Statistical significance: ***P0.001 when compared to UTP-treated cells. UTP induces biphasic MAPK phosphorylation Alloxazine To further study UTP stimulation of MAPKs, timeCcourse phosphorylation was investigated. remodeling of the ECM during nerve degeneration and regeneration , , , . 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 usually delayed but managed during nerve regeneration proximally and distally to the injury site, suggesting that MMP-2 functions to facilitate axonal extension along the nerve matrix . In spinal cord injury, the same pattern is observed: MMP-9 activity is usually 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 , , . 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 . MMP-9 also activates the Akt/ERK pathway and promotes migration by binding to the low-density lipoprotein receptor-related protein . 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) . 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 , , , . After injury, nucleotides released from cells activate the purinergic receptor-signaling pathway to mediate the response to injury . Nucleotide binding to P2Y receptors, which are coupled to the protein Gq, activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-biphosphate (PIP2) to diacylglycerol (DAG) and phosphoinositol tri-phosphate (IP3), resulting MUC1 in the release of intracellular Ca2+ from endoplasmic reticulum stores. The increase in cytosolic Ca2+ 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 , . 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 , , , . Accumulating evidence suggests that these MAPKs play a role in the migration of various cell types , , , . Even though 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 5-triphosphate (UTP) on Schwannoma cell migration and wound repair and to establish whether MMP-2 is usually involved in this effect. For the first time, we statement that UTP stimulates Schwannoma cell migration and wound repair through a MMP-2-dependent mechanism via P2Y2 receptors and MAPK pathway activation. Materials and Methods Reagents Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin, streptomycin, and glutamine were purchased from PAA (Linz, Austria). Donor bovine serum (DBS) was purchased from Gibco (Rockville, MD, USA). Suramin, PBS, Hoechst 33342, trypan blue, forskolin, pituitary extract, protease and phosphatase inhibitor cocktails, SB203580, SP600125, U0126, and UTP were purchased from SigmaCAldrich (St Louis, MO, USA). GM6001 was purchased from Merck Millipore (Billerica, MA, USA). All other Alloxazine reagents used were of analytical grade. Schwann cell collection cultures The rat Alloxazine schwannoma cell collection RT4-D6P2T was purchased from the European Collection of Cell Cultures (#93011415; ECACC, Salisbury, UK) and managed 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% CO2 humidified atmosphere at 37C..