Cyclin D2 Compensates for the Loss of Cyclin D1 in Estrogen-Induced Mouse Uterine Epithelial Cell Proliferation
Bo Chen and
Jeffrey W. Pollard
Departments of Developmental and Molecular Biology and Obstetrics, Gynecology and Womens Health, Center for the Study of Reproductive Biology and Womens Health, Albert Einstein College of Medicine, New York, New York 10461
Address all correspondence and requests for reprints to:Jeffrey W. Pollard, Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, New York, New York 10461. E-mail: pollard{at}aecom.yu.edu; bochen{at}aecom.yu.edu.
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ABSTRACT
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The cell cycle-regulatory protein, cyclin D1, is the sensor that connects the intracellular cell cycle machinery to external signals. Given this central role in the control of cell proliferation, it was surprising that mice lacking the cyclin D1 gene were viable and fertile. Fertility requires 17ß-estradiol (E2)-induced uterine luminal epithelial cell proliferation. In these cells E2 causes the translocation of cyclin D1/cyclin-dependent kinase 4 (CDK4) from the cytoplasm into the nucleus with the consequent phosphorylation of the retinoblastoma protein. In cyclin D1 null mice, E2 also induces retinoblastoma protein phosphorylation and DNA synthesis in a normal manner. CDK4 activity was slightly reduced in the D1 null mice compared with wild-type mice. This CDK4 activity was due to complexes of cyclin D2/CDK4. Cyclin D2 was translocated into the nucleus in response to E2 in the cyclin D1-/- mice to a much greater degree than in wild-type mice. This cyclin D2/CDK4 complex was also able to bind p27kip1 in cyclin D1-/- uterine luminal epithelial cells, allowing for the activation of CDK2. Our data show that in vivo cyclin D2 can completely compensate for the loss of cyclin D1 and reinforces the conclusions that cyclin Ds are the central regulatory point in the proliferative responses of epithelial cells to estrogens.
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INTRODUCTION
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IN THE MAMMALIAN cell cycle, the major rate-limiting step is at the G1/S boundary and is often called the restriction point (R), a point after which progression to the next G1 phase becomes independent of extracellular growth factors (1). There is substantial evidence that the transition through this R point is controlled by the state of phosphorylation of the retinoblastoma (Rb) family of proteins, pRb, p107, and p130 (also referred to as pocket proteins). The complexes capable of phosphorylating RB proteins are heterodimers, consisting of cyclins and their catalytic partners, the cyclin-dependent kinases (CDKs) (2). The first of these to act in G1 are the D-type cyclins and their associated kinases, CDK4 and CDK6, and these kinases are at least partially responsible for the initiation of Rb phosphorylation (3, 4, 5, 6, 7). Inhibition of cyclin D1-regulated activity in tissue culture showed that this was an essential regulatory point in the cell cycle. The cyclin D-induced phosphorylation of Rb proteins prepares these proteins for subsequent phosphorylation by another kinase complex containing cyclin E and CDK2, the activity of which reaches a peak in late G1 (8, 9). Hyperphosphorylation of Rb results in the release and consequent activation of members of the E2F transcription factor family (10, 11, 12). This results in the up-regulation of cyclin A, another partner of CDK2, which is required for the maintenance of S phase (13).
CDK activities are negatively regulated by cyclin-dependent kinase inhibitors (CKI) (14, 15). These are categorized into two major families: INK4 and cyclin interacting protein/CDK inhibitory protein (CIP/KIP). The CIP/KIP family comprises three members: p21, p27, and p57 (16, 17, 18, 19). They have a broad range of specificities and can inhibit all of the G1/S CDKs through physical binding with the cyclin/CDK complexes. The other INK family includes p16, p18, p19, and p15 (20, 21, 22, 23, 24, 25), proteins that exclusively bind with CDK4 and CDK6 kinases, and prevents their association with cyclin D (26).
There are three different D-type cyclins, D1, D2, and D3, that act in the early response to growth factor stimulation (27, 28). The genes are located on different chromosomes but share substantial homology and are expressed in a tissue-specific but sometimes overlapping manner (29, 30, 31). The functions of the cyclin Ds have been studied in different D-type cyclin-deficient mice. Loss of cyclin D1 impairs mammary gland and retinal development and causes some neurological abnormalities (32, 33). Cyclin D2-/- mice show defects in ovarian granulosa cell and testicular cell function (34) as well as cerebellar defects (35). Given the tissue culture experiments that showed the predominant role of cyclin D1 in regulating the cell cycle, it was very surprising that the null mutant mice showed such a limited range of cell proliferation defects and were viable and fertile.
Fertility in female mice requires the proper functioning of the ovarian steroid hormones, 17ß-estradiol (E2) and progesterone, which act sequentially and synergistically to prepare the uterus for embryo implantation. In adult female mice, E2 produced at estrus stimulates cell proliferation (36, 37). This proliferation is inhibited by exposure to progesterone (P4) (38). The cellular dynamics in the uterus observed during the estrous cycle and early pregnancy can be faithfully reproduced in ovariectomized mice by exogenous sex steroid hormone treatment. Using this system, a single sc injection of E2 to ovariectomized mice can stimulate a synchronized wave of cell proliferation in the mouse uterine epithelium (39, 40), which displays kinetics and biochemical responses similar to that observed after growth factor is added back to serum-starved cells in culture. After this hormonal treatment, the mouse uterine epithelial cells can be isolated to greater than 95% purity with their biochemical activities intact (41). In our earlier studies, we found that E2 stimulates cell proliferation by causing the nuclear accumulation of cyclin D1 and the activation of cyclin E and cyclin A/CDK2 kinase complexes. Subsequently, these kinase/holoenzyme complexes fully phosphorylate RB proteins and the cells progress into S phase (42). These events in the uterine epithelial cells were inhibited by P4, resulting in hypophosphorylation of pRb, cell cycle arrest, and differentiation to an embryo-receptive state.
In addition to the roles in regulating cell proliferation, cyclin D1 has been shown to exert actions independent of its association with CDK4 particularly on estrogen signaling. Several groups have reported a physical association between cyclin D1 and the estrogen receptor (43, 44). This interaction can stimulate estrogen receptor-mediated transcriptional activity due to an enhanced affinity between estrogen response elements and its cognate receptor.
Given the importance of estrogen in the regulation of the estrous cycle and pregnancy, and the central role of estrogen-induced cell proliferation in this process, together with the important functions of cyclin D1 in regulating cell proliferation and estrogen receptor-mediated transcription, it is perplexing that female cyclin D1 knockout mice are fertile. This implies that uterine cell proliferation is not impaired by the loss of cyclin D1. To examine the possibility that the action of cyclin D1 is compensated for by another cyclin, we investigated the regulation of cell proliferation in cyclin D1-deficient uterine epithelial cells in response to E2. In our studies, we demonstrated that cyclin D2 compensates for the loss of cyclin D1, and its activity results in phosphorylation of Rb proteins and, consequently, normal cell cycle progression. These data show that the cell cycle machinery can compensate for the loss of a component in vivo and emphasize the central role of cyclin Ds in the E2 regulation of uterine epithelial cell proliferation.
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RESULTS
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Absence of Cyclin D1 Does Not Inhibit E2-Induced Cell Proliferation in the Uterine Epithelium
Treatment of ovariectomized adult female mice with exogenous E2 induces cell proliferation in the uterine luminal and glandular epithelium (40, 46). Our earlier studies have suggested that cyclin D1 is one of the major downstream targets of E2, propelling the entry of these epithelial cells into DNA synthesis (42). To determine whether loss of cyclin D1 affected this E2-stimulated cellular proliferation, we compared cell cycle progression in the uterine epithelium of wild-type and cyclin D1 knock out mice using bromodeoxyuridine (BrdU) incorporation as an index of DNA synthesis. Mice treated with E2 at different time points were injected ip with BrdU 2 h before death. BrdU-positive cells were detected by immunohistochemistry in transverse uterine sections and counted. In untreated mice, there was a low level of DNA synthesis in the luminal and glandular epithelium with approximately 3% cells in S phase. An increase in BrdU-positive cells was observed at 5 h after E2 stimulation, which reached about 5060% of the luminal epithelial cells by 14 h after injection (Fig. 1
). These kinetics are similar to those observed in outbred mouse strains (40, 42). The kinetics of induction of DNA synthesis in the uterine epithelium was identical in cyclin D1-/- mice compared with wild-type mice both in timing and magnitude (Fig. 1
, A and B). Thus, cyclin D1 is dispensable for E2-induced DNA synthesis in vivo in the luminal epithelium.

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Figure 1. Cell Proliferation in Response to E2 Treatment in Wild-Type and Cyclin D1-/- Uteri
A, Ovariectomized mice were given BrDU ip 2 h before they were killed at the specified time points. Transverse uterine sections from these mice were immunostained with anti-BrdU antibody. Brown cells are those undergoing DNA synthesis. B, Graph showing the percentage (mean ± SD) of BrdU-positive luminal epithelial cells at different time points after E2 treatment.
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The Rb Family of Proteins Are Phosphorylated in Response to E2 in the Cyclin D1-/- Uterine Epithelium
The Rb pocket protein family is considered the central regulator of the G1/S cell cycle transition. The members, p107 and Rb, are hyperphosphorylated in response to E2 in mouse uterine epithelial cells with maximal phosphorylation being observed 78 h after treatment (42). To test whether cyclin D1 is required for Rb phosphorylation in the mouse uterine epithelial cells, we analyzed the patterns of Rb protein phosphorylation by Western blotting in cell extracts from epithelial cells 7 h after E2 treatment.
In wild-type and cyclin D1-/- mice, in the absence of hormone treatment, Rb and p107 protein had a very low level of phosphorylation with the hypophosphorylated form identified as the faster-migrating band representing greater than 95% of the protein (Fig. 2
, top panel). Seven hours after E2 treatment, Rb showed a very substantial increase in phosphorylation (Fig. 2
, top panel). The ratio of the slower moving hyperphosphorylated Rb to the faster moving hypophosphorylated Rb band was similar between cyclin D1-deficient and wild-type mice, showing that there was an equal level of Rb phosphorylation (Fig. 2
, top panel). Very similar results were observed for the phosphorylation of p107 that showed an increased level of phosphorylation in response to E2 treatment, which was not different between wild-type and mutant mice (Fig. 2
, middle panel).

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Figure 2. Phosphorylation Status of pRb and p107 before and after E2 Treatment
Epithelial extracts from the uteri of wild-type (WT) or cyclin D1-/- (D1-/-) mice treated for 7 h or untreated with E2 were subjected to Western blotting with anti-Rb, anti-p107, and antiphospho-Rb (Ser 807/811) antibodies. The hypo- (pRb, p107) and hyperphosphorylated (ppRB and pp107) bands are indicated by arrows.
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Phosphorylation of Rb and p107 can be effected by several CDKs with their coordinate cyclin partners. For example, cyclin E/CDK2 kinase complex is sufficient to effectively phosphorylate Rb proteins in the absence of cyclin D1 (7, 47). Although these experiments were performed by the introduction of supraphysiological levels of cyclin E into cells, it cannot be ruled out that cyclin E/CDK2 could compensate for the loss of cyclin D1 in vivo. To address this question, we used a phospho-specific antibody, which specifically recognizes Rb proteins phosphorylated at Ser807/811, the sites at which phosphorylation is catalyzed by CDK4/6 kinases (48). Essentially no phosphorylation could be detected in untreated mice with this antibody, while 7 h after E2 treatment, there was a substantial increase in the amount of pRb phosphorylated at Ser807/811 in both the wild-type and cyclin D1 null epithelial cell extracts (Fig. 2
, bottom panel). These data suggest that CDK4 activity is normal in cyclin D1-/- mouse uterine epithelial cells.
CDK4 Is Catalytically Active in the Cyclin D1-/- Mouse Uterine Epithelium
To test the above conclusion, we examined CDK4 activity in an immune complex CDK4-associated kinase assay using Rb as a substrate. Consistent with the above phosphorylation studies, the majority of the CDK4-associated kinase activity was maintained in cyclin D1 null mutant mice 7 h after estrogen treatment when compared with wild-type mice (Fig. 3A
). The expression level of CDK4 protein was not changed by estrogen treatment in either wild-type, as previously reported (42), or mutant mice. Furthermore, the cellular concentration of this protein in the cyclin D1 null uterine epithelial cells was the same as in the wild-type cells (Fig. 3B
). To ensure the true absence of cyclin D1 in the aforementioned CDK4 kinase assay, a Western blot of the immunocomplexes was performed. Cyclin D1 could readily be detected in the CDK4 complexes from wild-type cells, whereas it was absent in the null mutant cells (Fig. 3A
). Because CDK4 was active in the absence of cyclin D1, these data imply that, in response to E2, another member of the cyclin D family may bind and activate CDK4 to compensate for cyclin D1 loss. To examine this possibility, a Western blot analysis was performed to determine the expression of cyclin D2 and cyclin D3 in the epithelial extracts. In Western blots of uterine epithelium, we were able to detect only the expression of cyclin D2 and not D3 with the available antibodies. This analysis showed that cyclin D2 had a constant level of expression in uterine epithelial cells with or without E2 exposure and that the levels were similar between genotypes (Fig. 3B
).

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Figure 3. Cyclin D/CDK4 Status in Wild-Type and Cyclin D1-/- Mice after E2 Treatment
A, Epithelial cell lysates were harvested 7 h after E2 injection and subjected to immunoprecipitation with either an anti-CDK4 antibody or with mock preimmune serum [normal rabbit serum (NRS)]. Immune complexes were either assayed for the kinase activities using p56Rb as substrate (top) or Western blotted for cyclin D1 to detect the presence of cyclin D1 and its association with CDK4 (bottom). B, Western blots of epithelial cell lysates from control and 7 h E2-treated uteri probed with anti-CDK4, -cyclin D2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies.
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Cyclin D2 Compensates for the Loss Of Cyclin D1 in the Uterine Epithelium
We next asked whether cyclin D2 is responsible for the CDK4 kinase activity. To test the physical association between cyclin D2 and CDK4, cell extracts from uterine epithelium were subjected to immunoprecipitation either with a mouse monoclonal antibody to cyclin D2 or normal mouse serum. The immunoprecipitate was separated by electrophoresis and probed with antibody to CDK4 after blotting to a nylon membrane. Cyclin D2 was found in a complex with CDK4 regardless of whether or not the mice had been treated with E2. However, a small increase in this association was observed 7 h after E2 treatment. (Fig. 4A
). Significantly more CDK4 coprecipitated with cyclin D2 in the extracts from cyclin D1-/- uterine epithelial cells compared with the extracts from wild-type mice at 7 h after E2 exposure (Fig. 4A
).

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Figure 4. Cyclin D2-Associated CDK4 Activity in Wild-Type and Cyclin D1-/- Uterine Epithelium
A, Uterine epithelial lysates collected from control (untreated) and after 7 h of E2 exposure were immunoprecipitated with either normal mouse IgG [normal mouse serum (NMS)] or anticyclin D2 antibody. Immunoprecipitates were separated by SDS-PAGE and subjected to Western blotting with anti-CDK4 antibody. B, Uterine epithelial lysates from mice 7 h after E2 treatment were either preimmunoprecipitated with anticyclin D2 antibody (Depleted, Dep) or not (No Dep.). The resultant immune complexes were assayed for CDK4 kinase activity using the p56Rb substrate. In the NMS lane, the nondepleted lysate was immunoprecipitated with normal mouse IgG. The relative levels of kinase activities of each lane were determined by densitometric quantitation (mean ± SD) and expressed relative to the control nondepleted level. Lane 1, Wild-type uterine lysate without depletion; lane 2, wild-type uterine lysate with anticyclin D2 antibody depletion; lane 3, cyclin D1-/- uterine lysate without depletion; lane 4, cyclin D1-/- uterine lysate with anticyclin D2 antibody depletion.
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To determine whether the cyclin D2/CDK4 complex was catalytically active, we attempted an immune complex kinase assay after immunoprecipitation with anti-cyclin D2 antibody. Unfortunately, this approach failed, probably because the catalytic active site of the kinase was blocked by the antibody. To circumvent this problem, cell extracts were immunodepleted with anti-cyclin D2 antibody, followed by subsequent CDK4 kinase assay with the supernatant. Before performing this experiment, we first tested the efficiency of the immunodepletion. Using cell extracts from wild-type uterine epithelium 7 h after E2 treatment, we titered the anti-cyclin D2 antibody until we obtained conditions when essentially all of the cyclin D2 was removed (data not shown). Using this protocol, cyclin D2-immunodepleted supernatants from both the cyclin D1-/- and wild-type epithelial extracts were subjected to the CDK4 kinase assay with Rb as substrate. In the extracts from uterine epithelium treated with E2 for 7 h without cyclin D2 depletion, the CDK4 kinase activity in the cyclin D1-/- lysates was approximately 60% of wild-type levels as described above (Fig. 4B
). After immunodepletion of cyclin D2, more than 80% of CDK4 kinase activity remained in the wild-type uterine extract compared with less than 10% in the cyclin D1-/- lysate (Fig. 4B
). This result, plus the detected association between cyclin D2 and CDK4, strongly suggests that in the normal mouse uterine epithelium most of the CDK4 kinase activity comes from the cyclin D1/CDK4 kinase complex, whereas in the cyclin D1 null mice the cyclin D2/CDK4 kinase complex is the main contributor to CDK4 kinase activity.
Cyclin D2 Is Nuclear Localized in the Uterine Epithelium of Cyclin D1-/- Mice after E2 Treatment
The data above indicated that the epithelial cellular concentration of cyclin D2 does not change either in response to E2 or to the loss of cyclin D1. Given our previous studies that showed the nuclear accumulation of cyclin D1 in response to E2 treatment, we analyzed the subcellular localization of cyclin D2 after E2 stimulation. Transverse sections of paraffin- embedded uteri were immunostained with anti-cyclin D2 antibody (Fig. 5A
). Cyclin D2 protein in the control epithelium of wild-type and cyclin D1 null mutant mice was mainly cytoplasmic (Fig. 5A
). Seven hours after hormone treatment, in wild-type mice, it was still predominantly in the cytoplasm, although some weak nuclear staining was observed. In contrast, many nuclei were strongly stained by anticyclin D2 antibody in cyclin D1-/- mice with the signal in the cytoplasm correspondingly reduced (Fig. 5A
). This nuclear translocation of cyclin D2 in cyclin D1-deficient mice was confirmed by Western blotting of a nuclear fraction harvested from uterine epithelial cells (Fig. 5B
, upper panel). Nuclear lamins A and C (Fig. 5B
, bottom panel) were used as a protein loading control and were comparable between samples. In both mutant and wild-type mice there was significantly more cyclin D2 in the nuclear fraction 7 h after treatment (Fig. 5B
). However, the cyclin D2 concentration in the nuclear fraction derived from cyclin D1 null mutant mice was much higher than that seen in the wild-type mice 7 h after E2 induction, consistent with the result from immunohistochemistry. These data strongly suggest that, in the absence of cyclin D1, cyclin D2 is able to bind CDK4 and be translocated into the nucleus. Thus, the nuclear accumulation of cyclin D2 in the cyclin D1 null mice is similar to that observed for cyclin D1 in wild-type mice (42), and this mechanism allows the access of cyclin D2/CDK4 to its substrates, the Rb family of proteins.

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Figure 5. Localization of Cyclin D2 in the Mouse Uterus after E2 Injection
A, Localization of cyclin D2 determined by immunohistochemistry of cross-sections of uteri from wild-type (WT) and cyclin D1-/- (D1-/-) mice that were either untreated (E0) or exposed to 50 ng of E2 for 7 h (E7). B, Western blotting analysis of nuclear fractions of epithelial cells using antibody to cyclin D2 (top) or lamin A and C (bottom).
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In a similar fashion, we also analyzed the distribution of CDK4, the catalytic partner for cyclin Ds. In control mice of both genotypes, CDK4 was present in both the nuclei and cytoplasm of epithelial cells. However, treatment with E2 greatly enhanced the signal in the nucleus with a corresponding diminution in the cytoplasm of the epithelial cells in both wild-type and cyclin D1-/- mice (Fig. 6
).

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Figure 6. Immunostaining of Transverse Sections of Uteri for CDK4 with or without Cyclin D1 at 7 h after E2 Treatment
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CDK2 Kinase Activity Is Unaffected by the Loss of Cyclin D1
We had previously shown in the uterine epithelial cells of CD1 mice that CDK2 activity was up-regulated by E2. This elevation was confirmed by immune-complex CDK2 kinase assay in this strain of mice used in the present experiments (data not shown). At 7 h after E2 treatment, CDK2 activity was evident in wild-type uterine epithelial extracts. This was similar to the CDK2 kinase activity in the epithelial extracts from cyclin D1 null mice (Fig. 7A
). Western blotting analysis showed that the loss of cyclin D1 did not change the expression of CDK2 when compared with estrogen-treated control mice (Fig. 7B
).

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Figure 7. CDK2 Activity in Uterine Epithelial Lysates from Wild-Type and Cyclin D1-/- Mice
A, CDK2-associated kinase activities were assessed using HH1 as substrate in the lysate derived from uteri treated with E2 for 7 h. B, The cellular concentration of CDK2 in epithelial lysates was analyzed by Western blotting. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was used to determine GAPDH concentrations as a control for protein loading.
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Cyclin E and A are the two major CDK2 partners in the formation of the catalytically active kinase complex (49, 50). Therefore, we examined the expression patterns of these two cyclins by Western blot analysis and their associated CDK2 kinase activities by immune-complex kinase assay. Seven hours after E2 stimulation, the cellular concentration of cyclin E in epithelial lysates of both genotypes was not changed compared with control (Fig. 8A
). In contrast to the lack of E2 effect on cyclin E expression, there was a dramatic elevation in cyclin A expression in both wild-type and cyclin D1 null epithelial cells after hormone induction. It was induced approximately 3-fold 7 h after E2 treatment and reached the maximal level of about 10-fold 15 h after hormone treatment (Fig. 8A
). We confirmed this result by immunostaining for cyclin A in transverse sections of uteri. In the untreated uteri of both genotypes there were few cyclin A-positive cells, data that are consistent with the Western blotting results. However, 15 h after E2 induction, the majority of the cyclin A subcellular localization was in the nucleus. The number of positive cells was the same regardless of the genotype. Using an immune-complex kinase assay with histone H1 (HH1) as the substrate, the level of cyclin E-associated CDK2 kinase activity at 7 h and cyclin A/CDK2 activity at 15 h after E2 treatment was similar between genotypes (Fig. 8B
).

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Figure 8. Cyclin E and Cyclin A Expression and Their Associated CDK2 Kinase Activity
A, The cellular concentration of cyclin E and A was detected by Western blotting (top and middle) at the specified time points after E2 treatment in the genotypes of mice as described above. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was used to determine GAPDH concentrations as a control for protein loading. For the loading control of cyclin E, please refer to Fig. 7 (the same sample batch was used for these two Western blots). Cellular localization of cyclin A as detected by immunohistochemistry (bottom). B, Cyclin E (left)- and cyclin A (right)-associated CDK2 kinase activities were measured using HH1 as a substrate after immunoprecipitation with the indicated antibodies. In the cyclin E-associated CDK2 kinase assay, cell lysates were collected 7 h after E2 treatment, and in the cyclin A-associated kinase assay, mice were killed 15 h after E2 injection.
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P27kip1 Expression and Its Association with CDK4 and CDK2 in Cyclin D1-/- Mice
The activities of CDKs are negatively regulated by two families of CKIs (14, 27). In wild-type mice, p27kip1 was detected at a significant cellular concentration, and its expression was down-regulated by E2 treatment (42). This was confirmed in the present experiments in this strain of mice (Fig. 9A
). The down-regulation by E2 treatment was not altered by cyclin D1 loss because its expression level was similar between the two samples derived from the different genotypes. As p27kip1 can inhibit all cyclin/CDK kinase complexes, another role for the cyclin D1/CDK4 complex is to titrate p27kip1 away from the cyclin E/CDK2 complex, which results in the activation of this kinase holoenzyme (51, 52). To test whether cyclin D2 can replace cyclin D1 in this function, we analyzed the association of p27kip1 with the different cyclin/CDK complexes. Epithelial cell lysates from mice of both genotypes treated with E2 were immunoprecipitated with anti-p27 antibody and the precipitates were blotted with either anti-CDK2 or -CDK4 antibodies. The amount of CDK2 or CDK4 associated with p27kip1 (Fig. 9B
) was similar in the cyclin D1 null and wild-type uterine epithelial extracts. These findings suggest that cyclin D2 can compensate for the loss of cyclin D1 in its role in redistribution of the p27kip1 pool.

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Figure 9. p27kip1 Association with CDK2 and CDK4
p27kip1 expression in mouse uterine epithelial lysates from wild-type and cyclin D1-/- mice. Cell lysates were analyzed by Western blotting on the samples collected from different time points after E2 treatment as indicated. B, Cell lysates were immunoprecipitated with antibody to p27kip1, and then immunoblotted with antibody to CDK2 (top) or CDK4 (bottom) to detect the association between p27kip1 and CDK2 or CDK4.
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Estrogen Receptor Transcriptional Activity in Cyclin D1-/- Mice
Cyclin D1 can interact with the estrogen receptor and stimulate its transcriptional activity, an action that is independent of CDK4 (44). To test the effect of the loss of cyclin D1 on the regulation of estrogen receptor-mediated transcription, we choose the progesterone receptor as an indicator of E2-induced transcription because this protein is rapidly induced after E2 treatment as a result of transcriptional activation (52A ). There was a similar pattern of progesterone receptor expression between the wild-type and cyclin D1-/- mouse uteri 5 h after E2 treatment. Expression was highest in the luminal epithelium, and this could not be distinguished between genotypes (Fig. 10
).

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Figure 10. Immunohistochemistry for the Progesterone Receptor in Transverse Sections of Cyclin D1-/- and Wild-Type Uteri (E0: without E2 Treatment; E5: 5 h after E2 Treatment)
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DISCUSSION
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In mammals, estrogens are essential hormones for female fertility (37). In the uterus, E2 acting through its receptor transcription factors, regulates epithelial cell proliferation during estrus and interacts with progesterone to prepare the uterus for implantation, an event that requires stromal cell proliferation (37). Cell culture experiments with breast cancer cell lines indicate that E2 regulates cyclin D1 expression (53). In contrast to these cell culture experiments, our studies of the action of E2 in the uterine epithelium in vivo, demonstrated that this hormone only modestly regulates the overall level of cyclin D1 and not until late in G1, but instead, causes cyclin D1 relocalization from the cytoplasm to the nucleus (42). In this location, together with its partners CDK4 and 6, it has access to and phosphorylates Rb and pl07. Progesterone, a hormone that acts to inhibit E2-induced cell proliferation early in G1, completely prohibits this nuclear translocation, thereby blocking Rb phosphorylation (42). These data strongly suggest that the regulation of cyclin D1/Cdk4 and -6 activities are central to estrogen action. Thus, in vivo as in tissue culture, cyclin D1 appears to be the intracellular sensor for alterations of cell proliferation rates in response to changing environments. Given this conclusion and the requirement for epithelial cell proliferation and its subsequent inhibition for fertility, it was perplexing that the cyclin D1 null mutant mice are fertile, data that indicate the correct responsiveness of the uterus to E2 and P4.
Many experiments in cultured cells indicated that the phosphorylation of the Rb family proteins, and particularly Rb itself, by cyclin-dependent kinases is central to the regulation of the mammalian cell cycle (50, 54). It was surprising, therefore, when targeted ablation of these genes in mice revealed fairly modest phenotypes. For example, Rb null mutant embryo, after crossing with ID2 mutants, can survive to term (55), whereas cyclin D1 null mutant mice (32, 33), although smaller than wild-type mice with specific defects in the retina and mammary gland, are viable and fertile. Most of these cell cycle-regulatory proteins are members of gene families that at least in biochemical assays can substitute for one another. For example, there are three members of the Rb and cyclin D families. Consequently, an interpretation of the phenotypes in the null mutant mice is that the members of the family can functionally substitute for one another. A caveat for this interpretation is that the patterns of expression of many of these genes are not coincident. For example, the cyclin Ds have widely different expression patterns in the embryo and adult (27). Thus, an alternative explanation is that downstream targets of these genes are up-regulated, and this can compensate for the loss of the upstream regulatory factor. Data to support this latter view came from the rescue of the cyclin D1 null phenotype by the knock-in of cyclin E into the cyclin D1 locus in mice (56). Although cyclin E in partnership with CDK2 is downstream of cyclin D/CDK4 in the sequential phosphorylation of Rb, the early expression of cyclin E through the use of cyclin D1-regulatory elements resulted in complete compensation for the loss of cyclin D1. However, in cells that have not been manipulated to aberrantly express downstream targets, there is still the problem of how the extracellular signal, such as estrogen stimulation, could be sensed in a timely fashion by the cyclin E locus so that it could be responsive to environmental changes in an appropriate manner.
In this study, we showed that the uterus responds to sex steroid hormones with the appropriate pattern of cell proliferation in the absence of cyclin D1. In fact, the kinetics of entry in S-phase in the luminal epithelium in response to E2 was identical between cyclin D1-/- mice and their littermate controls and similar to other strains of mice that we have previously studied (42). The epithelial cell proliferation in cyclin D1-/- mice was also inhibited by P4, a hormonal treatment that permits the E2 induction of stromal cell proliferation (data not shown). Consistent with these observations, E2 induced cyclin A expression in these epithelial cells with similar kinetics in both mutant and wild-type mice. Cyclin A binds to and activates CDK2, which, together with the cyclin E/CDK2 complex, are required for the cell cycle progression through late G1 and S phase. The activity of these cyclin/CDK2 complexes was also comparable in these epithelial cells from cyclin D1-/- and wild-type mice. The equal expression and kinase activity between the wild-type and cyclin D1-/- samples suggest the compensation of cyclin D1 loss may not come from an up-regulation of the CDK2 complexes. To investigate this further, we analyzed CDK4 activity in the mutant epithelial cells. This was 6080% of levels found in wild-type lysates. This level appeared to be sufficient to give full Rb phosphorylation at the CDK4-specific sites, serine 807 and 811. Phosphorylation of these CDK4-specific sites enables Rb to be further phosphorylated by cyclinE/CDK2 complexes such that it becomes fully phosphorylated, and cells can progress through G1 into S phase. The degree of Rb phosphorylation in wild-type and cyclin D1-deficient mice is comparable after E2 treatment, which is consistent with the cell cycle data reported above.
CDK4 requires a cyclin D partner. We have shown that cyclin D2 is expressed in the uterine epithelium at similar levels in cyclin D1-/- and wild-type mice. Immunoprecipitation of cyclin D2 revealed that CDK4 was in complex with this protein and this association was increased by E2. However, significantly more cyclin D2 was associated with CDK4 in the cyclin D1-/- null uterine epithelium, suggesting a competition between these two proteins. Using immunodepletion of cyclin D2 from the epithelial lysates, we demonstrated that in contrast to wild-type cells where only a small fraction of the CDK4 activity was cyclin D2 associated, essentially all of this activity was provided by cyclin D2 in the cells lacking cyclin D1. From these data it can be concluded that cyclin D2 in the absence of D1 binds to and activates CDK4 to a sufficient degree to permit normal E2-induced uterine luminal epithelial cell proliferation. Although cyclin D1 and cyclin D2 have similar sequences that interact with CDK4 (57), the competition between the two proteins with CDK4 suggests that either the subtle sequence difference between cyclin D1 and D2 results in a higher affinity of cyclin D1 for CDK4 or that cyclin D1 is significantly more abundant than cyclin D2 in these cells.
Other than initiating and propagating phosphorylation of the Rb protein, cyclin D1 also plays a noncatalytic role in regulating G1 progression. In complex with CDK4 it binds the CIP/KIP CKIs, p27, p21, and p57, thus causing the redistribution of those CKIs from cyclin E/CDK2 pool to the cyclin D/CDK4 pool (14, 15, 58). This releases the cyclin E/CDK2 kinase complex from inhibition and results in their activation. Because p27kip1 was the major CKI in the mouse uterus, we examined the possibility that cyclin D2 can compensate this function of cyclin D1 in the absence of cyclin D1. After immunoprecipitation with anti-p27kip1 antibody, there were equal amounts of CDK2 and CDK4 bound to p27kip1 in both mutant and wild-type cells. This indicates that cyclin D2 can rescue cyclin E/CDK2 from p27kip1 inhibition. Consequently, we also found similar CDK2 kinase activities in the two different genotypes as well as the same cyclin E-associated CDK2 kinase activity.
A recent publication revealed that the tissue- specific expression pattern of D cyclins was altered after the loss of cyclin D1. These genetic experiments showed up-regulation of an alternative D-type cyclin in specific tissues during development, and this provided an explanation for the viability of the cyclin D1 null mutant mice (59). Consistent with this alternative regulation of cyclin D expression, cyclin D3 was shown to compensate for CD40-induced cell proliferation in cyclin D2-/- lymphocytes, although the lag period and extent of cell proliferation were considerably reduced (60). However, in the mouse uterine epithelium, cyclin D2 expression was not changed in response to cyclin D1 loss. Instead, it substituted for cyclin D1 in the translocation to the nucleus, and after hormone treatment the extent of cyclin D2 nuclear accumulation was much greater in the D1 null than wild-type cells. This suggests that, under normal conditions, cyclin D1 and D2 compete for the nuclear "transporter" mechanism. The ability of cyclin D2 to substitute for D1 in this translocation results in full Rb phosphorylation and the appropriate cell proliferation response. Several studies have shown that cyclin D compartmentalization is a mechanism that regulates cyclin D activity (61, 62). Our studies indicate that this mechanism has substantial physiological significance and that the ability of cyclin D2 to substitute for cyclin D1 provides the explanation for the fertility of cyclin D1-/- mice.
In studies in vitro, the interaction between cyclin D1 and estrogen receptor can enhance estrogen receptor-mediated transcription. Using the highly estrogen-responsive progesterone receptor gene as an index of E2-induced gene expression, we were able to demonstrate that this gene is equally expressed in the uterus of cyclin D1-/- and wild-type mice. Thus, if this positive effect of cyclin D1 on estrogen receptor transcriptional activity occurs in vivo, these data indicate that E2-induced gene expression can also be completely rescued by cyclin D2. This further demonstrates the interchangeable role of cyclin D2 and cyclin D1 in the uterine response to E2.
Overall, our data provide compelling in vivo evidence of a compensatory mechanism in the cell cycle machinery that is sufficient, at least in the uterus, to maintain hormone-induced cell proliferation. Cyclin D1 mice are smaller than wild-type mice, and variable levels of cell cycle compensation might result in this phenotype. However, in the uterus there is complete rescue by cyclin D2 such that the mice are fertile, and a similar mechanism in other cell types may explain their viability.
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MATERIALS AND METHODS
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Animal Treatment
Cyclin D1-/- mice were a kind gift from Pitor Sicinski (Dana-Farber Cancer Institute, Boston, MA). The mice are 129 sv backcrossed to C57BL/6 and randomly bred in a closed colony. The female mice were maintained for 1012 wk before being ovariectomized via a dorsal incision under tribromoethanol anesthesia. Two weeks after the surgery, mice were primed with 100 ng of E2 (Sigma, St. Louis, MO) in peanut oil by sc injection for 2 d before the experiment. Six days later, groups of three to five mice were killed at various time points after single sc injection of 50 ng of E2 in peanut oil, a dose that results in maximal cell proliferation in the uterine luminal epithelium. Control mice received a similar injection of vehicle alone. All experiments reported below were performed in duplicate and repeated at least once.
Preparation of Epithelial Cell Extracts
After hormone treatment, uteri were removed and split longitudinally and an epithelial extract was prepared as described (41). Lysates were sonicated and clarified by centrifugation. For each experiment, equal amounts of protein, measured by Bradford assay (45) (Bio-Rad Laboratories, Inc., Hercules, CA), were used. The extraction buffer contained 10 mM HEPES-KOH (pH 7.5), 0.1 M NaCl, 1 mM EDTA, 2.5 mM EGTA, 10 mM ß-glycerophosphate, 10% glycerol, 1 mM dithiothreitol (DTT), 1 mM NaF, 0.1 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg aprotinin/ml, 10 µg leupeptin/ml, 10 µg pepstatin A/ml. The washing buffer contained 90 mM HEPES-KOH (pH 7.5), 0.2 M NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.2% Tween 20, 10% glycerol, 10 mM ß-glycerophosphate, 1 mM DTT, 1 mM NaF, 0.1 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg aprotinin/ml, 10 µg leupeptin/ml, 10 µg pepstatin A/ml.
Immunoblotting and Antibodies
Cellular lysates were boiled in gel sample buffer containing sodium dodecyl sulfate and separated by electrophoresis, transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA), and blotted with the appropriate antibodies. The following rabbit antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA): anti-cyclin A (sc-596 and sc-571), anti-cyclin D2 (sc-593), anti-cdk2 (sc-163), anti-cdk4 (sc-260), anti-p27 (sc-528), anti-p107 (sc-528), and anti-progesterone receptor (sc-538). The mouse monoclonal antibodies against cyclin D1 (DCS-06) and cyclin D2 (DSC-3.1) were purchased from Neomarker. Antibody against pRb (G3-245) was acquired from PharMingen (San Diego, CA), and the anti-phospho-pRB (Ser807/811) antibody was obtained from New England Biolabs, Inc. (Beverly, MA).
Immunoprecipitation and Kinase Assay
The cellular lysates for CDK4 kinase assays were prepared in extraction buffer (see above) followed by the addition of an equal amount of washing buffer. The composition of extraction buffer for the CDK2 kinase assay was: 10 mM Tris (pH 7.4), 100 mM EDTA, 5 mM EDTA. Lysates were clarified, followed by incubation with 1 µg of antibody for 12 h at 4 C with shaking. The immunocomplexes were collected by adding protein A beads (Santa Cruz Biotechnology, Inc.). After washing with a stringent buffer (extraction buffer containing 150 mM NaCl), the beads were subsequently collected by centrifugation. The immunoprecipitates were then either subjected to SDS-PAGE followed by Western blotting or used for the kinase assay.
In the kinase assay using histone H1 as a substrate, the reaction was performed in 50 µl CDK2 kinase buffer [20 mM Tris (pH 7.4), 7.5 mM MgCl2, 1 mM DTT] containing 2 µg HH1, 30 µM ATP, and 3 µCi [
-32P]ATP (Amersham Pharmacia Biotech, Arlington Heights, IL) for 30 min at 30 C. For Rb kinase assay, CDK4 kinase buffer contained 50 mM HEPES-KOH (pH 7.5), 10 mM MgCl2, 1 mM DTT, 2.5 mM EGTA, 10 mM glycerophosphate, 0.1 mM Na3VO4, 1 mM NaF. The kinase reaction was started by the addition of 30 µl of Rb-kinase mix (kinase buffer with 20 µM ATP), recombinant truncated Rb protein (p56Rb; amino acids 379928; QED Bioscience, San Diego, CA), and 10 µCi of [
-32P]ATP).
Immunohistochemistry
Transverse 5-µm paraffin sections of the uterus were stained for BrdU incorporation using anti-BrdU antibody as described in the cell proliferation kit from Oncogene Science, Inc. (Manhasset, NY). The other sections to be immunostained for cyclin A, cdk4, and cyclin D2 were deparaffinized and subjected to antigen retrieval (except for the progesterone receptor immunostaining) by boiling the samples in 0.01 M sodium citrate buffer (pH 6.0) for 10 min. Nonspecific binding was blocked by incubating sections with 10% normal goat serum for 30 min. After the sections were incubated with the appropriate antibody, they were washed and exposed to biotin-conjugated secondary antibodies (Vector Laboratories, Inc., Burlingame, CA) for 30 min, followed by incubation with avidin DH-biotinylated horseradish peroxidase H complex as described (42). Sections were counterstained with hematoxylin (Sigma).
Preparation of a Nuclear Fraction from Uterine Epithelium
Uterine epithelial cell lysates were prepared as described before except that a sucrose-based extraction buffer containing 10 mM HEPES (pH 7.5), 50 mM NaCl, 0.5 M sucrose, 1 mM EDTA, 0.25 mM EGTA, 1 mM DTT, 0.6 mM spermidine, and protease inhibitors cocktail (Roche, Indianapolis, IN) was used. After filtration, Nonidet P-40 was added to a final concentration of 0.7%. The lysates were vortexed for 20 sec followed by repeated passing through a 22-gauge needle. Nuclei were collected by centrifugation at 800 x g for 10 min at 4 C and washed once with extraction buffer. The nuclei were then subjected to Western blotting analysis.
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ACKNOWLEDGMENTS
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We especially thank Dr. Pitor Sicinski for generously providing the cyclin D1 mutant mice for breeding, Liyin Zhu for the histological preparations and scientific discussion, and Jim Lee for his technical assistance in the mouse facility. We gratefully acknowledge Drs. Wei Tong and Haifan Zhang for their assistance.
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FOOTNOTES
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This work was supported by NIH Grants RO1 CA-89617 (to J.W.P.) and the Cancer Center Core Grant P30-CA-13330. J.W.P. is the Betty and Sheldon E. Feinberg senior faculty scholar in cancer research. J.W.P is Director of the "Center for the Study of Reproductive Biology and Womens Health," Albert Einstein College of Medicine.
Abbreviations: BrdU, Bromodeoxyuridine; CDK, cyclin- dependent kinase; CIP, cyclin interacting protein; CKI, cyclin-dependent kinase inhibitor; DTT, dithiothreitol; E2, 17ß-estradiol; KIP, CDK inhibitory protein; Rb, retinoblastoma.
Received for publication February 4, 2003.
Accepted for publication March 6, 2003.
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