From the Department of Biochemistry and Molecular
Biology, Medical College of Ohio, Toledo, Ohio 43699-0008 and the
¶ Departments of Surgery and Human Biological Chemistry & Genetics, The University of Texas Medical Branch,
Galveston, Texas 77555-0542
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The 5
1 integrin, a
fibronectin receptor, has been implicated in the control of cell growth
and the regulation of gene expression. We report that disruption of
ligation between
5
1 and fibronectin by
integrin
5 subunit or fibronectin monoclonal antibodies
stimulated DNA synthesis in growth-arrested FET human colon carcinoma
cells. This stimulation only occurred when monoclonal antibody was
added in the early G1 phase of the cell cycle after
release from quiescence by fresh medium. Stimulation of DNA synthesis
by
5 or fibronectin antibody was concentration- and
time-dependent. FET cells expressed
4
1 integrin (another fibronectin
receptor); however, addition of anti-human integrin
4
monoclonal antibody had no effect on DNA synthesis. Treatment with
5 monoclonal antibody led to a marked increase in the
expression of CDK4 in G1 phase of the cell cycle and
consequently increased the phosphorylation of retinoblastoma protein.
5 monoclonal antibody treatment increased both cyclin A-
and cyclin E-associated kinase activity which was accompanied by
increased protein levels of CDK2 and cyclin A. Western blotting of
immunoprecipitates demonstrated increased CDK2-cyclin E and CDK2-cyclin
A complexes in cells treated with
5 monoclonal antibody. Furthermore, disruption of
5
1/fibronectin
ligation activated mitogen-activated protein kinase p44 and p42
(extracellular signal-regulated kinase 1 and 2). Pretreatment of the
cells with a specific inhibitor of MEK-1, PD98059, blocked the
5 monoclonal antibody-induced mitogen-activated protein
kinase activity. In addition PD98059 prevented
5
monoclonal antibody-induced DNA synthesis. Since
5
1 ligation to fibronectin is associated
with decreased growth parameters, our results indicate that ligation of
5
1 integrin to fibronectin results in
suppressed mitogen-activated protein kinase activity which in turn
inhibits cyclin-dependent kinase activity in
growth-arrested cells.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Integrins are a large family of cell-surface glycoproteins that
mediate cell-cell and cell-extracellular matrix adhesion (1). Integrins
are heterodimers consisting of an subunit and a
subunit. The
subunit is non-covalently associated with the
subunit. Both
and
subunits are transmembrane proteins with large extracellular
domains that interact with extracellular matrix (ECM)1 proteins and
relatively small cytoplasmic domains that interact with cytoskeletal
proteins (2-4). Therefore, integrins can act as signaling receptors
and transmit growth regulatory signals from the extracellular matrix to
the interior of the cell (5). It has been shown that, upon ligand
binding, integrins regulate many intracellular signaling pathways that
involve cytoplasmic alkalinization, intracellular Ca2+
fluctuation, inositol lipid metabolism, protein kinase C,
mitogen-activated protein (MAP) kinases, and phosphatidylinositol
kinase (5-11).
Integrin 5
1, a fibronectin (FN) receptor,
has been implicated in the regulation of gene expression, cell growth,
and tumorigenicity. Overexpression of the
5
1 integrin in tumorigenic Chinese
hamster ovary cells leads to decreased tumorigenicity (12). A variant of K562 erythroleukemia cells selected for increased ability to attach
to fibronectin showed a 5-fold up-regulation of
5
1 expression and displayed significantly
reduced growth in vitro as well as reduced turmorigencity
(13). In contrast, loss of
5
1 expression in Chinese hamster ovary cells increased tumorigenicity (14). Recent
studies showed that integrins may also play an important role in the
control of gene expression. Exposure of rabbit synovial fibroblasts to
fibronectin fragments or anti-FN receptor antibody induces the
expression of metalloproteinase, stromelysin, and a 92-kDa gelatinase
(15-18). Overexpression of
5
1 in human
colon carcinoma HT29 cells induces the transcription of growth
arrest-specific gene 1 (GAS-1) and blocks the transcription
of immediate early genes c-FOS, c-JUN, and
JUN-B (19). Treatment of non-transformed FA-K562 cells
overexpressing
5
1 integrin with a
synthetic peptide ligand results in an increase in
CDC-2-dependent kinase activity (20, 21). Induction of cell
cycle progression by disruption of ligation indicates that the growth
inhibitory function of
5
1 integrin may
act through suppression of cell cycle progression. However, the signals
and their subsequent effects on control of cell cycle progression
resulting from disruption of
5
1 ligation have not been determined.
Cyclin-dependent kinases (CDK) complexed to regulatory cyclin subunits are key regulators of the cell cycle. Important cyclin-CDK complexes in mammalian cells are cyclin D-CDK4/CDK6, cyclin E-CDK2, and cyclin A-CDK2, acting primarily in G1 phase, the G1/S transition, and S phase, respectively (22-24). CDK activity can be regulated by changes in expression of cyclins and CDKs and by phosphorylation or dephosphorylation. In addition, CDK activity can be regulated by CDK inhibitors (i.e. p21 and p27) (25-32). Adhesion to substratum is required for cell cycle progression through G1 and into S phase in non-transformed fibroblasts. Adhesion-dependent cell cycle progression has been linked to the expression of cyclin D, cyclin A, and activation of cyclin E-CDK2 kinase (33-35). Activation of CDK2 resulted from decreased p21 and/or p27 (35). Cell adhesion is largely mediated by the interaction of ECM proteins with integrins; however, it is unclear which integrin(s) is involved and how cell cycle progression is altered by specific integrins.
The mitogen-activated protein (MAP) kinases are a family of highly conserved serine/threonine kinases activated by various extracellular signals (36, 37). It has been shown that MAP kinase activation is necessary for the induction of DNA synthesis by growth factors in fibroblasts (38, 39). Integrin-mediated cell adhesion can regulate MAP kinase activity (10). However, the contribution of the activation of MAP kinase pathway to DNA synthesis mediated by modulation of integrin/ECM interaction has not yet been defined.
Previous studies in our laboratory demonstrated that treatment of
HT1080 cells with anti-human integrin 5 subunit mAb or FN mAb stimulated DNA synthesis after cells were released from quiescence (40). Thus, it appears that ligation of some integrins may
promote cell cycle progression while others, such as
5
1, may inhibit cell cycle progression;
however, the mechanism by which
5 mAb stimulates DNA
synthesis is unclear.
We now show that increased DNA synthesis and CDK2 activity stimulated
by disruption of 5
1 ligation to FN is the
result of an increase in expression of CDK2 and cyclin A. In addition,
5 mAb also stimulated CDK4 expression and promoted pRb
phosphorylation. Disruption of
5
1/FN
ligation activated MAP kinase activity. This stimulation is essential
for
5 mAb-induced DNA synthesis and CDK activity.
Therefore, this is the first report demonstrating that disruption of
the interaction between FN and
5
1
integrin by
5 mAb stimulated expression of CDK2 and CDK4
kinase activity in a MAP kinase-dependent manner and
indicates that FN/
5
1 integrin interactions may suppress cell cycle progression by maintaining low
levels of CDK4 and CDK2 activity through repression of MAP kinase
activity.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture and Reagents--
The FET human colon carcinoma
cell line was originally established in vitro from a primary
human colon tumor (41). Cells were maintained in a chemically defined
McCoy's 5A serum-free medium supplemented with growth factors as
described previously (40). To study the effect of 5 mAb
on DNA synthesis after release from quiescence, cells were cultured to
confluence in serum-free medium and then rendered quiescent by growth
factor and nutrient deprivation as described previously (42). Release
from quiescence was achieved by addition of fresh supplemental McCoy's
5A medium (SM). Anti-
2, -
4,
-
5, and FN antibodies and mouse IgG were purchased from
Life Technologies Inc. Fab fragments were prepared from the same
5 mAb utilized throughout this study by proteolytic digestion using a kit from Pierce. Removal of Fc fragment or undigested IgG was accomplished by a protein A column. The purity of Fab fragments
was evaluated by SDS-polyacrylamide gel. Anti-CDK2, CDK4, cyclin A,
cyclin E, p21, p27, pRb, phospho-JNK kinase, goat anti-rabbit, and goat
anti-mouse antibodies were from Santa Cruz (Santa Cruz, CA).
Anti-extracellular signal-regulated kinase (Erk) 2, phospho-Erk1
kinase, phospho-p38 antibodies, and MEK1 inhibitor (PD98059) were
purchased from New England Biolabs (Beverly, MA).
[3H]Thymidine Incorporation Assay--
Cells were
inoculated into 24-well plates at a density of 3 × 104 cells per well, grown to confluence, and rendered
quiescent as described above. Fresh SM medium was used to release cells
from quiescence. Various concentrations and types of antibodies were added to cells after release from quiescence for different periods as
indicated in specific experiments. [3H]Thymidine (7 µCi) (Amersham Corp.) was added into triplicate wells. DNA was then
precipitated with 10% trichloroacetic acid after 1 h, and
[3H]thymidine was determined as described previously
(40). Growth-arrested cells were treated with 5 mAb in
the presence or absence of increasing concentrations of PD98059 to
determine the effect of the inhibitor on
5 mAb-induced
DNA synthesis. [3H]Thymidine incorporation was measured
at 22 h after release from arrest as described above.
Western Blot Analysis and Immunoprecipitation-- Cells were lysed for 30 min at 4 °C with lysis buffer (150 mM NaCl, 0.5% Nonidet P-40, 50 mM Tris, pH 6.8) containing 25 µg/ml leupeptin, 25 µg/ml aprotinin, 25 µg/ml trypsin inhibitor, 5 mM NaF, 1 mM sodium orthovanadate, 1 mM dithiothreitol, and 1 mM phenylmethysulfonyl fluoride. Cell lysates were cleared by centrifugation at 15,000 rpm for 10 min at 4 °C and quantitated by Bio-Rad protein assay. Fifty µg of total protein was subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes (Amersham Corp.). The membrane was incubated in blocking solution (Tris-buffered saline containing 5% non-fat dried milk and 0.05% Tween 20) for 1 h at room temperature followed by 1 h of incubation with primary antibody and 1 h of incubation with secondary antibody. Protein was detected using an enhanced chemiluminescence method according to the manufacturer's instructions (Amersham Corp.). Phosphorylation of Erk was assessed using an antiserum that specifically recognizes phosphorylated Erk1 and -2. Total protein (200 µg) was precipitated with different antibodies as indicated in specific experiments for immunoprecipitation. Immunocomplexes were absorbed by protein A-agarose for 1-2 h, subjected to 12% SDS-PAGE, transferred to nitrocellulose membranes, and blotted with various antibodies as described in specific experiments.
CDK2 Kinase Activity Assay--
Cell lysates were prepared as
described above, 50 µg of total protein was exposed to anti-CDK2,
anti-cyclin A, or anti-cyclin E antibodies for 2-3 h by agitation
followed by incubation with protein A- or G-agarose for 1-2 h. Beads
were then washed 3 times with lysis buffer followed by 3 washes with
kinase buffer (20 mM Tris, pH 7.5, and 4 mM
MgCl2) and resuspended in 10 µl of reaction buffer
containing 10 µCi of [-32P]ATP (3000 Ci/mmol, NEN
Life Science Products), 1.6 µg of histone H1 (Sigma), and 2 µl of
2 × kinase buffer. The reaction mixtures were incubated at
37 °C for 30 min and stopped by addition of 12 µl of 2 × loading buffer (62.5 mM Tris at pH 6.8, 1% SDS, 10% glycerol, and 5%
-mercaptoethanol). The phosphorylated histone H1
were analyzed on 10% SDS-PAGE and visualized by autoradiography.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Stimulation of DNA Synthesis by 5 mAb in FET
Cells--
Previously we demonstrated that human colon carcinoma FET
cells express cell surface
5
1 integrin as
well as fibronectin (41). To determine whether
FN/
5
1 integrin ligation contributed to
the control of DNA synthesis, FET cells were rendered quiescent by
growth factor and nutrient deprivation and then released with fresh SM
medium in the presence of anti-human integrin
5 subunit mAb for 8, 12, 18, 22, 24, and 26 h. Addition of fresh nutrients (SM medium) stimulated cells to re-enter the cell cycle as evidenced by
[3H]thymidine incorporation into DNA (Fig.
1). DNA synthesis peaked at 20-24 h
after stimulation. Addition of
5 mAb in addition to nutrients resulted in a 2-fold increase in [3H]thymidine
incorporation over cells treated with nutrients alone at the antibody
concentration employed for this experiment. Thus, disruption of
5
1 ligation to endogenous fibronectin
produced by FET cells resulted in the stimulation of DNA synthesis and implies that
5
1 integrin interactions
with fibronectin act as an impediment to DNA synthesis by human colon
carcinoma FET cells. These data are consistent with previous results
showing that disruption of
5
1-fibronectin
ligation stimulates DNA synthesis by quiescent HT 1080 cells (40).
|
Concentration Effects of 5 mAb or FN mAb on DNA
Synthesis--
The
4
1 integrin is a
fibronectin receptor that has been shown to be involved in modulation
5
1 control of collagenase gene expression
(43). Since FET cells express cell surface
4
1 protein (data not shown), it was
important to know whether disruption of ligation of
4
1 to fibronectin would also affect DNA
synthesis in FET cells. Quiescent FET cells were stimulated to re-enter the cell cycle by treatment with nutrients in addition to antibodies to
the
4 integrin subunit, the
5 subunit, or
the
4 subunit together with
5 subunit. As
shown in Fig. 2A,
4 mAb had no effect on DNA synthesis, whereas
5 mAb enhanced DNA synthesis approximately 2-fold at the
antibody concentration employed. Cells treated with both
4 and
5 mAb showed the same level of
enhanced DNA synthesis as cells treated with
5 mAb
alone. Addition of a monoclonal antibody to human integrin
2 subunit or a mouse IgG control antibody did not
enhance DNA synthesis. These results demonstrated that
2
1 and
4
1
integrins do not control DNA synthesis in FET cells and that the
stimulation of DNA synthesis was specific to
5 mAb. When
quiescent FET cells were treated with various concentrations of
5 mAb in addition to fresh medium, there was a
dose-dependent stimulation of [3H]thymidine
incorporation into cells (Fig. 2B). The highest
concentration of
5 mAb employed resulted in a 3-fold
increase in DNA synthesis. Treatment of FET cells with different
concentrations of FN mAb also resulted in a dose-dependent
increase in DNA synthesis (Fig. 2C). Fig. 2D
shows that treatment of cells with a Fab fragment prepared from the
same
5 mAb utilized throughout this study also generated
a 2-fold increase in DNA synthesis. Since there were no increases in
DNA synthesis when quiescent cells were plated on FN-coated plates
(data not shown) and since anti-FN antibody had a similar effect on DNA
synthesis as
5 mAb, it appears that this stimulation was
not simply due to the binding of the
5 mAb to
5
1 integrin but was due to the disruption
of binding by endogenously produced FN from FET cells to the
5
1 integrin.
|
Stimulation of DNA Synthesis by 5 mAb in Early
G1 Phase--
Antibody treatment was delayed until various
times after release of quiescent FET cells with fresh medium, and DNA
synthesis was measured at 22 h after initial stimulation with
medium for each antibody treatment to identify at which point in the
cell cycle
5 mAb exerts its effects after stimulation to
re-enter the cell cycle. There was a gradual loss of the ability of
5 mAb to stimulate DNA synthesis when the antibody
treatment was delayed 4-12 h after nutrient release. Addition after
12 h resulted in the complete loss of its stimulatory effect on
DNA synthesis (Fig. 3). These results
indicate that cells were only sensitive to
5 mAb during
early G1.
|
Alteration of Phosphorylation of pRb by Treatment with
5 mAb--
Stimulation of DNA synthesis by disruption
of
5
1/FN ligation by
5 mAb
was most effective only when antibody was added in the early
G1 phase of the cell cycle (Fig. 3). Therefore, we
hypothesized that
5 mAb might alter the phosphorylation
of pRb which takes place at the restriction point in G1 as
a result of induction of cyclin-dependent kinase components
in early G1. Western blots of cell lysates derived from
5 mAb-treated or untreated cells were probed with an
antibody to pRb, and the hypo- and hyperphosphorylated forms of pRb
were distinguished by their respective mobilities on SDS-PAGE. Fig.
4A shows that both forms of
pRb were low in quiescent FET cells and increased in cells released
with fresh medium. However, the hypophosphorylated form of pRb was
decreased in the cells released with fresh medium in the presence of
5 mAb for 6, 9, 12, and 16 h. These results
indicate that effects of perturbation of the interactions between
5
1 integrin and fibronectin on the cell
cycle are associated with the modulation of phosphorylation of
retinoblastoma protein. It has been shown that CDK4/CDK6 which
complexes with D-type cyclins is capable of phosphorylating pRb (44).
Therefore, we determined whether changes in phosphorylation of pRb were
associated with changes in expression levels of CDK4 in cells treated
with
5 mAb. Fig. 4B shows that protein levels
of CDK4 in cells released with fresh medium plus
5 mAb
were higher than those of cells released with fresh medium alone.
Cyclin D1 protein was slightly increased by
5 mAb
treatment (Data not shown). The results indicate that disruption of the
interaction of
5
1 integrin with
fibronectin increased the expression of CDK4 and, consequently,
decreased levels of the hypophosphorylated form of pRb.
|
Effect of 5 mAb Treatment on Cyclin E-associated
Kinase Activity--
Progression of cells through the cell cycle
requires sequential assembly and activation of
cyclin-dependent kinases (20). Cyclin E associates with
CDK2 to form complexes that control G1 phase progression
and G1-S transition (24). The kinetics of induction of
kinase activity were determined to ascertain whether
5
mAb treatment stimulated cyclin E-associated kinase as a function of
cell cycle progression. Cell lysates were prepared from quiescent cells
that were stimulated to reenter the cell cycle with fresh medium in the
presence or absence of
5 mAb and immunoprecipitated with
anti-cyclin E antibody. The resultant immunocomplexes were assayed for
kinase activity using histone H1 as a substrate. When cells were
released with fresh medium in the presence of
5 mAb, cyclin E-associated kinase activity was increased at 6, 9, and 12 h but then, as expected, declined at 16 and 22 h when cyclin A
kinase activity became more prominent (Fig.
5, A and B). Thus, treatment with
5 mAb stimulated cyclin E-associated
kinase activity during G1 prior to S phase entry. Since the
changes in kinase activity were expected to result from changes in CDK2
complex formation, we compared the expression levels of cell cycle
components and their complex formation in lysates from cells treated
with and without
5 mAb treatment.
5 mAb
treatment had no effect on the expression of cyclin E (Fig.
6 and Fig.
7B) and
cyclin-dependent kinase inhibitors p21 and p27 but did
increase the expression of CDK2 at 12 h (data not shown) and
22 h (Fig. 6) after cells were released from quiescence. The
increased levels of CDK2 resulted in increased levels of cyclin
E-associated CDK2 at 12 h after release (Fig. 7B). As
expected,
5 mAb had no effect on cyclin E-CDK2 complex
formation at 22 h. Since there were no changes in CDK2 complexed
to p21 (data not shown) and p27 (Fig. 7A), the increased
cyclin E-associated kinase activity was primarily due to increased
levels of cyclin E complex formation with CDK2.
|
|
|
Effect of 5 mAb on Cyclin A-associated Kinase
Activity--
Cyclin A-CDK2 complexes play a critical role in
G1-S transition and S phase progression (20); therefore,
the effect of
5 mAb treatment on cyclin A-associated
CDK2 kinase activity was also examined. Stimulation of cyclin
A-associated kinase activity was observed from 6 to 22 h after
release from quiescence in the presence of
5 mAb (Fig.
5, A and B). Immunoprecipitation with CDK2
antibody followed by Western blot analysis with cyclin A or CDK2
antibody was performed using cell lysates released with fresh medium in
the presence or absence of
5 mAb to determine whether
increased cyclin A-associated kinase activity was due to increased
CDK2-cyclin A complex formation. Fig. 7A reveals that
5 mAb increased levels of CDK2 complexed to cyclin A as well as total cellular CDK2 levels at 22 h after release from quiescence. Since
5 mAb had no effect on CDK2-associated
p21 (Data not shown) and p27 levels (Fig. 7A), the increased
cyclin A-associated kinase activity resulted from increased cyclin
A-CDK complex formation that was due to increased expression of cyclin A and CDK2 protein levels.
Stimulation of MAP Kinase Activity by 5 and FN
mAbs--
Integrin-mediated cell adhesion is involved in the
regulation of MAP kinases (10). Consequently, it was determined whether disruption of FN/
5
1 ligation could also
regulate MAP kinase activity. Cell lysates obtained from cells released
with fresh medium in the presence or absence of
5
monoclonal antibody were analyzed by Western blots using antibodies
specific for the phosphorylated forms of p42 (Erk2) and p44 (Erk1) MAP
kinases, JNK kinase, and p38 (Fig. 8).
Cells treated with
5 monoclonal antibody showed increased Erk1 and Erk2 kinase activity at 10, 20, and 30 min after
release from quiescence (Fig. 8, upper panel), although
5 mAb treatment had no effect on Erk1 and Erk2 kinase
activity at 6, 9 (Fig. 8, bottom panel), or 12 h (Fig.
8, upper panel). Stimulation was specific for the Erks as
antibody treatment had no effect on JNK kinase and p38 kinase
activities (data not shown). Immunoblotting with an antibody against
Erk, which recognizes both Erk1 and Erk2, revealed that
5 mAb had no effect on the expression of Erk1 and Erk2
protein levels (Fig. 8, middle panel). Fig. 8B
shows that FN mAb also activated Erk1 and Erk2 kinase activities.
Western analysis and DNA synthesis assays demonstrated that
5 mAb did not activate the epidermal growth factor
receptor signal transduction pathway (data not shown), indicating that
5 mAb activates MAP kinase activity exclusively through
an integrin-mediated pathway under these conditions.
|
Role of Activation of MAP Kinase in 5 mAb-induced
DNA Synthesis--
MAP kinase activation may provide the link between
cytoplasmic and nuclear signaling events. Therefore, we examined the
contribution of the Erk activation to
5 mAb-stimulated
DNA synthesis in FET cells using PD98059, a recently identified
compound that selectively inhibits MEK-1 activation (45, 46). Quiescent
cells were released with fresh medium alone or fresh medium plus
5 mAb in the absence or presence of different
concentrations of PD98059. Fig.
9A shows that PD98059
inhibited DNA synthesis in a dose-dependent manner, and 15 µM PD98059 completely blocked DNA synthesis induced by
5 mAb. Therefore, the activity of the
MEK-1-dependent Erk kinases is essential for
5 mAb-induced DNA synthesis. The primary substrates of
MEK are the p42 and p44 MAP kinase isoforms (Erk1 and Erk2, respectively). Quiescent cells were pretreated with PD98059 to examine
whether the inhibitor blocked the
5 mAb-induced
activation of the two MAP kinases. PD98059 had the expected effect of
inhibiting
5 mAb-induced phosphorylation of both Erk1
and Erk2 at 30 min (Fig. 9B, upper panel), but it had no
effect on JNK kinase and p38 kinase activities (Fig. 9B, middle
upper and middle lower panel). Western blot analysis
performed with an anti-Erk antibody revealed that PD98059 had no effect
on the expression of Erk1 and Erk2 protein levels (Fig. 9B,
bottom panel).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Treatment of human colon carcinoma FET cells with an anti-human
integrin 5 subunit antibody enhanced DNA synthesis after cells were released from quiescence. This was consistent with our
previous finding in HT1080 cells (40). FET cells express
5
1 integrin as well as
2
1 and
3
1
integrins (47); however, addition of
2 mAb to cells had
no effect on DNA synthesis (Fig. 2A). Therefore, it is
unlikely that stimulation of DNA synthesis by
5 mAb is
due to general changes in cell shape resulting from nonspecific
disruption of cell adhesion. The specificity of
5 mAb-induced DNA synthesis was further confirmed by the determination that this stimulation was concentration-dependent (Fig.
2, B and C). Cell attachment through
5
1 integrin enhances the expression of
specific metalloproteinase genes, whereas
4
1 can suppress these effects in rabbit
fibroblasts (43). FET cells express
4
1;
however, disruption of fibronectin binding to
4
1 by addition of an anti-human integrin
4 subunit antibody did not alter DNA synthesis (Fig.
2A), indicating that
4
1 had no
effect on the
5
1 controlled signal
transduction in these cells.
Established human fibroblasts undergo cell cycle arrest in the
G1 phase of the cell cycle when cultured in suspension
(48-50). Beyond a defined time point in G1, cells are no
longer dependent on adhesion to complete the cell cycle (51, 52). Our
studies indicate that 5 mAb was most effective in its
stimulatory effects in the early G1 phase of the cell cycle
(Fig. 3). The hyperphosphorylation of retinoblastoma protein that is
catalyzed by cyclin-dependent kinases occurs in
G1 phase (53). We have found that
5 mAb not only stimulated phosphorylation of pRb but also increased expression levels of CDK4 (Fig. 5, A and B). D-type cyclins
complexed to CDK4/CDK6 are major pRb kinases (53). Thus, the increased
CDK4 protein appears to be responsible, at least in part, for the
phosphorylation of pRb. A study by Guadagno et al. (34)
demonstrated that adhesion-dependent cell cycle progression
is linked to expression of cyclin A kinase activity. Our studies have
extended this work to demonstrate that
5 mAb increased
cyclin A-CDK2 complex formation through induction of both cyclin A and
CDK2 protein levels. Thus, it appears that cyclin A-CDK2 complexes
might be a common target of the integrin-mediated signals that control
cell proliferation.
Cell adhesion may lead to activation of cyclin E-CDK2 kinase activity
by decreasing expression of CDK2-associated inhibitors, p21 and p27
(33, 54). The disruption of 1 integrin contact with ECM
triggers a loss of G1 kinase activity resulting from decreased expression of cyclin D1 and cyclin E and increased expression of p21 and p27 (54). We did not observe any changes in levels of p27
and p21. The basal level of p21 in FET is almost undetectable, even in
growth-arrested quiescent cells. A recent study by Koyama et
al. (55) showed that an anti-
2 integrin Fab
fragment was able to stimulate p21 and p27 expression and inhibit the
cyclin E-associated kinase activity. In contrast, our study
demonstrates that
5 mAb was capable of stimulating CDK2
kinase activity and increasing hyperphosphorylation of pRb by induction
of CDK2 and CDK4 expression. This apparent discrepancy might be
reconciled by the explanation that signal transduction pathways
mediated by
5
1 in FET cells are different
from those mediated by
2
1 in other
systems. Indeed,
2 mAb treatment has no effect on FET DNA synthesis (Fig. 2A). These investigators found that
polymerized collagen, whose effect is mimicked by
2 mAb,
could also suppress p70 S6 kinase activity but had no effect on MAP
kinase activity. We have noted that
5 mAb stimulates MAP
kinase activity in FET cells. The importance of the MAP kinase
signaling pathway in proliferation has been shown in several cell types
for many different mitogens (56). Although integrin-mediated cell
adhesion is able to regulate the MAP kinase pathway (10), there have
been no reports demonstrating that stimulation of DNA synthesis is
dependent upon MAP kinase activity resulting from activation of
integrin signaling pathways. We have examined the effect of the MAP
kinase pathway on the stimulation of DNA synthesis induced by
5 mAb disruption of
5
1/FN
ligation. Addition of PD98059, a specific inhibitor of MEK activation,
blocked
5 mAb-induced DNA synthesis and Erk activation
(Fig. 9). Thus, stimulation of Erk is essential for
5
mAb-induced DNA synthesis. Recently, Wary et al. (57)
reported that treatment of cells in suspension with an
5
mAb causes phosphorylation of Shc, which is essential for
antibody-induced activation of MAP kinase and cell cycle progression.
These investigators concluded that antibody-mediated clustering of
5
1 caused activation of MAP kinase. In
our studies, we found that attached cells treated with an
anti-
5 blocking antibody showed increased DNA synthesis.
We have demonstrated that a Fab fragment of an
5
monoclonal antibody stimulated DNA synthesis as effectively as an
intact antibody. Since a Fab fragment is unable to cause receptor
clustering, the observed enhanced DNA synthesis was not due to receptor
clustering induced by antibody. This conclusion was also supported by
the ability of a FN blocking antibody to induce DNA synthesis and MAP
kinase activity by disruption of
5
1
ligation to endogenously produced cellular FN (Fig. 2C). All
the antibodies used in these experiments were capable of blocking FET
cell adhesion to their respective ligand-coated plates. Thus, our
results indicate that disruption of FN/
5
1
ligation, but not clustering of
5
1
integrin, causes the enhanced DNA synthesis and activation of MAP
kinase in FET cells. This conclusion differs from that arrived at by
Wary et al. (57), since they showed that clustering of
receptors on the cell surface resulted in activation of the MAP kinase.
This difference may well arise from the use of a normal cell model in
the study by Wary et al. (57) as opposed to the transformed
cells used in our study, as normal cells require a signal from the
extracellular matrix for proliferation, whereas transformed cells can
undergo anchorage-independent growth. Thus, the pathways for MAP kinase
activation may respond to different signals in the transformed
cells.
Our studies provide new insights into the nature of the interaction
between 5
1 integrin and FN as their
ligation appears to exert cell cycle control through repression of MAP
kinase-dependent mechanisms. This conclusion is based on
several lines of evidence indicating that
5
overexpression has an inhibitory effect on cell proliferation, whereas
disruption of
5
1/FN ligation stimulates DNA synthesis. Thus it appears that disruption of
5
1 ligation leads to a cascade of events
mediated by activation of Erk1 and -2 that in turn leads to activation
of both CDK4 and CDK2 kinase activity necessary to promote cell cycle
progression and ultimately to DNA synthesis. It should be noted that
despite the disruption of
5
1/FN ligation,
the cells remain adhered to their tissue culture substrate since the
adherence functions carried out by other integrins (including
4
1) remain intact. However, the mechanism of stimulation of cell cycle progression depends upon up-regulation of
CDK2 and cyclin A as opposed to the down-regulation of CDK inhibitors
seen in cells that have been restored to the adherent state from
suspension (33, 54, 55).
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants CA 38173 and CA50457 (to M. G. B.) and CA64191 (to T. C. K.).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.
§ This work was performed in partial fulfillment of the requirements for a Ph.D. degree at the Medical College of Ohio.
To whom correspondence should be addressed. Tel.:
419-381-4324; Fax: 419-382-7395.
1 The abbreviations used are: ECM, extracellular matrix; FN, fibronectin; pRb, retinoblastoma protein; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; CDK, cyclin-dependent kinase; MAP kinase, mitogen-activated protein kinase; SM, supplemental McCoy's 5A; Erk, extracellular signal-regulated kinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|