Uridine 5′-Triphosphate Promotes In Vitro Schwannoma Cell Migration through Matrix Metalloproteinase-2 Activation

In response to peripheral nerve injury, Schwann cells adopt a migratory phenotype and modify the extracellular matrix to make it permissive for cell migration and axonal re-growth. Uridine 5′-triphosphate (UTP) and other nucleotides are released during nerve injury and activate purinergic receptors expressed on the Schwann cell surface, but little is known about the involvement of purine signalling in wound healing. We studied the effect of UTP on Schwannoma cell migration and wound closure and the intracellular signaling pathways involved. We found that UTP treatment induced Schwannoma cell migration through activation of P2Y2 receptors and through the increase of extracellular matrix metalloproteinase-2 (MMP-2) activation and expression. Knockdown P2Y2 receptor or MMP-2 expression greatly reduced wound closure and MMP-2 activation induced by UTP. MMP-2 activation evoked by injury or UTP was also mediated by phosphorylation of all 3 major mitogen-activated protein kinases (MAPKs): JNK, ERK1/2, and p38. Inhibition of these MAPK pathways decreased both MMP-2 activation and cell migration. Interestingly, MAPK phosphorylation evoked by UTP exhibited a biphasic pattern, with an early transient phosphorylation 5 min after treatment, and a late and sustained phosphorylation that appeared at 6 h and lasted up to 24 h. Inhibition of MMP-2 activity selectively blocked the late, but not the transient, phase of MAPK activation. These results suggest that MMP-2 activation and late MAPK phosphorylation are part of a positive feedback mechanism to maintain the migratory phenotype for wound healing. In conclusion, our findings show that treatment with UTP stimulates in vitro Schwannoma cell migration and wound repair through a MMP-2-dependent mechanism via P2Y2 receptors and MAPK pathway activation.


Introduction
Peripheral nerve injury initiates a sequence of events through which macrophages and Schwann cells clear damaged axons and potentiate axonal regeneration and nerve remyelination [1]. During Wallerian degeneration, de-differentiating Schwann cells secrete growth factors and neurite-promoting factors to guide the growing axon, as well as proteolytic enzymes to remodel extracellular matrix (ECM) and facilitate cell migration [2,3,4,5] One important class of proteases are matrix metalloproteinases (MMPs), a large family of zinc-and calcium-dependent enzymes that act on a wide number of extracellular and cell surface receptors, ligands, and adhesion and structural proteins [6,7]. MMP upregulation has been linked to the pathogenesis of peripheral nerve damage [8,9,10,11]. Specifically, MMP-2 and MMP-9 have been involved in remodeling of the ECM during nerve degeneration and regeneration [12,13,14,15]. MMP-2 and MMP-9 are highly expressed after sciatic nerve injury: MMP-9 activity increases acutely at the site of injury some hours after nerve crush, whereas MMP-2 activity is delayed but maintained during nerve regeneration proximally and distally to the injury site, suggesting that MMP-2 acts to facilitate axonal extension along the nerve matrix [10]. In spinal cord injury, the same pattern is observed: MMP-9 activity is highly increased 12 to 24 hours after injury to facilitate leukocyte infiltration while MMP-2 increases its activity 5 to 14 days after injury to facilitate nerve recovery and limit the formation of a glial scar [16,17,18]. In Schwann cells, MMP-9 is required for insulin-like growth factor (IGF) release and the subsequent activation of the MEK/ERK pathway via IGF-1 and ErbB receptors [19]. MMP-9 also activates the Akt/ERK pathway and promotes migration by binding to the low-density lipoprotein receptor-related protein [20]. Taking into account these findings, the modulation of MMP activity may be a relevant target to enhance regeneration in demyelinating diseases of the peripheral nervous system (PNS) [17].
There is a growing body of evidence implicating purinergic P2Y receptors in cell communication, migration, and wound repair in response to injury in many cell types [21,22,23,24]. After injury, nucleotides released from cells activate the purinergic receptorsignaling pathway to mediate the response to injury [25]. Nucleotide binding to P2Y receptors, which are coupled to the protein Ga q , activates phospholipase C b (PLCb). PLCb cleaves phosphatidylinositol 4,5-biphosphate (PIP 2 ) to diacylglycerol (DAG) and phosphoinositol tri-phosphate (IP 3 ), resulting in the release of intracellular Ca 2+ from endoplasmic reticulum stores. The increase in cytosolic Ca 2+ induces a myriad of alterations in the tyrosine phosphorylation of proteins ranging from adhesion molecules to members of the mitogen-activated protein kinase (MAPK) family [26,27]. MAPKs such as c-Jun N-terminal protein kinase (JNK), extracellular signal-regulating kinase (ERK), and p38 transduce extracellular signals into various cellular responses such as proliferation, differentiation, and migration [28,29,30,31]. Accumulating evidence suggests that these MAPKs play a role in the migration of various cell types [32,33,34,35].
Although the activation of P2Y purinergic receptors is known to activate a MAPK signaling cascade, the role of the purinergic signaling pathway in relation with Schwann cell migration and wound repair has not yet been described. The present study aimed to determine the effect of extracellular uridine 59-triphosphate (UTP) on Schwannoma cell migration and wound repair and to establish whether MMP-2 is involved in this effect. For the first time, we report that UTP stimulates in vitro Schwannoma cell migration and wound repair through a MMP-2-dependent mechanism via P2Y 2 receptors and MAPK pathway activation.

Schwann cell line cultures
The rat schwannoma cell line RT4-D6P2T was purchased from the European Collection of Cell Cultures (#93011415; ECACC, Salisbury, UK) and maintained in DMEM high glucose medium supplemented with 2 mM L-glutamine, 50 U/mL penicillin, 50 mg/L streptomycin, and 10% (v/v) DBS. Cultures were incubated in a 5% CO 2 humidified atmosphere at 37uC. Cells were seeded at a density of 1.2610 5 cells/cm 2 and starved in 1% (v/v) DBS for 24 h before nucleotide treatment.

Schwann cell primary cultures
Schwann cells were isolated from the sciatic nerves of Sprague-Dawley rats on postnatal days 7 to 10 as previously described [36]. After chemical and mechanical dissociation, cells were cultured on dishes in DMEM high glucose medium supplemented with 10% (v/v) DBS, 5 mM forskolin, 20 mg/mL pituitary extract, 2 mM glutamine, 100 U/mL penicillin and 50 mg/L streptomycin. To eliminate fibroblasts, cells were replated on poly-L-lysine and laminin-coated culture dishes or wells according to the Kreider group [37]. In the resulting cultures, Schwann cells had a purity of more than 83% (Hoechst and S100 immunoreactivity). Cells were used for experiments at 7 to 10 days in culture.

Gelatin zymography
Conditioned culture medium was collected and centrifuged at 15,000 rpm for 5 min at 4uC. The cleared medium was concentrated 10-fold using Centricon with a 10-kDa pore size (Merck Millipore). Samples (20 mg protein) of Schwann cellconditioned media were then resolved on 10% SDS-PAGE gels containing 0.1% (w/v) gelatin as the protease substrate. After electrophoresis, gels were washed with 100 mM Tris-HCl (pH 7.5) and 2.5% (v/v) Triton X-100 for 30 min and subsequently incubated for 16 h at 37uC in digestion buffer containing 100 mM Tris-HCl (pH 7.5), 5 mM CaCl 2 , and 200 mM NaCl. Bands corresponding to activity were visualized by negative staining using 0.25% (w/v) Coomassie Brilliant Blue R-250 (Bio-Rad, CA, USA) for 6 hours followed by destaining for 30 min in a solution of 10% (v/v) acetic acid and 25% (v/v) isopropanol. To quantify the MMP-2 activity, the intensities of negative stained bands were analyzed using Image J software (National Institutes of Health, USA). MMP-2 activity was expressed as the active MMP-2 intensity per total MMP-2 intensity (pro-MMP-2 plus active MMP-2).

Gelatinase activity
Gelatinase activity was determined with in situ zymography using DQ-Gelatin fluorescein conjugate as a substrate. Schwannoma cells cultured on coverslips were washed with 0.5 M Tris-HCl (pH 7.6), 1.5 M NaCl, 50 mM CaCl 2 , and 2 mM NaN 3 . Cells were then treated with DQ-Gelatin (100 mg/mL) at 37uC for 2 h (EnzCheck Gelatinase Assay, Molecular Probes, USA). Finally, coverslips were mounted with the anti-fading medium Fluoromount (Sigma) and fluorescence microscopy images were obtained using a laser confocal microscope (Leica DM IRB). The number of fluorescence spots in each cell was counted in different regions and the results were expressed as number of positive MMP-2 Schwann cells per area.

Wound -healing assay
Schwann cells were seeded at 2.5610 5 cells/cm 2 and incubated for 48 h to allow for the formation of a monolayer. A wound was created in the monolayer with a 1000 mL pipette tip. The cells were then washed with PBS (pH 7.4) and replaced with DMEM high glucose medium supplemented with 2 mM L-glutamine, 50 U/mL penicillin, 50 mg/L streptomycin, and 1% (v/v) DBS. Cultures were incubated in a 5% CO 2 humidified atmosphere at 37uC. Images of 4 random fields along the wound were taken at different times using a Nikon camera connected to a Nikon inverted light microscope. Wound closure was quantified by measuring the percentage of the occupied wound area at different times using the TScratch v1.0 program (Matlab, Zurich, Switzerland). The rate of migration (velocity) was expressed as occupancy (%) per hour.

Migration assay
Schwann cell migration assays were performed using BD BioCoat cell culture inserts with 6.5-mm-diameter filters with an 8-mm pore size according to the manufacturer's instructions (BD Biosciences). Schwann cells at a density of 1.5610 5 cells/insert were seeded into the upper chamber in complete medium: DMEM high glucose medium supplemented with 2 mM Lglutamine, 50 U/mL penicillin, 50 mg/L streptomycin, and 1% (v/v) DBS. Complete medium with UTP was placed in the lower chamber as the chemoattractant. After incubation in a 5% CO 2 humidified atmosphere at 37uC, the migrated cells in the lower chamber were fixed with 70% (v/v) ethanol and stained with 0.2% (w/v) Crystal Violet. For each membrane, the number of cells was counted in 4 blindly chosen random fields under light microscopy. The fold increase in cell migration was expressed by taking the control value as 1.

Quantitative reverse transcription polymerase chain reaction (RT-PCR)
The total RNA from 10 6 Schwann cells was isolated using TRIzol reagent (Invitrogen, Paisley, UK) and reverse transcribed using an iScript cDNA synthesis kit (Bio-Rad, Berkeley, CA, USA). The cDNA template underwent quantitative analysis of cDNA amplification, which was assessed using SYBR Green (Bio-Rad). Standard PCRs were performed with the following specific primers ( All amplified PCR products were verified by melting curve analysis. Quantification was performed by the DDC T method, and levels of mRNA expression were normalised to the housekeeping gene that encodes GAPDH.

Short hairpin RNA
Knockdown Schwannoma cells were generated using short hairpin RNA directed against the rat MMP-2 gene (shMMP-2) constructed in the pLVTHM-GFP plasmid vector (Addgene, Cambridge, USA). Sequence targeting for MMP-2 was selected based on the rules for RNA susceptibility proposed by Tuschl's group [38] and using the siRNA prediction program from the Whitehead Institute for Biomedical Research [39]. Two complementary DNA oligonucleotides (Roche, Berlin, Germany) were annealed to produce a double-stranded DNA fragment encoding a 19-nucleotide sense strand, a 9-nucleotide loop, and a 19-

Statistical Analysis
Results were expressed as the mean 6SD of results from at least 3 independent experiments. Statistical analyses were carried out using the GraphPad Prism 5.0 Software package (ANOVA analysis plus Newman-Keuls post-test: *P#0.05, **P#0.01, and ***P#0.001).

Ethics Statement
Animal experimental procedures were submitted and authorized by the Ethics Committee of the Universitat Internacional de Catalunya (CEREA-UIC 2009).

UTP enhances Schwann cell migration and wound repair
In response to injury, Schwann cells increase their migration to cover the damaged area. Previous studies on microglial, smooth muscle, and epithelial cells have demonstrated that nucleotides trigger wound repair [22,24,40]. To investigate the ability of the nucleotide UTP to increase Schwannoma RT4-D6P2T cell migration, dose-response and time-course studies were performed using the Transwell migration assay (Fig. 1A). Dose-response studies revealed that cell migration significantly increased after UTP treatment (12 h, 100-500 mM). As previously reported, the observed decrease in cell migration at 1 mM is due to UTP cytotoxicity [41]. As extracellular concentration of UTP is greatly enhanced after injury [42,43], a high UTP concentration (250 mM) was used in all the experiments. On the other hand, time-course analysis showed that cell migration significantly increased between 6 and 12 h after UTP treatment (250 mM).
The effect of UTP on wound repair was also confirmed in Schwann cell line and in primary cultured Schwann cells. Treatment of these cells with UTP (250 mM) significantly reduced the time required to close the wound completely (100%). RT4-D6P2T cells incubated with UTP filled the wound after 20 hours (Fig. 1B, d) compared to control cells, which occupied only 5668% of the wound area in that time (Fig. 1B, b). Likewise, primary cultured Schwann cells treated with UTP filled the wound after 36 hours (Fig. 1B, h), while untreated cells occupied only 57612% of the wound area in that time (Fig 1A, f). Quantitative analysis of the wound-healing assay represented in Figure 1C revealed that UTP treatment increases Schwann cell velocity 2.1fold (from 1.760.5% to 3.660.2% of occupancy/h) for primary Schwann cells and a 1.6-fold increase (from 2.960.5% to 4.560.2% of occupancy/h) for the RT4-D6P2T cell line.

MMP-2 activation by UTP is essential for Schwann cell wound repair
MMPs in their active form have the potential to promote cell migration and are involved in wound repair [16]. In order to determine whether UTP is able to induce MMP activation, the activity of MMP-2 and MMP-9 in the conditioned media was studied using gelatin zymography in RT4-D6P2T cells after UTP treatment (250 mM). A time-response study revealed that MMP-2 becomes active (proteolytically cleaved) after 12 h to 24 h of UTP treatment, whereas MMP-9 was not detected at any time during the study (Fig. 2A). In the case of MMP-2, the pro-form of the protein (67 kDa) showed similar levels in both controls and UTPconditioned media after 12 h to 24 h of incubation. By contrast, the activity of the cleaved form of MMP-2 (60 kDa) appeared to be clearly enhanced in UTP-conditioned medium ( Fig. 2A). After assessing UTP-induced MMP-2 activation in RT4-D6P2T cells, we investigated whether MMP-2 activation was induced by wound. Interestingly, gelatin zymography experiments showed that MMP-2 was activated during wound healing (Fig. 2B), with increased activation in the presence of UTP (250 mM, 12 h). This amplified signal of the active form of MMP-2 was observed in the Schwann cell line as well as the primary Schwann cell culture (Fig. 2B). These results suggest that either the UTP nucleotide or the conditioned medium from wounded cultures induce the activation of MMP-2. The mRNA levels in RT4-D6P2T and primary Schwann cells were also evaluated by quantitative RT-PCR. Results showed that UTP significantly increases MMP-2 expression in both cell models after 6-24 hours of treatment (Fig.  S2).
Immunocytochemistry studies corroborated that MMP-2 protein expression is increased in primary Schwann cells (Fig. 2C, b) treated with UTP (250 mM, 24 h) compared to control cells (Fig. 2C, a). Quantitative analysis showed that the MMP-2 immunofluorescence intensity was significantly increased (p = 0.018) in UTP-treated cells compared to untreated cells (Fig. 2C). Furthermore, endpoint analysis of fluorescencequenched gelatin degradation in RT4-D6P2T cells revealed a significant increase (p = 0.002) in the number of MMP-2 positive cells in UTP-treated cells compared to untreated control cells ( Figure 2D). These findings are in agreement with the results obtained by gelatin zymography and gene expression analysis and corroborate that UTP enhances MMP-2 activation and expression.
Finally, to evaluate the role of activated MMP-2 in Schwannoma cell wound repair, a short hairpin RNA (shRNA) directed against the rat MMP-2 gene (shMMP-2) was developed. Following shMMP-2 transfection, substantial knockdown of the MMP-2 expression was observed in RT4-D6P2T cells at both mRNA (81.360.2% decrease) and protein (52.360.7% decrease) levels compared with transfected (shRandom) and nontransfected control cells (see Supporting Information, Fig. S1). Wound healing and zymography assays showed a significant decrease in the rate of migration (P#0.001; Fig. 3A) and MMP-2 activation (P#0.05; Fig. 3B) in MMP-2-silenced RT4-D6P2T cells when compared to control cells. Altogether, these results strongly suggest that UTPactivated MMP-2 plays an important role in Schwann cell wound repair.

P2Y 2 receptor is involved in Schwannoma cell wound repair and MMP-2 activation
The expression of the uridine-sensitive P2Y receptors (P2Y 2 , P2Y 4 and P2Y 6 ) was determined by RT-PCR in RT4-D6P2T cells and in primary Schwann cell culture. As shown in Figure 4A, three DNA fragments of 339, 377, and 450 bp were amplified. These fragments matched the predicted sizes of the amplifications products for the P2Y 2 , P2Y 4 and P2Y 6 receptor subtypes, respectively. Given that P2Y 2 receptor has been involved in cell migration of several cell types, their implication in Schwann cell wound repair was studied by using a shRNA directed against the P2Y 2 gene (shP2Y 2 ). A significant decrease in the rate of migration (P#0.001; Fig. 4B) and MMP-2 activity (P#0.01; Fig. 4C) in P2Y 2 -silenced RT4-D6P2T cells was observed by wound healing and zymography assays. This data suggest the involvement of P2Y 2 receptor in the UTP-induced Schwann cell migration and MMP-2 activation.
To investigate whether the signaling pathway through P2Y receptors is implicated in Schwannoma cell migration and MMP-2 activation, and given that P2Y receptors have been reported to induce MAPK activity [25,44], we determined whether the P2Y-MAPK pathway mediated UTP-induced migration in Schwann cell line. RT4-D6P2T cells were pre-incubated for 20 min before UTP (250 mM) stimulation with various selective inhibitors: a) suramin, an antagonist at P2Y 1, 2, 3, 6, 11 receptors [45,46]; b) U0126, an inhibitor of MEK1/2 (upstream ERK1/2 kinase); c) SB203580, an inhibitor of P38; d) SP600125, an inhibitor of JNK; and e) the broad-spectrum MMP inhibitor GM6001. Results revealed a significant decrease (P#0.001) in the rate of cell migration ( Figure 5A) and MMP-2 activation ( Figure 5B) after UTP treatment when cells were pre-incubated with 1 of the inhibitors, in comparison with UTP-treated cells.

UTP induces biphasic MAPK phosphorylation
To further study UTP stimulation of MAPKs, time-course phosphorylation was investigated. RT4-D6P2T cells were treated with UTP and cell lysates were collected after various time intervals. Equal amounts of protein were resolved in SDS-PAGE and blotted with MAPK antibodies against phospho-JNK, phospho-ERK1/2, and phospho-P38. The same membranes were also blotted with antibodies against total (active and non-active) forms of MAPKs (JNK, ERK1/2 and P38). Stimulation of RT4-D6P2T cells with UTP (250 mM) resulted in a transient increase in all phosphorylated MAPK levels at 5 min (Fig. 6A: early phosphorylation), which decreased to near baseline levels at approximately 45 to 120 min after stimulation. A second wave of increased phosphorylated MAPK levels appeared at approximately 6 to 12 h and was sustained for up to 24 h (Fig. 6A: late phosphorylation). It was previously described that suramin was able to block the early UTP-induced phosphorylation [47]. Our data show that suramin pretreatment also blocked the late UTPinduced late phosphorylation (Fig 6B). These results suggest that UTP activates P2Y 2 receptors, initiating downstream signaling cascades and resulting in biphasic MAPK phosphorylation.

MMP-2 activation mediates late MAPK phosphorylation
To determine a possible relationship between MAPK phosphorylation and MMP-2 activation, MMP-2-silenced RT4-D6P2T cells were treated with UTP (250 mM) and cell lysates were collected at different times. Next, western blotting for 3 phosphorylated MAPKs was performed. Results showed that while early phosphorylation is similar to that in control cells, late MAPK phosphorylation is significantly downregulated in MMP-2silenced cells (Fig. 7A). To confirm the involvement of active MMP-2 in late MAPK phosphorylation, RT4-D6P2T cells were stimulated with UTP in the absence or presence of a broadspectrum MMP inhibitor (10 mM, GM6001) and MAPK phosphorylation levels were measured in cell extracts collected 5 min (early phosphorylation; Fig. 7B) or 12 h (late phosphorylation; Fig. 7C) after UTP treatment. GM6001 treatment did not inhibit the early increase but significantly (P#0.05) reduced the UTPstimulated late increase in phosphorylated MAPK levels. These results suggest that a UTP-induced late increase in MAPK phosphorylation is MMP-2-dependent.

Discussion
Growing evidence suggests that nucleotides that are released upon injury stimulate nucleotide P2 receptors and serve as endogenous signals to induce a rapid wound healing response in glial cells [48,49,50]. In analogy with other G-protein-coupled receptors (GPCR), a cross-talk between P2Y and growth factor receptors may occur at different levels of the signal transduction pathway depending on receptor subtypes and the cell type and their respective environments [51,52]. Extracellular nucleotides are known to function as chemotactic agents for microglial cells, smooth muscle cells, and epithelial cells [22,24,40,53]. However, these studies did not explain the implication of the nucleotide UTP, a selective P2Y agonist, in injured Schwann cells. We hypothesize that UTP secreted to the medium during nerve injury binds P2Y receptors in the neighboring Schwann cells to induce MMP-2 activity and subsequent cell migration and wound recovery.
In the PNS, MMP-2 activity has been shown to increase after nerve injury and is associated with cell migration, axonal outgrowth, nerve regeneration, and remyelination [14,17,54]. Our results demonstrate that cell migration and wound repair significantly increased after UTP treatment in Schwann cells, and that the velocity of these processes depends directly on MMP-2 activation. We have also described that UTP significantly increases MMP-2 gene expression and that knockdown of MMP-2 is  enough to completely suppress UTP-induced cell migration and MMP-2 activation in the extracellular media. Intracellular calcium measurements clearly indicate that UTPrecognizing P2Y 2 and P2Y 4 receptors are functional in Schwann cells [41]. Given that UTP is hydrolyzed by ectonucleotidases to UDP, which is an agonist at P2Y 6 receptor, we first identified the P2Y receptor subtype that mediates Schwann cell migration and MMP-2 activation. To that end, we used a pharmacological approach (P2Y 2 is suramin-sensitive) and P2Y 2 -knowdown of Schwannoma cells, which suggested a predominant role of P2Y 2 receptors in this migratory effect. Although UTP and ATP are equivalent in stimulating P2Y 2 receptor, we decided to study the effect of UTP in order to avoid possible cross-signalling pathway induced by the activation of P2X receptors by ATP [55]. Then, our findings are consistent with previous reports demonstrating that P2Y 2 receptors are involved in cell migration in several cell types, providing further evidence of the functional role of P2Y 2 receptor during injury [56,57,58,59,60]. Indeed, P2Y 2 receptors have been shown to be upregulated in a variety of tissues in response to injury [61]. All together, our data support the hypothesis that activation of P2Y 2 receptors by extracellular UTP might regulate a reparative mechanism in response to wound injury.
Our previous work, and that of other authors, showed that P2Y receptor activation by UTP increases the expression of Ncadherin, an adhesion molecule related to Schwann cell migration, and that this increase is mediated by MAPK activation [47,62]. In the present study, we also demonstrate that UTP-induced MMP-2 activation is dependent on MAPK signaling pathways. Indeed, after nerve injury, increased activity in multiple pathwaysincluding the ERK/MAPK, JNK/c-Jun, Notch, and JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathways-can be detected in Schwann cells [63,64]. Furthermore, some authors suggest that ERK and JNK activity defines the state of Schwann cell differentiation. Whereas basal levels are necessary for the differentiation of precursors, elevated ERK and JNK activities drive dedifferentiation and migration [65,66,67,68]. By contrast, the role of p38 MAPK in Schwann cells is controversial; it has been associated with the regulation of myelination [69], but also with a negative regulatory role in differentiation and myelination [70].
As previously described in many growth factors and cytokines, UTP treatment also results in a biphasic phosphorylation of MAPKs [71,72]. This kinetic profile has recently been proposed as a model to allow a common signaling system to play distinct regulatory roles depending on the temporal duration of activation [73]. Although P2Y activation by UTP is well known to be involved in early MAPK phosphorylation, the role of UTP in late MAPK phosphorylation has not been described until now. Our data suggests that the initial transient phase of MAPK activation in Schwannoma cells is mediated by P2Y receptors [25,47], whereas the late sustained phosphorylation is also mediated by MMP-2 activation. One possible explanation for late MAPK phosphorylation is that active MMP-2 in the extracellular space cleaves a membrane-anchored growth factor on Schwannoma cells. Many proteins released by proteolysis of the ECM proteins are known to control cell response and may act as effectors for Schwann cell migration [74]. Multiple lines of evidence indicate that growth factors, such as neuregulin-1 (NRG1), are involved in Schwann cell migration and myelination [75]. Furthermore, NRG1 expression and its putative receptors, ErbB2 and ErbB3, are known to be induced in injured sciatic nerves in rats, promoting Schwann cell proliferation and dedifferentiation in injured nerves [76,77]. Thus, the cleavage of a growth factor (NRG1 type III) by MMP-2 and the subsequent phosphorylation of the ErbB transmembrane tyrosine kinase receptor could explain the late MAPK phosphorylation observed in Schwannoma cells after UTP treatment. This proposed hypothesis is based on previous observations demonstrating that MMP-9 regulates MAPK pathways through IGF-1 and ErbB receptors [19]. Nevertheless, the relationship between MMP-2 activation and late MAPK   (30 mg) were resolved in SDS-PAGE. Western blots were performed using antibodies against phosphorylated and total MAPK (ERK1/2, JNK, and P38). The ratio of phosphorylated MAPK to total MAPK was calculated by densitometry in each sample, and a value of 1 was given to the control cells. Representative western blots for each kinase are shown above the graphs. Blots are representative of 3 independent experiments. Statistical significance: *P#0.05 compared to control cells. (B) RT4-D6P2T cells were preincubated (30 min) with suramin (100 mM; P2Y 2 receptor antagonist). After UTP treatment (12 h, 250 mM), the ratio of phosphorylated MAPK to total MAPK was calculated by densitometry in each sample. Representative images are shown below the corresponding graphs. Each bar represents the mean 6 SD using 3 independent experiments. Statistical significance: ***P#0.001 when compared to control cells; ##P#0.005, and ###P#0.001 when compared to UTP-treated cells. doi:10.1371/journal.pone.0098998.g006 In light of these findings, we conclude that extracellular UTP (which mimics nerve injury) stimulates Schwannoma cell migration and wound repair through a MMP-2-dependent mechanism via P2Y 2 receptors and the activation of MAPK pathways. Moreover, evidence that P2Y 2 receptors mediate the migratory response of Schwann cells evoked by PNS injury indicates that selective P2Y 2 receptor agonists may provide new therapeutic opportunities for Schwann cell migration and peripheral nerve regeneration.