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First published online 27 May 2008
doi: 10.1242/jcs.025817

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p190B RhoGAP regulates endothelial-cell-associated proteolysis through MT1-MMP and MMP2

¹ Institut Européen de Chimie-Biologie, 2 rue Robert Escarpit, 33600 Pessac, France
² INSERM, U441, Université Victor Segalen Bordeaux 2, Avenue du Haut-Lévêque, 33600 Pessac, France
³ INSERM, U862, Institut François Magendie, 146 Rue Léo Saignat, 33077 Bordeaux Cedex, France
⁴ INSERM, U889, Université Victor Segalen Bordeaux 2, 146 Rue Léo Saignat, 33076 Bordeaux, France

View larger version (43K): [in this window] [in a new window]   Fig. 1. (A) Specific siRNAs inhibit the expression of p190 isoforms in HUVECs. HUVECs were transfected with the indicated siRNAs targeting either p190A (A1), p190B (B1) or both (A1+B1). After 65 hours, protein extracts were analysed by immunoblotting using specific antibodies. The expression of endogenous β-actin remained unchanged. (Graph) The effect was quantified by measuring the relative amounts of p190 isoforms in each condition. Each bar represents the mean±s.e. of five independent experiments for A1 and B1, and of four independent experiments for A1+B1. (B,C) Specific siRNAs inhibit the expression of p190 isoforms in HUVECs and do not alter the actin cytoskeleton. HUVECs were transfected with the indicated siRNAs, fixed and stained for F-actin and p190A (B) or p190B (C). Scale bars: 10 µm. (D) Silencing p190A and p190B does not modify RhoA activity in HUVECs. Cells treated with 2 µg/ml of lysophosphatidic acid (LPA) for 15 minutes or transfected with the indicated siRNAs were lysed and active RhoA GTPase was affinity-precipitated with GST-RBD-rhotekin, eluted from the beads and analysed by immunoblotting using anti-RhoA antibodies. For each condition, a fraction of the lysate was run to monitor the RhoGAP knockdowns and the amount of GTPase before precipitation. The graph shows the quantification of bands from two independent experiments; error bars represent the mean±s.e. RhoA activity was quantified in comparison to the negative control (control-siRNA-transfected cells). In p190-KD cells, basal levels of RhoA were not altered. LPA, which is a potent activator of RhoA, was used as a positive control in this experiment.

View larger version (35K): [in this window] [in a new window]   Fig. 2. Silencing p190B results in a decrease in MT1-MMP cell-surface presentation and MMP2 activation. (A) Cell lysates from HUVECs transfected with the indicated siRNAs and subsequently treated with PMA for 30 minutes were analysed by gelatin zymography. MMP2 was activated upon PMA treatment. The active form of MMP2 (62 kDa) became apparent, revealing the processing of pro-MMP2. In p190B-KD cells, MMP2 activation was decreased. No significant alteration of MMP2 activation was observed in the p190A-KD. The effect was quantified by measuring the percentage of active MMP2 relative to control conditions after PMA treatment. Each bar represents the mean±s.e. of three independent experiments. *P<0.005 in Student's t-test when compared with control. (B) Analysis of MT1-MMP expression in protein extracts from HUVECs transfected with the indicated siRNA by immunoblotting. β-actin expression was used as loading reference. Target knockdown was controlled using p190A- and p190B-specific antibodies. Specificity of the anti-MT1-MMP antibody was controlled using a previously described siRNA targeting MT1-MMP. (C) Quantification of MT1-MMP protein levels normalised using β-actin and relative to control (control-siRNA-transfected cells) is represented in the bar graph. Each bar represents the mean±s.e. (n=4 for A1 and B1, n=3 for A2 and B3). *P<0.001 in Student's t-test when compared with control. (D) MT1-MMP surface expression is altered in p190-KD cells. Biotinylated cell-surface proteins and non-biotinylated intracellular proteins were isolated from p190A- and p190B-KD cells and analysed by immunoblotting using anti-MT1-MMP, anti-β-actin and anti-β1-integrin antibodies. (E) The graph represents the quantification of four independent experiments. The amount of MT1-MMP present in cell-surface fractions significantly increased in p190A-KD cells and decreased in p190B-KD cells. *P<0.05 Student's t-test when compared with control.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Effect of p190 knockdown on MMP mRNA expression. Quantitative RT-PCR analysis of the effects of p190A and p190B knockdown on mRNA levels of p190A, p190B, MT1-MMP, MMP2, TIMP2 and β-actin in HUVECs. Each bar represents the mean±s.e. of four (A1, A2 and B1) or three (B3) independent experiments. *P<0.002; #P<0.02 in Student's t-test when compared with control.

View larger version (52K): [in this window] [in a new window]   Fig. 4. Effect of p190 knockdown on podosome formation and function. (A) p190A and p190B localised to podosomes in HUVECs. F-actin was labelled using rhodamine-phalloidin and p190 isoforms were labelled using anti-isoform-specific antibodies and secondary Alexa-Fluor-488-conjugated antibodies in HUVECs treated with PMA. Arrows indicate podosome structures. Scale bar: 10 µm. (B) Knockdown of p190B did not alter podosome formation, but knockdown of p190A increased the number of cells showing podosomes. HUVECs were transfected with siRNA designed against p190A or p190B for 65 hours, treated for 1 hour with PMA and stained for F-actin. Cells showing podosomes were counted and data are presented as a percentage compared with the control (control-siRNA-transfected cells). Error bars represent the mean±s.e. of ten (A1 and B1) or three (A2 and B3) independent experiments. *P<0.0005; #P<0.05 in Student's t-test when compared with control. (C) Alteration of in situ proteolytic activity in p190-KD cells. HUVECs transfected with the indicated siRNA were seeded on FITC-gelatin-coated coverslips. Half of the coverslips were treated with PMA for 1 hour to induce podosome formation. Cells were fixed and stained for F-actin. `Black holes' representative of gelatin-degradation zones were recorded as described in the Materials and Methods section and are presented as a percentage of the control response obtained with untreated cells. Each bar represents the mean±s.e. of four independent experiments. The effects obtained were statistically significant (#P<0.05; *P<0.01 in Student's t-test) for p190A- and p190B-KD cells.

View larger version (41K): [in this window] [in a new window]   Fig. 5. Knockdown of p190B prevents PMA- and FGF-induced capillary tube formation in Matrigel. (A) HUVECs transfected with control, A1, A2, B1, B3 or MT1-MMP siRNA were seeded on Matrigel. The formation of PMA- and FGF-induced capillary tubes was quantified after 6 hours at 37°C by counting the number of tubes. The means and s.e. of the percentage of tubes per field relative to the control are shown (n=3). The formation of tubes induced by PMA or FGF was significantly inhibited (P<0.05 in Student's t-test) in p190B- and MT1-MMP-KD cells when compared with control (control-siRNA-transfected cells). (B) Representative photomicrographs of capillary-like structures from control-, A1- or B1-siRNA-transfected HUVECs cultured on Matrigel for 6 hours in the presence of PMA are shown.

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