help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2003-0890
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
144/12/5450    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rider, V.
Right arrow Articles by Seifert, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rider, V.
Right arrow Articles by Seifert, C.
Endocrinology Vol. 144, No. 12 5450-5458
Copyright © 2003 by The Endocrine Society

Transit of Rat Uterine Stromal Cells through G1 Phase of the Cell Cycle Requires Temporal and Cell-Specific Hormone-Dependent Changes on Cell Cycle Regulators

Virginia Rider, Eric Thomson and Clinton Seifert

Department of Biology, Pittsburg State University, Pittsburg, Kansas 66762

Address all correspondence and requests for reprints to: Dr. Virginia Rider, Pittsburg State University, 1701 South Broadway, Pittsburg, Kansas 66762. E-mail: vrider{at}pittstate.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Progesterone pretreatment increases the number of synchronously proliferating stromal cells in the ovariectomized rat uterus, but estrogen is necessary to stimulate reentry into the cell cycle. To investigate the mechanisms underlying differential hormone actions, sexually mature ovariectomized rats were injected with progesterone (2 mg) for three consecutive days. Estradiol 17-ß (0.6 µg) was administered to initiate cell proliferation. Uterine samples were collected at timed intervals. Cell entry into DNA replication was monitored by injecting 5-bromo-2'-deoxyuridine (1 mg/100 g body weight) 2 h before necropsy. Demicolchicine (400 µg) was injected 30 min before necropsy to assess transit into M phase. Temporal progress through G1 was determined by spatial changes in cyclin D1/D3 proteins. Total cyclin D1/D3 protein and mRNA was measured by Western and Northern blotting. Estrogen increased the number of 5-bromo-2'-deoxyuridine-positive stromal cells (P < 0.05), compared with the number in rats treated with progesterone alone. An increase (P < 0.05) in the number of M-phase cells occurred at 12 h post estrogen. There was no evidence for epithelial cell proliferation in response to steroid treatments. Cyclin D1/D3 mRNA was expressed in the uteri of ovariectomized and hormone treated rats. The D-type cyclin proteins, however, were not evident in stromal cells without estrogen treatment. Progesterone pretreatment inhibited estrogen-dependent epithelial cell proliferation while redirecting D-type cyclin expression to the uterine stroma. Stromal cell transit through G1 required nongenomic steroid-dependent action on signal transduction pathways that control the nuclear localization and cell type-specific expression of the D-type cyclin proteins.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GROWTH AND function of the mammalian uterus is controlled by the sex steroids estrogen and progesterone (reviewed in Refs. 1, 2, 3). These hormones exert their effects primarily through intracellular receptors that operate as transcription factors and alter the rates of target gene expression (4). In the endometrium of the mouse (1, 5) and rat (6), estrogen stimulates the proliferation of luminal and glandular epithelial cells during the first few days of pregnancy. With development of the corpus luteum, however, proliferation switches from the epithelial compartments to the endometrial stroma (1, 6, 7, 8). This proliferative switch correlates with increased expression of Hoxa-10 in the endometrial stroma of the mouse (9). Mutant mice lacking normal Hoxa-10 expression show defective progesterone-dependent stromal cell proliferation (10), suggesting Hoxa-10 is required for the proliferative switch. The signal transduction networks that stimulate cell-specific proliferation and the subsequent differentiation (decidualization) of these cells into the decidua are key events in the preparation of the endometrium of many mammals, including the human female, for pregnancy (11, 12). Progesterone is essential for these changes because progesterone receptor antagonists block the proliferative switch in both the rat (6) and mouse (13), and decidualization fails in the uteri of mice lacking the progesterone receptor by targeted mutagenesis (14).

Separating the differential effects of estrogen vs. progesterone on cell proliferation and differentiation in the uterine stroma is difficult because estrogen receptors increase the expression of progesterone receptors in stromal cells (15). Uterine stromal cells can be induced to differentiate into decidual cells in the absence of estrogen, but the cells must be exposed to progesterone pretreatment followed by mechanical trauma such as crushing the uterine horn with a hemostat (16). The molecular mechanism by which progesterone pretreatment prepares stromal cells to enter into the differentiation pathway in response to a traumatic stimulus is not known. However, mechanical stimulation increases the number of progesterone receptors in stromal cells by what appears to be a systemic effect (15). Because differentiation pathways are intimately connected with cell proliferation, progesterone may exert its effects on either the expression or activity of cell cycle regulatory proteins. The hormone can stimulate differentiation, however, only if there are sufficient progesterone receptors for the cells to respond.

Much of the mechanistic information about the steroid control of cell proliferation has come from studies of breast cancer cell lines (17, 18). Such studies have clearly shown that sex hormones directly control G1 cell cycle regulatory proteins and stimulate transit from G1 into S phase. Estrogen stimulates both c-myc and cyclin D1 expression resulting in the activation of cyclin E-Cdk2 complexes. These complexes are activated because estrogen down-regulates the cyclin-dependent kinase inhibitor p21, which becomes depleted from the active cyclin E-Cdk2 complex (18). Previous research (17, 19, 20, 21) suggests that sex hormones exert their regulatory effects on proteins that control both early and late events in G1 phase of the cell cycle. In T47D breast cancer cells, progesterone transiently increases c-fos and c-myc (17), both of which are considered early gene responses to proliferation signals. Progesterone plus growth factors with tyrosine kinase activity, such as epidermal growth factor, basic fibroblast growth factor, and transforming growth factor-{alpha}, stimulate increased expression of cyclin D1 in cultured rat uterine stromal cells (19). Stromal cell lines cultured with progesterone plus basic fibroblast growth factor show accelerated entry into S phase and the timing for transit through G1 into S phase is sustained (19). Increased expression of cyclin D3 in the pregnant mouse uterus is associated with proliferating uterine stromal cells (22), whereas the cell type and nuclear-specific induction of cyclin D1 and p27 occur in the endometrium of ovariectomized rats given estrogen (23). Progesterone pretreatment of ovariectomized rats followed by a single injection of estrogen increases 3- to 5-fold the number of synchronously proliferating uterine stromal cells (6, 21). Together, these studies support the idea that the early targets for hormone control of uterine cell proliferation include proteins that regulate progress through G1 and entry into S phase of the cell cycle.

This present study takes advantage of previous reports (6, 21) showing that progesterone pretreatment of ovariectomized rats stimulates a 3- to 5-fold increase in the number of synchronously proliferating uterine stromal cells in response to estrogen. This model system has been exploited in an attempt to clarify the mechanisms by which progesterone pretreatment of the uterine stroma increases the number of proliferating cells. Moreover, a second goal was to delineate the molecular mechanisms by which progesterone synchronizes reentry of stromal cells into the cell cycle in response to estrogen signaling. The results indicate that progesterone exerts cell-specific effects by inhibiting estrogen-dependent proliferation in the epithelium while promoting estrogen-dependent proliferation in the stroma. This proliferative response involves the cell-specific nuclear expression of cyclin D1 and D3 proteins in uterine stromal cells that occurs through estrogen-dependent posttranscriptional control.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and hormone treatments
Sexually mature (150–175 g body weight) Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) were bilaterally ovariectomized and rested for 10 d. Rats were housed on a 14-h light, 10-h dark cycle at Pittsburg State University and provided rodent chow and water ad libitum. Animals were treated in accordance with the principles and procedures outlined in the National Institutes of Health Guidelines for the Care and Use of Experimental Animals. Protocols for the care and use of animals were approved by Pittsburg State University Animal Care Committee. To promote stromal cell proliferation, ovariectomized rats were injected sc with progesterone (2 mg; Sigma Chemical Co., St. Louis, MO) dissolved in sesame oil daily for 3 consecutive days. On the fourth day, estradiol 17-ß (0.6 µg; Sigma) was injected sc. This hormone regimen increases the number of synchronously proliferating stromal cells 3- to 5-fold, compared with normally pregnant animals (6, 21). The uterine horns were removed at various time periods (times 0–24 h) after estradiol injection (Fig. 1Go).



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 1. Steroid regimen to induce synchronous proliferation of rat uterine stromal cells. Ovariectomized rats were injected sc with progesterone (2 mg) daily for 3 consecutive days (72 hP). On the fourth day (0 hE), estradiol-17ß (0.6 µg) was administered sc. The uterine tissues were collected for immunocytochemical analysis of BrdU incorporation, mitotic spindle formation, and cyclin D1/D3 spatial distribution after 72 h progesterone pretreatment (72 hP) and at timed intervals after estrogen injection. Uterine cyclin D1/D3 mRNA and total protein were measured from pooled samples from ovariectomized animals and those treated with progesterone at the indicated times and after estrogen administration at the indicated times.

 
Indirect immunoperoxidase analysis
Uterine tissue was removed under anesthesia, and the uterine horns were fixed in 4% paraformaldehyde and embedded in paraffin using methods standard in our laboratory (24). Sections (~8 µm) were cut on a microtome and placed on Superfrost Plus slides (Fisher Scientific, Hanover Park, IL). Sections were treated with 0.1 M glycine in PBS for 2 h. To remove endogenous peroxidase activity, tissue sections were quenched in 0.3% hydrogen peroxide (Sigma) in methanol at 22 C for 30 min. Samples were blocked for 18 h in a blocking buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.2% gelatin, 0.05% Tween 20, 0.5% (wt/vol) powdered milk] at 4 C. The slides were washed in PBS and reacted with cyclin D1 (40 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA) and cyclin D3 (2 µg/ml; C-16, Santa Cruz Biotechnology) antibodies for 18 h at 4 C. To evaluate specificity of the reaction, some sections were incubated without primary antibody. Primary antibody was reacted with biotinylated affinity-purified antimouse (cyclin D1) and antirabbit (cyclin D3) secondary antibodies (Vector Laboratories, Burlingame, CA) for 30 min at 22 C. Slides were exposed to the Vectastain ABC reagent, washed in PBS, and reacted for 2 min with equal volumes of 1 µg/ml diaminobenzidine (Aldrich, Milwaukee, WI) dissolved in 0.1 M Tris (pH 7.2), and 0.1% (vol/vol) hydrogen peroxide diluted in PBS. Slides were counterstained with 1% methyl green dye in deionized water. The uterine horns from at least three separate animals at each time point were examined. Representative sections were photographed using a BX41 microscope (Olympus, Tokyo, Japan) equipped with a digital camera.

Mitotic activity
Fifteen minutes before necropsy, demicolchicine (400 µg, Sigma) was injected sc to arrest mitotic activity (6). Uterine horns were removed under anesthesia at 6-h intervals from 0 to 24 h after ß-estradiol injection fixed in 4% paraformaldehyde and embedded in paraffin. Transverse sections of the uterus were deparaffinized in xylenes and rehydrated. Slides were submerged in hematoxylin (Accustain, Sigma Diagnostics) for 9 min and rinsed in water for 1 min. The slides were dipped twice in an acid alcohol solution, rinsed in water, and then dipped twice in ammonia water and rinsed. The slides were submerged for 2 min in eosin followed by dehydration in ethanol and xylenes. Coverslips were applied using Permount (Fisher Scientific). Cells with mitotic spindles were quantitated across the entire tissue section at each time point. The field for each tissue section was divided into quadrants at lower magnification. The positive cells were identified and confirmed at x100 magnification (Olympus BX41 microscope). The counts of positive cells were made by two independent observers on at least five sections from each rat and at least three animals for each time point.

Bromodeoxyuridine detection
Two hours before necropsy, 5-bromo-2'-deoxyuridine (BrdU) was diluted 1:1 (vol:vol) in Evans Blue dye (2% wt/vol) and injected (1 mg/100 g body weight) into the lateral tail vein. The uterine horns were removed under anesthesia, fixed in 4% paraformaldehyde, and embedded in paraffin using standard methods (24). Transverse sections of the uterus were deparaffinized in xylenes and rehydrated in PBS. The sections were incubated in 10% normal goat serum to block nonspecific binding. Primary BrdU antibody (mouse monoclonal antibody in PBS/glycerin) was diluted (1:10) in incubation buffer (66 mM Tris buffer, 0.66 mM MgCl2, 1 mM 2-mercaptoethanol) and applied to the sections for 30 min at 37 C in a humidified chamber. Secondary antibody (sheep antimouse Ig-alkaline phosphatase in triethanolamine buffer) was diluted (1:10) in PBS and applied for 30 min at 37 C in a humidified chamber. Sections were reacted with a color-substrate solution containing nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in substrate buffer (100 mM Tris-HCl buffer, 100 mM NaCl, 50 mM MgCl2, pH 9.5) for 20 min at 22 C according to the manufacturer’s protocol. To reduce nonspecific absorption by endogenous alkaline phosphatases, levamisole (0.01 mM, Sigma) was added to the color substrate. Coverslips were applied using Kaiser’s glycerin-mounting media (Roche, Indianapolis, IN). Counts of BrdU-positive cells within the entire stromal compartment were made at a magnification of x400 (Olympus BX41 microscope) on at least five sections from each animal and three rats for each time period.

Northern blots
Total RNA was isolated and pooled from the uterine horns of two rats at each time point using a single-step guanidine method (25). Samples (25 µg) of total RNA were dried under vacuum centrifugation, suspended in an RNA denaturing solution (0.4 M 3-N-morpholinopropanesulfonic acid, 0.1 M sodium acetate, 0.01 M EDTA, 2.2 M formaldehyde, 50% formamide), and loaded onto a 2.2 M formaldehyde-1% agarose gel. RNA was transferred by diffusion onto a nylon membrane (Micron Separation Inc., Westboro, MA) for 18 h. The membrane was baked for 2 h at 60 C under vacuum. Hybridization probes were prepared by random prime labeling (Ready-To Go DNA labeling beads, Amersham Biosciences, Piscataway, NJ) of the full-length murine cyclin D1, murine cyclin D3, and rat ß-actin cDNAs. Northern blots were hybridized in 5x SSPE (0.75 M NaCl, 0.05 M NaH2PO4·H2O, 5 mM EDTA), 5x Denhardt’s, 0.5% (wt/vol) sodium dodecyl sulfate (SDS) and 100 µg/ml sheared salmon testes DNA (Sigma) at 65 C for 18 h. Blots were washed using high stringency in 5x SSPE [0.75 M NaCl, 0.05 M NaH2PO4, 5 mM EDTA (adjusted to pH 7.4 with NaOH)]-0.5% SDS at 22 C, 1x SSPE-0.5% SDS at 37 C, and 0.1x SSPE-1% SDS at 65 C. Blots were exposed to x-ray film (Fuji, Fisher Scientific) using intensifying screens for 3–4 d at -80 C. The amount of cyclin D1 and cyclin D3 mRNA was determined by scanning densitometry using the NIH image software (Scion Image). Values for target mRNA were adjusted for assay variation by dividing the integrated OD of the respective cyclin mRNA by the integrated OD of ß-actin mRNA on the same blot.

Western blots
The uterine horns were removed and pooled from ovariectomized rats (n = 4 per experiment) without further treatment. Ovariectomized rats (n = 8 per experiment) were treated with progesterone (2 mg) for 3 consecutive days. The uterine horns were removed and pooled from four rats without further treatment (0 h estradiol, 0 hE; see Fig. 1Go). The other four animals were injected with estradiol, and the uterine horns were removed and pooled 6 h later (6 hE). Total uterine proteins were obtained by homogenization of the uterine horns in 5 volumes of homogenization buffer containing protease inhibitor cocktails as described by us previously (26). Protein extracts (500–1000 µl) containing similar amounts of protein for each of three independent assays were incubated with cyclin D1 or cyclin D3 antibody (1 µl antibody per 100 µl of extract) for 18 h at 4 C. The antigen-antibody complex was collected by immunoprecipitation with protein A (50 µl) for 3 h at 4 C. The samples were reprecipitated with an equal amount of primary antibody to determine whether cyclin D1and cyclin D3 were removed by the initial immunoprecipitation (19). Samples were centrifuged and washed three times in PBS containing 0.1 M NaCl. The samples were heated at 95 C for 3 min in SDS-sample buffer, cooled to 22 C, and the proteins size fractioned by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane using standard methods (19). The membrane was blocked in Tris-buffered saline (0 mM Tris, pH 7.4; 150 mM NaCl) containing gelatin (0.2%), powdered milk (5%), and Tween 20 (0.05%) at 4 C for 14 h. The proteins were reacted with primary antibody (1:250 cyclin D1, 1:200 cyclin D3) for 90 min at 22 C. The blots were washed and reacted with species-specific alkaline phosphatase secondary antibodies (Sigma) diluted 1:1000. After washing, bound antibody was detected by incubating the blots with 0.3 mg/ml nitroblue tetrazolium and 0.15 mg/ml 5-bromo-4-chloro-3-indolyl phosphate in 0.1 M Na HCO3, 1.0 mM MgCl2, pH 9.8. The size of reactive bands was determined from prestained molecular size standards (Bio-Rad Laboratories, Hercules, CA), and the relative amount of reactive species was quantified using scanning densitometry (Scion image).

Statistical analysis
Differences among treatments were examined by ANOVA, and differences among means were determined through Scheffé’s post hoc tests. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stromal cell entry into S phase
Hormonally treated rats were injected with BrdU 2 h before necropsy. The presence of BrdU was indicated by a blue color in positive cells (data not shown). Sections treated without primary BrdU antibody showed no reaction (data not shown). None of the glandular or luminal epithelial cells at any time after estrogen administration showed evidence of DNA replication (data not shown). Moreover, both the longitudinal and circular smooth muscle cells did not show evidence of BrdU incorporation. The number of positive stromal cells was low in the endometrium from rats in the absence of estrogen treatment (time 0 hE, Fig. 1Go) with an average of 0.8 BrdU-positive cells per section (Fig. 2Go). The number of positive cells increased (P < 0.05) at 6 h with an average of 49 BrdU-positive cells per uterine section (Fig. 2Go). Positive stromal cells in the endometrium at this time were distributed primarily in the periluminal region although some positive cells were scattered throughout the stromal extracellular matrix. A weakly reactive group of positive cells was identified in the antimesometrial region of the endometrium (data not shown). This spatial distribution of positive cells changed at 12 h such that there was a more even dispersal of reactive cells throughout the stromal compartment, compared with those at 6 h (data not shown). The number of positive cells (31 per section, Fig. 2Go) at 12 h was greater (P < 0.05) than at time 0 (0 hE). At 18 h after estrogen exposure, the number of BrdU-positive cells declined (P < 0.05), compared with the number at 6 h, with an average of 14 cells per section (Fig. 2Go). In the endometrium at 18 h post estrogen injection, the positive cells were dispersed evenly both around the uterine lumen and scattered throughout the stromal compartment (data not shown). At 24 h after estrogen administration, the number of BrdU-positive cells continued to decline (P < 0.05), compared with the maximum number at 6 h, with an average of 0.21 positive cells per section. Taken together, these data suggest a peak of transit into S phase between 6 and 12 h after estrogen administration.



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 2. Estrogen stimulates stromal cell entry into S phase. Ovariectomized rats were treated with progesterone (2 mg) for 3 consecutive days (72 hP). At d 4 (0 hE) estradiol-17ß (0.6 µg) was injected to stimulate entry into the cell cycle, and the uterine horns were removed at timed intervals (6–24 hE). BrdU was injected 2 h before necropsy and incorporation was detected by immunocytochemistry. Data are the mean ± SEM from at least five sections taken from three separate rats at each time point. *, P < 0.05, compared with progesterone treatment alone (0 hE). a vs. c and a vs. d, P < 0.05, Scheffé’s post hoc test.

 
Stromal cell entry into M phase
To assess transit through DNA replication and entry into M phase, the number of cells with colchicine-blocked mitotic spindles were counted in uterine sections after staining with hematoxylin-eosin. None of the glandular or luminal epithelial cells at any time after estrogen administration showed evidence of mitosis (data not shown). Uterine tissues from animals without estrogen exposure (0 hE) contained an average of 0.65 mitotic stromal cells per section (Fig. 3Go). The mitotic cells were scattered at random throughout the uterine stroma (data not shown). At 6 h after injection of estrogen, there was an average of 54 mitotic stromal cells per section (Fig. 3Go). Mitotic stromal cells were evident in the periluminal stroma except at the apical region of the mesometrial aspect of the endometrium (data not shown). Within the antimesometrial region, the greatest number of positive cells were located in the periluminal stroma. At 12 h post estrogen, there was an increase (P < 0.05) in the number of mitotic cells in the stroma, with an average of 190 cells containing mitotic spindles per uterine section (Fig. 3Go). The concentration of mitotic cells was most evident at the antimesometrial aspect of the endometrium (data not shown). At 18 h after estrogen administration, the number of mitotic stromal cells declined (P < 0.05), compared with the number at 12 h such that there was an average of 21 mitotic cells per section (Fig. 3Go). The majority of cells displaying mitotic spindles were located in the antimesometrial stroma, but some mitotic cells were also evident at the mesometrial aspect (data not shown). At 24 h after estrogen injection, there was a continued decline (P < 0.05) in mitotic cells with an average of 10.6 mitotic stromal cells per section. The distribution of cells blocked at M phase was generally within the periluminal stroma, similar to those at 6 h after estrogen.



View larger version (11K):
[in this window]
[in a new window]
  		FIG. 3. Estrogen stimulates stromal cell entry into M phase. Ovariectomized rats were treated with progesterone (2 mg) for 3 consecutive days (72 hP). At day 4 (0 hE) estradiol-17ß (0.6 µg) was injected to stimulate entry into the cell cycle, and the uterine horns were removed at timed intervals (6–24 hE). To detect mitotic spindle formation, as evidence for entry into M phase, colchicine was injected 30 min before necropsy. The number of mitotic spindles in uterine stromal cells were counted as detailed in the text. Data are the mean ± SEM from at least five sections taken from three separate rats at each time point. *, P < 0.05, compared with progesterone treatment alone (0 hE). (b vs. c, b vs. d, c vs. d, and c vs. e), P < 0.05, Scheffé’s post hoc test.

 
Temporal and spatial expression of cyclin D1 protein
By immunocytochemical analysis, cyclin D1 protein was expressed in the luminal and glandular epithelial cells of rats treated with progesterone for 72 h (0 hE, Fig. 4AGo). There was no evidence of cyclin D1 protein in the uterine stromal cells at this time. At 6 h after estrogen administration stromal cells that were distributed primarily in the periluminal region expressed cyclin D1 (Fig. 4BGo). Immunoreactive protein was distributed in the cytoplasm of the luminal and glandular epithelial cells, whereas in the stromal cells, cyclin D1 localized to the nuclei (Fig. 4BGo). At 12 h post estrogen, cyclin D1 was detected in the stromal cells extending from the periluminal region into the extracellular matrix of the stroma, particularly in the antimesometrial region (Fig. 4CGo). This spatial distribution of cyclin D1 expression continued at 18 h after estrogen administration, although there was a greater concentration of positive cells in the periluminal region (data not shown). At 24 h post estrogen, expression of cyclin D1 was notably concentrated in the periluminal stromal cells (data not shown). Uterine sections lacking primary antibody showed no reactivity (Fig. 4DGo).



View larger version (157K):
[in this window]
[in a new window]
  		FIG. 4. Estrogen stimulates the cell-specific expression of cyclin D1 protein. Ovariectomized rats were injected with progesterone (2 mg) daily for 3 consecutive days. At d 4 estradiol 17-ß (0.6 µg) was administered to stimulate stromal cell entry into the cell cycle. In the absence of estrogen (0 hE, A) cyclin D1 was absent from uterine stromal cells. At 6 h post estrogen (6 hE, B) cyclin D1 was expressed in the nuclei of periluminal uterine stromal cells. Nuclear expression of cyclin D1 was evident at 12 h post estrogen (12 hE, C), and the distribution of positive cells extended out from the periluminal stroma. In the absence of primary antibody (D), cyclin D1 immunoreactivity was absent. Arrows indicate representative cyclin D1-positive stromal cells. Original magnification, x400.

 
Temporal and spatial expression of cyclin D3 protein
Cyclin D3 protein was not evident in the endometrium of rats treated with progesterone alone for 72 h (0 hE, Fig. 5AGo). The myometrium did not react with cyclin D3 antibody after progesterone or progesterone plus estrogen treatment (data not shown). The cytoplasm of the luminal epithelium was strongly immunoreactive at 6 h post estrogen administration. At this same time, cyclin D3 was also evident in the periluminal stromal cells. Some stromal cells showed cytoplasmic expression, whereas in other cells the protein localized in the nucleus (Fig. 5BGo). At 12 h after estrogen exposure, immunoreactive stromal cells were found primarily in the periluminal stroma (Fig. 5CGo) and in stromal cells of the antimesometrial region. The luminal epithelium was negative at this time, but a strong immunoreactive band that appeared to be associated with the basil lamina was evident (Fig. 5CGo). At 18 h post estrogen, cyclin D3 expression was detected in the uterine stromal cells, and its distribution was either nuclear or cytoplasmic (Fig. 5DGo). The luminal epithelial cells were negative, and the immunoreactive band that was associated with the basil lamina was no longer evident. A similar expression pattern to that at 18 h was observed at 24 h after estrogen injection (data not shown). In the absence of primary antibody, no immunoreactivity was evident (Fig. 5EGo).



View larger version (154K):
[in this window]
[in a new window]
  		FIG. 5. Estrogen stimulates the cell-specific expression of cyclin D3 protein. Ovariectomized rats were injected with progesterone (2 mg) daily for 3 consecutive days. At d 4 estradiol 17-ß (0.6 µg) was administered to stimulate stromal cell entry into the cell cycle. In the absence of estrogen (0 hE, A), cyclin D3 was absent from uterine stromal cells. At 6 h after estrogen exposure (6 hE, B) cyclin D3 was expressed in the cytoplasm of luminal epithelial cells and the nuclei of stromal cells (shown by arrows). At 12 h post estrogen (12 hE, C), the epithelial cells no longer expressed cyclin D3, whereas strong nuclear expression was still evident in uterine stromal cells (shown by arrows). At 18 h after estrogen administration (18 hE), cyclin D3 expression was absent from the epithelium and fewer cells in the stroma were reactive. In the absence of primary antibody, no immunoreactivity was detected (E). Proliferating human tonsil cells (F) showed a similar immunoreactive pattern to uterine stromal cells. Original magnification, x1000.

 
Hormonal priming is not required for cyclin D1 mRNA expression
Northern blot analysis of rat uterine total RNA revealed transcripts for cyclin D1 in the rat endometrium (Fig. 6AGo). A single-sized mRNA consistent with the 3.7-kb size of rat cyclin D1 (27) was expressed in the uteri of ovariectomized rats and after progesterone and estrogen injections (Fig. 6AGo). When cyclin D1 mRNA was adjusted to rat ß-actin, there was approximately 2-fold less cyclin D1 mRNA at 6 h post estrogen (6 hE), compared with ovariectomized rats (Fig. 6BGo). Although there was some variation in the amount of mRNA at the time points sampled after steroid treatment, there were no statistically significant (P > 0.05) differences before and after hormone treatments.



View larger version (52K):
[in this window]
[in a new window]
  		FIG. 6. Hormonal priming is not required for cyclin D1 mRNA expression. Ovariectomized rats were injected with progesterone (2 mg) daily for 3 consecutive days. At d 4 estradiol 17-ß (0.6 µg) was administered to stimulate stromal cell entry into the cell cycle. Uterine horns were removed at the times indicated, and total RNA was isolated from the uteri of two animals at each time point. Cyclin D1 mRNA was analyzed by Northern blotting. The same blot was rehybridized with rat ß-actin to control for assay variation. A, Cyclin D1 mRNA was present in ovariectomized (OVX) rats and those treated with progesterone for 24, 48, and 72 h. Cyclin D1 mRNA was also present in the uteri of rats pretreated with progesterone followed by estradiol 17-ß at timed intervals (6–24 h). B, Steady-state cyclin D1 mRNA levels were measured from autoradiographs of Northern blots. Values were adjusted using ß-actin mRNA to control for assay variation as described in the text. Data shown are the mean ± SEM adjusted cyclin D1 mRNA levels from triplicate samples and two independent experiments. No differences in cyclin D1 mRNA were measured.

 
Hormonal priming is not required for cyclin D3 mRNA expression in the rat uterus
A single-sized mRNA consistent with the 1.8-kb size of rat cyclin D3 (28) was present in ovariectomized rats, those pretreated with progesterone, and rats primed with progesterone and injected with estrogen (Fig. 7AGo). The level of cyclin D3 mRNA decreased approximately 2-fold in rats 6 h after estrogen (6 hE) treatment, compared with the amount in ovariectomized and progesterone-treated animals (Fig. 7BGo). Although there was some variation in the amount of cyclin D3 mRNA among the various time points, the differences were not statistically significant (P > 0.05).



View larger version (55K):
[in this window]
[in a new window]
  		FIG. 7. Hormonal priming is not required for cyclin D3 mRNA expression. Ovariectomized rats were injected with progesterone (2 mg) daily for 3 consecutive days. At d 4 estradiol 17-ß (0.6 µg) was administered to stimulate stromal cell entry into the cell cycle. Uterine horns were removed at the times indicated, and total RNA was isolated from the uteri of two animals at each time point. Cyclin D3 mRNA was analyzed by Northern blotting. The same blot was rehybridized with rat ß-actin to control for assay variation. A, Cyclin D3 mRNA was present in ovariectomized (OVX) rat uteri and the uteri of those treated with progesterone for 24, 48, and 72 h. Cyclin D3 mRNA was also present in the uteri of rats pretreated with progesterone followed by estradiol 17-ß at timed intervals (6–24 h). B, Steady-state cyclin D3 mRNA levels were measured from autoradiographs of Northern blots. Values were adjusted using ß-actin mRNA to control for assay variation as described in the text. Data shown are the mean ± SEM adjusted cyclin D3 mRNA levels from triplicate samples and two independent experiments. No differences (P > 0.05) in cyclin D3 mRNA were measured.

 
Estrogen increases the expression of cyclin D1 and D3 proteins
A single reactive species consistent with the size of cyclin D1 (34 kDa) was immunoprecipitated from uterine extracts of steroid-treated rats. Cyclin D1 protein was not detected in uterine extracts obtained from ovariectomized rats (data not shown). The amount of cyclin D1was low in the uteri of rats treated with progesterone for 72 h (Fig. 8Go, P). At 6 h post estrogen administration, the amount of protein increased more than 2-fold (Fig. 8Go, P+E). Cyclin D3 (34 kDa) was immunoprecipitated from uterine proteins of steroid-treated rats (Fig. 8Go). The protein was not detected in ovariectomized rats (data not shown). The protein was evident in rats treated for 3 consecutive days with progesterone (Fig. 8Go, P). The amount of cyclin D3 increased more than 2-fold at 6 h post estrogen exposure (Fig. 8Go, P+E). Similar results were obtained from three independent assays.



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 8. Cyclin D1/D3 protein is detected in the endometrium of steroid-treated rats. Uterine proteins were isolated from rats pretreated with progesterone (P) for 3 d, and the uterine horns were collected. Some progesterone-pretreated rats were injected with estradiol 17-ß (0.6 µg), and the uterine horns were collected 6 h later (E). Cyclin D1 and D3 were immunoprecipitated and analyzed by Western blotting as described in the text.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cell proliferation in the mammalian uterus is controlled by the female sex hormones estrogen and progesterone. In the present study, we used a model system in which progesterone pretreatment followed by a physiological dose of estrogen increases the number of synchronously proliferating rat uterine stromal cells. This model offers important advantages for the study of sex steroid control of G1 transit because the number of synchronously dividing stromal cells increases 3- to 5-fold over that in normally pregnant rats (6). As shown in this study, progesterone pretreatment followed by a single injection of estrogen results in the temporal transit of stromal cells through G1 into S phase. This progression is accompanied by changes in the temporal appearance and spatial distribution of cyclin D1 and D3 proteins. Two important differences in response to sex steroids between epithelial and stromal cells emerged during this study. First, progesterone pretreatment of the endometrium prevents nuclear localization of the D-type cyclins in the epithelial cells and the cells do not proliferate in response to estrogen signaling. Second, progesterone pretreatment of the uterus inhibits stromal cell expression of D-type cyclin proteins until the cells are exposed to estrogen. The consequence of estrogen-dependent signaling on the stroma is nuclear localization of cyclin D1 and D3 proteins. Importantly, this nuclear expression correlates with cellular entry into S phase (compare Figs. 2–4GoGoGo).

Mitogenic signals, including those arising from steroid hormone action on target cells, stimulate signal transduction pathways that promote entry into the cell cycle and induce the D-type cyclins to assemble with their catalytic partners (reviewed in Ref. 29). The D-type cyclins complex with cyclin dependent kinases (CDK) 4/6 and enter the nucleus in which they must be phosphorylated to initiate the kinase signaling cascade necessary to activate the numerous genes involved in DNA replication. In the endometrium of ovariectomized rats, estrogen is a mitogenic signal for epithelial cell proliferation (1, 12, 30, 31). However, in response to a physiological dose of estrogen, we now show that epithelial cells in the progesterone-pretreated endometrium cannot incorporate BrdU, form mitotic spindles, or show the morphology characteristic of epithelial cells stimulated by estrogen to proliferate as detailed by us (6) and others (32). Furthermore, progesterone pretreatment of the rat uterus does not inhibit cyclin D1 expression in the epithelial cells because the protein is present in the cytoplasm of uterine epithelial cells pretreated with progesterone (0 hE) and at all time points after estrogen administration (see Fig. 4Go). Thus, progesterone pretreatment inhibits the ability of the epithelial cells to respond to the normal mitogenic signal transmitted by estrogen. This signal seems to be critical for the nuclear localization of the D-type cyclins.

The D-type cyclins act as sensors and convert incoming signals through signal transduction cascades that stimulate cellular entry into DNA replication. Early events in response to a mitogenic signal such as estrogen include the assembly of the D-type cyclins with CDK4/6 followed by transport of the complexes into the nucleus (reviewed in Ref. 29). This study shows that despite estrogen signaling, progesterone pretreatment inhibits the nuclear localization of cyclin D1 and D3 in the epithelial cell nuclei and proliferation is blocked. Previous findings (33) showed that progesterone inhibits epithelial proliferation in the mouse uterus even if the endometrium is primed with estrogen before progesterone exposure. Similar to our observations, Tong and Pollard (33) reported that cyclin D1 was expressed in the cytoplasm of epithelial cells, suggesting that progesterone blocked cyclin D1 translocation from the cytosol to the nucleus. This postulated mechanism was supported further by immunoprecipitation analysis of epithelial extracts that confirmed cyclin D1-CDK complexes assembled, but the complexes seemed unable to shuttle from the cytoplasm to the nucleus (33). Together with the present findings, these results suggest that progesterone blocks estrogen signaling events on the uterine epithelium, and the block is effective without or with progesterone pretreatment. Furthermore, the results of this study suggest the signaling mechanism behind the block prevents the nuclear localization of the D-type cyclins and thereby inhibits G1-related events.

Evidence in other cell types suggests possible mechanisms by which cell-specific effects of sex steroids could occur on epithelium vs. stroma. We propose that cyclin D1/CDK complexes form and enter the nucleus. The turnover of this complex is generally rapid and requires the phosphorylation of a threonine residue (Thr-286) located near the carboxyl terminus of cyclin D1. Phosphorylation of Thr-286 is mediated by glycogen synthase kinase-3ß (GSK-3ß), and cells that express high levels of GSK-3ß rapidly turn over cyclin D1, ensuring export from the nucleus and degradation of the protein in the absence of mitogen (34). Inhibition of GSK-3ß action through activation of the Ras signal transduction pathway prevents the phosphorylation of cyclin D1 (34). In the context of the current study, nuclear localization of cyclin D1 in uterine stromal cells may occur because estrogen-dependent signaling activates the Ras signal transduction pathway. Estrogen has been shown to activate Ras and stimulate Akt (protein kinase B) phosphorylation in neuronal cells (35, 36). Such activation would block GSK-3ß-mediated phosphorylation of cyclin D1, and the complex would be retained in the nucleus. This potential mechanism is supported from studies of a mutant version of cyclin D1 that cannot be phosphorylated and remains in the nucleus throughout the cell cycle (34). Moreover, inhibition of GSK-3ß synthesis results in the accumulation of cyclin D1 in the nuclei of estrogen-stimulated MCF-7 breast cancer cells (37). Our data are also consistent with the idea that cyclin D1 in the uterine epithelium is hyperphosphorylated, perhaps because the epithelial cells contain high levels of GSK-3ß, leading to a rapid turnover of the protein and consistent cytoplasmic localization. Rapid turnover of cyclin D3 may account for the immunoreactive band associated with the basil lamina at 12 h, which is followed by nondetectable expression of the protein in the epithelium at 18 h (compare Fig. 5Go, C and D).

GSK-3ß activation is also regulated by cyclin-dependent kinase inhibitors (CKIs). Evidence suggests that CKIs actually play a pivotal role in the positive regulation of G1 transit because these inhibitors regulate GSK-3ß activation. Both of the CKIs, p21 and p27, promote the nuclear accumulation of cyclin D1 complexes by inhibiting nuclear export (38). This is mediated by p21 (and presumably p27) inhibition of GSK-3ß-dependent cyclin D1 nuclear export. Estrogen has been shown to increase the expression of p27 in rat uterine stromal cells but not in epithelial cells (23). Further studies on the cell-specific effects of steroid hormones on the regulation of CKIs and GSK-3ß in the uterus are now warranted.

The results from this study indicate that sex steroids not only affect the cell-specific localization of cell cycle regulators but also the appearance of the D-type cyclin proteins in uterine stromal and epithelial cells. It is of interest that one of the downstream targets in the Ras signal transduction pathway is eukaryotic initiation factor 4E-binding protein 1 (39). Activation of the Ras signal transduction pathway by estrogen is expected to lead to the phosphorylation of Akt, an established intermediate in the signal transduction response of neuronal cells to estrogen (35, 36). Estrogen signaling could stimulate translational regulation by stimulating Ras and thereby increase the phosphorylation of 4E-binding protein 1, leading to more efficient eIF4E-dependent translation (40, 41). Overexpression of eIF4E has been shown to augment the translation of cyclin D1 mRNA (41). This unexpected role of steroid hormones in regulating gene expression by translational mechanisms has not been widely reported. The present results, coupled with previous evidence (42, 43), suggest that future analysis of this gene regulatory mechanism will be important for more fully understanding the response of target cells to sex hormone action.

The findings from the present study are consistent with the idea that estrogen action can activate signal transduction pathways that control the appearance of some cell cycle regulatory proteins from previously localized transcripts independent of transcriptional regulation. These effects are cell specifically regulated in the uterine stromal cells. Moreover, estrogen signaling stimulates nuclear localization of these proteins in the stromal cells, but the progesterone-dependent block on uterine epithelial cells prevents nuclear localization of the D-type cyclins and proliferation is inhibited. Together, these findings indicate that some estrogen-dependent effects on target cells occur through nongenomic regulation of specific signal transduction pathways. Activation of these pathways stimulates the cell-specific expression and localization of G1 cell cycle regulators.


   Acknowledgments
 
The murine cyclin D1 and cyclin D3 cDNAs were gifts from Dr. Charles Sherr (St. Jude’s Children Hospital, Nashville, TN). We thank Dr. Michael Soares for the rat ß-actin cDNA. We acknowledge Malcolm Turner (Pittsburg State University) and Jim Swafford for help with the figures.


   Footnotes
 
This work was supported in part by a grant from the National Science Foundation (IBN-0091504) and the NIH Grant RR-16475 from the Biomedical Research Infrastructure Network Program of the National Center for Research Resources. C.S. is a K-BRIN scholar.

Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; CDK, cyclin-dependent kinase; CKI, cyclin-dependent kinase inhibitor; GSK-3ß, glycogen synthase kinase-3ß; SDS, sodium dodecyl sulfate.

Received July 17, 2003.

Accepted for publication August 21, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Finn CA, Martin L 1967 Patterns of cell division in the mouse uterus during early pregnancy. J Endocrinol 39:593–597
  2. Rider V 2001 Progesterone and the control of uterine cell proliferation and differentiation. Front Biosci 7:D1545–D1555
  3. Clarke CL, Sutherland RL 1990 Progestin regulation of cellular proliferation. Endocr Rev 11:266–301[Medline]
  4. Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  5. Quarmby VE, Korach KS 1984 The influence of 17-ß estradiol on patterns of cell division in the uterus. Endocrinology 114:694–702[Abstract]
  6. Rider V, Psychoyos A 1994 Inhibition of progesterone receptor function results in loss of basic fibroblast growth factor expression and stromal cell proliferation during uterine remodeling in the pregnant rat. J Endocrinol 140:239–249[Abstract]
  7. Galassi L 1968 Autoradiographic study of the decidual cell reaction in the rat. Dev Biol 17:75–84[CrossRef][Medline]
  8. Finn CA 1966 Endocrine control of endometrial sensitivity during the induction of the decidual cell reaction in the mouse. J Endocrinol 36:239–248[Medline]
  9. Lim H, Ma L, Ma W-G, Maas RL, Dey SK 1999 Hoxa-10 regulates uterine stromal cell responsiveness to progesterone during implantation and decidualization in the mouse. Mol Endocrinol 13:1005–1017[Abstract/Free Full Text]
  10. Yao MWM, Lim H, Schust DJ, Choe SE, Farago A, Ding Y, Michaud S, Church GM, Maas RL 2003 Gene expression profiling reveals progesterone-mediated cell cycle and immunoregulatory roles of Hoxa-10 in the preimplantation uterus. Mol Endocrinol 17:610–627[Abstract/Free Full Text]
  11. Noyes RW, Hertig AT, Rock J 1950 Dating of the endometrial biopsy. Fertil Steril 1:3–25
  12. Martin L, Finn CA 1968 Hormonal regulation of cell division in epithelial and connective tissues of the mouse uterus. J Endocrinol 41:363–371[Medline]
  13. Cullingford RW, Pollard JW 1988 RU486 completely inhibits the action of progesterone on cell proliferation in the mouse uterus. J Reprod Fertil 83:909–914[Abstract]
  14. Lyndon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery Jr CA, Shyamala G, Conneely OM, O’Malley BW 1995 Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev 9:2266–2278[Abstract/Free Full Text]
  15. Kurita T, Lee K-J, Saunders PTK, Cooke PS, Taylor JA, Lubahn DB, Zhao C, Makela S, Gustafsson J-A, Dahiya R, Cunha GR 2001 Regulation of progesterone receptors and decidualization in uterine stroma of the estrogen receptor-{alpha} knockout mouse. Biol Reprod 64:272–283[Abstract/Free Full Text]
  16. Shelesnyak MC 1933 The production of deciduomata in spayed immature rats after estrin and progestin treatment. Anat Rec 56:211–217[CrossRef]
  17. Musgrove EA, Sutherland RL 1994 Cell cycle control by steroid hormones. Cancer Biol 5:381–389
  18. Doisneau-Sixou SF, Sergio CM, Carroll JS, Hui R, Musgrove EA, Sutherland RL 2003 International Congress on Hormonal Steroids and Hormones and Cancer: estrogen and anitestrogen regulation of cell cycle progression in breast cancer cells. Endocr Relat Cancer 10:179–186[Abstract]
  19. Jones SR, Kimler BF, Justice WM, Rider V 2000 Transit of normal rat uterine stromal cells through G1 phase of the cell cycle requires progesterone-growth factor interaction. Endocrinology 141:637–648[Abstract/Free Full Text]
  20. Rider V, Kimler BF, Justice WM 1998 Progesterone-growth factor interactions in uterine stromal cells. Biol Reprod 59:464–469[Free Full Text]
  21. Tachi C, Tachi S, Lindner HR 1972 Modification by progesterone of oestradiol-induced cell proliferation, RNA synthesis and oestradiol distribution in the rat uterus. J Reprod Fertil 31:59–76[Medline]
  22. Tan J, Raja S, Davis MK, Tawfik O, Dey SK, Das SK 2002 Evidence for coordinated interaction of cyclin D3 with p21 and cdk6 in directing the development of uterine stromal cell decidualization and polyploidy during implantation. Mech Dev 111:99–1113[CrossRef][Medline]
  23. Zhuang Y-H, Sarca D, Weisz A, Altucci L, Cicatiello L, Rollerova E, Tuohimaa P, Ylikomi T 2001 Cell type-specific induction of cyclin D and cyclin-dependent kinase inhibitor p27kip1 expression by estrogen in rat endometrium. J Steroid Biochem Mol Biol 78:193–199[Medline]
  24. Rider V, Piva M, Cohen ME, Carlone DL 1995 Alternative splicing and differential targeting of fibroblast growth factor receptor 1 in the pregnant rat uterus. Endocrinology 136:3137–3145[Abstract]
  25. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  26. Rider V, Peterson CJ 1991 Activation of uteroglobin gene expression by progesterone is modulated by uterine-specific promoter-binding proteins. Mol Endocrinol 5:911–920[Abstract]
  27. Tamura K, Kanaoka Y, Jinno S, Nagata A, Ogiso Y, Shimizu K, Hayakawa T, Nojima H, Okayama H 1993 Cyclin G: a new mammalian cyclin with homology to fission yeast. Oncogene 8:2113–2118[Medline]
  28. Hosokawa Y, Onga T Nakashima K 1994 Induction of D2 and D3 cyclin-encoding genes during promotion of the G1/S transcription by prolactin in rat Nb2 cells. Gene 147:249–252[CrossRef][Medline]
  29. Sherr CJ, Roberts JM 1999 CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13:1501–1512[Free Full Text]
  30. Chen B, Pollard JW 2003 Cyclin D2 compensates for the loss of cyclin D1 in estrogen induced mouse uterine epithelial cell proliferation. Mol Endocrinol 17:1368–1381[Abstract/Free Full Text]
  31. Hastings JM, Licence DR, Burton GJ, Charnock-Jones DS, Smith SK 2003 Soluble vascular endothelial growth factor receptor 1 inhibits edema and epithelial proliferation induced by 17ß-estradiol in the mouse uterus. Endocrinology 144:326–334[Abstract/Free Full Text]
  32. Gunin AG, Emelianov VY, Tolmachev AS 2003 Expression of estrogen receptor-{alpha}, glucocorticoid receptor, ß-catenin and glycogen synthase kinase-3ß in the uterus of mice following long-term treatment with estrogen and glucocorticoid hormones. Eur J Obstet Gynecol Reprod Biol 107:62–67[CrossRef][Medline]
  33. Tong W, Pollard JW 1999 Progesterone inhibits estrogen-induced cyclin D1 and cdk4 nuclear translocation, cyclin E- and cyclin A-cdk2 kinase activation, and cell proliferation in uterine epithelial cells in mice. Mol Cell Biol 19:2251–2264[Abstract/Free Full Text]
  34. Diehl JA, Cheng M, Roussel MF, Sherr CJ 1998 Glycogen synthase kinase-3ß regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 12:3499–3511[Abstract/Free Full Text]
  35. Ivanova T, Mendez P, Garcia-Segura LM, Beyer C 2002 Rapid stimulation of the PI3-kinase/Akt signaling pathway in developing midbrain neurons by oestrogen. J Neuroendocrinol 14:73–79[CrossRef][Medline]
  36. Akama KT, McEwen BS 2003 Estrogen stimulates postsynaptic density-95 rapid protein synthesis via the Akt/protein kinase B pathway. J Neurosci 23:2333–2339[Abstract/Free Full Text]
  37. Hamelers IH, van Schaik RF, Sipkema J, Sussenback JS, Steenbergh PH 2002 Insulin-like growth factor I triggers nuclear accumulation of cyclin D1 in MCF-7S breast cancer cells. J Biol Chem 277:47645–47652[Abstract/Free Full Text]
  38. Alt JR, Gladden AB, Diehl JA 2002 p21 (Cip1) promotes cyclin D1 nuclear accumulation via direct inhibition of nuclear export. J Biol Chem 277:8517–8523[Abstract/Free Full Text]
  39. Gingras AC, Kennedy SG, O’Leary MA, Sonenberg N, Hay N 1998 4EBP1, a repressor of mRNA translation, is phosphorylated and inactivated by the Akt (PKB) signaling pathway. Genes Dev 12:502–513[Abstract/Free Full Text]
  40. Meric F, Hunt KK 2002 Translation initiation in cancer: a novel target for therapy. Mol Cancer Therapeut 1:971–979[Abstract/Free Full Text]
  41. Rosenwald IB, Kaspar R, Rousseau D, Gehke L, Leboulch P, Chen JJ, Schmidt EV, Sonenber N, London IM 1995 Eukaryotic translation initiation factor 4E regulates expression of cyclin D1 and transcriptional and post-transcriptional levels. J Biol Chem 270:21176–21180[Abstract/Free Full Text]
  42. Mora GR, Mahesh VB 1999 Autoregulation of the androgen receptor at the translation level: testosterone induces accumulation of androgen receptor mRNA in the rat ventral prostate polyribosomes. Steroids 64:587–591[CrossRef][Medline]
  43. Whelly SM, Barker KL 1974 Early effect of estradiol on the peptide elongation rate by uterine ribosomes. Biochemistry 13:341–346[Medline]



This article has been cited by other articles:


Home page
 J EndocrinolHome page
V. Rider, K. Isuzugawa, M. Twarog, S. Jones, B. Cameron, K. Imakawa, and J. Fang
Progesterone initiates Wnt-{beta}-catenin signaling but estradiol is required for nuclear activation and synchronous proliferation of rat uterine stromal cells
J. Endocrinol., December 1, 2006; 191(3): 537 - 548.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
L. M Walter, P. A W Rogers, and J. E Girling
The role of progesterone in endometrial angiogenesis in pregnant and ovariectomised mice
Reproduction, June 1, 2005; 129(6): 765 - 777.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
144/12/5450    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rider, V.
Right arrow Articles by Seifert, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rider, V.
Right arrow Articles by Seifert, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals