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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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UTP enhances Schwann cell migration and wound repair.(A) Transwell migration assay for RT4-D6P2T cells. Cells were seeded in the upper side of a transwell membrane and treated with UTP (250 µM) at different times (2, 4, 6, 8, 10, and 12 h), or incubated for 12 h with various UTP concentrations (0, 10, 100, 250, 500, and 1000 µM). Representative images (objective magnification ×10) of the dose–response and time–course transwell migration studies are shown. Migrated cells were stained with crystal violet and counted. Data were expressed as the fold increases of cell migration when compared to untreated cells. Values were calculated as the mean ± SD using 3 independent experiments. Statistical significance: *P≤0.05, **P≤0.01, and *** P≤0.001. (B) Wound-healing assay for Schwann cell line and for primary Schwann cells. Monolayer cells for both Schwann cultures were scraped (a, c, e, and g) and either untreated (b and f) or treated with 250 µM UTP (d and h). Micrographs are representative of at least 3 independent experiments (objective magnification ×10). (C) Quantitative analysis of the rate of migration (velocity) calculated as percentage of wound occupancy per hour. Values were calculated as the mean ± SD of 3 independent experiments. Statistical significance: *** P≤0.001.
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pone-0098998-g001: UTP enhances Schwann cell migration and wound repair.(A) Transwell migration assay for RT4-D6P2T cells. Cells were seeded in the upper side of a transwell membrane and treated with UTP (250 µM) at different times (2, 4, 6, 8, 10, and 12 h), or incubated for 12 h with various UTP concentrations (0, 10, 100, 250, 500, and 1000 µM). Representative images (objective magnification ×10) of the dose–response and time–course transwell migration studies are shown. Migrated cells were stained with crystal violet and counted. Data were expressed as the fold increases of cell migration when compared to untreated cells. Values were calculated as the mean ± SD using 3 independent experiments. Statistical significance: *P≤0.05, **P≤0.01, and *** P≤0.001. (B) Wound-healing assay for Schwann cell line and for primary Schwann cells. Monolayer cells for both Schwann cultures were scraped (a, c, e, and g) and either untreated (b and f) or treated with 250 µM UTP (d and h). Micrographs are representative of at least 3 independent experiments (objective magnification ×10). (C) Quantitative analysis of the rate of migration (velocity) calculated as percentage of wound occupancy per hour. Values were calculated as the mean ± SD of 3 independent experiments. Statistical significance: *** P≤0.001.

Mentions: 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 µM). 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 µM) 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 µM).


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)

UTP enhances Schwann cell migration and wound repair.(A) Transwell migration assay for RT4-D6P2T cells. Cells were seeded in the upper side of a transwell membrane and treated with UTP (250 µM) at different times (2, 4, 6, 8, 10, and 12 h), or incubated for 12 h with various UTP concentrations (0, 10, 100, 250, 500, and 1000 µM). Representative images (objective magnification ×10) of the dose–response and time–course transwell migration studies are shown. Migrated cells were stained with crystal violet and counted. Data were expressed as the fold increases of cell migration when compared to untreated cells. Values were calculated as the mean ± SD using 3 independent experiments. Statistical significance: *P≤0.05, **P≤0.01, and *** P≤0.001. (B) Wound-healing assay for Schwann cell line and for primary Schwann cells. Monolayer cells for both Schwann cultures were scraped (a, c, e, and g) and either untreated (b and f) or treated with 250 µM UTP (d and h). Micrographs are representative of at least 3 independent experiments (objective magnification ×10). (C) Quantitative analysis of the rate of migration (velocity) calculated as percentage of wound occupancy per hour. Values were calculated as the mean ± SD of 3 independent experiments. Statistical significance: *** P≤0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098998-g001: UTP enhances Schwann cell migration and wound repair.(A) Transwell migration assay for RT4-D6P2T cells. Cells were seeded in the upper side of a transwell membrane and treated with UTP (250 µM) at different times (2, 4, 6, 8, 10, and 12 h), or incubated for 12 h with various UTP concentrations (0, 10, 100, 250, 500, and 1000 µM). Representative images (objective magnification ×10) of the dose–response and time–course transwell migration studies are shown. Migrated cells were stained with crystal violet and counted. Data were expressed as the fold increases of cell migration when compared to untreated cells. Values were calculated as the mean ± SD using 3 independent experiments. Statistical significance: *P≤0.05, **P≤0.01, and *** P≤0.001. (B) Wound-healing assay for Schwann cell line and for primary Schwann cells. Monolayer cells for both Schwann cultures were scraped (a, c, e, and g) and either untreated (b and f) or treated with 250 µM UTP (d and h). Micrographs are representative of at least 3 independent experiments (objective magnification ×10). (C) Quantitative analysis of the rate of migration (velocity) calculated as percentage of wound occupancy per hour. Values were calculated as the mean ± SD of 3 independent experiments. Statistical significance: *** P≤0.001.
Mentions: 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 µM). 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 µM) 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 µM).

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