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Institute of General Pathology and Oncology, Faculty of Medicine and Surgery, Second University of Naples, Naples, Italy
Address all correspondence and requests for reprints to: Alessandro Weisz, Istituto di Patologia Generale e Oncologia, Facolta’ di Medicina e Chirurgia, Seconda Universita’ di Napoli, Larghetto S. Aniello a Caponapoli, 2, I-80138 Naples, Italy. E-mail: a.weisz{at}area.ba.cnr.it
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The best known regulators of G1 progression in mammalian
cells are represented by the three D-type cyclins (D1-3), whose
concentrations in the cell fluctuate characteristically during
progression through G1 in response to mitogenic stimuli
(10, 11, 12). They act predominantly by associating with the catalytic
subunits of specific cyclin-dependent kinases (cdks), namely
cdk 2, 4, 5, and 6, and show considerable structural and
functional homologies with each other. The significance of this
redundancy is not clear at present, although it is conceivable that in
certain instances the cdks can complement each other
functionally (10, 11). Assembly of D-type cyclins into holoenzymes with
p34cdk 4 and p38cdk 6 is
particularly important for these enzymes to function as positive
regulators of G1 progression, whereas cyclin
D-p32cdk 2 complexes are generally found to be
inactive during G1, with cdk 2 required
later, during S phase progression and S-G2 transition, in
association with cyclins A and E (13). Also, the functional role of the
cyclin D-p31cdk 5 complexes that can be detected
in vitro is not fully understood at present, as a clear role
for cdk 5 has been identified to date only in neurons, where
it associates with a p35 regulatory subunit and phosphorylates
neurophilament and Alzheimer’s 
proteins (14, 15). Association of
cyclins with the catalytic subunits of cdks is not
sufficient to endow the resulting holoenzymes with protein kinase
activity, as cdk-activating kinases (CAKs) are essential at
this stage (16, 17, 18, 19). The nature of CAKs is less defined, but they also
are likely to be targets for regulation by extracellular stimuli and to
include catalytic as well as regulatory subunits (20). Furthermore,
negative regulators of the cdk holoenzymes (cdk
inhibitors: ckis) have been identified, adding a further
degree of complexity to the regulatory pathways converging on these
enzymes (11, 12, 21). ckis exert multiple regulatory
functions, including assembly and stabilization of
cyclin-cdk complexes and modulation of substrate specificity
of cdk holoenzymes (10, 11, 13, 21). Finally, cyclins and
cdks, apart from their essential role as cell cycle
regulators, are involved in other key processes in eukaryotic cells,
including meiosis, differentiation, phosphate metabolism, apoptosis,
and transcription initiation (13, 22, 23). Indeed, an essential role
for p27Kip1 in uterine physiology was recently found, as
female mice carrying targeted disruption of the p27Kip1
gene show impaired uterine functions (24, 25, 26), including sterility,
ascribed in part to a block of development of implanted embryos due to
a defect intrinsic to the uterus (26).
We investigated and report here the effects of estrogen on cdk activity, cyclin messenger RNA (mRNA) expression, and cki protein content in the adult rat uterus in vivo. Results show that 17ß-estradiol induces in this organ early activation of cdk 4 and 6, as well as cdk 5, while at the same time increasing in a timely and sequential fashion expression of D-type, E and A cyclin mRNAs and of cdk inhibitors p15Ink4b and p27Kip1. Cdk 2 activity was unaffected by estrogen, suggesting that either this enzyme is not hormone responsive in this case, or alternatively, that cdk 2-inactivating molecules are present in the extracts and interfere with cdk 2 enzyme assays in vitro. These results establish for the first time a functional link between cdk activity, cyclins, and ckis and estrogen actions in the mammalian uterus.
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Materials and Methods |
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Immunoprecipitation, immunoblotting, and kinase assays
For immunoprecipitation, duplicate aliquots of cytosol
corresponding to 2.5 mg protein extract were incubated with 2–4 µg
each of preimmune or specific Igs for 1 h at 4 C. Immunocomplexes
were adsorbed to 25 mg protein A-Sepharose for 1 h at 4 C, before
washing with lysis buffer. Four fifths of each sample was washed twice
in kinase buffer (50 mM HEPES, 10 mM
MgCl2, 10 mM dithiothreitol, and 0.1
mM ATP), resuspended in 25 µl reaction buffer containing
kinase buffer, 10 µCi [
-32P]ATP (3000 Ci/mmol;
Amersham, Arlington Heights, IL), and 2 µg histone H1 (Boehringer
Mannheim, Indianapolis, IN) or 0.3 µg purified GST-pRb [purified
from bacterial extracts as described previously (27)]. Kinase
reactions were carried out at 30 C for 30 min and blocked by the
addition of 25 µl 2 x SDS-electrophoresis sample buffer and
boiling for 2 min. Samples were then fractionated in 10%
polyacylamide, gels, fixed, stained, and autoradiographed; protein
bands corresponding to the substrate were excised and counted in a
liquid scintillation counter. Data reported represent the average of
results obtained in two or three separate experiments performed in
duplicate. For immunoblotting, one fifth of each immunoprecipitate was
resuspended in SDS sample buffer, denatured, and fractionated by
electrophoresis, proteins were then electrotransferred to Hybond-ECL
filters (Amersham), and the filters were processed for Western blotting
as described previously (27). For direct immunodetection, 30 µg total
protein extracts from rat uterus were analyzed. The following antisera
were used: 
-CDK2 (sc-163), -CDK4 (sc-260), -CDK5 (sc-173),
-CDK6 (sc-177), -p15 (sc-613), -p16 (sc-468 and sc-759), and
-p21 (sc-472); all, including the related competitor peptides, were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and were used
according to the manufacturer’s instructions. Furthermore, the
following antisera were also used: -CDK2, -CDK4, and -CDK6 [a
gift from A. Giordano (Thomas Jefferson University, Philadelphia, PA)
and C. J. Sherr (St. Jude Children’s Hospital, Memphis, TN)], -p16
(a gift from S. Dauvois, Imperial Cancer Research Fund, London, UK),
-p21 (a gift from W. Harper, Baylor College of Medicine, Houston,
TX), cod AB-1 from Oncogene Science, -p27 (a gift from H. Toyoshima
and T. Hunter, The Salk Institute, San Diego, CA), and
-p67ER [against the estrogen receptor (ER) isoform;
a gift from C. Abbondanza, Second University of Naples, Naples,
Italy].
Northern blot RNA analysis
Ten micrograms of uterine poly(A)+ RNA or 40 µg
total endometrial RNA were analyzed by Northern blotting as described
previously (6). The following complementary DNA (cDNA) probes were
used: mouse cyclin D1, D2, D3, cdk 4, cdk 5,
cdk 6, human cyclin E, cyclin A, -actin, rat 1A, hamster
ß-actin, and v-fos. Quantitative data (average of results
obtained in multiple independent experiments) are expressed as
densitometric units after normalization on the basis of 1A mRNA
concentration (estrogen unresponsive) in each lane. Unless otherwise
specified, the autoradiograms reported refer to filters used only once,
as rehybridization with a different probe often showed high background
and reduced intensity of the specific signals.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and indicate
that each antibody selected was indeed specific and recognized a
protein of the expected molecular mass (sizes, in kilodaltons, reported
in the figure), with the notable exception of the anti-cdk 2
antibodies, that recognized an additional protein of about 40 kDa, its
position marked by an arrow on the right side of
the relevant part of the figure. The nature of this protein is
presently under evaluation, but it is likely to represent one of the
cdk homologs that have been characterized in other cases
(11, 13). The additional band observable in the anti-cdk 4
immunoprecipitates challenged with the specific competing peptide
represent a high mol wt immunoreactive contaminant present in the
cdk4 peptide solution, as it could also be observed upon
direct Western blot analysis of this (data not shown). Furthermore, the
additional, low mol wt band that can be observed in the
anti-cdk 5 blots represents the light chain of the
antibodies used for immunoprecipitation, whereas that present in the
anti-cdk 6 blots was not detected in all experiments and
might represent a degradation product of the Ig heavy chain or a
contaminating species. The efficiency of the immunoprecipitation
procedure, controlled each time by comparing the relative amount of
each protein present in the crude and purified fractions, was
comparable in different experiments and was found to be relatively
constant; about 25–40% of the cdk proteins present in
crude extracts were recovered after this partial purification step
(data not shown). Only for cdk 4 was this value lower,
ranging from about 10–20%. This was not due to selective interaction
of only a subset of these molecules to the antibodies, but, instead, to
the loss of antibody-bound protein during washing of the agarose beads,
as assessed by Western blot analysis of residual anti-cdk 4
immunoreactive species in the supernatants obtained after
immunoprecipitation (data not shown). Three different
anti-cdk 4 antisera were tested, and all gave comparable
results, indicating that antibodies raised against human cdk
4 might bind the rat enzyme with relatively low affinity (data not
shown).
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). Results show significant hormonal
activation of cdk 4 (up to 3-fold stimulation between 10–25
h), cdk 5 (3.5- to 5-fold increase, reaching a maximum after
15 h of estrogen stimulation), and cdk 6 (2.5- to
3-fold stimulation, detectable by 2.5–5 h and remaining elevated
thereafter). Treatment of the animals with a suboptimal dosage of
estrogen (0.01 µg/100 g BW E2) or injection of
vehicle solution alone (10% ethanol-90% saline) were not accompanied
by significant changes in cdk enzyme activity in the uterus
after 2.5 or 15 h (data not shown). Addition of an excess of the
specific peptide competitor to the immunoprecipitation reactions
reduced phosphorylation of the substrates to background levels in each
case (data not shown). The increase in cdk 4 and 6
activities reflected for the most part activation of each holoenzyme,
as the uterine concentrations of the corresponding catalytic subunits
showed only minor and nonreproducible variations when analyzed in
separate experiments (see Western blots of Fig. 1B and data not shown).
On the other hand, estrogen increased the uterine concentration of the
catalytic subunit of cdk 5 during the first 2.5–5 h of
stimulation (Fig. 1B). Comparative analysis of the
p31cdk 5 concentration in the
immunoprecipitates, however, indicated that the increase in the
concentration of the catalytic subunit did not exceed 1.5- to 2-fold
(data not shown) and, for this reason, could not account in full for
the hormonal effects on enzyme activity reported in Fig. 1B. The H1
kinase activity of cdk 2, instead, was unaffected by the
hormone for up to 25 h (Fig. 1B). This result was confirmed using
two different anti-cdk 2 antisera raised against human and
mouse cdk 2 (data not shown). For possible explanations of
this result, see Discussion. When nonimmune rabbit serum was
used, only background phosphorylation of either histone H1 or pRb was
detected, which did not change significantly after treatment of the
animals with E2 (top panels in Fig. 1B).
Increased expression of cyclin mRNA in adult rat uterus and
endometrium after stimulation with E2
Cyclins are the best characterized regulatory partners of
cdks. Attempts to measure cyclin protein levels
in rat uterine extracts by immunoprecipitation and Western blotting
using various antisera showed a number of nonspecific abundant protein
bands that cross-reacted with these antibodies in vitro and
prevented correct assessment of the cyclin concentration in the samples
(data not shown), as described previously in immature rat uterus by
Lundeen and Gorski (28). For this reason, cyclin mRNA expression in
estrogen-stimulated rat uterus was investigated by Northern blot
analysis of poly(A)+ RNA and is shown in Fig. 2. D1 and D3 mRNAs were readily detectable in RNA
extracted from ovariectomized rat uterus, and their relative
concentrations increased progressively after estrogen stimulation, with
a 3-fold increase peaking at 8 h and a 2-fold increase between
8–20 h, respectively, compared to the concentration of control 1A mRNA
(29) in the same lanes. Basal D2 mRNA concentration, instead, was
relatively low, but underwent a transient 7-fold increase within the
first 2 h of stimulation and decreased thereafter. The labeled
mouse cyclin D2 cDNA probe hybridized weekly with cyclin D3 mRNA. The
two bands, however, migrated differently and could be easily
distinguished, as reported in Fig. 2 (left panel). The
cyclin E mRNA concentration, at the lower limit of detection in these
samples, increased only slightly and reached a maximum (
2-fold
increase) after 16–20 h of estrogen. It is worth mentioning that the
labeled human cyclin E cDNA probe used for the hybridization reactions
also recognized a larger RNA species (see upper band in the
autoradiogram obtained with the cyclin E probe and reported in the
left panel of Fig. 2), whose nature cannot be defined at
present. Finally, cyclin A mRNA was detectable only in
estrogen-stimulated uterus and showed considerable accumulation
(>9-fold increase) in whole uterine mRNA between 16–30 h. The
kinetics of accumulation of this RNA most likely mark the S phase in
cycling uterine cells. Indeed, this result corresponds to that obtained
by [3H]thymidine labeling of S phase cells in this organ
under comparable experimental conditions (30) (marked as S phase at the
top of the left panel in Fig. 2). For comparison,
the relative levels of other RNAs were determined under the same
conditions, including cytoskeletal ß-actin mRNA, estrogen responsive,
and induced during the delayed early uterine response to the hormone
(31) and cdk 4, 5, and 6 mRNAs (left panel in
Fig. 2). The steady state concentration of ß-actin mRNA starts to
increase significantly by 1–2 h, reaching a peak after 4 h,
indicating a good response of the uterine cells to hormonal stimulation
under these conditions, whereas all other RNAs were expressed at
comparable levels before or after hormone treatment compared to 1A mRNA
hybridization signals in the same blots, with the exception of
cdk 5 mRNA, which accumulated in estrogen-treated uteri
(compare 0 with 2, 12, 20, and 24 h in the lower
autoradiographs of the left panel of Fig. 2).
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-actin mRNA, which was undetectable in these endometrial RNA samples
(data not shown). As reported in Fig. 3, both D1 and D3
mRNAs could be detected and were found to be induced by the hormone in
the endometrium, with kinetics comparable to those observed in whole
uterine RNA (compare data reported in Fig. 3 with those in Fig. 2). D1
and D3 mRNA induction was preceded by early and transient accumulation
of the c-fos mRNA, encoded by a primary estrogen target gene
in growth-responsive cells (4, 6, 7, 30, 32) that is expressed only in
the endometrial epithelial cells of estrogen-stimulated adult rat
uterus (33). D2 mRNA, instead, was undetectable in these samples (data
not shown), indicating that either its levels were below the limits of
detection (compare results obtained with cyclin D1 and D3 in Figs. 2 and 3 and with c-fos in Fig. 3 and Refs. 6, 8, and 31) or,
alternatively, that accumulation of this mRNA in response to estrogen
did not occur in the endometrial cells analyzed here.
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and 2), expression of the best characterized
ckis, including p15Ink4b, 16Ink4a,
p21Cip1-Waf1-Sdi1-Cap20, and p27Kip1, was also
assessed here by Western blotting. As shown in Fig. 4, p15, almost undetectable in uterine extracts from ovariectomized rats,
accumulated and was clearly detected after 15–30 h of estrogen
stimulation (>10-fold induction, with a biphasic increase peaking at
15 and 25–30 h; upper blot in Fig. 4). More complex was the
molecular analysis of p16, as all of the different antibodies available
also cross-reacted with p15 and the concentration of p16 in extracts
from whole uterus was very low under all conditions tested. Preliminary
data indicate, however, that this inhibitor is also likely to
accumulate in estrogen-treated uteri (data not shown). It was not
possible to assess p21 levels in uterine extracts, as this protein was
undetectable under all experimental conditions tested. This was
confirmed using different anti-p21 antibodies, all capable of
recognizing rat p21 (data not shown). On the other hand, p27 was easily
detectable in uterine extracts, and its concentration was clearly
influenced by E2. Uterine levels of p27, in fact,
increased by 1–2.5 h of estrogen stimulation (3- to 5-fold), decreased
slightly thereafter, and increase again by 30 h (middle
blot in Fig. 4).
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isoform of the ER
(p67ER) was measured in the same extracts using a specific
mouse monoclonal antibody (AER317) (34). Contrary to p15, p16, or p27,
the p67ER content in the extracts decreased by about
40–50% after 10–30 h of treatment with E2
(lower blot in Fig. 4). This result is in agreement with
previous reports indicating estrogen-mediated down-regulation of the ER
mRNA concentration in rat uterus in vivo (35) and of ER mRNA
and protein levels in human breast cancer cells in culture (36, 37).
Furthermore, it validates the results obtained in the same samples with
the anti-ckis antibodies. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We monitored here the effects of E2 on
cdk activity in ovariectomized rat uterus, and the results
show that cdk 4, 5, and 6 are significantly activated within
the first few hours of hormonal stimulation. Based on the data reported
in Fig. 1B, it is possible that cdk 4, 5, and 6 are involved
in estrogen control of mid to late G1 progression in
growth-responsive uterine cells. Consistent with this possibility are
the kinetics of enzyme activation reported here and the known role of
these enzymes during G1 progression and G1 to S
transition. The best characterized activatory partners of
cdk 4, 5, and 6 are D-type cyclins (D1-3); after
accumulation in the cell during G1 in response to
extracellular stimuli, these proteins form stable complexes with the
catalytic subunits of cdks, and these, in turn, become the
target for phosphorylation by CAKs (10). Cyclin levels are increased by
mitogens via transcriptional activation of the corresponding genes in a
variety of cell types, although posttranscriptional regulation has also
been demonstrated. In view of the known effects of estrogen on cell
cycle regulatory gene expression in rat uterus (3) and on cyclin D1 and
cdk 4 activity in human breast cancer cells (27, 39), it is
possible to assume that one of the mechanisms that underlie
E2 stimulation of cdk activity in rat
uterine cells could reside in regulation of D-type cyclin levels. This
is supported by the finding that estrogen, while inducing activation of
cdks, increases the expression of all D-type G1
cyclin mRNAs in the uterus. In this respect, a notable difference was
found between D1 or D3 and D2, as only the latter did not appear to be
induced in luminal endometrial cells, suggesting that this cyclin may
not be involved in the mitogenic actions of estrogen in this uterine
compartment or, alternatively, that if indeed regulation by the hormone
occurs in the endometrium, this could directly target the protein. In
addition to their effects on cyclins, it is possible that estrogen
exerts a positive effect on CAK activity, thereby controlling multiple
components of the cdk cascades. Further studies will be
required, however, to determine precisely the expression of the
catalytic subunits of cdks and the related molecules within
the different cell types of the uterus, accompanied by a detailed
biochemical analysis of holoenzyme composition, activity, and
phosphorylation status before and after hormonal stimulation, after
tissue fractionation, or after primary culture of uterine cells. This
applies in particular to cdk 5, which is also regulated by
estrogen in the uterus, as this kinase is thought to play a specific
role in postmitotic cells. It will be informative to determine the
eventual presence and hormonal regulation of p35, the only known
specific activator of this enzyme and to date found only in neurons
(14, 15). When reliable synthetic inhibitors of cdk activity
become available, it will be possible to assess other functional roles
of cdk holoenzymes in the uterus by determining the
consequences of specific cdk blockades on different
physiological parameters in this organ. The data reported here open the
way for such studies and are particularly significant, as this is an
ideal model system to analyze cdk activities and regulation
in normal, nontransformed cells.
Contrary to the other cdks studied, cdk 2 activity was unaffected by E2. Although this could indicate the lack of response of this enzyme to estrogen, the possibility that uterine extracts include inhibitor or inactivating molecules, preventing a correct assessment of this enzyme activity in vitro, cannot be ruled out at present. Alternatively, cdk 2 activation could occur, but be restricted to a limited cell population, for example endometrial epithelial cells, and thus escape detection in extracts from the whole organ. In this respect, cdk 2 could differ from all other kinases analyzed here, and this would suggest multiple roles for cdk 4, 5, and 6 in the variegated uterine response to estrogen.
Analysis of the cki content in uterine extracts indicates that p15Ink4b, p27Kip1, and, to a lesser extent, 16Ink4a are all expressed in the uterus after estrogen stimulation. Apparently, this is in contrast with the effects of the hormone on cdk activity. However, it is possible to assume that the concentrations of these proteins change only in specific cell types, and this could help determine the specificity of the cellular responses to the mitogenic action of estrogen in the mature uterus. Alternatively, timed expression of ckis could mediate the differentiation of subsets of proliferating cells and/or balance, in time, the mitogenic action of estrogen. Finally, of particular interest is the rapid and significant effect of estrogen on the p27Kip1 concentration. This protein, in fact, has been recently shown to exert a key role in the control of uterine functions; p27-deficient female mice are sterile and show impaired ovarian functions, with prolonged estrous phases and an inability of ovarian granulosa cells to differentiate into luteal cells (25, 26). However, sterility is also consequent to impairment of a still undefined uterine cell function(s) that prevents the development of the implanted embryo (26). The data reported here, indicating estrogen control of p27 expression in the uterus, suggest that these hormones could exert a specific regulatory role on the functions controlled by this protein. Following the indications provided by this study, a detailed mapping of cki-expressing cell types before and after stimulation with hormones, including not only estrogen but also progesterone, will help to settle these important questions.
In conclusion, the data reported here establish a link among cdk, cyclins, cki activities, and estrogen actions in rodent uterus, offering a novel experimental approach to investigate the mechanisms that underlie the regulation of cdk activity in nontransformed cells and the roles of these enzymes and the related regulatory molecular partners in uterine physiology.
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Acknowledgments |
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Footnotes |
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Received August 19, 1996.
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