GSK-3B Mediates in the Progesterone Inhibition of Estrogen Induced Cyclin D2 Nuclear Localization and Cell Proliferation in Cyclin D1À/À Mouse Uterine Epithelium

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FEBS Letters 581 (2007) 3069–3075

GSK-3b mediates in the progesterone inhibition of estrogen induced cyclin D2 nuclear localization and cell proliferation in cyclin D1À/À mouse uterine epithelium

Yuxiang Wanga, Hui Fengb, Cheng Bia, Liyin Zhuc, Jeﬀrey W. Pollardc, Bo Chena,* a School of Life Science and National Key Laboratory of Genetic Engineering, Fudan University, Shanghai 200433, PR China b Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA c Department of Developmental and Molecular Biology and Center for the Study of Reproductive Biology and Women’s Health, Albert Einstein College of Medicine, Bronx, NY 10461, USA

Received 3 May 2007; revised 25 May 2007; accepted 25 May 2007

Available online 4 June 2007

Edited by Jesus Avila

(D1, D2, and D3) and their kinase partners Cdk4 and Cdk6 Abstract We report that glycogen synthase kinase (GSK)-3b is phosphorylated at ser9 and inactivated in uterine epithelial cells act early in G1 phase. Although their expression show some- what over-lapping patterns and can compensate for each from E2-treated cyclin D1 null mutant mice. Simultaneous administration of P4 together with E2 blocked this effect. Phar- other, biochemical assays and the different D-type cyclin defi- macological inhibition of GSK-3b activity in mice treated with cient mice reveal functional differences among them [7]. P4E2 reversed the nuclear exclusion of cyclin D2 in the uterine As an important gate-keeper in G1–S phase transition, the epithelial cells and this caused phosphorylation of Rb protein activity of cyclin Ds is tightly controlled. In cultured cells, it and progression of cells towards S-phase. Our results indicate is generally accepted that extracellular growth signals includ- that GSK-3b is a major target of E2 and P4 in regulation of cy- ing E2, cause an elevation in cyclin D1 concentration that acts clin D2 localization in the mouse uterine epithelium. to activate Cdk4/6 by forming complexes that also titer away 2007 Federation of European Biochemical Societies. Published the cell cycle inhibitor p27kip1 from the cyclin E/Cdk2 com- by Elsevier B.V. All rights reserved. plexes. However, in contrast to these studies in tissue culture, Keywords: Cyclin D2; GSK-3b; Estrogen; Progesterone we found that in uterine epithelial cells in vivo, E2 only induces the nuclear accumulation of cyclin D1, which can be prevented by P4 pretreatment [3,8]. Our previous studies also demonstrated that cyclin D2 can compensate for the loss of cyclin D1 in estrogen-induced epithelial cell proliferation in the 1. Introduction mouse uterus and in these circumstances, cyclin D2 accumu- lates in the nucleus and the cells proceed normally into DNA The actions of ovarian steroid hormones estradiol-17b (E2) synthesis [8]. Although our studies in the mouse uterine epithe- and progesterone (P4) in the regulation of cell cycle has been lium revealed that E2 and P4 regulates GSK-3b activity to con- analyzed in in vivo and in vitro systems [1–3]. In the normal trol cyclin D1 localization [9], it is unclear how the localization uterus, ovarian E2 causes the uterine epithelial cells to undergo of cyclin D2 is regulated in vivo. rounds of cell proliferation [4]. In contrast, P4 inhibits this E2 GSK-3b is implicated in many fundamental processes induced cell proliferation and induces cell differentiation [5]. among species, including glycogen and glucose metabolism, The cellular dynamics in the uterus can be faithfully repro- embryonic cell fate determination, tumorigenesis, and apopto- duced by administration of exogenous female sex hormones sis [10], suggesting a pivotal role of GSK-3b in cellular regula- to adult female ovariectomized mice in regimens that are sim- tion. Studies in mice and cell lines suggest there is a negative ilar to their physiological secretion level [6]. correlation between GSK-3b and cyclin D2. B-cell receptor The sequential action of cyclins with their cyclin dependent signaling causes an inhibitory phosphorylation of GSK-3b, kinase (Cdk) partners regulates orderly cell cycle progression and promotes cell progression, that is cyclin D2 dependent during the G1–S phase transition. These cyclin and CDK holo- [11,12]. Furthermore, inhibition of GSK-3b by LiCl can stim- enzymes include the cyclin D-Cdk4 and -Cdk6 complexes act- ulate cerebellar granule cell proliferation [13]. Consistent with ing in G1 and the cyclin E- and cyclin A-CDK2 complex acting this, genetic disruption of cyclin D2 in mice leads to decrease in at the G1–S transition. Both kinase complexes inactivate Rb granule cell number in the cerebellum [14], indicating the family of proteins by phosphorylation. The D-type cyclins requirement of cyclin D2 in granule cell proliferation. In this paper, we investigated the regulation of cyclin D2 localization in cyclin D1-deficient uterine epithelial cells in response to E2 and P4. Disruption of GSK-3b function resulted * Corresponding author. Fax: +86 13141361446. in an inhibition of the response to P4 in mouse uterine epithe- Q1 E-mail addresses: [email protected], [email protected] lial cells in cyclin D1 deficient mouse. These data show that (B. Chen). GSK-3b is one of the major regulators of cyclin D2 in vivo and further emphasize the importance of GSK-3b in E and Abbreviations: GSK, glycogen synthase kinase; E2, estradiol-17b;P4, 2 progesterone; CDK, cyclin dependent kinase P4 mediated signaling.

0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.05.072 3070 Y. Wang et al. / FEBS Letters 581 (2007) 3069–3075

2. Materials and methods 3. Results

2.1. Animal treatment 3.1. Phosphorylation and activity of GSK-3b is regulated by E À/À 2 Cyclin D1 (Cdnd1) mice were a kind gift from Pitor Sicinski and P in cyclin D1À/À mouse uterine epithelium (Dana-Farber Cancer Institute, Boston, MA). The mice were ovariec- 4 tomized via a dorsal incision under tribromoethanol anesthesia. Mice To investigate whether the nuclear accumulation of cyclin were primed with 100 ng of E2 (Sigma, St. Louis, MO) for 2 d before D2 induced by E2 in the cyclin D1À/À mouse uterine epithe- the experiment. Six days later, groups of three to five mice were killed lium which is inhibited by pre-treatment with P4, is regulated at various time points groups as described [9]. All experiments reported by GSK-3b, we first asked whether GSK-3b is regulated by here were repeated at least two to three times. the steroid hormones in this mouse genetic background. Uterine epithelial cellular extracts from ovariectomized cyclin 2.2. Preparation of epithelial cell extracts After hormone treatment, uteri were removed and an epithelial ex- D1À/À mouse that had been treated with E2 or P4E2 for 5 h tract of greater that 95% purity was prepared as described [15]. were subjected to Western blot analysis. As shown in Fig. 1, GSK-3b protein concentration did not change in response to 2.3. Western blot and antibodies the different hormone treatment. With the phosphor-specific Polyclonal antibodies for cdk2 (sc-163), cdk4 (sc-260), cdk6 (sc-177), antibody against GSK-3b, only a low level of GSK-3b phos- p27 (sc-528), cyclin A (sc-597), and cyclin E (sc-481) were obtained phorylation at ser9 could be detected in the control uterine epi- from Santa Cruz Biotechnology, Inc. Monoclonal antibodies for pRb (G3-245) were obtained from Pharmingen; cyclin D2 (DCS-3.1) thelial extract of mice without any hormone treatment. E2 was obtained from Neomarkers; and that for PCNA was obtained treatment, however, induced a substantial level of ser9 phos- from Boehringer Mannheim. The anti-phospho-Rb(ser807/811) and phorylation, whereas pretreatment with P4 reduced the phos- anti-phospho-b-catenin(Ser33/37/Thr41) antibodies were acquired phorylation of GSK-3b back to the level observed in control from Cell Signaling. Western blotting was performed as described be- condition (Fig. 1). fore. Quantitative analysis of at least three samples was performed by densitometry of linearly exposed films and expressed relative to the b-catenin, a major substrate of GSK-3b, is phosphorylated GAPDH control. by this enzyme at Ser33/37/Thr41[16]. We used its phosphorylation status as an indicator for GSK-3b activity. Phos-b-Cate- 2.4. Immunohistochemistry nin at Ser33/37/Thr41 was detectable in the epithelium of Transverse 5-lm paraffin sections of the uterus were immunostained control un-treated cyclin D1À/À mice (Fig. 1). In contrast, for PCNA, cyclin D2 was subjected to antigen retrieval by boiling the E almost completely abolished phosphorylation at these sites, samples in 0.01 M sodium citrate buffer (pH 6.0) for 10 min. They were 2 washed and exposed to biotin-conjugated secondary antibodies, fol- and this was reversed by pre-treatment with P4. However, the lowed by incubation with avidin DH-biotinylated horseradish peroxi- total expression level of b-catenin remained constant. GSK-3b dase H complex. Sections were counterstained with hematoxylin. activity is therefore reciprocally regulated by E2 and P4.

2.5. Preparation of a nuclear fraction from uterine epithelium 3.2. Inhibition of GSK-3b leads to cyclin D2 nuclear Uterine epithelial cell lysates were prepared as described [8]. The lysates were vortexed for 20 s followed by repeated passing through a 22- accumulation gauge needle. Nuclei were collected by centrifugation at 800 · g for The phosphorylation pattern and activity of GSK-3b 10 min. strongly correlated with cyclin D2 localization in uterine

Fig. 1. P4 inhibited E2-induced GSK-3b phosphorylation and activity in cyclin D1À/À mouse uterine epithelial cell lysates harvested 5 h after hormone treatment were separated by SDS–PAGE, transferred to membranes and probed with antibodies against GSK3-b, Ser9-phosphorylated form of GSK-3b. b-Catenin, and Ser33/37/Thr41-phosphorylated form of b-catenin as indicated. Detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. Relative values of pGSK-3b and pb-catenin are shown for three determinations by densitometric analysis (means ± S.E. of the means). *P value from Student’s t-test was tested (P < 0.0011). Y. Wang et al. / FEBS Letters 581 (2007) 3069–3075 3071

Fig. 2. Cyclin D2 sub-cellular localization in the cyclin D1À/À mouse uterine epithelium following different treatments. (A) Expression level of b-catenin, and Ser33/37/Thr41-phosphorylated form of b-catenin in the uterine epithelium following either P4E2 treatment alone or in combination with LiCl for 7 h was analyzed by Western blotting using corresponding antibodies. (B) 1–6, Localization of cyclin D2 as determined by immunohistochemistry with an anti-cyclin D2 antibody of cross section of uteri from cyclin D1À/À mice treatment as indicated. The brown color represents the positive staining and the columnar cells are the luminal epithelium. (C) Western blotting analysis of total cellular or nuclear fractions of uterine epithelial cells from cyclin D1À/À mice treated as indicated above and probed with antibodies against cyclin D2 or lamin A/C as the loading control. Relative values are shown for three determinations by densitometric analysis (means ± S.E. of the means). *P value from Student’s t-test was tested (P < 0.0007). (D) Localization of cyclin D2 as determined by immunohistochemistry with an anti-cyclin D2 antibody of cross section of uteri from cyclin D1À/À mice treatment as indicated. (E) Western blotting analysis of total cellular or nuclear fractions of uterine epithelial cells from cyclin D1À/À mice treated as indicated above and probed with antibodies against cyclin D2 or lamin A/C as the loading control. 3072 Y. Wang et al. / FEBS Letters 581 (2007) 3069–3075 epithelial cells of mice treated with different hormones as we described before [9]. This result suggests that the female sex steroid hormones may exert their function through GSK-3b to regulate cyclin D2 sub-cellular localization in cyclin D1À/À mouse uterine epithelium. To test this possibility, we used the chemical inhibitor of GSK-3b, LiCl, to explore the role of GSK-3b.50ll of LiCl in a plutonic gel at the concentration of 20 mM was injected into the uterine lumen of mice 2 h before E2 or P4E2 treatment. As assessed by immunoblotting of uterine epithelial cell lysates from cyclin D1À/À mice 7 h after the P4E2 treatment for phosphorylated form of b-catenin at Ser33/37/Thr41, essentially all of GSK-3b activity was inhibited by LiCl (Fig. 2A). We next examined the subcellular localization of cyclin D2 in response to LiCl treatment by immunostaining. In control mice without any treatment, cyclin D2 in cyclin D1 null mice was predominantly in the cytoplasm (Fig. 2B1). However, LiCl treatment alone already induced nuclear localization of cyclin D2 in 50% of uterine epithelium cells (Fig. 2B2). Seven hours Fig. 3. Phosphorylation Status of pRb in cyclin D1À/À uterine epithelium. Extracts of the epithelial cells from mice treated as after E2 stimulation, most of the uterine epithelial cell nuclei were strongly stained by the anti-cyclin D2 antibody indicated were subjected to Western blotting with anti-Rb and anti- phospho-Rb (Ser807/811) antibodies. The hypo (pRb) and hyperphos- (Fig. 2B3). LiCl did not further enhance cyclin D2 accumula- phorylated (ppRb) bands are indicated by arrows. The histogram tion in E2 treated cyclin D1 null uteri (Fig. 2B4). In contrast, indicates densitometric determination of the ratio of ppRb with pRb although administration of P4 and E2 coincidently inhibited for three independent determinations (means ± S.E. of the means). * nuclear localization of cyclin D2, blocking GSK-3b by LiCl re- P value from Student’s t-test was tested (P < 0.003). versed the localization of cyclin D2 back into the nucleus (Fig. 2B5 and B7). To assure the nuclear accumulation of cyclin D2 by LiCl was not due to the side effect of the increased contributed this Rb phosphorylation, we used a phospo-spe- intra-luminal pressure, we also injected PBS to the P4E2 treated cyclin D1À/À mice. As expected, this treatment did not cific antibody, which specifically recognized Rb proteins phos- change the cytoplasmic localization of cyclin D2 (Fig. 2B6). phorylated at Ser807/811, the sites whose phosphorylation is The nuclear accumulation of cyclin D2 in the epithelium of cy- catalyzed by CDK4/6 kinases [18]. Essentially, no phosphory- clin D1 null mice with different treatment was confirmed by lation of Rb at Ser807/811 could be detected in uterine epithe- Western blotting of a nuclear fraction from uterine epithelial lium of cyclin D1 null mice treated with P4E2, while intra- cells against cyclin D2 (Fig. 2C, upper panel). However, in luminal administration of LiCl substantially increased this all of the cyclin D1 null mice under different treatments, the to- phosphorylated form of Rb. These data suggest that by inhib- tal cyclin D2 cellular concentration was similar (Fig. 2C). To iting GSK-3b function, cyclin D2 is relocalized to the nucleus, further confirm the cyclin D2 localization after disruption of therefore can access and phosphorylate its substrate, Rb pro- GSK-3b activity, we used TDZD-8 from Merck [17], another tein. specific inhibitor against GSK-3b at the concentration of 10 uM, which demonstrated a similar effect to LiCl (Fig. 2D 3.4. Inhibition of GSK-3b counteracts progesterone inhibition of and E). These data strongly support the hypothesis that cell cycle progression in cyclin D1À/À mice GSK-3b mediates E2 and P4E2 regulated cyclin D2 subcellular E2 treatment of adult ovariecotmized mice results in cell pro- localization in the uterine epithelium of mice. liferation in the luminal epithelium, which peaks 15 h after treatment [6].P4 completely suppresses this E2 induced prolif- 3.3. Cyclin D2 nuclear localization is sufficient for Rb eration as well as the basal epithelial cell growth [5]. Rb phos- phosphorylation in the uterine epithelium of cyclin D1 null phorylation by LiCl in P4E2 treated cyclin D1À/À mouse mice suggests this would drive cell progression from G1 into S To address the consequence of LiCl triggered cyclin D2 nu- phase. Therefore, we used proliferating cell nuclear antigen clear accumulation in the P4E2-treated cyclin D1 null mice, we (PCNA) as an index of cell progression. In the untreated mice, tested whether Rb protein phosphorylation status was chan- there were a small number of PCNA positive cells in luminal ged. Cellular extracts from cyclin D1 null uterine epithelium epithelium (Fig. 4A). Addition of LiCl into the uterine lumen isolated from mice following the different treatments for 7 h of the control untreated cyclin D1À/À mice induced 50% of were subjected to analysis by Western blotting with antibody epithelial cells to become PCNA positive. Fifteen hours after against Rb protein. The slower migrating phosphorylated E2 treatment, the majority of the luminal epithelial cells were form of Rb was distinct in the E2 treated sample. In the PCNA positive (Fig. 4A). P4 pretreatment completely abol- P4E2 treated sample, only the faster migrating hypophosph- ished the E2-induced as well as basal level of nuclear PCNA orylated form of Rb. However, inactivation of GSK-3b by in cyclin D1 null uterine epithelium, but it stimulated stromal LiCl significantly elevated the amount of the hyperphospho- cell nuclear PCNA accumulation. Inhibition of GSK-3b activ- rylated form of Rb protein to a level comparable that detected ity in P4E2 treated mice induced cells to be PCNA positive in in the uterine epithelial cells of cyclin D1 null mice treated both epithelium and stroma (Fig. 4A). GSK-3b inhibitor, with E2 (Fig. 3). To further confirm that cyclin D2/CDK4 TDZD-8, demonstrated similar effect to LiCl (Fig. 4B). Y. Wang et al. / FEBS Letters 581 (2007) 3069–3075 3073

Fig. 4. Uterine PCNA nuclear accumulation in response to the various hormones and LiCl treatments (A) and (B), Immunohistochemistry with anti- PCNA antibody followed by hematoxylin staining of transverse sections of uteri isolated from cyclin D1À/À after the treatments as indicated. Brown color represents the positive signal.

3.5. Cyclin D2 nuclear accumulation is responsible for cell cycle expression levels did not change in response to steroid hor- progression in the GSK-3b inhibited cyclin D1 null uterine mones or the treatments of combinations of these hormones epithelium treated with P4E2 with LiCl. Similarly, Western blotting of nuclear extract of To test if the cell cycle progression in GSK-3b inhibited cy- uterine epithelial cells from the cyclin D1 null mice 6 h after clin D1 null mice treated with P4E2 was attributed to alter- treatment, revealed that LiCl injection to the animals treated nated expression of other cyclins, CDKs or CKIs in G1/S with P4E2 did not significantly change the expression patterns transition, we analyzed the uterine epithelial cellular concen- of the other cell cycle proteins (Fig. 5C). Therefore, the reason tration of the cell cycle proteins. CDK2 and CDK4 were ex- why inhibition of GSK-3b function can counteract the P4 neg- pressed at a constant level regardless of the treatment of ative effect on cell cycle progression in the cyclin D1 null uter- mice with steroid hormones or LiCl (Fig. 5A and B). E2 injec- ine epithelial cells is mainly due to the restoration of cyclin D2 tion had little effect on the cyclin E level. However, simulta- nuclear accumulation. neously administration of P4 with E2 decreased cyclin E expression to about 50%. Inhibition of GSK-3b in P4E2 treated cyclin D1 null mice did not restore the cyclin E level. Cyclin A 4. Discussion expression was induced by E2 in the cyclin D1À/À uterine epithelial cells. P4 injection inhibited this E2induction, which can- In some breast cancer cell lines, E2 stimulate cyclin D1 not be restored by administration of LiCl to the uterine lumen. expression, while P4 inhibit this event [19]. A recent publica- Among the different CKIs, we can only detect p27, whose tion reveals that GSK-3b mediates in the PTEN regulation 3074 Y. Wang et al. / FEBS Letters 581 (2007) 3069–3075

Fig. 5. Expression patterns of different cell cycle proteins in cyclin D1À/À mouse after different treatments. (A) Uterine epithelial cell lysates from cyclin D1À/À mice treated with various hormone and LiCl regimens as indicated, were harvested at 0, 3 and 9 h after treatment. These were subjected to Western blot analysis with antibodies against cyclin A, cyclin E, CDK2, CDK4 and p27kip1. GAPDH was used as loading control in this assay. (B) Densitometric analysis of the expression of CDK4, CDK2, cyclin E, p27 at different time points after the hormonal treatments as indicated. Data shown are the means ± S.D. of three experiments. (C) Western blotting analysis of nuclear fractions of uterine epithelial cells from cyclin D1À/À mice treated as indicated above and probed with antibodies against c cyclin A, cyclin E, CDK2, CDK4 and p27kip1. GAPDH was used as loading control in this assay, lamin A/C was used as loading control.

of cyclin D2 expression at transcriptional level in NIH3T3 cells mains to be explored but is likely to be important in the con- [20]. However, in uterine epithelial cells in ovariectomized cy- trol of uterine cell proliferation. clin D1 null mice, GSK-3b activity only affects the cellular Compensatory mechanisms for the loss of one protein in the localization of cyclin D2 without changing its expression in re- cell cycle field has not only been demonstrated within the same sponse to different hormone treatment. Although increased family member proteins, but also among the different cyclins or levels of cyclin D occur in different cancer types, many studies CDKs [23]. However, LiCl injection into cyclin D1 null mice suggest these may not be the only reason for the transforming did not significantly change the expression patterns of different ability of cyclin Ds. Indeed the importance of nuclear retention G1–S cell cycle proteins. Therefore, the reason why inhibition of cyclin D1 has been shown in that the mutant non-phospho- of GSK-3b function can counteract the P4 negative effect on cell rylatible form of cyclin D1 that is refractory to nuclear export cycle in cyclin D1 null uterine epithelial cells is mainly due to is highly potent in transforming NIH3T3 cells [21]. In addition, the restoration of cyclin D2 nuclear accumulation. These data the mechanisms of stimulation of cell proliferation without underlines the importance of cyclin D proteins in the regulation changing the expression level of cyclin D1 by E2 in this of the whole cell cycle machinery in response to hormones in vivo uterine system has been investigated and shown to in vivo. In this uterine epithelial system, P4counteracts E2 by act via GSK-3b [9]. Our findings further support the previous inhibiting phosphorylation of GSK-3b, which consequently data showing regulation of the subcellular localization of cy- inactivates cyclin D and stops cell cycle progression. clin Ds by steroid hormones and suggest that this may be a In summary, we show the mechanism that is responsible for key event in the malignant transition to hormone dependent cyclin D2 nuclear accumulation in an in vivo physiologically tumors. relevant system. These studies have further verified the impor- Interestingly, we found that the phosphorylation of b-cate- tance of GSK-3b in the regulation of cell cycle progression. nin is inhibited in E2 treated mice and reversed by P4E2 and Studies form the Roberts group have also reported that this is enacted by GSK-3b. In many systems the nuclear local- GSK-3b kinase can regulate cell proliferation through CDK2 ization of b-catenin is regulated by the Wnt pathway acting by controlling cyclin E degradation [24]. Therefore, these data through GSK-3b. Wnt signaling has been shown to be impor- suggest that targeting GSK-3b pathway might be a good strat- tant in steroid hormone regulated uterine growth [22]. How- egy for cancer therapy. ever, in our previous studies we have shown that the sex steroid hormone regulation of GSK-3b activity is exclusively Acknowledgements: We especially thank Dr. Pitor Sicinski for gener- through a PI-3kinase/AKT pathway [9] suggesting that this ously providing the cyclin D1 mutant mice. This work was supported b-catenin phosphorylation is regulated by this route and not by Chinese NSFC Grant 30600316 (to B.C.) and NIH RO1CA 89617 the Wnt one. The relationship between these two pathways re- (to J.W.P). Y. Wang et al. / FEBS Letters 581 (2007) 3069–3075 3075

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