European Journal of Pharmacology 595 (2008) 35–38

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European Journal of Pharmacology

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Short communication

Pleiotrophin prevents cocaine-induced toxicity in vitro

Esther Gramage, Luis F. Alguacil, Gonzalo Herradon ⁎Lab. Pharmacology and Toxicology, Univ. CEU San Pablo, 28668 Boadilla del Monte, Madrid, Spain

⁎ Corresponding author. Lab. Pharmacology and ToxiCEU, Urb. Montepríncipe, 28668 Boadilla del Monte3724700; fax: +34 91 3510475.

E-mail addresses: herradon@ceu.es, gherradon@yaho

0014-2999/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.07.067

a b s t r a c t

a r t i c l e i n f o

Article history:

Pleiotrophin is a cytokine i Received 3 June 2008Received in revised form 29 July 2008Accepted 31 July 2008Available online 13 August 2008

Keywords:PleiotrophinCocaineNeurotoxicitySurvivalDopaminergic

nvolved in differentiation, survival and repair processes in the central nervoussystem. Pleiotrophin is upregulated in the brain after administration of different drugs of abuse, thussuggesting a protective role of this cytokine on drug-induced toxicity. We have tested this hypothesis in vitrousing NG108-15 cells, a line widely used for neurotoxicity studies. It was found that pleiotrophin (3 and 6 μM)significantly prevents cocaine (5 mM)-induced cytotoxicity as measured by the neutral red test. Similarresults were obtained in PC12 cells, which were found to endogenously express both pleiotrophin and itsmain target, receptor protein tyrosine phosphatase (RPTP) β/ζ. Blockade of pleiotrophin signaling using anti-pleiotrophin antibodies (2 μg/ml) did not potentiate cocaine-induced toxicity; interestingly, incubation ofPC12 cells only with anti-pleiotrophin antibodies significantly reduced cellular viability, thus suggesting animportant role of endogenous pleiotrophin signaling in cell survival. The data suggest that pleiotrophinoverexpression in response to drugs of abuse may be relevant to prevent drug-induced toxicity.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Pleiotrophin is a secreted, highly conserved 136-amino acidcytokine (Milner et al., 1989; Li et al., 1990) that is expressed indiscrete loci that correspond at the same time to peaks of growth andearly differentiation of neurons and glia (Silos-Santiago et al., 1996;Deuel et al., 2002). Although expression of pleiotrophin is limited toonly a few cell types of the central nervous system in adults (Li et al.,1990; Silos-Santiago et al., 1996), it has been shown to be highlyupregulated at sites of injury and repair (Yeh et al., 1998; Ezquerraet al., 2008). Therefore, pleiotrophin signaling may be critical fordifferentiation of different cells both in development and in woundrepair. Pleiotrophin binds to the receptor protein tyrosine phospha-tase (RPTP) β/ζ (Meng et al., 2000), inactivating its phosphataseactivity and, as a result, increasing the phosphorylation levels of thesubstrates of RPTP β/ζ, β-catenin (Meng et al., 2000), GIT1/Cat-1(Kawachi et al., 2001), Fyn (Pariser et al., 2005a) and β-adducin(Pariser et al., 2005b,c). Anaplastic lymphoma kinase (ALK) has alsobeen proposed as a receptor for pleiotrophin (Stoica et al., 2001),however it has been recently determined that ALK is also a substrate ofRPTPβ/ζ (Perez-Pinera et al., 2007).

Pleiotrophin exhibits a trophic effect on dopaminergic neurons invitro (Hida et al., 2003) and induces the differentiation of catechola-minergic neurons from embryonic stem cell-derived nestin-positivecells (Jung et al., 2004). These findings were consistently linked with

cology, Universidad San Pablo, Madrid, Spain. Tel.: +34 91

o.es (G. Herradon).

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pleiotrophin overexpression in striatum of patients with Parkinson'sdisease (Marchionini et al., 2007), leading the authors to suggest atrophic effect of pleiotrophin in dopaminergic neurons in thisneurodegenerative disease. Pleiotrophin could be also expected toextend its protective role to other kind of cellular damage, i.e. drug-induced toxicity. Interestingly, pleiotrophin is upregulated in thenucleus accumbens after acute administration of amphetamine and inthe cingulate cortex, fronto-parietal cortex and caudate–putamenafter delta-9-tetrahydrocannabinol injection (Mailleux et al., 1994; LeGreves, 2005). Despite these findings, the possible effects ofpleiotrophin on the neurotoxicity induced by drugs of abuse havenot been studied before.

To fill this gap in knowledge, we have tested the effects ofpleiotrophin on cocaine-induced toxicity in different cell cultures. Wefirst used NG108-15 cells since they are classically used in neurotoxi-city studies in vitro (see for example Schelman et al., 2004). Then, wetried to confirm and extend our results in PC12 cells, a linewidely usedto evaluate the effects of drugs of abuse in vitro (Cunha-Oliveira et al.,2006) that is known to express high levels of dopamine transporter,the pharmacological target of cocaine (Kadota et al., 1996).

2. Material and methods

2.1. Cell cultures

NG108-15 (neuroblastoma×glioma hybrid) cells were cultured in 96-well plates (10,000 cells/well) with Dulbecco's Modified Eagle's Medium(DMEM) supplemented with 10% fetal bovine serum (FBS). PC12 (ratpheochromocytoma) cells were cultured with RPMI-1640 Mediumsupplemented with 10% FBS in 96-well plates (10,000 cells/well).

36 E. Gramage et al. / European Journal of Pharmacology 595 (2008) 35–38

2.2. Cellular viability

Cellular viability was studied with the neutral red test (Invittox IP-64, European Center for the Validation of Alternative Methodsdatabase) as previously described (Morales et al., 2008). To test theeffects of pleiotrophin (Sigma, Spain) on cocaine (Alcaliber, Spain)-induced toxicity in cell cultures, NG108-15 and PC12 cells wereincubated for 24 h with cocaine (5 mM) and/or pleiotrophin (3 and6 μM). In addition, we aimed to test the possibility that endogenouspleiotrophin could protect PC12 cells against cocaine-induced toxicity.To block pleiotrophin signaling in cocaine-treated cells, we supple-mented the cultures with anti-pleiotrophin antibodies, an experi-mental strategy previously used to disrupt pleiotrophin-inducedneuritogenesis in cultures (Bao et al., 2005) and to block pleiotrophinsignaling in cultured cancer cells (Chen et al., 2007). We incubatedPC12 cells with cocaine (5 mM) for 24 h in the presence or absence ofgoat anti-pleiotrophin antibodies (Santa Cruz biotechnology, SantaCruz, CA) (2 μg/ml) or goat IgGs (2 μg/ml) (Invitrogen, Spain) ascontrol. All determinations were carried out in 3–6 replicates, and atleast three independent experiments were performed.

2.3. Western blots

Protein samples from PC12 cell lysates (six 60-mm plates) weremixedwith loading buffer (60mMTris pH 6.8,10% glycerol, 5% sodiumdodecyl sulfate (SDS), 0.65% β-mercaptoethanol, and 0.01% bromo-phenol blue), boiled for 5 min, and loaded onto 4–12% polyacrylamidegels (Invitrogen, Carlsbad, CA) as appropriate. The gels weretransferred to nitrocellulose membranes, blocked with 50 mM Tris,150 mMNaCl, 0.1% Tween-20 (TBS-T), and 5% non-fat milk for 1 h, andthen incubated with goat anti-pleiotrophin antibodies (1:1000) in 5%bovine serum albumin (BSA) overnight and rat anti-RPTP β/ζantibodies (1:3000) (R&D Systems, Minneapolis, MN) in TBS-T with5% non-fat milk overnight. Membranes were incubated for 1 h withappropriate secondary antibodies (Santa Cruz biotechnology, Santa

Fig. 1. Effects of pleiotrophin on cocaine-induced toxicity in NG108-15 and PC12 cells. (Apleiotrophin (PTN; 3, 6 μM) for 24 h. (B) NG108-15 cells cultured with media supplemesupplemented with cocaine (5 mM) and pleiotrophin (PTN; 3 μM) for 24 h. (D) PC12 cells cultwas assessed by the neutral red test in all cases. Pleiotrophin is abbreviated as PTN. Results

Cruz, CA) conjugated with horseradish peroxidase (1:5000) in TBS-Twith 5% non-fat milk for 30 min. The immunoreactive proteins werevisualized using the ECL Enhanced method according to themanufacturer's instructions (Amersham, San Francisco, CA).

2.4. Statistical analysis

The results of cytotoxicity studies were analysed by one-wayanalysis of variance (ANOVA) and Tukey post-hoc tests. Significancewas considered at the 0.05 level.

3. Results

3.1. Pleiotrophin prevents cocaine-induced toxicity in NG108-15 andPC12 cells

To test the effects of pleiotrophin on cell cultures incubated with ahigh concentration of cocaine (5 mM), we first used NG108-15 cells. Itwas found that low concentrations of pleiotrophin (3 and 6 μM)significantly increased the cellular viability of NG108-15 cell culturesincubated with cocaine (5 mM) for 24 h as measured by the neutralred test (Fig. 1A). In contrast, pleiotrophin (3 and 6 μM) was devoid ofsignificant effects by itself (Fig. 1B).

To find out if these findings were extendable to other cell types, wetested the effects of pleiotrophin (3 μM) on cocaine (5 mM)-inducedtoxicity in PC12 cells. Similarly, we found that pleiotrophin (3 μM)significantly prevented the decrease of cellular viability induced bycocaine in PC12 cells (Fig. 1C). Again, we did not observe significanteffects in PC12 cells incubated exclusively with pleiotrophin (3 μM) for24 h (Fig. 1D).

3.2. Effects of pleiotrophin on PC12 cell survival

Since pleiotrophin is known to be expressed at high levels in neuralprogenitor cells (Jung et al., 2004), we tested the protein levels of

) NG108-15 cells cultured with media supplemented with cocaine (Coc; 5 mM) andnted with pleiotrophin (PTN; 3, 6 μM) for 24 h. (C) PC12 cells cultured with mediaured with media supplemented with pleiotrophin (PTN; 3 μM) for 24 h. Cellular viabilityare expressed as mean±S.E.M. ⁎Pb0.05 vs Cocaine.

Fig. 2. Effects of anti-pleiotrophin antibodies on PC12 cells. (A) Pleiotrophin and RPTPβ/ζ protein levels were measured by Western blot in non-treated PC12 cells. (B) Cellularviability of PC12 cells cultured with media supplemented with cocaine (Coc; 5 mM)and/or anti-pleiotrophin antibodies (2 μg/ml) and/or goat IgGs as control (2 μg/ml) for24 h was measured by the neutral red test. Pleiotrophin is abbreviated as PTN. Resultsare expressed as mean±S.E.M. ⁎Pb0.05 vs Control. #Pb0.05 vs IgGs.

37E. Gramage et al. / European Journal of Pharmacology 595 (2008) 35–38

pleiotrophin and its receptor, RPTP β/ζ in Western blots of PC12 cells.As expected, we found readable detectable levels of expression of bothpleiotrophin and RPTP β/ζ in PC12 cells (Fig. 2A). This result suggestedthe possibility that endogenous pleiotrophin could protect, at leastpartially, PC12 cells from cocaine-induced cytotoxicity. To test thishypothesis, we blocked pleiotrophin signal in cocaine-treated cultureswith anti-pleiotrophin antibodies. Incubation with anti-pleiotrophinantibodies (2 μg/ml) did not potentiate cocaine-induced toxicity inPC12 cells (Fig. 2B). Surprisingly, we found that exclusive incubationwith anti-pleiotrophin antibodies significantly reduced cellularviability when compared with goat IgGs-treated cells and control(untreated) cells (Fig. 2B).

4. Discussion

Pleiotrophin expression has been found to be upregulated indifferent brain areas in response to drugs of abuse such as delta-9-tetrahydrocannabinol and amphetamine (Mailleux et al., 1994; LeGreves, 2005). Interestingly, midkine the only other member of thisfamily of heparin-binding growth factors (see review by Deuel et al.,2002) is also upregulated in the rodent brain and in humans inresponse to alcohol, nicotine and morphine (Flatscher-Bader andWilce, 2006; Ezquerra et al., 2007), suggesting that this family ofsecreted cytokines may modulate the toxicity induced by drugs ofabuse in vivo. Pleiotrophin and midkine are highly redundant infunction (Herradon et al., 2005). For example, both of them exhibitsurvival effects on catecholaminergic neurons and induce thedifferentiation of progenitors to catecholaminergic neurons, increas-ing their levels of tyrosine hydroxylase, the rate-limiting enzyme ofcatecholamine biosynthesis (Kikuchi et al., 1993; Hida et al., 2003;Jung et al., 2004). Taking into account that catecholaminergic neuronsare main targets for many drugs of abuse, we hypothesized thatupregulation of these growth factors could limit drugs neurotoxiceffects. The data presented here strongly support this hypothesis sincethey demonstrate that pleiotrophin efficiently protects NG108-15 cellcultures from cocaine insult. Importantly, we have also demonstratedthat the effects of pleiotrophin on cocaine-induced toxicity areextendable to other cell type, PC12 cells, a catecholaminergic neuronalmodel traditionally used to study the neurotoxic effects of cocaine invitro (see for example Cunha-Oliveira et al., 2006).

The mechanisms involved in the protective role of pleiotrophin inthe central nervous system remain to be established, but they probablyinvolve an interactionwith RPTP β/ζ. Pleiotrophin inactivates RPTP β/ζincreasing the phosphorylation levels of RPTP β/ζ substrates (Meng et

al., 2000). As mentioned in the Introduction, among these substratesare β-catenin (Meng et al., 2000), Fyn (Pariser et al., 2005a), β-adducin(Pariser et al., 2005b,c) and anaplastic lymphoma kinase (Perez-Pineraet al., 2007). Increased tyrosine phosphorylation of these substratescontribute to disrupt homophilic cell–cell adhesion and cytoskeletalstability, facilitating as a result an epithelial mesenchymal transition(see review by Perez-Pinera et al., 2006), all of them steps needed indifferentiation and wound repair processes.

Interestingly, blockade of endogenous pleiotrophin with anti-pleiotrophin antibodies did not potentiate cocaine-induced toxicity inPC12 cells, suggesting that endogenous pleiotrophin expression levelsare probably too low to protect cells from the toxic effect of highconcentrations of cocaine. Surprisingly, exclusive incubation withanti-pleiotrophin antibodies significantly reduced PC12 cell viabilityto ∼50% compared with control cells, suggesting that pleiotrophin is asurvival factor for PC12 cells in culture. This finding clearly correlateswith the ability of pleiotrophin to exert survival effects on catecho-laminergic neurons in primary cultures (Hida et al., 2003).

In summary, our results demonstrate that pleiotrophin preventscocaine-induced toxicity in NG108-15 and PC12 cell cultures,suggesting that pleiotrophin overexpression in different brain areasin response to drugs of abuse may be relevant to prevent drug-induced toxicity in vivo. In addition, our data support a survival role ofendogenous pleiotrophin for PC12 cells in culture.

Acknowledgements

This work has been supported by grant SAF2007-61528 fromMinisterio de Educación y Ciencia of Spain.

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