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Endocrinology, doi:10.1210/en.2004-0199
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Endocrinology Vol. 145, No. 12 5862-5874
Copyright © 2004 by The Endocrine Society

Insulin-Induced Cell Cycle Progression Is Impaired in Chinese Hamster Ovary Cells Overexpressing Insulin Receptor Substrate-3

Yasushi Kaburagi, Ryo Yamashita, Yuzuru Ito, Hitoshi Okochi, Ritsuko Yamamoto-Honda, Kazuki Yasuda, Hisahiko Sekihara, Takehiko Sasazuki, Takashi Kadowaki and Yoshio Yazaki

Departments of Metabolic Disorder (Y.K., R.Y., Y.I., K.Y., T.S., Y.Y.) and Tissue Regeneration (H.O.), Research Institute, International Medical Center of Japan, Shinjuku-ku, Tokyo 162-8655; Department of Endocrinology and Metabolism (R.Y., Y.I., H.S.), School of Medicine, Yokohama City University School of Medicine, Yokohama 236-0004; Department of Metabolic Disease (R.Y.-H., T.K.), Graduate School of Medicine, University of Tokyo, Tokyo 113-8655; and The Institute for Diabetes Care and Research (R.Y.-H.), Asahi Life Foundation, Tokyo 100-0005, Japan

Address all correspondence and requests for reprints to: Yasushi Kaburagi, M.D., The Department of Metabolic Disorder, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. E-mail: kaburagi{at}ri.imcj.go.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To analyze the roles of insulin receptor substrate (IRS) proteins in insulin-stimulated cell cycle progression, we examined the functions of rat IRS-1 and IRS-3 in Chinese hamster ovary cells overexpressing the human insulin receptor. In this type of cell overexpressing IRS-1 or IRS-3, we showed that: 1) overexpression of IRS-3, but not IRS-1, suppressed the G1/S transition induced by insulin; 2) IRS-3 was more preferentially localized to the nucleus than IRS-1; 3) phosphorylation of glycogen synthase kinase 3 and MAPK/ERK was unaffected by IRS-3 overexpression, whereas that of protein kinase B was enhanced by either IRS; 4) overexpressed IRS-3 suppressed cyclin D1 expression in response to insulin; 5) among the signaling molecules regulating cyclin D1 expression, activation of the small G protein Ral was unchanged, whereas insulin-induced gene expression of c-myc, a critical component for growth control and cell cycle progression, was suppressed by overexpressed IRS-3; and 6) insulin-induced expression of p21, a cyclin-dependent kinase inhibitor, was decreased by overexpressed IRS-3. These findings imply that: 1) IRS-3 may play a unique role in mitogenesis by inhibiting insulin-stimulated cell cycle progression via a decrease in cyclin D1 and p21 expressions as well as suppression of c-myc mRNA induction in a manner independent of the activation of MAPK, protein kinase B, glycogen synthase kinase 3 and Ral; and 2) the interaction of IRS-3 with nuclear proteins may be involved in this process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN INITIATES ITS biological effects by binding the cell surface insulin receptor and activating its endogenous tyrosine kinase (1). Activation of the receptor tyrosine kinase leads to tyrosine phosphorylation of endogenous substrates including insulin receptor substrate (IRS) proteins, Grb2-associated binder 1, p60dok, Cbl, adaptor protein containing pleckstrin and SH2 domains, and Src homology and collagen (1, 2, 3, 4, 5). Subsequent to tyrosine phosphorylation, these proteins bind various Src homology 2 domain-containing proteins, such as the regulatory subunits of phosphatidylinositol (PI) 3-kinase (p85{alpha}, p55{alpha}, p50{alpha}, p85ß, and p55PIK), growth factor receptor-bound protein 2, and SH2 domain-containing tyrosine phosphatase 2, which mediate various biological processes (6). Four members of the IRS family have been cloned (2, 3, 4, 5, 7), each of which contains a pleckstrin homology domain interacting with phospho-lipids, a phosphotyrosine-binding domain binding to the tyrosine-phosphorylated region of the insulin receptor and the C-terminal portion containing a number of tyrosine phosphorylation sites that can bind various Src homology 2 domain-containing proteins (1, 8). Despite the structural similarities between these IRSs, analyses of the IRS knockout mice demonstrated that IRS proteins have different functions in development and metabolism (9, 10, 11, 12, 13), although the molecular mechanisms of these functional differences among IRSs have not been analyzed in detail.

Several studies have shown that IRS proteins play a role in transmitting mitogenic signals in various cell types. Murine hematopoietic 32D myeloprogenitor cells, which do not express any of the IRS proteins, showed restoration of the mitogenic response to insulin, IGF-I, and IL-4 via overexpression of IRS-1 (14, 15). The expression of antisense IRS-1 RNA impaired insulin-induced mitogenesis in hepatoma cells and Chinese hamster ovary (CHO) cells (16, 17). IRS-1-deficient 3T3 embryonic fibroblast cell lines exhibited impaired IGF-I-induced cell cycle progression, which was restored by the overexpression of IRS-1, whereas IRS-2 overexpression had a minor mitogenic effect, indicating that the signals mediated by IRS-1 may not be completely restored by other IRS proteins (18). In contrast, analyses of hematopoietic cell lines revealed that IRS-2 is also involved in transmitting mitogenic signals in response to insulin and IL-4 in these cells (14, 19). IRS-2 was also shown to play a role in promoting cell proliferation in pancreatic ß-cell lines (20, 21), which may be inversely correlated with the marked reduction of ß-cell mass in mice lacking IRS-2 (22). Although IRS-4 was reportedly expressed at lower levels in a few tissues and organs (23), analyses of NIH3T3 cells and 32D cells overexpressing IRS-4 revealed that IRS-4 plays a role in the mitogenic pathways mediated by insulin, IGF-I, and IL-4 (24, 25). In contrast, an analysis of embryonic fibroblast cell lines from wild-type and IRS-1 null mice demonstrated that IRS-3 and IRS-4 might act as negative regulators of the IGF-1 signaling pathway by suppressing the functions of IRS-1 and IRS-2 at several steps, partly caused by the decreased mRNA and protein levels of IRS-2 (26). These findings collectively imply that IRS proteins may play distinct roles in transmitting proliferative signals depending on the cellular context.

In our previous study, we analyzed insulin-induced glucose transport in primary adipocytes from IRS-1 null mice (27). Both IRS-1 and IRS-3 are expressed in comparable amounts in primary adipocytes from wild-type mice (27), whereas the lack of IRS-1 led to a 50% decrease in insulin-induced glucose transport, indicating that IRS-3 partially contributes to this process (27). We also showed that overexpression of IRS-3, as well as IRS-1, protects CHO cells overexpressing human insulin receptors (CHO-IR) from staurosporine-induced apoptosis via the PI 3-kinase/protein kinase B (PKB)/Forkhead cascade (28). In this study, to analyze the roles of IRS-1 and IRS-3 in insulin-induced cell proliferation, we examined the effects of rat IRS-1 or IRS-3 overexpression on insulin-induced cell cycle progression in CHO-IR cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies
Mouse anti-Rb monoclonal antibody (G3–245) was obtained from PharMingen (San Diego, CA), mouse anti-cyclin E monoclonal antibody (Ab-1) from Calbiochem (San Diego, CA), goat anti-IRS-3 polyclonal antibodies (R-20), rabbit anticyclin D1 polyclonal antibodies (H-295), mouse anticyclin-dependent kinase (CDK) 2 monoclonal antibody (D-12), rabbit anti-CDK4 polyclonal antibodies (C-22), rabbit anti-CDK6 polyclonal antibodies (C-21), rabbit anti-p21 polyclonal antibodies (M-19), rabbit antihemagglutinin polyclonal antibodies [{alpha}HA, Y-11], rabbit anti-p44 MAPK/ERK polyclonal antibodies, which also recognize p42 MAPK/ERK2 (K-23), and rabbit antiinsulin receptor ß-chain polyclonal antibodies (C-19) from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-IRS-1 polyclonal antibodies (06–248) and a Ral activation assay kit were purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit antiphospho-PKB (S473) polyclonal antibodies (no. 9271), rabbit anti-PKB polyclonal antibodies (no. 9272), rabbit antiphospho-p44/42 MAPK polyclonal antibodies (no. 9101), rabbit antiglycogen synthase kinase-3 (GSK3){alpha}/ß polyclonal antibodies (no. 9331) and rabbit anti-GSK3ß polyclonal antibodies (no. 9332) were obtained from Cell Signaling Technology (Beverly, MA).

Cell lines analyzed in this study
CHO-IR and CHO-IR cells overexpressing HA-tagged rat IRS-1 or IRS-3 (CHO-IR/IRS-1 or CHO-IR/IRS-3) were previously described (28, 29). The amount of IRS-1 expressed in CHO-IR/IRS-1 or CHO-IR/IRS-3 cells was 166 ± 1.7 and 103 ± 2.8% of that in CHO-IR cells, respectively (Fig. 1Go). Although a previous study demonstrated that IRS-3 transcripts are present not only in adipocytes but also in liver, lung, kidney, and heart (30), IRS-3 expression was undetectable by anti-IRS-3 immunoblotting in parental CHO-IR cells as well as CHO-IR/IRS1 cells (Fig. 1Go), whereas IRS-2 expression was not altered in each cell line, compared with CHO-IR cells (data not shown).



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  		FIG. 1. CHO-IR cells overexpressing IRS-1 or IRS-3 (CHO-IR/IRS1 or CHO-IR/IRS3 cells) analyzed in this study. Upper panel, expression of the HA-tagged IRS proteins in CHO-IR cell lines. Lower panel, expression of the IRß subunit, IRS-1, and IRS-3 in CHO-IR cell lines. Total cell lysates from the established cell lines were subjected to immunoblotting with {alpha}HA, {alpha}IRß, {alpha}IRS-1, or {alpha}IRS-3, as described in Materials and Methods.

 
Flow cytometric analysis
Cells were harvested by trypsin (0.5 mg/ml) and EDTA (0.02 mg/ml) treatment, washed twice with PBS (pH 7.4), and centrifuged at 800 x g for 5 min at 4 C. Cells were then incubated in 0.25 mg/ml RNase (Sigma, St. Louis, MO) for 30 min at 37 C and stained with 50 mg/ml propidium iodide (Sigma) for 30 min at 4 C. Fluorescence of labeled cell nuclei was measured by flow cytometry using EPICS ALTRA (Beckman Coulter, Palo Alto, CA) with a minimum of 10,000 events for each sample. Data were analyzed using WinCycle software (Beckman Coulter). To examine the inhibition of insulin-induced cell cycle progression by the PI 3-kinase inhibitor LY294002 (Sigma) or MAPK kinase (MEK) inhibitor PD980059 (Sigma), 10 µM LY294002 or 50 µM PD980059 were added to serum-starved cells for 1 h, and the cells were subsequently incubated with the kinase inhibitors during a 12-h insulin stimulation in some experiments.

Detection of IRS proteins localized in the nuclei by immunostaining
For immunostaining, CHO cells were plated on 35-mm glass base dishes (Iwaki, Tokyo, Japan) and cultured for 48 h at 37 C. The cells were washed with ice-cold PBS three times, followed by 30 min treatment with PBS containing 4% paraformaldehyde for fixation. Then the cells were permeabilized by treating with ChemMate antibody diluent (Dako- Cytomation, Glostrup, Denmark) for 30 min and were incubated with {alpha}HA (1:100) at 4 C overnight, washed with ice-cold PBS containing 0.05% Tween 20, incubated with tetramethylrhodamine isothiocyanate-conjugated antibody against rabbit IgG (1:100) at 37 C for 1 h, and washed with ice-cold PBS containing 0.05% Tween 20, followed by TO-PRO-3 (1:2000, Molecular Probes, Eugene, OR) labeling for 5 min at room temperature. The stained sections were mounted in Gel/Mount (Biomeda, Foster City, CA), and overexpressed IRS proteins were visualized and imaged using an immunofluorescence microscope (Carl Zeiss, Esslingen, Germany). In some experiments, to evaluate the effect of neomycin on nuclear localization of overexpressed proteins, 100 µM neomycin (Sigma) were added to the media 6 h before the experiment.

Western blot analysis
CHO cells were plated at a density of 5 x 105 cells/100-mm dish or 2 x 105 cells/60-mm dish and cultured for 24 h at 37 C. CHO cells were initially serum-starved for 24 h and treated with 10 nM insulin for 6 h. The cells were washed twice with ice-cold PBS and then solubilized for 10 min with ice-cold lysis buffer [1% Triton X-100, 50 mM NaCl, 25 mM HEPES, 10 mM NaF, 1 mM Na3VO4, 0.2 mM Na2MoO4, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/ml leupeptin (pH 7.4)]. Insoluble materials were removed by centrifugation at 13,000 x g for 10 min. To evaluate the nuclear distribution of CDKs, nuclear extracts were isolated using nuclear/cytosol fractionation kit (BioVision, Mountain View, CA) in some experiments. Extracted proteins were subjected to SDS-PAGE followed by Western blotting with the indicated antibodies and visualized using a BM chemiluminescence Western blotting kit (Roche Molecular Biochemicals, Mannheim, Germany). Protein band density was analyzed with National Institutes of Health image.

Determination of Ral activity
GTP-bound Ral from CHO cells treated or untreated with 100 nM insulin for 1 min was evaluated by a pull-down assay using the Ral activity assay kit according to the manufacturer’s protocol.

Northern blot analysis
Total RNA was isolated from the cells with Trizol (Invitrogen, Carlsbad, CA). Then 30–40 µg RNA were loaded onto a 0.66 M formaldehyde-containing 1.0% agarose gel in 1x 3[N-morpholino]propanesulfonic acid buffer followed by electrophoresis. RNA was transferred overnight to an Immobilon-Ny+ membrane (Millipore, Bedford, MA), and UV cross-linked. The membrane was hybridized with digoxigenin-labeled c-myc or glyceraldehyde-3-phosphate dehydrogenase cDNA probes overnight. After hybridization, the membrane was washed, followed by detection using a digoxigenin luminescent detection kit (Roche Molecular Biochemicals). The density of the hybridized band was analyzed with NIH image.

Statistical analysis
Where appropriate, data were expressed as the means and SE of three independent experiments. Statistical significance was assessed using Student’s t test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell cycle analysis
To examine the roles of IRS proteins in regulating the cell cycle, we established CHO-IR cell lines overexpressing IRS-1 or IRS-3. The flow cytometric analysis of parental CHO-IR cells revealed that treatment with 10 nM insulin, a concentration at which maximal mitogenic response was reportedly observed in CHO-IR cells (27), produced a maximal increase in the S+G2/M phase percentage at 12 h, whereas it did not significantly change the S+G2/M phase percentage at 6 or 24 h as compared with 0 h (data not shown). Therefore, we chose 10 nM insulin treatment for 12 h as the experimental condition for cell cycle analysis. However, the comparison of insulin-treated and -untreated cells revealed that insulin stimulation for 6 h (P < 0.01), as well as that for 12 h (P < 0.001), significantly increased the S+G2/M phase percentage as compared with unstimulated control (data not shown), indicating that the cells exhibited cell cycle progression induced by insulin treatment for 6 as well as 12 h. In the analysis of CHO cells overexpressing IRS proteins, the treatment of CHO-IR or CHO-IR/IRS-1 cells with insulin increased the percentage in S phase, whereas the effect of insulin was much less in CHO-IR/IRS-3 cells (Fig. 2AGo). The percentages of CHO-IR and CHO-IR/IRS-1 cells in S+G2/M phase was 74.3 ± 3.4 and 74.7 ± 2.8%, respectively, although there was a significant decrease to 63.2 ± 1.2% in CHO-IR/IRS-3 cells as compared with CHO-IR cells (Table 1Go). Treatment of cells with insulin increased the percentage of cells in G2/M phase by 26.2 ± 4.0 and 25.2 ± 2.1% over control in CHO-IR and CHO-IR/IRS-1 cells, respectively (Fig. 2BGo), whereas in CHO-IR/IRS-3 cells, it was significantly reduced to 9.6 ± 1.1% as compared with CHO-IR cells (P < 0.05) (Fig. 2BGo). These observations indicate that IRS-3 plays a unique role in cell cycle regulation by suppressing the insulin-induced G1/S transition.



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  		FIG. 2. Effects of insulin on cell cycle progression in CHO-IR cells overexpressing IRS proteins. A, Histogram representative of three experiments. B, Percentages of cells in S+G2/M. CHO cells were initially serum starved for 24 h and treated with 10 nM insulin for 12 h. Cell cycle distribution was determined by flow cytometric analysis of DNA content in propidium iodide-stained cells as described in Materials and Methods. Data are expressed as a percent above control and shown as the means ± SE of three independent experiments. *, P < 0.05 vs. CHO-IR.

 

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  		TABLE 1. Effect of insulin on cell cycle distribution in CHO-IR, CHO-IR/IRS-1, and CHO-IR/IRS-3 cells

 
Nuclear localization of overexpressed IRS proteins
Recently IRS-3 was shown to localize to not only the plasma membrane but also the nucleus, in which it acts as a transcriptional coactivator (31). Other IRS proteins including IRS-1 and IRS-2 have been also found to localize, under certain conditions, to the nucleus in which they interact with nuclear proteins (32). To test the possibility that the nuclear distribution of IRS-3 may play some role in IRS-3-specific cell cycle inhibition, we examined the subcellular localization of overexpressed IRS-1 or IRS-3 in CHO cells by immunostaining (Fig. 3Go). Whereas most of IRS-1 was detected in cytosol with only faint staining in the nucleus, a marked nuclear staining of IRS-3 was observed, which was resistant to neomycin, an inhibitor of nuclear translocation of some proteins (33). These findings suggest that IRS-3 may play a distinct role in the nucleus by interacting with IRS-3-specific nuclear proteins.



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  		FIG. 3. Nuclear localization of overexpressed IRS proteins in CHO-IR cell lines. CHO cells plated on glass base dishes were cultured for 48 h at 37 C. Six hours before the experiment, the cells were treated or untreated with 100 µM neomycin. Overexpressed IRS proteins were visualized by immunostaining with {alpha}HA. TO-PRO-3 labeling was also shown to confirm the localization of the overexpressed proteins in the nuclei. The experiments were performed at least three times independently. Representative results are shown.

 
Effects of IRS protein overexpression on PKB and MAPK pathways
To examine the effects of IRS overexpression on the major insulin-signaling pathways, we evaluated activation phosphorylation of PKB and MAPK/ERK, both of which have been shown to be involved in G1 to S phase progression (34, 35, 36, 37, 38). In fact, the analysis of CHO-IR cells with the PI 3-kinase inhibitor LY294002 and the MEK inhibitor PD980059 showed that both of the kinase inhibitors significantly impaired the insulin-induced cell cycle progression and cyclin D1 expression in this parental cell line (data not shown). We also examined time-course data of PKB or MAPK phosphorylation, which revealed that both of them increased at 6 h, comparable with those at 12 h (data not shown). Based on these data, we selected 6 h insulin treatment as the experimental condition for signaling analysis.

In the analysis of PKB phosphorylation, we measured the ratio of phosphorylated to total PKB because we observed that PKB expression in CHO-IR/IRS-3 cells was significantly decreased as compared with CHO-IR cells (28) (Fig. 4Go). Overexpression of both IRS-1 and IRS-3 significantly elevated the phosphorylated to total PKB ratio in insulin-treated and -untreated states, although the elevation of the ratio was much greater in CHO-IR/IRS-3 cells (Fig. 4Go). The analysis of MAPK phosphorylation and expression showed that a 10 nM insulin treatment for 6 h caused a marked increase in phosphorylation of p42 and p44 MAPKs in all cell types, although no significant differences in MAPK phosphorylation were observed among the three cell lines (Fig. 5Go). As IRS-3 overexpression did not inhibit the activation of these protein kinases (Figs. 4Go and 5Go), which are involved in the regulation of insulin-induced cell cycle progression in parental CHO-IR cells (data not shown), these findings imply that IRS-3 may impair cell cycle progression induced by insulin in a manner independent of the PKB cascade and the MAPK cascade.



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  		FIG. 4. Effects of IRS protein overexpression on PKB expression and phosphorylation in CHO-IR cell lines. CHO cells were initially serum starved for 24 h and treated with 10 nM insulin for 6 h. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting using anti-PKB antibody or antiphospho-PKB antibody. Blots were quantified by scanning densitometry. Data are the means ± SE from three independent experiments. *, P < 0.05 vs. CHO-IR; **, P < 0.01 vs. CHO-IR.

 


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  		FIG. 5. Effects of IRS protein overexpression on MAPK expression and phosphorylation in CHO-IR cell lines. CHO cells were initially serum starved for 24 h and then treated with 10 nM insulin for 6 h. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting using anti-MAPK antibody or antiphospho-MAPK antibody. Blots were quantified by scanning densitometry. Data are the means ± SE from three independent experiments.

 
Effects of IRS protein overexpression on GSK3 phosphorylation
To examine the effects of IRS overexpression on GSK3, which is involved in a wide-range of cellular processes including cell cycle regulation (39), we evaluated inactivation phosphorylation of GSK. Insulin-induced phosphorylation of either GSK3{alpha} or GSK3ß was not significantly different among the three cell lines (Fig. 6Go). We also observed that basal phosphorylation of GSK3{alpha}, but not that of GSK3ß, was significantly increased in CHO-IR/IRS-3 cells as compared with CHO-IR cells (P < 0.05) (Fig. 6Go), reflecting the up-regulation of basal activity of PKB, which phosphorylates GSK3 and inhibits its activity (28, 39). Because IRS-3 overexpression did not impair the phosphorylation of GSK3 in response to insulin (Fig. 6Go), these findings indicate that GSK3 does not take part in the inhibition of insulin-induced cell cycle progression mediated by overexpressed IRS-3.



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  		FIG. 6. Effects of IRS protein overexpression on GSK3 expression and phosphorylation in CHO-IR cell lines. CHO cells were initially serum starved for 24 h and then treated with 10 nM insulin for 6 h. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting using anti-GSK3ß antibody or antiphospho-GSK3{alpha}/ß antibody. Blots were quantified by scanning densitometry. Data are the means ± SE from three independent experiments. *, P < 0.05 vs. CHO-IR.

 
Expressions of CDKs and cyclins
Because cyclin D1 is thought to form the protein complex with CDKs in response to growth factors, which causes Rb phosphorylation triggering cell cycle progression into the S phase and DNA synthesis (40), we attempted to determine whether the expressions of CDKs and cyclin D1 are modulated by insulin stimulation in CHO-IR, CHO-IR/IRS-1, or CHO-IR/IRS-3 cells. The expression levels of CDK2, CDK4, and CDK6 in both unstimulated and insulin-treated cells were not significantly different in any of the cell lines examined (Fig. 7AGo). The analysis of nuclear extracts also showed that overexpression of IRS proteins does not significantly change the distribution of CDK proteins as compared with CHO-IR cells except for CDK4 in insulin-unstimulated CHO-IR/IRS-3 cells (Fig. 7BGo). Although cyclin D1 expression did not significantly differ in unstimulated states among these cell lines, insulin treatment increased cyclin D1 expression 5.3- and 5.2-fold in CHO-IR and CHO-IR/IRS-1 cells, respectively, whereas the insulin-induced increase in cyclin D1 expression was significantly reduced to 2.4-fold in CHO-IR/IRS-3 cells as compared with CHO-IR cells (P < 0.05) (Fig. 8Go). These findings indicate that IRS-3 inhibits insulin-stimulated cyclin D1 expression, which may be one of the reasons for insulin-induced cell cycle progression being impaired in CHO-IR/IRS-3 cells.



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  		FIG. 7. Expression and nuclear/cytosol distribution of CDK proteins in CHO-IR cells overexpressing IRS proteins. A, Expression of CDKs. B, Nuclear/cytosol distribution of CDK proteins. CHO cells were serum starved for 24 h and then treated with 10 nM insulin for 6 h. Total cell lysates (A) or nuclear/cytosol fractions (B) were separated by SDS-PAGE and analyzed by immunoblotting using anti-CDK2, CDK4, or CDK6 antibody. Blots were quantified by scanning densitometry. Data are the means ± SE from three independent experiments. **, P < 0.01 vs. CHO-IR.

 


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  		FIG. 8. Expression of cyclin D1 in CHO-IR cells overexpressing IRS proteins. CHO cells were initially serum starved for 24 h and then treated with 10 nM insulin for 6 h. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting using anticyclin D1 antibody. Blots were quantified by scanning densitometry. Data are the means ± SE from three independent experiments. *, P < 0.05 vs. CHO-IR.

 
Phosphorylation of Rb
To investigate whether IRS overexpression affects Rb, the downstream effector of cyclin D1-CDK4/6 kinase (40), phosphorylation states of Rb was analyzed by immunoblotting with anti-Rb antibody, which is able to detect the active hypophosphorylated form of Rb as well as the inactive slowly migrating hyperphosphorylated form of the protein. In serum-starved cells, phosphorylated Rb was found in an active and inactive form in each CHO-IR cell line. When the ratios of the amount of the active hypophosphorylated form of Rb to that in untreated CHO-IR cells were calculated, insulin treatment decreased the hypophosphorylated Rb from 100 to 58 ± 9.3% in CHO-IR cells, 99.3 ± 12.1 to 44.0 ± 4.9% in CHO-IR/IRS-1 cells, and 96.0 ± 16.9 to 94.3 ± 12.5% in CHO-IR/IRS-3 cells, respectively (Fig. 9Go). These data suggest that IRS-3, but not IRS-1, impaired insulin-stimulated Rb phosphorylation, which may be one of the causes of the IRS-3-specific cell cycle inhibition.



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  		FIG. 9. Insulin-induced Rb phosphorylation in CHO-IR cells overexpressing IRS proteins. CHO cells were initially serum starved for 24 h and then treated with 10 nM insulin for 6 h. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting using anti-Rb antibody. The experiments were independently performed three times. Representative results are shown.

 
Insulin-induced Ral activation
To examine whether IRS-3 protein could impair activation of Ral, a Ras-related GTPase that plays a role in stimulating cyclin D1 transcription in a MAPK- and PKB-independent manner (41), Ral activity in untreated or insulin-treated CHO cells was evaluated. The GTP-bound form of Ral was not significantly altered by the overexpression of either IRS-1 or IRS-3 in untreated cells (Fig. 10Go). Neither IRS-1 nor IRS-3 overexpression significantly impairs an increase in the amount of the GTP-bound form of Ral in response to 100 nM insulin treatment for 1 min (Fig. 10Go). Thus, these findings indicate that Ral does not take part in the impaired cell cycle progression via the suppressed cyclin D1 expression in the setting of IRS-3 overexpression.



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  		FIG. 10. Insulin-induced Ral activation in CHO-IR cells overexpressing IRS proteins. CHO cells were serum starved for 24 h and then treated with 100 nM insulin for 1 min. The amount of GTP-loaded Ral (GTP-Ral) was determined as described in Materials and Methods. Data are the means ± SE from three independent experiments.

 
Insulin-induced c-myc gene expression
To assess the mechanism underlying the inhibition of cyclin D1 expression in IRS-3 overexpressing cells, we performed a Northern blot analysis of c-myc, the gene of c-Myc, which regulates G1 CDK activity by multiple steps including transcriptional control of cyclin D1 (42, 43). Stimulation with 10 nM insulin for 6 h increased the c-myc mRNA level by 1.8- and 1.6-fold in CHO-IR and CHO-IR/IRS-1 cells, respectively, whereas the response of CHO-IR/IRS-3 cells to insulin was significantly blunted as compared with CHO-IR cells (P < 0.05) (Fig. 11Go). These data indicate that IRS-3 inhibits insulin-stimulated c-myc mRNA induction, which may be one of the causes of the suppressed cyclin D1 expression in response to insulin.



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  		FIG. 11. Insulin-induced c-myc mRNA expression in CHO-IR cells overexpressing IRS proteins. CHO cells were serum starved for 24 h and then treated with 10 nM insulin for 6 h. Total RNA was extracted and analyzed for the expression of c-myc and glyceraldehyde-3-phosphate dehydrogenase mRNA by Northern blotting. c-myc was normalized to glycerol-3-phosphate dehydrogenase mRNA and the amount of c-myc from untreated CHO-IR cells was arbitrarily set at 1. Data are the means ± SE from three independent experiments. *, P < 0.05 vs. CHO-IR. GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

 
Insulin-induced p21expression
The CDK inhibitors including p21 have been shown to interact with cyclin-CDK complexes and block G1/S transition through the cell cycle, although recent studies revealed these proteins act as positive regulators of cyclin D-dependent kinases (44). Moreover, both in vivo and in vitro experiments demonstrated that p21 promotes the formation of active cyclin D/CDK4 kinase complex as well as nuclear targeting of this protein complex (45). To examine whether these CDK inhibitors play a role in IRS-3-specific inhibition of cell cycle progression, we evaluated the relevant protein expressions in insulin-treated and -untreated cells (Fig. 12Go). In unstimulated cells, overexpression of IRS-3, but not IRS-1, significantly reduced the expression of p21, as compared with CHO-IR cells (P < 0.01). Treatment of the cells with 10 nM insulin for 6 h significantly enhanced p21 expression 1.3- and 1.3-fold in CHO-IR and CHO-IR/IRS-1 cells as compared with unstimulated cells (P < 0.05 and P < 0.05, respectively), whereas p21 expression in CHO-IR/IRS3 cells was significantly suppressed even in the presence of insulin as compared with CHO-IR cells (P < 0.05) (Fig. 12Go). These data indicate that IRS-3 inhibits insulin-stimulated p21 expression, which may affect the formation of cyclin D1/CDK4 complex leading to IRS-3-specific inhibition of cell cycle progression in response to insulin.



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  		FIG. 12. The expression of p21 in CHO-IR cells overexpressing IRS proteins. CHO cells were serum starved for 24 h and then treated with 10 nM insulin for 6 h. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting using anti-p21 antibody. Data are the means ± SE from three independent experiments. *, P < 0.05 vs. control; **, P < 0.01 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we analyzed the role of IRS-3 in regulating insulin-stimulated cell cycle progression. IRS-1 and IRS-3, which are expressed in comparable amounts in primary adipocytes (4, 27), mediate insulin-induced glucose transport (27). Several groups (14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26) reported the relationship between IRS proteins and mitogenesis. However, the effect of IRS-3 on mitogenic signals has not been delineated in detail. Therefore, to investigate the roles of IRS proteins in mitogenesis, we analyzed CHO cells stably expressing human IR and rat IRS-1 or IRS-3 (28). In this study, we observed the protein level of IRS-1 to be unaffected by overexpressed IRS-3 in CHO-IR cells (Fig. 1Go). We also showed insulin-induced cell cycle progression to be impaired by IRS-3 overexpression (Table 1Goand Fig. 1Go), whereas insulin-induced phosphorylation of MAPK was not affected and PKB phosphorylation was elevated (Figs. 4Go and 5Go). Moreover, we demonstrated that IRS-3 was markedly targeted to the nuclei as compared with IRS-1 (Fig. 3Go). These results suggest that IRS-3 acts as a negative regulator of mitogenesis without affecting the major insulin-signaling pathway in the CHO cell line and that there exists novel IRS-3-specific antiproliferative pathways independent of the kinase cascades regulated by PKB or MAPK, one of which may be mediated via the interaction with IRS-3-specific nuclear proteins. The IGF-I receptor, which shares a high degree of homology with the insulin receptor, is involved in tumor progression and apoptosis inhibition and has recently emerged as a new target for anticancer drugs (46, 47). As a signaling protein downstream of the IGF-I receptor, IRS-1 also plays a crucial role in inhibiting differentiation and initiating malignant transformation (47). Therefore, the proteins, which inhibit IRS-1-mediated mitogenic signals via the IRS-3-specific antiproliferative signal independent of the kinase cascades regulated by PKB or MAPK, may be a promising target for antitumor drugs with only minor side effects, given the lack of effects on the major pathways downstream of IRS. To identify these proteins, further studies using proteomic analysis of the proteins specifically expressed in IRS-3-overexpressing cells and IRS-3-interacting proteins in the nucleus may be required.

Cell proliferation requires a successful transition through the restriction point. Cyclin D is a rate-limiting step for transition through this restriction point (37). Thus, cyclin D is positioned at an important point in the cell cycle program at which growth factor-stimulated signaling acts. Growth factors promote phosphorylation of Rb by regulating the synthesis and stability of cyclin subunits (35, 36, 37, 38). Accumulation of cyclin D1 results in the assembly of cyclin D1/CDK4, six complexes that in turn regulate cyclin D1-CDK4/6 kinase activity (37). In this study, we demonstrated insulin increases cyclin D1 protein level in CHO-IR and CHO-IR/IRS1 cells (Fig. 8Go). However, this insulin-induced increase in cyclin D1 expression was significantly suppressed in CHO-IR/IRS-3 cells as compared with CHO-IR cells, suggesting that IRS-3 may impair the insulin-induced expression of cyclin D1, resulting in decreased cyclin D1-CDK4/6 kinase activity (Fig. 8Go). We also showed that insulin stimulation decreased the active hypophosphorylated form of Rb in CHO-IR and CHO-IR/IRS-1 cells, whereas in CHO-IR/IRS-3 cells, the hypophosphorylated form of Rb was not apparently decreased in response to insulin, indicating that insulin-induced cyclin D1-CDK4/6 kinase activation is impaired by overexpressed IRS-3 (Fig. 9Go). These data suggest that IRS-3 blocks insulin-induced cell cycle progression by suppressing cyclin D1 expression and its downstream phosphorylation inactivation of Rb.

It has recently been shown that three Ras effector pathways control cyclin D1 protein levels in a cell growth-dependent manner requiring the cooperative actions of these three pathways for maximal stimulation (43). These are the Raf/MEK/MAPK cascade (48), the Ral GTPase signaling pathway (41) and the PI 3-kinase/PKB pathway, which increases the stability of cyclin D1 protein via inhibition of GSK3 (36, 39, 49). To determine which of these Ras-downstream pathways are inhibited by IRS-3, we first investigated the contribution of the MAPK and PKB pathways in regulating cyclin D1 expression. MAPK phosphorylation did not appear to play a role in the distinct pattern of cyclin D1 expression mediated by overexpressed IRS proteins because the extent of maximum MAPK phosphorylation did not differ among CHO-IR, CHO-IR/IRS-1, and CHO-IR/IRS-3 cells (Fig. 5Go). Although the expression of PKB was decreased in CHO-IR/IRS3 cells, overexpression of both IRS-1 and IRS-3 significantly elevated the phosphorylated to total PKB ratio in both insulin-treated and -untreated states, although the elevation of the ratio was much greater in CHO-IR/IRS-3 cells (Fig. 4Go), indicating that PKB was not responsible for the suppression of insulin-stimulated cyclin D1 expression in CHO-IR/IRS-3 cells either. We also observed that phosphorylation of neither GSK3{alpha} nor GSK3ß in response to insulin was affected by overexpressed IRS proteins (Fig. 6Go). As for the Ral GTPase signaling pathway, neither IRS-1 nor IRS-3 significantly affected insulin-induced activation of Ral as compared with CHO-IR cells (Fig. 10Go). These findings indicate that IRS-3 directly or indirectly inhibits cell cycle progression through signaling pathways, other than these Ras effector pathways, thereby suppressing insulin-induced cyclin D1 expression in CHO-IR/IRS-3 cells. Although {rho}-family proteins have recently been shown to play a critical role in growth factor-dependent cell cycle progression (reviewed in Ref. 50), insulin-stimulated activation of Rac, CDC42, or {rho} was undetectable in our pull-down assays (data not shown).

The protooncogene c-myc, a key regulator of cell proliferation and apoptosis, encodes c-Myc, which has been shown to be regulated by both the MAPK and PI3-kinase/PKB pathways and to induce CDK activity by a direct transcriptional activation of cyclin D1 (43). Moreover, the analyses of Myc or cyclin D null cells have demonstrated the cyclin D/CDK complexes to be the targets of Myc-mediated cell cycle progression (51, 52). In c-myc null cells, the major defect was impaired activation of cyclin D1/CDK4 and cyclin D1/CDK6 complexes during G1/S transition, whereas more cyclin D1 protein was expressed than in wild-type cells (50). Overexpression of cyclin D1 in c-myc null cells restored the kinetics of Rb phosphorylation but did not reverse cell proliferation retardation, indicating that c-Myc not only regulates the function of cyclin D1 but also plays a role at multiple steps in promoting cell cycle progression (50). In mouse embryo fibroblasts lacking cyclin D1 or D2, overexpressed Myc did not activate the cyclin E/CDK2 complex, which is also essential for G1/S transition and failed to promote cell proliferation (51). Overexpression of cyclin D1 or D2 in cyclin D1 null cells restored Myc-induced cell cycle progression by sequestrating the G1 cell cycle inhibitors p27 and p21 from the cyclin E/CDK2 complex via cyclin D/CDK complexes, thereby activating the cyclin E/CDK2 complex (51). In CHO-IR/IRS3 cells, the gene expression of c-myc and induction of cyclin D1 protein in response to insulin were suppressed, both of which may cause cyclin D/CDK4 inhibition (Figs. 8Go and 11Go). Although the induction of CDK4 was shown to be inhibited in c-Myc-deficient RAT1 cells (53), the CDK4 expression level was unchanged in CHO-IR/IRS3 cells as compared with parental CHO-IR cells, indicating that the CDK4 protein level is apparently not a limiting step in cell cycle progression in CHO-IR/IRS3 cells (Fig. 7Go).

We also observed that p21 expression is significantly decreased by overexpressed IRS-3 in both insulin-treated and -untreated states (Fig. 12Go). This CDK inhibitor has been recently shown to act as a positive regulator of cyclin D-dependent kinases (44). Moreover, both in vivo and in vitro experiments demonstrated that p21 promotes the formation of active cyclin D/CDK4 kinase complex via concomitant binding of cyclin D and p21 to CDK4 and that this CDK inhibitor targets the protein complex to the nucleus (45). In addition, the analysis of MCF-7 breast cancer cells treated with IGF-I also demonstrated the role of p21 as a positive regulator of cyclin D-dependent kinases (54). In our analysis, insulin-induced expression of p21 was enhanced in CHO-IR and CHO-IR/IRS1 cells, whereas the expression of p21 in CHO-IR/IRS3 cells was suppressed both in insulin-untreated and -treated states (Fig. 12Go), indicating that suppressed expression of p21 may play a role in IRS-3-mediated cell cycle inhibition in response to insulin.

Taken together, the analysis of CHO-IR cells overexpressing IRS-1 or IRS-3 showed that: 1) overexpressed IRS-3, but not IRS-1, suppresses the G1/S transition induced by insulin, whereas insulin-induced phosphorylation of PKB, MAPK and GSK3, as well as Ral activation, is not impaired by either IRS protein; 2) overexpressed IRS-3, but not IRS-1, was preferentially localized to the nucleus; 3) overexpression of IRS-3, but not IRS-1, suppresses cyclin D1 expression in response to insulin; 4) transcription of c-myc is also suppressed by overexpressed IRS-3; and 5) IRS-3 overexpression decreases insulin-induced expression of p21. From these data, the molecular mechanism of IRS-3-mediated cell cycle inhibition may be deduced as shown in Fig. 13Go, implying that IRS-3 plays a unique role in the inhibition of insulin-stimulated cell cycle progression, which may be attributable, at least in part, to impaired induction of c-myc mRNA and suppressed expression of cyclin D1 and p21. The preferential localization of IRS-3 to the nucleus suggests that the interaction of IRS-3 with nuclear proteins may be also involved in this process.



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  		FIG. 13. A model for molecular mechanism of insulin-induced cell cycle progression inhibited by IRS-3 overexpression.

 


   Acknowledgments
 
We thank Yasuhiko Nagasaka (Beckman Coulter) for helping us with the flow cytometric analysis. We are grateful to Drs. Natoshi Sugimoto and Yoh Takuwa (Department of Physiology, Kanazawa University School of Medicine, Kanazawa) for helping us with the small G protein activity assays. We also appreciate the helpful suggestions and support provided by Ms. Yuko Fujiwara, Ms. Keiko Hamada, Ms. Kanoko Miura, (International Medical Center of Japan), Dr. Mitsuhiko Noda, and Ms. Kumiko Kimura (Institute for Diabetes Care and Research, Asahi Life Foundation, Tokyo).


   Footnotes
 
This work was supported by a grant for diabetes research (MF-4) from the Organization for Pharmaceutical Safety and Research (to Y.K.). This work was also supported in part by a grant for medical research from Takeda Pharmaceutical Co., Ltd. (to. Y.K.).

Y.K., R.Y., and Y.I. contributed equally to this work.

Abbreviations: CDK, Cyclin-dependent kinase; CHO, Chinese hamster ovary; CHO-IR, CHO cells expressing the human insulin receptor; CHO-IR/IRS1, CHO-IR cells overexpressing IRS-1; CHO-IR/IRS3, CHO-IR cells overexpressing IRS-3; GSK, glycogen synthase kinase; HA, hemagglutinin; IRS, insulin receptor substrate; MEK, MAPK kinase; PI, phosphatidylinositol; PKB, protein kinase B.

Received February 17, 2004.

Accepted for publication August 18, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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