Institute of Signaling, Developmental Biology and Cancer Research CNRS-UMR6543, Centre Antoine Lacassagne, 33 Avenue de Valombrose, 06189 Nice, France
Author for correspondence (e-mail: vanobber{at}unice.fr)
Accepted 26 May 2004
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Summary |
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Key words: Integrin-linked kinase, Endothelial cells, Fibronectin fibrillogenesis, Adhesion, Migration
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Introduction |
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Functional studies of ILK mutants in Drosophila (Zervas et al., 2001) and Caenorhabditis elegans (Mackinnon et al., 2002
) have revealed a crucial role for ILK in linking the actin cytoskeleton and the plasma membrane at sites of integrin-mediated adhesion. More recently, conditional or complete disruption has been reported of the mouse ILK-encoding gene (Grashoff et al., 2003
; Sakai et al., 2003
; Terpstra et al., 2003
). Mice lacking this gene die during early embryonic development owing to a defect in epiblast polarization with an abnormal distribution of F-actin beneath the plasma membrane (Sakai et al., 2003
). Chondrocyte-specific disruption of ILK in the mouse leads to chondrodysplasia and respiratory distress causing death of the mice at birth (Grashoff et al., 2003
; Terpstra et al., 2003
). Cellular defects in ILK-deficient fibroblasts and chondrocytes include impaired adhesion and spreading, altered cytoskeletal organization and a reduced rate of proliferation. Altogether, these findings confirm the key function of ILK as modulator of integrin function.
In cultured mammalian cells, ILK has been localized to cell-matrix adhesion sites by immunofluorescence staining (Li et al., 1999). Several studies have identified ILK interaction with focal adhesion components such as the adaptor protein paxillin, the LIM domain proteins PINCH 1 and 2, the actin-binding proteins CH-ILKBP/actopaxin/
-parvin and ß-parvin/affixin (for review, see Wu and Dedhar, 2001
), and, more recently, the FERM domain protein UNC-112/mig-2 (Mackinnon et al., 2002
). In addition, ILK associates with a serine/threonine protein phosphatase of the PP2C family, referred to as ILKAP (ILK-associated phosphatase) (Leung-Hagesteijn et al., 2001
). The existence of multiple diverse ILK partners suggests that many regulatory mechanisms converge at the level of ILK to mediate integrin signal transduction.
Here, we set out to characterize the role of ILK in regulation of endothelial cell morphology and function by using a specific, effective RNA-interference approach. Our results demonstrate that ILK is an essential cytoplasmic partner of 5ß1 integrin required for recruitment of this integrin to fibrillar adhesions and their transformation into competent matrix-forming structures.
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Materials and Methods |
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Cell culture
Bovine aortic endothelial cells (BAECs), kindly supplied by H. Drexler (Max-Planck-Institute, Bad Nauheim, Germany), were grown in Dulbecco's modified Eagle's medium (DMEM) with low glucose (Invitrogen Life Technologies, Cergy Pointoise, France) containing 5% heat-inactivated fetal calf serum. For collection of conditioned media, cells (2.5x105) were seeded in six-well culture plates 24 hours after the second transfection with RNA duplexes. After 12 hours, cells were washed in serum-free medium for 2 hours then incubated for an additional 24 hours in serum-free medium containing 0.1% bovine serum albumin (BSA). Conditioned medium was harvested, clarified and concentrated tenfold on a Microcon centrifugal filter (10,000 molecular-weight cutoff).
Antibodies
Rabbit polyclonal anti-ILK antibody (DI) was directed against a glutathione-S-transferase (GST)-ILK ankyrin-domain fusion protein. Mouse monoclonal anti-ILK (clone 65.1.9) and anti-cortactin (clone 4F11) antibodies were from Upstate Biotechnology (Charlottesville, VA). Anti-AKT and anti-phospho-AKT (pS473) antibody polyclonal antibodies were from Cell Signaling Technology (Beverly, MA). The anti-RhoA monoclonal antibody (clone 26C4) was from Santa Cruz Biotechnology (Santa Cruz, CA) and the monoclonal anti-fibronectin (clone 10) and anti-paxillin (clone 165) antibodies were from BD Biosciences (San Diego, CA). The mouse monoclonal antibody against cyclin D1 (clone DCS-6) was from NeoMarkers (Fremont, CA) and rabbit polyclonal anti-pFAK (pY397) from BioSource International (Nivelles, Belgium). Mouse monoclonal antibodies directed against the integrins vß3 (LM609) and
5ß1 (HA5), and the rabbit polyclonal anti-
5 integrin antibody were from Chemicon International (Temecula, CA). The rabbit polyclonal anti-ERK antibody (E1B4) that preferentially recognizes ERK2 was raised against a C-terminal ERK1 peptide. Rabbit polyclonal anti-ß1 and -ß3 antibodies were kindly provided by C. Albiges-Rizzo (Institut Albert Bonniot, Grenoble, France) and N. Kieffer (University Center, Luxembourg), respectively. Anti-phospho-PAK1 polyclonal antibody (Shamah et al., 2001
) was a kind gift of S. Shamah and M. Lin (M. Greenberg's laboratory, Harvard Medical School, Boston). Secondary antibodies coupled to horseradish peroxidase or alkaline phosphatase were from Promega France (Charbonnières-les-Bains, France) and New England BioLabs (Beverly, MA), respectively. Fluorescently labeled secondary antibodies were purchased from Molecular Probes (Eugene, OR).
siRNAs and double-stranded RNA transfection
Short interfering RNAs (siRNAs) were purchased from Eurogentec (Serang, Belgium). Equal amounts of complementary RNA oligonucleotides were combined to a final concentration of 20 µM and annealed according to the protocol supplied by the manufacturer. RNA interference in BAECs was achieved by performing two transfections of double-stranded RNA, the first 7 hours after plating and the second 24 hours later, using a modified calcium-phosphate protocol. Unless otherwise stated, cells were analysed between 24 hours and 48 hours after the second transfection. The siRNA sequences used in this study to target ILK (5'-CCCGGCUCAGGAUUUUCUCTT and 3'-TTGAGAAAAUCCUGAGCCGGG) correspond to a region in the human ILK gene of identical sequence in bovine ILK cDNA (S. Sauze-Fernandez and E.V.O.-S., unpublished). As control, an RNA duplex designed to target the Drosophila ILK transcript (GenBank accession number AF226669) was used (5'-CCCAAGCUCCGCAUCUUUUCTT and 3'-TTGAAAAGTUGCGGAGCUUGGG). The efficiency of ILK silencing, determined by western-blot analyses in each experiment, ranged between 70% and 80%.
Immunofluorescence and deconvolution microscopy
BAECs plated on glass coverslips coated with the indicated matrix protein were fixed in a solution of 3% paraformaldehyde containing 2% sucrose for immunofluorescence staining and mounted in Gel/Mount (Biomedia, Foster City, CA). Fluorescence was observed on a Zeiss inverted microscope (Axiovert 200M) equipped with a CoolSnap HQ cooled charge-coupled-device camera (Roeper Scientifique, Every, France). Image acquisition and analysis was performed using the MetaMorph Imaging System (Universal Imaging Corp.). Deconvolution microscopy using the constrained iterative algorithm (Softworx 2.5, Applied Precision) was performed with the Applied Precision DeltaVision system (Applied Precision, Issaquah, WA) built on an Olympus IX-70 base.
Cell-adhesion assay
The adhesion assay was performed in 96-well microtiter plates coated overnight at 4°C with the indicated doses of matrix proteins or poly-L-lysine, as control. After coating, the wells were washed with PBS and blocked with 0.1% fatty-acid-free BSA in DMEM for 1 hour at 37°C. Cells were detached with trypsin-EDTA, resuspended in serum-free DMEM containing 0.1% fatty-acid-free BSA and plated on the indicated substrates (1.5x104 cells per well; 1x105 in the case of vitronectin-coated wells). After 40 minutes, non-adherent cells were removed by extensively washing with PBS and remaining cells were fixed and stained with Giemsa. Cell adhesion was determined on triplicate wells by counting attached cells in three different fields. Image analyses were performed using NIH Image software (http://rsb.info.nih.gov/nih-image/default.html). Data are presented as means ± s.e.m.
Migration assays and tube-like structure formation
For the wound healing assay, confluent cells were scraped with a white (10 µl) Gilson pipet tip. Following wounding, the culture medium was changed to remove detached and damaged cells, and wound closure was monitored microscopically 6 hours later. Three different fields of the wound were photographed at the beginning and at the end of the experiment. The wound size was then determined and migration was expressed as the percentage of maximum wound size. Haptotactic migration was analysed on transwell filters (6.5 mm diameter, 8 µM pore size) from Corning (Corning, NY). Trypsinized cells (1.5x104) resuspended in DMEM supplemented with 0.1% fatty-acid-free BSA were placed on the upper filter and medium containing chemoattractant filled the lower chamber. After 4 hours, the transwells were rinsed in PBS to eliminate non-adherent cells and remaining cells in the upper chamber were removed with a cotton bud (Q-Tip®). After an additional wash in PBS, cells were fixed in methanol for 15 minutes at room temperature and stained with 0.1% crystal violet. Three different fields of each transwell were photographed and cells were counted.
Capillary-like structure formation was analyzed on a bed of Matrigel (Beckton Dickinson) (300 µl Matrigel in a 35 mm diameter well). The gel was polymerized at 37°C and 2 x105 cells in complete Endothelial Growth Medium (PromoCell, Heidelberg, Germany) were seeded on the matrix. Morphological changes were microscopically monitored.
Western blotting and northern analysis
Western blots were performed on whole-cell lysates or conditioned culture medium, separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Saclay, France). Proteins were detected by enhanced chemiluminescence using horseradish-peroxidase- or alkaline-phosphatase-based substrates from Pierce (Rockford, IL) and New England BioLabs, respectively. Where indicated, immunoblots were quantified using the GeneGnome chemiluminescent imaging system (Syngene, Frederick, MD). Total RNA was extracted from cells using the Qiagen (Courtaboeuf, France) RNeasy Kit, separated on 1% agarose gels and transferred to hybridization membranes. Polymerase chain reaction (PCR)-generated fragments of the indicated plasmids were radiolabeled using the rediprime II random prime kit (Amersham Pharmacia Biotech). Plasmids carrying the wild-type human ILK coding region (1353 bp), the complete wild-type human ß1A and the human ß3 sequences were kindly provided by S. Dedhar (University of British Colombia, Vancouver, Canada), A. Sonnenberg (The Netherlands Cancer Institute Amsterdam, The Netherlands) and N. Kieffer (University Center, Luxembourg), respectively. A plasmid containing a fragment (fibronectin repeats 6-10) of the human fibronectin cDNA was a gift of K. Yamada (NIH, Bethesda, Maryland, USA).
Fluorescence-activated cell sorting analysis
Surface expression of integrins was determined by flow cytometry using a FACScalibur (Becton Dickinson, Bedford, MA) following detachment of cells. Cells were incubated with anti-vß3 (4 µg ml1) or anti-
5ß1 (4 µg ml1) antibodies for 1 hour at 4°C, rinsed three times with ice-cold PBS and incubated with an anti-mouse antibody coupled to FITC. After three additional washes, cells were fixed with 3.7% formaldehyde and analysed. DNA content was determined according to the protocol provided by Becton Dickinson. Briefly, cells were fixed in ethanol and labeled with propidium iodide (10 µg ml1) just before the analysis by flow cytometry.
RhoA pull-down assay
Activation of RhoA was measured in a pull-down assay (Ren et al., 1999), as previously described (Vouret-Craviari et al., 2002
).
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Results |
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Silencing ILK expression in endothelial cells affects cell-matrix adhesions and actin dynamics
To dissect the role of ILK in regulation of endothelial cell adhesion, motility and differentiated function, we used an RNA-interference approach to reduce the expression of the protein selectively in transfected cells. Knock-down experiments were performed in BAECs in which post-transcriptional gene silencing proved to be highly efficient and reproducible. On western blots, ILK migrates as single band of approximately 52 kDa. Transfection of double-stranded RNA molecules complementary to the ILK transcript (ILK siRNA) suppressed ILK expression in these cells by up to 80% compared with control siRNA- or non-transfected cells, as determined by western blotting of cell lysates (Fig. 2A). Northern analysis revealed the total loss of ILK mRNA transcripts under these conditions (Fig. 4C).
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Immunofluorescence images confirmed the substantial reduction in ILK levels in ILK siRNA-transfected cells and also revealed differences in the shape of residual ILK-containing structures (Fig. 2B). Whereas ILK staining in control cells highlighted elongated fibrillar adhesions over the entire ventral surface of cells, staining in ILK-deficient cells was restricted to shorter plaques. ILK depletion also had a marked effect on the distribution of paxillin. Thus, the staining of paxillin in fiber-like structures disappeared following ILK suppression and staining became restricted to classical focal adhesions (Fig. 2C). We verified that depletion of ILK did not alter the levels of paxillin expression (data not shown). These findings suggest that ILK might be involved in regulating maturation or turnover of cell-matrix adhesions.
Loss of ILK also incurred changes in the actin-based cytoskeleton characterized by an increase in immunostaining of short, parallel actin stress fibers (Fig. 2C). Under sparse culture conditions, control BAECs extended broad lamellipodial protrusions all around cells, whereas ILK siRNA-transfected cells remained compact and projected structures were smaller (Fig. 2D, top; see supplementary Fig. S1 (http://jcs.biologists.org/cgi/content/full/117/19/4559/DC1)]. Kymograph analyses were performed to examine the effect of ILK depletion on the dynamics of membrane protrusive activity. In the representative kymographs depicted in Fig. 2D, bottom, obtained from phase-contrast time-lapse recordings of cells 2 hours after plating on fibronectin-coated dishes, it can be seen that control cells (left) displayed periodic extension and retraction of lamellipodia. By contrast, the protrusive activity at the edge of ILK-depleted cells (right) remained low. This difference in cell spreading was confirmed by quantitative morphometric analysis of control and ILK siRNA-transfected cells 24 hours after plating on fibronectin-coated coverslips. Indeed, ILK depletion resulted in a twofold reduction in the surface area of spread cells (Fig. 2E). Thus, ILK appears to be involved in regulation of molecular events that drive spreading and the extension of membrane protrusions.
ILK silencing alters the distribution of 5ß1 integrin and fibronectin matrix assembly
Fibrillar adhesions represent 5ß1-rich sites at which integrins bind to fibronectin fibrils and direct matrix assembly. Consistent with a disappearance of these structures in ILK-depleted cells, we noted a striking alteration in the distribution of the
5ß1 fibronectin receptor following ILK silencing. Staining of this integrin (visualized with both an anti-
5 polyclonal antibody and an anti-
5ß1 monoclonal antibody) appeared largely punctate with occasional localization in small plaque-like structures (Fig. 3). By contrast, ILK silencing had little or no effect on the staining pattern of the vitronectin receptor
vß3 in cells plated on vitronectin. The
vß3 integrin is associated with short plaques in both control and ILK siRNA-transfected cells [see supplementary Fig. S2 (http://jcs.biologists.org/cgi/content/full/117/19/4559/DC1)]. Thus, the presence of ILK is required for recruitment of
5ß1 into cell-matrix adhesions, whereas it is dispensable for recruitment of
vß3 to adhesive structures.
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The absence of fibrillar adhesions and the perturbed localization of 5ß1 in ILK-deficient endothelial cells prompted us to investigate the effect of ILK silencing on fibronectin fibrillogenesis, because
5ß1-rich fibrillar adhesions are known to play an important role in this process. Thus, following siRNA transfection, cells were plated on uncoated coverslips for 32 hours before immunostaining without permeabilization for detection of extracellular fibronectin fibrils. In control cultures, a well-defined network of fibrillar fibronectin matrix was associated with most cells (Fig. 4A). By contrast, only weak staining of surface fibronectin could be detected following ILK silencing. To elucidate the mechanism by which ILK silencing impairs fibrillogenesis, we compared the levels of total cellular fibronectin, fibronectin mRNA expression and secretion of the protein in control and ILK siRNA-transfected cells. As shown in Fig. 4A, bottom, immunostaining of fibronectin in permeabilized cells fixed 2.5 hours after plating (to avoid the production of an extensive extracellular fibronectin matrix) revealed that the protein was not preferentially sequestered inside ILK-depleted cells. Thus, the defect in fibrillogenesis in these cells could not be attributed to an intracellular accumulation of fibronectin. An organized extracellular fibronectin matrix could already be detected only 2.5 hours after plating in some control cells, as opposed to ILK siRNA-transfected cells. Accordingly, western blotting showed a dose-dependent decrease in cell-associated fibronectin following ILK siRNA transfection, rather than an augmentation (Fig. 4B). Finally, the levels of fibronectin mRNA were not attenuated in ILK siRNA-transfected cells, excluding the possibility of a suppressive effect of ILK silencing on fibronectin gene transcription or mRNA stability (Fig. 4C). Nonetheless, we did detect a twofold increase in the amount of soluble fibronectin in conditioned medium from ILK-deficient cells (Fig. 4D), consistent with our observation that exogenous fibronectin was unable to rescue this phenotypic trait (not shown). Altogether, these findings support the hypothesis that the defect in fibronectin fibrillogenesis in ILK-depleted cells stems from a deficiency in ILK-dependent fibrillar adhesions.
ILK silencing enhances cell adhesion
We next compared the adhesive properties of control and ILK siRNA-transfected cells. As shown in Fig. 5, reduction of ILK expression had a positive effect on cell adhesion measured 40 minutes after plating. This effect was also observed at earlier times (10 minutes), before spreading occurred (data not shown). Enhanced adhesion of ILK-depleted cells was detected on plates coated with fibronectin, type-I collagen, fibrinogen or vitronectin (Fig. 5). This pleiotypic effect was integrin-dependent, because no significant difference was detected upon adhesion of cells to poly-L-lysine-coated plates, and it was found at all concentrations of matrix proteins tested (0.1-10 µg ml1, data not shown). It is noteworthy that ILK-depleted cells were more resistant to detachment than control cells, as quantified using time-lapse images of trypsin-treated cells (data not shown).
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Because changes in cell adhesion could simply reflect changes in integrin expression levels, we determined the surface expression of 5ß1 and
vß3 heterodimers on BAEC by fluorescence-activated cell sorting (FACS) analysis. These two fibronectin-binding integrins are of particular importance in vascular development and angiogenesis. Indeed, the specific mean of fluorescence (which corresponds to the increase in fluorescence intensity relative to second antibody alone) was higher for both integrins in ILK-depleted cells (Fig. 6A). This increase was estimated to be 1.86 times for
5ß1 and 2.45 times for
vß3 integrin following ILK siRNA transfection. Total expression of the ß1 subunit protein and mRNA was unchanged following ILK silencing, as determined by western blotting of equal amounts of cell lysates, and northern analyses (Fig. 6B,C). Western analysis did reveal a slightly higher level (1.4 times) of the
5 subunit in ILK-deficient cells, which could contribute to the increased surface expression of
5ß1 integrin (Fig. 6B). Interestingly, ß3 mRNA and protein levels were higher in ILK siRNA-transfected cells than in control cells, indicating that ILK expression can affect ß3 transcription and/or mRNA stability differently.
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ILK silencing inhibits cell migration and tube-like structure formation
Coordinated regulation of cell adhesion and adhesion complex remodeling are crucial for cell movement. Thus, we sought to evaluate the effect of ILK silencing on BAEC migration. First, an in vitro wound-healing assay was used in which confluent monolayers were disrupted with a pipet tip. After 6 hours, the distance between the parallel migration fronts on either side of the wound was determined at three locations. As shown in Fig. 7A, ILK silencing reduced cell migration into the wound. Whereas the wound size was reduced by 50% in control siRNA-transfected cells, it was reduced by only 10% in ILK-targeted cultures. Decreased cell motility of ILK-depleted cells was confirmed in a modified Boyden-chamber assay. In this case, we observed an inhibition of haptotactic migration through a transwell filter towards fibronectin or collagen (Fig. 7B). Furthermore, following ILK knock-down, cells were insensitive to the chemotactic effect of S1P, a potent stimulator of endothelial cell migration (Fig. 7C).
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We next assessed the differentiated phenotype of endothelial cells with reduced ILK expression, as determined by their ability to assume a spindle-shaped morphology and organize into capillary-like structures on a Matrigel basement membrane matrix. Within 6 hours, control siRNA-transfected cells aligned and formed honeycomb structures that became thicker and more homogeneous with time (Fig. 7D). In ILK-deficient cells, this process was significantly delayed, consistent with the observed decrease in their motility. By 20 hours, ILK-depleted cells became organized into a mesh. Interestingly, a striking difference could be seen between the meshwork formed in ILK-deficient and control cultures. The capillary-like structures in ILK-depleted cultures were composed of single-cell alignments, whereas, in control cultures, they were multilayered, as mentioned above. These results would suggest that ILK deficiency results in reduced intercellular cohesion on a mixed Matrigel matrix.
Integrin signaling in ILK-deficient cells
RhoA has been found to play a role in the formation of focal adhesions from which stress fibers radiate (Clark et al., 1998; Nobes and Hall, 1995
; Rottner et al., 1999
) and, more recently, in fibronectin fibrillogenesis (Danen et al., 2002
; Zhong et al., 1998
). Accordingly, we assessed the possible impact of ILK repression on Rho activity following adhesion of cells to fibronectin-coated plates. The activation status of the GTPase was determined by measuring the levels of GTP-bound RhoA using a pull-down assay. As shown in Fig. 8A, RhoA was activated by adhesion in control siRNA-transfected cells and levels of the GTP-loaded form remained elevated for at least 60 minutes. Thus, 60 minutes after plating, Rho-GTP levels were an average of 2.5 times the basal values (in three independent experiments). In ILK-depleted cells, we observed adhesion-induced RhoA activation, but the extent and persistence of activation was reduced during spreading compared with control cells.
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To determine the consequences of ILK silencing on outside-in signaling, we analysed the activation of integrin-stimulated signaling cascades in cells following adhesion. One such cascade involves the protein tyrosine kinase FAK, which is autophosphorylated upon the binding of most integrins to extracellular matrix ligands at a docking site (Tyr-397) involved in recruitment of SH2-containing proteins and activation of the Ras-Raf-ERK cascade (Parsons et al., 2000). Using antibodies that recognize phospho-FAK (pY397), we found no significant difference in the level of FAK phosphorylation between ILK siRNA- and control siRNA-transfected cells following adhesion to fibronectin or collagen (Fig. 8B).
Similar to FAK, no differences were observed in basal or adhesion-stimulated phosphorylation of PAK between control and ILK-depleted cells, as shown in Fig. 8B. Importantly phosphorylation of PAK, a downstream effector of Rac and Cdc42, has previously been shown to reflect changes in Rac1 activity that are below the limits of detection in pull-down assays (Shamah et al., 2001). Thus, by this criterion, ILK silencing does not appear to impair Rac activation. Accordingly, translocation of cortactin from the cytosol to the rim of membrane ruffles in response to S1P, another Rac-dependent event in endothelial cells (Vouret-Craviari et al., 2002
), was observed in control and ILK-deficient cells challenged with the agonist (Fig. 8C).
Finally, because ILK has been found to induce phosphorylation of the protein kinases AKT/PKB and GSK3 following adhesion of cells to fibronectin or in response to agonists that activate phosphoinositide-3-kinase, we examined the consequences of ILK silencing on this pathway. As shown in Fig. 9A, we were unable to detect a significant decrease in AKT phosphorylation on Ser-473 in exponentially growing BAECs following ILK siRNA transfection. Further, insulin-stimulated levels of phospho-AKT were comparable in control and ILK siRNA-transfected cells (not shown). However, cyclin-D1 expression was suppressed in ILK-deficient cells. Cell-cycle analysis by FACS (Fig. 9B) revealed that this decrease was accompanied by an accumulation of cells in G0/G1 and a decrease in G2/M. It can be seen that sub-G1 DNA content was similar in both control and ILK-depleted cells, indicating that ILK silencing had little or no effect on cell survival.
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Discussion |
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ILK depletion had a marked effect on the formation of cell-matrix adhesions, including focal adhesions and extracellular matrix adhesions, recently renamed fibrillar adhesions (for reviews, see Critchley, 2000; Geiger et al., 2001
; Yamada and Geiger, 1997
; Zamir and Geiger, 2001
). Focal adhesions are typically flat plaques at the tips of actin stress fibers rich in paxillin, vinculin and tyrosine-phosphorylated proteins. ILK depletion in BAECs increased the staining of short paxillin- and vinculin-positive adhesions, suggesting that ILK might be involved in regulating focal-adhesion disassembly or maturation. Similar to ILK-depleted cells, FAK- and Src-deficient cells display an increase in the number of focal adhesions as well as decreased motility. It has been shown that these tyrosine kinases play important roles in the turnover, rather than the assembly of focal adhesions (Ilic et al., 1995
; Volberg et al., 2001
).
Fibrillar adhesions are elongated structures that arise from focal adhesion and participate in the organization of the extracellular fibronectin matrix. In monolayer cultures, fibrillar adhesions have been shown to contain 5ß1 integrin, tensin,
-parvin and little or no phosphotyrosine-containing proteins (Zamir and Geiger, 2001
). Under these 2D conditions, paxillin is absent from fibrillar adhesions in fibroblastic cells. However, when cells are cultured in a 3D matrix environment, paxillin has been found to colocalize with
5ß1 along fibronectin-containing extracellular fibers in what have been termed 3D-matrix adhesions (for a review, see Cukierman et al., 2002
). Interestingly, we found that paxillin colocalizes with
5ß1 integrin in fibrillar-type adhesions in control BAECs plated on fibronectin, reminiscent of its localization in 3D-matrix adhesions. Furthermore, ILK (a known paxillin binding partner) is present in these structures. Indeed, 2D cultures of BAECs accumulate an extensive fibronectin network that might mimic a 3D environment. In BAECs, we have not been able to detect the endogenous fibrillar adhesion protein tensin with currently available tools, although molecular characterization of ILK-dependent adhesions in these cells is under way.
Our finding that fibrillar structures are virtually absent from BAECs when ILK expression is suppressed indicates that ILK might participate in the formation of fibrillar adhesions and/or the transformation of focal adhesions into fibrillar adhesions. Although several integrins bind to fibronectin, the principal integrin responsible for fibronectin polymerization is 5ß1. This integrin binds soluble fibronectin at the ventral surface in the case of endothelial cells and, through a cell-driven process involving acto-myosin contractility, cryptic self-assembly sites in the fibronectin molecule are unmasked and spontaneous polymerization occurs. An elegant study of matrix assembly dynamics in human fibroblasts revealed that occupied
5ß1 integrin translocates from focal contacts along stress fibers to fibrillar adhesions and this is tightly coupled to fibronectin fibrillogenesis (Pankov et al., 2000
). Thus, the fact that ILK is required for association of
5ß1 with fibrillar adhesive structures suggests that ILK might be required for this translocation process. Interestingly, ILK-depleted BAECs plated on fibronectin-coated coverslips form
5ß1-positive focal adhesions soon after plating. This organization of focal adhesions in ILK siRNA-transfected cells occurs more rapidly than in control cells [see supplementary Fig. S3, 30 minutes after adhesion (http://jcs.biologists.org/cgi/content/full/117/19/4559/DC1)], in accordance with our finding that ILK silencing enhances cell adhesion. However, in absence of ILK, the
5ß1-containing plaques disappear within 2-5 hours and staining of the integrin becomes dispersed (Fig. 3, 24 hours after adhesion). Although the mechanisms underlying this phenomenon remain to be elucidated, it is possible that ILK regulates: binding parameters between soluble fibronectin to
5ß1; the cell-driven exposure of self-association sites in fibronectin; lateral diffusion and multimerization of the integrin in the membrane; and/or the organization of
5ß1-containing adhesion complexes competent for cytoskeletal coupling, a prerequisite for fibronectin matrix assembly. Importantly, we have shown that there is no defect in fibronectin expression or secretion in ILK-deficient cells. Thus, although the absence of ILK does not impair integrin binding to plastic-adsorbed fibronectin (cell adhesion assay),
5ß1 is not competent for matrix assembly without ILK-dependent localization to fibrillar adhesions. It is noteworthy that adhesion to fibronectin in both control and ILK-depleted BAECs was mediated by
5ß1 integrin, as determined by antibody inhibition assays, whereas adhesion to vitronectin-coated plates occurred via
vß3 (data not shown). This finding would exclude the possibility that a switch from
5ß1- to
vß3-mediated cell adhesion and spreading accounts for the observed defects in matrix assembly in ILK-depleted cells.
Fibronectin matrix plays important roles in many biological processes including morphogenesis and tumorigenesis. Indeed, a role has been proposed for ILK in fibronectin matrix assembly in epithelial cells (Wu et al., 1998). More recently, ILK overexpression has been implicated in the pathogenesis of diabetic glomerulosclerosis via mesangial matrix expansion (Guo et al., 2001
). Our finding that ILK silencing inhibits endothelial cell cohesion in an in vitro tubular morphogenesis assay suggest a new role for ILK-dependent fibronectin matrix deposition in regulation of cellular associations required for vascular network formation. Although the molecular mechanism involved in this process is not known, ILK binding to its partners including PINCH (Tu et al., 1999
) and CH-ILKBP (Tu et al., 2001
), an actin-binding component of fibrillar adhesions [also known as
-parvin/actopaxin (Nikolopoulos and Turner, 2000
; Olski et al., 2001
)], is required for localization to cell/extracellular-matrix contacts (Zhang et al., 2002
) and fibronectin matrix deposition in mesangial cells (Guo and Wu, 2002
).
In light of the observed defects in fibronectin matrix assembly, it was tempting to speculate that ILK acts by somehow impinging on the Rho pathway. It is noteworthy that robust activation of RhoA by the soluble agonist LPA was observed in both control and ILK siRNA-transfected cells (data not shown). In addition, LPA had similar effects on the actin cytoskeleton in both cell types. Although we did not detect a modification of basal or LPA-stimulated RhoA activity in adherent cells following ILK depletion, we did observe a reduction in the persistence of RhoA activation upon adhesion to fibronectin. The mechanisms responsible for this difference and its implications for adhesion, spreading and fibronectin fibrillogenesis remain to be determined.
Outside-in signaling to FAK was intact following ILK silencing. This finding is not surprising, because vinculin-positive focal adhesions are abundant in ILK-depleted cells. These highly tyrosine-phosphorylated structures containing FAK and downstream signaling components (Parsons et al., 2000) are thought to be the major site of integrin signal transduction. Likewise, the Cdc42/Rac effector PAK is efficiently phosphorylated following adhesion of ILK-depleted cells. In adherent cells, both FAK and PAK are activated in response to serum, independent of ILK expression, and cortactin is translocated to the plasma membrane upon S1P stimulation; this last response is indicative of a functional Rac pathway (Vouret-Craviari et al., 2002
). It is noteworthy that, although cortactin translocation takes place in ILK-deficient cells, the projected structures do not have the same morphology [see supplementary Movies 1 and 2 (http://jcs.biologists.org/cgi/content/full/117/19/4559/DC1)]. Thus, in the absence of ILK, some Rac-dependent but ILK-independent events might occur (i.e. cortactin translocation), whereas others (broadening/maturation of lamellipodia) might require both Rac and ILK.
The enhanced adhesion of ILK-depleted endothelial cells to ß1 and ß3 integrin ligands suggests that, in control cells, ILK might destabilize cell-matrix interactions, at least during the earliest stages of cell adhesion, that were examined in this study. Consistent with this concept, previous studies reported that ILK overexpression significantly impairs cellular adhesion to fibronectin, vitronectin and laminin in epithelial cells (Hannigan et al., 1996), and to collagen matrix in glomerular podocytes (Kretzler et al., 2001
). Similarly, in mononuclear leukocytes, overexpression of ILK was found to negatively regulate ß1-integrin/VCAM-1-dependent firm adhesion to endothelial cells (Friedrich et al., 2002
). The enhanced adhesion observed in ILK-depleted endothelial cells could be attributed to one or more factors including increased integrin expression, modulation of the integrin activation state and/or increased adhesion receptor clustering. At present, our finding that ILK-depleted cells present an increased surface density of
5ß1 and
vß3 offer a possible explanation for this effect. In the case of epithelial cells that manifested impaired adhesion upon ILK overexpression (Hannigan et al., 1996
), surface expression of integrins was not altered, and it was suggested that ILK plays a role in inside-out integrin signaling. Indeed a considerable body of evidence indicates that the activation state of integrins is controlled by interaction of their cytoplasmic domains with other proteins that behave as intracellular activators. The ability of ILK to modulate integrin affinity states or to affect integrin aggregation on the plasma membrane, a closely coupled event, remains to be further investigated. It is noteworthy that treatment of ILK-deficient cells with the divalent cation Mn2+, which is known to induce a high-affinity conformation and to mimic integrin activation, increases adhesion to fibronectin similar to control cells (data not shown). From these findings we can conclude that the removal of ILK per se does not confer a pre-activated conformation on fibronectin-binding integrins.
There are certain discrepancies between our findings and the reported cellular defects in deficient fibroblasts and chondrocytes, including impaired adhesion. It is difficult to explain the discrepancies between these findings and our observation that ILK enhances adhesion in endothelial cells following RNA interference. In the reports by Sakai et al. (Sakai et al., 2003), Grashoff et al. (Grashoff et al., 2003
) and Terpstra et al. (Terpstra et al., 2003
), ILK depletion did not significantly modify integrin expression in cultured cells. It is possible that differences in cellular context (e.g. integrin repertoire and culture conditions) contribute to the divergent effects on cell adhesion. Our findings that ILK-depleted BAECs display reduced cyclin-D1 expression and an increase in G0/G1 arrested cells agree with the findings in ILK-deficient fibroblasts and chondrocytes, although the defect in cell proliferation was only modest in the case of endothelial cells. Further, we did not note any significant decrease in the levels of phospho-AKT present in exponentially growing BAECs following ILK siRNA transfection, consistent with the papers of Sakai and Grashoff (Grashoff et al., 2003
; Sakai et al., 2003
). For reasons that are presently unclear, these latter results are at variance with reports by Terpstra et al. and Troussard et al. (Terpstra et al., 2003
; Troussard et al., 2003
).
In conclusion, our work reported here identifies ILK as a key regulator of the endothelial cell phenotype. Further, it provides novel insights into the mechanisms underlying fibrillar adhesion assembly and matrix remodeling that have important implications for integrin function in endothelial network formation.
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Acknowledgments |
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Footnotes |
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* These authors contributed equally to this work
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References |
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