Dictyostelium Differentiation-inducing Factor-3 Activates Glycogen Synthase Kinase-3beta and Degrades Cyclin D1 in Mammalian Cells*

Fumi Takahashi-YanagaDagger §, Yoji TabaDagger , Yoshikazu MiwaDagger , Yuzuru Kubohara, Yutaka Watanabe||, Masato Hirata**, Sachio MorimotoDagger , and Toshiyuki SasaguriDagger

From the Dagger  Department of Clinical Pharmacology, Graduate School of Medical Sciences and ** Department of Molecular and Cellular Biochemistry, Graduate School of Dental Sciences, Kyushu University, Fukuoka 812-8582, Japan,  Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan, and the || Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790-8577, Japan

Received for publication, June 11, 2002, and in revised form, October 30, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In search of chemical substances applicable for the treatment of cancer and other proliferative disorders, we studied the signal transduction of Dictyostelium differentiation-inducing factors (DIFs) in mammalian cells mainly using HeLa cells. Although DIF-1 and DIF-3 both strongly inhibited cell proliferation by inducing G0/G1 arrest, DIF-3 was more effective than DIF-1. DIF-3 suppressed cyclin D1 expression at both mRNA and protein levels, whereas the overexpression of cyclin D1 overrode DIF-3-induced cell cycle arrest. The DIF-3-induced decrease in the amount of cyclin D1 protein preceded the reduction in the level of cyclin D1 mRNA. The decrease in cyclin D1 protein seemed to be caused by accelerated proteolysis, since it was abrogated by N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor. DIF-3-induced degradation of cyclin D1 was also prevented by treatment with lithium chloride, an inhibitor of glycogen synthase kinase-3beta (GSK-3beta ), suggesting that DIF-3 induced cyclin D1 proteolysis through the activation of GSK-3beta . Indeed, DIF-3 dephosphorylated Ser9 and phosphorylated tyrosine on GSK-3beta , and it stimulated GSK-3beta activity in an in vitro kinase assay. Moreover, DIF-3 was revealed to induce the nuclear translocation of GSK-3beta by immunofluorescent microscopy and immunoblotting of subcellular protein fractions. These results suggested that DIF-3 activates GSK-3beta to accelerate the proteolysis of cyclin D1 and that this mechanism is involved in the DIF-3-induced G0/G1 arrest in mammalian cells.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Differentiation-inducing factors (DIFs)1 were identified in Dictyostelium discoideum as the morphogens required for stalk cell differentiation of Dictyostelium (1). In the DIF family, DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) was the first to be identified, and DIF-3, the monochlorinated analogue of DIF-1, is a natural metabolite of DIF-1 in Dictyostelium (2). However, the actions of DIFs are not limited to Dictyostelium. They also have strong effects on mammalian cells. DIF-1 and/or DIF-3 strongly inhibit proliferation and induce differentiation in several leukemia cells, such as the murine erythroleukemia cell line B8, human leukemia cell line K562, and human myeloid leukemia cell line HL-60 (3, 4). DIF-3 has been reported to have the most potent antiproliferative effect on mammalian leukemia cells among DIF analogues examined to date (5). Recently, we found that DIF-1 strongly inhibits proliferation and induces differentiation in human vascular smooth muscle cells, indicating that cells sensitive to DIFs are not limited to transformed cells (6).

However, the target molecule (receptor) of DIFs is unknown, and it is not clear even in Dictyostelium how DIFs induce an antiproliferative effect and cell differentiation. DIFs are small hydrophobic molecules and are therefore expected to be able to cross cell membranes without requiring channels or carriers. Also, the rapidity with which DIFs induce prestalk cell-specific gene expression suggests that they directly regulate gene expression. Therefore, the target molecule(s) for DIFs may be located in cytoplasm or nucleus (7). Although the precise mechanisms underlying their antiproliferative and differentiation-inducing effects are not yet known, we found that DIF-1 induces cell cycle arrest at G0/G1 phase by suppressing the expression of cyclin D1 (6). Cyclin D1 is synthesized early in G1 phase and plays a key role in the initiation and progression of this phase. When cells enter the S phase, cyclin D1 is rapidly degraded by ubiquitin-proteasome-dependent proteolysis (8).

Therefore, in the present study, we investigated the mechanism underlying the DIF-induced inhibition of cyclin D1 expression. We particularly paid attention to the possible involvement of glycogen synthase kinase-3beta (GSK-3beta ), because this serine/threonine protein kinase has been shown to regulate not only cyclin D1 gene transcription by phosphorylating beta -catenin but also cyclin D1 proteolysis by directly phosphorylating cyclin D1 itself (9-12). Here we show for the first time that DIF-3, which is more effective than DIF-1 in inhibiting cell proliferation, induces the rapid degradation of cyclin D1 and the activation of GSK-3beta .

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals-- DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) was purchased from Affiniti Research Products. DIF-3 (1-(3-chloro-2, 6-dihydroxy-4-methoxyphenyl)-1-hexanone) was synthesized by Toyama Chemical Co. (Tokyo, Japan). U0126 was purchased from Cell Signaling Technology. N-acetyl-Leu-Leu-norleucinal and wortmannin were purchased from Sigma.

Cell Culture and Transfection-- HeLa cells and bovine aortic endothelial cells (BAECs) were grown in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 0.1 µg/ml streptomycin. The cells were plated on plastic tissue culture dishes or coverslips. Human umbilical vein endothelial cells were plated on 0.1% gelatin-coated dishes and maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 20% fetal bovine serum, 5 ng/ml bovine fibroblast growth factor, 100 units/ml penicillin G, and 0.1 µg/ml streptomycin. Wild-type human cyclin D1 cDNA was provided by Dr. K. Tamai (Medical and Biological Laboratories Co., Nagano, Japan) and subcloned into pcDNA3 (Invitrogen). Transfection was carried out using TransIT-LT1 (Mirus), and transfected cells were maintained in growth medium for 16 h before stimulation.

Cell Proliferation Assay-- The cells were plated on 24-well plates (0.5 × 104 cells/well) and treated with or without various amounts of DIF-1 or DIF-3 for given periods. Cells were harvested by the trypsin/EDTA treatment and enumerated.

Flow Cytometry-- Cells harvested by the trypsin/EDTA treatment were suspended in hypotonic fluorochrome solution containing 50 µg/ml of propidium iodide, 0.1% sodium citrate, and 0.1% Triton X-100 (13). Cells (5 × 103) for each sample were analyzed for fluorescence by a Becton-Dickinson FACScalibur (Franklin Lakes, NJ).

mRNA Expression Analysis-- Total cellular RNA was extracted with Isogen (Nippon Gene). Using 1 µg of the RNA, the expression of cyclin D1 mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) using Ready-To-Go RT-PCR Beads (Amersham Biosciences) (6).

Purification of Nucleic and Cytoplasmic Proteins-- Nucleic and cytoplasmic proteins were purified from cells cultured in 100-mm plates using NE-PERTM nuclear and cytoplasmic extraction reagents (Pierce). Five µg of each sample was subjected to Western blot analysis.

Immunoblotting-- Samples were separated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane using a semidry transfer system (1 h, 15 V). After blocking with 5% skim milk or 5% bovine serum albumin for 1 h, the membrane was probed with a first antibody. For the polyclonal anti-cyclin D1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), the polyclonal anti-cyclin D2 antibody (Santa Cruz Biotechnology), the polyclonal anti-phospho-GSK-3beta (Ser9) antibody (Cell Signaling Technology), the polyclonal anti-phospho-Akt (Ser473) antibody (Cell Signaling Technology), and the polyclonal anti-phospho-p90RSK (Ser380) antibody (Cell Signaling Technology), the incubation was carried out overnight at 4 °C. For the monoclonal anti-phosphotyrosine antibody (PY-20; Santa Cruz Biotechnology), the monoclonal anti-GSK-3beta antibody (BD Transduction Laboratories), and the monoclonal anti-cyclin D3 antibody (Santa Cruz Biotechnology), the incubation was carried out for 1 h at room temperature. The membrane was washed three times and incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Bio-Rad) for 1 h. Immunoreactive proteins on the membrane were visualized by treatment with a detection reagent (LumiGLO, Cell Signaling Technology). An optical densitometric scan was performed using Science Lab 99 Image Gauge Software (Fuji Photo Film).

Immunoprecipitation-- HeLa cells (1 × 106 cells) were incubated with or without DIF-3 for the periods indicated. Cells were lysed on ice for 1 h in 1 ml of the lysis buffer (50 mM NaCl, 5 mM NaF, 2 mM Na3VO4, 10 mM Tris/HCl, pH 7.4, 1 mM EDTA, 1% (v/v) Triton X-100, and 2 mM phenylmethylsulfonyl fluoride), and insoluble cell debris was removed by centrifugation at 5,000 rpm for 3 min. The cell lysate was precleared with protein A-Sepharose CL-4B (Amersham Biosciences) and then incubated with an anti-GSK-3beta antibody (1 µg) and protein A-Sepharose CL-4B at 4 °C for 3 h. After incubation, proteins bound to the antibody/protein A-Sepharose complex were precipitated by centrifugation at 15,000 rpm for 5 min and washed three times with the lysis buffer.

GSK-3beta Activity Assay-- For the in vitro kinase assay, immunoprecipitated samples were washed twice with lysis buffer and twice with a kinase assay buffer (20 mM Tris/HCl, pH 7.4, 5 mM MgCl2, and 1 mM dithiothreitol). Kinase activity was measured by mixing immunoprecipitated GSK-3beta with 50 µl of kinase assay buffer containing 20 mM Tris/HCl (pH 7.4), 5 mM MgCl2, 1 mM dithiothreitol, 250 µM ATP, 5 µCi of [gamma -32P]ATP (Amersham Biosciences), and 10 µM GSK-3beta substrate peptide (Upstate Biotechnology, Inc., Lake Placid, NY). The samples were incubated at 30 °C for 30 min, and the reaction was terminated by adding 10 µl of 50% trichloroacetic acid. Samples were then centrifuged at 15,000 rpm for 5 min, and 40 µl of supernatant was spotted on P81 phosphocellulose filter paper (Whatman). The filters were washed five times with 0.75% phosphoric acid and twice with acetone and analyzed by scintillation counting.

Fluorescence Microscopy-- Cells plated on CELL-TAKR (Collaborative Biomedical Products)-treated coverslips were incubated with or without DIF-3 (30 µM) for given periods and washed with phosphate-buffered saline. The cells were fixed and permeabilized in ice-cold methanol/acetone (1:1) for 15 min at -20 °C and then washed twice with phosphate-buffered saline. After blocking with 2% bovine serum albumin in phosphate-buffered saline for 30 min, the coverslips were incubated with polyclonal or monoclonal anti-GSK-3beta antibody (1:200) overnight at 4 °C. The coverslips were washed twice with phosphate-buffered saline and incubated with anti-mouse IgG or anti-rabbit IgG + IgA + IgM-biotin (Nichirei) for 1 h at room temperature followed by streptavidin-fluorescein isothiocyanate conjugate (1:50 dilution; Invitrogen) for 1 h at room temperature. The cells were examined under a fluorescence microscope (Olympus).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DIFs Inhibited HeLa Cell Proliferation-- DIFs exhibit powerful antiproliferative effect in leukemia cells (3-5). In the present study, we first examined whether DIFs also inhibit the proliferation of HeLa cells. As shown in Fig. 1, A and B, DIF-1 and DIF-3 both strongly inhibited HeLa cell proliferation in a dose-dependent fashion, suggesting that DIFs are also effective in solid tumors. These antiproliferative effects were unlikely to be caused by cytotoxicity, because the number of dead cells indicated by the trypan blue exclusion test was not increased by the treatment with DIFs (data not shown). Consistent with the result obtained in leukemia cells (5), DIF-3 was more effective than DIF-1 in HeLa cells. Therefore, we used DIF-3 in the subsequent experiments. We next examined the cell cycle distribution using flow cytometry. Although the cell populations in S and G2/M phases decreased after the treatment with DIF-3, the population in G0/G1 phase significantly increased (Fig. 1C), indicating that DIF-3 induced G0/G1 arrest in HeLa cells. This result was consistent with our previous study as to the effect of DIF-1 on vascular smooth muscle cell cycle (6).


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Fig. 1.   Antiproliferative effect of DIF-1 and DIF-3 on HeLa cells. HeLa cells seeded on a 24-well plate (0.5 × 104 cells/well) were incubated with various concentrations of DIF-1 or DIF-3. Cells were harvested by the trypsin/EDTA treatment at the times indicated and enumerated. A, the time course of the increase in cell numbers. B, the increase in cell numbers at 72 h are shown as percentages of the control cell number. Values are means ± S.E. for three independent experiments. *, p < 0.005; **, p < 0.001 compared with the control (Student's t test). C, flow cytometry. HeLa cells were incubated with DIF-3 (30 µM) for the indicated periods and then harvested by the trypsin/EDTA treatment. After the trypsin/EDTA treatment, cells were stained with propidium iodide (PI), and fluorescence of nuclei was measured. The percentages of cell number in the cell cycle phases are also shown. The results are means ± S.E. of six independent experiments.

DIF-3 Suppressed the Expression of Cyclins D1, D2, and D3 in HeLa Cells-- We reported that DIF-1 induced G0/G1 arrest, suppressing the expression of cyclins D1, D2, and D3 in human vascular smooth muscle cells (6). In HeLa cells, DIF-3 also reduced both the mRNA and protein levels of cyclin D1 (Fig. 2, A and B). RT-PCR analyses showed that the cyclin D1 mRNA level was not significantly affected after a 1-h incubation with DIF-3 (Fig. 2A). Although it began to slowly decrease from 3 h, a considerable amount of cyclin D1 mRNA was still expressed until 6 h. Despite this slow decrease in the level of mRNA, DIF-3 rapidly reduced the protein level of cyclin D1 (Fig. 2B). Cyclin D1 protein markedly decreased after 1 h of incubation with DIF-3 and nearly completely disappeared by 3 h. This rapid decrease in the amount of protein was not explained by suppression of mRNA expression. The protein levels of cyclins D2 and D3 also significantly decreased after 1 h of treatment with DIF-3 and almost disappeared after 6 h of treatment (Fig. 2C).


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Fig. 2.   The effect of DIF-3 on D-type cyclin expression. HeLa cells were incubated with or without DIF-3 (30 µM) for the periods indicated. A, RT-PCR analysis for cyclin D1. Total RNA (1 µg) was subjected to RT-PCR for cyclin D1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCR cycle numbers were 24 for cyclin D1 and 20 for glyceraldehyde-3-phosphate dehydrogenase. The result demonstrated is representative of three other experiments. B, Western blot analysis for cyclin D1. Samples were separated by 12% SDS-PAGE, and immunoblot analysis was performed using an anti-cyclin D1 antibody. Demonstrated are the results representative of three other experiments. C, Western blot analysis for cyclins D2 and D3. Samples were separated by 12% SDS-PAGE and immunoblotted using an anti-cyclin D2 antibody and an anti-cyclin D3 antibody. Demonstrated are the results representative of three other experiments.

DIF-3-induced G0/G1 Arrest Was Rescued by the Overexpression of Cyclin D1-- To clarify whether the overexpression of cyclin D1 is able to rescue cells from cell cycle arrest induced by DIF-3, wild-type human cyclin D1 cDNA was transfected to BAECs, since transfection efficiency was the highest in BAECs among mammalian cell species we examined including HeLa cells. As shown in Fig. 3A, the expression levels of cyclin D1 in untransfected cells and in cells transfected with empty pcDNA3 were reduced after 24 h of incubation with DIF-3, demonstrating that DIF-3 showed the same effect in BAECs as in HeLa cells. However, the expression level of cyclin D1 in cells transfected with pcDNA3/cyclin D1 was not significantly changed by DIF-3 treatment. We then examined the cell cycle distribution using untransfected and transfected cells. Although DIF-3 induced G0/G1 arrest in untransfected and pcDNA3-transfected cells, there was no significant difference in pcDNA3/cyclin D1-transfected cells between the absence and presence of DIF-3 treatment (Fig. 3B). Therefore, DIF-3 was likely to induce cell cycle arrest by reducing the expression level of cyclin D1.


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Fig. 3.   Overexpression of cyclin D1 disabled DIF-3 from inhibiting cell cycle. A, BAECs untransfected or transfected with the plasmids indicated were incubated with or without DIF-3 (30 µM) for 24 h. Protein samples were subjected to immunoblot analysis using an anti-cyclin D1 antibody. The pcDNA3/cyclin D1-transfected cells expressed an ~15-fold excessive amount of cyclin D1 compared with untransfected cells. Demonstrated are the results representative of three other experiments. B, cell cycle distribution. Untransfected and transfected BAECs were incubated with or without DIF-3 (30 µM) for 12 h. Cells were then stained with propidium iodide (PI), and the nuclear fluorescent levels were measured with a flow cytometer. The percentages of cell number in the cell cycle phases are shown as means ± S.E. of three independent experiments performed in duplicate.

DIF-3 Induced Cyclin D1 Proteolysis-- To determine the mechanism for the decrease in cyclin D1 protein, we used N-acetyl-Leu-Leu-norleucinal, which inhibits ubiquitin-proteasome-dependent degradation of cyclins (14). As shown in Fig. 4A, N-acetyl-Leu-Leu-norleucinal prevented the DIF-3-induced loss of cyclin D1. We then performed a chase experiment to determine whether the turnover rate of cyclin D1 is changed by the treatment with DIF-3. As shown in Fig. 4B, DIF-3 significantly decreased the half-life of cyclin D1 in the presence of cycloheximide. These results indicated that DIF-3 rapidly reduced the amount of cyclin D1 protein by accelerating ubiquitin-proteasome-dependent proteolysis.


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Fig. 4.   DIF-3 induced proteolysis of cyclin D1. A, the effect of a proteasome inhibitor. Cells pretreated with or without N-acetyl-Leu-Leu-norleucinal (20 µM) for 3 h were incubated in the presence or absence of DIF-3 (30 µM) for 3 h, and immunoblot analysis was performed using an anti-cyclin D1 antibody. The result is representative of three other experiments. B, the rate of cyclin D1 degradation. HeLa cells were incubated with or without DIF-3 (30 µM) in the presence of cycloheximide (5 µM). Cells were lysed in SDS sample buffer at the times indicated and subjected to immunoblot analysis for cyclin D1. The expression levels of cyclin D1 were quantified by densitometry and statistically analyzed. Values are means ± S.E. of three independent experiments. *, p < 0.01 compared with the value at time 0 (Student's t test).

Lithium Inhibited DIF-3-induced Effects-- It has been reported that cyclin D1 undergoes ubiquitin-proteasome-dependent proteolysis upon phosphorylation by GSK-3beta (12). To investigate whether GSK-3beta is involved in the DIF-3-induced degradation of cyclin D1 and inhibition of cell proliferation, we examined the effect of lithium chloride on these DIF-3-induced effects, because lithium ion is a specific inhibitor for GSK-3beta (15). As shown in Fig. 5A, lithium chloride (20 mM) completely inhibited the degradation of cyclin D1 induced by DIF-3. We next examined the effect of lithium chloride on the inhibition of cell proliferation induced by DIF-3 (Fig. 5, B and C). Since 20 mM lithium chloride strongly inhibited cell proliferation on its own probably due to cytotoxicity, we reduced the concentrations of lithium chloride to 2-5 mM. Although 5 mM lithium chloride inhibited cell proliferation by about 30%, it partially prevented the effect of DIF-3 (Fig. 5B). Fig. 5C shows more clearly that lithium chloride dose-dependently inhibited the effect of DIF-3. Based on these results, we hypothesized that DIF-3 activates GSK-3beta to induce proteolysis of cyclin D1 and antiproliferative effect.


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Fig. 5.   Lithium chloride inhibited the effects of DIF-3. A, the effect of lithium chloride on DIF-3-induced cyclin D1 degradation. HeLa cells were pretreated with or without lithium chloride (20 mM) for 3 h and then incubated in the presence or absence of DIF-3 (30 µM) for 3 h. Protein samples were separated by 12% SDS-PAGE and immunoblotted for cyclin D1. The result demonstrated is representative of three other experiments. B, the effect of lithium chloride on cell proliferation. Cells were treated with or without lithium chloride (20 mM) for 3 h and then incubated in the presence or absence of DIF-3 (30 µM) for 24 h. Cells were enumerated after harvested by the trypsin/EDTA treatment. Values are means ± S.E. for three independent experiments. C, data presented in B are differently expressed. The numbers of cells treated with DIF-3 are shown as percentages of the cell numbers obtained in the absence of DIF-3. Values are means ± S.E. for three independent experiments. *, p < 0.05; **, p < 0.001 (Student's t test).

DIF-3 Activated GSK-3beta -- The activity of GSK-3beta was measured using an in vitro kinase assay. DIF-3 (30 µM) elevated GSK-3beta activity by 1.9-fold after 30 min incubation, and GSK-3beta was still activated at 3 h (Fig. 6A). In the presence of lithium chloride, however, DIF-3 was not able to activate GSK-3beta (Fig. 6B). Since GSK-3beta is activated by the dephosphorylation of Ser9 (9-11), the level of GSK-3beta Ser9 phosphorylation was examined using an anti-phospho-GSK-3beta (Ser9) antibody. As shown in Fig. 6C, DIF-3 dramatically reduced the phosphorylation level of Ser9 on GSK-3beta after the incubation with DIF-3 for 30 min, and the phosphorylation level of Ser9 was slowly recovered, looking like a mirror image of the time course of GSK-3beta activity (Fig. 6A). Further, we examined the tyrosine phosphorylation level of GSK-3beta , since the activity of this enzyme has been reported to be increased by phosphorylation of Tyr216 (9-11). As shown in Fig. 6D, DIF-3 significantly elevated the tyrosine phosphorylation level of GSK-3beta by 2.0-fold after the incubation with DIF-3 for 30 min. These results strongly indicated that DIF-3 activates GSK-3beta .


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Fig. 6.   DIF-3 activates GSK-3beta . HeLa cells were incubated with or without DIF-3 (30 µM) for the periods indicated. A, in vitro kinase assay. GSK-3beta was immunoprecipitated from cell lysates and measured for kinase activity using a substrate peptide derived from the sequence of glycogen synthase. The results are means ± S.E. of three independent experiments performed in duplicate. *, p < 0.01 compared with the control at time 0 (Student's t test). B, the effect of lithium chloride on GSK-3beta activity. GSK-3beta immunoprecipitated from cell lysates was measured for kinase activity using a substrate peptide derived from the sequence of glycogen synthase in the presence or absence of 10 mM lithium chloride. The results are means ± S.E. of three independent experiments. C, phosphorylation of Ser9 on GSK-3beta . Cell lysates were subjected to immunoblot analysis using an anti-phospho-GSK-3beta (Ser9) antibody. The levels of Ser9 phosphorylation on GSK-3beta were quantified by densitometry and shown as percentages of the levels in the control cells. Values are means ± S.E. of three independent experiments. *, p < 0.01 compared with the control (Student's t test). D, tyrosine phosphorylation of GSK-3beta . Immunoprecipitated GSK-3beta was subjected to immunoblot analysis using an anti-phosphotyrosine antibody (PY-20). The membrane was reprobed with an anti-GSK-3beta antibody. The levels of tyrosine-phosphorylated GSK-3beta were quantified and shown as percentages of the levels in the control cells. Values are means ± S.E. of three independent experiments. *, p < 0.01 compared with the control (Student's t test).

DIF-3 Induced Nuclear Translocation of GSK-3beta -- GSK-3beta is a cytosolic protein; however, it is translocated into the nucleus when activated (9-11). GSK-3beta thereby accumulated in the nucleus phosphorylates cyclin D1 and excludes it from nucleus, resulting in its degradation in the cytoplasm (12). To test whether DIF-3-activated GSK-3beta is able to target cyclin D1, we examined the subcellular distribution of GSK-3beta after stimulation with DIF-3. Immunofluorescent staining for GSK-3beta revealed that GSK-3beta was most present in the cytoplasm and that there was only a small amount in nuclei in unstimulated cells; however, it was markedly translocated into nuclei after stimulation with DIF-3 (Fig. 7A). Importantly, the time course of GSK-3beta translocation into nuclei was similar to that of cyclin D1 degradation. As shown in Fig. 7B, a similar result was obtained using another anti-GSK-3beta antibody. The nuclear translocation of GSK-3beta was confirmed by immunoblotting of subcellular protein fractions. As shown in Fig. 7C, the treatment with DIF-3 markedly increased GSK-3beta in the nuclear fraction. Its reduction in the cytosolic fraction may not be very clear, because we applied 5 µg of each protein sample, although the amounts of cytosolic proteins extracted were ~5 times those of nuclear proteins. These results clearly demonstrated that cytosolic GSK-3beta was translocated into the nucleus in response to DIF-3.


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Fig. 7.   DIF-3 induces GSK-3beta nuclear accumulation. A and B, immunofluorescence. Cells were plated on coverslips and incubated with or without DIF-3 (30 µM) for the periods indicated. Immunofluorescent staining with a monoclonal (A) or a polyclonal (B) anti-GSK-3beta antibody was performed as described under "Materials and Methods." The results are representative of three other experiments. C, Western blot analysis. Nuclear and cytoplasmic proteins were purified from HeLa cells incubated with or without DIF-3 (30 µM) for 2 h. Western blot analysis was carried out using a monoclonal anti-GSK-3beta antibody. The results are representative of three other experiments.

The Phosphatidylinositol 3-Kinase (PI3K)/Akt Pathway and the Mitogen-activated Protein Kinase (MAPK) Cascade Were Not Involved in DIF-3-induced Cyclin D1 Degradation-- GSK-3beta has been reported to be phosphorylated on Ser9 by several protein kinases (e.g. Akt, which is activated by PI3K, and p90 ribosomal S6 kinase (p90RSK), which is activated by the MAPK cascade) (16). We therefore examined the effect of DIF-3 on the phosphorylation levels of Akt and p90RSK to determine whether DIF-3 inhibits these kinases. Although DIF-3 did not affect the phosphorylation level of Akt, it significantly induced phosphorylation on Ser380 of p90RSK (Fig. 8A). We also examined the effects of a PI3K inhibitor wortmannin and a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor U0126 on cyclin D1 degradation induced by DIF-3. However, as shown in Fig. 8B, neither prevented the effect of DIF-3 on cyclin D1. Therefore, the PI3K/Akt pathway and the MAPK cascade did not seem to be involved in DIF-3-induced cyclin D1 degradation.


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Fig. 8.   Involvement of the PI3K/Akt pathway and the MAPK cascade in DIF-3 signaling. A, the effect of DIF-3 on the phosphorylation of Akt and p90RSK. Cells were treated with or without 30 µM DIF-3 for the periods indicated. Protein samples were subjected to immunoblot analysis using anti-phospho-Akt (Ser473) or anti-phospho-p90RSK (Ser380) antibody. The results are representative of three other experiments. B, the effects of a PI3K inhibitor and a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 inhibitor on cyclin D1 degradation induced by DIF-3. Cells were treated with 300 nM of wortmannin (PI3K inhibitor) for 30 min or 10 µM of U0126 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 inhibitor) for 2 h and then incubated with or without DIF-3 (30 µM) for 3 h. Protein samples were subjected to immunoblot analysis using an anti-cyclin D1 antibody. The results are representative of three other experiments.

The Effect of DIF-3 on Untransformed Mammalian Cells-- In human umbilical vein endothelial cells as well, DIF-3 inhibited cell proliferation (Fig. 9A), induced G0/G1 arrest (Fig. 9B), and induced the degradation of cyclin D1 and the activation of GSK-3beta (Fig. 9C), indicating that these effects of DIF-3 are not limited to transformed cells but common between transformed and untransformed cells.


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Fig. 9.   The effect of DIF-3 on vascular endothelial cells. A, cell number. Human umbilical vein endothelial cells were enumerated after incubated with or without DIF-3 (30 µM) for 24 h. The increases in cell number are shown as means ± S.E. of three independent experiments. B, cell cycle distribution. Endothelial cells were incubated with DIF-3 (30 µM) for 24 h and analyzed for cell cycle by flow cytometer. The percentages of cell number in the cell cycle phases are shown as means ± S.E. of three independent experiments performed in duplicate. C, the effect of DIF-3 on the levels of cyclin D1 and phospho-GSK-3beta . Endothelial cells were incubated with or without DIF-3 (30 µM) for the periods indicated and subjected to immunoblot analysis. The results are representative of three other experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we showed that DIF-3 accelerated the degradation of cyclin D1 by activating GSK-3beta mainly using HeLa cells. However, this effect of DIF-3 was also found in other cell species including transformed and normal (untransformed) cells. As an example, we have demonstrated the results obtained in vascular endothelial cells. Therefore, DIF-3 may ubiquitously activate GSK-3beta in the wide variety of cell types. Since HeLa cells express human papilloma virus antigens E6 and E7, which inactivate retinoblastoma protein (8), it is not clear whether cyclin D1 is required for their cell cycle progression. However, lithium chloride prevented both the degradation of cyclin D1 and the inhibition of cell proliferation induced by DIF-3. Therefore, cyclin D1 may be required for proliferation, and the disappearance of cyclin D1 may be responsible for the anti-proliferative effect of DIF-3 in HeLa cells. Furthermore, overexpression of cyclin D1 prevented DIF-3-induced cell cycle arrest in untransformed endothelial cells. Taken together, cyclin D1 degradation seemed to be a main mechanism for DIF-3 to induce cell cycle arrest.

DIF-3, naturally generated from DIF-1 as its first metabolite, is much weaker than DIF-1 in the ability to induce stalk cell differentiation in Dictyostelium (2). However, in contrast, the antiproliferative effect of DIF-3 was significantly stronger than that of DIF-1 in HeLa cells, consistent with a previous report that DIF-3 is more effective than DIF-1 at inhibiting proliferation in K562 human leukemia cells (5). This species difference in the sensitivity to DIFs may be caused by a difference in the nature of the target molecule (such as the affinity for DIFs) between mammalian and Dictyostelium cells.

We found that DIF-3 not only elevated the activity of GSK-3beta but also induced nuclear translocation of the kinase. GSK-3beta was initially considered to be a soluble protein expressed in cytoplasm; however, GSK-3beta would have no access to nuclear proteins such as cyclin D1 if it resided in cytoplasm. Recent evidence has indicated that GSK-3beta in nucleus phosphorylates nuclear proteins, such as cyclin D1 (12), nuclear factor of activated T-cells (17), heat shock factor-1 (18), and cAMP-response element-binding protein (19). Indeed, GSK-3beta has been identified from the nuclei of cell cycle-arrested NIH-3T3 cells (12), cardiomyocytes stimulated with endothelin-1 (20), and heat shock- and staurosporine-treated SH-SY5Y human neuroblastoma cells (21). Therefore, DIF-3 seems to make it possible for GSK-3beta to phosphorylate cyclin D1 by translocating GSK-3beta into the nucleus.

Not only a rapid proteolysis but also a reduction in cyclin D1 mRNA expression was induced by DIF-3, although this latter effect took much longer time. Cyclin D1 gene expression is activated by beta -catenin, the degradation of which is also initiated by GSK-3beta (22). Therefore, activation of GSK-3beta was expected to lead to a reduction in both protein and mRNA levels of cyclin D1 through independent pathways. Our results agreed well with this rationale. The proteolysis and mRNA reduction both seemed to be explained by DIF-3-induced GSK-3beta activation.

The target molecule for DIFs is still unknown even in Dictyostelium. Since DIF-3 activates GSK-3beta , a target for DIF-3 may be a protein closely related to the regulation of GSK-3beta activity. The activity of GSK-3beta is decreased by phosphorylation of Ser9 and increased by phosphorylation of Tyr216 (9-11). We found that DIF-3 decreased the phosphorylation level of Ser9 and increased the phosphorylation level of Tyr216 on GSK-3beta . Akt activated by PI3K and p90RSK activated by the MAPK cascade are the candidate molecules to modulate GSK-3beta activity by Ser9 phosphorylation (16). Although DIF-3 did not affect the Ser473 phosphorylation of Akt, it strongly induced phosphorylation on Ser380 of p90RSK. However, we were not able to elucidate the role of DIF-3-induced p90RSK activation, since this kinase did not seem to be involved in cyclin D1 degradation induced by DIF-3 (Fig. 6B). DIF-3 also enhanced tyrosine phosphorylation on GSK-3beta . Recently, a novel nonreceptor tyrosine kinase, ZAK-1, has been found to directly activate GSK-3 in Dictyostelium (23). However, a ZAK-1 counterpart in mammals has not yet been discovered. A target molecule for DIF-3 might be a novel protein kinase or phosphatase controlling GSK-3beta activity in mammalian cells. Recently, it has been reported that not only phosphorylation but also distribution regulates GSK-3beta activity (21). Thus, DIF-3-induced accumulation of GSK-3beta in nuclei might have an important role to regulate GSK-3beta activity. In addition, if mammalian cells have a target molecule for DIFs as Dictyostelium, one could hypothesize that mammals also produce DIF-like substances to control growth and differentiation.

The Wnt signaling pathway is essential for embryonic development, cell proliferation, cell differentiation, microtubule dynamics, and cell motility. Several mutations have been identified in the components of the Wnt pathway in a variety of malignant tumors. For instance, most human colon cancers have mutations in the APC gene that result in the accumulation of beta -catenin (24). Mutations in beta -catenin, which amplify the accumulation of beta -catenin itself, also have been identified in colon cancers (25), malignant melanomas (26), prostate cancers (27), and hepatocellular carcinomas (28). beta -Catenin accumulated to an abnormal level would produce an excessive amount of cyclin D1 mRNA and promote tumor growth (29). Therefore, chemicals that activate GSK-3beta , such as DIFs, may be useful for the treatment of several cancers.

Recently, it has been reported that proapoptotic stimuli, such as heat shock and staurosporine, activate GSK-3beta and induce its accumulation in nucleus (21). However, cytotoxic or proapoptotic agents damage not only cancer cells but also normal cells, which would cause severe adverse drug events. Distinct from staurosporine and other proapoptotic agents, DIF-3 did not induce the activation of caspase-3 (data not shown). Therefore, the antiproliferative effect of DIF-3 did not seem to be caused by the induction of apoptosis. DIF-3 is a unique compound that activates GSK-3beta but does not induce apoptotic cell death.

    ACKNOWLEDGEMENTS

We thank Katsuyuki Tamai (Medical and Biological Laboratories Co., Nagano, Japan) for kindly providing human cyclin D1 cDNA, Hiroki Yoshida (Medical Institute of Bioregulation, Kyushu University) for technical advice, and Toyama Chemical Co. for synthesizing DIF-3.

    FOOTNOTES

* This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology (a grant-in-aid for Scientific Research) and Uehara Memorial Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Clinical Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. Fax: 81-92-642-6084; E-mail: yanaga@clipharm.med.kyushu-u.ac.jp.

Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M205768200

    ABBREVIATIONS

The abbreviations used are: DIF, differentiation-inducing factor; GSK-3beta , glycogen synthase kinase-3beta ; BAEC, bovine aortic endothelial cell; RT-PCR, reverse transcription-PCR; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; RSK, ribosomal S6 kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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