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INTRODUCTION |
p27 is a member of the Kip family of G1
cyclin-cdk1 inhibitors
(reviewed in Refs. 1 and 2). In cycling cells, p27 targets cyclin-cdk2
complexes for inactivation, is associated with cyclin D2/3-cdk4/6
complexes in a non-inhibitory fashion (3, 4), and is an assembly factor
for cyclin D1-cdk4 complexes (5). Together the assembly functions and
sequestration of p27 in cyclin D-cdk4 complexes can titrate the amount
of p27 from cyclin E-cdk2, allowing cells to progress through
G1-phase and enter S-phase (6). However, because
accumulation of p27 is biphasic with a maximal amount in quiescent
non-cycling cells and a lower intermediate amount in G1
cells, the initial reduction of p27 might be essential for progression
from G0 to G1.
Although multiple processes conspire to determine the threshold amount
of p27 at each stage of the cell cycle, including regulation at the
level of transcription (7-12), protein synthesis (13-15), protein
degradation (16, 17), sequestration (4), and cellular localization
(18), the major contributions to the threshold in
G0/G1 cells come from a combination of
cdk2-independent proteolysis (16) and synthesis rate (14). Following
entry into S-phase, p27 levels reach a nadir because of
cdk2-dependent ubiquitin-dependent proteolysis
(14). Because p27 abundance is important to the decision to enter and
exit the cell cycle, we expected that the rate of p27 synthesis as
cells transit the G0-G1 period might be a
target of mitogenic signals, so we set out to determine the signaling
pathways that control translation of p27 mRNA.
Here we report that the amount of p27 mRNA translation was
sensitive to the activity of GTPases of the Rho family, being maximally induced when Rho is inactivated in a G0 cell. Cell cycle
arrest induced by inhibition of either the PI3 or p70S6 kinase pathways or simply reducing the amount of activated Rho or activated Erk without
inducing cell cycle arrest failed to affect the amount of p27 mRNA
translation. We have shown that this aspect of
Rho-dependent regulation required an element within the
last 300 nucleotides of the mRNA. We also established that
RhoAN19, a dominant negative allele of Rho, could inhibit
RasV12-induced foci formation in wild-type cells and cells
lacking either Rb or p16 but not those lacking p27. However, p27 status
did not affect the ability of RhoAN19 to interfere with
stress fiber formation. Combining these observations we suggest a model
wherein Rho-dependent changes in p27 synthesis rates may
impact progression during the G0/G1 interval of
the cell cycle.
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EXPERIMENTAL PROCEDURES |
Plasmids--
The series of SvL vectors have been described
(19). The expression vectors pCMV-p27 (20), pCEFL-RhoAQ63L
and pCEFL-RhoAN19 (21, 22), and pBabe-Ras V12
(23) have been described.
To generate retroviruses expressing RhoAN19, an N-terminal
hemagglutinin tag was added by PCR to the cDNA of
RhoAN19 from a pCEFL derivative, and the product was
subcloned into pWZL-hygro (23).
Cell Culture, Transfection, and Drug Treatments--
MDA468
cells were grown in Dulbecco's modified Eagle's high
glucose:F-12 supplemented with non-essential amino acids, 10% fetal
bovine serum, and 2 mM glutamine. Transient transfections were carried out for MDA468 cells essentially as described (19). Cells
were plated at 106 cells per 10-cm dish, grown overnight,
and then transfected using the calcium phosphate method with 5 µg of
luciferase reporter plasmid, 2.5 µg of pCMV-
(CLONTECH), and carrier DNA to a total of 20 µg
per dish, unless otherwise stated. Sixteen hours after transfection
precipitates were washed out with fresh medium. Where indicated,
replicate dishes, transfected with the same DNA mixtures, were
drug-treated 8-10 h after the washes. Luciferase and -galactosidase activities were assayed 24 or 48 h afterward as described (19) Luciferase activity was normalized for efficiency
(luciferase/-galactosidase ratio) in each transfection. To calculate
the change in expression by each treatment and construct, we divided
the normalized activity in treated cells by the value obtained in
untreated cells, and we expressed this relative to the change seen in
expression from constructs lacking any UTR sequences.
We measured the amount of RNA expressed from each reporter construct by
RNase protection assay as described (19). Quantitation was performed
using an analyzer Fuji Film Bas 2500 and the MacBas version 2.5 software.
Lovastatin (Merck) was activated as described previously (24). We
obtained PD98059 from Biomol and LY294002 and rapamycin from
Calbiochem. The amount and duration of treatment are indicated in the
legends to the figures.
Cell Cycle Analysis--
Cell cycle phase distribution was
determined by flow cytometry of propidium iodide-stained cells. Whole
cell suspensions were washed in PBS, fixed in 70% ethanol, stained in
50 µg/ml propidium iodide, 1 mg/ml RNase, 0.1% Triton X-100, and
analyzed using the Multicycle software.
For analysis of transfected populations, an expression vector of the
CD19 surface marker was included in the DNA transfection mixture.
CD19-positive transfected cells were stained as described (25); cells
were detached in PBS, 0.1% EDTA, washed, and stained with a
FITC-conjugated anti-CD19 antibody (BD PharMingen). After fixation in
75% ethanol, cell pellets were washed in PBS, 1% bovine serum
albumin, 0.5% Tween 20 and stained in 5 µg/ml propidium iodide, 200 µg/ml RNase, 0.1% Triton X-100. DNA profiles were obtained from the
FITC-positive population.
Immunoblotting--
For immunodetection of activated forms of
signaling molecules after drug treatments (Fig. 1), cells in the dishes
were washed with ice-cold PBS prior to addition of lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol,
1% Triton X-100, 5 mM EGTA, 1.5 mM
MgCl2) containing phosphatase and protease inhibitors (20 mM Na3VO4, 1 mM NaF, 4 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of
aprotinin and leupeptin). Cells were scraped out of the dishes and
incubated for 15 min on ice. Otherwise, cell pellets were lysed in RIPA
buffer (20 mM Hepes, pH 7.4, 100 mM NaCl, 1%
Triton X-100, 10% glycerol, 0.1% SDS, 0.1% deoxycholate) containing
protease inhibitors (4 mM phenylmethylsulfonyl fluoride and
10 µg/ml each of aprotinin and leupeptin) for 15 min on ice and then
sonicated. All lysates were clarified by centrifugation at 13,000 × g for 15 min. Protein concentration was determined by
Bradford assay (Bio-Rad). After SDS-PAGE, proteins were transferred onto PVDF membranes and detected with antibodies using ECL detection system. The affinity-purified anti-p27 antibody has been described (4).
Antibodies are as follows: to MAPK (06-182, Upstate Biotechnology, Inc.), p70S6k (9202, New England Biolabs), phospho-MAPK (9101, New England Biolabs), phospho-Akt (9271, New England Biolabs), phospho-p70S6k (9204, New England Biolabs), anti-hemagglutinin epitope
(clone 12CA5, Roche Molecular Biochemicals), and 
-tubulin (T9026, Sigma).
Foci Formation Assay--
Mouse embryo fibroblasts were isolated
from 13.5-day post-coitum embryos generated in intercrosses of
p27+/
(26), Rb+/ (27), or Ink4a/ (28) mice. After harvesting
the embryos, heads and red organs were removed and used for genotyping
by both PCR and Western blot. Carcasses were cut into pieces and then digested in tyrosine at 37 °C for 30 min. Cell pellets from single embryos were resuspended in Dulbecco's modified Eagle's high
glucose medium supplemented with 2 mM glutamine, 10% fetal
bovine serum, gentamicin, penicillin, and streptomycin, plated in a
10-cm dish, and considered as passage 0. Immortal derivatives were
established following a 3T3 protocol.
3T3 fibroblasts of different genotypes were plated at 106
cells per 10-cm dish and transfected by the calcium phosphate method with 2 µg of either pBabe-puro or pBabe-RasV12 and 18 µg of either pWZL-hygro or pWZL-RhoN19 per dish. After an overnight
incubation, the precipitates were washed out. Cells were grown in
complete medium for 24 h and then selected in the presence of 75 µg/ml hygromycin for 4-5 days. Selected cells were counted, mixed
with wild-type primary MEFs (3 × 103 drug-resistant
cells plus 3 × 105 wild-type cells), and plated at
3 × 105 cells per 60-mm dish. The medium was changed
every other day. After 14 days, foci were stained with Giemsa. Foci
were counted on a Bio-Rad Image Analyzer using the Bio-Rad Quantity One
Analysis Software and confirmed by visual counting under microscope.
RhoN19-expressing MEFs--
Retroviral supernatants were
generated by transfection of 293T cells (5 × 106
cells/10 cm dish) with 15 µg of retroviral vector and 15 µg of ecotropic helper virus. Supernatants were collected 48 h after transfection, pooled from replicate dishes, and filtered through a
0.45-µm filter.
MEFs were plated at 8 × 105 cells/10-cm dish the
night before infection and exposed to viral supernatants (4 ml per
dish) containing 4 µg/ml Polybrene for 24 h. Cells were grown in
complete medium for 24 h and then selected in the presence of 75 µg/ml hygromycin for 4 days.
Infected MEFs plated in coverslips were fixed in 4% paraformaldehyde
for 30 min, permeabilized in 1 M Hepes, pH 7.5, 300 mM sucrose, 50 mM NaCl, 3 mM
MgCl2, 0.5% Triton X-100 for 4 min on ice, and then
stained with 2 µg/ml FITC-phalloidin (Sigma). Counts were performed
on captured images.
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RESULTS |
A Lovastatin-induced Translational Element Is Contained within a
300-Nucleotide Sequence at the 3'-End of p27 mRNA--
To
determine whether specific signaling pathways affected the translation
of p27 mRNA, we used an assay developed to measure the
translational efficiency of p27 mRNA (19). This assay takes advantage of a reporter plasmid that expresses the luciferase gene
product under the control of the SV40 promoter, SV40 polyadenylation signal, and the untranslated regions of the p27 mRNA. Following co-transfection of the reporter vector with a vector expressing -galactosidase from a cytomegalovirus promoter into MDA468 cells, we
treated them with an assortment of drugs that affected different signaling pathways. We used an inhibitor of Mek1 (PD98059), an inhibitor of PI3 kinase (LY294002) which affects both the activity of
p70S6 kinase and Akt, an inhibitor of p70S6 kinase (rapamycin), or an
inhibitor of protein prenylation (lovastatin). As measured by trypan
blue staining the toxicity of these treatments was insignificant (data
not shown).
PD98059 had only a modest effect on cell cycle progression, whereas
cells treated with the other drugs accumulated with a G0/G1 content of DNA (Fig.
1A). We then prepared extracts
from these cells and blotted for Erk1/2, phosphorylated Erk1/2, p70S6k, phosphorylated p70S6k, phosphorylated Akt, and p27 (Fig.
1B). The amount of phosphorylated Erk1/2 was decreased in
PD98059-treated and lovastatin-treated cells but not in LY294002- or
rapamycin-treated cells. The amount of phosphorylated S6 kinase was
decreased in rapamycin-, LY294002-, and lovastatin-treated cells but
not in those treated with PD98059. The amount of phosphorylated Akt was decreased in LY294002- and lovastatin-treated cells but not in PD98059-
or rapamycin-treated cells. The p27 protein accumulated in cells
treated with lovastatin, rapamycin, or LY294002 but not PD98059 (Fig.
1C).
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Fig. 1.
Lovastatin induces the translation of p27
mRNA. MDA468 cells were transfected with reporter constructs,
and 24 h later they were treated with either 10 or 50 µM PD98059 (PD), LY294002 (LY), 10 or 50 nM rapamycin (RAP), or 30 µM
lovastatin (LOV) for additional 24 h prior to analysis.
DMS (DMSO), Me2SO. Replicate dishes were used to
assess cell cycle distribution (A), efficiency of the
treatment on their respective targets (B), p27 protein
accumulation (C), and expression from the reporter plasmids
(D and E). A, cell cycle
distribution. Samples were analyzed for DNA content by flow cytometry.
The samples are indicated above each panel, and the percent
of cells in each phase of the cell cycle for a representative
experiment is shown. The experiment was repeated at least three
times for each drug. B, extracts were prepared as
described under "Experimental Procedures" and 40 µg resolved by
electrophoresis on SDS-polyacrylamide gels (8% for p70S6k and 10% for
MAPK and Akt), transferred to PVDF, and the amount of MAPK,
phosphorylated MAPK (phospho-MAPK), p70S6kinase (p70S6K),
phosphorylated p70S6kinase (phospho-p70S6K), and the phosphorylated
form of Akt (phospho-Akt) was determined. The treatments are indicated
above each lane, and the respective antibodies used for
immunoblotting are to the right of each panel. This
experiment was repeated at least three times for each drug.
C, p27 accumulation. Extract was prepared in RIPA
buffer and 50 µg was resolved by electrophoresis on 12%
SDS-polyacrylamide gels, transferred to PVDF, and probed with a
p27-specific antibody. The drug treatment is indicated above
each lane. The experiment was repeated at least three times.
D, only lovastatin induces luciferase accumulation from
the reporters. Lysates were prepared from transfected cells, and
luciferase and -galactosidase activity was measured as described
(19). The experiment was repeated at least three times.
E, RNase protection assay analysis. 5 µg of
total RNA prepared from transfected and drug-treated cells was
subjected to RNase protection assay using the probes in the
left-hand lane. Below each lane, we indicate the
ratio of luciferase (luc) mRNA to -galactosidase
(-gal) mRNA determined by PhosphorImager
quantitation. The experiment was repeated at least three times, and RNA
was used at both 5 and 10 µg in each case to ensure linearity of the
assay.
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Similar to the increase of endogenous p27, lovastatin induced
luciferase accumulation (Fig. 1D). Lovastatin is known to
increase the synthesis of p27 (15) and decrease the turnover of p27
(29). Increased synthesis doubles the available p27, and the reduction in proteolysis must make up the rest to achieve the 3-4-fold increase in steady state observed in the treated cells. Rapamycin and LY294002 did not increase reporter expression consistent with previous reports
(30, 31) that these agents affect the rate of p27 turnover. The steady
state amounts of luciferase and -galactosidase mRNA were not
differentially affected by the drugs used (Fig. 1D). This suggested that increased translation of p27 would
not be a general effect of G1 arrest, although an increase
in p27 protein might be.
To map the sequences responsible for the
lovastatin-dependent induction of p27 synthesis, we
examined the effect of lovastatin treatment on different mutant
constructs. Constructs containing only the 3'-UTR alone had negligible
activity, consistent with our previous report (19) that the 5'-UTR
contains sequences required for both basal and induced translation of
the p27 mRNA. The reporters containing only the 5'-UTR were not
affected by lovastatin, suggesting that the 3'-UTR was required for the
lovastatin-dependent induction of p27 (Fig.
2). We found that deleting nucleotides 1680-1978 of the 3'-UTR eliminated the lovastatin-inducible element. This loss was not due to the length of the 3'-UTR because expression from an even shorter construct lacking two-thirds of the 3'-UTR, but
including this 300-nucleotide region (
638-1680), was induced by
lovastatin treatment (Fig. 2). The yield of mRNA was equivalent in
all the mutants (data not shown). We conclude that the ability of
lovastatin to enhance p27 translation lies in an element within the
most distal 300 nucleotides of the 3'-UTR.
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Fig. 2.
The lovastatin response element is in the
last 300 nucleotides of the 3'-UTR. Cells were transfected with
the reporters indicated below each bar and the accumulation
of luciferase, normalized for -galactosidase, was measured in
non-treated (white bars) and lovastatin-treated cells
(black bars). This experiment was repeated five times, with
three independent preparations of plasmid DNA. LOV,
lovastatin.
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The Lovastatin Effect Is Mediated by Inactivating
Rho--
Lovastatin blocks the metabolic pathway leading to protein
prenylation by inhibiting hydroxymethylglutaryl-CoA reductase. This
causes mislocalization of several membrane-anchored proteins and can
have effect on the activity of Erk, Akt, and p70S6 kinases as well as
other proteins. As the inactivation of Erk, Akt, and p70S6 kinase did
not enhance synthesis individually (Fig. 1D), we began to
look at the Rho family of GTPases. Because both the Rho proteins, by
C-terminal geranylgeranylation, and their GTP exchange factors,
by lipid interactions with the pleckstrin homology domain (32), are
membrane-localized, lovastatin will block the prenylation of Rho
required for its membrane localization and its subsequent activation.
Consistent with this, cells treated with lovastatin showed typical
changes in morphology as well as redistribution of Rho from membranes
to the cytosolic fraction (data not shown).
We examined whether direct inhibition of Rho would enhance synthesis
from the reporters, similar to the lovastatin-dependent localization failure. The toxin C3 exoenzyme is a highly specific inhibitor of RhoA, RhoB, and RhoC that only weakly affects other GTPases (33). C3 catalyzes ADP-ribosylation at asparagine 41, preventing interactions of these Rho family members with their effectors (33). Cells co-transfected with vectors expressing C3 toxin,
a CD19+ cell surface marker expression vector, and the reporters showed
increased luciferase activity similar to lovastatin-treated cells (Fig.
3A). Co-expression of p27 in
place of C3 toxin did not increase reporter expression (Fig.
3A), despite the fact that both the toxin and p27 caused
growth arrest in the CD19+ population of cells (data not shown).
Co-transfection of either C3 or p27 did not affect the relative amounts
of luciferase and -galactosidase mRNA produced in a manner
consistent with the change in activity (Fig. 3). Together these data
indicate that inactivation of Rho enhances the synthesis of p27.
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Fig. 3.
Rho activity is a determinant of translation
of p27 mRNA as measured by the reporters. A,
the accumulation of luciferase from the reporter correlates with
changes in Rho activity. The experiment is similar to that described in
the legend to Fig. 1D. Samples are indicated
below each bar. The experiment was performed at least
four times. B, RNase protection assay
analysis. RNA was prepared from cells transfected with either p27
Kip1 or C3 exoenzyme (pEVX) as indicated
above each lane. The amount of total RNA hybridized to the
probes (in the left-hand lane) is indicated above
each lane. Below each lane, we indicate the ratio of
luciferase (luc) mRNA to -galactosidase
(-gal) mRNA as determined by PhosphorImager
quantitation. RPA was repeated at least twice for each sample.
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Next we asked if we could overcome the effect of lovastatin on reporter
expression by using a GTPase-deficient, constitutively active mutant of
RhoA, RhoAQ63L. There is a disagreement in the literature
as to the specific requirements of membrane localization for activity;
however, many groups (34-37) have shown that constitutively active
alleles of Rho act in the absence of proper membrane localization. This
may be attributed to the overexpression of the mutant, and high levels might allow it to fulfill some of the activated Rho functions simply by
mass action. We co-transfected RhoAQ63L together with the
reporter vectors prior to addition of lovastatin. The active form of
Rho prevented the increase in translation caused by treatment with
lovastatin (Fig. 3), suggesting that inactivation of Rho is a required
event. This allele does not abrogate the G1 arrest induced
by lovastatin (data not shown), indicating that Rho inactivation is
only part of the cellular response to lovastatin. Additionally,
p27/ mouse fibroblasts arrest in response to lovastatin, arguing that changes in p27 are only part of the cellular response to
the drug (data not shown). We did not attempt to ask if the RhoAQ63L allele would reverse the effect of C3 exoenzyme
because ADP-ribosylation would interfere with the ability of the
RhoQ63L to associate with effector proteins. Thus, we
concluded that the lovastatin induced increase in reporter synthesis,
and the ability of the activated mutant of Rho to suppress this is
consistent with the notion that Rho could affect p27 accumulation
directly, independent of cyclin E-cdk2-initiated proteolysis as shown
previously (38) in IIC9 cells.
Finally we asked if inhibiting Rho activity would be sufficient to
induce p27 synthesis. We were unable to induce synthesis from the
reporters when co-transfected with a vector expressing RhoAN19, a dominant negative mutant of Rho into MDA468
cells (data not shown). On the other hand, we could not document that
the amount of RhoAN19 expressed was sufficient to inhibit
endogenous Rho activity. We next tried NIH3T3 cells. These cells,
unlike MDA468 cells, have a well organized cytoskeleton with stress
fibers easily detected by phalloidin staining. Stress fiber formation
is a reflection of Rho activity (32). NIH3T3 cells were transfected
with RhoAN19 and reporter activity scored and correlated to
the disruption of the cytoskeleton. Although the cytoskeleton was
disrupted, cells failed to arrest growth, and we failed to
observe a change in reporter activity (data not shown). Consequently,
from this and the data above, we conclude that Rho activity is
necessary but not sufficient in cycling cells to induce changes in p27
synthesis rate.
Inhibition of Rho Activity Suppresses RasV12-induced
Transformation in a p27-dependent Manner--
Rho function
had been shown to be necessary for the transforming activity of
RasV12 (39-42). Thus we tested if RhoAN19
would interfere with RasV12 transformation in a
p27-dependent manner.
To overcome the Arf/p53-dependent growth arrest that occurs
when introducing RasV12 into primary mouse fibroblasts (28,
43), we immortalized p27-deficient and Rb-deficient cells by continued
passage on a strict 3T3
protocol.2 Additionally, we
used Ink4a/ MEFs, deficient for p16Ink4a
(and p19Arf1) (28). The Arf co-deletion confers
immortality. We also used NIH3T3 cells as a prototype model cell line.
Most of the 3T3 clones were still dependent on serum for growth and
became quiescent when grown to confluence (data not shown). There was
an increase in the spontaneous transformation rate of
Rb/ cells (Footnote 2 see foci formation in Fig.
4A as an example).
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Fig. 4.
RhoN19 inhibits transformation by
RasV12 in a p27-dependent manner.
A, representative experiment showing foci formation by
Giemsa staining 2 weeks after plating. Populations of wild-type, Rb
null, p16/p19 null, and p27 null mouse embryo fibroblasts were
transfected with the vectors indicated to the left,
selected, and 3,000 of these cells were plated with 300,000 wild-type
MEFs and grown 14 days prior to staining and counting of foci. Foci
were counted on a Bio-Rad Image Analyzer using the Bio-Rad Quantity One
Analysis software. B, the average values of duplicate
plates from at two to three independent experiments are shown in
tabular format. C, the amounts of RasV12
and RhoAN19 were determined by direct immunoblotting of the
selected cells or immunoprecipitation/immunoblotting with hemagglutinin
antibodies, respectively. This was repeated twice for each clone.
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These four lines were co-transfected with retroviral constructs
encoding RasV12, RhoAN19, both, or the
respective empty vectors, and cells were selected for drug resistance.
For each line, 3,000 resistant cells were pooled and combined with
300,000 wild-type primary mouse fibroblasts and plated into 60-mm
dishes. Foci were scored 2 weeks later after Giemsa staining. The
number of foci is representative of the number of cells transformed by
RasV12; they are no longer arrested by growth to
confluence. A representative experiment is shown in Fig. 4A.
In determining the number of foci in Rb/ cells, we have
subtracted the number of colonies arising spontaneously and suppressed
by RhoAN19 co-expression. The extent of transformation by
RasV12 was nearly the same in all genotypes, but colony
size was different, with Rb-deficient cells producing fairly large
colonies relative to the Ink4/, p27/, and
NIH3T3 cells (Fig. 4, A and B). Overall, in two
independent experiments with two independent clones of each genotype,
we find that the steady state amount of RasV12 was always
lower in Rb-null cells (Fig. 4C). We do not know why.
Co-expression of RhoAN19 with RasV12 could
reduce foci formation in all except for the p27/ cells,
where it had no effect (Fig. 4, A and B). The
amount of Rho expressed was equivalent in all four genotypes (Fig.
4C). Similar Rho-dependent repression effects on
RasV12 were observed in passage 2 fibroblasts from
wild-type and p27/ cells co-transfected with
RasV12 and SV40 large T antigen which eliminates both Rb
and p53 functions (data not shown). Taken together, these results
indicate that the ability of RhoAN19 to interfere with the
transforming activity of oncogenic Ras is dependent on the presence of p27.
Inhibition of Rho Induces Cytoskeletal Changes Independently of p27
Status--
The transformation studies above indicate that p27, but
not Rb or p16, were targets of Rho activity associated with
transformation by RasV12. The ability of p27 deficiency to
alter the requirement for Rho signaling might have reflected a general
change in the activity of Rho in these cells. Stress fibers are a
measure of the activity of Rho (32). We examined the appearance of
stress fibers by phalloidin staining to assess the basal activity of
Rho in p27/ cells and cells expressing the dominant
negative mutant. We transduced both wild-type and p27-deficient passage
2 primary mouse embryo fibroblasts (MEF) with a retroviral construct
expressing RhoAN19 and a hygromycin resistance gene from a
bicistronic message. Pools of cells were isolated following drug
selection. Because of the selection, cells in these pools were expected
to contain RhoAN19, at levels that would not prevent their
proliferation. We confirmed this by measuring thymidine incorporation
during a short pulse and by examining DNA content by flow cytometry in
passage 4 cells (data not shown). Moreover, we did not detect a
significant increase in the steady state amount of p27, the related cdk
inhibitor p21, or cyclin E at passage four (Fig.
5A). In wild-type cells at
passage 4, there was a small increase (30%) in the synthesis of p27 as measured by immunoprecipitable material following a 30-min pulse with
[35S]methionine in the presence of LLnL, an inhibitor of
the proteasome (data not shown). The amount of RhoAN19
expressed was nearly equivalent in the wild-type and
p27/ cells, perhaps being a bit higher in the
p27/ cells (Fig. 5A). Stress fibers were
readily observed in the control-infected p27/ cells and
clearly disrupted in p27-deficient cells expressing RhoAN19
(Fig. 5B). These results were quantitated for 500 cells in
three independent transductions where control wild-type fibroblasts expressing RhoAN19 were also examined (Fig. 5B).
As the disruption of actin stress fibers was quantitatively similar,
the data suggest that p27-deficient cells do not have a significantly
higher level of basal Rho activity and that the dominant negative Rho
protein can effectively function, at least with regards to its role in
cytoskeleton organization.
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Fig. 5.
RhoN19 alters stress fiber
formation in p27/ mouse embryo fibroblasts.
Wild-type (WT) and p27/ mouse embryo
fibroblasts were transfected with retroviruses encoding a
hemagglutinin-tagged RhoAN19, selected in hygromycin for 4 days, and grown an additional week prior to making extracts and
examining the actin cytoskeleton by phalloidin staining. A,
immunoblots. 50 µg of extract were resolved by 12% SDS-PAGE and
transferred to PVDF membranes and probed with the antibodies indicated
to the right of each panel. The source of extract is
indicated above each lane. WZL represents cells
infected by the retrovirus lacking an insert. RhoAN19
represents cells infected by the WZL retrovirus expressing
RhoAN19. This is a representative experiment of three
independent transductions on three individual primary cultures.
B, RhoAN19 affects stress fiber formation in a
p27-independent manner. Primary mouse fibroblasts were stained with
FITC-phalloidin, and representative fields are shown. The transduced
virus is indicated above each field. Four to five fields
from three independent transductions of wild-type and
p27/ cells (indicated by a hatch mark) were
used to determine the percentage of cells with stress fibers, and these
results are plotted in the accompanying graph.
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DISCUSSION |
There is accumulating evidence that G1 progression is
impacted by the activation status of Rho family GTPases. Rho has been linked to transcription of p21 (44), the transcription of cyclin D1
(45), and perhaps directly to the activation of cyclin E-cdk2 and the
ubiquitin-dependent degradation of p27 (38). Furthermore, transformation by RasV12 can be suppressed by dominant
negative Rho (40-42), and this at least partially involves the
activation of ROCK (46). Here we show that p27 synthesis can be
modulated in a Rho-dependent fashion in growth-arrested
cells, higher in cells with inactive Rho and lower in cells with active
Rho. Additionally, we show that the suppression of RasV12
transformation by dominant negative Rho is dependent on p27 but not on
either Rb or p16.
Rho and Control of p27 Synthesis--
Here we establish that Rho
activity can affect synthesis of p27 in non-cycling cells through a
cis-element contained in the last 300 nucleotides of the 3'-UTR of the
mRNA. In breast epithelial cells (above) and NIH3T3 cells (data not
shown), we have shown that inactivation of the Rho signaling pathway by
a generic inhibitor of protein prenylation (lovastatin) and by a
specific inhibitor (C3 exoenzyme) induce synthesis from a p27 reporter
without altering the available steady state amount of message from the
reporter. Consistent with the activity of Rho being the target for
lovastatin-induced synthesis, we find that we can overcome the effect
by introducing a constitutively active mutant of Rho,
RhoAQ63L. This mutant cannot efficiently hydrolyze GTP but
can still bind GTP and interact with effectors without the requirement
for membrane localization. Furthermore, other signaling pathways
inactivated by lovastatin, including the Mek1, PI3-kinase, and S6
kinase pathways, individually cannot induce synthesis. We did not
examine combinatorial effects and thus cannot state that the
lovastatin-induced increase in synthesis is entirely due to Rho and is
not assisted by some other signaling pathway being modulated (see below).
How might Rho repress translation of the p27 mRNA? There are many
effectors of Rho, ROCK and LIM kinases being the best defined. Which,
if any, of these signaling pathways is utilized by Rho to repress p27
synthesis needs to be investigated further. We have found that neither
cytochalasin D which disrupts the cytoskeleton nor plating cycling
cells in the absence of integrin signaling was sufficient to induce p27
reporter activity in our assay.2 Because both of these
treatments would affect the activity of the ROCK kinase, this suggests
that the Rho linkage to p27 synthesis may not be associated with
signals that measure cytoskeletal events. Once we narrow down the
sequences required for the Rho-dependent effect on
translation and identify proteins that interact there, in a manner
dependent on Rho signaling, we should be able to make some progress in
elucidating this novel translational regulation pathway of Rho.
Rb and p27 Are on Different Pathways with Respect to Transformation
of 3T3 Cells by RasV12--
Others have shown that Rho can
increase transcription of cyclin D1 (45) and decrease the transcription
of p21 (44) in various model cell systems. Both effects are in some
fashion dependent on cells with a high persistent level of Erk
signaling. Rho is essential for the repression of p21 transcription in
cells that are subject to a hyperactive Erk signal. Cyclin D1 is
induced by such signals but only when maintained in the presence of Rho activity. Interestingly, the relationship of cyclin D to cell cycle
control is dependent on the presence of Rb. Established evidence
indicates that in the absence of this tumor suppressor, fibroblasts are
no longer dependent on cyclin D-cdk4 activity for cell proliferation
(47-49). Consequently, one might have expected that Rb-negative cells
would be refractory to the proliferative effects of inhibiting Rho.
However, we report that the transformation of Rb/ cells,
as well as p16-deficient cells (cells in which a reduction of the Ink4
inhibitor could increase cyclin D-cdk4 complex formation), was still
modulated by the expression of dominant negative Rho. On the other
hand, the transformation of p27/ cells was not
attenuated by the dominant negative Rho. This could not be attributed
to an increase in Rho activity as measured by the formation of stress
fibers in those cells and their disruption in the cells expressing
dominant negative Rho. There is other supporting evidence for
independent functions for p27 and Rb in transformation. We reported
previously (50) that Rb and p27 could cooperate to enhance tumor
development in a pituitary model independently of enhancement in the
number of proliferating cells. Consequently, Rho is acting
through a mechanism involving p27. This is discrete, although possibly
overlapping, from the p16/Rb axis when participating in transformation
by RasV12.
p27 and p21 in G1 Progression--
Singularly, neither
p27 nor p21 has been implicated as an important mediator of
G1 progression in mouse fibroblasts except in the case of
NIH3T3 cells that when treated with antisense oligonucleotides gain a
degree of serum independence (51, 52). Primary cultures of p27- or
p21-deficient mouse embryo fibroblasts were not even partially
serum-independent (53-55).2 These cells, when
immortalized, retain serum dependence and are no more prone to
spontaneous transformation as wild-type cells.2 The rate at
which these cells enter S-phase from a serum-starved state is
indistinguishable from wild-type cells. This is similar to observations
in p16 null fibroblasts that have normal growth characteristics and
remain susceptible to Ras-induced senescence (56, 57).
Nevertheless, the notion that p27 is an important protein for the
regulation of the cell cycle during serum-induced entry into S-phase is
supported by the observation that cdk4-deficient mouse embryo
fibroblasts induced to re-enter the cell cycle from serum starvation
have a prolonged G1-phase, a prolongation that was almost
completely eliminated by p27 deficiency. Similar analysis has not been
carried out on p21/ cells. We have noted that p27 deficiency eliminates a modest delay in S-phase entry in primary passage 2 mouse fibroblasts expressing dominant negative
Rho,2 consistent with the possibility that the
G1 function of Rho is affecting accumulation of p27, but
clonal variations in expression level and the resultant delay make the
analysis of this problematic without a good single cell assay. Rho
activity can affect progression of cells from G0 into
S-phase in at least four of the following ways: it can reduce synthesis
of p27 (data here); it can reduce the transcription of p21 (39);
it can increase the transcription of cyclin D1; and it can affect
cyclin E-cdk2 activity. Ultimately, the only thing the cell needs is to
suppress the available p27 (or p21) that can interact with cyclin
E-cdk2. During the G0 to G1 transition, the
amount of p21 in a cell coupled with both cdk2-independent proteolysis
of p27 (16) and a decrease in synthesis of p27 would establish the
threshold to which cyclin D-cdk4 would need to accumulate. Once cyclin
D-cdk4 has passed this threshold, active cyclin E-cdk2 can accumulate,
allowing the cells to progress from G1 into S-phase and the
cdk2-dependent turnover of p27. In support of the role of
Rho suppressing cdk-inhibitor levels during this transition, we find
that p21/ cells are also resistant to the effect of RhoAN19 in the RasV12 foci formation assay
(data not shown).
Conclusions--
In conclusion, our data indicate a genetic
relationship between Rho and p27 as related to the ability of Rho to
participate in transformation by RasV12 in immortalized 3T3
cells. It suggests that this is not identical to the function of
inactivating Rb, as Rb-deficient cells retain sensitivity to Rho
inhibition in this assay. We speculate that at least part of the
Rho-p27 connection is associated with modulating the synthesis of p27
protein, because both lovastatin and C3 exoenzyme increase
translational efficiency of p27 mRNA. Nevertheless, the amount of
p27 synthesis is not appreciably increased in NIH3T3 cells transfected
with RhoAN19 at levels sufficient to disrupt stress fiber
formation, indicating that inactivating Rho while necessary is not
sufficient for the translational effect (data not shown). One
possibility is that other pathways must be simultaneously impaired to
allow a translational effect of Rho inactivation. We have tried to
co-transfect reporters with RhoAN19 and either a p27
expression vector or a RasV12 expression vector, or
transfect reporters with RhoAN19 and treat cells with
either LY294002 or rapamycin (data not shown). However, in no case
could we recapitulate the translational enhancement we saw with
lovastatin or C3 exoenzyme.2 Alternatively, the inability
of RhoAN19 to function in translation enhancement is
somehow associated with the fact that it is a competitive inhibitor of
Rho signaling, and both lovastatin and C3 exoenzyme act in a
non-competitive manner. Lovastatin is a strong inhibitor of prenylation
thus affecting both the overall activity of Rho family members and the
activities of many other molecules involved in the activation of Rho
family members. C3 exoenzyme acts catalytically to inactivate the RhoA, RhoB, and RhoC family members.
However, p27 deficiency can attenuate the ability of
RhoAN19 to suppress foci formation. It is important to note
that taking cells co-transfected with RasV12 and
RhoAN19, which do not have enhanced reporter activity (data
not shown), is not identical to the additional selective pressures
evident in a foci formation assay. Only a small proportion (~1%) of
cells transduced with RasV12 are able to overcome the
contact inhibition growth arrest signal. We do not know why this is,
but it could be related to both the amount of signal a cell needs to
become transformed and the amount that it can tolerate, as well as
additional cryptic mutations that might arise in the population. It is
under this condition where we find the connection between Rho and p27.
How much of that connection is associated with p27 translation is not
yet clear; nevertheless, p27 synthesis is in some way affected by Rho
status, and Rho status is less important in p27/ cells
transformed by RasV12.
§,
TOP
ABSTRACT
INTRODUCTION
Former Pew Scholar of Biomedical Sciences and presently a
Hirschl Scholar. To whom correspondence should be addressed: RRL917D, Box 207, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New
York, NY 10021. Tel.: 212-639-2354; Fax: 646-422-2062; E-mail: a-koff@ski.mskcc.org.
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