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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 regulates MMP-2 activity and expression.(A) Time–course gelatin zymography analysis for Schwann cell line. Conditioned media from 2.5×106 RT4-D6P2T cells that were either treated or untreated with UTP (250 µM) at different times (6, 12, and 24 hours) were loaded on SDS-PAGE gel containing gelatin. The zymogram is representative of 3 independent experiments. (B) Gelatin zymograms for a Schwann cell line and primary Schwann cells after UTP and wound healing treatments. Conditioned media from Schwann cells were collected after 12 h of UTP (250 µM) or wound healing treatments. Equal amounts of protein (20 µg) were loaded on SDS-PAGE gel containing gelatin. (C) Representative dual-fluorescence labeling of MMP-2 (green) and nuclei (blue) using specific antibody against MMP-2 and Hoechst staining and quantitative analysis for primary Schwann cells (a and b, objective magnification ×40). Scale bar: 50 µm. Statistical significance: *P≤0.05 compared to control cells. (D) In situ zymography images and quantitative analysis of gelatinase activity in RT4-D6P2T cells. Scale bar: 50 µm. Statistical significance: **P≤0.01 compared to control cells.
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pone-0098998-g002: UTP regulates MMP-2 activity and expression.(A) Time–course gelatin zymography analysis for Schwann cell line. Conditioned media from 2.5×106 RT4-D6P2T cells that were either treated or untreated with UTP (250 µM) at different times (6, 12, and 24 hours) were loaded on SDS-PAGE gel containing gelatin. The zymogram is representative of 3 independent experiments. (B) Gelatin zymograms for a Schwann cell line and primary Schwann cells after UTP and wound healing treatments. Conditioned media from Schwann cells were collected after 12 h of UTP (250 µM) or wound healing treatments. Equal amounts of protein (20 µg) were loaded on SDS-PAGE gel containing gelatin. (C) Representative dual-fluorescence labeling of MMP-2 (green) and nuclei (blue) using specific antibody against MMP-2 and Hoechst staining and quantitative analysis for primary Schwann cells (a and b, objective magnification ×40). Scale bar: 50 µm. Statistical significance: *P≤0.05 compared to control cells. (D) In situ zymography images and quantitative analysis of gelatinase activity in RT4-D6P2T cells. Scale bar: 50 µm. Statistical significance: **P≤0.01 compared to control cells.

Mentions: MMPs in their active form have the potential to promote cell migration and are involved in wound repair [16]. In order to determine whether UTP is able to induce MMP activation, the activity of MMP-2 and MMP-9 in the conditioned media was studied using gelatin zymography in RT4-D6P2T cells after UTP treatment (250 µM). A time–response study revealed that MMP-2 becomes active (proteolytically cleaved) after 12 h to 24 h of UTP treatment, whereas MMP-9 was not detected at any time during the study (Fig. 2A). In the case of MMP-2, the pro-form of the protein (67 kDa) showed similar levels in both controls and UTP-conditioned media after 12 h to 24 h of incubation. By contrast, the activity of the cleaved form of MMP-2 (60 kDa) appeared to be clearly enhanced in UTP-conditioned medium (Fig. 2A). After assessing UTP-induced MMP-2 activation in RT4-D6P2T cells, we investigated whether MMP-2 activation was induced by wound. Interestingly, gelatin zymography experiments showed that MMP-2 was activated during wound healing (Fig. 2B), with increased activation in the presence of UTP (250 µM, 12 h). This amplified signal of the active form of MMP-2 was observed in the Schwann cell line as well as the primary Schwann cell culture (Fig. 2B). These results suggest that either the UTP nucleotide or the conditioned medium from wounded cultures induce the activation of MMP-2. The mRNA levels in RT4-D6P2T and primary Schwann cells were also evaluated by quantitative RT-PCR. Results showed that UTP significantly increases MMP-2 expression in both cell models after 6-24 hours of treatment (Fig. S2).


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 regulates MMP-2 activity and expression.(A) Time–course gelatin zymography analysis for Schwann cell line. Conditioned media from 2.5×106 RT4-D6P2T cells that were either treated or untreated with UTP (250 µM) at different times (6, 12, and 24 hours) were loaded on SDS-PAGE gel containing gelatin. The zymogram is representative of 3 independent experiments. (B) Gelatin zymograms for a Schwann cell line and primary Schwann cells after UTP and wound healing treatments. Conditioned media from Schwann cells were collected after 12 h of UTP (250 µM) or wound healing treatments. Equal amounts of protein (20 µg) were loaded on SDS-PAGE gel containing gelatin. (C) Representative dual-fluorescence labeling of MMP-2 (green) and nuclei (blue) using specific antibody against MMP-2 and Hoechst staining and quantitative analysis for primary Schwann cells (a and b, objective magnification ×40). Scale bar: 50 µm. Statistical significance: *P≤0.05 compared to control cells. (D) In situ zymography images and quantitative analysis of gelatinase activity in RT4-D6P2T cells. Scale bar: 50 µm. Statistical significance: **P≤0.01 compared to control cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098998-g002: UTP regulates MMP-2 activity and expression.(A) Time–course gelatin zymography analysis for Schwann cell line. Conditioned media from 2.5×106 RT4-D6P2T cells that were either treated or untreated with UTP (250 µM) at different times (6, 12, and 24 hours) were loaded on SDS-PAGE gel containing gelatin. The zymogram is representative of 3 independent experiments. (B) Gelatin zymograms for a Schwann cell line and primary Schwann cells after UTP and wound healing treatments. Conditioned media from Schwann cells were collected after 12 h of UTP (250 µM) or wound healing treatments. Equal amounts of protein (20 µg) were loaded on SDS-PAGE gel containing gelatin. (C) Representative dual-fluorescence labeling of MMP-2 (green) and nuclei (blue) using specific antibody against MMP-2 and Hoechst staining and quantitative analysis for primary Schwann cells (a and b, objective magnification ×40). Scale bar: 50 µm. Statistical significance: *P≤0.05 compared to control cells. (D) In situ zymography images and quantitative analysis of gelatinase activity in RT4-D6P2T cells. Scale bar: 50 µm. Statistical significance: **P≤0.01 compared to control cells.
Mentions: MMPs in their active form have the potential to promote cell migration and are involved in wound repair [16]. In order to determine whether UTP is able to induce MMP activation, the activity of MMP-2 and MMP-9 in the conditioned media was studied using gelatin zymography in RT4-D6P2T cells after UTP treatment (250 µM). A time–response study revealed that MMP-2 becomes active (proteolytically cleaved) after 12 h to 24 h of UTP treatment, whereas MMP-9 was not detected at any time during the study (Fig. 2A). In the case of MMP-2, the pro-form of the protein (67 kDa) showed similar levels in both controls and UTP-conditioned media after 12 h to 24 h of incubation. By contrast, the activity of the cleaved form of MMP-2 (60 kDa) appeared to be clearly enhanced in UTP-conditioned medium (Fig. 2A). After assessing UTP-induced MMP-2 activation in RT4-D6P2T cells, we investigated whether MMP-2 activation was induced by wound. Interestingly, gelatin zymography experiments showed that MMP-2 was activated during wound healing (Fig. 2B), with increased activation in the presence of UTP (250 µM, 12 h). This amplified signal of the active form of MMP-2 was observed in the Schwann cell line as well as the primary Schwann cell culture (Fig. 2B). These results suggest that either the UTP nucleotide or the conditioned medium from wounded cultures induce the activation of MMP-2. The mRNA levels in RT4-D6P2T and primary Schwann cells were also evaluated by quantitative RT-PCR. Results showed that UTP significantly increases MMP-2 expression in both cell models after 6-24 hours of treatment (Fig. S2).

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