UTP regulates MMP-2 activity.

<p>Wound healing and gelatin zymograms of RT4-D6P2T cells transfected with shRNA directed against the MMP-2 gene (shMMP-2) 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: *<i>P</i>≤0.05 and ***<i>P</i>≤0.001 when compared to control cells; #<i>P</i>≤0.05 and ###<i>P</i>≤0.001 when compared to UTP-treated cells.</p

Uridine 5′-Triphosphate Promotes <i>In Vitro</i> Schwannoma Cell Migration through Matrix Metalloproteinase-2 Activation

<div><p>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 <i>in vitro</i> Schwannoma cell migration and wound repair through a MMP-2-dependent mechanism via P2Y₂ receptors and MAPK pathway activation.</p></div

UTP regulates MMP-2 activity and expression.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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: *<i>P</i>≤0.05 compared to control cells. (<b>D</b>) In situ zymography images and quantitative analysis of gelatinase activity in RT4-D6P2T cells. Scale bar: 50 µm. Statistical significance: **<i>P</i>≤0.01 compared to control cells.</p

UTP induces biphasic MAPK phosphorylation.

<p>(<b>A</b>) 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: *<i>P</i>≤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: ***<i>P</i>≤0.001 when compared to control cells; ##<i>P</i>≤0.005, and ###<i>P</i>≤0.001 when compared to UTP-treated cells.</p

UTP enhances Schwann cell migration and wound repair.

<p>(<b>A</b>) 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: *<i>P</i>≤0.05, **<i>P</i>≤0.01, and *** <i>P</i>≤0.001. (<b>B</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). (<b>C</b>) 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: *** <i>P</i>≤0.001.</p

P2Y₂ receptors are necessary for Schwann cell line wound repair.

<p>(<b>A</b>) 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>B-C</b>) 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: **<i>P</i>≤0.01 and ***<i>P</i>≤0.001 when compared to control cells; ##<i>P</i>≤0.01 and ###<i>P</i>≤0.001 when compared to UTP-treated cells.</p