessed (i) the activation ofthe canonical pathway by co-transfecting the cells with areporter gene (luciferase) controlled by b-catenin/TCF/LEF-responsive elements and (ii) the activation of the calciumpathway by using the reporter gene controlled by NFAT-responsive elements. Dephosphorylation of NFAT by calci-neurin (a calcium-dependent protein phosphatase) allowsnuclear translocation and transcriptional activity of NFAT(Loh et al. 1996; Rao et al. 1997). Cells were transfectedwith different amounts of constructs encoding for Wnt1 orWnt7a (0.05, 0.1, or 0.2 lg/well). Expression of both Wntisoforms activated the canonical pathway (Fig. 4a). Wnt7awas apparently more efficacious than Wnt1, although anycomparison between cells transfected with different con-structs is elusive. Interestingly, however, the ratio of efficacybetween Wnt7a and Wnt1 on the stimulation of the calciumpathway in sister cultures was inverted. Wnt7a induced onlya modest activation of the calcium pathway (about twofoldincrease in luciferase expression), whereas Wnt1 induced alarger activation (almost fourfold increase in luciferaseexpression) (Fig. 4b). The ratio between maximal stimulationof the canonical pathway and maximal stimulation of thecalcium pathway (calculated as percentage increase ofluciferase expression above controls) was 2.78 for Wnt7aand 0.61 for Wnt1. We extended the analysis to the

expression of b-catenin in nuclear extracts of PC12 cellsexpressing either Wnt7a or Wnt1 for 24 or 48 h. At bothtimes, Wnt7a expression induced a greater increase of nuclearb-catenin levels than Wnt1 (Fig. 4c), which confirms thepreferential activation of the canonical pathway by Wnt7a.

Involvement of the calcium/protein kinase C and thecanonical pathways in the effect of Wnt1 and Wnt7a onPC12 cell proliferationWe next examined whether the ‘preferential’ activation of thecanonical or the calcium pathway could account for the

Table 2 Hoechst chromatin staining in PC12 cells transfected with a

control vector or with a vector encoding for Wnt1 or Wnt7a (1 lg/

0.5 mL) for 48 h

Percentage (%) of cells bearing chromatin

fragmentation or nuclear picknosis

Control vector 15.4 ± 0.7

Wnt1 14 ± 1.2

Wnt7a 16 ± 1.4

Values are means ± SEM of six determinations.

(a)

(b)

Fig. 3 Wnt7a fails to induce differentiation of PC12 cells. Phase-

contrast images of PC12 cells transfected with a control plasmid

(control vector), transfected with plasmids encoding for Wnt7a or Wnt-

1, or treated with 10 ng/mL nerve growth factor (NGF) are shown in

(a). Cells were examined 48 h after transfection or NGF application.

Note the presence of neuritis in cultures treated with NGF and the

absence in control cultures or in cultures expressing Wnt7a or Wnt-1.

Objective = 20·. Immunoblot analysis of the neuronal marker, b-

tubulin, and the cholinergic marker, ChAT, is shown in (b). The blot is

representative of two independent experiments with identical results.

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1592 | P. Spinsanti et al.

different regulation of cell proliferation by Wnt7a and Wnt1.First, we co-transfected PC12 cells with a vector encodingfor the Wnt antagonist, Dkk-1, which, as expected, reducedthe activation of the canonical pathway by both Wnt7a andWnt1 (Fig. 5a). In cells expressing Dkk-1, Wnt7a was lessefficacious in reducing [3H]thymidine incorporation, whereasthe stimulating action of Wnt1 on [3H]thymidine incorpora-tion was unaffected (Fig. 5b). We also challenged thecultures with lithium ions (10 mmol/L, added as LiCl),which mimic the activation of the canonical Wnt pathway byinhibiting GSK3b (Hedgepeth et al. 1997; van Noort et al.2002). Lithium alone reduced [3H]thymidine incorporation,and occluded the inhibitory action of Wnt7a. In contrast,Wnt1 was still able to enhance [3H]thymidine incorporationin the presence of lithium, although to a lesser extent than incontrol cultures (Fig. 6a). We carried out experiments inthe presence of the protein kinase C (PKC) inhibitor,bisindolylmaleimide (1 lmol/L). This drug slightly reducedbasal [3H]thymidine incorporation (by about 10–15%), butlargely attenuated the stimulation of [3H]thymidine incorpo-ration induced by Wnt1 (Fig. 6a). In contrast, the loweringactivity of Wnt7a was unaffected, with Wnt7a and bis-indolylmaleimide showing additive effects on [3H]thymidineincorporation (Fig. 6a). Finally, we transfected PC12 cellswith cDNA encoding for a constitutively active form ofb-catenin. Constitutively active b-catenin strongly activated

the canonical pathway, as shown by a large increase in TCF/LEF luciferase expression (Fig. 6b), and mimicked the actionof Wnt7a in reducing [3H]thymidine incorporation in PC12cells (Fig. 6c).

DiscussionWe have shown that Wnt1 and Wnt7a, two Wnt isoforms thatare widely involved in CNS development (Dickinson et al.1994; Ikeya et al. 1997; Megason and McMahon 2002;Hirabayashi et al. 2004; Schuller and Rowitch 2006),differentially affect proliferation of PC12 cells. We usedtwo methods to assess cell proliferation: (i) measurements of[3H]thymidine incorporation in cells transfected with Wnt7aor Wnt1 without previous starvation and (ii) assessment ofnuclear cyclin D1 and cyclin E levels in cells starved for 24 hprior to transfection, and then synchronized to proliferate byaddition of 10% serum after transfection. This was necessarybecause levels of cyclin D1 and cyclin E are known toincrease during mid-late G1 phase, and progressively declinein the following phases of the cell cycle (Arellano andMoreno 1997; Dynlacht 1997). We found that expression ofWnt1 increased, whereas expression of Wnt7a reduced cellproliferation. A proliferative action of Wnt1 has already beenreported in PC12 cells (Issack and Ziff 1998; Chou et al.2000), whereas the antiproliferative action of Wnt7a is novel.Interestingly, Wnt1 and Wnt7a differed in their ability to

(a) (b)

(c)

24 h0

10

20 *Control vectorWnt7aWnt1

*#

#

*

*

48 h

Fig. 4 Activation of the canonical pathway or the calcium pathway by

different amounts of plasmids encoding for Wnt1 and Wnt7a (0.05–

0.2 lg/0.2 mL) in PC12 cells. In (a) cells were co-transfected with a

reporter gene (luciferase) controlled by TCF/LEF-responsive ele-

ments. In (b) the reporter gene is controlled by NFAT-responsive

elements. Luciferase activity was assessed 24 h after cell transfection

and expressed as relative luminescence units (RLU) after normaliza-

tion with the internal standard (Renilla). Values are means ± SEM of

six determinations from two independent experiments. *p < 0.05 (one-

way ANOVA + Fisher’s PLSD) versus the respective controls (TCF

alone or NFAT alone). Immunoblot analysis of ß-catenin in cell nuclear

extracts is shown in (c). Analysis was carried out both at 24 and 48

after transfection. Densitometric values are means ± SEM from four

independent experiments. p < 0.05 (one-way ANOVA + Fisher’s PLSD)

versus the respective control values (control vector) (*) or versus the

respective Wnt7a values (#).

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Regulation of PC12 cell proliferation by Wnt1 and Wnt7a | 1593

activate the canonical Wnt pathway and the calcium pathwayin PC12 cells. In parallel experiments, Wnt1 was moreeffective than Wnt7a in increasing the calcium/PKC pathway(as detected by NFAT-mediated gene expression), but lesseffective in activating the canonical b-catenin/TCF-LEFpathway. Calcium has been implicated in at least threeb-catenin-independent Wnt pathways: the calcium/calmodu-lin-dependent kinase-II/PKC pathway, the G-protein/phos-pholipase C pathway, and the Rho/Jun-N-terminal kinase(JNK) pathway (Kohn and Moon 2005). The first twopathways converge into PKC activation (Kohn and Moon2005). Wnt1 fails to activate JNK in PC12 cells (Chou andHoward 2002). Pharmacological inhibition of PKC abro-gated the proliferative action of Wnt1, but did not affect theantiproliferative action of Wnt7a in PC12 cells. In contrast,the action of Wnt7a was occluded by lithium and wassensitive to the Wnt inhibitor, Dkk-1. Lithium mimics theactivation of the canonical Wnt pathway by inhibitingGSK3b (Hedgepeth et al. 1997; van Noort et al. 2002),although it produces pleiotropic effects in excitable cells.Dkk-1 is a secreted protein that interacts with LRP5/6 and anadditional receptor named Kremen, induces a rapid endocy-tosis of LRP5/6, and therefore inhibits the canonical Wntpathway (Bafico et al. 2001; Mao et al. 2001; Semenovet al. 2001; Kawano and Kypta 2003). More remarkably, aconstitutively active form of b-catenin, which activated TCF/LEF-dependent gene expression, mimicked the action ofWnt7a in inhibiting cell proliferation. Thus, our data suggestthat Wnt1 enhances PC12 cell proliferation by preferentiallyactivating one of the two calcium/PKC pathways, whereasWnt7a reduces PC12 cell proliferation by preferentiallyactivating the canonical b-catenin-dependent pathway. Thebalance between activation of these pathways might becritical for the regulation cell proliferation in PC12 cells.Accordingly, the proliferative action of Wnt1 was largely

Fig. 5 Dkk-1 attenuates the antiproliferative activity of Wnt7a in PC12

cells. Inhibition of the canonical Wnt pathway (assessed by co-trans-

fecting the cells as in Fig. 2a) is shown in (a), where data are mean-

s ± SEM of six to nine values from two to three independent

experiments. p < 0.05 versus TCF alone (*) or versus the respective

value without Dkk-1 (i.e. Wnt1 or Wnt7a alone) (#) (one-way

ANOVA + Fisher’s PLSD). Cells were transfected with 0.2 lg/0.2 mL of

plasmids encoding for Wnt1, Wnt7a, or Dkk-1. Modulation of [3H]thy-

midine incorporation by Wnt1 or Wnt7a in the absence or presence of

Dkk-1 is shown in (b), where data are means ± SEM of 9–12 deter-

minations from three to four independent experiments. p < 0.05 ver-

sus control vector (*) or versus the respective value without Dkk-1 (i.e.

Wnt1 or Wnt7a alone) (#) (one-way ANOVA + Fisher’s PLSD). Cells

were transfected with 0.4 lg/0.2 mL of plasmids encoding for Wnt1,

Wnt7a, or Dkk-1.

Fig. 6 Modulation of [3H]thymidine incorporation by Wnt1 and Wnt7a

in PC12 cells treated with the PKC inhibitor, bisindolylmaleimide (Bis,

1 lmol/L), or with lithium ions (Li, 10 mmol/L, added as LiCl) is shown

in (a). Bis and lithium ions were added at the time of cell transfection

and maintained in the medium for 48 h. Values are means ± SEM of

nine determinations from three independent experiments. p < 0.05

versus the respective control vector (*) or versus the respective values

obtained in the absence of Bis or Li (#) (one-way ANOVA + Fisher’s

PLSD). Cells were transfected with 0.4 lg/0.2 mL of plasmids

encoding for Wnt1 or Wnt7a. Activation of TCF/LEF-dependent gene

expression in cultures expressing a constitutively active form of b-

catenin (c.a. ß-catenin) or Wnt7a is shown in (b). Data of [3H]thymidine

incorporation are shown in (c). Values are means ± SEM of four to five

determinations. *p < 0.05 (one-way ANOVA + Fisher’s PLSD) versus

control values (control vector).

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1594 | P. Spinsanti et al.

reduced, albeit still present, when the antiproliferativecomponent mediated by the canonical pathway was amplifiedby lithium.

Our findings have a number of potential implicationswhich derive from the nature of PC12 cells. These are tumorcells that originate from the neural crest and show the abilityto differentiate in neurons in response to nerve growth factor(Levi et al. 1988; Vaudry et al. 2002). Activation of the cellcycle in neurons contributes to neurodegeneration in avariety of CNS disorders, including Alzheimer’s disease (seeIntroduction and references therein). It has been hypothe-sized that b-amyloid enables a potential mitogenic activity ofWnt mediated by the activation of the canonical pathway indegenerating neurons (Caricasole et al. 2003a). If our data onPC12 cells are predictive of what may occur in differentiatedneurons, then we expect that Wnt is involved in theactivation of an unscheduled mitotic cycle, but this maycritically depend on the Wnt isoforms, and may be mediatedby the calcium/PKC pathway. This can be easily investigatedin cultured neurons exposed to b-amyloid or other insults incombination with various Wnt isoforms.

A second implication is in oncology because a deregula-tion of Wnt signaling is associated with different types oftumors (Fukuchi et al. 1998; de La Coste et al. 1998; Rimmet al. 1999). The evidence that b-catenin controls theexpression of proto-oncogenes and cell cycle proteins, suchas c-myc and cyclin D1 (Tetsu and McCormick 1999; Barkeret al. 2000), supports the general belief that activation of thecanonical pathway promotes tumor cell proliferation.Nuclear b-catenin accumulation is frequently observed intumor cells, and mutations of proteins that lie along thecanonical Wnt pathway (e.g. adenomatous polyposis coli,axin, or b-catenin) are associated with different types oftumors, including colon carcinoma, medulloblastoma, hepa-tocellular carcinoma, and endometrial adenocarcinoma(Miyaki et al. 1990; Zurawel et al. 1998; Huang et al.2000; Dahmen et al. 2001; Darnell 2002; Kikuchi 2003; Leet al. 2004; Hendrix et al. 2006). However, recent evidencesuggests that the role of Wnt in tumor growth may not beunivocal. Mutant mice lacking Wnt5a develop lymphoidmalignancies (Liang et al. 2003), suggesting that Wnt5a actsas a tumor suppressor. In addition, Wnt7a inhibits trans-formed growth in non-small cell lung cancer cells (Winnet al. 2005, 2006). The latter effect does not involve thecanonical pathway, but is mediated by the activation of JNK(Winn et al. 2005) or type-5 extracellular regulated kinase(Winn et al. 2006). Our data challenge the view thatactivation of the canonical pathway invariably support tumorcell proliferation because the antiproliferative action ofWnt7a in PC12 cells is apparently mediated by canonicalpathway, although we did not extend the analysis to JNK ortype-5 extracellular regulated kinase. The hypothesis thatdifferent Wnt isoforms produce differential effects on tumorgrowth and that the balance between the calcium/PKC

pathway and the canonical pathway is critical for theregulation of cell proliferation is attractive and warrantsfurther investigation.

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