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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.(A) RT-PCR amplification with specific primers against P2Y2, P2Y4, and P2Y6 receptor subtypes was performed in RT4-D6P2T cells and in primary Schwann cells after the isolation of total RNA. PCR products were separated on a 1% agarose gel and visualized with ethidium bromide. (B-C) Wound healing and gelatin zymograms of RT4-D6P2T cells transfected with shRNA directed against the P2Y2 gene (shP2Y2) and control cells (non-transfected cells or cells transfected with shRandom sequence). Representative images (objective magnification ×10) of wound healing and gelatin zymograms and quantitative analysis of the rate of migration (velocity) and MMP-2 activity are shown. Values were calculated as the mean ± SD using 3 independent experiments. Statistical significance: **P≤0.01 and ***P≤0.001 when compared to control cells; ##P≤0.01 and ###P≤0.001 when compared to UTP-treated cells.
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pone-0098998-g004: P2Y2 receptors are necessary for Schwann cell line wound repair.(A) RT-PCR amplification with specific primers against P2Y2, P2Y4, and P2Y6 receptor subtypes was performed in RT4-D6P2T cells and in primary Schwann cells after the isolation of total RNA. PCR products were separated on a 1% agarose gel and visualized with ethidium bromide. (B-C) Wound healing and gelatin zymograms of RT4-D6P2T cells transfected with shRNA directed against the P2Y2 gene (shP2Y2) and control cells (non-transfected cells or cells transfected with shRandom sequence). Representative images (objective magnification ×10) of wound healing and gelatin zymograms and quantitative analysis of the rate of migration (velocity) and MMP-2 activity are shown. Values were calculated as the mean ± SD using 3 independent experiments. Statistical significance: **P≤0.01 and ***P≤0.001 when compared to control cells; ##P≤0.01 and ###P≤0.001 when compared to UTP-treated cells.

Mentions: The expression of the uridine-sensitive P2Y receptors (P2Y2, P2Y4 and P2Y6) 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 P2Y2, P2Y4 and P2Y6 receptor subtypes, respectively. Given that P2Y2 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 P2Y2 gene (shP2Y2). A significant decrease in the rate of migration (P≤0.001; Fig. 4B) and MMP-2 activity (P≤0.01; Fig. 4C) in P2Y2-silenced RT4-D6P2T cells was observed by wound healing and zymography assays. This data suggest the involvement of P2Y2 receptor in the UTP-induced Schwann cell migration and MMP-2 activation.


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.(A) RT-PCR amplification with specific primers against P2Y2, P2Y4, and P2Y6 receptor subtypes was performed in RT4-D6P2T cells and in primary Schwann cells after the isolation of total RNA. PCR products were separated on a 1% agarose gel and visualized with ethidium bromide. (B-C) Wound healing and gelatin zymograms of RT4-D6P2T cells transfected with shRNA directed against the P2Y2 gene (shP2Y2) and control cells (non-transfected cells or cells transfected with shRandom sequence). Representative images (objective magnification ×10) of wound healing and gelatin zymograms and quantitative analysis of the rate of migration (velocity) and MMP-2 activity are shown. Values were calculated as the mean ± SD using 3 independent experiments. Statistical significance: **P≤0.01 and ***P≤0.001 when compared to control cells; ##P≤0.01 and ###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-g004: P2Y2 receptors are necessary for Schwann cell line wound repair.(A) RT-PCR amplification with specific primers against P2Y2, P2Y4, and P2Y6 receptor subtypes was performed in RT4-D6P2T cells and in primary Schwann cells after the isolation of total RNA. PCR products were separated on a 1% agarose gel and visualized with ethidium bromide. (B-C) Wound healing and gelatin zymograms of RT4-D6P2T cells transfected with shRNA directed against the P2Y2 gene (shP2Y2) and control cells (non-transfected cells or cells transfected with shRandom sequence). Representative images (objective magnification ×10) of wound healing and gelatin zymograms and quantitative analysis of the rate of migration (velocity) and MMP-2 activity are shown. Values were calculated as the mean ± SD using 3 independent experiments. Statistical significance: **P≤0.01 and ***P≤0.001 when compared to control cells; ##P≤0.01 and ###P≤0.001 when compared to UTP-treated cells.
Mentions: The expression of the uridine-sensitive P2Y receptors (P2Y2, P2Y4 and P2Y6) 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 P2Y2, P2Y4 and P2Y6 receptor subtypes, respectively. Given that P2Y2 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 P2Y2 gene (shP2Y2). A significant decrease in the rate of migration (P≤0.001; Fig. 4B) and MMP-2 activity (P≤0.01; Fig. 4C) in P2Y2-silenced RT4-D6P2T cells was observed by wound healing and zymography assays. This data suggest the involvement of P2Y2 receptor in the UTP-induced Schwann cell migration and MMP-2 activation.

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