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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 induces biphasic MAPK phosphorylation.(A) Western blot analysis of time–course MAPK phosphorylation induced by UTP. RT4-D6P2T cells were incubated with UTP (250 µM) at the indicated times and equal amounts of protein (30 µg) were resolved in SDS-PAGE. Western blots were performed using antibodies against phosphorylated and total MAPK (ERK1/2, JNK, and P38). The ratio of phosphorylated MAPK to total MAPK was calculated by densitometry in each sample, and a value of 1 was given to the control cells. Representative western blots for each kinase are shown above the graphs. Blots are representative of 3 independent experiments. Statistical significance: *P≤0.05 compared to control cells. (B) RT4-D6P2T cells were preincubated (30 min) with suramin (100 µM; P2Y2 receptor antagonist). After UTP treatment (12 h, 250 µM), the ratio of phosphorylated MAPK to total MAPK was calculated by densitometry in each sample. 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 control cells; ##P≤0.005, and ###P≤0.001 when compared to UTP-treated cells.
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pone-0098998-g006: UTP induces biphasic MAPK phosphorylation.(A) Western blot analysis of time–course MAPK phosphorylation induced by UTP. RT4-D6P2T cells were incubated with UTP (250 µM) at the indicated times and equal amounts of protein (30 µg) were resolved in SDS-PAGE. Western blots were performed using antibodies against phosphorylated and total MAPK (ERK1/2, JNK, and P38). The ratio of phosphorylated MAPK to total MAPK was calculated by densitometry in each sample, and a value of 1 was given to the control cells. Representative western blots for each kinase are shown above the graphs. Blots are representative of 3 independent experiments. Statistical significance: *P≤0.05 compared to control cells. (B) RT4-D6P2T cells were preincubated (30 min) with suramin (100 µM; P2Y2 receptor antagonist). After UTP treatment (12 h, 250 µM), the ratio of phosphorylated MAPK to total MAPK was calculated by densitometry in each sample. 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 control cells; ##P≤0.005, and ###P≤0.001 when compared to UTP-treated cells.

Mentions: To further study UTP stimulation of MAPKs, time–course phosphorylation was investigated. RT4-D6P2T cells were treated with UTP and cell lysates were collected after various time intervals. Equal amounts of protein were resolved in SDS-PAGE and blotted with MAPK antibodies against phospho-JNK, phospho-ERK1/2, and phospho-P38. The same membranes were also blotted with antibodies against total (active and non-active) forms of MAPKs (JNK, ERK1/2 and P38). Stimulation of RT4-D6P2T cells with UTP (250 µM) resulted in a transient increase in all phosphorylated MAPK levels at 5 min (Fig. 6A: early phosphorylation), which decreased to near baseline levels at approximately 45 to 120 min after stimulation. A second wave of increased phosphorylated MAPK levels appeared at approximately 6 to 12 h and was sustained for up to 24 h (Fig. 6A: late phosphorylation). It was previously described that suramin was able to block the early UTP-induced phosphorylation [47]. Our data show that suramin pretreatment also blocked the late UTP-induced late phosphorylation (Fig 6B). These results suggest that UTP activates P2Y2 receptors, initiating downstream signaling cascades and resulting in biphasic MAPK phosphorylation.


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 induces biphasic MAPK phosphorylation.(A) Western blot analysis of time–course MAPK phosphorylation induced by UTP. RT4-D6P2T cells were incubated with UTP (250 µM) at the indicated times and equal amounts of protein (30 µg) were resolved in SDS-PAGE. Western blots were performed using antibodies against phosphorylated and total MAPK (ERK1/2, JNK, and P38). The ratio of phosphorylated MAPK to total MAPK was calculated by densitometry in each sample, and a value of 1 was given to the control cells. Representative western blots for each kinase are shown above the graphs. Blots are representative of 3 independent experiments. Statistical significance: *P≤0.05 compared to control cells. (B) RT4-D6P2T cells were preincubated (30 min) with suramin (100 µM; P2Y2 receptor antagonist). After UTP treatment (12 h, 250 µM), the ratio of phosphorylated MAPK to total MAPK was calculated by densitometry in each sample. 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 control cells; ##P≤0.005, and ###P≤0.001 when compared to UTP-treated cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048211&req=5

pone-0098998-g006: UTP induces biphasic MAPK phosphorylation.(A) Western blot analysis of time–course MAPK phosphorylation induced by UTP. RT4-D6P2T cells were incubated with UTP (250 µM) at the indicated times and equal amounts of protein (30 µg) were resolved in SDS-PAGE. Western blots were performed using antibodies against phosphorylated and total MAPK (ERK1/2, JNK, and P38). The ratio of phosphorylated MAPK to total MAPK was calculated by densitometry in each sample, and a value of 1 was given to the control cells. Representative western blots for each kinase are shown above the graphs. Blots are representative of 3 independent experiments. Statistical significance: *P≤0.05 compared to control cells. (B) RT4-D6P2T cells were preincubated (30 min) with suramin (100 µM; P2Y2 receptor antagonist). After UTP treatment (12 h, 250 µM), the ratio of phosphorylated MAPK to total MAPK was calculated by densitometry in each sample. 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 control cells; ##P≤0.005, and ###P≤0.001 when compared to UTP-treated cells.
Mentions: To further study UTP stimulation of MAPKs, time–course phosphorylation was investigated. RT4-D6P2T cells were treated with UTP and cell lysates were collected after various time intervals. Equal amounts of protein were resolved in SDS-PAGE and blotted with MAPK antibodies against phospho-JNK, phospho-ERK1/2, and phospho-P38. The same membranes were also blotted with antibodies against total (active and non-active) forms of MAPKs (JNK, ERK1/2 and P38). Stimulation of RT4-D6P2T cells with UTP (250 µM) resulted in a transient increase in all phosphorylated MAPK levels at 5 min (Fig. 6A: early phosphorylation), which decreased to near baseline levels at approximately 45 to 120 min after stimulation. A second wave of increased phosphorylated MAPK levels appeared at approximately 6 to 12 h and was sustained for up to 24 h (Fig. 6A: late phosphorylation). It was previously described that suramin was able to block the early UTP-induced phosphorylation [47]. Our data show that suramin pretreatment also blocked the late UTP-induced late phosphorylation (Fig 6B). These results suggest that UTP activates P2Y2 receptors, initiating downstream signaling cascades and resulting in biphasic MAPK phosphorylation.

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