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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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MMP-2 activation mediates late MAPK phosphorylation.(A) Western blot analysis of time–course MAPK phosphorylation induced by UTP. RT4-D6P2T cells transfected with shRNA against MMP-2 were incubated with UTP (250 µM) at the indicated times. (B-C) Schwannoma cells were preincubated for 30 min with a broad-spectrum MMP inhibitor (GM6001, 10 µM) and then incubated with UTP (250 µM) at either 5 min (early phosphorylation) or 12 hours (late phosphorylation). Proteins from cell lysates (30 µg) were resolved by SDS-PAGE and blotted against either phosphorylated or total MAPKs. Representative western blots for each kinase are shown above the graphs. Blots are representative of 3 independent experiments. Statistical significance: *P≤0.05, **P≤0.005, and ***P≤0.001 when compared to control cells; #P≤0.05, ##P≤0.005, and ###P≤0.001 when compared to UTP-treated cells.
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pone-0098998-g007: MMP-2 activation mediates late MAPK phosphorylation.(A) Western blot analysis of time–course MAPK phosphorylation induced by UTP. RT4-D6P2T cells transfected with shRNA against MMP-2 were incubated with UTP (250 µM) at the indicated times. (B-C) Schwannoma cells were preincubated for 30 min with a broad-spectrum MMP inhibitor (GM6001, 10 µM) and then incubated with UTP (250 µM) at either 5 min (early phosphorylation) or 12 hours (late phosphorylation). Proteins from cell lysates (30 µg) were resolved by SDS-PAGE and blotted against either phosphorylated or total MAPKs. Representative western blots for each kinase are shown above the graphs. Blots are representative of 3 independent experiments. Statistical significance: *P≤0.05, **P≤0.005, and ***P≤0.001 when compared to control cells; #P≤0.05, ##P≤0.005, and ###P≤0.001 when compared to UTP-treated cells.

Mentions: To determine a possible relationship between MAPK phosphorylation and MMP-2 activation, MMP-2-silenced RT4-D6P2T cells were treated with UTP (250 µM) 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-2-silenced 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 broad-spectrum MMP inhibitor (10 µM, 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 UTP-stimulated late increase in phosphorylated MAPK levels. These results suggest that a UTP-induced late increase in MAPK phosphorylation is MMP-2-dependent.


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)

MMP-2 activation mediates late MAPK phosphorylation.(A) Western blot analysis of time–course MAPK phosphorylation induced by UTP. RT4-D6P2T cells transfected with shRNA against MMP-2 were incubated with UTP (250 µM) at the indicated times. (B-C) Schwannoma cells were preincubated for 30 min with a broad-spectrum MMP inhibitor (GM6001, 10 µM) and then incubated with UTP (250 µM) at either 5 min (early phosphorylation) or 12 hours (late phosphorylation). Proteins from cell lysates (30 µg) were resolved by SDS-PAGE and blotted against either phosphorylated or total MAPKs. Representative western blots for each kinase are shown above the graphs. Blots are representative of 3 independent experiments. Statistical significance: *P≤0.05, **P≤0.005, and ***P≤0.001 when compared to control cells; #P≤0.05, ##P≤0.005, and ###P≤0.001 when compared to UTP-treated cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098998-g007: MMP-2 activation mediates late MAPK phosphorylation.(A) Western blot analysis of time–course MAPK phosphorylation induced by UTP. RT4-D6P2T cells transfected with shRNA against MMP-2 were incubated with UTP (250 µM) at the indicated times. (B-C) Schwannoma cells were preincubated for 30 min with a broad-spectrum MMP inhibitor (GM6001, 10 µM) and then incubated with UTP (250 µM) at either 5 min (early phosphorylation) or 12 hours (late phosphorylation). Proteins from cell lysates (30 µg) were resolved by SDS-PAGE and blotted against either phosphorylated or total MAPKs. Representative western blots for each kinase are shown above the graphs. Blots are representative of 3 independent experiments. Statistical significance: *P≤0.05, **P≤0.005, and ***P≤0.001 when compared to control cells; #P≤0.05, ##P≤0.005, and ###P≤0.001 when compared to UTP-treated cells.
Mentions: To determine a possible relationship between MAPK phosphorylation and MMP-2 activation, MMP-2-silenced RT4-D6P2T cells were treated with UTP (250 µM) 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-2-silenced 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 broad-spectrum MMP inhibitor (10 µM, 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 UTP-stimulated late increase in phosphorylated MAPK levels. These results suggest that a UTP-induced late increase in MAPK phosphorylation is MMP-2-dependent.

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