L1CAM-erbB interaction enhances neuregulin induced phosphorylation of erbB3. a

<p>) Upper panel: MCF-7 cells were transiently transfected with pcDNA3-L1CAM or pcDNA3 empty vector. 24 h later, cells were serum starved and stimulated with recombinant NRG1 (50 nM) for 15 min. Then, cells were harvested and lysed. Extracts were submitted to SDS-PAGE and blotted with anti-p-Tyr monoclonal antibody or anti-erbB3 polyclonal antibody. This experiment was repeated three times. A representative experiment is shown. Lower panel: the same approach was used in cells transfected with the ΔIg-L1CAM truncated construct. This experiment was repeated twice. One of them is shown. <b>b</b>) Quantification of western blots by densitometry. The normalized amount of phosphorylated 180 kDa band is increased in cells that express the full length but not the truncated ΔIg-L1CAM protein, suggesting that the physical interaction of L1CAM and erbB3 is needed for the enhancing effect on neuregulin receptor activation. Bars represent standard errors <b>c</b>) Proposed model: the interaction with L1CAM sensitizes erbB receptors to the activation by neuregulins. Removing the Ig-like rich region of L1CAM prevents the interaction and avoids receptor sensitization. For simplicity, only <i>cis</i>-interactions are depicted in the model.</p

Different Ig-like domains can support L1CAM physical interaction with erbB receptors.

<p><b>a</b>) Truncated proteins ΔIg1-3L1CAM and ΔIg4-6L1CAM are normally expressed and distributed when transfected into COS-7 cells. High magnification confocal images of cells transiently transfected with the indicated constructs are shown. L1CAM was detected with an anti-L1CAM monoclonal antibody (green) and erbB3 with a polyclonal antibody (red). Nuclei were counterstained with the Hoechst stain (blue). As is shown both deletion mutants of L1CAM co-localize with erbB3 (white). <b>b</b>) Ablation of Ig-like domains 1 to 3 or 4 to 6 does not abrogate L1CAM interaction with erbB3: HEK293 cells were transfected with pcDNA3-erbB3 and the different truncated forms of L1CAM. Cell extracts were immunoprecipitated with anti-L1CAM antibody and blotted against erbB3. As shown, erbB3 was pulled down when co-expressed with ΔIg1-3L1CAM, ΔIg4-6L1CAM and full-length constructs but not when co-expressed with the ΔIg-L1CAM construct. As expected, anti-L1CAM antibody does not immunoprecipitate erbB3 in cells transfected with pcDNA3-erbB3 exclusively. Input lanes demonstrate the expression of erbB3 in the extracts. An aliquot of the immunoprecipitated was probed with anti-L1CAM to verify the adequate expression and immunoprecipitation of the truncated proteins. IgG bands show that a similar amount of immunoprecipitated was loaded. This experiment was repeated twice. A representative experiment is shown.</p

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

The Ig-like domains but not the fibronectin repeats of L1CAM mediate the physical interaction with erbB receptors.

<p>a) Ablation of Ig-like domains abrogates L1CAM interaction with erbB3: HEK293 cells were transfected with pcDNA3-erbB3 and the different truncated forms of L1CAM. Cell extracts were immunoprecipitated with anti-L1CAM antibody and blotted against erbB3. As is shown, erbB3 was pulled down when co-expressed with the full length and ΔFn-L1CAM constructs, but not when was co-expressed with the ΔIg-L1CAM construct. As expected, anti-L1CAM antibody does not immunoprecipate cells transfected with pcDNA3-erbB3 exclusively. Input lanes demonstrate the expression of erbB3 in the extracts. An aliquot of the immunoprecipitate was probed with anti-L1CAM to verify the adequate immunoprecipitation of the truncated proteins. IgG bands show that a similar amount of immunoprecipitate was loaded. This experiment was repeated three times. A representative experiment is shown. b) To rule out sorting problems that could explain the absence of co-IP, L1CAM and deleted constructs were co-transfected with erbB3 in COS-7 cells. As is shown, the distribution of L1CAM, ΔIg-L1CAM and ΔFn-L1CAM is similar when transfected into HEK293 cells, being detectable in the plasma membrane. c) The co-localization of the deleted constructs and full length L1CAM with erbB3 was nearly complete, ruling out sorting defects for the mutant proteins. L1CAM was detected with the anti-L1CAM monoclonal antibody (green) and erbB3 with a polyclonal antibody (red). Nuclei were counterstained with the Hoechst stain (blue). Co-localization (white) was revealed with the ImageJ software and the Co-localization Finder plugin.</p

Physical interaction of L1CAM with erbB receptors. a

<p>) L1CAM co-immunoprecipitates with erbB1 (EGFR): a pcDNA3 plasmid containing the cDNA encoding for human L1CAM and the pcDNA6A-EGFR construct were transiently co-transfected into the HEK293 cells. 48 h later cells were homogenized and L1CAM immunoprecipitated (IP). Immunoprecipitates were resolved by SDS-PAGE and blotted with anti-myc antibody to detect EGFR. As is shown, EGFR was pulled down only in L1CAM expressing cells. EGFR expression was similar in both extracts (input). Immunoblot with anti-L1CAM shows that this protein was correctly immunoprecipitated. <b>b</b>) Reverse co-immunoprecipitation. IP with anti-myc antibody pulls down L1CAM only in EGFR transfected cells. L1CAM expression was similar in both extracts (input). Immunoblot with anti-myc shows that the EGFR was correctly immunoprecipitated. <b>c</b>) L1CAM co-immunoprecipitates with erbB2: pcDNA3-L1CAM and the pcDNA3-erbB2 were transiently co-transfected into the HEK293 cells. Extracts were immunoprecipitated with the anti-L1CAM antibody. erbB2 was pulled down only in L1CAM expressing cells. erbB2 expression was similar in both extracts (input). Anti-L1CAM immunoblot shows that this protein was correctly immunoprecipitated. <b>d</b>) Reverse co-immunoprecipitation. IP with anti-erbB2 antibody pulls down L1CAM only in erbB2 transfected cells. L1CAM expression was similar in both extracts (input). Anti-erbB2 WB shows that erbB2 was correctly immunoprecipitated. <b>e</b>) erbB3 co-immunoprecipitates with L1CAM: pcDNA3-L1CAM and the pcDNA3-erbB3 were transiently co-transfected. Extracts were immunoprecipitated with the anti-L1CAM antibody. erbB3 was pulled down only in L1CAM expressing cells. erbB3 expression was similar in both extracts (input). Anti-L1CAM immunoblot shows that this protein was correctly immunoprecipitated. <b>f</b>) Reverse co-immunoprecipitation. IP with anti-erbB3 antibody pulls down L1CAM only in erbB3 transfected cells. L1CAM expression was similar in both extracts (input). Anti-myc WB shows that erbB3 was correctly immunoprecipitated. <b>g</b>) Proximity ligation assay showing L1CAM-erbB3 <i>in vivo</i> interaction. HEK293 cells were enforced to express L1CAM and erbB3. To identify the transfected cells, a plasmid encoding GFP was included. As is shown, only the transfected cells (green) were positive for the PLA signal (red). Note that the interaction signal can be detected in cells that are not in contact with other transfected cells, showing that the interaction between L1CAM and erbB3 is produced in <i>cis</i>. Scale bars represent 20 µm. <b>h</b>) L1CAM (red) co-localizes with EGFR (erbB1), erbB2 and erbB3 (green) in growing axons during brain development (at E14). Images at the right correspond to the co-localization channel (white). Co-localization is evident in cortical projections. Poor co-localization of L1-CAM was detected with Notch 2, used as a control for specificity. Co-localization was revealed with the ImageJ software and the Co-localization Finder plugin (for co-localization at P3 stage see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040674#pone.0040674.s001" target="_blank">Figure S1</a>). Images show coronal sections of E14 mouse brain incubated with the indicated antibodies and acquired at low magnification wide-field fluorescence (at left) or higher magnification under the confocal microscope. Scale bars correspond to 100 µm. <b>i</b>) L1CAM physically interacts with erbB3 <i>in vivo</i>. Whole brains of two days old rats were homogenized in RIPA buffer clarified by centrifugation and cross-linked with DTBP. Supernatants were immunoprecipitated with the anti-erbB3 antibody and blotted with anti-L1CAM. As a control of specificity an aliquot of the extract was immunoprecipitated with a non-specific anti-IgG. As shown, L1CAM was pulled down when immunoprecipitation was performed with the anti-erbB3 but not with the anti-IgG. Input shows that L1CAM is abundantly expressed in the P2 rat brains. IgG bands demonstrate a similar loading of immunoprecipitated proteins. This experiment was repeated 5 times. A representative experiment is shown. <b>j</b>) A similar result was obtained with the receptor erbB2.</p

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 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

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

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