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Originally published In Press as doi:10.1074/jbc.M306340200 on September 3, 2003

J. Biol. Chem., Vol. 278, Issue 47, 46862-46868, November 21, 2003
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Vitamin D Inhibits G1 to S Progression in LNCaP Prostate Cancer Cells through p27Kip1 Stabilization and Cdk2 Mislocalization to the Cytoplasm*

Eddy S. Yang and Kerry L. Burnstein{ddagger}

From the Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33136

Received for publication, June 16, 2003 , and in revised form, August 14, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
1,25-(OH)2 vitamin D3 (1,25-(OH)2D3) exerts antiproliferative effects via cell cycle regulation in a variety of tumor cells, including prostate. We have previously shown that in the human prostate cancer cell line LN-CaP, 1,25-(OH)2D3 mediates an increase in cyclin-dependent kinase inhibitor p27Kip1 levels, inhibition of cyclin-dependent kinase 2 (Cdk2) activity, hypophosphorylation of retinoblastoma protein, and accumulation of cells in G1. In this study, we investigated the mechanism whereby 1,25-(OH)2D3 increases p27 levels. 1,25-(OH)2D3 had no effect on p27 mRNA levels or on the regulation of a 3.5-kb fragment of the p27 promoter. The rate of p27 protein synthesis was not affected by 1,25-(OH)2D3 as measured by luciferase activity driven by the 5'- and 3'-untranslated regions of p27 that regulate p27 protein synthesis. Pulse-chase analysis of 35S-labeled p27 revealed an increased p27 protein half-life with 1,25-(OH)2D3 treatment. Because Cdk2-mediated phosphorylation of p27 at Thr187 targets p27 for Skp2-mediated degradation, we examined the phosphorylation status of p27 in 1,25-(OH)2D3-treated cells. 1,25-(OH)2D3 decreased levels of Thr187 phosphorylated p27, consistent with inhibition of Thr187 phosphorylation-dependent p27 degradation. In addition, 1,25-(OH)2D3 reduced Skp2 protein levels in LNCaP cells. Cdk2 is activated in the nucleus by Cdk-activating kinase through Thr160 phosphorylation and by cdc25A phosphatase via Thr14 and Tyr15 dephosphorylation. Interestingly, 1,25-(OH)2D3 decreased nuclear Cdk2 levels as assessed by subcellular fractionation and confocal microscopy. Inhibition of Cdk2 by 1,25-(OH)2D3 may thus involve two mechanisms: 1) reduced nuclear Cdk2 available for cyclin binding and activation and 2) impairment of cyclin E-Cdk2-dependent p27 degradation through cytoplasmic mislocalization of Cdk2. These data suggest that Cdk2 mislocalization is central to the antiproliferative effects of 1,25-(OH)2D3.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3)1 exerts important effects on cellular proliferation and differentiation (1–4). 1,25-(OH)2D3 activates the vitamin D receptor (VDR), a ligand-dependent transcription factor that binds cis-acting DNA sequences known as vitamin D response elements (5). Several established human prostate cancer cell lines, as well as primary cultures of benign and cancerous prostatic tissue, express functional VDRs and are growth inhibited by 1,25-(OH)2D3 (6–12).

Our previous studies established that the initial growth inhibition of LNCaP and its androgen-independent derivative LNCaP-104R1 by 1,25-(OH)2D3 correlates with an increase in the levels of the cyclin-dependent kinase inhibitors (CKIs) p21WAF1,CIP1 and p27Kip1, a profound decrease in cyclin-dependent kinase 2 (Cdk2) activity, hypophosphorylation of pRb, and accumulation of cells in the G1 phase of the cell cycle (12, 13). 1,25-(OH)2D3 also decreased transcriptional activity of the E2F transcription factor family, which regulates the expression of genes necessary for S phase entry (12).

The CKI p27 appears to play a more central role than p21 in growth inhibition mediated by 1,25-(OH)2D3 and its analogs (13, 14). In several prostate cancer cell lines, 1,25-(OH)2D3 treatment results in persistent up-regulation of p27, whereas p21 is only induced transiently (12–14). In addition, 1,25-(OH)2D3-mediated growth inhibition of the androgen-independent LNCaP-derivative LNCaP-104R1 cells occurs without induction of p21 (13).

Loss of p27 expression correlates with prostate cancer recurrence, a more aggressive phenotype, and decreased patient prognosis and survival (15–22). Reduced p27 protein, however, is not the result of p27 gene mutations, which are quite rare in cancers (23–25). Activation of oncogenic signaling pathways ultimately results in accelerated p27 proteolysis and decreased p27 levels (26–29). Understanding the mechanisms whereby p27 levels are regulated may yield new targets for potential anticancer agents. Because of the emerging role of p27 in controlling prostate cancer growth, we further investigated the mechanism of 1,25-(OH)2D3 regulation of this CKI in LNCaP cells.

p27 is an important regulator of the G1 to S phase transition. p27 binds and inhibits cyclin E/Cdk2 and thereby negatively regulates S phase entry. To traverse G1, cellular p27 levels must decrease. A major mechanism to achieve this regulation involves ubiquitin-dependent p27 proteolysis (30, 31). Two rate-limiting steps for this process include phosphorylation at Thr187 by Cdk2 and recognition of Thr187-phospho-p27 by the SCFskp2 ubiquitination system (17, 32–36). Recent studies have revealed a novel pathway for p27 degradation at the G0/early G1 phase of the cell cycle that is independent of Thr187 phosphorylation (37–39). Although not well understood, this pathway is thought to be activated by mitogenic signaling during early G1 prior to Cdk2 activation. This initial phase of p27 degradation then facilitates Cdk2 activation, which results in further p27 degradation in late G1 and ultimately entry into S phase. Although this pathway still involves the ubiquitin-proteasome system, it is independent of Thr187 phosphorylation and may be Skp2-independent (37–39). The exact mechanisms of this novel pathway for p27 degradation remain to be resolved.

Evidence also exists for transcriptional (40, 41) and translational (31, 42–45) regulation of p27. Synthesis of p27 mRNA is governed by the Forkhead transcription factor family (46, 47). These transcription factors can be negatively regulated by the phosphatidylinositol 3-kinase/Akt growth signaling pathway, which is dysregulated in many cancers, including prostate (27). In addition, 1,25-(OH)2D3 can activate the p27 promoter by enhancing the binding of NF-Y and Sp1 transcription factors to the p27 promoter (41).

The 5'-untranslated region (UTR) of the p27 mRNA regulates the translation of p27. In quiescent cells, p27 translation is enhanced despite an overall decrease in the synthesis of most other proteins (31, 42–45). The translation of most proteins involves recognition of the mRNA 5' 7-methylguanosine cap by the eukaryotic initiation factor 4E. Eukaryotic initiation factor 4E activity is regulated by mitogenic stimulation (reviewed in Ref. 44). In the absence of mitogenic signaling, this cap-dependent translation is decreased through down-regulation of eukaryotic initiation factor 4E activity. The 5'-UTR of p27, however, contains an internal ribosomal entry site, which allows cap-independent translation of p27. This is enhanced by binding of HuR, a member of the embryonic lethal, abnormal vision (ELAV) family of RNA-binding proteins and binding of heterogeneous nuclear ribonucleoproteins C1 and C2, factors implicated in mRNA stability and processing, to the 5'-UTR of p27 (42–45, 48).

To elucidate the mechanism of 1,25-(OH)2D3-mediated increase in p27 protein levels, we investigated 1,25-(OH)2D3 regulation of p27 transcription, translation, and degradation in LNCaP cells. The 1,25-(OH)2D3-mediated increase in p27 was post-translational, via an increase in p27 protein half-life. 1,25-(OH)2D3 treatment decreased Thr187 phosphorylation of p27, a critical phosphorylation event that targets p27 for Skp2-mediated proteolysis (36). In addition, 1,25-(OH)2D3 decreased the nuclear localization of Cdk2, the kinase that phosphorylates p27 at Thr187. Thus, p27 up-regulation by 1,25-(OH)2D3 results from increased p27 protein half-life via decreased Thr187 phosphorylation. 1,25-(OH)2D3-mediated decreases in nuclear Cdk2 translocation may directly reduce cyclin E- and A-dependent Cdk2 activity because of reduced nuclear Cdk2 pools available for cyclin binding. Cytoplasmic Cdk2 mislocalization would also contribute to reduced Thr187 phosphorylation of p27. Thus, a determinant of growth regulation by 1,25-(OH)2D3 may be the capacity of 1,25-(OH)2D3 to decrease the nuclear localization of Cdk2, thereby preventing activation of this kinase.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Cell culture media (RPMI 1640 and Dulbecco's modified Eagle's medium-high glucose) were obtained from Invitrogen, and fetal bovine serum was from Hyclone (Logan, UT). 1,25-(OH)2D3 was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Mouse anti-actin antibody (1378 996) was obtained from Roche Applied Science. Anti-human phospho-Thr187-p27 antibodies were obtained from Zymed Laboratories (San Francisco, CA). Anti-human p27, Cdk2, Skp2, protein A-agarose beads, and anti-rabbit IgG, anti-goat IgG, and anti-mouse IgG antibodies with horseradish peroxidase conjugate were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescence-conjugated secondary antibodies, mAb414 antibodies, and 4,6-diamidino-2-phenylindole were generously provided by Dr. Beatriz Fontoura (University of Miami School of Medicine, Miami, FL).

Cell Culture—LNCaP-FGC cells (ATCC) were passaged and maintained in RPMI medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 100 µg/ml L-glutamine. All of the cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2.

[3H]Thymidine Uptake Assays—LNCaP cells were treated with either ethanol vehicle or 10 nM 1,25(OH)2D3. Following the 48-h treatment period, the media were replaced with [3H]thymidine-containing medium, and the cells were incubated for 18 h. Acid soluble tritium was removed by a trichloroacetic acid wash; the cells were then lysed, and [3H]thymidine uptake was determined by scintillation counting.

Reporter Plasmids and Luciferase Assay—The reporter plasmids p27PFLuc and control (41) were the generous gifts of Dr. Toshiyuki Sakai (Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji). The reporter plasmids 5'-SvL-3' and controls (43) were provided by Dr. Andrew Koff (Memorial Sloan-Kettering Cancer Center, New York). For luciferase assays, the cells were transfected with 5.0 µg of reporter plasmid and 1.0 µg of cytomegalovirus-{beta}-galactosidase vectors and treated with either ethanol vehicle or 10 nM 1,25-(OH)2D3 or serum-starved for the appropriate time periods. Following the treatment period, the cell lysates were collected, and luciferase activity was observed. {beta}-Galactosidase activities were measured to normalize for transfection efficiency.

Pulse-Chase Analysis—One day after plating, the cells were treated with either ethanol vehicle or 10 nM 1,25-(OH)2D3 for 24 h. Following the treatment period, the medium was replaced with Dulbecco's modified Eagle's medium (–met) and 10% dialyzed fetal bovine serum for 1 h. Next, the cells were pulsed for 1 h with 500 µCi of [35S]Met (PerkinElmer Life Sciences). Following the pulse, the cells were then chased for 0, 1, 3, and 6 h with Dulbecco's modified Eagle's medium (–met) medium containing 40 mM cold methionine and 10% dialyzed fetal bovine serum. After the chase times, the cells were lysed in sample buffer containing 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.5% Nonidet P-40, 10 µg/ml aprotonin, 10 µg/ml leupeptin, 50 mM NaF, 0.1 mM sodium orthovanadate. Extracts (500 µg protein) were precleared with 1 µg of normal rabbit IgG preadsorbed with protein A-agarose beads. Precleared extracts were then incubated with 1 µg of polyclonal p27 antibody and agitated overnight at 4 °C. The samples were then equilibrated with lysis buffer and 25 µl of packed volume of protein A-agarose beads for 4 h at 4 °C with agitation. Following three washes with lysis buffer, immune complexes were eluted by boiling in 20 µl of Laemmli gel loading buffer. The samples were then subjected to SDS-PAGE, transferred to nitrocellulose membrane filters, and exposed to autoradiography.

Western Blot Analysis—The cells were treated with ethanol vehicle, 10 nM 1,25-(OH)2D3, or serum-starved for appropriate times, washed, and lysed in sample buffer. The protein concentrations were determined by the Bio-Rad Dc Protein Assay according to the manufacturer's instructions. 50 µg of cell extract proteins were subjected to standard SDS-PAGE and transferred to nitrocellulose membrane filters. The filters were processed for Western blotting using standard procedures. Briefly, the filters were incubated overnight at 4 °C in blocking solution (5% dry milk, 0.1% Tween in 1x wash buffer (20 mM Tris, 50 mM NaCl, 2.5 mM EDTA)) followed by incubation with the primary antibody for 1 h. For phospho-specific antibodies, the filters were blocked overnight in 5% bovine serum albumin, 0.1% Tween in PBS. Actin antibody (Roche Applied Science) was used at 0.5 µg/ml. p27 and Cdk2 antibodies were used at 1.0 µg/ml. After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibody, and the proteins were visualized using the ECL system (Amersham Biosciences) following the supplier's instructions.

In Vitro Cdk2 Kinase Assay—LNCaP cells plated at 40–50% confluency were treated with either ethanol vehicle or 10 nM 1,25-(OH)2D3. Following the treatment period, the cells were washed in PBS and solubilized in TNE buffer (50 mM Tris, pH 7.5, 140 mM NaCl, 5 mM EDTA) containing 1% Nonidet P-40, 1:100 dilution of the protease inhibitor mixture (Sigma), 50 mM NaF, and 0.1 mM sodium orthovanadate. The protein concentrations of the lysates were determined as described above. 200 µg of proteins were then incubated with 3 µg of rabbit anti-human Cdk2 or rabbit anti-human cyclin E antibodies for 1 h at 4 °C. Following this incubation, the samples were adsorbed with 40 µl of anti-rabbit IgG-agarose beads (Sigma) for 1 h at 4 °C. After washing once with TNE buffer and three times with kinase buffer (50 mM Tris, pH 7.4, 10 mM MgCl2), immune complexes were incubated in 30 µl of kinase buffer containing 1 µg of histone H1, 25 µM ATP, and 10 µCi of [{gamma}-32P]ATP for 30 min at 30 °C. The reactions were stopped by the addition of 4x Laemmli buffer. After 5 min of boiling, the samples were then subjected to SDS-PAGE and transferred to a nitrocellulose membrane. Phosphorylated histone H1 was visualized by autoradiography. After the autoradiography, the membrane was subjected to Western blotting for Cdk2 and cyclin E.

Subcellular Fractionation—The cells were treated with ethanol vehicle, 10 nM 1,25-(OH)2D3 or serum-starved for appropriate times. Following the treatment periods, the cells were washed in PBS and lysed in transport buffer (20 mM HEPES, pH 7.4, 110 mM potassium acetate, 2 mM MgCl2) containing 15 µg/ml digitonin (Calbiochem, Cambridge, MA) and 10 µg/ml aprotonin, 10 µg/ml leupeptin, 50 mM NaF, and 0.1 mM sodium orthovanadate. The lysates were then centrifuged for 5 min at 800 x g at 4 °C, and the supernatant was collected (cytosolic fraction). The nuclear fraction was obtained by sonication of the pellet in transport buffer. The protein concentrations of both fractions were determined as described above. 25 µg of cytosolic and 50 µg nuclear proteins were then subjected to SDS-PAGE and transferred to nitrocellulose membrane filters. The filters were processed for Western blotting of Cdk2, p27, nucleolin (for fractionation purity), and actin (loading control).

Immunofluorescence—One day after plating on coverslips, the cells were treated with ethanol vehicle, 10 nM 1,25-(OH)2D3, or serum-starved for appropriate times. Following the treatment period, the cells were fixed in 2% formaldehyde in PBS for 10 min. Next, the cells were permeablized in PBS containing 1% bovine serum albumin, 0.1% Triton X-100 for 5 min. The cells were then incubated for 1 h with rabbit anti-Cdk2 (1:50 dilution in PBS) and mouse anti-nucleoporin antibodies (mAB414), washed in PBS, and incubated for 1 h with Cy3-conjugated goat anti-rabbit (1:1000) and fluorescein isothiocyanate-conjugated donkey anti-mouse antibodies. Following three additional washes, the coverslips were incubated with 4,6-diamidino-2-phenylindole for 5 min and mounted on glass slides in anti-fade medium. The images were then collected using confocal microscopy (Zeiss).

Quantification and Statistical Analysis—Western blot data were quantified using densitometry. The data were analyzed via either a two-tailed unpaired t test or a one-way analysis of variance followed by a Bonferroni post test using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA). For confocal image analysis, fluorescence intensities were determined using ImageJ (rsb.info.nih.gov/ij/). For the 24-h time point, 85 control cells and 81 treated cells were quantified. For the 48-h time point, 108 control cells and 89 treated cells were quantified. Statistical analysis was performed using an unpaired two-tailed t test (*, p < 0.05; **, p < 0.001).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
1,25-(OH)2 Vitamin D3 Does Not Regulate the Transcription or Translation of p27—We previously reported that 1,25-(OH)2D3 inhibits the growth of the LNCaP prostate cancer cell line (11, 12). [3H[Thymidine incorporation in 1,25-(OH)2D3-treated LNCaP cells was decreased ~85% of control (Fig. 1A). This decrease in proliferation by 1,25-(OH)2D3 is associated with an increased percentage of LNCaP cells in G1 and a concomitant decrease in S and G2/M (Fig. 1B). Consistent with inhibition of G1 to S progression, 1,25-(OH)2D3 increases the levels of the CKI p27Kip1, enhances the association of this CKI with Cdk2, and decreases Cdk2 activity in LNCaP cells (Refs. 12–14 and Fig. 1C). The mechanism of 1,25-(OH)2D3-mediated up-regulation of p27, however, is not known.



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  		FIG. 1.
Vitamin D-mediated decreases in LNCaP proliferation involve enhanced association of p27 with cyclin E/Cdk2 and reduced Cdk2 activity. LNCaP cells were treated with either ethanol vehicle or 10 nM 1,25-(OH)2D3 for 48 h. A, to assay proliferation in these cells, [3H]thymidine uptake assays were conducted as described under "Experimental Procedures." The data represent two experiments performed in triplicate. Statistical significance was determined using a two-tailed unpaired t test. *, p < 0.01. B, cell cycle distribution was assessed by flow cytometry of propidium iodide-stained cells. Statistical significance was determined using one-way analysis of variance with a Bonferroni post test. **, p < 0.001 versus vehicle-treated cells. C, to measure Cdk2 activity, the cell lysates were immunoprecipitated with antibodies specific to cyclin E. Immune complexes were then subjected to a kinase assay using [{gamma}-32P]ATP and histone H1. Phosphorylated histone H1 was then visualized using autoradiography. Levels of cyclin E, Cdk2, and p27 in the immune complexes were determined by Western blot analysis. Vit D, vitamin D.

 
To test whether 1,25-(OH)2D3 regulates p27 at the transcriptional level, we utilized a reporter gene construct in which the luciferase reporter is driven by a 3.5-kb fragment of the p27 promoter (41). It was previously shown that 1,25-(OH)2D3 activates this construct in U937 leukemic cells despite the apparent lack of vitamin D response elements in the p27 promoter (41). However, although serum starvation of LNCaP cells enhanced luciferase activity, 1,25-(OH)2D3 did not increase luciferase activity driven by this fragment of the p27 promoter (Fig. 2). The time points utilized to assay p27 promoter activity following 1,25-(OH)2D3 treatment were within the range in which p27 protein is up-regulated (Ref. 13 and Figs. 3B and 5). Northern blot analysis confirmed this finding because no changes in p27 mRNA levels were observed in response to 1,25-(OH)2D3 (data not shown). These data show that 1,25-(OH)2D3-mediated up-regulation of p27 protein was not due to increased p27 transcription or increased p27 mRNA levels.



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  		FIG. 2.
Vitamin D does not enhance p27 promoter activity. LNCaP cells were transfected with either the p27PFLuc (–3568 to –12 position of p27 promoter) or pGVB2Luc (empty vector) reporter construct and the {beta}-galactosidase gene. Twenty-four h later, the cells were treated with either ethanol vehicle or 10 nM 1,25-(OH)2D3 for 4 and 24 h. Luciferase and {beta}-galactosidase activities were assessed in the cell lysates. Normalized luciferase activity was calculated by dividing luciferase activity by {beta}-galactosidase activity. The data represent two experiments each performed in triplicate. Statistical significance was determined using one-way analysis of variance with a Bonferroni post test. **, p < 0.001. Vit D, vitamin D.

 



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  		FIG. 3.
Vitamin D does not increase p27 protein synthesis. A, LNCaP cells were transfected with either SvL control or 5'-3' SvL luciferase reporter constructs (luciferase activity governed by the 5'- and 3'-untranslated regions of p27) and treated with either vehicle or 10 nM 1,25-(OH)2D3. The cells were harvested 24 h later, and luciferase activity was assessed. As a positive control, transfected cells were also subjected to serum starvation, a known stimulator of p27 protein synthesis. B, parallel dishes of LNCaP cells were treated with either vehicle or 10 nM 1,25-(OH)2D3 or subjected to serum starvation for 24 h. The cell extracts were subjected to immunoblot analysis with antibodies against p27. Actin was used as a loading control. The data in A represent two experiments performed in triplicate. Luciferase activity in serum-starved cells was significantly different from vehicle-treated cells (p < 0.05). Vit D, vitamin D.

 



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  		FIG. 5.
Vitamin D decreases phospho-Thr187-p27 levels in LN-CaP. A, LNCaP cells were treated with either vehicle or 10 nM 1,25-(OH)2D3. Following the indicated treatment period, the proteins were harvested and subjected to immunoblot analysis with antibodies specific to Thr187 phosphorylated p27, total p27, and actin (loading control). B, three (8- and 48-h time points) or two (24-h time point) independent experiments were quantified using densitometry. The data represent the average phospho-Thr187-p27 levels as percentages of time-matched controls. Statistical analysis was performed using an unpaired two-tailed t test. *, p < 0.01 versus time matched control. Vit D, vitamin D.

 
The rate of p27 translation is regulated by the 5'- and 3'-UTRs of the p27 mRNA (31, 42–45). Utilizing a reporter gene construct in which the luciferase gene is flanked by the 5'- and 3'-UTRs of p27, we assessed whether 1,25-(OH)2D3 can enhance the rate of p27 translation. 1,25-(OH)2D3 did not enhance translation stimulated by the 5'- and 3'-UTRs of p27, whereas serum starvation, a known inducer of p27 protein synthesis (42–45), did (Fig. 3A). As a further control for p27 protein up-regulation, parallel cell lysates were subjected to Western blot analysis for p27, and cell cycle distribution was determined. Despite a lack of any apparent 1,25-(OH)2D3 induction of translation driven by the p27-UTRs, p27 protein was increased in both 1,25-(OH)2D3-treated and in serum-starved cells (Fig. 3B). In addition, following 24 h of 1,25-(OH)2D3 treatment, LNCaP cells accumulated in G1 (data not shown). These data are consistent with 1,25-(OH)2D3-mediated increases in p27 protein levels occurring through a mechanism other than increased translation.

1,25-(OH)2 Vitamin D3 Stabilizes p27 Protein via Decreased Cdk2 Phosphorylation of Thr187—To determine whether 1,25-(OH)2D3-mediated increases in p27 protein are due to decreased p27 degradation, pulse-chase analysis was performed. As seen in Fig. 4, 1,25-(OH)2D3 increased the half-life of p27 protein from ~4.5 h in asynchronously proliferating vehicle-treated cells to greater than 6 h in 1,25-(OH)2D3-treated cells. The half-lives of p27 in both treated and untreated cells are comparable with those found in other studies for growth arrested and asynchronous cells (31, 49).



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  		FIG. 4.
Vitamin D increases the half-life of p27 in LNCaP. A, LNCaP cells were treated with either ethanol vehicle or 10 nM 1,25-(OH)2D3 for 24 h. The cells were then pulse-labeled with [35S]methionine for 1 h and chased for the indicated times. The cell lysates were obtained and immunoprecipitated with antibodies specific for p27. Immune complexes were then subject to SDS-PAGE and transferred onto a nitrocellulose membrane. Labeled p27 was then detected via autoradiography. B, quantification of three independent experiments using densitometry. The data shown represent average percentages of labeled p27 ± S.E. compared with the amount of labeled p27 at chase time 0 for each treatment group. Statistical analysis was performed using a one-way analysis of variance followed by Bonferroni post-test. *, p < 0.01; **, p < 0.001. Vit D, vitamin D.

 
For cells to enter S phase, p27 levels must decline. This process is regulated in part by phosphorylation of p27 at Thr187 by cyclin E/Cdk2 complexes. This phosphorylation event targets p27 for degradation by allowing its recognition by the SCFskp2 ubiquitin ligase complex. To investigate further the 1,25-(OH)2D3-mediated regulation of p27 proteolysis, the levels of phospho-Thr187-p27 were assessed. As seen in Fig. 5, 1,25-(OH)2D3 treatment at 24 and 48 h results in decreased phospho-Thr187-p27, whereas total p27 was increased at these two points. These results affirmed that 1,25-(OH)2D3-mediated upregulation of p27 in LNCaP cells was due to decreased targeting of p27 for proteolysis.

1,25-(OH)2 Vitamin D3 Treatment Results in Decreased Skp2 Levels—The SCFskp2 ubiquitin ligase complex directs Thr187 phosphorylation-dependent p27 proteolysis. It was recently reported that the vitamin D analog EB1089 mediates up-regulation of p27 in AT-84 head and neck squamous carcinoma cells via down-regulation of Skp2 and decreased association of p27 with Skp2, which leads to p27 stabilization (50). We found that Skp2 protein levels were decreased by 1,25-(OH)2D3 after 24 and 48 h of treatment (Fig. 6). Because Skp2 is destabilized as cells exit from the cell cycle (51), the down-regulation of Skp2 may be the result of growth arrest by 1,25-(OH)2D3. Thus, we examined a more proximal event of p27 degradation, namely the localization and activation of Cdk2.



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  		FIG. 6.
Vitamin D decreases Skp2 levels in LNCaP. A, LNCaP cells were treated with either vehicle or 10 nM 1,25-(OH)2D3. The proteins were harvested at the indicated times and subjected to immunoblot analysis with antibodies specific to Skp2 and actin (loading control). B, three independent experiments were quantified using densitometry. The data represent the average Skp2 levels as percentages of time-matched controls. Statistical analysis was performed using an unpaired two-tailed t test. *, p < 0.001 versus time matched control. Vit D, vitamin D.

 
1,25-(OH)2 Vitamin D3 Stabilization of p27 Correlates with Decreased Nuclear Localization of Cdk2—The late G1 phase of p27 degradation that increases at the G1 to S phase transition is dependent on Cdk2 phosphorylation of p27 at Thr187. For Cdk2 to be fully activated, newly synthesized Cdk2 must translocate into the nucleus to be phosphorylated by Cdk-activating kinase (CAK) and to be dephosphorylated by the phosphatase Cdc25A. CAK phosphorylates Cdk2 at Thr160, and Cdc25A dephosphorylates Cdk2 at Thr14 and Tyr15 (52, 53). Regulation of Cdk2 nucleocytoplasmic trafficking is a poorly understood process. To test whether 1,25-(OH)2D3-mediated effects on p27 degradation might involve regulation of Cdk2 localization, vehicle- and 1,25-(OH)2D3-treated LNCaP cells were subjected to immunofluorescence staining of Cdk2. Examination of these cells using confocal microscopy revealed decreased Cdk2 nuclear staining following 24 and 48 h of 1,25-(OH)2D3 treatment (Fig. 7). Subcellular fractionation and Western blot analysis confirmed this shift to predominantly cytoplasmic Cdk2 localization (Fig. 8). These results suggest that 1,25-(OH)2D3-mediated growth inhibition involves decreased Cdk2 nuclear localization, which leads to the inhibition of cyclin-Cdk2 activity and the impairment of Cdk2-dependent p27 proteolysis.



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  		FIG. 7.
Vitamin D decreases nuclear localization of Cdk2. LNCaP cells were cultured on coverslips and treated with either vehicle (A) or 10 nM 1,25-(OH)2D3 (B) for 48 h. The cells were stained with antibodies to Cdk2 (orange) and nucleoporins (mAB414) (green). To stain the nuclei, the cells were also incubated with 4,6-diamidino-2-phenylindole (DAPI, blue). Coverslips were then mounted on slides and viewed using a confocal microscope. The data are representative of two independent experiments. C and D, quantification of Cdk2 fluorescence following 24-h (C) or 48-h (D) treatments. Fluorescence intensities were determined using ImageJ (rsb.info.nih.gov/ij/). Statistical analysis was performed using an unpaired two-tailed t test. *, p < 0.05; **, p < 0.001. Vit D, vitamin D; mAb, monoclonal antibody; Nuc, nuclear; Cyt, cytoplasmic.

 



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  		FIG. 8.
Vitamin D decreases nuclear levels of Cdk2. A, LNCaP cells were treated with either vehicle or 10 nM 1,25-(OH)2D3 for 48 h. The cells were then fractionated into nuclear and cytosolic fractions. Proteins from each respective fraction were subjected to immunoblot analysis with antibodies to Cdk2 or p27. Actin was used as a loading control. To assess the purity of the fractions, immunoblot analysis for the nucleolar protein nucleolin was performed. The data are representative of three independent experiments. B, quantification of Cdk2 levels was performed via densitometry. Statistical analysis was performed using an unpaired two-tailed t test. *, p < 0.01 versus vehicle-treated cells. Vit D, vitamin D; Nuc, nuclear; Cyt, cytosolic.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
1,25-(OH)2D3 has been shown to decrease the growth of many cancers, including prostate. The mechanism of the initial growth inhibitory events is by blocking the G1 to S phase transition in the cell cycle (10, 12–14). 1,25-(OH)2D3-mediated growth inhibition has also been attributed to the induction of apoptosis (programmed cell death) (54). However, the dose and timing of 1,25-(OH)2D3 treatment as well as the cell cycle stage and expression levels of cell cycle genes may dictate the response to hormone.

In this study, we show that 1,25-(OH)2D3-mediated induction of p27 protein levels in LNCaP prostate cancer cells occurred through a reduction in the rate of p27 degradation. 1,25-(OH)2D3-mediated stabilization of p27 correlated with a decrease in Thr187 phosphorylation of p27, the critical phosphorylation event necessary for targeting p27 for cyclin E-Cdk2-dependent proteolysis in late G1. We made the novel observation that 1,25-(OH)2D3 decreased nuclear localization of Cdk2. Because full activation of Cdk2 by the regulatory CAK and Cdc25A requires nuclear translocation of Cdk2, cytoplasmic sequestration of Cdk2 would prevent cyclin E-Cdk2 activation and reduce the overall activity of this complex. Thus, in 1,25-(OH)2D3-treated cells, the decreased Cdk2 nuclear localization may also act to inhibit p27 degradation and may be a key event in 1,25-(OH)2D3-mediated growth inhibition of LNCaP cells.

1,25-(OH)2D3 acts through its cognate receptor, the VDR, which is a ligand-activated transcription factor. Growth inhibition of prostate cancer cells by 1,25-(OH)2D3 requires the VDR; however, VDR expression is not sufficient to cause this effect (9, 11). p27 regulation by 1,25-(OH)2D3, however, occurs post-translationally through effects on p27 degradation. Transcriptional regulation of p27 by 1,25-(OH)2D3 occurs in the myelomonocytic cell line U937 despite the p27 promoter lacking canonical vitamin D response elements (41). p27 up-regulation in these cells is a result of increased binding of Sp-1 and NF-Y factors to the p27 promoter, leading to enhanced p27 transcription. Because the expression and regulation of transcription factors vary from cell to cell, 1,25-(OH)2D3 regulation of these factors may depend on cell-specific mechanisms that may be absent in LNCaP cells.

Translational regulation of p27 by 1,25-(OH)2D3 has not been reported but does occur with other growth arresting agents such as lovastatin (43). The rate of p27 protein synthesis is governed by heterogeneous nuclear ribonucleoproteins C1, C2, and the embryonic lethal, abnormal vision (ELAV) family of RNA-binding proteins, which all have been implicated in the enhanced polysomal association of p27 mRNA (43–45, 48). Currently, no evidence exists for 1,25-(OH)2D3-mediated regulation of heterogeneous nuclear ribonucleoproteins.

Our results showing p27 stabilization by 1,25-(OH)2D3 are in accord with studies utilizing G1 arresting agents that up-regulate p27 via post-translational mechanisms (31, 50, 55). p27 degradation is a complex process and involves phospho-Thr187-dependent and -independent mechanisms (30, 31, 37–39). As cells traverse the G1 to S phase transition, p27 levels decrease because of both decreased translation and mitogen-mediated Cdk2-independent p27 proteolysis in early G1. This initial reduction in p27 leads to cyclin E/Cdk2 activation. In late G1 and early S phase, p27 degradation is governed by Cdk2-dependent phosphorylation of p27 at Thr187. Phosphorylation of p27 at Thr187 allows p27 recognition by the ubiquitin ligase SCFskp2 complex, which ubiquitinates p27 and targets p27 for proteolysis. In LNCaP cells, 1,25-(OH)2D3 appears to regulate the Cdk2-dependent phase of p27 proteolysis via sequestration of Cdk2 to the cytoplasm.

Similar to the actions of the vitamin D analog EB1089 on AT-84 head and neck squamous carcinoma cells, 1,25-(OH)2D3 decreased Skp2 protein levels in LNCaP cells. Although the reduction in Skp2 levels may play a role in 1,25-(OH)2D3-mediated p27 stabilization in LNCaP, this effect may be a result of G1 accumulation. Skp2 is rapidly degraded during the G0/G1 phase of the cell cycle (51). Also, the lowered Skp2 levels cannot account for the 1,25-(OH)2D3-mediated decline in Thr187 phosphorylation of p27. In contrast, decreased nuclear Cdk2 available for cyclin binding and activation would result in decreased phospho-Thr187-p27 targeted for degradation. Thus, decreased nuclear Cdk2 may be the more critical event in 1,25-(OH)2D3-mediated growth inhibition of LNCaP cells.

The nucleocytoplasmic trafficking of Cdk2 is not well understood. Reports suggest a possible role of the ERK signaling pathway in the regulation of Cdk2 translocation (52, 53, 56). The decrease in nuclear Cdk2 following 1,25-(OH)2D3 treatment may occur via decreased nuclear import and/or increased nuclear export of Cdk2. Import of the cyclin E-Cdk2 complex into the nucleus occurs via the importin {alpha}/{beta} system (57). The mechanism of Cdk2 nuclear export and its possible dependence on the nuclear export protein Crm1, however, is not known. Cytoplasmic Cdk2 mislocalization may be a key event in 1,25-(OH)2D3-mediated growth inhibition of LNCaP cells because this leads to decreased cyclin E/Cdk2 activity. Therefore, G1 accumulation would be due to decreased Cdk2 availability in the nucleus for cyclin binding and decreased activation of cyclin/Cdk2 complexes by CAK and Cdc25A. In addition, mislocalization of Cdk2 would impair the Cdk2-dependent phase of p27 proteolysis. Thus, regulation of Cdk2 localization may be an important mediator of the fate of cells during the G1 to S phase transition of the cell cycle.


   FOOTNOTES
 
* This work was supported by NIEHS, National Institutes of Health Fellowship F30 ES05910-02 and the NIDDK, National Institutes of Health Grant DK45478. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Molecular and Cellular Pharmacology (R-189), University of Miami School of Medicine, P.O. Box 016189 (R-189), Miami, FL 33136. E-mail: kburnste{at}miami.edu.

1 The abbreviations used are: 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; VDR, vitamin D receptor; CKI, cyclin-dependent kinase inhibitor; Cdk2, cyclin-dependent kinase 2; UTR, untranslated region; PBS, phosphate-buffered saline; CAK, Cdk-activating kinase. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Toshiyuki Sakai, Andrew Koff, Joyce Slingerland, and Beatriz Fontoura for generosity in providing various materials and suggestions. We are grateful to Dr. Slingerland for many helpful comments on the manuscript. We also appreciate the assistance of Carol Maiorino, Leah Lyons, and Drs. Sen-Hong Zhuang and Jennifer McCafferty.



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