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Molecular and Cellular Biology, February 2001, p. 1319-1328, Vol. 21, No. 4
Departments of Developmental and Molecular
Biology and Obstetrics and Gynecology and Women's Health, Center
for the Study of Reproductive Biology and Women's Health, Albert
Einstein College of Medicine, New York, New York 10461
Received 17 July 2000/Returned for modification 7 September
2000/Accepted 14 November 2000
The cell cycle of cultured cells appears to be regulated by
opposing actions of the cyclins together with their partners, the
cyclin-dependent kinases (Cdk), and their inhibitors (Cki). Consistent
with this situation null mutations in the genes for cyclin D1 and Cki
p27Kip1 in mice give opposite phenotypes of dwarfism and
gigantism. To test their genetic interactions, we generated mice
nullizygous for both genes. Correction of cyclin D1 or p27 null to
wild-type phenotypes was observed for many but not all traits. These
included, for cyclin D1 Retinoblastoma protein (Rb)
phosphorylation is thought to be central to the control of the
eukaryotic cell cycle. Its phosphorylation is regulated by cyclins and
their partners, the cyclin-dependent kinases (Cdks). The G1
phase cyclin-Cdk complexes are cyclin D (D1, D2, and D3)-Cdk4 and -Cdk6
and cyclin E-Cdk2. Cyclin D is tightly regulated by extracellular
signals and initiates a chain reaction leading to activation of
internal signals. Cyclin E is, at least in part, responsible for
conferring this internal signal. Cyclin D, once induced in early
G1, binds and activates Cdk4 and -6 and initiates
phosphorylation and inactivation of Rb. Cyclin E associated with Cdk2
can further phosphorylate Rb in mid-G1, albeit at different
sites (12). Rb is sequentially inactivated to release its
negative influence on transcription factors such as E2Fs and on histone
deacetylase (8). These processes result in elevated
transcription of cyclin E and A as well as of many G1
progression and S-phase genes. This positive-feedback role of cyclin
E-Cdk2 can ensure full phosphorylation of Rb and irreversible progression of the cell cycle (reviewed in reference 18).
The prevailing view of the sequential phosphorylation of Rb by cyclin D- and cyclin E-associated kinase complexes is challenged by the fact
that overexpression of human cyclin E can bypass the Rb/E2F pathway and
drive the cell cycle (10). This suggests that cyclin E
might act downstream of the cyclin D1/Rb pathway. Therefore, the
interrelationship of these cell cycle-regulatory molecules in vivo
remains to be further explored.
Cyclin D-Cdk4 and -Cdk6 complexes are subjected to negative regulation
by both the Ink4 and the Cip/Kip families of inhibitors, while cyclin
E-Cdk2 complexes are negatively controlled only by the Cip/Kip family.
Cip/Kip inhibitors, such as p21Cip/Waf1,
p27Kip1, and p57Kip2, are balanced between
cyclin D-Cdk4 and -Cdk6 and cyclin E-Cdk2 complexes. When cyclin D
protein levels are increased by mitogens, more p27Kip1 is
bound to cyclin D1, resulting in the redistribution of
p27Kip1 from the cyclin E-Cdk2 complex to a cyclin D-Cdk4
or -Cdk6 complex, thereby releasing cyclin E-Cdk2 from the negative
control of p27. Therefore, apart from their-kinase function in
phosphorylating Rb, the cyclin D-Cdk4 complexes indirectly stimulate
cyclin E-Cdk2 complexes by titrating out their inhibitors (19,
20). Recently p21 and p27 have also been shown to positively
regulate cyclin D-Cdk4 complexes at low concentration by facilitating
their assembly and possibly their nuclear translocation
(2). Consistent with this, mouse embryo fibroblasts (MEFs)
lacking p21 and p27 showed little cyclin D-Cdk4 assembly or activity,
together with reduced cyclin D1 levels and nuclear localization
(2). However, interestingly, the p21- and p27-deficient
MEFs did not exhibit overall cell cycle defects.
In contrast to the well-documented Rb-centered pathway elucidated in
cultured cells, the roles and the relationships of these cell
cycle-regulatory molecules in mice are more enigmatic. Null mutations
in the activators and inhibitors of the same pathway would be expected
to produce similar or opposite phenotypes. This is generally not the
case. Some induced null mutations in these genes resulted in embryonic
lethality (26, 27); others produced minimal phenotypes
(3), and in some cases only specific cell lineages were
grossly affected (22). In the last group were null
mutations in p27Kip1 and cyclin D1
genes, which resulted in mice whose tissues are globally affected. Mice
homozygous for a null mutation in the cyclin D1 gene are
small, with all organs affected; in particular, the retina and the
pregnant mammary gland exhibit severe hypoplasia (4, 21).
p27Kip1 Cyclin D1 and p27 are both highly expressed in the proliferating neural
retina (15, 21, 28). Consistent with this expression pattern, retinas from cyclin D1 Female infertility in the p27 Altogether these data suggest opposing actions of cyclin D1 and p27 in
regulating rates of cell proliferation in vivo. To test the hypothesis
that these genes are interacting, we generated mice that carried null
mutations in both genes. In these double-mutant mice we found that
overall growth rates were restored to wild-type levels. In addition,
loss of cyclin D1 corrected the implantation defect of
p27 Generation of p27 Histology.
Eyes were removed from the mice and fixed in 10%
formalin at 4°C overnight. After the first 2 h of fixation, two
holes were pierced in the lens side of each eye with a 26-gauge needle
to ensure fixation of the interior of the eyes. Eyes were processed for
cross sections through the optical nerve. The slides were stained with
hematoxylin-eosin for histology.
Dark-field image and TUNEL assay.
Retinas were dissected in
phosphate-buffered saline (PBS) in petri dishes, and the dark-field
images of intact retinas were taken using a Zeiss Stemi SV11 microscope
with the photoreceptor side facing up. For the terminal
deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling
(TUNEL) assay of the whole retinas, retinas were dissected in PBS and
fixed in fresh 4% paraformaldehyde on ice for 1 h. TUNEL was
performed according to the manufacturer's instructions (Promega).
Briefly, after fixation, the retinas were permeabilized by 0.2% Triton
X-100 in PBS for 5 min and then were end labeled with
fluorescein-12-dUTP for 1 h. The retinas were mounted onto glass slides
with 70% glycerol and coverslips, and pictures were taken with a Zeiss
Axioskop microscope.
Implantation analysis.
To induce superovulation, 3- to
12-week-old mice were sequentially injected intraperitoneally with 5 U
of follicle-stimulating hormone (Calbiochem) and, 46 to 48 h later,
with 5 U of human chorionic gonadotropin (Sigma), followed by mating
with males of proven fertility. Plugs were detected the following
morning, which was designated embryonic day 0.5 (e0.5). Some of the
mice were mated with males sterilized by vasectomy, and e0.5 fertilized eggs were transferred to the oviducts or e3.5 blastocysts were transferred to the uteri. At e5 to e17, the pregnant mice were dissected for histology. At e5, intravenous injection of Pontamine sky-blue dye was used to facilitate observation of the implanted sites.
Pontamine sky-blue dye is a high-molecular-weight dye that can only
infiltrate through the blood vessel into the tissue during intensive
vascularization, and this "blue-spotting" method is used as a
marker of the implantation site at early stages of pregnancy (5 to 6 days postcoitum [dpc]). For pregnancy after e6, visual observation of
the uterus was used to identify implantation sites followed by
histology to examine the success of the pregnancy.
BrdU incorporation in the ovaries.
Mice were superovulated
as described above. Three days after mating, female mice were injected
intraperitoneally with bromodeoxyuridine (BrdU) (100 µg per g of body
weight). After being labeled with BrdU for 2 to 3 h, the ovaries
were dissected out, fixed, and processed for paraffin sections. BrdU
staining was performed according to the manufacturer's instruction
(Calbiochem, Oncogene Research)
Biochemical analysis of retinas.
Neonatal retinas were
dissected in PBS containing 0.2 mM phenylmethylsulfonyl fluoride (PMSF)
and frozen at Phenotypic characterization of p27 and cyclin D1 double-null mutant
mice.
cyclin D1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.4.1319-1328.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Genetic Evidence for the Interactions of Cyclin
D1 and p27Kip1 in Mice
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ mice, body weight, early
lethality, retinal hypoplasia, and male aggressiveness and, for
p27/ mice, body weight, retinal hyperplasia, and embryo
implantation. p27/ traits that were not corrected were
the aberrant estrus cycles, luteal cell proliferation, and
susceptibility to pituitary tumors. This mutual correction of these
phenotypes is the first genetic demonstration of the interaction of
these inhibitory and stimulatory cell cycle-regulatory molecules in
vivo. The molecular basis for the correction was analyzed in the
neonatal retina. Retinal cellularity was rescued in the cyclin D1 null
mouse by loss of p27 with only a partial restoration of phosphorylation
of retinoblastoma protein (Rb) and Cdk4 activity but with a dramatic
elevation of Cdk2 activity. Our data provide in vivo genetic validation
of cell culture experiments that indicated that p27 acts as a negative
regulator of cyclin E-Cdk2 activity and that it can be titrated away by
cyclin D-Cdk4 complexes. It also supports the suggestion that the
cyclin E/Cdk2 pathway can largely bypass Rb in regulating the cell
cycle in vivo.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ mice exhibit multiorgan hyperplasia (5,
9, 15). The corresponding gigantism and dwarfism of the two null
mutant mice support the biochemical data that cyclin D1 and p27 are
counteracting forces acting in the same pathway.
/ mice exhibit decreased
cell numbers in all cell layers (4, 21) owing to both a
decreased rate of cell proliferation and increased apoptosis
(13). In contrast, p27Kip1 -deficient retinas
appear to have normal size. However, they show a marked disorganization
of the cellular pattern, although in a partially penetrant fashion
(15; A. Koff, personal communication), with patches of the
outer nuclear layer invading the rod-and-cone (RC) layer until they
reach the pigment epithelium.
/ mice is due to abnormal
estrous cycles, a poor ovulation rate, and a failure to produce
nidatory estrogen required for embryo implantation (23).
In contrast, cyclin D1 null mutant mice appear normally
fertile although they are unable to nurse their litters due to a
failure of lobuloalveolar development of their mammary glands during
pregnancy (22). The ovarian defect in p27/
mice is correlated with prolonged cell proliferation of corpus luteum
(CL) cells compared to those of wild-type mice. Cyclin D2 is expressed
in the proliferating granulosa cells, but this expression is lost when
the granulosa cells differentiate into CL cells (23).
However a low level of cyclin D1 could be detected in luteal cells
(data not shown) (A. Koff, personal communication) in the first 48 h after ovulation (7), suggesting that in the absence of
p27 this cyclin, together with cyclin D3, might support prolonged cell proliferation.
/ females and also almost completely
rescued the disorganization of the retinal structures, while loss of
p27 restored the cellularity of the cyclin
D1/ retinas. These studies indicate that in many
but not all tissues mutations in cyclin D1 and p27 are mutually
suppressing and suggest that the removal of one gene product allows the
unencumbered activation of the other gene product.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ cyclin D1/
mice.
The p27/ and cyclin
D1/ mice were kind gifts from Andrew Koff
(Memorial Sloan-Kettering Cancer Center, New York, N.Y.) and Piotr Sicinski (Dana-Farber Cancer Institute, Boston, Mass.), respectively. Both of the mice are 129sv backcrossed to C57BL/6 and randomly bred in
a closed colony. The mice deficient for both p27 and
cyclin D1 genes were produced by intercrossing the
p27+/ and cyclin D1+/ mice. Genotyping of
the mice was performed as described previously (9, 21).
70°C in 100 µl of 10 mM HEPES-0.1% (vol/vol)
Tween 20 buffer (24). Retinal lysates were made either in
Tween 20 buffer (50 mM HEPES-KOH [pH 7.5], 0.15 M NaCl, 1 mM EDTA,
2.5 mM EGTA, 0.1% [vol/vol] Tween 20, 10% [vol/vol] glycerol, 10 mM 
-glycerophosphate, 0.1 mM Na3VO4, 1 mM
NaF, 0.2 mM PMSF, and 10 µg of aprotinin, leupeptin, and pepstain
A/ml) or in NP-40 buffer (50 mM Tris-HC1 [pH 7.4], 0.25 M NaCl, 5 mM
EDTA, 0.5% [vol/vol] NP-40, 50 mM NaF, and proteinase inhibitors as
described above). Proteins were extracted from the retinas by
sonication. Protein concentration was measured using the Bradford
reagent (Bio-Rad). Equal amounts of proteins were used for all the
biochemical studies as described before (24). Briefly,
cyclin E-associated kinase activity was measured in immunoprecipitates in NP-40 buffer using histone H1 (Boehringer Mannheim) as a substrate, while Cdk4- and Cdk6-associated kinase activity was measured in immunoprecipitates in Tween 20 buffer using p56Rb (amino
acids 379 to 928; QED Bioscience, San Diego, Calif.) as a substrate.
One hundred to 200 µg of protein was used for the kinase assays.
Antibodies against cyclin E (M-20), cyclin A (C-19), Cdk2 (M-2), Cdk4
(C-22), Cdk6 (C-21), p107 (C-18), and p27 (C-19) were obtained from
Santa Cruz Biotechnology. Anti-Rb (G3-245) was obtained from
Pharmingen, anti-cyclin D1 (DCS-6 and Ab-3) and D3 (DCS-22) were
obtained from Neomarkers, and anti--tubulin was obtained from Sigma.
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ mice are smaller,
while p27/ mice are larger, than their
wild-type littermates (4, 5, 9, 15, 21). In mice
nullizygous for both cyclin D1 and p27 genes,
these phenotypes are suppressed and the mice displayed a normal adult
body weight. In fact, a single wild-type allele of p27
(p27+/ cyclin D1/)
can partially rescue the growth of cyclin
D1/ mice (Fig. 1).
All experiments reported in this study were carried out in a randomly
bred closed colony to minimize the possibility that a segregating gene
in the mixed 129/Bl6 background could explain the modification of a
phenotype. This conclusion of the lack of a modifying gene affecting
the phenotype is strongly reenforced by the heterozygous effect of the
loss of a single allele of p27, giving an
intermediate-growth phenotype. Given this breeding strategy, it can be
concluded that loss of p27 suppresses the growth deficiency caused by
loss of cyclin D1 and that the absence of cyclin D1 suppresses the
gigantism of p27/ mice. However, the growth
rates were not exactly the same as those of the wild-type mice (Fig.
1). Initially, the p27/ cyclin
D1/ mice weighed less than the wild-type mice, but
they caught up with them by puberty (females) or afterwards (males). In
fact, female mice ended up having a significantly elevated body weight compared to wild-type mice (Student t test; P < 0.005) although their weight was lower than that of the
p27/ mice (Student t test;
P < 0.01) (Fig. 1B). In contrast double-null males
were not significantly larger than the wild-type mice by 9 weeks
(Student t test; P > 0.05).
View larger version (22K):
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FIG. 1.
Loss of p27 and cyclin D1 suppresses the gigantism and
the dwarfism of cyclin D1 / and
p27/ mice, respectively. Body weights of
wild-type (WT; n = 24 for males, n = 31
for females), p27/ (n = 24
for males, n = 17 for females), cyclin
D1/ (D1/; n = 9
for males, n = 8 for females),
p27+/ cyclin D1/
(n = 16 for males, n = 21 for females),
and p27/ cyclin
D1/ (n = 19 for males,
n = 11 for females) were measured weekly beginning 2 weeks after the mice were born. Values are means ± standard
errors of the means. (A) Male growth curve; (B) female growth curve.
*, WT males are not significantly different from p27/
cyclin D1/ males in weight at 9 weeks of age; #,
p27/ cyclin D1/ females are
significantly heavier than WT females (Student t test;
P < 0.005) while significantly lighter than
p27/ females (Student t test; P < 0.0001) at 9 weeks of age.
/ pups that were fostered onto surrogate
mothers. By this method, we could exclude the possibility that the poor
viability of the cyclin D1/ mice was simply
due to competition for nutrition from their heterozygous littermates
during postnatal development. Of the 11 pups produced in these litters,
despite the fact that they had higher body weights than the usual
cyclin D1 homozygous null mutant mice of similar age, 3 died within 2 months and 3 more died between 2 and 4 months. This suggests that the
increased mortality in cyclin D1/ mice was
not solely due to the decreased body size. In contrast, of more than 30 p27/ cyclin D1/
mice, none exhibited premature mortality and all lived through to the
end of the study (5 to 12 months) in a fashion similar to that of
p27/ mice. The early lethality of cyclin D1
null mutant mice was therefore corrected by the loss of p27.
Cyclin D1/ mice show behavior that strongly suggests a
neurological abnormality. They display a leg-clasping reflex by
retracting their limbs toward their trunks when lifted by their tails,
and the male mice are very aggressive and fight with each other even when caged with their littermates (4, 21). The p27 and
cyclin D1 double-null mutant mice still exhibit the leg-clasping
defect, but the males have normal wild-type aggressiveness. This shows a partial correction of the neurological defects of cyclin D1 null
mutant mice. The p27 null mice also show pituitary hyperplasia and
adenoma. In studies of five double-mutant mice older than 1 year two
had pituitary adenomas. Although with this small cohort subtle
differences in frequency between mice having p27-deficient genotypes could not be determined, since none of the wild-type mice of
comparable age had tumors, it can nevertheless be concluded that the
pituitary dysplasia of p27/ mice was not
rescued in the double-mutant mice.
Partial rescue of the female fertility of p27/
cyclin D1/ mice.
Female mice homozygous for the
p27 null mutation have a very extended and intermittent
estrous cycle with a diestrus-like delay (9). This was not
corrected by introduction of the cyclin D1 null mutation, and the
double-null mutant mice had prolonged intermittent cycles. These data
suggest that the hypothalamic-pituitary-ovarian axis remained defective
in the double-nullizygous mice. However, both genotypes respond to
superovulation regimens. Therefore, we used this method to obtain
enough mice to analyze implantation. Implantation was scored either by
the appearance of implantation sites if the pregnancy was carried for
over 6 days or by the Pontamine sky-blue dye method (17)
if the pregnancy was between 5 and 6 days. cyclin D1 null
females have normal fertility; therefore this study excluded analysis
of this genotype. In accord with our previous observation
(23), among 12 p27/
cyclin D1+/+ females tested, only 1 (8%) showed
any embryo implantation, with approximately 20 implantation sites being
detected in this mouse. These sites appeared to be smaller than the
corresponding wild-type implantation sites (data not shown). This
implantation failure could be rescued as shown previously
(23). When 10 ng of 17-estradiol was given to serve as
a source of nidatory estrogen at day 3.5 of pregnancy to the
p27/ mice just before implantation, 88%
(seven out of eight) of mice showed many implantation sites (9 to 30 implants per mouse). However, in the p27/
cyclin D1/ females there was a significant
improvement (P < 0.05) in the rate of implantation
even without this E2 supplement, with 44% (8 out of 18) of
the mice displaying implantation sites. This was not significantly
different (P = 0.084 by Fisher's exact test) from the
enhancement in implantation rate for p27/
mice due to treatment with nidatory estrogen.
/ mice was rescued in the
p27/ cyclin D1/
mice. We performed superovulation on the mice of various genotypes and
mated them with wild-type males. Three days after ovulation, we
injected BrdU intraperitoneally 3 h before sacrifice.
Immunohistochemistry was performed using anti-BrdU antibodies on ovary
sections to determine the cohort of cells undergoing DNA synthesis. We
found that the wild-type ovaries and ovaries from
p27+/ cyclin D1/
mice display mature CLs with few BrdU-positive cells, while
p27-deficient and p27- and cyclin D1-deficient ovaries showed a
remarkable increase in the BrdU-positive cells in the CL (Fig.
2A). Six days after ovulation, ovaries
from all genotypes, including the wild type, p27/, and p27/
cyclin D1/, exhibited very few cells
positive for BrdU (Fig. 2B). These data show that the delayed exit from
the cell cycle in the CLs of p27/ mice was
not rescued in the double-nullizygous females even though the estrogen
production is corrected.
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The retinal phenotype of p27/
cyclin D1/ mice.
It has been
previously reported that cyclin D1/ mice
display a dramatic reduction in cell numbers in all layers of the
neural retina (4, 21). This is due to decreased cell
proliferation and increased apoptosis during retinal development
(13). It also has been shown that p27-deficient retinas
exhibit dysplasia in the photoreceptor layer (15). Because
of this and the ability to explore the biochemical mechanism in this
tissue, we investigated retinal development in p27 cyclin D1
double-nullizygous mice.
/ mice had retinas of normal thickness.
However, 60% of these mice (12 out of 20) also showed protrusions of
the photoreceptor cell layer into the RC layer, among which 8 showed
severe dysplasia (Fig. 3b), as well as some disorganization of the
pigment epithelium and RC layers. All cyclin D1-deficient retinas
displayed hypocellularity and a disorganized development of all retinal
layers, with scattered acellular areas ("holes") in the
photoreceptor layer, as had been reported before (Fig. 3c)
(15). In contrast, the retinas from mice deficient for
both cyclin D1 and p27 genes generally showed normal retinal development and cellular organization (Fig. 3e) and were
similar in structure to the wild-type retinas (Fig. 3a). Some (21%; 9 out of 43) of these mice still possessed a disorganization of the
photoreceptor cells similar to that of p27/
mice, although only 3 showed a severe defect. The general severity was
much reduced compared to that observed in
p27/ retinas, with thin threads of cells
being found in the RC layer but without the columns of cells
characteristic of p27-deficient retinas (Fig. 3f). Therefore, the loss
of p27 can rescue the hypoplastic phenotype of cyclin D1-deficient
retinas, and loss of cyclin D1 can restore an organized structure to
p27-deficient retinas (significantly different from the single-null
mutants, respectively, by Fisher's exact test; P < 0.005). Retinas from the p27+/
cyclin D1/ mice showed an appearance similar
to that of those from the cyclin D1/ mice (Fig. 3d).
|
/ retinas display a
much higher level of apoptosis in the photoreceptor layer, with a peak
of cell death occurring between the second and the fourth postnatal
weeks, which is not observed in the wild-type mice. This photoreceptor
cell death exhibits a unique pattern: the death is first observed in
scattered clusters and then expands to the neighboring cells,
eventually forming extensive holes (13). We therefore
studied cell death in retinas derived from mice of the various
genotypes. We isolated the neural retinas from adult mice free of the
pigmented epithelial cell layer and observed the backs of the retinas
by dark-field microscopy. The wild-type retinas showed smooth
photoreceptor layers (Fig. 4Aa), as did the retinas from p27/ mice (Fig. 4Ab). In contrast,
cyclin D1-deficient retinas displayed numerous holes on the
photoreceptor side (Fig. 4Ac) as previously described
(13). However, retinas from
p27/ cyclin D1/
mice had a normal appearance (Fig. 4Ad). Retinas from
p27+/ cyclin D1/
mice appear similar to cyclin D1-deficient retinas (data not shown).
The holes in the cyclin D1-deficient mice can also be observed by
examination of cross sections of the whole eyes, as can their
correction by the loss of p27 (Fig. 3).
|
/ mice are due to
apoptosis in the photoreceptor layers and can be examined by TUNEL
staining. In contrast to wild-type mice, where there was little
apoptosis (Fig. 3Ba), intense TUNEL-positive signals were observed in
the cyclin D1/ retinas between 1 and 3 weeks
postnatally, when the holes were expanding three dimensionally. The
TUNEL-stained cells showed a ring pattern either at the edge of the
expanding holes (Fig. 4Bc), (13) or within the holes (data
not shown). The p27+/ cyclin
D1/ retinas exhibited a similar apoptotic
phenotype (data not shown). Interestingly the p27-deficient retinas
also showed positive signals for TUNEL staining in the photoreceptor
layers but in a different pattern from that for retinas from cyclin
D1-deficient mice. They displayed many apoptotic cells dispersed
throughout the retinas (Fig. 4Bb). Dark-field images of the same field
indicate that the apoptotic cells are located within the protruding
patches of cells (data not shown). Notably, most retinas from
p27/ cyclin D1/ mice showed no positive
cells by TUNEL staining (Fig. 4Bd) and appeared to be similar to those
of wild-type mice (Fig. 4Ba). A few also exhibited scattered apoptotic
cells, but this condition was much less severe than that found in the
p27/ retinas (data not shown).
Molecular mechanism of the rescue of the cyclin D1/
phenotype in the retina.
The rescue of the overall growth and the
retinal phenotypes in the double-null mutant mice suggests the opposing
actions of the two molecules in cell proliferation throughout
development. However, surprisingly, MEFs isolated from cyclin D1 null
mice grew normally under standard culture conditions (4,
16). This was also the case with the MEFs that we isolated. In
one report (1) proliferation was slower when the cells
were seeded at low density, although this was not found by us or others
(4, 16). These data suggest that there might be variations
in MEF isolates and that growth abnormalities are not a general
property of these cyclin D1 null cells. However, since there was a
profound effect on the retinal phenotypes and since this tissue is
suitable for biochemical analysis, we examined the tissue to establish mechanisms for the reciprocal correction of phenotypes. To investigate this, we isolated the neural retinas from day 1 postpartum mice for
biochemical analysis.
/ mice exhibited comparable ratios (1.3:1
to 1.4:1) of hyperphosphorylated Rb (ppRb) to hypophosphorylated Rb
(pRb). In contrast, cyclin D1-deficient retinal extracts showed only a
very low ratio (0.06:1) of ppRb to pRb and the Rb protein level was
significantly reduced. In extracts from p27/ cyclin
D1/ retinas there was a slight but significant increase
in this ratio (0.3:1) and in protein level (Fig.
5A). These data were confirmed by use of
an antibody that recognizes specifically the Cdk4-specific phosphorylation at serine 780 of Rb. Both wild-type and
p27/ mice showed abundant levels of phosphorylation on
both the upper and lower Rb bands corresponding to ppRb and pRB,
respectively (Fig. 5A). Cyclin D1 null retinas had very little
phosphorylation of Ser-780 and only of the lower pRb band, while the
double-null mutant, although somewhat variable from mouse to mouse
(three mice showing the extremes are illustrated in Fig. 5A), had
increased Ser-780 phosphorylation compared to the cyclin
D1/ retina level, but this was also largely restricted
to the lower band. Nevertheless, this was still very significantly
reduced from wild-type levels (Fig. 5A). These data showed that loss of p27 alone did not cause overphosphorylation of Rb in the
p27/ retinas, consistent with the normal
thickness of the retinas, while removal of p27 from the retinas of
cyclin D1/ mice rescued the cell cycle progression with
increased phosphorylation of Rb, but only to ~20% of the wild-type
level. A similar result was found for p107, another member of the Rb
family of proteins. It was strongly expressed in the wild-type and
p27/ retinal cells but was expressed at very low levels
in cyclin D1/ retinal lysates. In the double-null
mutant there was a small but significant increase in p107 levels over
that of the cyclin D1/ retina but not to wild-type or
p27/ levels (Fig. 5B).
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-tubulin
antibodies. To avoid repetition, representative Western blots for
-tubulin of two lysates per genotype are shown in Fig. 5B and
demonstrate that the changes in kinase activities (Fig. 5C) observed in
the same lysates were largely due to changes in the specific activities
of the kinases.
Immunoblotting with anti-cyclin D1 and anti-p27 antibodies confirmed
the genotyping of the mice since bands corresponding to cyclin D1 were
only detected in wild-type and p27/ mice (Fig. 5B),
while p27 could not be detected in any of the p27 null mutant mice.
Cyclin D2 was at or below the level of detection of Western blotting in
this tissue in mice of all genotypes (data not shown). In contrast,
cyclin D3 was detected in all retinal lysates, with an elevated level
found only in cyclin D1 null retinas; this level returned to the
wild-type level following removal of the p27 gene (Fig. 5B). Cdk4
levels were reduced in the retinas from both cyclin
D1/ and p27/
cyclin D1/ mice but were similar in
wild-type and p27/ retinal lysates (Fig.
5B). However, Cdk6 protein levels showed a reciprocal response to Cdk4,
with elevated concentrations observed in the absence of cyclin D1 (Fig.
5B). Cdk4-associated kinase activities were dramatically reduced in the
cyclin D1-deficient retinas compared to those in wild-type retinas
(Fig. 5C). The retinal extracts from the double-knockout mice also
displayed very low Cdk4 activities, but these were consistently higher
than those detected in cyclin D1/ retinas
(Fig. 5C). Interestingly, the p27-deficient retinas exhibited significantly higher Cdk4 activities than wild-type retinas. This result was consistently found in six independent lysates made from
p27-deficient retinas (Fig. 5C). In contrast, no difference in Cdk6
kinase activity was observed in retinas with any of these genotypes,
even though the protein concentration was higher in retinas lacking
cyclin D1.
Cyclin E-Cdk2-associated kinase activities were significantly increased
in the retinas from p27/ cyclin
D1/ mice. This elevation of cyclin E-Cdk2
activities was comparable to that observed in p27-deficient retinas.
The activity of cyclin E-Cdk2 in the cyclin D1-deficient retinas was
very low compared to that of the wild type (Fig. 5C). Cyclin E levels
were similar in the retinas from mice with all genotypes, indicating
that these effects were not due to altered cyclin E levels (Fig. 5B). A
similar pattern of results was obtained when Cdk2 activities were
directly measured (Fig. 5C). The total protein levels of Cdk2 and the
activated form of Cdk2 in cyclin D1-deficient retinas, determined from
the faster-migrating threonine 160 phosphorylated form, are markedly reduced compared to those from mice with other genotypes (Fig. 5D),
consistent with the low Cdk2 activity. In contrast, wild-type, p27/, and p27/ cyclin
D1/ retinas showed similar amounts and phosphorylation
status of Cdk2 (Fig. 5D). Consistent with this pattern of Cdk2
activity, its other partner, cyclin A, which is also considered a
marker of cell proliferation, was significantly reduced in the retinas of cyclin D1 null mice but was markedly restored in the double-null mutant retinas (Fig. 5B).
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Cyclin D-Cdk4 and -Cdk6 and cyclin E-Cdk2 and cyclin A-Cdk2
complexes sequentially and differentially phosphorylate and inactivate Rb. Cyclin D-Cdk4 and -Cdk6 also serve as a "sink" to titrate out
the Cip/Kip class of Cdk inhibitors, so that cyclin E-Cdk2 complexes
are further activated. Cyclin E-Cdk2 and cyclin A-Cdk2 ensure the
maintenance of full phosphorylation and inactivation of Rb, thereby
inducing E2F downstream targets and relieving Rb repression of target
genes through the release of histone deacetylase (8, 19).
A low concentration of p27 can also facilitate the assembly and nuclear
translocation of cyclin D1-Cdk4 complexes (2). To explore
the interactions between cyclin D1 and p27 in vivo, we generated cyclin
D1 p27 double-null mutant mice. Mice nullizygous for both cyclin
D1 and p27 genes display a reciprocal rescue of
phenotypes in many but not all cases. The double-knockout mice showed
normal body weight and mortality, and the males were less aggressive.
The retinas of p27 cyclin D1 null mutant mice exhibited
normal thickness and were devoid of extensive apoptosis in the
photoreceptor layer. Loss of cyclin D1 can also rescue the hyperplastic
retinal phenotype and the implantation defect in
p27/ mice. These data strongly suggest
interactions between cyclin D1 and p27 in some tissues. However, the
failure to rescue all phenotypes, especially those associated with p27
deficiency, such as the failure of CL cells to appropriately exit the
cell cycle, the estrus cyclicity, the incidence of pituitary adenoma,
and the leg-clasping reflex, suggests independent actions of these molecules in other tissues.
Unfortunately, the lack of a detailed understanding of the underlying
basis of many of the defects at the present time precludes the analysis
of these independent actions. However, it is known that embryo
implantation in mice is triggered by a burst of estrogen production
(nidatory estrogen) synthesized by the CL just before implantation and
that in p27/ females the implantation defect
could be rescued by a single injection of E2 administered
to mimic this nidatory estrogen (23). Since the
implantation defect of p27/ mice could be
transferred to wild-type mice by ovary transplantation, the failure to
produce E2 was organ autonomous. Analysis of CL cell
proliferation in p27/ mice showed that these
cells failed to exit from the cell cycle appropriately, suggesting that
this might interfere with their differentiation and the ability to
synthesize nidatory E2 on cue (23). Removal of
cyclin D1 corrected the implantation defect of
p27/ mice but, surprisingly, not the
inability of the CL cells to exit the cell cycle appropriately. Recent
studies have indicated that cyclin D1 is expressed in granulosa cells
but is down-regulated upon the luteal transition and is virtually
absent by 72 h postovulation, while cyclin D3 is up-regulated in
the cells (7). This suggests that cyclin D3 is important
in maintaining the proliferation of these cells in the absence of p27
but that cyclin D1 plays a novel role in regulating the capacity of
luteal cells to synthesize nidatory estrogen.
In cyclin D1 p27 double-mutant mice, even though the implantation
defect was restored, pregnancies still could not be carried to term and
appeared to terminate around midgestation with embryos that had
undergone turning and had well-formed placentae. This revealed another
previously unappreciated function for p27 later in pregnancy since
cyclin D1-deficient mice can carry litters to term. This could be due
to the dysregulation of ovarian hormone production or a defect in
uteroplacental function after implantation. The possibility of a defect
in the hypothalamic-pituitary-ovarian axis is consistent with the fact
that estrous cyclicity was not restored in the
p27/ cyclin D1/
mice. Cyclin D1/ mice also display a failure
of lobuloalveolar development in mammary gland development during
pregnancy. However, analysis of mammary gland development in
p27/ and the double-null mice during
pregnancy was not possible due to the failure of these mice to carry
pregnancies to term. The mammary gland phenotype in response to
exogenous hormones in ovariectomized mice or following transplantation
needs to be analyzed to determine the interaction of p27 and cyclin D1
in this tissue.
Ablation of p27 in cyclin D1/ mice rescued
the hypocellularity of the cyclin D1/
retinas. In the cyclin D1-deficient retinas cell death occurs in
clusters of cells resulting in holes in the photoreceptor layers. This
apoptosis was also inhibited in the double-nullizygous mice. The rescue
of the hypocellularity in the cyclin D1/
retinas by loss of p27 led us to determine the molecular basis of this
rescue. In both the cyclin D1-deficient and p27- and cyclin D1-deficient retinas, Cdk4 levels are significantly reduced, suggesting that Cdk4 is unstable without its partner, cyclin D1. In the cyclin D1-deficient retinas, the reduced activities of both Cdk4 and cyclin
E-Cdk2 explain the hypophosphorylation of Rb and the reduced cell
proliferation. Cdk4 activity in the double-null retinas is low but
consistently higher than that in the cyclin D1/
retinas. Cyclin D2 is not up-regulated in cyclin D1-deficient retinas
(6), but cyclin D3 is expressed and is even up-regulated in cyclin D1-deficient retinas. This elevation of cyclin D3 expression in the cyclin D1/ retinas might account for the
residual Cdk4 activity and the presence of a retinal structure in these
mice. It also suggests that Cdk4, presumably in complex with cyclin D3
and alleviated from p27 repression, in the double-null retinas leads to
partial correction of Rb phosphorylation, which may contribute to
rescue of retinal cellularity. An interesting phenomenon observed in these studies that was not entirely explainable with current models was
that we saw no change in Cdk6 activity among retinas with different
genotypes. This suggests that Cdk6 is not a major kinase phosphorylating Rb in the retina. The data also show cyclinD-Cdk4 and
-Cdk6 activity even in the absence of p27. Since p21 was below the
level of detection in the retinal lysates (data not shown), this
indicates that the Cip/Kip inhibitors are not required for these cyclin
D-Cdk4 and -Cdk6 complexes to form and be functional in vivo as was
found by Cheng et al. (2) in cultured cells.
The relatively small increase in pRb phosphorylation in the double-null
mutant might facilitate G1-to-S progression. Thus, to
analyze downstream targets of Rb, we examined the expression of cyclin
A and E. The cyclin A level is very low in the cyclin D1 null retinas,
consistent with the hypocellularity of this tissue. But cyclin A
expression is markedly rescued in the double-null mutant, a result that
is in line with the recovered cellularity of this tissue. This rescue
of cyclin A expression and of cellularity in the absence of full Rb
phosphorylation suggests that loss of p27 may activate another
mechanism that obviates the need for cyclin D1 and the full
inactivation of Rb by phosphorylation. Indeed in these mice the role of
cyclin D1-Cdk4 or -Cdk6 in redistributing p27 from cyclin E-Cdk2 to its
own complex is no longer needed due to loss of p27. Consistent with
this was the markedly increased activity of cyclin E-Cdk2 complexes in
the p27/ cyclin D1/ retinal extracts,
up to a level that was comparable to that observed in
p27/ extracts. However, this elevated activity does not
result in dramatically increased Rb phosphorylation. This may be
because of the failure of the prerequisite Rb phosphorylations
performed by cyclin D-Cdk4 (12).
The above data suggest that in the p27- and cyclin D1-deficient retinas
cyclin E-Cdk2 bypasses the requirement of full Rb inactivation and
directly promotes cell cycle progression. Although the possibility that
the small increase in pRb phosphorylation in the double-null mutant is
sufficient to release enough E2F to transactivate the cyclin A gene and
stimulate cell proliferation cannot be discounted, it seems more likely
that cyclin E-Cdk2 acts independently to perform these tasks through
another mechanism. This is consistent with the studies of Lukas et al.
(11), who showed that cell proliferation could be rescued
by overexpression of cyclin E in the presence of a form of Rb incapable
of being phosphorylated and without activation of E2F. It is also in
line with the results from Geng et al. (6), who showed
that the retinal defect in cyclin D1/ mice
can be rescued by replacing the mouse cyclin D1 locus with human cyclin
E without fully phosphorylating Rb (6). Furthermore, it
has recently been shown that cyclin E-Cdk2 phosphorylates p220(NPAT) and that this stimulates histone gene transcription, giving a direct
link between cyclin E-Cdk2 and essential events in S phase (14,
29).
In summary, our biochemical studies of the neonatal retinas revealed that loss of p27 rescued the hypocellularity of the cyclin D1-deficient retinas in the presence of only a 20% restoration of Rb phosphorylation but with a dramatically increased activation of cyclin E-Cdk2. These whole-mouse experiments support the biochemical data derived from cell culture experiments of the titration of p27 away from cyclin E-Cdk2 complexes by cyclin D1-Cdk4 and -Cdk6 complexes and analysis of Cdk4 null mutant MEFs in the presence and absence of p27 (25). The mutual suppression of many but not all phenotypes of the individual null mutant mice also indicated that p27 and cyclin D1 function to antagonize each other genetically and biochemically. This is also the first genetic demonstration in vivo of interactions between a cell cycle inhibitor and an activator, p27 and cyclin D1, respectively.
| |
ACKNOWLEDGMENTS |
|---|
We thank Andy Koff and Martine Roussel for their critical comments on the manuscript and Liang Zhu for helpful discussions. We thank A. Koff and P. Sicinski for generously providing the mice for breeding, Liyin Zhu and Bo Chen for the histological preparations of the mouse pituitaries, and James Lee for genotyping the mice.
J.W.P. is a Betty and Sheldon Feinberg senior scholar in cancer research. This work was supported in part by an NCI grant to the Albert Einstein Cancer Center, P30-13330.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Center for the Study of Reproductive Biology and Women's Health, Departments of Developmental and Molecular Biology and Obstetrics and Gynecology and Women's Health, Albert Einstein College of Medicine, 1300 Morris Park Ave., New York, NY 10461. Phone: (718) 430-2090. Fax: (718) 430-8972. E-mail: pollard{at}aecom.yu.edu.
Present address: Whitehead Institute, Massachusetts Institute of
Technology, Cambridge, MA 02142.
| |
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Materials and Methods
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Discussion
References
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Molecular and Cellular Biology, February 2001, p. 1319-1328, Vol. 21, No. 4
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.4.1319-1328.2001
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