(Received for publication, November 25, 1996, and in revised form, February 28, 1997)
From the Department of Medical Biophysics, University of Toronto and Cancer Biology Research, Sunnybrook Health Science Centre, Toronto, Ontario M4N 3M5, Canada
Cell adhesion to substratum has been shown to
regulate cyclin A expression as well as cyclin D- and
E-dependent kinases, the latter via the up-regulation of
cyclin D1 and the down-regulation of cyclin-Cdk inhibitors p21 and p27,
respectively. This adhesion-dependent regulation of cell
cycle is thought to be mediated by integrins. Here we demonstrate that
stable transfection and overexpression of the
integrin-linked kinase (ILK), which
interacts with the 1 and
3 integrin cytoplasmic domains, induces
anchorage-independent cell cycle progression but not serum-independent
growth of rat intestinal epithelial cells (IEC18). ILK overexpression
results in increased expression of cyclin D1, activation of Cdk4 and
cyclin E-associated kinases, and hyperphosphorylation of the
retinoblastoma protein. In addition, ILK overexpression results in the
expression of p21 and p27 Cdk inhibitors with altered electrophoretic
mobilities, with the p27 from ILK-overexpressing cells having reduced
inhibitory activity. The transfer of serum-exposed IEC18 cells from
adherent cultures to suspension cultures results in a rapid
down-regulation of expression of cyclin D1 and cyclin A proteins as
well as in retinoblastoma protein dephosphorylation. In marked
contrast, transfer of ILK-overexpressing cells from adherent to
suspension cultures results in continued high levels of expression of
cyclin D1 and cyclin A proteins, and a substantial proportion of the retinoblastoma protein remains in a hyperphosphorylated state. These
results indicate that, when overexpressed, ILK induces signaling pathways resulting in the stimulation of G1/S
cyclin-Cdk activities, which are normally regulated by cell adhesion
and integrin engagement.
Normal, untransformed epithelial cells require anchorage to a substratum for cell growth and survival. Adhesion to the extracellular matrix (ECM)11through the G1 and into the S phase of the cell cycle. When forced to remain in suspension, such cells arrest in the G1 phase of the cell cycle and undergo apoptosis (1-3). Oncogenic transformation frequently induces anchorage-independent growth, in vitro, and is a specific correlate of tumor growth in vivo (4, 5).
In fibroblasts, cell adhesion has recently been demonstrated to regulate cell cycle progression by inducing the expression of cyclin D1 (6), the activation of cyclin E-Cdk2 (6, 7), and phosphorylation of the retinoblastoma protein (Rb) (6). Fibroblast adhesion also results in the down-regulation of expression of the Cdk inhibitor proteins, p21 and p27 (6, 7). The combined, adhesion-dependent elevation in cyclin D1 and the decrease in the expression of p21 and p27 result in the stimulation of cyclin D-Cdk4 and cyclin E-Cdk2 activities, both of which can phosphorylate Rb. This latter event relieves the restriction of the entry of cells into S phase, presumably by the release of the transcription factor E2F from phosphorylated Rb (8, 9). In some cell types the expression of cyclin A is also regulated in an anchorage-dependent manner (3, 10, 11), and anchorage-independent growth induced by activated Ras has been shown to depend on cyclin A expression (11). However, in these latter experiments cyclin D1 expression (12) and cyclin E-dependent kinase activity (11) were also dependent on Ras activation. Although mitogens can also activate cyclin D- and cyclin E-dependent kinases, cell adhesion per se can regulate these activities. The regulation of G1 Cdks, therefore, requires the convergence of signals from both growth factors as well as from the ECM.
Anchorage of cells to the ECM is mediated to a large extent by
integrins, a large family of heterodimeric cell surface receptors (13,
14). The interaction of integrins with ECM ligands results in the
transduction of intracellular signals leading to stimulation of
tyrosine phosphorylation (15, 16), turnover of phosphoinositides (17),
and activation of the Ras-mitogen-activated protein kinase (MAPK)
pathways (18-21). The activation of MAPK by cell adhesion is dependent
on the presence of an intact actin cytoskeleton (22), as well as
activated p21rho (23). Presumably the
adhesion-dependent stimulation of cyclin A expression and
of cyclin D1- and cyclin E-associated Cdk activities is also mediated
via integrins, although it is not clear as yet whether this requires
the activation of MAPK. The cytoplasmic domain of the integrin
1 subunit is required for many of the integrin mediated
signaling events (13, 24, 25).
Integrin-proximal events involved in the initiation of
integrin-mediated signal transduction are still poorly understood. However, a novel ankyrin-repeat containing serine-threonine protein kinase (ILK) has recently been demonstrated to associate with the
integrin 1 and
3 subunit cytoplasmic
domains (26) and may be involved in regulating integrin-mediated
signaling. Overexpression of ILK in intestinal epithelial cells results
in an altered cellular morphology, reduction in cell adhesion to ECM,
and also the stimulation of anchorage-independent growth in soft agar
(26). Such constitutively ILK-overexpressing cells are also tumorigenic
in nude mice.
We now report that overexpression of ILK in rat intestinal epithelial cells (IEC18) increases the expression of cyclin A, cyclin D1, and Cdk4 proteins. The activities of both cyclin D1-Cdk4 and cyclin E-Cdk2 kinases are also elevated, resulting in hyperphosphorylation of the Rb protein. In addition both p21 and p27, inhibitors of cyclin-Cdks, have altered electrophoretic mobilities and p27 from ILK-overexpressing cells has reduced inhibitory activity as compared with the p27 from the parental IEC18 cells. Furthermore, whereas cyclin A and cyclin D1 protein expression, and Rb phosphorylation, are down-regulated upon transfer of IEC18 cells to suspension culture, they are constitutively up-regulated in ILK-overexpressing cells kept in suspension. ILK overexpression in these epithelial cells thus overrides the adhesion-dependent regulation of cell cycle progression through G1 and into S phase, indicating that ILK maybe a key regulator of integrin-mediated cell cycle progression.
Three cell lines were used throughout this
study: IEC18, ILK13, and ILK14. IEC18 is an immortalized
non-tumorigenic rat intestinal epithelial cell line (27), cultured in
-minimal essential medium supplemented with 2 mM
L-glutamine (Life Technologies, Inc.), 3.6 mg/ml glucose
(Sigma), 10 µg/ml insulin (Sigma), and 5% fetal calf serum (Life
Technologies, Inc.). ILK13 cells were engineered to overexpress ILK by
stable transfection into the parental IEC18 as described previously
(26). ILK14 cells are the control transfectants (26). Both ILK13 and
ILK14 cell lines were grown under the same conditions as the parental
IEC18, with addition of 200 µg/ml G418 (Geneticin, Life Technologies,
Inc.) to maintain a selection pressure for ILK or control vector,
respectively. Two independently derived clones of each ILK13 (A1a3 and
A4a) and ILK14 (A2c3 and A2c6) were used.
IEC18, ILK13 (ILK-overexpressing cells) and
ILK14 (control transfectants) cells were harvested from tissue culture,
counted and 104 cells from each cell line were plated in
35-mm tissue culture plates (Nunc). Cells were grown in -minimal
essential medium as described above under different serum
concentrations (fetal calf serum, Life Technologies, Inc.) for various
number of days. At each time point, adherent cells were harvested with
5 mM EDTA/PBS (phosphate-buffered saline, pH 7.6) and
viable cells were then quantitated by trypan blue exclusion.
Asynchronously growing cells
were harvested from monolayer culture using 5 mM EDTA/PBS
and washed two times in PBS. Cells were then resuspended in -minimal
essential medium containing 5% fetal calf serum and transferred to
50-ml tubes. A short burst of CO2 was given to the cells
before tubes were capped. Suspension cells were incubated for 12 h, rotating on a nutator at 37 °C in 5% CO2.
Thereafter, cells were either fixed for fluorescence-activated cell
sorting (FACS) analysis or alternatively cell pellets were recovered,
washed twice in ice-cold PBS, and then lysed in Nonidet P-40 lysis
buffer.
Cells were collected, washed in ice-cold PBS (pH 7.6), fixed in 70% ethanol for 1 h on ice, rinsed with PBS, and DNA stained with 50 µg/ml propidium iodide in PBS containing 10 µg/ml RNase for 30 min at room temperature. Cell cycle profiles were analyzed by FACS using a Becton Dickinson FACScan analyzer, and the percentage of cells in the various phases of cell cycle was calculated using CellFit software.
ImmunoblottingCells grown in monolayer or in suspension
were lysed in ice-cold Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris, pH 7.6, 1 mM
EDTA) plus inhibitors (1.0 mM PMSF, 20 µg/ml aprotonin,
20 µg/ml leupeptin) or in ice-cold Tween 20 lysis buffer (0.1% Tween
20, 50 mM Hepes, pH 7.5, 150 mM NaCl, 2.5 mM EGTA, 1 mM EDTA) plus inhibitors (1 mM dithiothreitol, 1.0 mM PMSF, 20 µg/ml
aprotonin, 10 mM -glycerophosphate, 0.1 mM
sodium vanadate, 1 mM sodium fluoride).
Total protein extracts or immune complexes were resolved on SDS-PAGE and then transferred to Immobilon-P (Millipore). The membrane was first blocked in 5% milk in TBST (0.05% Tween 20 (Sigma) in Tris-buffered saline, pH 7.4) and then incubated with primary antibody. The following antibodies were used: anti-cyclin D1 (DCS-6, mouse monoclonal, from Dr. J. Bartek, Danish Cancer Society, Copenhagen, Denmark), anti-cyclin E (rabbit polyclonal, Santa Cruz), anti-cyclin A (rabbit polyclonal, Santa Cruz), anti-Cdk4 (rabbit polyclonal, Santa Cruz), anti-Cdk2 (rabbit polyclonal, Santa Cruz), anti-PSTAIRE (mouse monoclonal, a gift from Dr. S. Reed, The Scripps Research Institute, La Jolla, CA), anti-p27 (mouse monoclonal, Transduction Laboratories), anti-p21 (rabbit polyclonal, Santa Cruz), anti-retinoblastoma (mouse monoclonal, Pharmingen), and anti-ILK (affinity-purified rabbit polyclonal). Protein detection was carried out using secondary antibody (either anti-mouse-HRP (Jackson Laboratories or Pharmingen), anti-rabbit-HRP (Jackson Laboratories), or protein A-HRP (Amersham Life Science)) and the enhanced chemiluminescence (ECL) detection system (Amersham Life Science).
Kinase AssaysFor Cdk4-associated kinase activity,
asynchronous cells growing in monolayer culture were lysed in ice-cold
Tween 20 lysis buffer (0.1% Tween 20, 50 mM Hepes, pH 7.5, 150 mM NaCl, 2.5 mM EGTA, 1 mM
EDTA), containing the following inhibitors (1 mM
dithiothreitol, 0.1 mM PMSF, 20 µg/ml aprotonin, 10 mM -glycerophosphate, 0.1 mM sodium
vanadate, 1 mM sodium fluoride). Cell lysates were then sonicated. Protein A-Sepharose beads (Sigma Immunochemicals Co.) precoated with Cdk4 antibody (rabbit polyclonal, Santa Cruz) were used
to immunoprecipitate Cdk4. Cdk4-associated kinase activity was assayed
using the protocol of Matsushime et al. (28).
For cyclin E kinase assays, cells from asynchronous monolayer culture were lysed in 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris, pH 7.6) plus protease inhibitors (0.1 mM PMSF, 20 µg/ml aprotonin, 20 µg/ml leupeptin). Cyclin E was immunoprecipitated with polyclonal anti-cyclin E serum (gift from Dr. Steve Reed, The Scripps Research Institute, La Jolla, CA and also from Dr. D. Agrawal, M. Lee Moffit Cancer Center, Tampa, FL) and complexes collected on protein A-Sepharose beads (Sigma Immunochemicals Co.). Cyclin E-associated kinase reactions were carried out as described previously (29).
For both cyclin D1-Cdk4 and cyclin E-Cdk2 assays, kinase reaction products were resolved by SDS-PAGE and the incorporation of radioactivity in substrate was visualized by autoradiography (X-Omat AR (Eastman Kodak Co.) or REFLECTIONTM (DuPont)) and quantitated by PhosphorImager (Molecular Dynamics).
p27 Inhibitory AssayCell lysates (100 µg of protein) were recovered from asynchronously growing IEC18 or ILK-overexpressing cells using lysis buffer as for cyclin E-Cdk2 kinase assays with Nonidet P-40 at 0.1% concentration. Lysates were boiled for 5 min and clarified by centrifugation. p27 was immunoprecipitated (rabbit polyclonal anti-p27 serum provided by Dr. T. Hunter, Salk Institute, La Jolla, CA) from boiled lysates. Immune complexes were collected on protein A-Sepharose beads and then washed five times in 0.1% Nonidet P-40 lysis buffer. To release bound p27, the beads were resuspended in 200 µl of 0.1% Nonidet P-40 lysis buffer, containing protease inhibitors (1 mM PMSF and 20 µg/ml each aprotonin, leupeptin, and pepstatin), boiled for 5 min and supernatants recovered. Cyclin A-Cdk2 complexes immunoprecipitated from asynchronous ILK14 cells (control-transfected cells) were used as test substrate for inhibition by p27. Heat-stable p27 released from immune complexes was incubated at 30 °C for 30 min together with cyclin A-Cdk2. Cyclin A-Cdk2 kinase activity was assayed using histone H1 (Boehringer Mannheim) as a substrate and compared with the activity of cyclin A-Cdk2 complexes without added immunoprecipitated p27. As a negative control, non-immune serum immunoprecipitates were collected, boiled, and supernatant added to active cyclin A-Cdk2 test complexes. The p27 antiserum used in these assays does not cross-react with p21. Detection of radioactivity in kinase substrate was carried out as described for kinase assays.
We have shown
previously that overexpression of the ILK in normal rat intestinal
epithelial cells (IEC18) results in a less adherent phenotype and in
anchorage-independent growth in soft agar (26). When maintained in
suspension, IEC18 cells have been demonstrated to undergo programmed
cell death (30), which is suppressed by mutant c-Ha-ras
oncogene expression (30). Since we have found that ILK overexpression
in IEC18 cells induces anchorage-independent growth as well as
tumorigenicity in nude mice,2 we wanted to
determine whether ILK overexpression also suppresses suspension-induced
cell cycle arrest and cell death. ILK-overexpressing cell clones
(ILK13) are capable of anchorage-independent cell growth in soft agar
(26). This increased cell survival is reflected in the greater
proportion of ILK13 cells that are present in S phase after 12 h
in suspension, as compared with the control ILK14 cells, in which the
percentage of cells in S phase falls to 5% (Fig.
1A). Furthermore, a sub-G1 (<2
N) population is present in the control ILK14 cells after
12 h in suspension, consistent with the presence of apoptotic
cells. This population of cells is completely absent in the
ILK-overexpressing ILK13 clones (Fig. 1A). We next wished to
address whether ILK overexpression also induces serum-independent
growth in monolayer-adherent cultures. As shown in Fig. 1B,
the growth rate of ILK13 cells is not elevated when compared with the
IEC18 or the control ILK14 cells. In fact, the ILK-overexpressing
clones grow slightly more slowly than the parental IEC18 and the ILK14
control-transfected cells (Fig. 1B). In addition, ILK13
cells fail to survive in serum-free conditions similar to the IEC18 and
control ILK14 cells. These data demonstrate that ILK overexpression
selectively induces anchorage-independent growth but not serum
(mitogen)-independent growth.
ILK Overexpression Alters the Expression of Cell Cycle Regulators
Adhesion of fibroblasts to ECM has been shown to
induce the expression of cyclin D1 (6). Since overexpression of ILK in epithelial IEC18 cells induces cell survival and cell cycle progression in the absence of adhesion, we wanted to determine whether ILK overexpression altered the expression and/or activity of cell cycle
regulators. The expression of various cell cycle regulators was
examined in IEC18, ILK13, and ILK14 cells growing under standard tissue
culture conditions. As shown in Fig. 2A,
ILK-overexpressing cell clones (ILK13) (26) express substantially
higher levels of cyclin D1 protein than the parental IEC18, or
control-transfected ILK14 cells. In contrast, the level of expression
of cyclin E is not altered in ILK13 cells. The expression of cyclin A
was examined as well and was found to be elevated in ILK13 cells (data not shown in Fig. 2; see Fig. 4). Since the cyclins function in complex
with the cyclin-dependent kinases, Cdks, we also determined the expression of Cdk4 and Cdk2 kinases that complex with cyclin D1 and
cyclin E, respectively. Surprisingly the level of Cdk4 protein is also
elevated in the ILK13 cells, whereas Cdk2 is not altered (Fig.
2A). The kinase activities of Cdk4 and Cdk2 are also
regulated by inhibitor proteins, p21 and p27, and the expression of
these inhibitors is known to be enhanced in non-adherent (suspension) cells and decreases upon cell adhesion (6, 7). In ILK-overexpressing cells, both p21 and p27 are increased (Fig. 2A), and their
electrophoretic mobilities are clearly altered. The faster migrating
forms of p21 and p27 in ILK-overexpressing cells may reflect covalent
modification, or in the case of p27, the product of partial proteolytic
cleavage (31). For p27, at least, this alteration correlates with a
decreased inhibitory potential (see Fig. 3b).
Such faster migrating forms have also been observed after exposure of
fibroblasts to UV irradiation (32).
To demonstrate that the observed changes are mediated by ILK, we transfected IEC18 cells with an ILK expression vector under the control of a metal inducible promoter (33). As shown in Fig. 2B, induction of ILK expression with Zn2+/Cd2+ results in the stimulation of expression of ILK. Concomitantly, the expression of cyclin D1 is also induced in these cells (Fig. 2B). The data shown were confirmed in two independent clones, and the treatment of the parental IEC18 cells with Zn2+/Cd2+ had no effect on ILK or cyclin D1 expression (data not shown). These data demonstrate that increased ILK expression can induce the expression of cyclin D1 protein.
We next determined whether the complex formation between the cyclins, Cdks, and the p21/p27 inhibitors was also altered upon ILK overexpression. As shown in Fig. 2C, both cyclin D1 and Cdk4 are elevated in Cdk4 immunoprecipitates from ILK13 cells as compared with the parental IEC18 and control ILK14 cells. Although the amount of p27 is also higher in Cdk4 immunoprecipitates from ILK13 cells, quantification clearly demonstrates that the ratio of p27 to cyclin D1-Cdk4 is much higher in IEC18 and ILK14 cells than it is in the ILK13 cells (Table I). Furthermore, the p27 in Cdk4 immunoprecipitates from ILK13 clones has the faster electrophoretic mobility (Fig. 2C). The amount of cyclin E-associated Cdk2 did not differ between the parental IEC18 and ILK-overexpressing (ILK13) cells. However, in the ILK13 cells, although cyclin E-associated p27 was increased, p27 manifested the altered mobility seen in the Cdk4 complexes (Fig. 2D).
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Cyclin D1 and Cdk4 proteins are increased upon ILK overexpression, but those of cyclin E and Cdk2 are not (Fig. 2). To determine whether this translates into increased kinase activities, we carried out immune complex in vitro kinase assays for both cyclin D1-Cdk4 and also cyclin E-Cdk2 using recombinant Rb and histone H1 as substrates, respectively. As shown in Fig. 3a, the kinase activity of Cdk4 is dramatically increased in the ILK-overexpressing clones (ILK13). Although protein levels of cyclin E and Cdk2 are not elevated (Fig. 2), cyclin E-Cdk2 kinase activity is also increased in these cells (Fig. 3a). Cell adhesion in fibroblasts has been shown to stimulate Cdk2 activity (6, 7), without elevations in cyclin E or Cdk2 levels (6). This is thought to be brought about by the down-regulation of the Cdk inhibitors p21 and p27. In the ILK-overexpressing cells, the increased Cdk2 activity could result, at least in part, from the decreased inhibitory activity of p27 (see Fig. 3b). The net effect of the increased activities of cyclin D1-Cdk4 and cyclin E-Cdk2 in ILK13 cells is an increase in the retinoblastoma protein phosphorylation (Fig. 3a).
p27 from ILK-overexpressing Cells Is Altered and Has a Lower Cdk Inhibitory PotentialIt has been demonstrated previously that nonadherent fibroblasts express high levels of the Cdk inhibitor p27 and that down-regulation of p27 upon cell substratum adhesion increases cyclin E-Cdk2 activity (6, 7). Although ILK overexpression elevates cyclin E-Cdk2 activity, the levels of Cdk inhibitors, p21 and p27, are not decreased. In fact, they appear to be elevated as compared with the IEC18 and ILK14 control cells (Fig. 2). However, both p21 and p27 from ILK13 cells have an altered electrophoretic mobility (Fig. 2). We therefore determined whether the altered electrophoretic mobility of p27 correlated with an altered inhibitory potential of this protein and hence might contribute to the increased cyclin E-Cdk2 kinase activity. To analyze p27 activity, we immunoprecipitated p27 from IEC18 and ILK13 cells and assayed its ability to inhibit test cyclin A-Cdk2 kinase complexes. As shown in Fig. 3b, p27 from IEC18 cells inhibits cyclin A-Cdk2 in a dose-dependent manner. When compared with the activity of p27 from IEC18 cells, equivalent amounts of ILK13-derived p27 (Fig. 3b) showed significantly less inhibitory activity in this type of assay (Fig. 3b). This decreased p27 inhibitory activity could contribute to the higher cyclin E-Cdk2 activity present in the ILK13 cells. Thus although, p27 can complex with cyclin E-Cdk2 (Fig. 2) in the ILK13 cells, its inhibitory potential is reduced resulting in a net higher level of cyclin E-Cdk2 kinase activity.
Adhesion-independent Up-regulation of Cyclin D1 and Cyclin A Expression, and Rb Hyperphosphorylation in ILK-overexpressing CellsNon-adherent fibroblasts express low levels of cyclin D1 and have low cyclin D1-Cdk4 and cyclin E-Cdk2 activities. Untransformed fibroblasts and epithelial cells are also growth-inhibited in suspension and arrest in the G1 phase (1-3). Since ILK overexpression in IEC18 cells induces cell survival and promotes cell cycle progression in suspension, we wanted to determine whether the increased levels of cyclin D1 and Rb protein hyperphosphorylation were maintained in suspension. Furthermore, since the expression of cyclin A is regulated in an anchorage-dependent manner in some cells, we also examined adhesion-dependent regulation of cyclin A protein expression in IEC18 and ILK-overexpressing (ILK13) cells. Exponentially growing adherent cultures of ILK13 and the control, ILK14 cells were placed in suspension for 12 h. The cells were then lysed, and the expression of cyclin D1 and cyclin A and Rb phosphorylation were determined by immunoblotting. As expected, cyclin D1 and cyclin A proteins fall with increased duration in suspension in control (ILK14) cells (Fig. 4). However, in the ILK13 cells, the elevated cyclin D1 and cyclin A expression is maintained in suspension. Similarly, whereas Rb is rapidly dephosphorylated in control (ILK14) cells in suspension, a substantial proportion of Rb remains hyperphosphorylated in suspension ILK13 cells (Fig. 4). These data indicate that overexpression of ILK overcomes the adhesion-dependent regulation of cyclin D1 and cyclin A protein expression and Rb phosphorylation, suggesting that ILK is in the signaling pathway that mediates integrin-dependent regulation of the cell cycle.
Cell adhesion to components of the extracellular matrix is a requirement for cell growth and survival for a wide variety of cell types (1, 2, 4). Inhibition of cell adhesion results in growth arrest, and many epithelial and endothelial cells also undergo apoptosis (1-3). Cell adhesion to the ECM results in the activation of signaling pathways, which maintain cell cycle progression from G1 to S phase. The key components of the cell cycle machinery known to be regulated by cell adhesion to ECM are cyclin D1 and cyclin A expression, activation of cyclin D-Cdk4 and cyclin E-Cdk2 kinases, and retinoblastoma protein phosphorylation (3, 6-8, 10, 11). Determination of the molecular basis of this regulation is clearly important and may be central to our understanding of anchorage-independent cell growth and oncogenic transformation.
It is highly likely that integrins, as receptors for ECM components, initiate signaling events that activate the above mentioned cell cycle regulators. Integrin activation and ligation have been shown to activate MAPK via p21ras-dependent (18, 20, 34) and -independent (21) pathways. Activation of MAPK, in turn, can regulate the transcription (35), and translation of cyclin D1 mRNA, the latter by regulating the activity of PHAS-1 (36). The adhesion-dependent increase in cyclin D1 expression is also regulated, in part, at the level of mRNA translation (6), and therefore activation of MAPK may be crucial in adhesion-dependent cell cycle control. Anchorage-dependent expression of cyclin A has been shown to be regulated at the level of gene transcription (10). The integrin-proximal events responsible for the activation of downstream signaling pathways still need to be fully characterized.
We have recently identified a novel serine/threonine protein kinase
(ILK), which can associate directly with the cytoplasmic domain of
integrin 1 and
3 (26). Overexpression of
this kinase in epithelial cells induces anchorage-independent growth
(26) and oncogenic transformation.2 In this paper, we have
demonstrated that, when overexpressed, human ILK induces
adhesion-independent cell survival of rat intestinal epithelial cells,
increases both cyclin D1 and cyclin A protein levels, and stimulates
the activation of cyclin-dependent kinases. Specifically,
we have shown that the induction of ILK expression by stable
transfection (ILK13), or by inducible transfection, results in the
elevation cyclin D1 protein. Furthermore, the activity of Cdk4 is
substantially elevated in ILK13 clones when compared with parental
IEC18 cells or control ILK14 clones. In contrast, although the
expression of cyclin E and Cdk2 are unchanged, cyclin E-Cdk2 activity
is increased in the ILK13 clones. The combined activation of Cdk4 and
Cdk2 activities results in the hyperphosphorylation of the
retinoblastoma protein (Rb), the phosphorylation of which regulates the
entry of cells into S phase (8, 9). Surprisingly, ILK overexpression
also seems to increase the levels of both p21 and p27 Cdk inhibitors.
Since Cdk inhibition by KIP family proteins relies on an increase in
the molar ratio of p21 or p27 in the Cdk complex, the ratio of
p27:target cyclin-Cdk is important (37). The ratio of p27 to cyclin D1
in complex with Cdk4 is substantially higher in the IEC18 and ILK14
cells than it is in the ILK-overexpressing (ILK13) cells. Thus despite
the ILK-mediated increase in KIP proteins, the molar ratio (shown for
p27) of KIP:cyclin D-Cdk4 in ILK-13 cells is not increased.
Another interesting consequence of ILK induction is the expression of altered forms of both p21 and p27. These altered forms have faster electrophoretic mobilities as compared with p21 and p27 from the parental IEC18 cells and the control-transfected (ILK14) clones. The nature of this alteration is not clear as yet but could result from altered phosphorylation (38) or proteolytic degradation (31). However, the expression of different isoforms, for example, by alternative splicing cannot be ruled out. A potential functional consequence of this alteration appears to be decreased inhibitory activity, as demonstrated for p27. This decreased inhibitory activity could account for the increased cyclin E-Cdk2 activity observed in ILK-overexpressing cells.
Of significant importance to the oncogenic properties of ILK and its role in integrin-mediated signal transduction is the finding that ILK-overexpressing cells (ILK13) continue to cycle in serum-containing suspension cultures, whereas the control transfectant clones (ILK14) undergo cell cycle arrest and apoptosis, as described previously (30). In IEC18 and control ILK14 cells, inhibition of adhesion to ECM results in a rapid down-regulation of expression of both cyclin D1 and cyclin A proteins, Rb dephosphorylation, and G1 arrest. This is in marked contrast to ILK13 clones, in which cyclin D1 and cyclin A expression as well as Rb phosphorylation are maintained upon transfer to suspension cultures and there is no inhibition of cell cycle progression. ILK, like Ras, stimulates the expression of cyclin A and cyclin D1 resulting in Rb phosphorylation. However, unlike Ras, ILK does not induce serum-independent cell growth, indicating that anchorage-independent cell growth can be stimulated independently of serum-independent cell growth. Preliminary data indicates that overexpression of ILK does not activate Ras, but can activate MAPK,3 thus suggesting that ILK can activate a Ras-independent pathway capable of altering cell cycle control resulting in anchorage-independent cell growth. Ras activates, on the other hand, other cellular functions, which result in both anchorage-independent and serum-independent cell growth. Although one must be cautious in interpreting data from overexpression studies, our results indicate that ILK may have an important role in modulating anchorage-independent cell cycle progression. Whether the kinase activity of ILK is required for this regulation remains to be determined.
Elevated cyclin D1 expression is quite common in certain types of cancers, especially breast and esophageal carcinomas (39-41). Although in some cases the increased cyclin D1 expression is due to gene amplification (41), for the majority of the cases, the molecular basis of this increased expression is unclear (42). Since ras mutations are infrequent in breast carcinomas, it is unlikely that Ras plays an important role in the elevation of cyclin D1. Our results suggest that the altered expression of ILK might be involved in the elevated cyclin D1 expression seen in some cancers, and this will be the subject of future studies. Finally, our results suggest a role for ILK in specifically coupling anchorage-dependent growth and cell cycle regulation. Altered expression, and/or kinase activity, of ILK could have an important role in uncoupling cell cycle regulation by cell adhesion and may play a crucial role in pathogenesis of cancer and cardiovascular diseases.
We thank Mina Viscardi for secretarial assistance.