Disruption of Fibronectin Binding to the α5β1 Integrin Stimulates the Expression of Cyclin-dependent Kinases and DNA Synthesis through Activation of Extracellular Signal-regulated Kinase*

The α5α1 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 α5α1 and fibronectin by integrin α5 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 α5 or fibronectin antibody was concentration- and time-dependent. FET cells expressed α4β1 integrin (another fibronectin receptor); however, addition of anti-human integrin α4monoclonal antibody had no effect on DNA synthesis. Treatment with α5 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. α5 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 α5 monoclonal antibody. Furthermore, disruption of α5α1/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 α5 monoclonal antibody-induced mitogen-activated protein kinase activity. In addition PD98059 prevented α5monoclonal antibody-induced DNA synthesis. Since α5α1 ligation to fibronectin is associated with decreased growth parameters, our results indicate that ligation of α5α1 integrin to fibronectin results in suppressed mitogen-activated protein kinase activity which in turn inhibits cyclin-dependent kinase activity in growth-arrested cells.

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)(3)(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 Ca 2ϩ fluctuation, inositol lipid metabolism, protein kinase C, mitogen-activated protein (MAP) kinases, and phosphatidylinositol kinase (5)(6)(7)(8)(9)(10)(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)(16)(17)(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 G 1 phase, the G 1 /S transition, and S phase, respectively (22)(23)(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)(26)(27)(28)(29)(30)(31)(32). Adhesion to substratum is required for cell cycle progression through G 1 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)(34)(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). Antiextracellular signal-regulated kinase (Erk) 2, phospho-Erk1 kinase, phospho-p38 antibodies, and MEK1 inhibitor (PD98059) were purchased from New England Biolabs (Beverly, MA).
[ 3 H]Thymidine Incorporation Assay-Cells were inoculated into 24well plates at a density of 3 ϫ 10 4 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. [ 3 H]Thymidine (7 Ci) (Amersham Corp.) was added into triplicate wells. DNA was then precipitated with 10% trichloroacetic acid after 1 h, and [ 3 H]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. [ 3 H]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 g/ml leupeptin, 25 g/ml aprotinin, 25 g/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 g 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 g) 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 g of total protein was exposed to anti-CDK2, anticyclin 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 MgCl 2 ) and resuspended in 10 l of reaction buffer containing 10 Ci of [␥-32 P]ATP (3000 Ci/mmol, NEN Life Science Products), 1.6 g of histone H1 (Sigma), and 2 l of 2 ϫ kinase buffer. The reaction mixtures were incubated at 37°C for 30 min and stopped by addition of 12 l of 2 ϫ 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.

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 [ 3 H]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 [ 3 H]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 [ 3 H]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 G 1 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 G 1 .
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 G 1 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 G 1 as a result of induction of cyclin-dependent kinase components in early G 1 . Western blots of cell lysates derived from ␣ 5 mAbtreated 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 G 1 phase progression and G 1 -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 G 1 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 G 1 -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 FET cells were rendered quiescent (QUI) by growth factor and nutrient deprivation, and then treated with a 1:100 of dilution of ␣ 5 mAb for the indicated periods following release from quiescence. CDK2, cyclin A, and cyclin E antibodies were used to precipitate CDK2, cyclin A, and cyclin E, respectively, from 50 g of protein. The resultant immunocomplexes were assayed for histone H1 kinase activity as described under "Materials and Methods" and resolved by 10% SDS-PAGE. A, kinetics of the induction of cyclin A-and cyclin E-associated kinase activity. B, kinase assays of CDK2, cyclin A, and cyclin E at 22 h after release. 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 mAbstimulated 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 M PD98059 completely blocked DNA synthesis induced by ␣ 5 mAb. Therefore, the activity of the MEK-1dependent 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 G 1 phase of the cell cycle when cultured in suspension (48 -50). Beyond a defined time point in G 1 , 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 G 1 phase of the cell cycle (Fig. 3). The hyperphosphorylation of retinoblastoma protein that is catalyzed by cyclin-dependent kinases occurs in G 1 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 FIG. 7. Effect of ␣ 5 mAb treatment on the formation of CDK2 complexes. Cell lysates were prepared from cells treated with ␣ 5 mAb as described in Fig. 5. A, the association of cyclin A, cyclin E (a), or p27 (b) with CDK2 complexes was determined by immunoprecipitation of 200 g of protein with anti-CDK2 antibody followed by Western blot analysis with the indicated antibodies. B, cell lysates were harvested from cells at 12 h after release with fresh medium in the presence or absence of ␣ 5 mAb. 200 g of protein was subjected to immunoprecipitation with anti-cyclin E antibody followed by Western blot analysis with CDK2 and cyclin E antibodies, respectively. 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 G 1 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 appar-ent 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 antibodyinduced 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).