From the Samuel C. Johnson Medical Research Center,
Mayo Clinic Scottsdale, Scottsdale, Arizona 85259, the
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
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 5
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
5
1 in CHO cells
with endogenous
5
1 increased Fn
deposition in extracellular matrix (28), whereas CHO B2 cells that are
deficient in
5 (29) did not assemble plasma Fn into the
extracellular matrix (30). Reconstituting
5
1 integrin expression by transfecting
the CHO B2 cells with a full-length cDNA encoding the human
5 chain completely restored fibrillar Fn matrix assembly
(30). These studies established an important role of
5
1 integrin in supporting Fn matrix
assembly by CHO cells. In addition to
5
1
integrin, members of the
3 integrins
(
IIb
3 and
v
3) also initiate Fn matrix assembly (8, 24, 31), although some of the other Fn-binding integrins such as
4
1 (32) or
v
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
5 integrin null mutant embryos, assemble a Fn matrix in
the absence of
5
1 (34). The primary role
of
5
1 in Fn matrix assembly appears to
involve initiating the assembly, as Fn mutants lacking the
5
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 IIb
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 cytoplasmic domains of both
1 and
3 integrins, and phosphorylates the
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-5 integrin cytoplasmic domain antibody AB47 was
generated using a synthetic peptide representing the membrane distal
region of the
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
5 integrin antibody (HM
5-1) and
mouse anti-rat
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 -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
-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
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
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 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
5
1 integrins was estimated by
immunoprecipitation of surface-biotinylated cell lysates with a
polyclonal rabbit anti-
5
1 antibody as
described (41).
Immunofluorescent Staining--
Fn matrix assembly was analyzed
by immunofluorescent staining of cell monolayers (8). Cells were
suspended in the -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.
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 -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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
|
|
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.
|
|
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
5
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
5
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
5
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
5
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,
5
1 integrin, and Fn matrix.
|
|
|
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.
|
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.
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ILK is a novel ankyrin repeat-containing serine/threonine protein
kinase (41). ILK interacts with the 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 5
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
5
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
5
1 integrin (50) and in decreased Fn
matrix assembly in culture. Overexpressing
5
1 in CHO cells with endogenous
5
1 suppressed the in vivo
tumorigenicity of the cells (28) and CHO B2 cells that are deficient in
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
5
1 in HT29 colon carcinoma
cells results in the growth arrest of these cells and reduced
tumorigenicity, ligation of
5
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
-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
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 5
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
5
1 integrin by 8A2 activating antibody
resulted in occupancy of most cell surface of
5
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 5
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
-catenin and LEF-1
(70), or whether ILK directly activates
5
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 5
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
5
1-mediated cell survival (71). This
latter conclusion was derived from the use of wild type
5
1 and
5 cytoplasmic
deleted (
5
C
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; -MEM,
-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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|