1 Groupe du Conseil de Recherches Médicales sur le Développement Fonctionnel et la Physiopathologie du Tube Digestif, Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Quebec J1H 5N4, Canada; and 2 Centre de Biochimie-Centre National de la Recherche Scientifique, Université de Nice, 06108 Nice, France
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ABSTRACT |
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The present report delineates the critical pathway in the G1 phase involved in downregulation of p27Kip1, a cyclin-dependent kinase inhibitor, which plays a pivotal role in controlling entry into the S phase of the cell cycle. In resting CCL39 fibroblasts and IEC-6 intestinal epithelial cells, protein levels of p27Kip1 were elevated but dramatically decreased on serum stimulation, along with hyperphosphorylation of pRb and increased CDK2 activity. In both cell types, expression of ras resulted in an increase of basal and serum-stimulated E2F-dependent transcriptional activity and a reduction in p27Kip1 protein levels as well. The role of the mitogen-activated protein (MAP) kinase cascade in p27Kip1 reduction and S phase reentry was reinforced by the blockades of serum-induced E2F-dependent transcriptional activity and p27Kip1 downregulation with the MKK-1/2 inhibitor PD-98059. In both cell lines, downregulation of p27Kip1 was associated with a repression of its synthesis, an event mediated by the p42/p44 MAP kinase pathway. Using an antisense approach, we demonstrated that p27Kip1 may control cell cycle exit in both cell types. These data indicate that activation of the MAP kinase cascade is required for S phase entry and p27Kip1 downregulation in fibroblasts and epithelial cells.
growth control; cell cycle; G1-to-S phase transition; growth signaling
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INTRODUCTION |
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OVER THE LAST DECADE, major attention has been devoted
to our understanding of how growth factors transduce their signals across the plasma membrane. Although many aspects remain to be addressed, strong lines of convergence are emerging in growth signaling. At least three signaling networks, not yet fully elucidated, appear to be crucial for
G0-to-G1
phase progression: 1) the Ras Raf
MKK
p42/p44 MAP kinase cascade,
resulting in activation of a range of transcription factors such as
Elk-1, c-Ets-1, and c-Ets-2 (29, 54);
2) the lipid signaling pathways,
including phosphatidylinositol 3'-kinase (18), and subsequent
regulation of p70 S6 kinase (8); and
3) the small GTP-binding proteins of
the Rho family as a consequence of integrin complex assembly or actin
polymerization (21, 33).
Transduction of extracellular mitogenic signals culminates in the expression and assembly of different kinase holoenzymes, the cyclin-cyclin-dependent kinase (CDK) complexes. Progression through the cell cycle is thus governed by the sequential formation, activation, and subsequent inactivation of CDK complexes (48). The activation of CDKs is subjected to multiple levels of regulation: the synthesis of the cyclins and their assembly into cyclin-CDK complexes, the appropriate phosphorylation-dephosphorylation of the CDKs in these complexes, and the inhibitory action of the CDK inhibitors (CKIs) in these complexes (30, 49). CKIs constitute a new class of regulatory proteins that control cell cycle progression by binding to and inactivating the CDK complexes. Two gene families of CKIs have been identified in mammalian cells: the INK4 proteins, specific inhibitors of cyclin D-CDK4/CDK6 complexes (13), and the Kip/Cip inhibitors (p21Cip/p27Kip1/p57Kip2), with broader specificity (49). Overexpression of these inhibitors causes G1 arrest (9, 13, 14, 38, 42, 49).
p27Kip1 was initially discovered
as a CKI induced by an extracellular antimitogenic signal (38). It
accumulates in many situations in which cells are arrested in the
G0/G1
phase (37, 38, 49). Recent findings highlighted a major difference
between p21Cip and
p27Kip1 during the initial
response to growth factor stimulation (49). Indeed, whereas
p27Kip1 tends to accumulate in
quiescent cells and declines in response to mitogenic stimulation (32,
42, 49), p21Cip levels are
generally low in quiescent cells and rise in response to mitogenic
treatments (14, 49). Furthermore, various antimitogens, including
transforming growth factor- in mink epithelial cells (37), rapamycin
in T lymphocytes (32), and cAMP in macrophages (19), prevent
mitogen-induced p27Kip1
downregulation and, therefore, CDK activation and
G1 progression. We and others have
recently demonstrated that overexpression of p27Kip1 antisense cDNA allowed
cells to grow for several generations in medium supplemented with
insulin and transferrin (42) or in medium containing low concentrations
of serum (9). Conversely, it was recently demonstrated that targeted
disruption of the murine p27Kip1
gene enhanced growth of the mice and led to striking enlargement of
thymus, pituitary, adrenal, and gonadal organs (11, 22, 31). These data
suggest that p27Kip1 plays a major
role in controlling cell cycle exit, a fundamental step in growth control.
Considering the pivotal role of p27Kip1 in G1-to-S phase transition, regulation of its expression remains a key issue to understand growth control. Which critical pathway in the G1 phase does signal p27Kip1 downregulation? On the basis of the observation that prolonged p42/p44 MAP kinase activation was absolutely required to pass the R restriction point in the G1 phase of the cell cycle (35), we demonstrate that activation of p42/p44 MAP kinases is necessary for p27Kip1 downregulation and for cell cycle progression in the nontransformed hamster fibroblast cell line CCL39 and the intestinal epithelial cell line IEC-6.
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EXPERIMENTAL PROCEDURES |
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Materials
The enhanced chemiluminescence immunodetection system, [methyl-3H]thymidine, and [Methods
Cell culture.
The established Chinese hamster lung fibroblast line CCL39 and its
derivative [clones v-ras5C (46)
and PS200 (45)] were cultured in DMEM (GIBCO/BRL, Burlington, ON,
Canada) containing 7.5% FCS, penicillin (50 U/ml), and streptomycin
(50 µg/ml). The lack of
Na+/H+
exchange activity in PS200 was exploited to select positive clones for
various genes of interest in cotransfection experiments, with the
Na+/H+
antiporter gene (NHE1) as a
selective marker (42, 45). CCL39-derived Raf-1 estrogen
receptor-expressing (
Raf-1:ER) cells were cultivated in DMEM without
phenol red in the presence of 7.5% FCS and G-418 (400 µg/ml) (26).
The rat intestinal epithelial crypt cell line IEC-6 was obtained from
Dr. A. Quaroni (Cornell University, Ithaca, NY). This cell line and the
transformed cell line Ha-rasIEC-6 (5)
were cultured in DMEM containing 5% FCS, as previously described (41).
Constructs.
Plasmid E2F SV40-luc, which contains a high-affinity E2F binding site
from the dihydrofolate reductase (DHFR) promoter coupled to a
luciferase gene, was a gift of Dr. P. Farnham (University of Wisconsin,
Madison, WI) (50). MAP kinase phosphatase-1 (MKP-1) construct (kindly
provided by Dr. N. K. Tonks) was described previously (6).
Dominant-active and dominant-negative mutants of RhoA and Rac1 (kindly
provided by Dr. Marc Symons, Onyx Pharmaceuticals, Richmond, VA) have
previously been described (33, 39). C3 exoenzyme was kindly provided by
Dr. P. Boquet (4). The estradiol receptor/constitutively active Raf-1
chimeric construct (Raf-1:ER) was provided by Dr. M. McMahon (44).
The full-length mouse p27Kip1
[
EXlog(+), Novagen], provided by Dr. J. Massague
(Memorial Sloan-Kettering Cancer Center, New York, NY), was subcloned
into the expression vector pECE. A 1-kb-pair restriction fragment
(EcoR
I-Hind III) from
Exlog(+)p27Kip1 was inserted in
the reverse orientation, as previously described (42), in the pECE
expression vector to obtain a full-length antisense
p27Kip1.
Kinetics of thymidine incorporation. Cells were serum-deprived for 24 h and then stimulated with 10% FCS (CCL39 cells) or 5% serum (IEC-6 cells) for the indicated times. At each point the incorporation of radiolabeled thymidine ([methyl-3H]thymidine, 4 µCi/ml) into DNA was performed for 1 h during the last 0.5 h of serum incubation, as previously described (23). The reaction was stopped by rinsing the cells four times with 5% TCA. Then the cells were harvested with 0.1 M NaOH, and the radioactivity incorporated in the TCA-insoluble fraction was counted by liquid scintillation.
Protein expression and immunoblotting.
Cells were rendered quiescent (G0
arrested) by a 24-h incubation in serum-free DMEM. Cells were then
stimulated with 10% FCS (CCL39 cells) or 5% FCS (IEC-6 cells) for the
indicated times. Cells were lysed in SDS sample buffer [62.5 mM
Tris · HCl, pH 6.8, 2.3% SDS, 10% glycerol, 5%
-mercaptoethanol, 0.005% bromphenol blue, 1 mM phenylmethylsulfonyl
fluoride (PMSF)], and proteins (40 µg) from whole cell lysates
were separated by SDS-PAGE in 10% acrylamide gels. Proteins were
detected immunologically after electrotransfer onto nitrocellulose
membranes. The blots were incubated with primary antibody in blocking
solution for 2-4 h at 25°C and then incubated with horseradish
peroxidase-conjugated goat anti-mouse or anti-rabbit (1:1,000) IgG in
blocking solution for 1 h. The blots were revealed by the Amersham
enhanced chemiluminescence system. Protein concentrations were measured
using a modified Lowry procedure with BSA as standard (36).
CDK2 kinase assay.
Cells were serum starved for 24 h and restimulated by addition of serum
for the indicated times. Cells were lysed for 10 min on ice with 1 ml/dish of lysis buffer (150 mM NaCl, 1 mM EDTA, 40 mM Tris, pH 7.6 + 1% Triton X-100) supplemented with protease inhibitors (0.1 mM PMSF,
10 µg/ml leupeptin, 1 µg/ml pepstatin, 10 µg/ml aprotinin) and
phosphatase inhibitors (0.1 mM
o-vanadate, 20 mM
p-nitrophenyl phosphate, 40 mM
-glycerophosphate). Lysates cleared by centrifugation (10,000 g, 10 min) were incubated for 2 h at
4°C with protein A-Sepharose preincubated for 1 h with anti-CDK2.
Immunocomplexes were then washed four times with ice-cold lysis buffer
and three times with ice-cold kinase buffer (20 mM p-nitrophenyl phosphate, 10 mM
MgCl2, 1 mM dithiothreitol, in 30 mM HEPES, pH 7.4) before the kinase assay was performed. The kinase
reaction was started by incubating the immunocomplexes at 30°C in
the presence of the substrate histone H1 and
[
-32P]ATP at
20-100 µM, 1-5 µCi/assay. After 30 min the reaction was stopped by addition of hot Laemmli buffer. Radiolabeled substrates were
separated from immunocomplexes by SDS-PAGE and autoradiographed. Incorporation of 32P on histone H1
was linear over the course of the kinase assay (23).
p27Kip1 protein expression in transiently transfected PS200 cells. PS200 cells seeded at a density of 500,000 cells per well in six-well plates were cotransfected by the calcium phosphate technique with 5 µg of the selection vector pEAP (NHE1 cDNA), together with 5 or 15 µg of the relevant expression vector (25, 42, 45). Two days after transfection, cells were submitted to an acid-load selection. Cells were allowed to recover for at least 4 h before serum starvation for 24 h. Cells were then stimulated with 10% serum and lysed 18 h later. Expression of p27Kip1 was monitored by immunoblotting.
Transient transfections and luciferase assays.
CCL39 and IEC-6 cells were seeded in a 24-well plate and cotransfected
by calcium phosphate precipitation or by lipofection (Lipofectin,
GIBCO/BRL), respectively, with 0.1 µg of E2F SV40-luc reporter and
0.1 µg of the relevant expression vector (pCMV, pcDNAneo, or pECE)
containing MKP-1 (6), dominant-active and dominant-negative mutants of
RhoA, Rac (33, 40), p27Kip1 sense,
or p27Kip1 antisense (42).
Normalization was achieved by cotransfecting 0.1 µg of pCH110, a
-galactosidase reporter construct, as an internal control for the
transfection efficiency. Two days after transfection, luciferase and
-galactosidase activities were measured as previously described (25,
42).
Metabolic labeling of cells with [35S]methionine and immunoprecipitations. For labeling newly synthesized proteins, we used DMEM lacking cysteine and methionine supplemented with trans-35S label. During the last hour of incubation, cells were rinsed with cysteine- and methionine-free medium and incubated in 1.5 ml (for each 60-mm-diameter plate) of the same medium containing 100 µCi of trans-35S label. Labeling was allowed to proceed for 1 h. Cells were rinsed with PBS and lysed with the addition of 0.15 ml of boiling 1% SDS, 10 mM Tris · HCl, pH 7.4, and 1 mM PMSF. The lysates were boiled for an additional 5 min, sonicated briefly, and centrifuged for 5 min. Supernatants were then precipitated with 8% TCA and dissolved in 0.1 N NaOH, from which aliquots of 10 µl were counted by scintillation to assess total incorporation. Immunoprecipitation experiments were performed with equal amounts of precipitated material (3 × 106 cpm/sample) and resuspended in 1.5 ml of buffer A (1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 0.2 mM sodium vanadate, 0.2 mM PMSF). Antibodies were then added overnight at 4°C (dilution 1:500). After addition of the protein A-Sepharose, the samples were rocked for 1-2 h. Immune complexes were pelleted and washed four to five times in 1 ml of buffer A. The final pellets were recovered in gel loading buffer and separated on 10% SDS-PAGE. Gels were then fixed in 10% acetic acid for 30 min and dried for autoradiography. Autoradiograms were scanned for quantitation (Fuji PhosphoImager).
Data presentation. Assays were performed in duplicate or triplicate. The data are from representative experiments performed at least twice. Results obtained with the luciferase assays were analyzed by a Student's t-test. Results were considered significantly different at P < 0.05.
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RESULTS |
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Kinetics of S Phase Entry in CCL39 Fibroblasts and IEC-6 Epithelial Cells
Figure 1 shows the timing of entry into the S phase exhibited by CCL39 fibroblasts and IEC-6 epithelial cells after serum deprivation for 24 h followed by reactivation by growth factors. Thymidine incorporation in resting cells was low in both cell lines. As previously reported (23), in CCL39 fibroblasts, the replication of DNA began ~15 h after stimulation and reached a maximum at 24 h, as determined by [3H]thymidine incorporation (Fig. 1A). However, in IEC-6 cells, DNA synthesis significantly began ~18 h after serum addition and reached a maximum at 24 h (Fig. 1B).
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Downregulation of p27Kip1 in Response to Serum: Correlation With Hyperphosphorylation of pRb and CDK2 Activity
To demonstrate a correlation between S phase entry and downregulation of p27Kip1, the hyperphosphorylation of pRb, the CDK2 activity, and the expression of different CKIs were analyzed by Western blotting on cell lysates from serum-stimulated CCL39 and IEC-6 cells (Fig. 2). A specific antibody able to detect the active hypophosphorylated form of pRb (bottom band) as well as the inactive hyperphosphorylated form of the protein (top band) was used. In serum-deprived CCL39 and IEC-6 cells, pRb was exclusively found in its hypophosphorylated state. In CCL39 cells, pRb inactivation (hyperphosphorylated form) became apparent at 15 h after serum stimulation when CDK2 activity was stimulated (Fig. 2A). The expression of p15INK4B was not significantly affected on serum addition (data not shown), whereas p21Cip expression was reproducibly increased twofold. In contrast, stimulation with serum decreased p27Kip1 by 60, 63, and 81% after 15, 18, and 22 h, respectively (Fig. 2A). Similarly, in IEC-6 cells, Rb phosphorylation, p27Kip1 downregulation, and CDK2 activity became apparent 18 h after serum addition, with a significant 38% reduction in p27Kip1 levels (Fig. 2B). At 22 h after serum addition, p27Kip1 expression was significantly reduced by 57%. However, the expression of p15INK4B (data not shown) and p21Cip was not significantly affected on serum addition in this cell line. These data obtained in two different cell lines indicate a strict correlation between the hyperphosphorylation of pRb/CDK2 activity and the downregulation of p27Kip1 inhibitor.
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Reduction in p27Kip1 Expression in v-rasCCL39 and Ha-rasIEC-6 Cells
The ras protooncogene is a central component of the mitogenic signal transduction pathways (3, 27). Ras proteins with activating mutations are implicated in transformation of cells in vivo and in culture, indicating a central role for Ras-mediated signaling events during the proliferation of eukaryotic cells (3). To examine the contribution of Ras signaling to the downregulation of p27Kip1, we analyzed CCL39 and IEC-6 cells stably expressing v-ras and Ha-ras, respectively. Interestingly, as shown in Fig. 3, the basal levels of p27Kip1 observed in CCL39 cells stably expressing v-ras and in IEC-6 cells stably expressing Ha-ras were significantly reduced by 62 and 45%, respectively, compared with basal p27Kip1 levels observed in parental cells. As shown in Fig. 3, addition of serum to these cells expressing Ras proteins resulted in a more pronounced reduction in p27Kip1 levels.
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To determine whether reduced p27Kip1 expression correlates with the known growth-stimulatory properties of the ras-transformed cells (3), we examined the effect of ras overexpression on a late G1 transcriptional event that plays a role in S phase entry. E2F1 is a member of the recently described E2F family of transcription factors that are cell cycle regulated. Although undetectable in quiescent cells, E2F1 is serum induced during the late G1 phase (50). The DHFR and thymidine kinase genes, which are required for DNA synthesis and are transcribed at the G1-to-S transition, contain E2F-dependent binding sites in their promoters. In addition, microinjection of E2F into quiescent fibroblasts provokes S phase reentry, underscoring the importance of E2F in cell growth control (24). We therefore performed transient transfection assays with a plasmid construction containing the E2F-responsive DHFR promoter linked to a luciferase reporter gene (50). In transiently transfected CCL39 and IEC-6 cells, E2F-regulated gene expression increased more than five- and threefold, respectively, in response to serum stimulation. Interestingly, the basal E2F-dependent luciferase activity was strongly enhanced by 12- and 9-fold in v-rasCCL39 and Ha-rasIEC-6 cells, respectively. Addition of serum to these ras-expressing cells only slightly increased E2F-dependent transcriptional activity. Interestingly, cotransfection of p27Kip1 potently reduced basal and serum-stimulated E2F transcriptional activity in v-rasCCL39 cells and Ha-rasIEC-6 cells (Fig. 3).
Activation of the p42/p44 MAP Kinase Cascade Is Involved in Mitogen-Induced Downregulation of p27Kip1 and S Phase Entry in CCL39 and IEC-6 Cells
A sustained activation of the p42/p44 MAP kinase cascade is required for fibroblasts to pass the G1 restriction point and enter the S phase (35). Activation of p42/p44 MAP kinase requires the sequential activation of Ras, Raf-1, and MKK-1/2, with p42/p44 MAP kinase being the last step in this kinase cascade (29). To specifically determine the role of the p42/p44 MAP kinases, we analyzed p27Kip1 downregulation and E2F-dependent transcriptional activity using PD-98059, a specific inhibitor for MKK-1/2, the upstream activators of the p42/p44 MAP kinases (10). Indeed, as shown in Figs. 4A and 5A, addition of 20 µM PD-98059 to both cell lines potently inhibited stimulation of p42/p44 MAP kinase activities observed 5 and 120 min after serum addition. In IEC-6 cells the stimulatory effect of serum on p27Kip1 downregulation (Fig. 4B) and E2F-dependent transcriptional activity (Fig. 4C) was totally abolished by PD-98059. Furthermore, we also evaluated the effect of PD-98059 in Ha-ras-expressing IEC-6 cells. As shown in Fig. 4D, treatment of these cells with 20 µM PD-98059 potently reduced basal and serum-stimulated E2F-dependent transcriptional activity. These results suggest that p42/p44 MAP kinases are involved in p27Kip1 downregulation and G1-to-S phase transition in response to serum stimulation in epithelial cells.
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Treatment of CCL39 cells with PD-98059 significantly attenuated by 44% the downregulation of p27Kip1 induced by serum (Fig. 5B) and blocked E2F-dependent transcriptional activity (Fig. 5C). Along this line, we previously reported that expression of MKP-1 strongly reduced MAP kinase-dependent c-fos promoter activity (6), cyclin D1 expression (25), and DNA synthesis (6). Here, we show that MKP-1 expression blocked the serum-induced downregulation of p27 by ~35% (Fig. 5B) and abolished E2F-dependent transcriptional activity (Fig. 5C).
Moreover, when the endogenous p42/p44 MAP kinase activities were
inhibited by pretreatment of CCL39 fibroblasts with PD-98059 or by
ectopic expression of MKP-1,
p27Kip1 downregulation was only
partially attenuated. To better analyze the contribution of p42/p44 MAP
kinases in S phase reentry and p27Kip1 downregulation in
fibroblasts, we used the CCL39-derived cell line (CCL39-Raf-1:ER)
expressing an estradiol-dependent human Raf-1 protein kinase. In this
cell line the
Raf-1:ER chimera is rapidly activated in response to
estradiol, thereby activating MKK-1 and then p42/44 MAP kinases.
Previous characterization of CCL39-
Raf-1:ER cells has shown that
addition of estradiol to serum-starved cells stimulated p42/p44 MAP
kinases within minutes (26). CCL39-
Raf-1:ER cells were serum starved
for 24 h and then stimulated with 1 µM estradiol or 10% FCS for
various periods of time. As shown in Fig.
5D, the p42/44 MAP kinase activity
increased in response to estradiol, reaching a level comparable to the
maximal p42/44 MAP kinase activation measured in serum-stimulated CCL39 control cells, and remained elevated as long as estradiol was maintained. Activation of the Raf pathway alone slightly promoted Rb
phosphorylation, but not as much as we observed in serum-stimulated cells (Fig. 5E). Moreover, we could
not detect significant CDK2 activity in estradiol-stimulated cells.
Nevertheless, addition of estradiol over 15 h was sufficient to reduce
p27Kip1 levels by ~43%, whereas
serum reduced p27Kip1 levels by
80%, as measured by computer-assisted scanning. Figure 5E also indicates that an additive
effect on p27Kip1 downregulation
was observed when CCL39-
Raf1:ER cells were stimulated with estradiol
and serum. These data suggest that in fibroblasts at least two
mitogen-induced mechanisms are involved in
p27Kip1 downregulation, one being
regulated by the p42/p44 MAP kinase signaling pathway.
Involvement of RhoA Small GTPase in Mitogen-Induced Downregulation of p27Kip1 in Fibroblasts
In addition to the MAP kinase cascade, Ras is also known to control signaling pathways such as those linked to phosphatidylinositol 3-kinase (18) or Rac/Rho proteins (21, 33). A class of geranylgeranylated small G proteins termed the Rho small GTPases were proposed to be involved in the G1-to-S phase transition (16). We set up experiments to analyze the contribution, if any, of these small G proteins in the p27Kip1 downregulation process. We used previously characterized expression constructs encoding dominant-negative or dominant-active forms of RhoA and Rac (33, 39, 40) to modulate positively or negatively the endogenous activity of these small G proteins. As shown in Fig. 6, the overexpression of dominant-negative forms of Rac or RhoA in PS200 fibroblasts (a CCL39 derivative, see EXPERIMENTAL PROCEDURES) significantly attenuated serum-induced p27Kip1 downregulation (Fig. 6A) and abolished serum-stimulated E2F-dependent transcriptional activity (Fig. 6B). Moreover, overexpression of botulinum C3 exoenzyme, which specifically inhibits Rho proteins (4), completely blocked mitogen-induced p27Kip1 downregulation (Fig. 6A) and E2F-dependent transcriptional activity (Fig. 6B), further implicating RhoA in p27Kip1 regulation. Conversely, expression of the dominant-active form of RhoA in these PS200 fibroblasts was sufficient to significantly decrease p27Kip1 levels by 51% in the absence of any growth factors (Fig. 6A).
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It has been shown previously that Ras-dependent RhoA activity is essential for G1 progression in Chinese hamster embryo fibroblasts (55). We have shown in Fig. 3A that the basal levels of p27Kip1 were significantly reduced in CCL39 cells stably expressing v-ras. To further analyze the role of RhoA activity in G1 progression of CCL39 fibroblasts, we evaluated the effect of ectopic expression of the dominant-negative mutant of RhoA on E2F-dependent transcriptional activity in v-ras-expressing fibroblasts. As shown in Fig. 6C, ectopic expression of the dominant-negative form of RhoA potently reduced basal and serum-stimulated E2F transcriptional activity in v-rasCCL39 cells.
Prevention of Mitogen-Induced p27Kip1 Downregulation by Inhibition of Proteasome Function
Several studies reported that the amount of p27Kip1 proteins varied during progression of the cell cycle, whereas p27Kip1 mRNA levels remained unchanged (15, 34). Two different mechanisms were recently implicated in the regulation of p27Kip1 levels, namely, variations in the synthetic rate (1, 15) and in the half-life of the protein (34). To evaluate the role of the proteasome complex in the p27Kip1 downregulation process, we blocked proteasome-mediated proteolysis by using the specific peptide inhibitor N-acetyl-leucyl-leucylnorleucinal (LLnL) (43), added at various times after serum stimulation. As shown in Fig. 7A, LLnL prevented estradiol and serum-induced p27Kip1 downregulation in CCL39-
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p27Kip1 Neosynthesis Is Repressed by Activation of p42/p44 MAP Kinase Cascade in CCL39 and IEC-6 Cells
Two reports recently described a translational control of p27Kip1 accumulation during the cell cycle (1, 15). To determine which pathway could regulate amounts of p27Kip1 at the level of translation, we first measured in CCL39-Abrogation of p27Kip1 Expression Suppresses Quiescence in IEC-6 Cells and Reduces Growth Factor Requirement in CCL39 Fibroblasts
To evaluate the role of p27Kip1 in controlling the mitogen-sensitive G0/G1 growth arrest state, we transiently expressed a full-length p27Kip1 antisense construct in CCL39 and IEC-6 cells. This antisense was shown to reduce the expression of endogenous p27Kip1 in a dose-dependent manner (42). We first compared the effects of enforced or reduced p27Kip1 expression on E2F-dependent transcriptional activity. As shown in Fig. 8, E2F-dependent transcriptional activity was significantly stimulated by serum in both cell lines. Ectopic expression of p27Kip1 totally blocked serum-dependent activation in both cell lines. Conversely, expression of p27Kip1 antisense markedly increased basal and serum-stimulated E2F-dependent transcriptional activity in CCL39 cells (Fig. 8A). Furthermore, low serum concentration (2%) strongly stimulated E2F-dependent transcriptional activity in antisense-transfected CCL39 cells. Interestingly, as shown in Fig. 8B, depletion of p27Kip1 in IEC-6 cells increased basal E2F-dependent transcriptional activity by sevenfold. However, addition of serum did not further stimulate E2F-dependent luciferase activity. It is noteworthy that cotransfection of the sense p27 fully abolished the promoting effects induced by p27 antisense in both cell lines, emphasizing the specificity of the antisense approach.
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DISCUSSION |
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p27Kip1 maintains the quiescent state and regulates the G1 phase through its ability to inhibit the cyclin-CDK complexes that exert their function before and during the S phase. In the present report we demonstrated that the expression of p27Kip1 was high in quiescent fibroblasts and epithelial cells and decreased in response to mitogenic stimulation. Consistent with the previously reported role of p27Kip1 in integrating growth-inhibitory signals with the cell cycle machinery (9, 11, 22, 31, 42, 49), this decrease in p27Kip1 levels correlates with S phase entry, hyperphosphorylation of pRb, and stimulation of CDK2 activity. The biochemical pathways through which p27Kip1 protein levels are linked to the proliferation signals have not been described in detail. The present report delineates the critical pathways in the G1 phase that are involved in p27Kip1 downregulation in fibroblasts and epithelial cells.
Ras signaling is essential for cells to leave a quiescent state (G0) and to pass through the G1-to-S phase transition of the cell cycle (27). However, the mechanism by which Ras signaling regulates cell cycle progression is unclear. It was recently reported that the decrease in the intracellular level of p27Kip1 and the induction of cyclin D1 expression in response to growth factors were dependent on Ras signaling in NIH/3T3 and IIC9 fibroblasts (2, 55). In the present study we extended these observations by demonstrating that constitutive expression of Ras proteins in fibroblasts and epithelial cells was sufficient to induce a significant downregulation of p27Kip1 levels and to enhance E2F-dependent transcriptional activity in the absence of any growth factor.
The Ras GTP-binding proteins play a crucial role in mitogenic signaling
by coupling growth factor receptors to activation of the MAP kinase
cascade (3). In most cell types the mitogenic signal is relayed from
the cytoplasm into the nucleus by the nuclear translocation of the
ubiquitously expressed p42/p44 MAP kinase isoforms, resulting in
activation of transcription factors such as Elk-1, c-Ets-1, and c-Ets-2
(29, 54). Our previous studies showed that a sustained activation of
the p42/p44 MAP kinases was required for fibroblasts to pass the
G1 restriction point and enter the
S phase (35). Activation of the p42/p44 MAP kinase module was
sufficient to stimulate early gene expression (7), to induce cyclin D1
expression (25), and to reduce growth factor requirements for DNA
synthesis (7). Our data obtained in
ras-expressing cells indicate that
regulation of p27Kip1 is a
critical target of the Ras signaling cascade. However, transformation of cells by ectopic expression of oncogenic proteins may result in the
secretion of growth factors or hormones, which may then stimulate the
cell population in an autocrine/paracrine manner. To rigorously assess
the role of the MAP kinase cascade in the regulation of
p27Kip1 downregulation and S phase
entry, we used a CCL39-derived cell line expressing an
estrogen-dependent human Raf-1 protein kinase (CCL39-Raf1:ER) (26). We
demonstrated that the exclusive activation of the Raf MKK-1
p42/p44 MAP kinase cascade by estradiol was sufficient to
reduce by ~60% the p27Kip1
protein levels after 22 h. The involvement of p42/p44 MAP kinases in
p27Kip1 downregulation in CCL39
and IEC-6 cells was also reinforced by the MKK-1/2 inhibitor PD-98059,
which inhibited the downregulation of
p27Kip1 in response to serum and
the E2F-dependent transcriptional activity. These findings suggest that
the downregulation of p27Kip1
induced by activation of the MAP kinase cascade is involved in mitogen-induced cell cycle progression.
Our results can be reconciled with recent data (20, 56) showing that
activation of Raf-ER chimeric proteins in NIH/3T3 fibroblasts led to
an accumulation of cyclin D1 protein and a reduction in
p27Kip1 protein levels. Thus the
reduction in p27Kip1 protein
levels after induction of a constitutive Raf-1 activity could be an
important step in the Raf-1-mediated neoplastic transformation process.
However, despite a significant reduction in
p27Kip1 protein levels, we
(unpublished observations) and others (56) demonstrated that the large
majority of the Raf-ER cells remained in the
G1 phase and did not progress into
the S phase in response to estradiol, most likely as a result of a
strong induction in p21Cip protein
levels. It appears that a threshold for cell cycle arrest is defined by
the amount of Raf activity required for the induction of
p21Cip (56).
Given the recent demonstration that cyclin E-CDK2 directly phosphorylated p27Kip1 on threonine-187 and promoted its elimination from the cell (17, 47, 53), it is tempting to speculate that the difference in p27Kip1 downregulation observed in serum- or estradiol-treated cells is a reflection of a differential ability to activate CDK2. However, whereas CDK2 activity was unaffected, the downregulation of p27Kip1 in response to serum in Raf-ER cells was significantly increased by estradiol addition, suggesting the involvement of other signaling pathway(s) in the mitogen-induced p27Kip1 decrease in fibroblasts. Thus our data in CCL39 fibroblasts should not be interpreted as implying that the p42/p44 MAP kinase module is the only regulator of p27Kip1 in vivo.
Experiments with the hydroxymethylglutaryl-CoA reductase inhibitor
lovastatin showed that, among mevalonate metabolites, geranylgeranyl pyrophosphate was absolutely required for the elimination of
p27Kip1 in FRTL-5 cells (16). A
class of geranylgeranylated small G proteins termed Rho small GTPases
was proposed to be involved in the
G1-to-S phase transition (33, 39,
40). Expression of activated forms of Rac and Rho can transform
fibroblasts, whereas dominant-negative versions of Rac and Rho (33) can
inhibit transformation by oncogenic Ras, indicating that these small G
proteins are essential for Ras transformation. After the transient
expression of dominant-active and dominant-negative forms of RhoA and
Rac, we demonstrated that these G proteins were involved in
serum-induced p27Kip1
downregulation and E2F-dependent transcriptional activity in CCL39
fibroblasts. Moreover, ectopic expression of the dominant-negative form
of RhoA in v-ras-expressing
fibroblasts potently reduced E2F-dependent transcriptional activity.
These data are in total agreement with recent data demonstrating the
involvement of RhoA in Ras-induced
G1 progression and
p27Kip1 downregulation (55).
Furthermore, it has been recently demonstrated that RhoA regulates
p27Kip1 degradation through its
regulation of cyclin E/CDK2 (17). Interestingly, it has been reported
that cells expressing an activated form of Rac 1 showed enhanced growth
in low serum, a situation comparable to that observed in cells
expressing p27 antisense constructs (39). Hence, we predicted that a
sequential activation of the GTPases Ras Rac
Rho is
involved in inducing p27Kip1
elimination observed in response to mitogens in CCL39 fibroblasts. However, in IEC-6 epithelial cells, our data might suggest that the MAP
kinase cascade plays a crucial role in
p27Kip1 regulation and in Ras
transformation. Reports in the literature show that activation of MAP
kinase cascade can be involved (12, 56) or not involved (55) in
p27Kip1 downregulation. Genetic
differences in the cellular systems might be responsible for this discrepancy.
The abundance of p27Kip1 protein
is regulated by translational (1, 15) and posttranslational pathways
(proteolysis) (34) and, less commonly, at the level of transcription
(38). Addition of the proteasome inhibitor LLnL prevented
mitogen-induced p27Kip1
downregulation in CCL39 fibroblasts. In the present report we also
demonstrated that p27Kip1
synthesis was repressed on mitogenic stimulation and that activation of
the Raf MKK
p42/p44 MAP kinase pathway was sufficient
to elicit this repression within 3 h. This suggests that
p27Kip1 protein levels are
translationally regulated in fibroblasts. All these results indicate
that the mitogen-regulated decrease in
p27Kip1 expression occurred
through a mechanism of degradation in the proteasome and through a
translational mechanism controlled by the p42/p44 MAP kinase pathway.
However, because the serum-induced p27Kip1 downregulation was
abolished by proteasome inhibitors, it is reasonable to suggest that a
prior increase in the degradation rate is necessary for repression of
p27Kip1 synthesis. In this
respect, it was recently reported that Ras signaling mediated the
downregulation of p27Kip1 through
an inhibition of its synthesis and an increase in protein degradation
(51). Furthermore, involvement of the MAP kinase cascade in
p27Kip1 synthesis was also
confirmed in IEC-6 cells with PD-98059, which totally inhibited the
repressive effect of serum on
p27Kip1 neosynthesis. Thus, in
CCL39 fibroblasts, the mitogen-induced p27Kip1 downregulation seems to be
the result of a RhoA-dependent degradation of the inhibitor (17)
coupled with a MAP kinase-dependent repression of its synthesis. This
latter phenomenon may be most predominant in IEC-6 epithelial cells.
By forcing the expression of p27Kip1 in CCL39 fibroblasts and IEC-6 epithelial cells, we confirmed that this CKI is a potent growth suppressor recruiting cells into the G0 resting state. This dramatic effect of p27Kip1-induced inhibition on E2F-dependent transcriptional activity may be due to the previously noted p27Kip1-induced inhibition of the activities of cyclin D- and cyclin E-dependent kinases, which are rate limiting for G1 progression and S phase onset (30, 48). The antisense-mediated specific depletion of p27Kip1 demonstrated that p27Kip1 is a major player in growth control of fibroblasts and epithelial cells. Indeed, as we previously demonstrated (42), p27Kip1-depleted CCL39 fibroblasts are much more prone to induce E2F-dependent transcriptional activity at low growth factor concentration. In contrast, in IEC-6 epithelial cells, abrogation of p27Kip1 by cDNA antisense strongly enhanced basal E2F-dependent transcriptional activity at a level that was not further increased by growth factor addition. This observation suggests that p27Kip1 is tightly involved in controlling cell cycle exit in intestinal epithelial cells.
Finally, when the existence of several CKIs that could play a similar role in regulating entrance into the quiescent state is considered, it is remarkable that depletion of p27Kip1 alone in fibroblasts and epithelial cells had such a pronounced effect on E2F-dependent transcriptional activity. Moreover, it has been shown that reduced expression of p27Kip1 is a predictive factor of a poor prognosis for patients with breast and colorectal cancers (28, 52). It was also demonstrated that targeted disruption of the murine p27Kip1 gene led to larger animals with enlargement of thymus, pituitary, adrenal, and gonadal organs (22, 31). Our results clearly demonstrate the strong correlation between cell cycle progression and p27Kip1 downregulation in response to mitogens and identify the MAP kinase cascade as a major regulator of p27Kip1 expression in fibroblasts and epithelial cells.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Jean Morisset for critical reading of the manuscript, Dr. Jacques Pouysségur for judicious comments and fruitful discussions in the course of this work, and Pierre Pothier and Dominique Grall for technical assistance.
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FOOTNOTES |
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This work was supported by grants from the Centre National de la Recherche Scientifique, la Ligue Nationale Contre le Cancer et l'Association pour la Recherche contre le Cancer Grant ARC 9291, and Medical Research Council of Canada Group Grant GR-15186. N. Rivard is a chercheur-boursier from the Fonds de la Recherche en Santé du Québec.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: N. Rivard, Dept. d'Anatomie et de Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, QC, Canada J1H5N4 (E-mail: nrivard{at}courrier.usherb.ca).
Received 19 March 1999; accepted in final form 4 June 1999.
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