Integrin-linked Protein Kinase Regulates Fibronectin Matrix Assembly, E-cadherin Expression, and Tumorigenicity*

Chuanyue WuDagger §, Sarah Y. KeightleyDagger , Chungyee Leung-Hagesteijnpar , Galena Radevapar , Marc Coppolinopar , Silvia Goicoechea§, John A. McDonaldDagger , and Shoukat Dedharpar **

From the Dagger  Samuel C. Johnson Medical Research Center, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259, the par  Cancer Biology Research Program, Sunnybrook Health Science Centre, and Department of Medical Biophysics, University of Toronto, Toronto M4N 3M5, Ontario, Canada, and the § Department of Cell Biology and The Cell Adhesion and Matrix Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-0019

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Fibronectin (Fn) matrix plays important roles in many biological processes including morphogenesis and tumorigenesis. Recent studies have demonstrated a critical role of integrin cytoplasmic domains in regulating Fn matrix assembly, implying that intracellular integrin-binding proteins may be involved in controlling extracellular Fn matrix assembly. We report here that overexpression of integrin-linked kinase (ILK), a newly identified serine/threonine kinase that binds to the integrin beta 1 cytoplasmic domain, dramatically stimulated Fn matrix assembly in epithelial cells. The integrin-linked kinase activity is involved in transducing signals leading to the up-regulation of Fn matrix assembly, as overexpression of a kinase-inactive ILK mutant failed to enhance the matrix assembly. Moreover, the increase in Fn matrix assembly induced by ILK overexpression was accompanied by a substantial reduction in the cellular E-cadherin. Finally, we show that ILK-overexpressing epithelial cells readily formed tumors in nude mice, despite forming an extensive Fn matrix. These results identify ILK as an important regulator of pericellular Fn matrix assembly, and suggest a novel critical role of this integrin-linked kinase in cell growth, cell survival, and tumorigenesis.

    INTRODUCTION
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Introduction
Procedures
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Cell-extracellular matrix and cell-cell interactions play critical roles during many physiological and pathological processes including morphogenesis and tumorigenesis. Many of the cell-extracellular matrix and cell-cell interactions are mediated by cell adhesion molecules that are members of the integrin and cadherin families. Integrins are alpha beta heterodimeric transmembrane glycoproteins that interact with extracellular (or other cell surface) molecules and cytoplasmic molecules, including cytoskeletal and catalytic signaling proteins (1-5). Recent studies indicate that integrins not only receive signals from extracellular matrix but also actively participate in the assembly of extracellular matrix (5-8).

Fibronectin (Fn)1 is a major constituent of extracellular matrices deposited during embryogenesis and wound healing (9-11). The assembly of Fn matrix is a highly regulated cellular process in which soluble, dimeric Fn molecules are assembled into an insoluble, fibrillar pericellular matrix (5-7, 12). A common feature of many oncogenically transformed cells is that they lose the ability of assembling a Fn matrix (13). However, exceptions to the rule of neoplastic cells lacking Fn matrix clearly exist (9, 14). For example, Fn matrix assembly is dramatically enhanced in hairy cell leukemia cells (15, 16). Thus, although it is true that Fn matrix assembly is altered in most neoplastic cells, the specific phenotype (inhibition or stimulation of Fn matrix assembly) is probably determined by the origin of the neoplastic cells and the initial target of the oncogenic transformation. Because Fn matrix has a major impact on cell adhesion, migration, cell growth, and cell differentiation (1, 9, 17-20), an understanding of the molecular mechanism by which cells control Fn matrix assembly may provide important information on tumorigenicity and may lead to new ways of controlling tumor growth.

A number of studies have established that binding of Fn by specific integrins is critical in initiating Fn matrix assembly. Fn fragments containing the RGD-containing integrin binding site or antibodies recognizing the integrin binding site inhibited Fn matrix assembly in cultured cells and developing amphibian embryos (8, 21-24). In addition, antibodies to alpha 5beta 1 integrin reduced the deposition of Fn into extracellular matrix by fibroblasts (25-27). The participation of Fn-binding integrins in Fn matrix assembly has been extensively studied in Chinese hamster ovary (CHO) cells. Overexpressing alpha 5beta 1 in CHO cells with endogenous alpha 5beta 1 increased Fn deposition in extracellular matrix (28), whereas CHO B2 cells that are deficient in alpha 5 (29) did not assemble plasma Fn into the extracellular matrix (30). Reconstituting alpha 5beta 1 integrin expression by transfecting the CHO B2 cells with a full-length cDNA encoding the human alpha 5 chain completely restored fibrillar Fn matrix assembly (30). These studies established an important role of alpha 5beta 1 integrin in supporting Fn matrix assembly by CHO cells. In addition to alpha 5beta 1 integrin, members of the beta 3 integrins (alpha IIbbeta 3 and alpha vbeta 3) also initiate Fn matrix assembly (8, 24, 31), although some of the other Fn-binding integrins such as alpha 4beta 1 (32) or alpha vbeta 1 (33) do not. The ability of cells to use multiple integrins to support Fn matrix assembly provides the cells with a versatile mechanism for control of Fn matrix assembly. It may also explain why certain cells, such as fibroblastic cells derived from alpha 5 integrin null mutant embryos, assemble a Fn matrix in the absence of alpha 5beta 1 (34). The primary role of alpha 5beta 1 in Fn matrix assembly appears to involve initiating the assembly, as Fn mutants lacking the alpha 5beta 1 integrin binding site could not be assembled into Fn matrix unless in the presence of native Fn (35, 36).

We recently found that activation of specific Fn-binding integrins, either by mutations at the integrin cytoplasmic domains or using activating antibodies, dramatically stimulated Fn matrix assembly (8, 24). These studies indicate that the ability of a cell to assemble a Fn matrix is not only controlled by the types of integrins it expresses but also regulated by the Fn binding activity of the integrins. Previous studies have demonstrated that the extracellular ligand binding affinity of integrins can be controlled from within the cells (inside-out signaling) (3, 5, 38-40). Expression of a constitutively active R-Ras in CHO cells bearing alpha IIbbeta 3 integrin activated the integrin and resulted in increased Fn matrix assembly (37), whereas activation of Raf-1, probably via the extracellular signal-related kinase mitogen-activated protein kinase pathway, suppresses integrin activation and Fn matrix assembly (72). However, the molecular events that are immediately upstream of the integrins in the cellular control of Fn matrix assembly were not understood. A very attractive model is that cells control integrin activity and Fn matrix assembly via interactions of cytoplasmic regulatory proteins with integrin cytoplasmic domains.

One promising candidate that may be involved in regulating Fn matrix assembly is integrin-linked kinase (ILK), a newly identified ankyrin-repeat containing serine/threonine kinase (41). ILK binds to the beta  cytoplasmic domains of both beta 1 and beta 3 integrins, and phosphorylates the beta 1 cytoplasmic domain in vitro (41). We report here that overexpression of ILK in epithelial cells dramatically stimulated integrin-mediated Fn matrix assembly, down-regulated E-cadherin, and induced tumor formation in vivo. Our results identify ILK as an important regulator of pericellular Fn matrix assembly, and suggest a critical role for this integrin-linked kinase in cell-cell interactions and tumorigenesis.

    EXPERIMENTAL PROCEDURES
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Procedures
Results
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References

Reagents-- All organic chemicals were of analytic grade and were obtained from Sigma or Fisher unless otherwise specified. Media for cell culture were from Life Technologies, Inc. Fetal bovine serum was from HyClone Laboratories, Inc. (Logan, UT). Polyclonal rabbit anti-alpha 5 integrin cytoplasmic domain antibody AB47 was generated using a synthetic peptide representing the membrane distal region of the alpha 5 integrin cytoplasmic domain (LPYGTAMEKAQLKPPATSDA). Polyclonal rabbit anti-Fn antibody MC54 was raised against purified plasma Fn and purified with a protein A-Sepharose affinity column (30). Polyclonal rabbit anti-29-kDa fragment of Fn antibody was raised against the amino-terminal 29-kDa fragment of Fn and was further purified using Sepharose beads coupled with the 29-kDa fragment of Fn (42). Anti-ILK polyclonal antibody 91-4 was prepared in rabbits as described previously (41). Monoclonal hamster anti-rat alpha 5 integrin antibody (HMalpha 5-1) and mouse anti-rat beta 3 integrin antibody (F11) were from PharMingen (San Diego, CA). Monoclonal mouse anti-vinculin antibody (hVIN-1) and purified rabbit IgG were purchased from Sigma. The Fn fragments (110-kDa RGD-containing integrin-binding fragment, the 20-kDa and 70-kDa amino-terminal fragments, and the 60-kDa gelatin-binding fragment) were prepared as described previously (43).

cDNA Vectors, Transfection, and Cell Culture-- Rat intestinal epithelial cells (IEC-18) were maintained in alpha -MEM medium (Life Technologies, Inc.) supplemented with 5% FBS (Atlanta Biologicals, Norcross, GA), 3.6 mg/ml glucose, 10 µg/ml insulin, and 2 mM glutamine. The pRC/CMV and metallothionein promoter (MT)-driven expression vectors containing sense and antisense full-length ILK cDNA sequences were generated as described previously (41, 73). The expression vectors were transfected into IEC-18 cells using calcium phosphate, and the transfected cells were selected with G418 as described (41, 73). The expression of human ILK in IEC-18 cells transfected with the MT-ILK expression vectors (MT-ILK) was induced by growing the cells in alpha -MEM medium containing 100 µM ZnSO4 and 20 µM CdCl2 for 24 h or as specified in the experiments. The kinase-inactive ILK mutant (GH31R) was generated by a single point mutation (Glu right-arrow Lys) at amino acid residue 359 within the kinase subdomain VIII (41) using the Promega Altered Site II in vitro mutagenesis system. The mutated DNA was cloned into a pGEX expression system (Pharmacia Biotech Inc.) and expressed as a glutathione S-transferase fusion protein (41). Kinase assays were carried out using the recombinant protein as described previously (41), and the results showed that the Glu359 right-arrow Lys point mutation completely inactivated the kinase activity.2 The cDNA encoding the kinase-inactive mutant was cloned into a pcDNA3 expression vector (Invitrogen) and transfected into IEC-18 cells, and stable transfectants were selected as described previously (41).

Determination of ILK, E-cadherin, and beta 1 Integrin Levels-- The cellular levels of ILK and E-cadherin were determined by immunoblot using an affinity-purified polyclonal rabbit anti-ILK antibody 91-4 (41), and an anti-E-cadherin antibody (Upstate Biotechnologies, Inc., Lake Placid, NY). The cell surface expression of alpha 5beta 1 integrins was estimated by immunoprecipitation of surface-biotinylated cell lysates with a polyclonal rabbit anti-alpha 5beta 1 antibody as described (41).

Immunofluorescent Staining-- Fn matrix assembly was analyzed by immunofluorescent staining of cell monolayers (8). Cells were suspended in the alpha -MEM medium containing 5% FBS and other additives as specified in each experiment. Cells were plated in 12-well HTCR slides (Cel-Line, Inc., Newfield, NJ; 50 µl/well) at a final density of 2 × 105 cells/ml and cultured in a 37 °C incubator under a 5% CO2, 95% air atmosphere. Cells were fixed with 3.7% paraformaldehyde, and staining with the polyclonal rabbit anti-Fn antibody MC54 (20 µg/ml) and Cy3-conjugated goat anti-rabbit IgG antibodies (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA; 2.5 µg/ml). Stained cell monolayers were observed using a Nikon FXA epifluorescence microscope, and representative fields were photographed using Kodak T-Max 400 or Ektachrome 1600 direct positive slide film. To obtain representative images, exposure times for different experimental conditions were fixed, using the positive, e.g. matrix-forming cells, as the index exposure length.

In double staining experiments, 3.7% paraformaldehyde-fixed cells were permeabilized with 0.1% Triton X-100 in TBS containing 1 mg/ml BSA. The cells were then incubated with primary antibodies from different species as specified in each experiment. After rinsing, the bound primary antibodies were detected with species-specific Cy3- and fluorescein isothiocyanate-conjugated secondary antibodies. Stained cell monolayers were observed using a Nikon FXA epifluorescence microscope equipped with Cy3 and fluorescein isothiocyanate filters.

For inhibition studies, ILK13-A4a cells that overexpress ILK were plated in 12-well HTCR slides in the alpha -MEM medium containing 5% FBS and other additives as specified (2 µM anti-29-kDa Fn fragment antibody, 2 µM rabbit control IgG, or 4.2 µM of one of the following Fn fragments: 110-kDa RGD-containing integrin-binding fragment of Fn, 70-kDa amino-terminal fragment of Fn, or 60-kDa gelatin-binding fragment of Fn). The cells were cultured for 4 h, and then fixed and stained with the polyclonal rabbit anti-Fn antibody and the Cy3-conjugated goat anti-rabbit IgG antibodies as described above.

Isolation and Biochemical Characterization of Extracellular Matrix Fn-- To isolate and biochemically characterize extracellular matrix Fn, we cultured the cells in 100-mm tissue culture plates (Corning, Inc., Corning, NY) in alpha -MEM medium supplemented with 5% FBS, 2 mM L-glutamine, 3.6 mg/ml glucose, 10 µg/ml insulin, and other additives as specified in each experiment for two days. The cell monolayers were then washed three times with PBS containing 1 mM AEBSF and harvested with a cell scraper. The extracellular matrix fraction was isolated by sequential extraction of the cells with: 1) 3% Triton X-100 in PBS containing 1 mM AEBSF; 2) 100 µg/ml DNase I in 50 mM Tris, pH 7.4, 10 mM MnCl2, 1 M NaCl, 1 mM AEBSF; and 3) 2% deoxycholate in Tris, pH 8.8, 1 mM AEBSF (30). Fn in the deoxycholate-insoluble extracellular matrix fraction was analyzed by immunoblot with polyclonal rabbit anti-Fn antibody MC54 and an ECL detection kit as described previously (32). In addition, Fn in the matrix fractions was quantified by ELISA. In this assay, proteins in the matrix fractions derived from same number of cells were solubilized with 2% SDS in TBS (140 mM NaCl, 20 mM Tris-HCl, pH 7.4) containing 5 mM 2-mercaptoethanol and diluted 1:80 with 100 mM NaHCO3 (pH 9.2) before adding to wells (100 µl of matrix proteins corresponding to 31 µg of cellular proteins) of polystyrene 96-well ELISA plates (Corning). After incubation at 4 °C for 16 h, the remaining protein binding sites were blocked with 10 mg/ml BSA in 100 mM NaHCO3 (pH 9.2) at 37 °C for 2 h. The wells were rinsed three times with 0.1% (v/v) Triton X-100 in TBS, followed by incubation with 1 µg/ml of anti-Fn rabbit IgG (MC54) in TBS containing 0.1% (v/v) Triton X-100 and 10 mg/ml BSA at 37 °C for 90 min. At the end of incubation, the wells were rinsed four times with 0.1% (v/v) Triton X-100 in TBS. The wells were then incubated with an alkaline phosphate-conjugated goat anti-rabbit IgG (60 ng/ml, Jackson ImmunoResearch). After rinsing four times with 0.1% (v/v) Triton X-100 in TBS and twice with TBS, bound alkaline phosphate conjugate was detected colorimetrically with p-nitrophenyl phosphate at 405 nm using an ELISA microplate reader. The amounts of Fn were calculated from the A405 nm values based on a standard curve generated using purified bovine plasma Fn under identical experimental condition. The standard curve was linear within the range used.

Colony Formation in Soft Agar-- ILK13-A1a3 cells that overexpress ILK (3 × 105/well) and Ras-37 cells that overexpress Ha-RasVal-12 (2 × 103/well) were plated in 35-mm wells, in 0.3% agarose and assayed for colony growth after 3 weeks as described (41). Fn fragments were incorporated in the agar at the final concentrations indicated.

Tumor Formation in Athymic Nude Mice-- IEC-18, ILK14, or ILK13 cells were resuspended in PBS and inoculated subcutaneously into athymic nude mice (107/mouse). Six mice were inoculated per cell line. In situ tumor formation was assessed after 3 weeks.

Tyrosine Phosphorylation of p125FAK in ILK Cells-- ILK13-Ala3 and ILK14-A2C3 cells growing in monolayer culture were harvested using 5 mM EDTA/PBS (pH 7.6), and the cells were washed twice in PBS. Cells were resuspended in serum-free medium and then transferred to plain tissue culture plates (Nunc) or tissue culture plates precoated with 10 µg/ml Fn (Life Technologies, Inc.) or maintained in suspension. For the suspension control cells were kept in a 50-ml rocker tube. After 1 h of incubation at 37 °C in 5% CO2, cell monolayer (for the adherent controls) and cell pellet (for the suspension controls) were washed twice in ice-cold PBS and lysed in Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris, pH 7.4, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.2 unit/ml aprotonin, 2 µg/ml leupeptin, and 1 mM sodium vanadate). FAK was immunoprecipitated from 400-500 µg of total cell extract using 4 µg of mouse monoclonal anti-p125FAK antibody and protein A-agarose conjugate (Upstate Biotechnologies, Inc.). Immune complexes were washed three times in lysis buffer, boiled in SDS-polyacrylamide gel electrophoresis sample buffer, and run on a 7.5% gel. Resolved proteins were transferred to Immobilon-P (Millipore) and membrane blocked in 5% BSA (Sigma) in TBST (0.1% Tween 20 in Tris-buffered saline, pH 7.4). Tyrosine-phosphorylated FAK was detected using the RC20H recombinant antibody (horseradish peroxidase-conjugated, Transduction) and ECL detection system (Amersham Corp.).

    RESULTS
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Introduction
Procedures
Results
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References

Stimulation of Fn Matrix Assembly by ILK-- To determine whether ILK plays a role in regulation of Fn matrix assembly, we analyzed the ability of cells expressing different levels of ILK to assemble a Fn matrix. IEC-18 rat intestinal epithelial cells assembled a small amount of Fn matrix consisting of mostly short fibrils (Fig. 1A). ILK13-A1a3 cells, which were isolated from the IEC-18 cells stably transfected with a pRC/CMV expression vector containing full-length ILK coding sequence, express a much higher level of ILK than the parental IEC-18 cells (41). The ILK-overexpressing ILK13-A1a3 cells assembled an extensive Fn matrix resembling that formed by fibroblasts (Fig. 1B), whereas control transfectants (ILK14-A2C3), which express a level of ILK similar to that expressed by the parental IEC-18 cells, assembled a small amount of Fn matrix that is indistinguishable from that of the IEC-18 cells fibroblasts (Fig. 1C). To exclude the possibility that the observed effect depends on a specific clone, we analyzed 10 additional cell lines that were independently isolated from the cells transfected with the pRC/CMV-ILK expression vector (ILK13-A4a, A1d11, A4c, A4c3, and A4i) or the control vector (ILK14-A2C6, A2a3, A2g3, A2g8, and A3a1). Fn matrix assembly was dramatically increased in all six ILK-overexpressing cell lines (Table I). In contrast, all six control cell lines assembled a low level of Fn matrix resembling that of the parental IEC-18 cells. In marked contrast to overexpression of ILK, overexpression of an oncogenic Ha-Ras mutant in which the 12th amino acid residue is mutated (Ha-RasVal-12) in the IEC-18 cells abolished the assembly of Fn fibrils (Fig. 1E).


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Fig. 1.   Stimulation of Fn matrix assembly by ILK. The cells were cultured in the alpha -MEM medium containing 5% FBS for 24 h, and Fn was detected by indirect immunofluorescence as described under "Experimental Procedures." The ILK expression levels of cells shown in panels A-D were summarized in Table I. Panel E shows the IEC-18 cells that overexpress Ha-RasVal-12. Cells overexpressing the wild type ILK (B) are indicated by an asterisk.

                              
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Table I
Fn matrix assembly by cells expressing different levels of ILK
The ILK13 cell lines were independently isolated from IEC-18 rat intestinal epithelial cells that were stably transfected with a pRC/CMV expression vector containing full-length ILK coding sequence and they express a much higher level of ILK than the parental IEC-18 cells. The ILK14 cells were control transfectants (41). The MT-ILK1 (IIB8) cells were isolated from IEC-18 cells transfected with the sense ILK expression vector (MT-ILK1). The MT-ILK6 (E2) cells were isolated from IEC-18 cells transfected with the anti-sense ILK expression vector (MT-ILK6). The GH31R cells were isolated from IEC-18 cells transfected with a pCDNA3 expression vector encoding a ILK kinase-inactive mutant in which glutamic acid residue 359 was replaced with a lysine residue. The relative ILK expression levels were based on immunoblot analysis with anti-ILK antibodies (41). Fn matrix assembled by the cells was detected by immunofluorescent staining as described in Fig. 1.

To further confirm a regulatory role of ILK in Fn matrix assembly, we transfected IEC-18 cells with an ILK expression vector that was under the control of metallothionein promoter (MT-ILK). The expression of ILK in the MT-ILK transfectants increased when they were grown in the presence of Zn2+ and Cd2+ (Fig. 2A, lanes 1 and 2). Consistent with a critical role of ILK in Fn matrix assembly, these cells assembled more Fn matrix under the culture condition favoring the expression of ILK (Fig. 2, B and C). In control experiments, neither the ILK expression (Fig. 2A, lanes 3 and 4) nor the Fn matrix assembly (Fig. 2, D and E) of the parental IEC-18 cells was altered when they were cultured in the presence of Zn2+ and Cd2+. Thus, overexpression of ILK, either driven by a cytomegalovirus promoter or driven by a metallothionein promoter, stimulates Fn matrix assembly.


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Fig. 2.   ILK expression and Fn matrix assembly by cells transfected with an inducible ILK expression vector. IEC-18 cells transfected with the inducible ILK expression vector (MT-ILK1 cells, passage 17), and the parental IEC-18 cells as a control, were cultured in the alpha -MEM medium containing 5% FBS in the absence (-Zn++/Cd++) or presence (+Zn++/Cd++) of 100 µM ZnSO4 and 20 µM CdCl2 for 24 h. A, ILK expression. Equal amounts of cell lysates (25 µg of protein/lane) of MT-ILK1 cells (lanes 1 and 2) and IEC-18 cells (lanes 3 and 4) were separated by SDS-polyacrylamide gel electrophoresis, and ILK was detected by immunoblot using an affinity-purified polyclonal rabbit anti-ILK antibody 91-4 as described under "Experimental Procedures." B-E, Fn matrix assembly. The cells were cultured under conditions identical to panel A. Fn was detected by indirect immunofluorescence as described under "Experimental Procedures."

Involvement of Integrin-linked Kinase Activity in the Cellular Regulation of Fn Matrix Assembly-- To test whether the kinase activity is involved in the stimulation of Fn matrix assembly by ILK, we have overexpressed a kinase-inactive ILK mutant (GH31R) in the IEC-18 cells. Unlike cells overexpressing the wild type ILK (Fig. 1B), cells overexpressing the kinase-inactive ILK mutant did not assemble an increased amount of Fn into the extracellular matrix (Fig. 1D). Thus, the kinase activity is critical in the cellular signal transduction leading to the up-regulation of Fn matrix assembly.

Biochemical Characterization of Fn Matrix Assembled by Cells Overexpressing ILK-- The Fn matrix deposited by fibroblastic cells is characterized by insolubility in sodium deoxycholate (44). To determine whether Fn matrix induced by overexpression of ILK in the epithelial cells shares this characteristic, we extracted the cell layers with 2% sodium deoxycholate and analyzed the insoluble matrix fractions by immunoblotting. Fig. 3A shows that the cells overexpressing ILK (A1a3, A4a, and IIB8) assembled much more Fn into the deoxycholate-insoluble matrix than the cells that express relatively low level of ILK (A2C6, A2C3, and E2). By contrast, cells overexpressing Ha-RasVal-12 failed to deposit detectable amount of Fn into the detergent-insoluble matrix (Ha-Ras). The amount of matrix Fn deposited by cells expressing different levels of ILK was quantified by ELISA. The results showed that the ILK-overexpressing cells (A4a) deposited much more (>300%) Fn into the extracellular matrix than the control cells (A2C3) (Fig. 4). These results are consistent with the immunofluorescent staining data (Figs. 1 and 2). Taken together, they provide strong evidence supporting an important role of ILK in regulation of Fn matrix assembly.


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Fig. 3.   Characterization of Fn matrix. A, immunoblot analysis of extracellular matrix Fn. The cells that constitutively overexpress ILK (ILK13-A1a3, ILK13-A4a), control cells (ILK14-A2C6, ILK14-A2C3), and the cells that constitutively overexpress Ha-RasVal-12 (Ha-Ras) were cultured in the alpha -MEM medium. The IEC-18 cells that were transfected with the MT expression vector containing the sense ILK cDNA (MT-ILK1-IIB8), or the IEC-18 cells that were transfected with the MT expression vector containing the antisense ILK cDNA (MT-ILK6-E2) as a control, were cultured in the alpha -MEM medium supplemented with 125 µM ZnSO4 and 2.5 µM CdCl2. The extracellular matrix fractions were isolated from the cells, and Fn in the extracellular matrix fractions were analyzed by immunoblot with polyclonal rabbit anti-Fn antibody MC54 as described under "Experimental Procedures." Cells overexpressing ILK are indicated by asterisks. B-F, inhibition of ILK-induced Fn matrix assembly by the amino-terminal 70-kDa fragment and the RGD-containing 110-kDa fragment of Fn. ILK13-A4a cells overexpressing ILK were cultured in the presence of 2 µM anti-29-kDa Fn fragment antibody (B), 2 µM rabbit control IgG (C), 4.2 µM 70-kDa amino-terminal fragment of Fn (D), 4.2 µM 110-kDa RGD-containing integrin-binding fragment of Fn (E), or 4.2 µM 60-kDa gelatin-binding fragment of Fn (F) for 4 h. Fn was detected by indirect immunofluorescence as described under "Experimental Procedures." The scale bar in F equals 50 µm and applies to panels B-F.


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Fig. 4.   Quantification of Fn matrix by ELISA. Fn in the extracellular matrices deposited by cells overexpressing ILK (A4a), cells overexpressing Ha-RasVal-12 mutant (H-Ras), and control cells (A2C3) were quantified by ELISA as described under "Experimental Procedures." Bars represent mean ± S.D. of three replicates. The ILK-overexpressing A4a cells are indicated by an asterisk.

Participation of the RGD-containing Integrin-binding Domain and the Amino-terminal Domain of Fn in ILK-stimulated Fn Matrix Assembly-- Integrin-mediated Fn matrix assembly requires at least two discrete portions of Fn, the RGD-containing integrin-binding domain and the amino-terminal domain (8, 35, 43, 45, 46). To determine whether these domains also participate in Fn matrix assembly induced by overexpression of ILK, we utilized the 110-kDa RGD-containing fragment, the 70-kDa amino-terminal domain of Fn, and an antibody against the amino-terminal domain of Fn (anti-29-kDa). Both the antibody (Fig. 3B) and the Fn fragments (Fig. 3, D and E) decreased the Fn fibril formation induced by ILK. The inhibition was specific, as neither irrelevant rabbit IgG (Fig. 3C) nor a 60-kDa Fn Fragment lacking the amino terminus (Fig. 3F) inhibited the Fn matrix assembly. Thus, both the RGD-containing integrin-binding domain and the amino-terminal domain of Fn are involved in Fn matrix assembly promoted by overexpression of ILK.

Effect of ILK Overexpression on the Formation of Focal Adhesion and Matrix Contacts-- Cell adhesion to extracellular substrates is mediated by transmembrane complexes termed focal adhesions, which contain integrin, vinculin, and other cytoskeletal proteins. We previously showed that a connection between extracellular Fn and the intracellular actin cytoskeleton mediated by the integrins is required for the assembly of Fn fibrils (8). To begin to investigate whether the integrins are involved in the up-regulation of Fn matrix assembly by ILK, we analyzed the cell surface expression of the alpha 5beta 1 integrin and its co-localization with cytoskeleton-associated proteins such as vinculin in the cells that express different levels of ILK. Similar levels of cell surface alpha 5beta 1 integrins were expressed on the surface of the cells that express different levels of ILK (Fig. 5). In addition, abundant vinculin-containing focal adhesions were detected in the cells that express a relatively low level of ILK (Figs. 6C and 7C) as well as in the cell that overexpress ILK (Figs. 6A and 7A). However, only small amounts of alpha 5beta 1 integrin (Fig. 6D) and Fn (Fig. 7D) were co-localized with the focal adhesions in the cells that express a relatively low level of ILK. Overexpression of ILK promoted co-localization of alpha 5beta 1 integrin (Fig. 6, A and B) and Fn (Fig. 7, A and B) with vinculin. Thus, although cells expressing a relatively low level of ILK are not defective in the assembly of focal adhesion, a higher level of ILK promotes the assembly of complexes containing vinculin, alpha 5beta 1 integrin, and Fn matrix.


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Fig. 5.   Cell surface alpha 5beta 1 integrin expression. IEC-18, ILK13-A4a, ILK14-A2C3, MT-ILK-1 (IIB8), and MT-ILK-6 (E2) cells were surface-labeled with sulfo-NHS-biotin. Cell surface-biotinylated alpha 5beta 1 integrins were immunoprecipitated with a rabbit anti-alpha 5beta 1 antibody and detected by peroxidase-conjugated streptavidin and ECL as described (41).


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Fig. 6.   Effect of ILK overexpression on localization of alpha 5beta 1 integrin and vinculin. The ILK13-A4a cells that overexpress ILK (A and B) and the control ILK14-A2C3 cells (C and D) were incubated with a mouse monoclonal anti-vinculin antibody and a rabbit polyclonal anti-alpha 5 integrin antibody. The bound mouse and rabbit antibodies were detected with Cy3-conjugated anti-mouse IgG antibody (A and C) and fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody (B and D), respectively. ILK13-A4a cells overexpressing ILK are indicated by a asterisk. The scale bar in D equals 5 µm and applies to all panels.


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Fig. 7.   Effect of ILK overexpression on localization of Fn and vinculin. Fn and vinculin were detected in the ILK13-A4a (A and B) and ILK14-A2C3 (C and D) cells as described in Fig. 6, except that a mouse monoclonal anti-vinculin antibody and a rabbit polyclonal anti-Fn antibody were used as the primary antibodies.

Overexpression of ILK Down-regulates E-cadherin-- E-cadherin is an important epithelial cell adhesion molecule mediating cell-cell interactions. Because overexpressing ILK in epithelial cells disrupted the characteristic "cobble-stone" epithelial morphology of the epithelial cells (41), we studied the effect of ILK expression on the cellular level of E-cadherin. The level of E-cadherin in cells expressing different amount of ILK was determined by immunoblot using an anti-E-cadherin antibody. The parental IEC-18 epithelial cells expressed abundant E-cadherin (Fig. 8A, IEC-18). Overexpression of Ha-RasVal-12 in IEC-18 cells reduced the level of E-cadherin (Fig. 8A, Ras-37). Strikingly, E-cadherin was completely eliminated in ILK13-A1a3 and A4a cells that overexpress ILK, whereas it was present at a normal level in ILK14-A2C3 and A2C6 cells that express a similar level of ILK to the parental IEC-18 cells (Fig. 8A). These results indicate an inverse correlation between the level of ILK and that of E-cadherin.


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Fig. 8.   Effects of ILK overexpression on E-cadherin expression and tyrosine phosphorylation of pp125FAK. A, E-cadherin expression. The cellular levels of E-cadherin in the parental IEC-18 cells, the ILK13-A1a3 and ILK13-A4a cells that overexpress ILK, the ILK14-A2C6 and ILK14-A2C3 cells that were transfected with a control vector, and the Ras-37 cells that overexpress Ha-RasVal-12 were determined by immunoblot using an anti-E-cadherin antibody as described under "Experimental Procedures." B, phosphorylation of focal adhesion kinase. ILK13-Ala3 and ILK14-A2C3 cells were resuspended in medium and then transferred to a plain tissue culture plate (Plastic), tissue culture plate precoated with 10 µg/ml Fn (Fibronectin), or maintained in suspension (Suspension). After 1 h of incubation at 37 °C in 5% CO2, cells were washed twice in ice-cold PBS and lysed in Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris, pH 7.4, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.2 unit/ml aprotonin, 2 µg/ml leupeptin, and 1 mM sodium vanadate). FAK was immunoprecipitated from total cell extract using anti-pp125FAK antibody, and tyrosine-phosphorylated pp125FAK was detected by immunoblot using an anti-phosphotyrosine antibody as described under "Experimental Procedures."

In contrast to E-cadherin level, overexpression of ILK did not alter the ability of the cells to phosphorylate focal adhesion kinase (pp125FAK) in response to cell adhesion to Fn (Fig. 8B), indicating that tyrosine phosphorylation of pp125FAK does not transduce the signals leading to the alterations observed upon ILK overexpression, and in particular tyrosine phosphorylation of pp125FAK does not play a regulatory role in ILK-induced Fn matrix assembly.

Induction of in Vivo Tumorigenesis by Overexpression of ILK-- To assess a potential role of ILK in tumorigenesis, we injected cells expressing varying levels of ILK into athymic nude mice subcutaneously. Tumors arose within 3 weeks in 50-100% of the mice injected with the ILK13 cells (107 cells/mouse) that overexpress ILK, whereas no tumors were detected in the mice that were injected with the same number of the IEC-18 or ILK14 cells expressing lower levels of ILK (Table II). Thus, overexpression of ILK in these epithelial cells promotes tumor formation in vivo.

                              
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Table II
Tumorigenicity of ILK overexpressing IEC-18 cells
Athymic nude mice were inoculated subcutaneously with the cells expressing high (ILK13-A1a3 and A4a) or low (IEC-18 and ILK14-A2C3) levels of ILK (107 cells/mouse in PBS). The mice were monitored for tumor formation at the site of inoculation after 3 weeks.

Inhibition of ILK-induced Cell Growth in Soft Agar by Amino-terminal Fragments of Fn That Inhibit Matrix Assembly-- One of the hallmarks of tumor-forming cells is that their growth is less dependent on anchorage as measured by their ability to grow in soft agar culture. Similar to cells overexpressing Ha-RasVal-12, cells overexpressing ILK were able to grow in soft agar (41). However, in marked contrast to the Ha-RasVal-12-overexpressing cells, ILK-overexpressing cells assembled an abundant Fn matrix (Table I). We therefore began to test whether the ability of the ILK-overexpressing cells to grow in soft agar culture is related to the elevated level of Fn matrix assembly. We cultured the cells overexpressing ILK and the cells overexpressing Ha-RasVal-12, respectively, in soft agar either in the presence or absence of the 70-kDa Fn amino-terminal fragment, which inhibits the ILK-induced Fn matrix assembly (Fig. 3D). The 70-kDa Fn fragment significantly inhibited the ILK-induced "anchorage-independent" growth in soft agar (Fig. 9A). Similar inhibition was observed with the 29-kDa fragment of Fn (Fig. 9A), another known inhibitor of Fn matrix assembly. In contrast, the Ha-RasVal-12-induced anchorage-independent growth in soft agar was not inhibited by the 70-kDa Fn fragment (Fig. 9B). Moreover, the ILK-induced cell growth in soft agar was not inhibited by the 60-kDa Fn fragment (Fig. 9A), which does not inhibit the Fn matrix assembly induced by ILK (Fig. 3F). These results suggest that Fn matrix likely plays an important role in the ILK-induced, but not the Ha-Ras-induced, cell growth in soft agar.


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Fig. 9.   Inhibition of ILK-induced cell growth in soft agar by the amino-terminal fragments of Fn. ILK13-A4a cells that overexpress ILK (A) and Ras-37 cells that overexpress Ha-RasVal-12 (B) were plated in 35-mm wells, in 0.3% agarose (untreated) or 0.3% agarose containing the Fn fragments, and assayed for colony growth after 3 weeks as described (41). 0.3 mg/ml 70-kDa Fn amino-terminal fragment (+70 kDa), 0.3 mg/ml 29-kDa Fn amino-terminal fragment (+29 kDa), or 0.3 mg/ml 60-kDa Fn fragment as a control, was included in wells as indicated. Colonies were counted and scored per field (d = 1 cm) in duplicate, and defined as a minimum aggregate of 50 cells. Bars represent mean ± S.D. of duplicate determinations.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

ILK is a novel ankyrin repeat-containing serine/threonine protein kinase (41). ILK interacts with the beta 1 cytoplasmic domain via the carboxyl-terminal region of the kinase catalytic domain, leaving the ankyrin-like repeat region to interact with other as yet unidentified proteins. The effect of overexpressing ILK protein in intestinal epithelial cells is quite dramatic. The overexpressing cells assume a vastly different cellular morphology from the "cobble-stone" epithelioid morphology of the parental cells. Cell adhesion to ECM proteins is altered, and the cells can grow in an anchorage-independent manner in soft agar (41).

The altered cellular morphology and the anchorage-independent growth of ILK-overexpressing cells prompted us to examine three prominent features of malignant transformation. These include loss of expression or function of E-cadherin, tumorigenicity in vivo, and alterations in Fn matrix assembly. We have demonstrated in this paper that the overexpression of ILK results in a loss of E-cadherin protein expression, offering a possible explanation for the loss of cell-cell contact in these cells. Indeed, losses of cell-cell adhesion have been implicated in tumorigenicity in vivo (47-49). We have also shown that ILK-overexpressing cells are tumorigenic in nude mice in contrast to the parental IEC-18 intestinal epithelial cells and the control transfected clones. Thus, ILK can be considered to be a proto-oncogene. Another important, and somewhat surprising, finding is the apparent involvement of ILK in Fn matrix assembly. Overexpression of ILK in IEC-18 cells stimulated Fn matrix assembly. This is a property of transfected cell clones constitutively overexpressing ILK, and also of transfected clones in which ILK expression is induced using a metallothionein-inducible promoter.

The ILK-stimulated Fn matrix assembly was inhibited by the amino-terminal domain of Fn, as well as the RGD-containing integrin-binding domain of Fn, suggesting that RGD-binding integrins mediate ILK functions in Fn matrix assembly. Due to the unavailability of anti-integrin function blocking antibodies against rat integrins, it has not been possible to identify directly the specific integrin(s) involved in the enhanced Fn binding and matrix assembly. However, using immunofluorescence analysis, we found that ILK overexpression promoted the co-localization of the alpha 5beta 1 integrin (Fig. 6, A and B) and Fn (Fig. 7, A and B) with vinculin, whereas in the parental IEC-18 cells and control-transfected cells, vinculin-containing focal adhesion plaques were not co-localized with the alpha 5beta 1 integrin (Fig. 6, C and D) and Fn (Fig. 7, C and D). Our preliminary studies also indicate that cell surface Fn binding activity, but not cell surface integrin expression level, was increased in cells overexpressing ILK.3 These findings are consistent with our previous observations that a connection between extracellular Fn and the actin cytoskeleton is required for the assembly of Fn fibrils (8) and further suggest that ILK overexpression likely enhances both the extracellular Fn-integrin interaction and the intracellular integrin-cytoskeleton interactions.

The kinase activity of ILK is clearly important in the stimulation of Fn matrix assembly, as overexpression of a kinase-inactive ILK mutant failed to enhance Fn matrix assembly. However, because ILK has potential binding sites for integrins and probably other intracellular signaling molecules (41), and because Fn matrix assembly can be regulated by post ligand occupancy events (8), it is possible that other activities of ILK may also play important roles in the stimulation of Fn matrix assembly. Delineation of signaling pathways leading to the regulation of Fn matrix assembly is an important area of future studies.

Oncogenic transformation often results in decreased expression of alpha 5beta 1 integrin (50) and in decreased Fn matrix assembly in culture. Overexpressing alpha 5beta 1 in CHO cells with endogenous alpha 5beta 1 suppressed the in vivo tumorigenicity of the cells (28) and CHO B2 cells that are deficient in alpha 5 exhibited increased tumorigenicity in vivo (51). However, there are many exceptions to this paradigm, and it should be noted that many tumor cell lines have elevated levels integrins and can organize Fn matrices in culture to variable extents (9, 14, 52). It has been shown that, whereas increased expression of the integrin alpha 5beta 1 in HT29 colon carcinoma cells results in the growth arrest of these cells and reduced tumorigenicity, ligation of alpha 5beta 1 integrin on these cells by cell attachment to a Fn substrate reverses the growth inhibition induced by overexpression of the integrin (53, 54). These results suggest that cell growth and tumorigenicity are controlled by signaling pathways that can be regulated by the levels of free and Fn-ligated integrins. Indeed, the ability to form a Fn matrix is important for the anchorage-independent growth of transforming growth factor beta -treated fibroblasts (55, 56), and the ability of mammary carcinoma cells (SP1) to grow in soft agar depends on Fn matrix assembled by the cells (74). In a recent study, Weaver et al. (75) have demonstrated that treatment of human breast cancer cells in a three-dimensional culture with inhibitory beta 1 integrin antibody leads to a striking morphological and functional reversion to a normal phenotype. Our observations that the 29-kDa and 70-kDa amino-terminal fragments of Fn inhibited the ILK-induced anchorage-independent growth in soft agar are consistent with these findings and raise an interesting possibility that ILK-induced Fn matrix assembly could contribute to the promotion of the anchorage-independent growth and tumor formation by ILK. However, although it is possible that an alteration in Fn matrix assembly could contribute to abnormal cell growth and consequently tumor formation, and it is clear that ILK plays important roles in Fn matrix assembly, anchorage-independent cell growth, and tumor formation, direct evidence for a causal relationship between the ILK-stimulated Fn matrix assembly and the anchorage-independent cell growth or tumor formation has yet to be obtained. Future studies investigating the roles of ILK in regulation of Fn matrix assembly in three-dimensional culture and in vivo, and an understanding of the molecular mechanisms by which ILK regulates Fn matrix assembly and cell growth will likely provide valuable information on this important subject.

It is intriguing to compare the effects of overexpression of ILK with those induced by overexpression of Ras. Aside from the dramatically difference in the effect on Fn matrix assembly, the expression of activated Ras results in the disregulation of multiple signaling pathways and typically renders cells serum-independent, as well as anchorage-independent for cell growth (57). On the other hand, the overexpression of ILK does not result in serum-independent cell growth (73), but induces anchorage-independent cell growth. These results indicate that ILK normally regulates adhesion-dependent signaling pathways and that the disregulation of ILK (e.g. by overexpression) induces anchorage-independent cell growth specifically. Thus, it is likely that ILK-mediated signaling may be involved in the regulation of integrin inside-out signaling, as activated integrins are required for Fn matrix assembly (8).

We have shown previously that the attachment of the ILK-overexpressing ILK13 cells to surface coated with exogenous Fn is reduced when compared with the wild type or control ILK14 cells (41). This perceived reduction of attachment to exogenous Fn could be due to the increased Fn matrix produced by the ILK13 cells resulting in the occupancy of most alpha 5beta 1 integrins on the cell surface, and consequently decreased adhesion to the Fn-coated surface. The "integrin activation-induced reduction of cell adhesion to Fn-coated surface" had been observed previously. For example, Faull et al. (58) reported that activation of alpha 5beta 1 integrin by 8A2 activating antibody resulted in occupancy of most cell surface of alpha 5beta 1 integrin by Fn, which resulted in a reduction of cell adhesion to Fn-coated surface. The inhibition of cell adhesion to Fn induced by overexpression of ILK may also result from events independent of Fn binding, as cell adhesion clearly can be modulated by intracellular signaling pathway without affecting integrin ligand binding activity (59, 60). In this regard, it is worth noting that cell adhesion to other extracellular proteins such as laminin and vitronectin was also inhibited by overexpression of ILK (41).

The ability to assemble an extensive Fn fibrillar matrix is a property of mesenchymal cells, and it is intriguing that the stimulation of this activity by ILK overexpression in the epithelial cells is accompanied by a dramatic down-regulation of cellular E-cadherin expression. Numerous previous studies have established that cellular E-cadherin level or activity is down-regulated during epithelial-mesenchymal transition (61-64). Moreover, in a recent study, Zuk and Hay (65) demonstrated that inhibition of alpha 5beta 1 integrin, which is a substrate of ILK, significantly inhibited epithelial-mesenchymal transition of lens epithelium. It is now also widely accepted that many invasive carcinomas exhibit a loss of E-cadherin expression (47, 48, 66, 67), and E-cadherin gene has been found to be a tumor/invasion-suppressor gene in human lobular breast cancer (68). The tumor suppressor gene fat in Drosophila is also homologous to cadherins (69). ILK may therefore be involved in coordinating cell-matrix adhesion and cell-cell adhesion in epithelial-mesenchymal transition, and overexpression of ILK may drive epithelial cells toward a mesenchymal phenotype and oncogenic transformation. It is unclear at present as to whether ILK is directly involved in the down-regulation of expression of E-cadherin, a consequence of which would be the stimulation of mesenchymal properties via the interaction of beta -catenin and LEF-1 (70), or whether ILK directly activates alpha 5beta 1 integrin, resulting in increased Fn matrix assembly and as a consequence, decreased E-cadherin level. The data presented here favor the latter possibility, although the former possibility deserves further investigation and the two mechanisms are not necessarily mutually exclusive.

The ILK-stimulated Fn matrix assembly may allow enhanced interaction of Fn with alpha 5beta 1. This integrin has recently been shown to be specific in supporting survival of cells on Fn, although no direct correlation was found between Fn matrix assembly and alpha 5beta 1-mediated cell survival (71). This latter conclusion was derived from the use of wild type alpha 5beta 1 and alpha 5 cytoplasmic deleted (alpha 5Delta Cbeta 1) mutants. It is likely that for cell survival, both receptor interaction with Fn as well as proper intracellular interactions are required. We have found recently that ILK overexpression in IEC-18 cells induces cell survival in suspension cultures largely due to the up-regulation of expression cyclin D1 and cyclin A proteins (73). Whether the ability of these cells to organize a Fn matrix is involved in the induction of cyclin D1 and cyclin A expression remains to be investigated. Future studies will focus on the molecular mechanisms by which ILK regulates gene expression, integrin activation, and matrix assembly.

Acknowledgments-- We thank Dr. J. Filmus for the Ras transformed IEC-18 cells (Ras-37), and Ka Chen and Tammy Brehm-Gibson for technical support.

    FOOTNOTES

* This work was supported in part by grants from the American Heart Association (to C. W.), the American Lung Association (to C. W.), the Arizona Disease Control Research Commission (to C. W. and J. A. M), and the National Cancer Institute of Canada (to S. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Edward Livingston Trudeau Scholar of the American Lung Association and Parker B. Francis Fellow in Pulmonary Research. To whom correspondence should be addressed: Dept. of Cell Biology and The Cell Adhesion and Matrix Research Center, University of Alabama at Birmingham, Birmingham, AL 35294-0019. Tel.: 205-975-2253; Fax: 205-934-7029; E-mail: cwu{at}bmg.bhs.uab.edu.

** Terry Fox Scientist of the National Cancer Institute of Canada.

1 The abbreviations used are: Fn, fibronectin; ILK, integrin-linked kinase; CHO, Chinese hamster ovary; FBS, fetal bovine serum; alpha -MEM, alpha -minimal essential medium; MT, metallothionein promoter; TBS, Tris-buffered saline; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; FAK, focal adhesion kinase; AEBSF, [4-(2-aminoethyl)benzenesulfonylfluoride, HCl].

2 A. Novak, S. Hsu, C. Leung-Hagesteijn, G. Radeva, J. Papkoff, R. Montesano, C. Roskelley, R. Grosschedl, and S. Dedhar, submitted for publication.

3 C. Wu, S. Y. Keightley, C. Leung-Hagesteijn, G. Radeva, M. Coppolino, S. Goicoechea, J. A. McDonald, and S. Dedhar, unpublished observations.

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Discussion
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