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Published as doi: 10.1096/fj.05-4579fje.
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(The FASEB Journal. 2006;20:1489-1491.)
© 2006 FASEB

Regulation of cell-matrix adhesion dynamics and Rac-1 by integrin linked kinase

Etienne Boulter, Dominique Grall, Sébastien Cagnol and Ellen Van Obberghen-Schilling1

Institute of Signaling, Developmental Biology and Cancer Research, CNRS-UMR6543, Centre Antoine Lacassagne, Nice, France

1Correspondence: Institute of Signaling, Developmental Biology and Cancer Research, CNRS-UMR6543, Centre Antoine Lacassagne, 33 Ave. de Valombrose, Nice 06189, France. E-mail: vanobber{at}unice.fr

ABSTRACT

Extracellular matrix (ECM) receptors of the integrin family initiate changes in cell shape and motility by recruiting signaling components that coordinate these events. Integrin-linked kinase (ILK) is one such partner of ß1 integrins that participates in dynamic rearrangement of cell-matrix adhesions and cell spreading by mechanisms that are not well understood. To further elucidate the role of ILK in these events, we engineered a chimeric molecule comprising ILK fused to a membrane-targeted green fluorescent protein (ILK-GFP-F). ILK-GFP-F is highly enriched in cell-matrix adhesions, and its expression in fibroblasts leads to an accumulation of focal adhesions (2–5 µm) and elongated adhesions (>5 µm). ILK-GFP-F enhances cell spreading on fibronectin and induces a constitutive increase in the levels of GTP-bound Rac-1. Conversely, ILK knock-down by siRNA transfection decreases active Rac-1. Endogenous ILK was found to associate with PKL (paxillin kinase linker) and the Rac/Cdc42 guanine nucleotide exchange factor ßPIX. Further, expression of a dominant negative ßPIX mutant reversed the increase in active Rac-1 levels of ILK-GFP-F-expressing cells, thus placing ßPIX in the pathway leading from ILK to Rac-1 activation. However, expression of constitutively active Rac only partially restores the spreading defects of ILK-depleted cells, suggesting that an additional ILK-dependent signal is required for cell spreading.—Boulter, E., Grall, D., Cagnol, S., and Van Obberghen-Schilling, E. Regulation of cell-matrix adhesion dynamics and Rac-1 by integrin linked kinase.


Key Words: Rho family GTPase • integrin signaling • cell spreading • RNA interference

ADHESION OF CELLS TO ECM components regulates not only shape and motility but also determines how cells respond to soluble mitogens, survival, and differentiation-inducing factors. Cell-matrix interactions are largely mediated by surface receptors of the integrin family, a well studied class of adhesion molecules composed of {alpha}ß heterodimers, devoid of catalytic activity (1) . Binding of integrins to their cognate ligand in the ECM leads to integrin activation and clustering that triggers the formation of large multiprotein complexes beneath the membrane, referred to as cell matrix adhesions (2) . These complexes physically link integrins to the actin cytoskeleton and functionally couple them to the appropriate intracellular signaling networks (3) . Over the past few years, the structural and functional diversity of cell-matrix complexes has become increasingly appreciated, and various types of cell-matrix adhesions have been identified in adherent cultured cells (2 , 4) . The most common cell-matrix adhesions include focal adhesions, focal complexes, and fibrillar adhesions, each displaying distinct molecular components, morphological properties, and subcellular distribution (5) . More than 50 different proteins have been reported to localize to cell matrix adhesions (2) , including proteins with structural or scaffolding functions, as well as proteins that possess intrinsic catalytic activity and participate in signal transmission. Whereas some of these proteins bind directly to integrin cytoplasmic tails, others are recruited to adhesive structures via protein-protein interactions with adaptor molecules (6 , 7) .

Integrin-linked kinase (ILK) is a widely expressed integrin adaptor, first isolated in a yeast 2-hybrid screen, which directly associates with the cytoplasmic tail of the ß1 and ß3 subunits (8) . ILK is a modular protein for which three domains have been described in the literature. An N-terminal region composed of four ankyrin repeats involved in protein-protein interactions is adjacent to a short pleckstrin-homology- (PH) like domain that binds phosphoinositol (PI) (3 , 4 , 5) P3 (9) , and a C-terminal kinase catalytic domain with high-sequence similarity to serine/threonine kinases. In addition to integrin ß subunits, several ILK interactors have been identified over the past few years, and the sites of binding of these interactors to the ILK protein have been defined to varying extents. The ankyrin repeat region binds to the LIM-only Domain proteins PINCH-1 and 2 (10) and to ILKAP (ILK associated phosphatase) (11) , a PP2C family protein phosphatase. The kinase domain of ILK contains a paxillin binding site (PBS) that interacts with the LD1 (LIM Domain 1) motif of paxillin (12) . Binding of {alpha}- and ß-parvin (alias CH-ILKBP/actopaxin and affixin, respectively) (13 14 15) and the Caenorhabditis elegans unc-112 gene product (16) , ortholog of the human FERM-domain-containing protein Mig-2, has been mapped to the kinase region of ILK as well. Understanding the molecular interactions that take place between ILK and its partners beneath clustered integrins in living cells and their functional significance remains a major challenge.

Since its discovery nearly 10 years ago, ILK has been established as an important regulator of integrin mediated cellular events including adhesion, spreading, migration, gene expression, survival, and matrix remodeling, in a variety of cell types (for recent reviews (17 , 18) ). The ILK protein is highly conserved throughout evolution and has been found to function downstream of integrins in Drosophila (19) and Caenorhabditis elegans (16) . In the mouse, ILK fulfills an essential role very early during embryonic development because the ILK null phenotype is characterized by peri-implantation lethality with impaired epiblast polarization (20) . Deregulation of ILK expression or activity has been observed in human pathologies with aberrant proliferation/survival status as in cancer (see (21) ), or with altered fibronectin matrix deposition as in diabetic glomerulosclerosis (22) .

We have recently shown by RNA interference that ILK participates in the regulation of adhesion, spreading, migration, capillary morphogenesis and cell matrix adhesion dynamics in endothelial cells. In particular, a role for ILK was established in the formation of fibrillar adhesions that drive fibronectin fibrillogenesis (23) . In the present study, we attempted to further define the role of ILK in these events by using a gain of function strategy that entails targeting ILK to the plasma membrane via C-terminal fusion with an H-Ras membrane localization signal. In the case of wild-type PAK1, simple membrane anchoring is sufficient for focal adhesion localization (24) and stimulation of downstream signaling events, including the activation of Rac-1 (25) . Similarly, overexpression of focal adhesion kinase (FAK) linked to the extracellular and transmembrane domains of IL2R promoted its phosphorylation and initiation of integrin signaling (26) . We show here in fibroblasts and endothelial cells that expression of membrane-anchored ILK affects cell-matrix adhesion dynamics, promotes cell spreading, and activates the small GTPase Rac-1 via the ß-PIX exchange factor.

MATERIALS AND METHODS

All reagents, unless specified, were from Sigma Chemical Co. (Saint Quentin Fallavier, France). Tissue culture plasticware was from Nunc (Roskilde, Denmark).

Cell culture
Bovine aortic endothelial (BAE) cells, kindly supplied by Dr. H. Drexler (Max-Planck-Institute, Bad Nauheim, Germany), were grown as described (23) . CCL39 cells were stably transfected with plasmids encoding ILK-GFP or ILK-GFP-F using a modified Ca2+ phosphate protocol and grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 7.5% FCS and 400 µg/ml Geneticin (G418). CCL39 S19E cells (clone 10.2), stably expressing the Tet repressor and TREX ILK-GFP-F have been described in (27) . The HEK293 Phoenix cell line (American Type Culture Collection) was cultured in DMEM containing 8% heat-inactivated FCS. HeLa cells were maintained in DMEM containing 5% heat-inactivated FCS.

Plasmids
The vectors pcDNA3.1-ILK-GFP and pcDNA3.1-ILK-GFP-F were engineered from pcDNA3.1-ILK wild-type (WT), kindly provided by Dr. S. Dedhar (University of British Columbia, Vancouver, Canada). Briefly, a single nucleotide was introduced in the pEGFP-F vector (BD Biosciences Clontech, Le Pont de Claix, France) using the Quickchange XL mutagenesis kit (Stratagene Europe, Amsterdam, The Netherlands) upstream of the GFP initiation codon in order to shift the reading frame. Subsequently, the enhanced GFP (EGFP) or EGFP-F sequence was inserted in frame in AgeI/PmeI sites at the 3' end of the V5 epitope tag in pcDNA3.1-ILK WT by enzymatic digestion with AgeI/BglII for ILK-GFP or AgeI/XbaI for ILK-GFP-F. Alternatively, the retroviral vector pLXSN was used to express ILK-GFP-F in BAE cells. The pLNGFP control plasmid was kindly provided by Dr. Jean-Claude Chambard (CNRS-UMR6543, Nice France). The vector encoding enhanced green fluorescent protein (GFP) fused to constitutively active Rac-1 in the pBabepuro vector were provided by Dr. P. Roux (CRBM, Montpellier) (28) . ßPIX DHm (mutations L238R, L239S in the DH domain of ßPIX (25) ) was a kind gift of Dr. Ivan De Curtis (San Raffaele Scientific Institute, Milan, Italy).

Antibodies
Rabbit polyclonal anti-ILK antibody (Ab) (DI) was directed against a glutathione S-transferase (GST)-ILK ankyrin domain fusion protein. Rabbit polyclonal anti-ILK antibodies were directed against both an internal and a C-terminal ILK peptide (E41). Mouse monoclonal, anti-ILK (clone 65.1.9), antiphospho-tyrosine (clone 4G10) and anti-Rac-1 (clone 23A8) were from Upstate Biotechnology (Charlottesville, VA). Anti-RhoA (clone 26C4) was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-AKT and antiphospho-AKT (Ser-473 polyclonal antibodies were from Cell Signaling Technology Inc, (Beverly, MA). The rabbit polyclonal anti-{alpha}5 integrin Ab and the polyclonal anti-ßPIX Ab were from Chemicon International (Temecula, CA). The mouse monoclonal antitransferrin receptor was from Zymed (CliniSciences, Montrouge, France). The rabbit polyclonal anti-ERK Ab (E1B4) has been described in (23) . The mouse monoclonal anti-GIT2/PKL (referred to as PKL) and antipaxillin antibodies were from BD Biosciences Transduction Labs (Le Pont de Claix, France). The mouse monoclonal antivinculin Ab was from Sigma Chemical Co. (Saint Quentin Fallavier, France) and the mouse monoclonal anticaspase 9 from MBL (Nagoya, Japan). Monoclonal anti-hemagglutinin Ab was from BabCO (Richmond, CA). Monoclonal anti-V5 Ab was from Invitrogen (Cergy Pontoise, France). TrueblotTM secondary antibodies coupled to horseradish peroxidase used for Western blot analysis after immunoprecipitation experiments were from eBioscience (San Diego, CA). Secondary antibodies coupled to horseradish peroxidase or alkaline phosphatase were from Promega France (Charbonnières-les-Bains, France). Fluorescently labeled secondary antibodies were purchased from Molecular Probes (Eugene, OR).

siRNAs and double-stranded RNA transfection
Small interfering RNAs (siRNAs) were purchased from Eurogentec (Serang, Belgium). Sequences of control and ILK (23) and Rac-1 (29) siRNA have been reported. Double-stranded RNA transfection of BAE cells was performed using a Ca2+ phosphate protocol, as described previously (23) . Unless otherwise stated, cells were analyzed 24 to 48 h after the second transfection. The efficiency of silencing was determined by Western blot analysis in each experiment.

Retroviral infection of bovine aortic endothelial cells
Retroviral packaging HEK 293 Phoenix cells were transfected using a modified Ca2+ phosphate protocol in the presence of 25 mM chloroquine. The next day, the medium was replaced with fresh growth medium. After 48 h, condition medium was harvested, filtered through a 0.45 µm filter, and polybrene was added to a final concentration of 4 µg/ml. For infection, BAE cells were seeded 4 h before the addition of the viral supernatant. After 2 rounds of overnight infection with freshly harvested viral supernatant, the BAE cells were split and grown for up to three passages in complete medium containing 400 µg/ml geneticin (GFP and ILK-GFP chimeras) or 3 µg/ml puromycin (GFP-RacV12) to select infected cells. The efficiency of viral infection was verified by immunofluorescence staining and Western blot analysis.

Immunofluorescence and Image Analysis
Cells were plated on glass coverslips coated with fibronectin 10 µg/ml for 24 h and fixed in a solution of 3% paraformaldehyde containing 100 mM sucrose. Cells were permeabilized, stained with the appropriate Ab, or Alexa-phalloidin, and mounted in Gel Mount (Biomedia Corp, Foster City, CA). Fluorescence was observed on a Zeiss inverted microscope (Axiovert 200 M) equipped with a CoolSnap HQ cooled charge-coupled device camera (Roeper Scientifique, Every, France). Image acquisition, processing and analysis were performed using MetaMorphTM Imaging software. Scaled images acquired from GFP-stained cells were thresholded in order to define objects corresponding to cell-matrix adhesions. A filter was applied to discard objects smaller than 0.5 µm (between three and four pixels at x400 magnification), which were considered as background noise. Then, filtered objects were counted, and the number of objects was divided by the cell surface area to obtain the density of cell-matrix adhesions. Alternatively, the "fiber length" (length of the object taking into account its curvature as defined by MetaMorph) of each object was measured and classified into three groups depending on their "fiber length". Cell surface area was measured on F-actin stained cells.

Pull-down assay
Determination of active Rac-1 and active RhoA levels in cell lysates were performed using GST-CRIB (PAK3) and GST-RBD (rhotekin) fusion proteins, respectively (as described in (30) ).

Immunoprecipitation and Western blot analysis
Cells were rinsed in ice-cold PBS and lysed in Nonidet P-40 lysis buffer (50 mM HEPES pH=7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 2 mM Na3VO4, 1 mM NaF, 2 mM ß-glycerophosphate). Lysates were centrifuged at 10,000 rpm for 10 min at 4°C and supernatants were immunoprecipitated overnight with indicated immune sera. Thirty microliters of a 50% slurry of Protein A- sepharose beads were then added for 1 hour, pellets were rinsed 5 times and resuspended in Laemmli loading buffer for SDS-PAGE and Western blotting, as described previously (23) . The presence of ILK in immunoprecipitates was analyzed by performing SDS-PAGE under nonreducing conditions to avoid comigration with immunoglobulin heavy chains.

Cell fractionation
Separation of the particulate membrane fraction from the cytosolic fraction of cells was performed as described by del Pozzo et al. (31) .

RESULTS

Membrane-targeted ILK increases cell spreading and modifies cell-matrix adhesion dynamics in fibroblasts
ILK has very close spatial and functional ties with the plasma membrane. In various cultured cell lines, ILK is localized in cell matrix adhesions (23 , 32) , and its kinase activity has been reported to be regulated both in vivo and in vitro by phosphatidylinositol-3,4,5-triphosphate (9) . Thus, we engineered a plasma membrane-anchored ILK in order to constitutively localize it to the cellular compartment where it is physiologically recruited. This was accomplished by fusing the C-terminus of ILK to a green fluorescent protein (GFP) fitted with the farnesylation sequence of H-Ras (ILK-GFP-F) (Fig. 1 A).


Figure 1
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  		Figure 1. Membrane-targeted ILK. A) Schematic representation of membrane-targeted integrin-linked kinase (ILK-GFP-F). The C-terminal of ILK was fused to a green fluorescent protein (GFP) fitted with the farnesylation sequence of H-Ras. B) ILK-GFP and ILK-GFP-F expression in CCL39 fibroblasts was monitored by Western blotting using an anti-ILK Ab (DI). C) Exponentially growing ILK-GFP and ILK-GFP-F cells were subjected to hypotonic lysis and isolation of the membrane (M) and soluble (S) fractions was performed. Ten micrograms of protein were separated by SDS-PAGE and analyzed by Western blotting using antitransferrin receptor as a marker for the membrane and anticaspase 9 as a marker for the cytosol. Results are representative of 2 independent experiments. Chimeric ILK proteins were detected with anti-V5 Ab, and endogenous ILK was detected with the DI anti-ILK Ab. D) The mean surface area (±SEM, n>60) was determined on F-actin-stained cells 20 h after plating on fibronectin-coated coverslips. E) CCL39-derived cells expressing EGFP-F, ILK-GFP, or ILK-GFP-F were plated on fibronectin-coated coverslips for 20 h then fixed, permeabilized and stained for paxillin. Digital overlay of images was performed using MetaMorph software. Scale bar = 20 µm.

To study the effects of membrane-targeted ILK on cell phenotype and integrin signaling, expression vectors encoding ILK-GFP-F, or ILK-GFP as control, were stably transfected in CCL39 hamster fibroblasts. Expression levels were monitored using a polyclonal anti-ILK Ab by Western blot analysis. As shown in Fig 1B , ILK-GFP and ILK-GFP-F each migrate as a single band of ~80 kDa on Western blots, consistent with the theoretical MW of the chimeras. ILK chimeras were not overexpressed, as compared to endogenous ILK, and expression of the endogenous protein was not modified in turn by the presence of the chimeras (Fig. 1B ). As expected, the construct harboring the H-Ras farnesylation sequence showed enhanced membrane localization, compared to the control ILK-GFP construct, as determined by cell fractionation experiments (Fig. 1C ). It is noteworthy that expression of the chimeric proteins did not modify the membrane to cytosol ratio of endogenous ILK.

The most striking phenotype of ILK-GFP-F-expressing fibroblasts was their increased size. The surface area of spread ILK-GFP-F cells plated on fibronectin was 1.82 fold larger than that of ILK-GFP cells (Fig. 1D ), suggesting that membrane targeting alone is able to activate the cell machinery involved in cell spreading.

We next examined the localization of the chimeras by fluorescent microscopy together with paxillin, a characteristic cell-matrix adhesion component that has been shown to physically interact with ILK (12) . In CCL39 cells expressing EGFP-F alone, paxillin was mainly cytosolic with some punctate staining in small focal adhesions (Fig. 1E , top). ILK-GFP was mostly cytosolic as well. However, in agreement with the interactions described between ILK and paxillin, we did observe some of the fluorescent protein colocalized with paxillin in focal complexes and focal adhesions (Fig. 1E , middle). In contrast, ILK-GFP-F localization was highly enriched in cell matrix adhesions of various sizes, distributed throughout the basal cell surface and along the periphery (Fig. 1E , bottom). This finding suggests that plasma membrane targeting is sufficient to induce ILK accumulation in cell matrix adhesions and demonstrates that a limiting step in ILK localization is likely to be its recruitment to the plasma membrane. Moreover, in ILK-GFP-F cells, paxillin staining was also enriched in the same cell-matrix adhesions as ILK-GFP-F (Fig. 1E , bottom), suggesting that ILK localization can direct paxillin recruitment to cell-matrix adhesions.

To determine the effect of membrane-targeted ILK expression on dynamic regulation of cell-matrix adhesions, we compared the formation of adhesive structures in ILK-GFP and ILK-GFP-F cells following their adhesion to fibronectin. The distribution of the fluorescent chimeras at various times is shown in Fig. 2 A. CCL39-derived cells begin to spread between 1 and 2 h after plating and they reached their fully spread size by 4 h. At all times examined, the ILK-GFP-F protein was primarily localized in cell-matrix adhesions, whereas the distribution of ILK-GFP was more diffuse, as mentioned above. Interestingly, expression of the membrane-anchored chimera induced a marked increase in the number of adhesions per cell. Furthermore, adhesive structures were longer in ILK-GFP-F cells than in ILK-GFP cells, and they accumulated in the center of cells. We verified that the adhesive structures observed with the ILK chimeras corresponded to integrin-based cell-matrix adhesions by double-labeling with an anti-{alpha}5 integrin Ab (Fig. 2B ). Quantification of the density of {alpha}5ß1 integrin-rich adhesions in spread cells (20 h after plating) revealed a 1.9-fold increase in ILK-GFP-F expressing cells (Fig. 2C ). Adhesions were classified according to their size, as the most common adhesive structures (i.e., focal complexes, focal adhesions and fibrillar adhesions) display characteristic lengths (see (5) ). The results summarized in Fig. 2D show that the percentage of focal complexes per cell (i.e., the smallest adhesive structures, 0.5–2 µm) was reduced by 14% in ILK-GFP-F cells, as compared to ILK-GFP cells (black bars). Indeed, the average number of focal adhesions (2–5 µm) and elongated adhesions(>5 µm) was increased by 1.74- and 2.24-fold, respectively, in ILK-GFP-F cells (gray and white bars). Altogether, these findings suggest that ILK-GFP-F expression enhances the formation of focal complexes and their maturation into longer, more mature adhesions. In light of the accumulation of elongated adhesions at later times, membrane retention of ILK may affect the turnover of focal adhesions as well.


Figure 2
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  		Figure 2. Characterization of cell-matrix adhesions in CCL39 cells expressing ILK-GFP or ILK-GFP-F. A) Cells were plated on fibronectin-coated coverslips in serum-free medium for the indicated times before detection of GFP-tagged chimeras. Scale bar = 30 µm. B) Localization of {alpha}5ß1 integrin was visualized by immunostaining with anti-{alpha}5 Ab (red). Digital overlay of images is shown on the right. Scale bar = 20 µm. C) Density of cell-matrix adhesions was quantified using MetaMorph software, as described in Materials and Methods, on cells plated for 20 h on fibronectin-coated glass coverslips. Results represent the mean ± SEM (n=20, P<0.001). D) Size distribution of {alpha}5-containing cell-matrix adhesions was determined for each cell and classified according to morphological features described in (5) (see Materials and Methods for details). Results are depicted as a percentage of total number of adhesions per cell (mean±SEM, n=20). Numbers on the right correspond to the fold increase in ILK-GFP-F cells relative to ILK-GFP cells.

Cell-matrix adhesions also display distinct molecular compositions and subcellular distribution. Paxillin localization is reportedly restricted to peripheral focal complexes and focal adhesions (5) . Intriguingly, ILK-GFP-F and paxillin colocalized in elongated cell-matrix adhesions that share morphological similarities and subcellular distribution with fibrillar adhesions. This unusual localization of paxillin prompted us to examine the components of cell matrix adhesions more closely and their distribution in these cells. To do so, cells were stained for the integrin {alpha}5 subunit of the fibronectin receptor {alpha}5ß1, paxillin, vinculin and phospho-tyrosine. In ILK-GFP cells, all proteins colocalized with the fluorescent chimera in focal complexes and focal adhesions and no elongated adhesions could be detected (Table 1 , left). Phospho-tyrosine staining was restricted to focal complexes. In contrast, ILK-GFP-F cells displayed both short and elongated adhesions in which all stained proteins colocalized with ILK-GFP-F. In the case of phospho-tyrosine, the pattern was slightly different and punctate staining was detected in the periphery and along elongated adhesions (Table 1 , right). Unfortunately, we were unable to detect tensin, a key fibrillar adhesion marker, in CCL39 cells. Further, there was no significant increase in fibronectin matrix assembly on ILK-GFP-F expression (not shown). Therefore, these elongated adhesions do not fulfill the molecular requirements to be considered as classical fibrillar adhesions (33) and are more likely to be elongated focal adhesions undergoing some maturation process.


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  		Table 1. Composition of cell-matrix adhesions

Membrane-targeted ILK increases Rac-1 activation in fibroblasts
In light of the increased cell spreading observed in ILK-GFP-F cells and the known role for Rac in this process (34) , we assessed the ability of ILK-GFP-F to control Rac-1 activity in CCL39 fibroblasts. To do so, levels of active Rac-1 were monitored in a pull-down assay using GST-CRIB (PAK3) to sequester GTP-bound Rac in lysates of ILK-GFP and ILK-GFP-F fibroblasts. Rac-1GTP levels were measured in exponentially growing cells, in detached cells, and in cells after plating on fibronectin for 10 min. As shown in Fig. 3 A, Rac-1 was regulated similarly in both cell types. Consistent with findings in fibroblasts (31) , basal levels of Rac-1GTP decreased on detachment of cells and fibronectin stimulated Rac-1 to levels observed in exponentially growing cells. However, Rac-1 activation was markedly higher in ILK-GFP-F cells than in ILK-GFP cells.


Figure 3
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  		Figure 3. Membrane-targeted ILK increases Rac-1 activity in CCL39 fibroblasts. Activation of Rac-1 was measured in a GST-CRIB (PAK3) pull-down assay on (A) exponentially growing ILK-GFP and ILK-GFP-F expressing attached cells (Att), cells maintained in suspension in serum-free medium for 15 min (Sus) or cells plated on fibronectin-coated dishes (10 µg/ml) for 10 min (FN). B) Active Rac-1 was measured in CCL39 fibroblasts expressing ILK-GFP-F under control of a tetracycline-sensitive promoter in serum-deprived nontreated (control) or tetracycline-induced cells (1 µg/ml, 20 h) (+Tet) after adhesion (10 min) to fibronectin (FN) or stimulation or adherent cells with 10 ng/ml PDGF. ILK-GFP-F expression was monitored by Western blot analysis using anti-V5 Ab (left panel). Immunoblots were quantified as described in Materials and Methods and results were expressed as fold increase relative to control. Results represent mean values (±SEM) from 3 independent experiments. C) Active RhoA was measured in CCL39 fibroblasts expressing ILK-GFP-F under control of a tetracycline-sensitive promoter in exponentially growing (Expo) or serum-deprived nontreated (control) cells. ILK-GFP-F expression was induced as indicated in B. Representative results from 3 independent experiments are shown.

To verify that the observed differences in active Rac-1 levels between ILK-GFP and ILK-GFP-F expressing cells was not due to clonal variations or indirect effects of long-term expression of the membrane-anchored protein, we expressed ILK-GFP-F under control of a tetracycline-dependent promoter in CCL39 cells and assayed Rac-1 activity under noninduced and induced conditions. As shown in Fig. 3B (left), expression of the chimeric protein is very weak or nondetectable in absence of tetracycline. After tetracycline treatment, the expression of ILK-GFP-F increased. Similar to the results obtained with stable clones, active Rac-1 levels were higher in cells expressing ILK-GFP-F under all conditions tested, that is, in serum-starved adherent cells, following adhesion to fibronectin and after stimulation with a soluble agonist, platelet-derived growth factor (PDGF). This is consistent with the fact that tetracycline-inducible CCL39 cells expressing ILK-GFP-F are phenotypically similar to stable ILK-GFP-F clones. It is noteworthy that no difference in active RhoA was observed following induction of ILK-GFP-F expression in growing cells. Nonetheless, in the absence of soluble growth factors, we did observe that active RhoA levels were higher in cells expressing ILK-GFP-F than in control cells (Fig. 3C ).

ILK regulates Rac-1 in endothelial cells
To extend these studies on the regulation of Rac-1 by ILK, BAE cells were used. as we previously found that ILK depletion resulted in defects in cell spreading and migration in this system (23) . Furthermore, in addition to the gain-of-function approach (heterologous expression of ILK-GFP-F), these cells are amenable to loss-of-function studies (ILK knock down). As shown in Fig. 4 A, ILK silencing had a major effect on levels of Rac-1-GTP present in lysates of exponentially growing cells. An 80% decrease in ILK expression, as determined by Western blot analysis, led to a 90% decrease in GTP-bound Rac-1 in cells. Conversely, the expression of ILK-GFP-F in these cells induced an increase in GTP-bound Rac-1, consistent with the effects of this chimera in CCL39 fibroblasts (Fig. 4B ). Moreover, constitutive membrane targeting of ILK in BAE cells resulted in enhanced membrane ruffling (Fig. 4C ) and increased surface area of spread cells (Fig. 4D ). As shown in Fig 4C , ILK-GFP-F BAE cells displayed broad membrane protrusions enriched in cortical actin, which was not organized in thick cables but rather assembled into thin branched networks. Similar to CCL39 cells, ILK-GFP-F localized to cell-matrix adhesions (not shown).


Figure 4
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  		Figure 4. Regulation of Rac-1 activity by ILK in BAE cells. GTP-bound Rac-1 levels were determined in a GST-CRIB (PAK3) pull-down assay. A) Efficiency of ILK silencing was determined by Western blot analysis. GTP-bound Rac-1 and total Rac-1 levels were measured 24 h after the second transfection with 50 nM control or ILK siRNA duplexes. B) (top) Expression of ILK-GFP-F in transduced cells. Levels of active GTP-bound Rac-1 and total Rac-1 in exponentially growing GFP- or ILK-GFP-F-expressing BAE cells are shown below. C) Immunostaining of F-actin in exponentially growing GFP- or ILK-GFP-F expressing BAE cells. Images (x400 magnification) were sharpened using the two-dimensional deconvolution function (no neighbor parameters) of MetaMorph. Scale bar = 20 µm. D) Morphometric analysis revealed a 1.58-fold increase (P<0.001) in mean surface area of ILK-GFP-F expressing BAE cells, relative to control (GFP) cells. Cells were plated on fibronectin-coated coverslips 1 h before fixation and staining with Alexa-phalloidin to label F-actin. Scale bar = 40 µm.

RacV12 expression partially restores spreading in endothelial cells
We next examined whether expression of an active form of Rac-1 could overcome the spreading defect in ILK-depleted cells. To do so, ILK silencing was performed on BAE cell populations expressing GFP-RacV12 or GFP alone as control (Fig. 5 A). First, it can be seen in Fig. 5B (top panels) that GFP-RacV12 expression in cells transfected with a control siRNA duplex induced a marked increase in the surface area of spread cells. One hour after plating on fibronectin-coated coverslips, the GFP-RacV12 cells were 2.1-fold larger than control cells. Consistent with our previous findings (23) , ILK silencing considerably reduced the size of spread BAE cells. Interestingly, this reduction was observed in both control GFP cells and in cells expressing constitutively active Rac-1 (Fig. 5B middle panel), suggesting that an ILK-dependent event, distinct from Rac-1 activation, contributes to cell spreading. However, it can also be noted that RacV12 expression partially restored spreading of ILK-depleted cells. Finally, ILK-depleted cells and Rac-1-depleted cells displayed a similar reduction in size (Fig. 5B , bottom panel), yet silencing of these two proteins led to distinct morphologies. Most notably, Rac-depleted cells were totally devoid of lamellipodial extensions.


Figure 5
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  		Figure 5. Constitutively active Rac expression in ILK-deficient cells. BAC cells expressing GFP or GFP-RacV12 were transfected with control, ILK-, or Rac-1 targeted siRNA duplexes (50 nM) before analysis of cell spreading. A) Western blot analysis of ILK and Rac-1 levels in the GFP-expressing cells shown in B. B) Staining of F-actin (x400 magnification) in GFP or GFP-RacV12 BAE cells 1 h after adhesion to fibronectin. Cells were fixed 24 h after the second transfection with 50 nM siRNA duplexes. Scale bar = 40 µm. Morphometric analysis was performed on F-actin-stained cells 1 h after plating on fibronectin-coated coverslips. The mean surface area of spread cells (±SEM, 50<n<80) is plotted.

Role for PKL and ßPIX in ILK-dependent activation of Rac-1
To identify a molecular link between ILK and Rac-1 activation, we examined the role of ßPIX, a Rac/Cdc42 guanine nucleotide exchange factor (GEF) of the COOL/PIX family. It has previously been shown that the ILK partner, paxillin, can bind directly to the Arf-GTPase activating protein (Arf-GAP) proteins GIT1 and GIT2/PKL (35 ,36) . PKL, in turn, binds to and activates the Rac/Cdc42 GEF, ßPIX (37) . For these experiments, HeLa cells were used because the various antibodies tested did not recognize PIX in CCL39 hamster fibroblasts or bovine endothelial cells. Further, the high transfection efficiency of HeLa cells allowed us to use a dominant negative approach to address this question. As shown in Fig. 6 A, endogenous ILK coimmunoprecipitated with both PKL and ßPIX, suggesting that they may associate in a functional complex in intact cells. To determine whether the GEF activity of ßPIX is implicated in ILK-dependent Rac activation, HeLa cells were transfected with a plasmid encoding a hemagglutinin (HA)-tagged DH domain mutant of ßPIX, devoid of GDP/GTP exchange activity, which has previously been shown to exert a dominant negative effect (25) . Thereafter, Rac-GTP levels in growing cells were evaluated in a pull-down assay. As shown in Fig. 6B , and similar to our findings in fibroblasts (Fig. 3) , membrane-targeted ILK expression had a stimulatory effect on Rac-1 in HeLa cells. When ILK-GFP-F was coexpressed with the DH-ßPIX mutant, Rac-1 activity decreased by a factor of 2. These results indicate that ßPIX, likely via the ILK/PKL/ßPIX complex, participates in Rac-1 regulation by ILK.


Figure 6
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  		Figure 6. A) Association of endogenous ILK with ßPIX and PKL. Immunoprecipitation was performed on HeLa cells using preimmune (PI) or anti-ILK serum (DI). Total lysates (Tot) or immunoprecipitates were analyzed for the presence of ßPIX, PKL, ILK, and ERK (negative control) by Western blot analysis. B) Rac-1 pull-down assays were performed on HeLa cells transiently transfected with expression vectors encoding ILK-GFP-F and/or an HA-tagged DH domain mutant (PIX DHm), as indicated (+). The expression of exogenous ßPIX was detected using an Ab directed against the HA epitope, and ILK chimers were detected with the monoclonal anti-V5 Ab. A representative experiment is presented (n=2); fold-induction of Rac-1 GTP levels over basal levels in control cells transfected with empty vectors alone are indicated below.

Membrane-targeting of ILK has no effect on serine 473 phosphorylation of Akt
Finally, in light of the proposed role for ILK in activation of the Akt/GSK3 pathway, we examined the impact of ILK-GFP-F expression on Akt phosphorylation. As shown in Fig. 7 , expression of ILK-GFP-F in the tetracycline-inducible CCL39 line did not promote any change in the phosphorylation of Akt on serine 473, under basal or serum-stimulated conditions. Similar results were obtained in BAE cells (data not shown). These findings suggest that membrane-targeting and enrichment of ILK in cell-matrix adhesions is not sufficient for this effect. It is noteworthy that ILK knockdown in BAE cells does not alter basal or stimulated phosphorylation of Akt, as described previously (23) .


Figure 7
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  		Figure 7. Basal and serum-stimulated Akt phosphorylation. Phosphorylation of Akt on serine 473 was monitored by Western blot analysis in CCL39 fibroblasts expressing ILK-GFP-F under the control of a tetracycline-sensitive promoter. Cells were grown in absence (-Tet) or presence (+Tet) of 1 µg/ml tetracycline for 20 h and then serum-staved overnight, in absence (-Tet) or presence (+Tet) of tetracycline, prior to stimulation with 10% serum (FCS) for the indicated times. Representative results from at least 3 independent experiments are shown.

DISCUSSION

In this study, we set out to examine the role of ILK in the regulation of cell-matrix adhesions, cell spreading, and Rac activation. This was performed using a GFP-tagged form of ILK constitutively directed to the membrane that could be visualized by immunofluorescence imaging of fixed or living cells. In cells expressing this construct, cell-matrix adhesions were found to be the major site of localization.

Cell-matrix adhesion targeting of ILK
Previous studies on ILK function have largely relied on overexpression strategies or ILK depletion approaches, such as gene knockout in the mouse, RNA interference in cultured cells, or classical genetics in invertebrate model organisms (for recent reviews, see (21 , 38) ). However, our study is the first to attempt to "activate" ILK by altering its subcellular localization. Importantly, the concentration of chimeric ILK expression in stable fibroblast clones was lower than that of endogenous ILK, suggesting that the observed effects were not due to overexpression of the chimeras. The control chimera, ILK-GFP, was present in both cell-matrix adhesions and the cytosol, suggesting that this construct, that contains a 33-amino acid spacer between ILK and GFP, behaves similarly to the endogenous protein. Surprisingly, ILK-GFP-F was restricted to cell-matrix adhesions and was not diffusively dispersed throughout the plasma membrane, as we observed with EGFP-F. These observations indicate that ILK contains all the information required to direct its localization in adhesive structures, once targeted to the membrane and that a limiting step in its localization to these structures is likely to be plasma membrane recruitment. Thus far, the molecular mechanisms by which ILK is directed to the membrane remain largely unknown. Previous studies have shown that localization of ILK to focal adhesions does not require a functional PH domain; rather, interactions with both PINCH1 and CH-ILKBP/{alpha}-parvin are important (32) . Indeed, ILK has been shown to form a tripartite complex with PINCH and {alpha}-parvin, and this complex, although necessary, is not sufficient for ILK localization to cell-extracellular matrix adhesions. Interestingly, a F438A mutation in subdomain XI of the kinase domain of ILK prevented its localization in cell-matrix adhesions without altering its binding to PINCH1 and {alpha}-parvin (32) . ILK recruitment to adhesive complexes has also been found to depend on a functional paxillin binding site (PBS) (12) . These results together with our findings suggest that some unknown partners and/or mechanisms, involving at least C-terminal sequences regulate ILK localization. In C. elegans, UNC-112/mig-2, UNC-52/perlecan, and ßPAT-3/integrin are necessary for ILK localization to nascent muscle cell attachments (16) . Thus, integrin ß1, which binds to ILK C-terminus (8) , Mig-2 or some other yet to be identified protein might be important for ILK recruitment to the plasma membrane and targeting to cell-matrix adhesions in mammalian cells. Additionally, in our experiments, paxillin distribution was found to be dependent on ILK, whereas the contrary is generally proposed (12) .

Dynamics of cell-matrix adhesions
Membrane targeting of ILK markedly changed the dynamics and the profile of cell-matrix adhesions. First, ILK-GFP-F induced a significant increase in the density of cell-matrix adhesions after plating on fibronectin, suggesting that ILK-GFP-F increases the de novo formation of adhesions. In addition to the expanded number of adhesive structures, the size of cell-matrix adhesions was increased in ILK-GFP-F cells. Interestingly, these elongated adhesions were morphologically similar to fibrillar adhesions that drive assembly of the extracellular fibronectin network. As defined in human fibroblasts, fibrillar adhesions contain the fibronectin receptor {alpha}5ß1, tensin, and extracellular fibronectin, and they exclude paxillin, vinculin, and tyrosine phosphorylated proteins (5) . In ILK-GFP-F expressing hamster fibroblasts, the molecular composition of these elongated adhesions (>5 µm) was essentially the same as that of shorter adhesions. Furthermore, they were not associated to extracellular fibronectin fibrils, for at least up to 48 h after plating (unpublished observations). Therefore, these atypical adhesions are not likely to be bona fide fibrillar adhesions. Rather, they may be focal adhesions undergoing some maturation and elongation process, possibly resulting from forced membrane retention of ILK. It is possible that membrane retention of ILK may be required but is not sufficient to drive the process of fibronectin fibrillogenesis. Interestingly, in Drosophila, Brown and coworkers have shown that blistery/tensin stabilizes integrin adhesive contacts and that ILK is required for proper localization of blistery/tensin to integrin adhesive contacts (39) . Therefore, it is attractive to hypothesize that in mammalian cells, ILK may be required for proper localization and function of tensin. Further analyses of ILK and tensin and their interaction will be necessary to clarify their respective roles in regulation of cell-matrix adhesion dynamics. Additionally, we observed increased RhoA activation in serum-deprived ILK-GFP-F fibroblasts suggesting that ILK-GFP-F may enhance intrinsic (adhesion-based) RhoA activation in the absence of soluble agonists. Thereby, ILK-GFP-F may increase local RhoA-GTP loading and isometric tensions in adhesions resulting in their maturation and elongation (40) .

ILK-dependent regulation of cell spreading and Rac-1 activity
The observed increase in Rac-1 activity detected in CCL39 fibroblasts and BAE cells that express ILK-GFP-F, as compared to ILK-GFP cells, is consistent with the highly spread morphology of these cells. Conversely, ILK silencing in BAE cells induced a strong decrease in both cell spreading (23) and Rac-1 activation, as previously shown in HeLa cells (41) . ILK- and Rac-1-depleted cells displayed the same cell surface area, consistent with a regulation of Rac-1 by ILK. Further, constitutively active Rac-1 expression increased the spread surface area of ILK-deficient cells. However, we found that RacV12 expression only partially rescued the spreading defect of ILK-deficient cells, as compared to control cells expressing the constitutively active GTPase. Nevertheless, under conditions of high Rac-1 activity (RacV12 expression) ILK silencing did reduce cell spreading, suggesting that ILK can regulate cell spreading both through Rac-1 activation and another unidentified pathway possibly involving the turn-over of cell-matrix adhesions.

We have previously shown that RhoA activation following adhesion to fibronectin is less sustained in ILK-depleted endothelial cells upon adhesion to fibronectin (23) . Similarly, ILK has been reported to positively regulate RhoA activation in osteosarcoma cells (42) However, in exponentially growing ILK-depleted cells, or ILK-GFP-F-expressing cells under conditions in which Rac-1 levels were altered, RhoA activation was not significantly modified (this study and data not shown), suggesting that the molecular players involved in the regulation of these GTPases by ILK are distinct. We did note a slight decrease (~25%) in the levels of GTP-bound Cdc42 (unpublished observations). The regulation of Rac-1 by ILK may proceed through various pathways, as ILK binds to several partners that have direct or indirect links to Rac. Most notably, paxillin is a key protein in the regulation of Rac activity. Tyrosine-phosphorylated paxillin can bind to Crk family proteins, which then recruit DOCK180/ELMO and C3G, GEFs for Rac-1 and Rap1, respectively (43 , 44) . Additionally, Rap1 can activate Rac-1 through relocalization of Vav2 and Tiam1 (45) . Alternatively, paxillin can activate Rac-1 by recruiting FAK and inducing downstream activation of PI-3K-dependent GEFs (46) or via the recruitment of a GIT2/ßPIX complex (37) . Strikingly, paxillin was also recently reported as a negative regulator of Rac-1 by controlling the levels of GTP-bound Arf6 with the Arf-GAP (GIT1) (47) . PIX family GEFs are very attractive intermediates for ILK-dependent Rac-1 stimulation since {alpha}PIX can bind to ß-parvin (48) and thus activate Rac-1 (49) . In the present study, we observed that endogenous ILK is associated with GIT2/PKL and ß-PIX in HeLa cells. Furthermore, expression of a GEF-defective ß-PIX, harboring a mutation in the DH domain, interfered with ILK-GFP-F-induced Rac-1 activation, demonstrating that at least ß-PIX nucleotide exchange activity participates in ILK-dependent Rac-1 activation. The activation of ß-PIX by ILK and the role of other partners, i.e., paxillin and/or parvins, in this process remains to be further investigated, as {alpha}-parvin silencing as been shown to increase Rac-1 activation (50) .

Finally, it is noteworthy that the effects of membrane-targeted ILK on Rac-1 activation, as well as on cell spreading and adhesion stability, were opposite to those observed on ILK silencing. These results suggest that recruitment of ILK to the plasma membrane in itself is sufficient to induce a gain of function. Along this line of reasoning, the ILK-GFP-F gain of function mutant did not affect basal or agonist-stimulated phosphorylation of Akt on residue S473, indicating that targeting of ILK to the plasma membrane and, in particular to adhesive structures in CCL39 fibroblasts or endothelial cells, is not sufficient for initiation of this signaling cascade. This tool should be useful for further elucidation of ILK-dependent integrin signaling events.

ACKNOWLEDGMENTS

We kindly acknowledge Pierre Roux and Ivan De Curtis for providing the plasmids encoding GFP-tagged Rac-1V12 and ßPIX DH domain mutant, respectively. We thank Valérie Vouret-Craviari for help and advice during the initial stages of this study and Gertraud Orend for critically reading the manuscript. The fellowship of E.B. was funded by the Association pour la Recherche contre le Cancer and La Ligue Contre le Cancer (Comité 06). Further support for this study was provided by the Association for International Cancer Research, the Centre National de la Recherche Scientifique and the University of Nice (UMR6543). We are grateful to the Emerald Foundation for the time lapse imaging station.

Received for publication July 19, 2005. Accepted for publication February 27, 2006.

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