The p53 transcriptional target gene wnt7b contributes to NGF-inducible neurite outgrowth in neuronal PC12 cells

Christopher Brynczka1,2,3 and B. Alex Merrick1,*

1National Center for Toxicogenomics, National Institute of Environmental Health Sciences, NationalInstitutes of Health, Research Triangle Park, North Carolina 27709

2Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NorthCarolina 27606

AbstractDifferentiation of PC12 cells by nerve growth factor (NGF) is characterized by changes in signaltransduction pathways leading to growth arrest and neurite extension. The transcription factor p53,involved in regulating cell cycle and apoptosis, is also activated during PC12 differentiation andcontributes to each of these processes but the mechanisms are incompletely understood. NGFsignaling stabilizes p53 protein expression which enables its transcriptional regulation of targetgenes, including the newly identified target, wnt7b, a member of the wnt family of secretedmorphogens. We tested the hypothesis that wnt7b expression is a factor in NGF-dependent neuriteoutgrowth of differentiating PC12 cells. Wnt7b transcript and protein levels are increased followingNGF treatment in a p53-dependent manner, as demonstrated by a reduction in wnt7b protein levelsfollowing stable shRNA-mediated silencing of p53. In addition, overexpressed human tp53 wascapable of inducing marked wnt7b expression in neuronal PC12 cells but tp53 overexpression didnot elevate wnt7b levels in several tested human tumor cell lines. Ectopic wnt7b overexpression wassufficient to rescue neurite outgrowth in NGF-treated p53-silenced PC12 cells which could beblocked by JNK inhibition with SP600125 and did not involve β-catenin nuclear translocation.Addition of sFRP1 to differentiation medium inhibited wnt7b-dependent phosphorylation of JNK,demonstrating that wnt7b is secreted and signals through a JNK-dependent mechanism in PC12 cells.We further identify an NGF-inducible subset of wnt receptors that likely supports wnt7b-mediatedneurite extension in PC12 cells. In conclusion, wnt7b is a novel p53-regulated neuritogenic factor inPC12 cells that in conjunction with NGF-regulated Fzd expression is involved in p53-dependentneurite outgrowth through noncanonical JNK signaling.

Keywordswnt7b; wnt; p53; PC12; NGF; neurite; JNK; Fzd

IntroductionThe wnt family of secreted lipid-modified signaling proteins are involved in a spectrum ofdevelopmental processes and are homologous to the Drosophila wingless and mouse int-1developmental control genes (Logan and Nusse, 2004). Within the nervous system, wnt family

*Address correspondence to: B. Alex Merrick, Ph.D., National Institutes of Environmental Health Sciences, P.O. Box 12233, ResearchTriangle Park, NC 27709, Tel. 919-541-1531; Fax. 919-541-4704; E-Mail: merrick@niehs.nih.gov3Present address: Massachusetts General Hospital and Harvard Medical School, Center for Cancer Research, 55 Fruit Street, Boston,MA 02114

NIH Public AccessAuthor ManuscriptDifferentiation. Author manuscript; available in PMC 2008 October 7.

Published in final edited form as:Differentiation. 2008 September ; 76(7): 795–808. doi:10.1111/j.1432-0436.2007.00261.x.

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signaling has been implicated in neuronal development (Ille and Sommer, 2005), including thedifferentiation of neural progenitor cells (Hirabayashi et al., 2004), dendritic development(Ciani and Salinas, 2005) and adult cellular homeostasis including neurogenesis (Lie et al.,2005). Germline loss of wnt1 results in profound central nervous system developmentaldefects, particularly in the midbrain and cerebellum (McMahon and Bradley, 1990; Thomasand Capecchi, 1990). Dysregulated wnt signaling has also been associated with cellulartransformation and tumorigenesis (Nusse, 2005). Individual members of the wnt gene familyhave been partially characterized based on their ability to induce in vitro cellular transformation(Wong et al., 1994) but recent evidence suggests a more complicated picture of wnt signalingbased on cell type (Yi et al., 2005) and receptor availability (Mikels and Nusse, 2006). Thesimplified model of wnt signaling involves wnt ligand-mediated activation of frizzled receptor(Fzd) and LDL related protein 5/6 receptor (Lrp5/6) heterodimers at the plasma membranesurface, which propagate an internal signaling cascade either resulting in β-catenin stabilizationand subsequent activation of the Tcf/Lef transcriptional complex (canonical model) oralternately involves the less-defined Ca2+- or c-Jun N-terminal kinase (JNK)-dependentnoncanonical signaling paradigm (Gordon and Nusse, 2006).

PC12 cells have been extensively studied as an in vitro model of nerve growth factor (NGF)-induced neuronal differentiation (Fujita et al., 1989), a process which involves tumorsuppressor p53 (tp53) activity for both cell cycle arrest and more recently, neurite extension(Hughes et al., 2000; Fabian et al., 2006; Brynczka et al., 2007; Poluha et al., 1997; Zhang etal., 2006). In order to understand the specific mechanism of p53 function within NGF-induceddifferentiation, our lab has recently identified a number of p53 transcriptional targets indifferentiating PC12 cells through a chromatin immunoprecipitation cloning strategy, in whichwe identified NGF-regulated p53 binding to the wnt7b locus (Brynczka et al., 2007). Previousreports have suggested that wnt7b is transcriptionally regulated during hindbrain developmentby Pax6 in mice (Takahashi et al., 2002) and by the TTF-1, GATA6, and Foxa2 transcriptionfactors in cultured lung epithelium from binding sites within the 5′ promoter region(Weidenfeld et al., 2002). In addition to these factors, we identified a region within the firstwnt7b intron, approximately 2 kB downstream from the transcriptional start site which wasoccupied by p53 in a NGF-dependent manner during PC12 cell differentiation.

Wnt7b-mediated signaling has been described as acting through both canonical (Wong et al.,1994; Wang et al., 2005) and noncanonical signaling pathways (Rosso et al., 2005) dependingupon both cell type and receptor availability, without inducing cellular transformation (Wonget al., 1994; Naylor et al., 2000). The developmental role of wnt7b has been partially describedthrough the generation of null mouse models. Wnt7b-/- mice are nonviable and die shortlyfollowing parturition due to extensive lung malformation and hemorrhage due to defects inmesenchyme proliferation (Shu et al., 2002) and are also deficient in chorion/allantois fusionduring placental development (Parr et al., 2001). In hippocampal neurons, wnt7b stimulatesdendritic development through noncanonical signaling (Rosso et al., 2005). Neurite outgrowthin differentiating PC12 cells is a p53-dependent process (Di Giovanni et al., 2006; Bacsi et al.,2005) and the aberration of p53 transcriptional activity leads to a generalized lack of neuriteextensions (Di Giovanni et al., 2006; Fabian et al., 2006). In the p53-silenced PC12 cell, botha generalized lack of neurite extension upon treatment with NGF and a decrease in expressionof the p53 target gene wnt7b have been described (Brynczka et al., 2007). These resultssuggested that wnt7b may be involved in a signaling mechanism through which p53-regulatedneurite outgrowth occurs.

NGF-dependent expression and activity of the wnt7b protein has not previously been describedwithin the wild-type PC12 cell (Erdreich-Epstein and Shackleford, 1998). We hypothesizedthat wnt7b function may be related to p53-dependent neurite outgrowth during PC12differentiation and aimed to characterize the expression and function of wnt7b within these

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cells. We now report that wnt7b is a p53-inducible target gene that is regulated in a time-dependent manner within PC12 cells upon NGF treatment. In combination with previouslypublished data (Brynczka et al., 2007), we collectively demonstrate that p53 regulates wnt7bat the promoter, RNA and protein level. We further identify that p53-regulated wnt7bexpression leads to NGF-induced neurite outgrowth through a noncanonical JNK-regulatedsignaling mechanism and define an NGF-inducible subset of wnt receptors through which thisprocess may occur.

MethodsCell culture

Rat PC12 cells (ATCC, Manassas, VA) were grown in RPMI 1640 medium supplemented with10% horse serum, 5% fetal calf serum, 4 mM L-glutamine and penicillin/streptomycinantibiotics (cell culture reagents from Invitrogen, Carlsbad, CA) in a humidified 37° Cincubator maintained at 5% CO2. PC12 cells which stably express anti-p53 shRNA (hereaftercalled p53sh#3 cells) have been described previously (Brynczka et al., 2007; Brynczka andMerrick, 2007) and were cultured as above. Briefly, p53sh#3 cells were generated by lentiviralinfection and stable selection of infected cells carrying the blasticidin positive selection marker.PC12 cells were plated on rat tail type I collagen (Sigma, St. Louis, MO) prior toexperimentation and were differentiated over indicated time intervals by the addition of 50 ng/mL NGF 2.5S (Chemicon, Temecula, CA) in RPMI 1640 medium supplemented with 1% horseserum and antibiotics. Kinase inhibitors targeting JNK (SP600125) and p38MAPK(SB202190) (Calbiochem, Darmstadt, Germany) were used at 1 μM and 25 μM, respectively.Recombinant sFRP1 (R&D Systems) was added directly to culture medium at a dose of 5 μg/mL for both control and wnt7b-expressing cells where indicated and incubated at 37° for 4hours prior to NGF addition.

The Homo sapiens normal lung fibroblast (IMR-90), fibrosarcoma (HT-1080) andosteosarcoma (Saos-2) cell lines were cultured in DMEM supplemented with 10% fetal calfserum, 4 mM L-glutamine and penicillin/streptomycin antibiotics in a humidified 37° Cincubator maintained at 5% CO2. All experiments were carried out using cells of less than 30passages.

TransfectionOverexpression studies were performed by transfection of either Rattus norvegicus (Rn)wnt7b cDNA (Genbank accession NM_001009695.1) cloned into the pDREAM 2.1 vector(Genscript, Piscataway, NJ) or Homo sapiens (Hs) tp53 cDNA (Genbank accessionNM_00546.3) cloned into the pCMV6-XL4 vector (Origene, Rockville, MD). Transfectionswere performed 24 hours prior to NGF treatment using either Lipofectamine 2000 (Invitrogen)or FuGene (Roche, Indianapolis, IN) reagents according to manufacturer’s recommendedprotocol.

Protein and RNA assaysqPCR was performed following total RNA isolation and on-column DNase treatment (Qiagen,Valencia, CA) from indicated sample types according to manufacturer’s protocol (Qiagen).RNA concentration was determined using a NanoDrop spectrophotometer (BioRad,Richmond, CA). 1.0 μg RNA from each sample was used for cDNA synthesis by theSuperScript II reverse transcriptase according to manufacturer’s instructions (Invitrogen).qPCR was carried out with 1/20th reaction volume of cDNA per sample, HotStart master mix(SuperArray, Frederick, MD) and 0.25 μM each primer (IDT, Coralville, ID) in a GeneAmp9700 PCR instrument (Applied Biosystems, Foster City, CA). PCR was carried out for anempirically identified cycle number in which each amplicon was measured within the linear

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phase of target amplification. Equivalent volumes of each PCR reaction were run on 2% TBEagarose gels containing ethidium bromide and photographed under UV illumination. RT-PCRwas performed on cDNA samples isolated as above using SYBR green-based detection(Applied Biosystems, Foster City, CA) in a GeneAmp 7900 real-time PCR instrument (AppliedBiosystems). Relative quantitation of gene expression was performed using the 2-ΔΔct methodand compared to GAPDH levels. Gene expression in the untreated cell was used as theendogenous control for indicated treatment groups as appropriate for individual cell types.Primer sequences were used as follows: Rn (Rattus norvegicus) wnt7b exon 1 forward 5′-CTGGGAGCCAACATCATCTG-3′, Rn wnt7b exon 1 reverse 5′-TGCCCAAAGACGGTCTTCTC-3′, Rn wnt7b exon 2 forward 5′-GAGGCTGCCTTCACATACG-3′, Rn wnt7b exon 2 reverse 5′-GCCTTCTGCCTGGTTGTAG-3′, Rn wnt7b exon 3 forward 5′-GTCGGGCTCATGTACTACC-3′, Rn wnt7b exon 3 reverse 5′-GTCCTCCTCGCAGTAGTTG-3′, Hs (Homo sapiens) wnt7b forward 5′-TATCCCAGAGAGCAAAGTG-3′, Hs wnt7b reverse 5′-TGTGTTAGTGCCGAGAATC-3′,Rn GAPDH forward 5′-ATCCCATCACCATCTTCCAG-3′, Rn GAPDH reverse 5′-CCTGCTTCACCACCTTCTTG-3′, Hs GAPDH forward 5′-GGACCTGACCTGCCGTCTAG-3′, Hs GAPDH reverse 5′-TAGCCCAGGATGCCCTTGAG-3′, Rn p53 forward 5′-CAGCCAAGTCTGTTATGTGC-3′,Rn p53 reverse 5′-GTCTTCCAGCGTGATGATG-3′, Hs p53 forward 5′-AATAGGTGTGCGTCAGAAG-3′, Hs p53 reverse 5′-CTTACATCTCCCAAACATCC-3′,Fzd1 forward 5′-CGCTCTTCGTCTATCTGTTC-3′, Fzd1 reverse 5′-GTAGTCTCCCCTTGTTTGC-3′, Fzd2 forward 5′-AGGGCACTAAGAAAGAAGG-3′,Fzd2 reverse 5′-TGTAGAGCACGGAGAAGAC-3′, Fzd3 forward 5′-CAGGCACAGTAGTTCTCATC-3′, Fzd3 reverse 5′-AGCAGTCACCACACATAGAG-3′,Fzd4 forward 5′-GTTGGAAAGGCTAATGGTC-3′, Fzd4 reverse 5′-AGTCATCTGCAGAATACCG-3′, Fzd5 forward 5′-CACAGCCACATTCACTATG-3′,Fzd5 reverse 5′-GTAGCGAGTTCAGGTTTTG-3′, Fzd6 forward 5′-CAAAGGTTCCACATCTCTG-3′, Fzd6 reverse 5′-GGTCGTCTCCAGTGTAGTG-3′, Fzd7forward 5′-GCTTTGTGTCTCTCTTTCG-3′, Fzd7 reverse 5′-CAGTTCTTTCCCTACCATG-3′, Fzd9 forward 5′-AGGTTTTGTGGCTCTCTTC-3′, Fzd9reverse 5′-AGGGGTCTGTCTTAGTCATG-3′, Fzd10 forward 5′-GGGAGGAGGTAAAAGAAGG-3′, Fzd10 reverse 5′-GTCCCAAACGAGTAGAACAC-3′,Lrp5 forward 5′-GAGCACGTGATTGAGTTTG-3′, Lrp5 reverse 5′-CTCGGTCCAGTAGATGTAGC-3′, Lrp6 forward 5′-TGCTATGTCCTTCACTGTTG-3′,Lrp6 reverse 5′-GCCTCGATTCTCACTAAGC-3′, Ror2 forward 5′-ACCTCTGTCTGCTTCATCC-3′, Ror2 reverse 5′-CCACCCTTGAATTACATACG-3′, Rykforward 5′-GAAAGGGTCACACTGAAAG-3′, Ryk reverse 5′-ATAGGAAGGAGGTTTCTGTG-3′.

Western blotting was performed on samples grown as above and treated as indicated. Proteinisolation, electrophoresis and transfer to nitrocellulose (Invitrogen) was performed as describedpreviously (McNeill-Blue et al., 2006). HRP-based detection was subsequently carried out witheither ECL (GEH Amersham, Piscataway, NJ) or SuperSignal (Pierce, Rockford, IL) reagentand used the following antibodies: wnt7b (Q-13, Santa Cruz Biotechnology, Santa Cruz, CA),wnt7a/b (H-40, Santa Cruz), wnt7a (K-15, Santa Cruz) actin (MAb1501R, Chemicon,Temecula, CA), phosphorylated cJun (9164, Cell Signaling, Danvers, MA), phosphorylatedJNK (9251, Cell Signaling), total JNK (9252, Cell Signaling), β-catenin (9581, Cell Signaling),donkey anti-rabbit IgG-HRP (GEH Amersham) and sheep anti-mouse IgG-HRP (GEHAmersham).

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MicroscopyIndirect immunofluorescence of wnt7b or β-catenin protein was performed after fixation ofcells with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA). Cells werepermeabilized with 0.4% Triton X-100 and incubated with either anti-wnt7b (Santa Cruz) oranti-β-catenin antibody (Cell Signaling, Danvers, MA). Protein localization was detected withgoat anti-rabbit Alexa 594-conjugated secondary antibody (Invitrogen). Samples weremounted using ProLong Gold reagent (Invitrogen) containing the nuclear counterstain DAPIand photographed using an Olympus IX70 inverted microscope (Olympus, Center Valley, PA)and appropriate filter sets. Image exposure time for each fluorophore was maintained acrossall samples for each experiment. Merging of images was performed using AxioVision software(Carl Zeiss, Oberkochen, Germany).

Morphologic analysis and image capture of PC12 and p53sh#3 cells treated as indicated wasperformed at 40x magnification using microscope and software as described above. Totalneurite outgrowth was measured in indicated samples by the scoring of neurites at least onecell diameter in length, with at least 100 cells counted per sample. Experiments for neuritenumber measurements were repeated at least twice and results from multiple experiments werecompiled for statistical analysis. Pairwise comparisons were performed between samples asindicated using Student’s t-test at significance level of p ≤ 0.05. Neurite length measurementswere performed by scoring neurite outgrowth relative to cell diameter of wnt7b transfectedcells and wild-type cells after 48 hours NGF treatment using same samples as above. Thesmallest recorded neurite length measurement was 0.5 cell diameters. Neurite length wasscored in at least 100 cells per sample and average lengths were compared using Student’s t-test with significance at p ≤ 0.05.

ResultsWnt7b is a p53-inducible target gene in NGF-treated PC12 cells

Wnt7b transcript levels were significantly elevated from basal (naïve) amounts within 3 daysof NGF exposure and were maintained at elevated levels for at least 7 days with continuedNGF stimulation (Figure 1A). Transcript levels were undetectably low in the mitotic cell aspreviously described (Erdreich-Epstein and Shackleford, 1998). Intracellular wnt7b proteinlevels were increased following NGF treatment within 8 hours (Figure 1B) and remained highlyelevated over the course of 7 days (Figure 1C). Intracellular wnt7b protein levels increasedrapidly with NGF treatment relative to mRNA levels, which suggested that wnt7b RNA wasefficiently translated within PC12 cells. Levels of wnt7b protein were similarly increasedrelative to total p53 protein levels upon NGF stimulation as previously reported (Brynczka etal., 2007; Brynczka and Merrick, 2007), where significant increases in p53 protein abovebaseline levels were also detected within 8 hours of NGF treatment.

Recent studies in our lab have characterized significantly elevated p53 occupancy of a bindingsite within the first wnt7b intron upon NGF treatment in PC12 cells with accompanied p53-dependent transcription of the wnt7b locus (Brynczka et al., 2007). We aimed to determinewhether wnt7b protein expression was also dependent upon p53 activity. Two constitutive anti-p53 shRNA-expressing PC12 cell lines were utilized in which p53 RNA and protein levelswere stably decreased relative to wild-type PC12 cells (Brynczka et al., 2007; Brynczka andMerrick, 2007) to explore the effect of reduced p53 protein levels on wnt7b expression. Asdescribed above, wnt7b protein was highly elevated within 24 hours of NGF treatment in wild-type cells compared to the untreated naïve cell. However, wnt7b levels following NGFtreatment in each p53-silenced cell line were significantly lower and approached the amountobserved in the untreated wild-type cell (Figure 2A). Silencing of p53 levels was more efficient

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in p53sh#3 than p53sh#2 cells (Brynczka et al., 2007), which was reflected in theproportionately graded reduction of wnt7b levels within each cell line.

Since mouse and human p53 appear to operate in an analogous functional manner despiteroughly 15% divergence in primary sequence (Luo et al., 2001), we aimed to determine whetherthe wnt7b gene could be also be effectively induced by Homo sapiens p53 within the Rattusnorvegicus PC12 and Homo sapiens Saos-2, HT1080 and IMR-90 cell lines. Constitutiveoverexpression of Hs tp53 cDNA in PC12 cells led to observable increases in p53 RNA levels,which could be distinguished from endogenous Rn p53 transcripts through primer selectivity(Figure 2B). Elevated Hs p53 led to significantly increased wnt7b transcript levels in theabsence of NGF treatment in PC12 cells, demonstrating that Hs p53 protein was capable ofinducing Rn wnt7b transcription. No changes in endogenous Rn p53 levels were observedfollowing Hs p53 overexpression in PC12 cells (Figure 2B), suggesting that increased wnt7bexpression was solely dependent upon exogenous Hs p53. Overexpression of Hs p53 cDNAin the human p53-null Saos-2 osteosarcoma cell line significantly increased tp53 RNA levelswithout observable changes in wnt7b levels (data not shown). Similarly, no changes inwnt7b levels were observed following Hs p53 overexpression in either the HT1080 or IMR-90cell lines (data not shown). Collectively, these data demonstrate that increased p53 proteinexpression was capable of initiating wnt7b transcription in PC12 neuronal cells.

Wnt7b is expressed as a 27 kDa protein in PC12 cellsThe wnt7b protein was identified as a single 27 kDa band via immunoblot (Figure 1C) withinrat PC12 cells. However, the calculated molecular weight of wnt7b derived from accessionNP_001009695 is 39.3 kDa which was observed by wnt7b immunoblot following cDNAoverexpression in COS monkey embryonal kidney cells (Burrus and McMahon, 1995). Wealso noted that a wnt7b variant with alternative first exon usage had been described within thedeveloping chick eye (Fokina and Frolova, 2006), a region in which p53 is highly expressed(Pokroy et al., 2002). Thus, we aimed to determine whether the observed lower molecularweight wnt7b protein was generated as a transcriptional variant regulated by p53 or as a full-length transcript post-translationally modified to generate the observed protein. Constitutiveexpression of full-length wnt7b cDNA lacking intronic sequence, and therefore lacking theidentified p53 binding site within the first intron (Brynczka et al., 2007), also produced a 27kDa product that we observed by immunoblotting (Figure 3A). Wnt7b protein expression wasincreased in mitotic cells transfected with pDREAM-wnt7b, while NGF treatment alone ledto elevated wnt7b levels that were further increased in transfected cells. No changes inmigration distance were detected between endogenous and ectopically-expressed wnt7b.

Using primers to selectively amplify each exon within wnt7b cDNA, it was determined thateach exon was represented in the expressed transcript (Figure 3B). Amplicon levels from eachexon were increased as expected upon NGF treatment but differences in intensity among exons1 through 3 were attributed to varying amplification efficiencies or PCR product sizes fromrelative differences in ethidium bromide signal output. Sequencing of a near full-length wnt7bcDNA amplicon determined no differences from the annotated full RNA sequence (accession#NM_001009695.1) for wnt7b when compared via pairwise alignment. Therefore, each of threeexons was represented in the wnt7b transcript upon NGF-induced differentiation of PC12 cellsdespite SDS-PAGE migration at 27 kDa in reducing conditions. The observation that neitherdecreased migration nor multiple bands were identified by immunoblot in transfected cellssuggested that wnt7b is post-translationally processed (i.e. proteolytic cleavage) in thedifferentiating PC12 cell.

Immunofluorescent measurement of wnt7b within mitotic and differentiating PC12 cellsdemonstrated nearly undetectable levels within mitotic cells, while transfection withpDREAM-wnt7b greatly increased wnt7b fluorescence (Figure 3C). In particular,

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overexpressed wnt7b was found to localize as dense foci either within or at the plasmamembrane surface of the mitotic PC12 cell. Following NGF treatment, wnt7b protein wasreadily detected and evenly distributed throughout the cytoplasmic compartment with localizednodal regions apparent upon transfection with pDREAM-wnt7b. Both primary antibody aloneand secondary (Alexa 594-conjugated) antibody alone did not produce backgroundfluorescence (data not shown). Because the antibody used was capable of detecting both wnt7aand 7b, we also analyzed wnt7a expression to verify the specificity of immunofluorescencemeasurements. No wnt7a immunoreactive bands were detected on immunoblot of PC12cellular lysate in either the mitotic or NGF-differentiated state (data not shown). Further, ourinability to detect wnt7a confirms previous reports describing a lack of wnt7a expression inwild-type PC12 cells (Erdreich-Epstein and Shackleford, 1998) and supports the antibodyselectivity for wnt7b detected in our immunofluorescence measurements.

Wnt7b recovers neurite outgrowth in p53-silenced cellsIn order to determine the physiological effect of wnt7b within PC12 cells, we studiedphenotypic changes upon overexpression in both the wild type and p53-silenced p53sh#3 cellline (Figure 4A). NGF treatment for 24 hours induced neurite extension in wild-type cells whileneurite formation was inhibited in p53sh#3 cells as previously reported (Brynczka et al.,2007) and as seen in both dominant negative (Di Giovanni et al., 2006; Eizenberg et al.,1996) and temperature-sensitive (Zhang et al., 2006) p53 expressing neuronal cells. Upontransfection with pDREAM-wnt7b, no observable changes in neurite growth were detected inmitotic cells. However, ectopic wnt7b overexpression was sufficient to recover neuriteoutgrowth in p53sh#3 cells in the presence of NGF (Figure 4A, results collated in Figure 6B).Significantly increased neurite length (Figure 4B) was also observed within 48 hours in wnt7boverexpressing cells concurrently treated with NGF as compared to control NGF-treated cells.

Wnt7b activates noncanonical JNK-dependent signalingBecause expression of wnt7b rescued neurite outgrowth in p53-silenced PC12 cells followingNGF treatment, we aimed to describe the signaling mechanism through which wnt7bcontributes to neuritogenesis. Canonical wnt signaling is known to induce the accumulationand nuclear localization of the Tcf/Lef transcriptional cofactor β-catenin (Akiyama, 2000).Subcellular localization and relative levels of β-catenin were visualized by indirectimmunofluorescence following NGF treatment and wnt7b overexpression in wild-type andp53sh#3 cells (Figure 5A). β-catenin was localized primarily at intercellular junctions aspreviously described (Perez-Moreno and Fuchs, 2006) in each sample group irrespective ofNGF stimulation, p53 silencing or wnt7b overexpression. Both primary antibody alone andsecondary (Alexa 594-conjugated) antibody alone did not produce fluorescence. Absoluteprotein levels of β-catenin were not stabilized following NGF treatment or wnt7boverexpression in comparison to naïve cells as determined via both immunofluorescence(Figure 5A) and immunoblotting (Figure 5C). Lack of nuclear β-catenin localization orstabilization following either NGF treatment alone or NGF treatment with concomitant wnt7boverexpression demonstrated that canonical wnt signaling was not activated by NGF or wnt7bwithin the tested time frame.

Wnt signaling may also proceed along a noncanonical pathway, propagated through cJun N-terminal kinase (JNK) activity and the subsequent post-translational phosphorylation ofdownstream molecular targets (Veeman et al., 2003). Wnt7b activation of noncanonicalsignaling was therefore studied as a putative mechanism through which neurite outgrowthcould be influenced by wnt7b. Phosphorylation of cJun, a direct downstream target of activatedJNK, was measured over time in wild-type PC12 cells following NGF treatment (Figure 5B).PC12 cells treated with NGF demonstrated a rapid spike in cJun phosphorylation at 1 hourrelative to those cultured in complete medium, with phosphorylation levels significantly

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decreased within 4 hours before becoming elevated again at 48 hours following NGF treatment.Notably, the time course of delayed cJun phosphorylation was similar to the observed increaseof wnt7b levels following NGF treatment (Figure 1C). In order to determine whether increasedJNK signaling activity was dependent upon wnt7b and whether wnt7b protein was secretedinto culture medium, we expressed wnt7b cDNA in PC12 cells concomitantly treated withNGF and recombinant secreted Frizzled-related protein 1 (sFRP1), an endogenous extracellularwnt7b antagonist (Rosso et al., 2005) (Figure 5C). NGF treatment alone for 24 hours resultedin observable phosphorylation of JNK, which could be increased further by overexpression ofwnt7b. JNK phosphorylation was nearly undetectable in NGF-treated p53sh#3 cells but wasmarked in cells concomitantly expressing wnt7b. Addition of sFRP1 directly to culture mediummarkedly reduced JNK phosphorylation in both wnt7b overexpressing and control wild-typeand p53sh#3 cells treated with NGF, demonstrating that both endogenous and ectopically-expressed wnt7b must be secreted in order to activate JNK phosphorylation. In addition, neitherexpression of wnt7b nor addition of sFRP1 to medium resulted in changes in β-catenin proteinlevels in either wild-type or p53sh#3 cells, demonstrating that the wnt7b signaling axis doesnot incorporate β-catenin stabilization.

In order to determine whether JNK activity contributed directly to neurite outgrowth, we treatedwnt7b-expressing wild-type and p53sh#3 cells with NGF and kinase inhibitors (Figure 6A).JNK and p38MAPK, an associated family member which is not recognized as a component ofwnt signaling and used as a negative control, were respectively inhibited with SP600125 andSB202190. Effects upon neuritogenesis following 24 hours NGF treatment, selective kinaseinhibition and wnt7b overexpression were scored in wild-type or p53sh#3 PC12 cells (Figure6A, compiled in 6B). Compared to high levels of neuritogenesis visible in both wild-type andp53sh#3 wnt7b-transfected cells treated with NGF (Figure 4A), neurite outgrowth andextension in both cell types were significantly attenuated following JNK inhibition (Figure 5A,5C). By comparison, p38MAPK inhibition did not significantly abrogate neurite outgrowth ineither cell type transfected with wnt7b. Attenuation of neurite outgrowth in wnt7b-expressingp53sh#3 cells following JNK, but not p38MAPK, inhibition demonstrated the functionalinvolvement of JNK signaling in the process of neurite outgrowth involving wnt7b.

NGF-inducible subset of Fzd receptorsWnt7b overexpression in p53sh#3 cells can rescue neurite outgrowth in the presence of NGF.However, we found that wnt7b expression without growth factor stimulation had no effect onneurite growth in PC12 cells. We hypothesized that NGF treatment caused induction of wntreceptor(s) through which the wnt7b ligand might potentially activate JNK signaling. In orderto identify putative NGF-inducible wnt7b receptors, expression levels were measured by RT-PCR over time at 4 to 48 hours for all annotated wnt-associated receptors identified within theRattus norvegicus genome (Figure 7). Compared to the untreated naïve wild-type cell,expression levels for two Fzd receptors from the 13 tested genes were significantly increasedover time following NGF treatment. We observed significantly elevated Fzd7 expressionwithin 4 hours of NGF treatment, with levels subsequently decreased over the course of 48hours (Figure 7). In addition, Fzd9 was also identified as an NGF-inducible wnt receptor withtranscript levels that were increased within 4 hours and were sustained over the course of 48hours (Figure 6A, 6B). Observed increases in Lrp6 (Figure 6A) were not recapitulated insubsequent qPCR (Figure 6B) experiments. Levels of Fzd10 were also modestly increasedwithin 24 hours following NGF treatment with levels near baseline at other tested time points.In contrast, we observed a significant reduction in transcript levels of Fzd5 and Fzd2.

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DiscussionThe current study demonstrates an expanding role for p53 signaling in cellular differentiation,specifically within NGF treated PC12 neuronal cells. In addition to its known roles in DNAdamage-induced growth arrest and apoptosis (Vousden, 2006; Vogelstein et al., 2000), p53appears to be significantly involved in neuronal differentiation. p53 does so by negativeregulation of the cell cycle (Hughes et al., 2000) and the subsequent positive induction ofneurite outgrowth through factors such as wnt7b and other target genes recently identified byour lab (Brynczka et al., 2007) as well as the recently described actin binding protein, coronin1b, and the GTPase rab13 transcriptional targets (Di Giovanni et al., 2006). Data presentedhere, along with previously published experimental data (Brynczka et al., 2007), demonstratedthat p53 can positively regulate wnt7b at the promoter, RNA and protein level in a mannerdependent upon NGF stimulation during differentiation of PC12 cells. Rescue of neuriteoutgrowth by ectopic wnt7b expression in p53sh#3 cells suggests that wnt7b-induced signalingis an important pathway through which p53 promotes neurite outgrowth during PC12 neuronaldifferentiation, and therefore represents a unique target gene of the p53 transcription factorthrough which neuronal morphology may be regulated. While these studies have described arole for p53 transcriptional activity in the differentiation of neuronal cells, the relationship isnot yet clear between differentiation and other recognized functions of p53 signaling thatinclude the DNA-damage response, cell cycle arrest and pro-apoptotic signaling. It isappropriate to consider how these other diverse functions of p53 might be integrated intoneuronal differentiation. Interestingly, components of the intrinsic apoptotic pathway havefunctions within neuronal developmental processes that include caspase activity in synapticplasticity (Chan and Mattson, 1999) and Bcl-XL/Bax expression in the determination ofneuronal lineage (Chang et al., 2007). p53-dependent transcriptional regulation of genesinvolved in both cell cycle arrest and differentiation may provide a mechanism through whichprogression of both these processes can proceed from a single pathway node (Bacsi et al.,2005), perhaps suggesting a method in which cell cycle arrest can be ensured prior toundergoing morphological changes during differentiation. Furthermore, defects in PC12differentiation following p53 silencing suggests that redundancy in these processes by a p53functional equivalent does not exist in this cell type. However, the larger role for p53 duringin vivo development remains to be determined, where the majority of p53-null mice aredevelopmentally viable (Donehower et al., 1992) with a subset exhibiting neuronalmalformations (Sah et al., 1995).

Wnt7b promoted neurite outgrowth and extension within differentiating PC12 cells through asignaling mechanism involving the noncanonical JNK pathway without activating canonicalβ-catenin nuclear translocation and signaling. The lack of neurite outgrowth in mitotic cellsoverexpressing wnt7b suggested that wnt7b alone was not sufficient for neurite development.Based on our gene expression studies, we propose that wnt7b expression cooperates with NGF-inducible Fzd receptor expression (Fzd9 and/or Fzd7) and decreased expression of otherreceptors such as Fzd5 to promote increased neurite number and length. While establishmentof wnt7b as a definitive ligand for either the Fzd9 or Fzd7 receptors has not yet been confirmed,the NGF-dependent time course of Fzd9 and Fzd7 expression suggests an enticing mechanismthrough which PC12-acquired competence to the wnt7b ligand could lead to the induction ofneurite outgrowth. Furthermore, data presented here argues against p53-dependent expressionof the receptors Fzd7 or Fzd9. Importantly, we have demonstrated that introduction of wnt7bcDNA into p53sh#3 cells results in the recovery of neurite outgrowth but this rescue occursonly in the presence of NGF. The data suggest that the signaling mechanism for neuriteoutgrowth can occur with NGF-dependent gene expression and wnt7b expression, withconcurrent p53 silencing. The lack of β-catenin involvement and the identified contribution ofJNK activity in the process of neurite outgrowth suggests that wnt7b is a noncanonical wntligand in the differentiating PC12 cell. Since Fzd receptor type is known to lend specificity to

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downstream signaling upon stimulation by wnt ligands (Mikels and Nusse, 2006), both the celltype and expression complement of Fzd receptors may be involved in selective activation ofdownstream pathways by wnt ligands. In this regard, wnt7b reportedly activates canonicalsignaling in epithelial and smooth muscle vascular cells (Wang et al., 2005) but promotesosteogenesis and dendritic development through noncanonical mechanisms involvingPKCdelta and JNK, respectively (Tu et al., 2007; Rosso et al., 2005). Similarly, the outcomeof Fzd receptor activation may be functionally divergent as a function of cell type in whichthey are expressed. We observed increased expression of Fzd7 and Fzd9 along with decreasedexpression of Fzd5 and Fzd2 wnt receptors. The Fzd7 and Fzd9 receptors identified in thisstudy are involved in developmental processes, where the Fzd7 receptor appears to beselectively expressed in glial precursor cells and Fzd9 in precursor neuronal cells of thedeveloping mouse midbrain (Rawal et al., 2006). Fzd7 influences cellular morphology (Vincanet al., 2005; Vincan et al., 2007; Chen and Gumbiner, 2006) and neural crest induction (Abu-Elmagd et al., 2006) while Fzd9 is involved in normal hippocampal and behavioraldevelopment (Zhao et al., 2005) and is also expressed in neural precursor cells within thedeveloping neural tube (Van Raay et al., 2001). Interestingly, we observed downregulation ofthe Fzd5 receptor, described as a receptor for the related wnt7b family member, wnt7a, in PC12cells (Caricasole et al., 2003), although we did not observe wnt7a expression in either theundifferentiated or differentiated state. The discrete subset of NGF-upregulated anddownregulated Fzd receptors suggests to us that differentiating PC12 cells may be primed torespond to wnt availability in a specific autocrine manner. Future studies concerning theactivation of these receptors by wnt7b ligand within the PC12 cell type should provideadditional information concerning the role of Fzd9 and Fzd7 in NGF-induced differentiation.

Wnt proteins are primarily regulated at the level of the endoplasmic reticulum by factors suchas the evolutionarily conserved porcupine chaperone, which aids in the processing of nascentwnt proteins (Tanaka et al., 2000) through post-translational glycosylation and palmitoylation(Tanaka et al., 2002; Hofmann, 2000) and ultimately enables the proper folding and transportof wnt proteins from the ER for secretion. Immunofluorescence experiments shown here uponNGF treatment demonstrate dispersed cytosolic localization of wnt7b, while overexpressionof wnt7b also resulted in continued cytosolic localization with a noticeable punctate,membrane-associated localization. These data suggest that wnt7b may be associated with theendoplasmic reticulum upon expression in PC12 cells prior to secretion. This observation issimilar to previous reports describing wnt ER localization during processing (Tanaka et al.,2002), followed by transport to membrane regions such as lipid rafts prior to secretion (Zhaiet al., 2004). Data presented here also demonstrate that processing of wnt7b in PC12 cellsapparently generates a 27 kDa protein, unlike the predicted higher theoretical molecular weightof wnt7b or the demonstrated size upon wnt7b cDNA expression in COS monkey kidney cells(Burrus and McMahon, 1995). Our data indicate that processing is likely attributable to post-translational cleavage and not alternative splicing events or transcription from an alternativep53-regulated downstream transcriptional start site. Multiple pieces of evidence presented heresupport this conclusion: 1) expression of wnt7b cDNA lacking the intronic p53 binding siteresulted in a protein of 27 kDa in size as determined via immunoblot, and 2) sequencing ofendogenous wnt7b cDNA demonstrated expression of the full-length annotated gene. Thesignificance of a truncated wnt7b protein within these cells is not yet clear but the molecularweight does not appear to be dependent upon p53-induced transcription.

Multiple reports have described tissue-specific regulation of gene loci by activated p53 (diMasi et al., 2006; Fei et al., 2002; Coates et al., 2003) which suggest that activation of targetssuch as wnt7b may be dependent upon transcriptional coactivators or signaling in a specificcell context. Evidence presented here supports this conclusion, as Homo sapiens p53 proteinwas capable of inducing transcription of the wnt7b gene in Rattus norvegicus neuronal PC12cells but not in human normal lung fibroblasts (IMR-90), fibrosarcoma (HT-1080) or

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osteosarcoma (Saos-2) cell lines. Transcriptional cofactors for p53 such as JMY, hnRNP K(Moumen et al., 2005), YY1 (Gordon et al., 2006; Sui et al., 2004) and the SWI/SNF complex(Lee et al., 2002) each have a specificity for participating with p53 in gene expression accordingto a specific activating stimulus and cell type. Differences in chromatin assembly andaccessibility for p53 binding may also play a large role in cell-type specific differences intranscriptional activity and gene expression. Accordingly, it is not yet possible to definitivelydetermine whether wnt7b is a p53-inducible target gene in human cells, where a wider samplingof cell types or conditions may be necessary to identify wnt7b transcriptional regulation.

Based on data presented in this study, we propose a signaling paradigm as shown in Figure 8through which the transcription factor p53 regulates PC12 neuronal differentiation followingNGF treatment. Activation of the p53 transcription factor results in its increased nuclearlocalization and the transcriptional activation of wnt7b and other target loci. Wnt7b processing(∼27 kDa) and concomitant p53-independent expression of cognate Fzd7 and/or Fzd9 receptorswith NGF treatment ultimately generates a competent PC12 cell, in which the wnt7b ligandstimulates JNK signaling activity via a noncanonical wnt pathway involving the putative wnt7breceptors Fzd9 and/or Fzd7. We conclude that noncanonical wnt-regulated JNK signalingpromotes the outgrowth of neurites and the morphological differentiation of the PC12 cell.Future studies should further refine the contribution of wnt7b to neurite outgrowth anddetermine its extensibility to other neuronal systems and species.

AcknowledgementsThis work was supported by the Intramural Research Program of the NIH, National Institute of Environmental HealthSciences. We thank Dr. Kevin E. Gerrish and Dr. Serena M. Dudek for critical review of this manuscript.

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Figure 1.Wnt7b is expressed in a time-dependent manner upon NGF treatment in PC12 cells. A) qPCRfor wnt7b cDNA following NGF treatment at 50 ng/ml for indicated intervals. Each reactionwas performed on 1 μL cDNA derived from 1 μg DNase-treated RNA. PCR was performedfor 34 cycles. Actin amplification is shown as loading control, and was amplified for 23 cycles.B) Immunoblot for wnt7b performed on 15 μg protein from PC12 whole cell lysate followingtreatment with NGF for indicated interval. Actin is shown as loading control. Protein sizemarkers are indicated adjacent to blot image. C) Immunoblot for wnt7b using 15 μg proteinfrom PC12 whole cell lysate following treatment with NGF for indicated interval. Actin isshown as loading control.

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Figure 2.Wnt7b is a p53 target gene in NGF-induced PC12 differentiation. A) Immunoblot for wnt7bin wild-type and p53-silenced shRNA-expressing PC12 cell lines, performed on 15 μg proteinfrom indicated cell lysate following 24 h NGF treatment. Actin levels are shown for loadingcontrol. B) qPCR depicting PC12 expression levels of indicated genes in both control cells andfollowing Hs tp53 transfection (pCMV6-tp53) after 24 h. 1 μg DNase-treated RNA was usedin each RT reaction. 1 μL of cDNA from each sample was amplified for 23-33 cycles,depending on cDNA target. A ‘No RT’ control is shown to demonstrate absence of genomicDNA, with GAPDH expression shown for loading control. C) Logarithmic scale plot depictingrelative gene expression of indicated targets in PC12 cells and Hs tp53-expressing PC12 cellsas determined by RT-PCR. GAPDH was used as calibrator and expression was normalized tonaïve untreated PC12 target gene expression as endogenous control. Error bars are S.D. andsignificance reported at p ≤ .05 (*). Statistical analysis was performed using Student’s t-testcomparing Hs tp53-transfected cellular gene expression levels to those of naïve cells.

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Figure 3.Wnt7b is expressed as a 27 kDa protein in PC12 cells. A) Immunoblot for wnt7b in controland pDREAM-wnt7b transfected PC12 cells both with and without NGF treatment asindicated. Cells were transfected with pDREAM-wnt7b and incubated for 6 hours prior to NGFtreatment. Undifferentiated and NGF-treated cells were allowed to incubate for 24 hoursfollowing NGF addition until protein isolation. Protein size markers are depicted adjacent togel image. Actin protein levels are shown as loading control. B) Exon-specific RT-PCR inwild-type PC12 cells was performed on 1 μL cDNA derived from 1 μg DNase-treated RNA.Cells were isolated in either the undifferentiated naïve state as indicated or following 5 daystreatment with 50 ng/ml NGF. PCR was performed for 38 cycles with primers designed toamplify discrete regions within each of three exons composing the wnt7b gene. GAPDH isshown for loading control along with no RT negative controls using the same GAPDH primerset, amplified for 25 cycles. C) Immunofluorescence for expressed wnt7b within either controlor pDREAM-wnt7b transfected PC12 cells both with and without 50 ng/ml NGF treatment for24 hours. Secondary antibody is conjugated to Alexa594 (red). Nuclei are stained with DAPI(blue). Images are taken under 100x magnification.

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Figure 4.Wnt7b expression rescues NGF-induced neurite outgrowth in p53-silenced PC12 cells. A) 40xmagnification of naïve or 24 hour treated PC12 cells transfected with pDREAM-wnt7b asindicated. Absence of neurites can be observed in NGF-treated p53sh#3 cells compared to thewild-type controls. Overexpressed wnt7b in p53sh#3 cells generates substantially increasedneurite numbers when concomitantly treated with NGF. Observations are summarized inFigure 5C. B) Average neurite length, measured in cell diameters in both wnt7b-overexpressingand control wild-type PC12 cells following NGF treatment for 48 hrs. Bars represent averageneurite length with a sample size of approximately 100 cells bearing neurites, where non-neurite bearing cells were not scored and are therefore not represented in this comparison. Errorbars are S.D. and significance is reported at p ≤ .05 (*).

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Figure 5.Wnt7b activates JNK signaling. A) Immunofluorescence for β-catenin within either control orpDREAM-wnt7b transfected PC12 cells both with and without 50 ng/ml NGF treatment for24 hours. Secondary antibody is conjugated to Alexa594 (red). Nuclei are stained with DAPI(blue). Images are taken under 100x magnification. B) Immunoblot for phosphorylated cJunin both control and NGF-treated PC12 cells over time. Cells were treated with NGF forindicated interval prior to harvesting and cell lysis. Actin levels are shown for loading control.C) Immunoblot for phosphorylated JNK, total β-catenin and total JNK levels in wild-type andp53sh#3 cells following sFRP1 treatment. Cells were transfected with pDREAM-wnt7b 24hours prior to NGF addition. The wnt antagonist sFRP1 was added to culture medium at aconcentration of 5 μg/mL at 4 hours prior to NGF addition. Cells were harvested 24 hours afteraddition of NGF. Total JNK levels are shown for loading control.

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Figure 6.JNK activity promotes PC12 neurite outgrowth. A) 40x magnification of 24 hour NGF-treatedwild type or p53sh#3 PC12 cells which were transfected with pDREAM-wnt7b and treatedwith inhibitors as indicated. The JNK inhibitor (SP600125) was used at 1 μM and p38MAPKinhibitor (SB202190) was used at 40 μM and added to culture medium 1 hour prior topDREAM-wnt7b transfection and 5 hours prior to NGF addition. B) Neurite outgrowth datawere compiled from Figure 4A and Figure 5B depicting average neurite number (out of at least100 counted cells in each sample) following indicated treatment. Neurites equal to or greaterthan 1/2 cell diameter in length were tallied for each sample condition. Error bars reflectstandard deviation and significance (p ≤ .05) for pairwise comparisons are labeled as describedin chart legend.

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Figure 7.NGF regulates Fzd gene expression. The bar chart depicts relative expression levels by RT-PCR for known and annotated wnt receptor proteins (Fzd, Lrp5/6, Ryk, Ror2). Untreated PC12cells (naïve) were compared to cells treated with 50 ng/ml NGF over time as indicated. GAPDHwas used as calibrator and expression was normalized to naïve untreated PC12 target geneexpression as an endogenous control. Error bars reflect standard deviation. Gel images in insetshow qPCR validation of wnt receptors following NGF treatment. PCR was performed for23-30 cycles depending upon cDNA target abundance. GAPDH served as a loading controland was amplified for 23 cycles.

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Figure 8.Schematic depicting the roles of p53 and wnt7b in proposed pathway for NGF-mediated neuriteoutgrowth in PC12 differentiation. Briefly, NGF through its receptor TrkA, promotes cytosolictranslocation and phosphorylating activation at Ser15 (and other sites) of the p53 transcriptionfactor during PC12 neuronal differentiation. P53 activates transcription of wnt7b and othertarget loci. Wnt7b processing and secretion occurs with increased concomitant expression ofcognate Fzd7 and Fzd9 receptors mediated by NGF activity (independent of p53) to ultimatelygenerate a competent cell. Wnt7b stimulates JNK signaling activity via a noncanonical wntpathway involving the putative receptors Fzd9 and/or Fzd7. Wnt-regulated JNK signalingpromotes the outgrowth of neurites and morphological differentiation of the PC12 cell. SeeDiscussion for further detail.

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