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First published online 27 May 2008
doi: 10.1242/jcs.025817
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Research Article |
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
* Author for correspondence (e-mail: violaine.moreau{at}inserm.fr)
Accepted 10 April 2008
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Summary |
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 Top Summary IntroductionResultsDiscussionMaterials and MethodsReferences |
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Key words: p190 RhoGAP, Metalloproteinase, Podosomes
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Introduction |
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TopSummaryIntroductionResultsDiscussionMaterials and MethodsReferences |
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), whereas mice lacking p190B display axonal-tract deficits and neuronal-differentiation defects, pointing to a role for p190B in normal brain development (Matheson et al., 2006). In addition, p190B-deficient mice are 30% reduced in size because of a defect in insulin and IGF1 signalling impacting on adipogenesis functions (Sordella et al., 2002; Sordella et al., 2003). In cells, p190 proteins are recruited to sites of integrin clustering, where they couple signals from the ECM with the actin cytoskeleton through RhoGTPases. Both p190A and p190B exhibit their catalytic activity preferentially towards RhoA. Nevertheless, several phospholipids have been shown to switch this specificity towards Rac (Ligeti et al., 2004). p190A was first described as a tyrosine-phosphorylated protein in a complex with p120 RasGAP in serum-stimulated cells (Ellis et al., 1990; Settleman et al., 1992) and was then identified as a substrate of the Src kinase (Roof et al., 1998). In contrast to p190A, p190B is not abundantly phosphorylated in Src-transformed Rat-1 fibroblasts (Matheson et al., 2006). So far, no difference between the two isoforms has been found at the molecular level. Both isoforms are involved in the same cellular pathways and both interact with p120 RasGAP, Rnd3 and TFII-I (GTF2I) (Burbelo et al., 1995; Jiang et al., 2005; Wennerberg et al., 2003). However, recent studies suggested that p190 RhoGAPs might play more-complex roles than previously thought, and are not solely limited to the regulation of RhoA activity (Matheson et al., 2006).
In addition to actin dynamics, ECM remodelling is also crucial for cell migration and invasion. Among the different proteolytic systems, the matrix metalloproteinases (MMPs) strongly contribute to the cleavage of ECM proteins during invasive processes such as tumour metastasis or angiogenesis. Insoluble fibrin is one of the barriers that angiogenic endothelial cells must penetrate for neo-vascularisation. MT1-MMP (also known as MMP14) is a membrane-associated MMP that plays a crucial role in angiogenesis by virtue of its pericellular fibrinolytic activity. Besides its important contribution to ECM cleavage and broad-spectrum proteolytic capacities, MT1-MMP also acts as a membrane-anchored activator of the soluble MMP MMP2 (Itoh and Seiki, 2006). Both MT1-MMP and MMP2 regulate local ECM degradation at podosomes in endothelial cells (Tatin et al., 2006). Expression of MMP genes is transcriptionally regulated by a variety of extracellular factors, including cytokines and growth factors, as well as signals from the ECM.
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), we examined the role played by p190 isoforms in endothelial cells. Here, we provide data showing that the two p190 isoforms play distinct roles associated with endothelial cell invasiveness. We show that p190B knockdown selectively affects MT1-MMP and MMP2 expression and activity. We also demonstrated that p190B is involved in local ECM degradation at podosomes and capillary tube formation by endothelial cells. |
Results |
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), we analysed the response of the actin cytoskeleton to p190 depletion. Again, no major alteration in the organisation of the actin cytoskeleton was observed when comparing phalloidin staining of HUVECs transfected with either control or p190-targeted siRNAs (Fig. 1B,C, supplementary material Fig. S1C). Analysis of adhesive structures did not reveal any obvious alteration in vinculin distribution at focal adhesions in cells treated with p190 siRNAs (supplementary material Fig. S1D). However, further analysis is required to fully exclude the absence of effect in F-actin and/or focal-adhesion turnover and stability in cells transfected with p190-targeted siRNAs. Moreover, as previously shown for p190–/– mouse fibroblasts (Barberis et al., 2005; Brouns et al., 2000), siRNA-transfected HUVECs behaved similarly to their control counterparts in proliferation assays (data not shown). A redundant function for the closely related p190 isoforms could explain the absence of phenotype, but this hypothesis was ruled out when both isoforms were simultaneously knocked down (Fig. 1A). Indeed, cell morphology and behaviour remained unaltered under these conditions (supplementary material Fig. S1C and data not shown). In addition, RhoA-GTP basal levels measured with the Rhotekin Rho-binding domain (RBD) assay were not increased in these cells, as previously observed for p190A–/– and p190B–/– 3T3 fibroblasts (Wennerberg et al., 2003) (Fig. 1D). This latter result suggests that, in HUVECs, basal levels of RhoA activity are not regulated by p190 proteins or that other RhoGAPs compensate for the deficit in p190 proteins.
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). Using gelatin zymography, HUVECs transfected with p190B-targeted siRNA (designated as p190B-KD cells) showed reduced levels of active MMP2 when compared with cells transfected with either control or p190A-targeted siRNA (designated as p190A-KD cells) in response to phorbol-12-myristate-13-acetate (PMA) treatment (Fig. 2A). Quantification of the results showed that p190B siRNA consistently inhibited MMP2 activation by 50% on average (Fig. 2A). Because processing of pro-MMP2 into active MMP2 is driven by the membrane-anchored MMP MT1-MMP, we also analysed the expression of MT1-MMP in HUVECs transfected with control, A1 or B1 siRNAs (Fig. 2B). Western blot experiments revealed that p190B-siRNA-mediated knockdown resulted in 40-55% reduction of MT1-MMP protein expression (Fig. 2C). By contrast, siRNA-mediated knockdown of p190A did not significantly alter MT1-MMP expression. Because the function of MT1-MMP requires its cell-surface presentation, we then examined surface expression of MT1-MMP in these cells. Biotinylated cell-surface proteins were affinity-purified on streptavidin beads from a total-cell extract; unbound proteins, such as β-actin, are defined as the intracellular fraction (Fig. 2D). Fig. 2D,E show a decreased level of MT1-MMP at the surface of p190B-KD cells compared with control cells, consistent with the reduced activation of MMP2 in those cells. By contrast, p190A-KD cells displayed an increased amount of MT1-MMP on the cell surface. These results, confirmed by FACS analysis (supplementary material Table S1), suggest that p190 proteins antagonistically influence MT1-MMP cell-surface expression. This variation of MT1-MMP protein level at the cell surface might be explained either at the transcriptional or post-transcriptional level, or by alteration of MT1-MMP trafficking.
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MT1-MMP and MMP2 are associated with local matrix degradation mediated by podosomes, and MMPs have been shown to alter podosome turnover (Goto et al., 2002). We therefore addressed the role of p190 in podosome formation and function. p190A has been shown to localise to podosomes in smooth muscle and porcine aortic endothelial cells (Burgstaller and Gimona, 2004; Moreau et al., 2006). We previously described that in HUVECs short-term exposure to PMA induced disruption of stress fibres followed by the appearance of podosomes and rosettes (Tatin et al., 2006); this defect was characterised by the presence of vinculin rings around actin dots (supplementary material Fig. S2). We first investigated p190A and p190B relocalisation in HUVECs. To visualise cytoskeletal structures, polymerised actin was stained with rhodamine-conjugated phalloidin. Co-staining of F-actin with p190 isoforms revealed that p190A and p190B, which both localised to lamellipodia in untreated HUVECs (Fig. 1B,C), relocalised to podosomes in response to PMA treatment (Fig. 4A). Moreover, similar results were observed in the two other podosome-forming cell lines, i.e. porcine aortic endothelial cells expressing constitutively active Cdc42 (Moreau et al., 2003) and A7r5 smooth muscle cells treated with phorbol esters (Hai et al., 2002) (supplementary material Fig. S3). We then analysed the impact of p190A or p190B knockdown on podosome formation in HUVECs. With this intention, p190A- or p190B-KD cells were treated for 1 hour with PMA to induce podosomes and were then stained for F-actin and the podosomal marker vinculin. The number of cells with podosomes was evaluated under the microscope. Fig. 4B shows the percentage of cells showing podosomes in p190-KD cells relative to control siRNA-transfected cells. Inhibition of p190A expression resulted in a significant increase in the number of cells with podosomes, whereas the absence of p190B had no effect on this parameter. To analyse the in situ proteolytic activity existing in these cells, fluorescent-matrix degradation assays were carried out. HUVECs were seeded onto glass coverslips pre-coated with FITC-conjugated crosslinked gelatin and treated or not for 1 hour with PMA. ECM degradation was quantified by counting the dark areas present in the fluorescent matrix (Fig. 4C). The absence of p190A significantly affected endogenous (no treatment) and podosome-induced (+PMA) degradation. The fact that p190A depletion modulates intrinsic matrix degradation suggests that the effect measured upon PMA addition reflects the effects of the knockdown both on podosome formation and function, i.e. matrix degradation. By contrast, the absence of p190B dramatically decreased matrix degradation in the absence (no treatment) or presence (+PMA) of podosomes. These experiments show that only p190A is involved in PMA-induced actin reorganisation and podosome formation, and that both p190 isoforms are required for matrix degradation in HUVECs, but in an antagonist manner. Thus, p190B might control matrix degradation, and it might do so via the regulation of MMP expression and activity.
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; Genis et al., 2006). Because p190B plays a role in the regulation of MT1-MMP expression and matrix proteolysis, we set up in vitro assays using Matrigel to evaluate capillary tube formation by siRNA-treated HUVECs in response to fibroblast growth factor (FGF) or PMA (Fig. 5A,B). The stimulation of tube assembly by FGF and PMA in HUVECs was consistently inhibited by p190B siRNA, without interfering with cell adhesion to the matrix (Fig. 5 and data not shown). p190A siRNAs had no such effect, thus strengthening the functional differences between the two p190 isoforms in endothelial cells. MT1-MMP siRNAs also decreased tube formation in this assay, consistent with the inhibitory effect reported for anti-MT1-MMP antibodies (Genis et al., 2006). Our data demonstrate that the p190B isoform is selectively involved in matrix remodelling and in the resulting formation of capillary tubes by endothelial cells. We believe that this novel function of p190B arises from its involvement in the regulation of MMP expression. |
Discussion |
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). As previously reported by Burgstaller and Gimona (Burgstaller and Gimona, 2004), modifying local equilibrium between inactive and active RhoA might affect podosome formation. Indeed, p190A knockdown increased the number of cells showing podosomes and also the associated degradation activity. Because it has been suggested that the degradation activity is directly correlated to the half-life of the structures (Linder, 2007), the observed increase of these two parameters might in fact reflect a reduction in podosome turnover in p190A-KD cells. The microtubule cytoskeleton was shown to be involved in podosome dynamics in macrophages (Kopp et al., 2006); however, we were not able to detect any alteration in the pattern of 
-tubulin in these modified cells. Thus, our data suggest that, even if p190A and p190B proteins share a high degree of homology and are involved in similar signalling cascades, they might control actin-cytoskeleton-based processes through distinct mechanisms.
The specific role of each p190 isoform was further demonstrated when the effects of siRNA on functions related to endothelial cell invasiveness were analysed. The activity of two matrix MMPs, MMP2 and MT1-MMP, which have both been implicated in endothelial cell function, was decreased only in the p190B-KD cells. Subsequently, MMP2 and MT1-MMP mRNA expression levels were found decreased. The decrease in MMP2 protein levels (pro-MMP2 + active MMP2) was not as high as the decrease in MMP2 mRNA levels, which might reflect a low turnover of the protein. Thus, during the time course of the experiment, mRNA levels decrease more rapidly than protein levels. In the case of MT1-MMP, the decrease in mRNA levels correlated with the decrease in protein levels. Thus, our results suggest that the two collaborative proteases MT1-MMP and MMP2 are regulated by the same signalling pathways and transcription factors, and that p190B is specifically involved. So far, it has only been demonstrated that two different transcription factors, GATA2 and EGR1 regulate the expression of MMP2 and MT1-MMP in response to a type I collagen matrix in endothelial cells (Haas, 2005), even if both can be activated by mitogen-activated protein kinase (MAPK). Our preliminary results suggest that Erk1/2 activity is not altered in p190-KD cells (V.M., unpublished data). It remains to be elucidated whether alterations in MMP2 and MT1-MMP expression can be explained by the regulation of the activity of a specific transcription factor by p190B. Recently, it was reported that p190 proteins are able to physically interact with the serum-responsive transcriptional regulator TFII-I (Jiang et al., 2005), but this interaction seems to occur with both isoforms and TFII-I has never been shown to regulate MMP transcription. Another possible link is the cAMP-responsive element-binding protein (CREB): CREB activity is reduced in p190B-deficient mice (Sordella et al., 2002) and its interaction with ATF1 plays a role in CRE-dependent expression of MMP2 in metastatic human melanoma cells (Xie et al., 1997).
The regulation of MMP activities by the p190B isoform in endothelial cells also reveals its crucial role in matrix degradation. This suggests that p190B might regulate cell invasion during angiogenesis in vivo. It is of interest to note that p190B has been shown to be required for cell invasion during virgin mammary gland development, another model of tissue remodelling and protease action. p190B might facilitate the invasion of the terminal end buds into the surrounding fat pad during ductal morphogenesis (Chakravarty et al., 2003), a process dependent on MMP2 activity (Wiseman et al., 2003). More recently, Vargo-Gogola et al. (Vargo-Gogola et al., 2006) demonstrated that overexpression of p190B in transgenic mice disrupts mammary gland architecture, showing a disorganised ductal tree with increased branching and altered stroma surrounding the mature ducts. Indeed, an increase in collagen is observed in the stroma surrounding the p190B-overexpressing terminal end buds, suggesting that p190B might also play a role in matrix deposition. Thus, this function of p190B in matrix remodelling does not seem to be restricted to the endothelium.
In this study, we showed that the knockdown of p190A did not produce the same effects as did the knockdown p190B on endothelial cells. Because the decrease in MT1-MMP at the cell surface in the intracellular fraction (quantification not shown) and in the total-cell extract were in the same range, our interpretation is that p190B knockdown alters the overall expression of MT1-MMP and not its trafficking and/or localisation at the plasma membrane. By contrast, on the basis of the result that p190A knockdown increased the amount of MT1-MMP at the cell surface and not in the total-cell extract, it is tempting to speculate that p190A might be involved in targeting MT1-MMP to the plasma membrane. This hypothesis is further strengthened by the fact that p190A has been found to interact with Arf6 in a high-throughput yeast two-hybrid screen mapping protein interactions in Drosophila melanogaster (Formstecher et al., 2005). Thus, it is striking to notice that some of the obtained effects turned out to be antagonistic. Indeed, whereas p190B knockdown decreased MT1-MMP level at the cell surface of HUVECs, p190A knockdown increased it. We also observed this antagonism when the effects of the two knockdowns on podosome function were analysed. We believe that further experiments, which are required to strengthen this observation, will provide new insights into the possible crosstalk between both isoforms. To date, out of the few studies that report the role of p190 proteins in endothelial cells, all focused on p190A, which was found to be mainly involved in the regulation of endothelial permeability (Harrington et al., 2005; Holinstat et al., 2006; Mammoto et al., 2007). This process, which is based on the regulation of the endothelial cytoskeleton, required p190A GAP activity. p190A has been shown to act downstream of: (1) PKC
to regulate basal endothelial barrier function (Harrington et al., 2005), (2) focal adhesion kinase (FAK) to restore endothelial barrier integrity after thrombin-mediated increase of endothelial permeability (Holinstat et al., 2006), and (3) angiopoietin-1 in order to protect against endotoxin-induced vascular leakage (Mammoto et al., 2007). In addition, it has also been demonstrated that p190A activity is necessary for directional retraction of endothelial cells in the presence of semaphorin (Barberis et al., 2005). In this latter situation, p190A might mediate local release of stable cell-substrate adhesions. In the present study, we demonstrate that p190B is involved in matrix remodelling, impacting matrix degradation at podosomes and endothelial cell assembly into tube-like structures in Matrigel. Therefore, it is now clear that both p190 isoforms play crucial but distinct roles in endothelial cell biology.
Interactions between cells and the ECM are mediated by: (1) integral membrane proteins – including integrins, which provide a link between the ECM and the actin cytoskeleton, and (2) extracellular proteinases and their inhibitors, which mediate local degradation of ECM components. Our work demonstrates that p190B represents a novel interface between actin-cytoskeleton and ECM remodelling. Because the acquisition of a motile and invasive phenotype is also an important step in the development of tumours and metastasis, it becomes worth considering p190B as a new player in cancer, involved not only in actin-based motility, but also in ECM remodelling in this context.
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Materials and Methods |
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-tubulin were from Sigma.
Cell culture and cell stimulation
HUVECs were cultured in 100-mm dishes coated with 0.2% gelatin in endothelial cell basal medium supplemented with `supplement pack' (Promocell). In all experiments, HUVECs were used between passages 2 and 5. Cell stimulation was achieved using PMA at 50 ng/ml. The porcine aortic endothelial (PAE) cell line expressing V12Cdc42 under the control of an IPTG-inducible promoter was established previously (David et al., 1999) and cultured in F12 medium (Ham F12; GIBCO BRL) supplemented with 10% heat-inactivated FCS, penicillin-streptomycin, 100 µM hygromycin B and 500 nM puromycin. V12Cdc42 expression was achieved by adding 0.1 mM IPTG. A7r5 rat smooth muscle cells (ATCC) were grown in low-glucose (1 g/l) DMEM supplemented with 10% heat-inactivated FCS and penicillin-streptomycin. Podosome induction was achieved by a 1-hour PMA (50 ng/ml) treatment. All cells were cultured at 37°C in a 5% CO2 humidified atmosphere.
Measurement of RhoGTPase activity
pGEX-2T constructs containing rhotekin-Rho-binding domain were kindly provided by Martin Schwartz (University of Virginia, Charlotteville, USA). HUVECs were grown in 140-mm dishes, treated with siRNA, lysed and protein extracts were used for pull-down assays as previously described (Ren et al., 1999; Sander et al., 1998). Antibodies against RhoA were from Santa Cruz.
siRNA transfection
siRNAs were chemically synthesised (Qiagen) and introduced into HUVECs (200 pmol) using a calcium-phosphate precipitation-transfection protocol. In order to reach maximum transfection efficiency in primary endothelial cells, two rounds of transfection were performed (24 hours apart). All experiments were then performed about 40 hours after the second transfection, i.e. 65 hours after the first one. The antisense-strand siRNA targeted against the human p190A isoform was based on published sequences (Barberis et al., 2005). For p190B, we designed and used 5'-AACGTGCAGCTGCATCTAAAT-3' and 5'-AATGAGAAGCATATCTGGTTA-3', named B1 and B3, respectively. For MT1-MMP, the siRNA sequence has been published previously (Tatin et al., 2006). As control, we used the AllStars negative-control siRNA from Qiagen.
Immunofluorescence microscopy
Cells plated onto glass coverslips were prepared for immunofluorescence microscopy as previously described (Moreau et al., 2003). Fluorescent images were recorded on an Eclipse Nikon microscope using a 63x oil-immersion lens. Confocal images were captured on a Zeiss LSM510 confocal microscope. The images were processed using Adobe Photoshop 5.5 (Adobe Systems). Quantification of cells showing podosomes was assessed in three independent experiments in which at least 200 cells were counted.
ECM degradation assay
HUVECs were seeded on FITC-gelatin-coated coverslips prepared as described previously (Bowden et al., 2001). Briefly, a thin layer of fluorescein-conjugated gelatin was deposited on coverslips and cross-linked with 0.5% glutaraldehyde for 15 minutes at room temperature. After a wash in PBS and a 2-minute incubation in 70% ethanol, coverslips were kept in serum-free medium at 37°C until cells were seeded on the gelatin. For podosome-formation stimulation, 50 ng/ml PMA was added to cells in complete growth medium for 1 hour at 37°C. Cells were fixed in PFA and processed for immunofluorescence microscopy as described above. Colocalisation between dark areas and podosomes was visualized after merging FITC and rhodamine-phalloidin images. To quantify ECM degradation, we first determined the percentage of cells that presented at least one degradation area relative to the total number of cells analyzed in each condition. Values were then expressed as the percentage of degrading cells relative to control conditions (without PMA, transfected with control siRNA).
Analysis of matrix metalloproteinase activity by zymography
MMP activity was detected in cell supernatants and extracts as previously described (Tatin et al., 2006).
Biotinylation of cell-surface proteins
Cell-surface proteins were biotinylated as previously described (Remacle et al., 2003). Cells at 80% confluence in a 100-mm dish were incubated with 3.8 ml of a 1.5 mg/ml sulfo-NHS biotin (Pierce) in PBS solution for 30 minutes at 4°C with gentle shaking. Following quenching of biotinylation in PBS plus 100 mM glycine, and further incubation for 30 minutes at 4°C with gentle shaking, glycine solution was aspirated and rinsed twice with PBS. Cells were then lysed and biotinylated cell-surface fractions and intracellular fractions were separated and analysed by immunoblotting.
FACS analysis of cell-surface proteins
siRNA-treated HUVECs were detached with PBS containing 20 mM EDTA at 37°C, washed and resuspended in PBS containing 0.5% BSA. In total, 2.5x105 cells were incubated with MT1-MMP (Lem-2/15) antibodies for 30 minutes at 4°C. Cells were washed with PBS and incubated with Alexa-Fluor-488-conjugated anti-mouse IgG. Finally, fluorescent samples were analysed in an EPICS-XL flow cytometer (Beckman COULTER).
Real-time PCR
RNA was collected from 6x105 cells using the RNeasy mini kit (Qiagen). Integrity check and sample quantitation were made on the 2100 bioanalyser (Agilent Technologies). cDNA was synthesised from 2 µg of total RNA with or without PowerScript reverse transcriptase (Clontech, Palo Alto, CA, USA) and with random primers (Invitrogen). Aliquots of cDNA were then subjected to PCR amplification on a DNA Engine Opticon2 fluorescence-detection system (MJ Research/Bio-Rad) with specific forward and reverse oligonucleotide primers (listed in Table 1). The DyNAmo SYBR Green qPCR kit (Finnzymes) was used with the following PCR amplification cycles: initial denaturation, 95°C for 15 minutes, followed by 40 cycles with denaturation, 95°C for 20 seconds and annealing-extension, 61°C for 35 seconds. A dissociation curve was generated to verify that a single product was amplified. The cycle threshold (Ct) value of each gene was normalised against that of GAPDH and the relative level of expression calculated using the comparative (2–
CT) method.
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In vitro angiogenesis assay
Growth-factor-reduced Matrigel basement-membrane matrix (Becton Dickinson) was diluted 1:2 in endothelial growth medium. Diluted Matrigel (200 µl) was plated into flat-bottomed 48-well tissue-culture plates and allowed to gel for 45 minutes at 37°C before cell seeding. Then, 2.5x104 cells were added atop the Matrigel in the presence of 20 ng/ml PMA or 10 ng/ml FGF. After 6 hours of incubation, images were taken with a phase-contrast microscope (Nikon TE 2000) using a 4x objective. Capillary tubes were defined as cellular extensions linking cell masses or branch points. The formation of capillary tubes was quantified by counting the numbers of tubes per field.
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Acknowledgments |
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Footnotes |
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References |
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