Adhesion to extracellular matrix (ECM) (
)regulates
cell growth, differentiation, development, and
tumorigenesis(1, 2, 3, 4) . Contact
with the ECM is in part mediated through a family of heterodimeric
surface molecules called integrins. A heterodimer composed of one of
several
subunits and a
1 subunit acquires the ability to
interact with collagen, fibronectin, and laminin in clusters on the
cell surface called focal adhesion sites(5, 6) . The
short cytoplasmic tails of integrins are associated with talin,
vinculin, and
actinin, and still other proteins probably link
integrins with actin-containing cytoskeletons(7, 8) .
Therefore, integrins, located between the extracellular matrix and the
cytoskeleton, transmit biological signals into the cell, thereby
influencing the modulation of intracellular pH (9) , activation
of T-lymphocytes (10) and neutrophils(11) , and
tumorigenicity(12) . Many studies have shown that tyrosine
kinases and protein kinase C play critical roles in signal transduction
through integrins. Tyrosine phosphorylation of very late activation
antigen (VLA)-5 decreased during retinoic acid-induced differentiation
of teratocarcinoma cells(13) , and tyrosine phosphorylation of
specific proteins increased in association with
IIb
3-mediated
platelet aggregation induced by thrombin(14) . Phorbol
ester-induced cell adhesion in a human leukemia cell line was triggered
by protein kinase C and mediated by VLA-5(15) . In addition,
cross-linking of integrins on the cell surface with antibodies (16) has been shown to cause tyrosine phosphorylation of
proteins, including focal adhesion kinase (17) ,
paxillin(17) , and tensin(18) , that are in contact
with the short cytoplasmic tail of integrin and the cytoskeleton at the
focal adhesion sites.
Colo 201, isolated from an ascites of an
individual with carcinoma of the colon(19) , is a unique cell
line in that it has lost its epithelial appearance and exhibits a
round, floating, non-adherent morphology. Cell-to-cell and
cell-to-matrix interactions have probably been lost in Colo 201 due to
disruption of the adhesion system and the connection between focal
adhesion sites and the cytoskeleton during tumor progression and
metastasis.
We have been investigating the effects of kinase
modulators on cellular adhesion and spreading and have found that
staurosporin, a broad spectrum kinase inhibitor, induced VLA-3-mediated
cell spreading in Colo 201. The cell spreading was accompanied by
elevation of tyrosine phosphorylation in a set of intracellular
proteins despite the presence of staurosporin.
EXPERIMENTAL PROCEDURES
Cell Lines and Reagents
A human colon
cancer cell line, Colo 201, was obtained from the Japanese Cancer
Research Resources Bank. HT1080 (human fibrosarcoma) was obtained from
the American Type Culture Collection. These cell lines were grown in
RPMI 1640 medium supplemented with 10% fetal bovine serum (Nalgene,
Victoris, Australia) and antibiotics. Bovine collagen type I and
polylysine were purchased from Collaborative Research, and human plasma
fibronectin and mouse laminin produced by Engelbreth-Holm-Swarm sarcoma
were from Life Technologies, Inc. A synthetic peptide, GRGDSP, was
kindly provided by Takara Shuzou Co., Ltd. (Kyoto, Japan).
Staurosporin, KT-5720, KT-5823, herbimycin, calphostin, and 2,5-MeC
were purchased from Kyowa Medex Co. (Tokyo, Japan). H-7, H-89, and W-7
were from Seikagaku Kogyo (Tokyo, Japan). Cycloheximide, actinomycin D,
phenylmethylsulfonyl fluoride, aprotinin, leupeptin, iodoacetamide, and
sodium orthovanadate were from Sigma.
Antibodies
Monoclonal antibodies used in
this study were as follows. Gi9 (anti-
2) and GoH3 (anti-
6)
were obtained from Immunotech (Marseille, France). P1B5 (anti-
3),
anti-Src, and anti-
actinin were purchased from Oncogene Science.
SG/17 (anti-
4), KH/72 (anti-
5), and SG/19 (anti-
1) were
kindly provided by Dr. K. Miyake(20) . Anti-phosphotyrosine was
generously supplied by Brian Drucker (Dana-Farber Cancer Institute,
MA)(21) . 2A7 (anti-focal adhesion kinase) was purchased from
Upstate Biotechnology. Anti-tensin and anti-paxillin were from Chemicon
International, Inc., and G1CL (anti-human IgG) was from Becton
Dickinson.
Cell Spreading and Monoclonal Antibody Inhibition
Assays
Well plates were coated in triplicate with type I
collagen, fibronectin, laminin, or polylysine. Substrates were diluted
in phosphate-buffered saline (PBS) and added to the wells at final
concentrations of 1.25, 2.5, 5.0, and 10.0 µg/ml. Following
overnight incubation of the well plates at 4 °C, the unbound
substrates were removed by washing three times with PBS. The wells were
then blocked with 1% bovine serum albumin (BSA) in PBS for 2 h at 37
°C. Cells (3
10
cells) that had been harvested
from culture in exponential growth were resuspended in RPMI 1640 medium
supplemented with 1% BSA and seeded in the matrix-coated wells.
Staurosporin was prepared as a 35 mM stock solution in
dimethyl sulfoxide and added at 1:1000 dilution for all the
experiments. After a 4-h treatment with staurosporin (35 nM),
the cells were photographed at random using phase contrast microscopy
(Nikon, Japan). Spread cells displayed an epithelia-like appearance and
were clearly distinguishable from round, floating, non-spread cells.
The percentage of spread cells was estimated from the counts of 500
cells photographed at several randomly selected fields. For inhibition
experiments, cells that had been harvested and suspended in RPMI 1640
medium supplemented with 1% BSA were incubated with each monoclonal
antibody at a concentration of 10 µg/ml or synthetic GRGDSP peptide
at concentrations of 0.2 mM, 1.0 mM, and 5.0 mM for 30 min at 37 °C. Thereafter, the cells were transferred to
collagen-coated (10 µg/ml), laminin-coated (5 µg/ml), and
fibronectin-coated (10 µg/ml) wells. Staurosporin was then added,
and the spreading assay was carried out as described above. Divalent
cation sensitivity was evaluated as previously described(22) .
In brief, cells were washed with PBS containing with 1 mM EDTA
to deplete pre-existing divalent cations in the medium. Next, cells
were resuspended in 10 mM Tris-HCl, pH 7.4, 135 mM NaCl, 5 mM KCl, 1.8 mM glucose, and 1% BSA and
supplemented separately with CaCl
, MgCl
, and
MnCl
. The cells were seeded in matrix-coated wells, and
cell spreading was assayed as described above. All assays were run in
triplicate and presented as mean values. Variation between assays was
less than 10%.
Immunoprecipitation and
Immunoblotting
Cells were harvested from culture, washed
with PBS, and lysed in cold lysis buffer consisting of Tris-buffered
saline (140 mM NaCl, 20 mM Tris-HCl, pH 7.8)
containing 0.1% sodium azide, 10% glycerol, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 0.15 units/ml aprotinin, 10 mM EDTA, 10 µg/ml leupeptin, 100 mM sodium fluoride, and
2 mM sodium orthovanadate at 4 °C for 20 min. Insoluble
material was removed by centrifugation at 15,000 rpm for 15 min at 4
°C. The protein content of the lysates was determined with a BCA
kit using bovine serum albumin as the standard. Cell lysates (20
µg) were absorbed with normal rabbit serum and protein G-Sepharose
beads (Pharmacia, Uppsala, Sweden) for 2 h at 4 °C. The lysates
were then incubated with anti-Src and protein G-Sepharose beads to
collect antigen-antibody complex. The immunoprecipitates were washed
five times with lysis buffer containing inhibitors of proteases and
phosphatases and boiled at 100 °C for 5 min in a sample buffer
containing 125 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and
5% 2-mercaptoethanol. The released proteins were subjected to 8%
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and
electrophoretically transferred onto a nitrocellulose membrane. The
following procedures were carried out at room temperature. Nonspecific
binding sites on the filter were blocked by incubating the membrane in
PBS containing 0.05% Tween 20 (PBS-T) supplemented with 3% BSA. The
filters were then probed overnight with anti-tyrosine phosphate
monoclonal antibody in PBS-T at a dilution of 1:5000, washed with PBS-T
several times, and incubated for 1 h with biotinylated horse anti-mouse
IgG (Vector Laboratories Inc.). The blots were washed as above and
incubated with avidin-horseradish peroxidase complex (Vector) for 30
min. After washing, the membranes were developed using enhanced
chemiluminescence (Amersham, United Kingdom) according to the
manufacturer's protocol. To completely remove the anti-tyrosine
phosphate antibody and avidin-biotin complex, the blots were stripped
for 1-2 h at 55 °C in 62.5 mM Tris-HCl (pH 6.7), 2%
SDS, and 100 mM 2-mercaptoethanol. The blots were then
re-equilibrated in PBS-T, blocked, and reprobed separately with
anti-Src antibody at a dilution of 1:2000 or with 2A7 (anti-focal
adhesion kinase), anti-paxillin, or anti-tensin at a dilution of 1:1000
in the manner described above.
Flow Cytometry
Colo 201 cells were
incubated in the presence or absence of staurosporin for 4 h at 37
°C. The cells were then harvested, washed three times with PBS,
resuspended, and stained for 30 min on ice with primary antibodies to
the integrin subunits (5 µg/ml). After washing two times with PBS
supplemented with 1% BSA and 0.1% sodium azide, cells were incubated
with either fluorescein isothiocyanate (FITC)-conjugated goat ant-mouse
immunoglobulin (Becton Dickinson) or FITC-conjugated MAR18.5
(monoclonal antibody against rat IgG) (Organon Teknika) as second
antibody for mouse monoclonal antibody and rat monoclonal antibody,
respectively. G1CL (anti-human IgG1) was used as the negative control.
Immunofluorescence Microscopy
Plastic
3.5-cm dishes with inserted glass coverslips at the bottom were coated
with either collagen type I or laminin (5 µg/ml) in PBS for 1 h at
37 °C and blocked with 1% BSA in PBS for 1 h at 37 °C. Colo 201
cells were harvested, washed three times with serum-free RPMI 1640
medium, and plated on the matrix-coated dishes. After treatment with
staurosporin (35 nM) and confirmation of cell spreading, the
dishes were rinsed with pre-warmed PBS and fixed with paraformaldehyde
(3% w/v in PBS) for 10 min at room temperature. All of the following
procedures were performed at room temperature. Residual aldehyde groups
were blocked by incubation with 50 mM ammonium chloride in PBS
for 20 min, and the cells were permeabilized with 1% Triton X-100 in
PBS for 10 min. After blocking with 1% normal rabbit serum in PBS,
cells were incubated for 2 h with monoclonal antibody against
2
(IgG1),
3 (IgG1),
6 (IgG2a), or anti-
actinin (IgM) in
1% normal rabbit serum in PBS. The cells were washed with PBS, and
labeled second antibody was added. After 1 h, the cells were washed,
semi-dried, and examined under epifluorescence (IIRS, Zeiss, Germany).
Because preliminary studies showed no cross-reaction between
FITC-conjugated antibody against IgG and phycoerythrin-conjugated
antibody against IgM (Kirkegaard & Perry Laboratories, Inc.), these
second antibodies were added simultaneously in double staining
experiments.
RESULTS
Effect of ECM and Divalent Cations on Cell
Spreading Induced by Staurosporin
The ability of
staurosporin to induce spreading of Colo 201 cells was examined in
non-coated wells (3% BSA blocking prior to the assay only) and in wells
coated with collagen type I (5 µg/ml), laminin (5 µg/ml), and
fibronectin (5 µg/ml) (Fig. 1, A, C, E, and G, respectively). On collagen and laminin,
approximately 95% of the cells were found to have spread after a 4-h
treatment with staurosporin, and these showed an epithelia-like
morphology with formation of micropodia. On fibronectin, about 20% of
the cells were spread after a similar treatment, but micropodia
formation was poor. In the absence of staurosporin, cells did not
spread on either the non-coated or the coated wells (Fig. 1, B, D, F, and H). Staurosporin did
not have any effect on cell viability during the 4-h incubation time.
We tested the abilities of untreated and staurosporin-induced cells to
spread on several substances, collagen type I, fibronectin, laminin,
and polylysine, at various concentrations (Fig. 2A).
Unless stated otherwise, cell spreading was evaluated after 4 h of
incubation with staurosporin. In the absence of staurosporin, cell
spreading was not observed in any of the wells. After incubation with
staurosporin, spreading was seen in more than 90% of the cells in wells
coated with 5 µg/ml collagen or 2.5 µg/ml laminin. In contrast,
spreading on fibronectin was very weak, reaching only 30% even on wells
coated with 100 µg/ml fibronectin. Staurosporin did not induce cell
spreading on polylysine, which occurs via a non-integrin pathway. The
effect of divalent cations on the cell spreading was examined after
depletion of pre-existing cations with EDTA (Fig. 2B).
In the presence of Mg
or Mn
, cell
spreading started after 2 h of incubation and reached a plateau after 3
h. In the presence of Ca
, cell spreading progressed
more slowly, and 8 h of incubation was necessary for the spreading to
reach a plateau. For this reason, the sensitivity of cell spreading to
divalent cations was assayed after an 8-h incubation with staurosporin.
The cell spreading showed a high dependence on Mn
,
with marked induction occurring at Mn
concentrations
of 0.1 mM or greater. Sensitivity to Mg
was
relatively low; at 0.1 mM Mg
, cell spreading
was not induced significantly compared with spreading in the absence of
Mg
. The reliance on Ca
was lower
still. At 10 mM Ca
, cell spreading was only
27.1% on collagen and 25.3% on laminin. In the absence of staurosporin,
cell spreading was not induced at the examined concentrations of the
three divalent cations.
Figure 1:
Staurosporin-induced cell spreading in
Colo 201. Cells were seeded on non-coated (A, B),
collagen-coated (C, D), laminin-coated (E, F), and fibronectin-coated (G, H) wells.
Cell morphology was observed after a 4-h incubation in the presence (A, C, E, G) or absence (B, D, F, H) of staurosporin. The
cells were observed and photographed at 100
.
Figure 2:
A, effect
of extracellular matrix on spreading. Collagen type I, laminin,
fibronectin, and polylysine were diluted to the concentrations
indicated and used to coat well plates. The extent of cell spreading in
the presence (
) and absence (
) of staurosporin was evaluated
and is represented as the mean value of triplicate experiments. B, after treatment with 1 mM EDTA, cells were
resuspended in Tris-buffered saline, supplemented separately with
Ca
, Mg
, and Mn
at
0.1, 1.0, and 10 mM, and seeded on collagen-coated (
,
) and laminin-coated (
,
) wells. The extent of cell
spreading in the presence (
,
) and absence (
,
) of staurosporin is indicated as the mean value of triplicate
experiments.
VLA-3 Was Involved in Cell Spreading
The
above results on the affinity of Colo 201 cells for ECM proteins and on
the dependence of cell spreading on divalent cations suggested that the
1 family of integrins might be in part responsible for this
spreading process. To determine which chains of integrin were involved
in cell spreading, we tested the ability of monoclonal antibodies to
suppress the spreading (Fig. 3). Cell spreading to collagen (panel A) and laminin (panel B) was equally blocked
by antibodies against the
3 and
1 chains of integrin at a
concentration of 10 µg/ml. Spreading to collagen and laminin was
not inhibited by antibodies against
2 and
6 at the same
concentration. In our preliminary study, cell spreading of HT1080 was
decreased from 92.4 to 22.6% on collagen and from 88.6 to 16.5% on
laminin by the antibody against
2 (Gi9). Similarly, the antibody
against
6 (GoH3) inhibited spreading of HT1080 cells on laminin
from 88.2 to 19.8%. Because VLA-3-mediated binding to fibronectin has
been reported to be RGD-dependent(22) , we tested both the
inhibitory effect of antibodies and the effect of RGD on
staurosporin-induced cell spreading on fibronectin. Cell spreading on
fibronectin was not significantly affected by monoclonal antibodies
against the
2,
3,
4,
5,
6, and
1 chains
of integrin or by the synthetic peptide GRGDSP. This indicated that
cell spreading on fibronectin was not dependent on the RGD sequence of
fibronectin and did not occur in a
1 integrin-dependent manner.
The above inhibition study demonstrated that VLA-3, not VLA-2 or VLA-6,
was involved in cell spreading on collagen and laminin. Next, the
expression of integrin molecules was analyzed by indirect
immunofluorescence (Fig. 4). Flow cytometric analysis indicated
that Colo 201 expressed
2,
3,
6, and
1 chains on
the cell surface and that the level of expression was not altered after
4 h of incubation with staurosporin. The
4 and
5 chains were
not expressed on the Colo 201 cells. To evaluate the effects of protein
synthesis and mRNA transcription on the staurosporin-induced cell
spreading, cells were first incubated on collagen and laminin for 4 h
with cycloheximide (20 µg/ml) or actinomycin D (20 µg/ml) and
subsequently maintained in cycloheximide or actinomycin D throughout
the 4-h spreading assay. Cell spreading was not affected by either
cycloheximide or actinomycin D, which indicated that the spreading
process was not accompanied by staurosporin-induced protein synthesis
or mRNA transcription. Taken together, the results of antibody
inhibition studies and flow cytometry revealed that
staurosporin-induced spreading on collagen and laminin was mediated by
VLA-3 and was not accompanied by quantitative changes in synthesis of
the VLA-3 protein or transcript.
Figure 3:
Effect of blocking antibody on cell
spreading. The effects of monoclonal antibodies to integrin subunits on
staurosporin-induced cell spreading were assayed on collagen-coated (10
µg/ml) and laminin-coated (5 µg/ml) well plates (panels A and B, respectively). The following monoclonal antibodies
were used at a final concentration of 10 µg/ml: Gi9 (anti-
2),
P1B5 (anti-
3), SG/17 (anti-
4), KH/72 (anti-
5), GoH3
(anti-
6), and SG/19 (anti-
1). G1CL (anti-human IgG1) was used
as the negative control. Panel C, for the inhibition assay in
fibronectin-coated (10 µg/ml) wells, the effect of GRGDSP (0.2, 1,
and 5 mM) was also examined. Values are the mean of
triplicate experiments.
Figure 4:
Flow cytometric analysis of
2,
3,
4,
5,
6, and
1 expression on Colo 201
cells. Staurosporin-treated and untreated cells were stained with a
primary antibody, followed by FITC-conjugated second antibody, as
described under ``Experimental Procedures.'' A total of
10,000 cells were analyzed with FACScan. The horizontal and verticalaxes represent fluorescence intensity in a
log scale and cell number, respectively.
Effect of Tyrosine Kinase on Staurosporin-induced
Cell Spreading
To identify the target of staurosporin in
this spreading process, we attempted at first to induce spreading of
Colo 201 cells using a combination of inhibitors for specific kinases.
Cell spreading was not observed after treatments with protein kinase
C-specific inhibitors H-7 (10
-10
M) and photoactivated calphostin
(10
-10
M)(23) , tyrosine kinase inhibitors herbimycin A
(10
-10
M) and
2,5-MeC (10
-10
M),
cAMP-dependent kinase inhibitors H-89
(10
-10
M) and
KT-5720 (10
-10
M),
cGMP-dependent kinase inhibitor KT5823
(10
-10
M), and
calmodulin/calcium-dependent kinase inhibitor W-7
(10
-10
M) used
alone or in combination. Cell spreading was suppressed when a
combination of staurosporin and herbimycin A (2 mM) or 2,5-MeC
(2 mM) was given to the cells. No inhibitory effect was
observed when cells were incubated with a combination of staurosporin
and protein kinase C- or protein kinase A-specific inhibitor. These
data suggested that a tyrosine kinase that was not inhibited by
staurosporin was involved in the cell spreading. Phosphorylation of
tyrosine in whole cell lysates was examined at 0, 30, 60, and 120 min
during the incubation with staurosporin. At 30 min using
collagen-coated wells, almost all of the cells appeared round and were
still easily moved by agitating the plates. Western blot analysis (Fig. 5, panel A) at this point revealed that two bands
(88 and 50 kDa) had lower levels of tyrosine phosphorylation than the
corresponding bands at 0 min. The tyrosine phosphorylation of other
bands was unchanged. At 60 min, cells remained round but adhered the
bottom of the collagen-coated chamber, based on the observation that
the cells were not moved easily by vigorous agitation. The profile of
tyrosine phosphorylation at 60 min showed enhanced phosphorylation of
several proteins with bands at 220, 175, 150, 98, 62, 58, and 50 kDa,
as indicated at the rightside of panel A (Fig. 5). Three proteins with bands at 72, 68, and 64 kDa
(indicated by arrows in panel A in Fig. 5)
showed decreased tyrosine phosphorylation compared with the
corresponding bands at 0 and 30 min. The proteins at 88 and 50 KDa were
found to be re-phosphorylated at 60 min. Western blot analysis at 120
min of staurosporin treatment, when cell spreading was observed in the
Colo 201 cells, showed the same tyrosine phosphorylation profile seen
at 60 min. The membranes were stripped and reprobed separately with
anti-Src and anti-tensin antibodies. Fig. 5, panel B,
shows that a 58-kDa protein was recognized by the anti-Src antibody (left), and a 220-kDa protein was recognized by the
anti-tensin antibody (right). These were identical to the
tyrosine-phosphorylated proteins of the same molecular masses in panel A. The stripped membranes were also probed separately
with anti-focal adhesion kinase and anti-paxillin antibodies. No bands
were found to be recognized by these antibodies. Panel C shows
that Src product and tensin precipitated from whole cell lysates (left and right, respectively) were tyrosine
phosphorylated at 30 min and that the level of tyrosine phosphorylation
remained elevated throughout the remainder of the 120-min incubation
with staurosporin. Use of anti-focal adhesion kinase or anti-paxillin
antibody in the immunoprecipitation procedure did not reveal any
tyrosine-phosphorylated product. Neither cell adhesion nor spreading
was observed during an 8-h incubation when Colo 201 cells in
collagen-coated wells were simultaneously treated with staurosporin and
herbimycin A (2 mM). Western blot analysis (panel D)
showed that tyrosine phosphorylation declined during this treatment.
The time course of cell spreading and the profile of tyrosine
phosphorylation were similar when Colo 201 cells were seeded in
laminin-coated wells and treated as described above. In summary,
several proteins including Src product and tensin were tyrosine
phosphorylated in association with cell adhesion by a tyrosine kinase
that was not sensitive to staurosporin.
Figure 5:
Time course of tyrosine phosphorylation
during the incubation with staurosporin. Cells seeded on
collagen-coated wells were harvested at 0, 30, 60, and 120 min during
treatment with staurosporin. Panel A, whole cell lysates were
separated by 8% SDS-PAGE, transferred onto nitrocellulose, and probed
with anti-tyrosine phosphate antibodies. Panel B, the same
membrane was stripped and reprobed with anti-Src antibody or
anti-tensin antibody (left and right, respectively),
as described under ``Experimental Procedures.'' Panel
C, proteins precipitated from whole cell lysates of Colo 201 using
anti-Src or anti-tensin antibody (upper left and upper
right, respectively) were separated by 8% SDS-PAGE, transferred
onto nitrocellulose, and probed with anti-tyrosine phosphate
antibodies. The membranes were then stripped and reprobed with anti-Src
and anti-tensin antibodies (lower left and lower
right, respectively) to show the amounts of precipitated protein
loaded. Panel D, Colo 201 cells treated simultaneously with
staurosporin (35 nM) and herbimycin A (2 µM) were
harvested at 0, 30, 60, and 120 min and probed with anti-tyrosine
phosphate antibodies. The profiles of tyrosine phosphorylation in panels A, C, and D were
reproducible in three independent experiments. The molecular masses of
marker proteins are indicated in
kilodaltons.
Analysis of Focal Localization of Integrins by
Immunofluorescence Microscopy
To examine the distribution
of integrin chains on the cell surface in the presence of staurosporin,
indirect immunofluorescence microscopy was performed. Focal adhesion
sites were identified by means of immunofluorescence microscopy
utilizing a monoclonal antibody against cytoskeleton-associated
proteins such as
actinin(24) . In the absence of
staurosporin treatment, the
2,
3, and
6 chains of
integrin were diffusely expressed on the surface of Colo 201 cells as
shown in Fig. 6A. After 4 h of staurosporin treatment,
3 chain was condensed into focal plaques that coincided precisely
with the staining of
actinin on the spread Colo 201 cells (Fig. 6B), while
2 and
6 chains remained
homogeneously distributed on the cell surface (Fig. 6C). The same result was obtained whether cells
were spread on laminin or on collagen.
Figure 6:
Indirect immunofluorescence microscopy of
Colo 201 cells seeded on collagen. A, cells in the absence of
staurosporin were stained with Gi9 (anti-
2, left), P1B5
(anti-
3, middle), or GoH3 (anti-
6, right). B, after a 4-h incubation with staurosporin, cells were
stained with P1B5 (anti-
3, left) or anti-
actinin (right). C, spread cells after treatment with
staurosporin were stained with Gi9 (anti-
2, left) or GoH3
(anti-
6, right). All images of fluorescence were obtained
at 400
.
DISCUSSION
VLA-3 (
3
1) is a member of the
1 integrin
family and mediates interaction of the cell with ECM(25) . A
wide range of ECM proteins have been shown to be ligands for VLA-3 by
antibody inhibition studies, affinity chromatography, and
immunofluorescence microscopy. Antibodies against VLA-3 have been
demonstrated to block cell attachment to collagen, laminin, and
fibronectin(22, 26, 27) , and the affinity of
VLA-3 for these proteins has been used in purification procedures for
the integrin(22, 26) . Recently, epiligrin/kallinin (28) , invasin(29) , and entactin (30) have
been added to the list of ligands for VLA-3. The expanding list of
specific ligands for VLA-3 is controversial because adhesion to VLA-3
may be obscured by other activated integrins coexisting on the cell
surface. The standard binding assay, which utilizes immobilized
proteins, may not reflect the physiological cell-to-matrix interaction.
In this study, staurosporin induced cell spreading through two
different pathways in Colo 201 cells. On collagen and laminin, cell
spreading was dependent on VLA-3, as demonstrated by the antibody
inhibition study. In contrast, cell spreading on fibronectin was
independent of
1-integrin and the RGD sequence of fibronectin.
Furthermore, the morphology of the spread cells on collagen and laminin
was different from that of spread cells on fibronectin in that the
former developed longer micropodia. Colo 201 cells could not spread on
polylysine-coated wells either in the presence or in the absence of
staurosporin, which indicated that the staurosporin-induced cell
spreading did not involve a non-integrin cell-to-matrix interaction.
Staurosporin-induced cell spreading on collagen and laminin
responded differently to different divalent cations. After depletion of
pre-existing divalent cations by EDTA, as little as 0.1 mM Mn
was enough for the recovery of
staurosporin-induced cell spreading in most Colo 201 cells. Recovery
progressed more slowly in the presence of Ca
. At 10
mM Ca
, spreading was observed in only 27% of
the cells. Sequence analysis of cDNA for the
3 subunit of human
VLA-3 has shown at least three possible divalent cation sites on the
3 chain(25) . These binding sites appear to have different
affinities for the three divalent cations examined in the order
Mn
> Mg
>
Ca
.
In a study utilizing
3-transfected
cells(31) , adhesion to collagen type I and laminin was weak,
even after addition of a stimulatory antibody against
1 chain. The
ability of the
3-transfected cells to bind fibronectin was not
precisely evaluated in the presence of VLA-4, although attachment to
fibronectin was elevated by the anti-
1 antibody. On Colo 201
cells, VLA-3 has the ability to attach to collagen and laminin and to
cause cell spreading. Neither the VLA-2 nor the VLA-6 expressed on the
Colo 201 cells was involved in spreading. The process of adhesion and
spreading requires redistribution of integrin molecules to focal
adhesion plaques and changes in linkage between integrin and
cytoskeleton-associated proteins. VLA-2 and VLA-6 did not form adhesion
plaques on the Colo 201 cells. The inability of these integrins to
mediate adhesion of Colo 201 cells to collagen and laminin as well as
the weak affinity of VLA-3 in
3-transfected cells for these ECM
proteins (31, 32, 33) may be due to poor
connections between integrins and cytoskeletal proteins such as
vinculin and
actinin.
Regarding VLA-5, several reports have
indicated that activation of protein kinase C by phorbol ester causes
VLA-5-mediated cell adhesion in NB4 cells, a human promyelocytic
leukemia cell line(15) , and Chinese hamster ovary
cells(34) . The VLA-5-mediated adhesion was specific to
fibronectin, blocked by calphostin, a protein kinase C-specific
inhibitor, and accompanied by increased phosphorylation of focal
adhesion kinase. This spreading started as early as 10 min into the
stimulation and reached a maximum within 1 h, significantly earlier
than the VLA-3-mediated spreading induced by staurosporin. Neither
spreading process was accompanied by a change in the number of integrin
molecules, and the slower spreading process with VLA-3 may reflect the
differences in ECM ligand and inducing agent.
Staurosporin has an
inhibitory effect on a variety of serine/threonine
kinases(35) , including protein kinase C, cAMP-dependent
protein kinase, cGMP-dependent protein kinase, myosin light chain
kinase, and calcium/calmodulin-dependent protein kinase II, each at a
similar IC
(50% inhibition concentration). Staurosporin
also inhibits the tyrosine kinase activities of
p60
(36) , epidermal growth factor
receptor(37) , platelet-derived growth factor
receptor(37) , and insulin receptor(38) . In our study,
staurosporin, a broad spectrum kinase inhibitor, induced VLA-3-mediated
cell spreading. Addition of tyrosine kinase inhibitor herbimycin A or
2,5-MeC inhibited the staurosporin-induced spreading, implying that a
certain tyrosine kinase that was inhibited not by staurosporin but by
herbimycin A and 2,5-MeC was involved in the spreading process. In
unstimulated cells, this tyrosine kinase might be inhibited by other
activated kinases.
Western blot analysis showed that during the
treatment with staurosporin, tyrosine phosphorylation was enhanced in
seven proteins beginning at the time of cell adhesion. The 220- and
58-kDa proteins were identified as tensin and Src product,
respectively. Tensin is localized at the focal adhesion
site(39, 40) . It has an SH2 domain by which it likely
interacts with phosphotyrosine residues of other proteins(41) .
Clinical observation has suggested that in human colon cancer cells and
cell lines, Src expression is involved in the pathway of
differentiation(42) . In addition, many investigations have
shown that expression of Src in cells results in the phosphorylation of
a group of proteins at the focal adhesion site, including focal
adhesion kinase and paxillin(17) . In our study, however,
expression and tyrosine phosphorylation of focal adhesion kinase and
paxillin could not be detected.
The kinase activity of Src is
regulated in two different ways. Phosphorylation of Tyr
results in the elevation of Src kinase activity(43) ,
while phosphorylation of Tyr
inactivates the kinase
activity(44) . Indeed, it is unclear whether the kinase
activity of a Src product is enhanced or decreased by tyrosine
phosphorylation during incubation with staurosporin. Nonetheless, Src
appears to play a role in VLA-3-mediated cell spreading as a substrate
for the tyrosine kinase involved in the spreading process. Further
investigation is needed to characterize the tyrosine kinase responsible
for staurosporin-induced cell spreading.