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Uridine 5'-triphosphate promotes in vitro Schwannoma cell migration through matrix metalloproteinase-2 activation.

Lamarca A, Gella A, Martiañez T, Segura M, Figueiro-Silva J, Grijota-Martinez C, Trullas R, Casals N - PLoS ONE (2014)

Bottom Line: Inhibition of these MAPK pathways decreased both MMP-2 activation and cell migration.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Sciences, Facultat de Medicina, Universitat Internacional de Catalunya, Sant Cugat del Vallès, Spain.

ABSTRACT
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.

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P2Y2 receptors are necessary for Schwann cell line wound repair.RT4-D6P2T cells were preincubated (30 min) with selective inhibitors of different proteins involved in the P2Y receptor signaling pathway: suramin (100 µM; P2Y2 receptor antagonist), SP600125 (20 µM; JNK inhibitor), SB203580 (10 µM; P38 inhibitor), U0126 (10 µM; ERK inhibitor), and GM6001 (10 µM; MMP inhibitor). After UTP treatment (12 h, 250 µM), the rate of migration and MMP-2 activity were determined. Representative images are shown below the corresponding graphs. Each bar represents the mean ± SD using 3 independent experiments. Statistical significance: ***P≤0.001 when compared to UTP-treated cells.
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pone-0098998-g005: P2Y2 receptors are necessary for Schwann cell line wound repair.RT4-D6P2T cells were preincubated (30 min) with selective inhibitors of different proteins involved in the P2Y receptor signaling pathway: suramin (100 µM; P2Y2 receptor antagonist), SP600125 (20 µM; JNK inhibitor), SB203580 (10 µM; P38 inhibitor), U0126 (10 µM; ERK inhibitor), and GM6001 (10 µM; MMP inhibitor). After UTP treatment (12 h, 250 µM), the rate of migration and MMP-2 activity were determined. Representative images are shown below the corresponding graphs. Each bar represents the mean ± SD using 3 independent experiments. Statistical significance: ***P≤0.001 when compared to UTP-treated cells.

Mentions: 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 µM) stimulation with various selective inhibitors: a) suramin, an antagonist at P2Y1, 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.


Uridine 5'-triphosphate promotes in vitro Schwannoma cell migration through matrix metalloproteinase-2 activation.

Lamarca A, Gella A, Martiañez T, Segura M, Figueiro-Silva J, Grijota-Martinez C, Trullas R, Casals N - PLoS ONE (2014)

P2Y2 receptors are necessary for Schwann cell line wound repair.RT4-D6P2T cells were preincubated (30 min) with selective inhibitors of different proteins involved in the P2Y receptor signaling pathway: suramin (100 µM; P2Y2 receptor antagonist), SP600125 (20 µM; JNK inhibitor), SB203580 (10 µM; P38 inhibitor), U0126 (10 µM; ERK inhibitor), and GM6001 (10 µM; MMP inhibitor). After UTP treatment (12 h, 250 µM), the rate of migration and MMP-2 activity were determined. Representative images are shown below the corresponding graphs. Each bar represents the mean ± SD using 3 independent experiments. Statistical significance: ***P≤0.001 when compared to UTP-treated cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048211&req=5

pone-0098998-g005: P2Y2 receptors are necessary for Schwann cell line wound repair.RT4-D6P2T cells were preincubated (30 min) with selective inhibitors of different proteins involved in the P2Y receptor signaling pathway: suramin (100 µM; P2Y2 receptor antagonist), SP600125 (20 µM; JNK inhibitor), SB203580 (10 µM; P38 inhibitor), U0126 (10 µM; ERK inhibitor), and GM6001 (10 µM; MMP inhibitor). After UTP treatment (12 h, 250 µM), the rate of migration and MMP-2 activity were determined. Representative images are shown below the corresponding graphs. Each bar represents the mean ± SD using 3 independent experiments. Statistical significance: ***P≤0.001 when compared to UTP-treated cells.
Mentions: 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 µM) stimulation with various selective inhibitors: a) suramin, an antagonist at P2Y1, 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.

Bottom Line: Inhibition of these MAPK pathways decreased both MMP-2 activation and cell migration.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Sciences, Facultat de Medicina, Universitat Internacional de Catalunya, Sant Cugat del Vallès, Spain.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus