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Endocrinology, doi:10.1210/en.2007-1443
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Endocrinology Vol. 149, No. 7 3729-3742
Copyright © 2008 by The Endocrine Society

Distinct Modes of Activation of Phosphatidylinositol 3-Kinase in Response to Cyclic Adenosine 3', 5'-Monophosphate or Insulin-Like Growth Factor I Play Different Roles in Regulation of Cyclin D1 and p27Kip1 in FRTL-5 Cells

Toshiaki Fukushima1, Taku Nedachi1, Hidenori Akizawa, Masakazu Akahori, Fumihiko Hakuno and Shin-Ichiro Takahashi

Departments of Animal Sciences and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Address all correspondence and requests for reprints to: Shin-Ichiro Takahashi, Ph.D., Laboratory of Cell Regulation, Departments of Animal Sciences and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. E-mail: atkshin{at}mail.ecc.u-tokyo.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bioactivities of IGFs in various cells are often potentiated in the presence of other hormones. In previous studies we showed that pretreatment of rat FRTL-5 thyroid cells with TSH or other cAMP-generating agents markedly potentiated DNA synthesis induced by IGF-I. Under these conditions we found that phosphatidylinositol (PI) 3-kinase was activated in response to either cAMP or IGF stimulus, and both activation modes were indispensable for the potentiation of DNA synthesis. The present studies were undertaken to elucidate how cAMP and/or IGF-I stimulus regulated the G1 cyclin-cyclin dependent kinase (CDK)-inhibitor system, and to determine the roles of PI 3-kinase activation by cAMP or IGF-I stimulus in this system. We found that cAMP pretreatment enhanced IGF-I-dependent increases in cyclin D1, due to synergistic increases in mRNA and elevation of translation rates. Furthermore, cAMP pretreatment enhanced IGF-I-induced protein degradation of the CDK inhibitor, p27Kip1. These changes well explained an increase in cyclin E, leading to marked activation of G1 CDKs, followed by retinoblastoma protein phosphorylation. Our results using a PI 3-kinase inhibitor showed that cAMP-dependent PI 3-kinase activation plays an important role in the increase in cyclin D1 translation. In contrast, IGF-I-dependent PI 3-kinase activation was required for the increase in cyclin D1 mRNA levels and degradation of p27Kip1. Together, the present study elucidates the role of cAMP and IGF-I in differentially activating PI 3-kinase as a mediator of multiple molecular events. These events converge in the regulation of cyclin D1 and p27Kip1, leading to cAMP-dependent potentiation of IGF-I-dependent CDK activation and DNA synthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGFs PLAY important roles in intrauterine and postnatal growth (1, 2, 3). In vitro, IGFs are essential for cell proliferation, differentiation, and survival, as well as maintenance of cell functions in many cell types (4). Recently, it has become clear that the mitogenic activities of IGFs are often potentiated in various cells and organs by other growth factors and hormones, including epidermal growth factor, platelet-derived growth factor, or fibroblast-derived growth factor (5, 6, 7, 8, 9, 10, 11), estrogen (12, 13, 14), androgen (12, 15), and tropic hormones (16). Therefore, to determine the physiological significance of IGFs, it is essential to elucidate the molecular mechanisms of potentiation of IGF bioactivities by other factors.

It has been shown in our laboratory and others that TSH potentiates a mitogenic activity of IGFs in cultured thyroid cells of humans, rodents, and dogs (17, 18, 19). In addition, TSH stimulated thyroid follicular cells to secrete IGFs, and secreted IGFs induced cell growth in an autocrine manner during postnatal thyroid growth (20, 21). Interestingly, Cheung et al. (22) reported that, in subjects with hypopituitarism who were deficient in both TSH and IGF-I, thyroid development was often suppressed, and GH treatment restored IGF-I levels but did not increase thyroid size in the absence of TSH. In addition, other studies showed that an excess of IGF-I induced thyroid hyperplasia in patients with acromegaly or transgenic mice overexpressing IGF-I and IGF-I receptor (23, 24, 25). All of these results indicated that in vitro as well as in vivo, IGF-I promotes thyroid cell proliferation in the presence of TSH.

We have investigated the molecular mechanism of synergistic cell proliferation induced by TSH and IGF-I using FRTL-5, which is a nontransformed line of rat thyroid follicular cells and is widely used as a model to study thyroid physiology. We found that prolonged pretreatment of cells with TSH or other agents that increased intracellular cAMP, potentiated DNA synthesis induced by IGF-I (18). Further studies showed that a prolonged cAMP stimulus induced phosphatidylinositol (PI) 3-kinase activation through a novel mechanism, and this activation was necessary for the potentiation of DNA synthesis induced by IGF-I (26). On the other hand, we showed that prolonged cAMP pretreatment enhanced IGF-I-dependent tyrosine phosphorylation of insulin receptor substrate (IRS)-2 and activation of PI 3-kinase bound to IRS-2, and this enhanced signal was also indispensable for synergistic cell proliferation induced by TSH and IGF-I (27). However, it remains unclear how each mechanism of PI 3-kinase activation in response to either cAMP or IGF-I stimulus is important for cell-cycle progression.

In mammalian cells, activation of cyclin D-dependent kinases, cyclin-dependent kinase (CDK) 4 and 6, and cyclin E-dependent kinase, CDK2 is essential for cell-cycle progression from G1 phase to S phase (28). These CDKs are activated by the association with G1 cyclins, such as cyclin Ds and E, and the dissociation from CDK inhibitors (CKIs), such as p21Cip1 and p27Kip1 (29, 30, 31). In general, mitogenic stimuli are believed to activate cyclin D-dependent kinases first by increases in cyclin Ds and decreases in CKIs, followed by phosphorylation of retinoblastoma protein (Rb) (32, 33). This causes dissociation of the E2F transcription factors from Rb (34), resulting in induction of cyclin E gene expression (35). Increased cyclin E induces cyclin E-dependent kinase activation, resulting in maximal phosphorylation of Rb. Finally, free E2Fs cause expression of genes that are necessary for DNA synthesis, such as DNA polymerase {alpha} (36), leading to G1-S transition (37).

Previous studies showed that in FRTL-5 cells, cyclin D1, cyclin D3, cyclin E, CDK2, and CDK4 increased, and p27Kip1 decreased by the simultaneous stimulation with TSH and IGF-I/insulin (38, 39, 40, 41, 42, 43). In addition, Yamamoto et al. (44) reported that TSH pretreatment increased protein levels of cyclin D1 and E, and further IGF-I induced their accumulation driving the cell cycle from G1 to S phase. The present studies were undertaken to elucidate the control of gene expression, translation, and protein stability of G1 cyclins and p27Kip1, as well as the activities of CDKs in response to cAMP and IGF-I stimuli. Moreover, we demonstrated that cAMP pretreatment or IGF-I treatment regulated the activity of PI 3-kinase in a different manner. Thus, we investigated the roles of each mode of activation of PI 3-kinase in the changes in these cell-cycle regulatory proteins. Based on our results, we propose that each mechanism of activation of PI 3-kinase plays a distinct role in the synthesis of cyclin D1 and degradation of p27Kip1, and that the combination of the different modes of regulation enables G1-S phase transition in FRTL-5 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Coon’s modified Ham’s F-12 (Coon’s F-12), transferrin, bovine insulin, and bovine TSH (1.23 U/mg) for culture were purchased from Sigma-Aldrich (St. Louis, MO). Newborn bovine serum was obtained from Nissui (Tokyo, Japan). Dibutyryl cAMP (Bt2cAMP) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Recombinant human IGF-I was kindly donated by Dr. Toshiaki Ohkuma (Fujisawa Pharmaceutical Co., Osaka, Japan). LY294002 was purchased from Sigma-Aldrich. MG132 was obtained from BIOMOL Intl., L.P. (Plymouth Meeting, PA). Leupeptin and pepstatin were kindly donated by Dr. Takaaki Aoyagi (Institute of Microbial Chemistry, Tokyo, Japan). Anti-cyclin D1 antibody (72-13G), anti-cyclin E antibody (M-20), anti-p27Kip1 antibody (M-197, monoclonal), anti-p110{alpha} PI 3-kinase catalytic subunit antibody (H-201), and anti-p110β PI 3-kinase catalytic subunit antibody (S-19) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-CDK2 antibody (c18520), anti-CDK4 antibody (c18720), and anti-p27Kip1 antibody (K25020, polyclonal) were purchased from Transduction Laboratories (San Diego, CA). Anti-p21Cip1 antibody (no. 05-345), anti-p85 PI 3-kinase regulatory subunit antibody (no. 06-195), and anti-IRS-2 antibody (no. 06-506) were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-Rb antibody and anti-phospho Rb (S807/S811) antibody were from Cell Signaling Technology, Inc. (Beverly, MA). Antiphosphotyrosine antibody (no. P4110) was purchased from Sigma-Aldrich. Horseradish peroxidase (HRP)-linked antimouse IgG antibody (NA931) and HRP-linked antirabbit IgG (NA934) antibody were purchased from GE Healthcare UK Ltd. (Buckinghamshire, UK). Full-length cDNA of mouse cyclin D1 and cyclin E were kindly provided by Dr. Jay Cross (University of Toronto, Toronto, Ontario, Canada). Rabbit p27Kip1 partial cDNA was amplified by RT-PCR using 5'-GCCCGAGTTCTACTACAGACCC-3' and 5'-TTTACGTCTGGCGTCGAAGGCC-3' as PCR primers, and cloned using the AdvanTAge PCR Cloning Kit (CLONTECH, Palo Alto, CA). Other chemicals were of the reagent grade available commercially.

Cell culture
Cells of a line of rat thyroid follicular FRTL-5 (45) (no. CRL8305; American Type Culture Collection, Manassas, VA) were kindly provided by Dr. Leonard Kohn (Ohio University and Edison Biotechnology Institute, Athens, OH) and the Interthyr Research Foundation (Baltimore, MD). Cells were routinely cultured in Coon’s F-12 medium containing 0.33 mg/ml glutamine, MEM nonessential amino acids (ICN Biochemicals, Costa Mesa, CA), penicillin, streptomycin, amphotericin B, and kanamycin supplemented with 5% newborn bovine serum and a mixture of three hormones (3H), including TSH (1 mU/ml), insulin (10 µg/ml), and transferrin (5 µg/ml). Cells were cultured in 150 cm2 flasks (IWAKI, Tokyo, Japan) at 37 C in an atmosphere of 95% air, 5% CO2 in a humidified incubator. Cells (passage no. 16-25) were sparsely seeded [2.5 x 104 cells per well of a 48-well plate (IWAKI)] for DNA synthesis assay and [3 x 106 cells per 100-mm dish (IWAKI)] for protein and RNA extraction. Five days later, the cells were washed twice with Hanks’ balanced salt solution (Nissui), and cultures were continued for an additional 48 h in Coon’s F-12 medium containing 0.1% BSA, for cells to become quiescent.

DNA synthesis assay
Quiescent cells on 48-well plates were cultured for 24 h in Coon’s F-12 medium with 0.1% BSA with or without Bt2cAMP (1 mM) in the absence or presence of 50 µM LY294002. After this pretreatment the cells were washed five times with Hanks’ balanced salt solution and then treated with or without IGF-I (100 ng/ml) in the absence or presence of 50 µM LY294002 for indicated hours. [Methyl-3H]thymidine (0.3 µCi/well, 1 µCi/ml; GE Healthcare UK Ltd.) was added to each well 4 h before the termination of each experiment. The labeling was stopped by adding 1 M ascorbic acid. The cells were washed twice with ice-cold PBS and twice with ice-cold 10% trichloroacetic acid. Trichloroacetic acid-precipitated materials were solubilized with 250 µl 0.2 N NaOH and 0.1% sodium dodecyl sulfate (SDS), mixed into 5 ml clear-sol II (Nacalai Tesque), and the radioactivity was measured by a liquid scintillation counter (Aloka, Tokyo, Japan).

Immunoblotting (IB)
Quiescent cells in 100-mm dishes were pretreated and treated with or without test agents indicated as described in DNA synthesis assay. Cells were lysed at 0 C in 400 µl lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM NaF, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 500 µM Na3VO4, 100 kallikrein-inactivating U/ml aprotinin, 20 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 5 µg/ml pepstatin. The lysates were centrifuged at 15,000 x g for 10 min at 4 C. The protein assay of the supernatant was performed using a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). Equal amounts of proteins (75 µg protein) of each sample were mixed with a half volume of 3x Laemmli’s buffer [30 mM Tris-HCl (pH 7.8), 9% SDS, 15% glycerol, 6% 2-mercaptoethanol, 0.05% bromophenol blue]. The mixtures were boiled for 5 min, subjected to SDS-PAGE, and transferred onto nitrocellulose membrane (BA-85; Schleicher & Schuell BioScience, Keene, NH). The indicated first antibodies and HRP-conjugated second anti-IgG antibodies were hybridized according to standard IB protocols. Chemiluminescence reactions were performed using the enhanced chemiluminescence kit (ECL kit; PerkinElmer Life Science, Inc. Boston, MA), and the luminescence was exposed onto x-ray film (X-Omat; Kodak, Tokyo, Japan). Densitometric analysis was performed using the ImageJ 1.37 program (http://rsb.info.nih.gov/ij/; National Institutes of Health, Bethesda, MD).

Immunoprecipitation (IP)
One milligram of total protein of cell lysates was mixed with indicated antibodies (their concentrations were as recommended by the manufacturer) and made up to 1 ml with the lysis buffer described previously. Samples were incubated at 4 C for 1–2 h, and then 10 µl Protein A-Sepharose or Protein G-Sepharose (GE Healthcare UK Ltd.) was added, and incubation was continued for 1 h. Precipitates were washed with the lysis buffer three times. When samples were to be analyzed by IB, they were diluted with 1x Laemmli’s buffer, boiled for 5 min, and subjected to SDS-PAGE.

Cyclin E-associated CDK assay
Immunoprecipitates with anti-cyclin E antibody were diluted with the reaction mixture to give a final concentration of 20 mM HEPES-NaOH (pH 7.4), 40 mM MgCl2, 1% NP-40, 50 µM ATP, 10 µCi [{gamma}-32P]ATP (3000 Ci/mmol), and 2 µg/ml histone H1. After 30 min incubation at 25 C, the reactions were terminated by boiling these samples with a half volume of 3x Laemmli’s buffer. These samples were then subjected to SDS-PAGE, and radioactivity incorporated into the substrates was detected by autoradiography.

RNA extraction and Northern blotting (NB)
Quiescent cells in three 100-mm dishes for each sample were pretreated and treated with or without test agents as indicated. Total RNA was isolated from cells according to the acid guanidinium phenol-chloroform method. RNA (30 µg) was fractionated by electrophoresis through a 1.5% agarose gel containing formaldehyde, transferred to nylon membranes (GeneScreen Plus; PerkinElmer Life Science), and cross-linked by UV light. The membranes were hybridized at 42 C overnight with random primed 32P-labeled cDNAs produced by the Megaprime DNA labeling Kit (GE Healthcare UK Ltd.), then washed at 65 C and subjected to autoradiography.

Polysome fractionation followed by NB
Quiescent cells in nine 100-mm dishes for each sample were pretreated and treated with or without test agents as indicated. The cells were treated with Coon’s F-12 medium with 0.1% BSA containing 100 µg/ml cycloheximide (CHX) for 5 min at 37 C, stripped with PBS containing 0.08 M trisodium citrate, resuspended in ice-cold PBS containing 100 µg/ml CHX and 1 mM phenylmethylsulfonyl fluoride, and pelleted by centrifugation. The pellets were swollen for 2 min in 375 µl low-salt buffer [20 mM Tris (pH 9.0), 10 mM NaCl, 3 mM MgCl2] containing 1 mM dithiothreitol and 200 U RNAsin (Promega Corp., Madison, WI). The cells were lysed by the addition of 125 µl ice-cold low-salt buffer containing 0.2 M sucrose and 1.2% Triton N-101 (Sigma-Aldrich), followed by 10 strokes with a Dounce homogenizer. Lysates were centrifuged at 10,000 rpm (15,000 x g) for 1 min, and the supernatants (cytoplasmic extracts) were collected and mixed with 1:10 volume of solutions containing 10 mg/ml heparin (Sigma-Aldrich) and 1.5 M NaCl. The extracts were layered over 0.5–1.5 M sucrose gradients and centrifuged at 40,000 rpm (284,000 x g) in a Hitachi P40ST rotor (Hitachi, Ltd., Tokyo, Japan) for 90 min at 4 C. Gradients were fractionated with monitoring of 254-nm absorbance, and mixed with 1:10 volume solutions containing 5% SDS and 1 mg/ml proteinase K. After incubation for 30 min at 37 C, RNA was prepared with two phenol/chloroform (1:1) extractions and one chloroform extraction, followed by ethanol precipitation using glycogen (final concentration: 20 µg/ml) as carrier, and subjected to NB analysis.

Protein degradation assay of cyclin D1
To analyze cyclin D1 protein stability, quiescent cells were pretreated with or without 1 mM Bt2cAMP and treated with 100 ng/ml IGF-I for 12 h. A total of 10 µg/ml CHX was then added into the medium, and the incubation was continued for indicated times. The cell lysates were subjected to IB analysis using anti-cyclin D1 antibody.

Polyubiquitination assay of p27Kip1
Cells were pretreated with or without 1 mM Bt2cAMP and then treated with 100 ng/ml IGF-I in the presence of 10 µM MG132 for the indicated time. The cell lysates were then subjected to IP with anti-p27Kip1 polyclonal antibody, and the immunoprecipitates were subjected to IB with anti-ubiquitin antibody.

PI 3-kinase assay
Immunoprecipitates with anti-p110{alpha} antibody or anti-p110β antibody were washed with the lysis buffer, LiCl buffer [100 mM Tris-HCl (pH 7.5), 500 mM LiCl], distilled water, Tris-NaCl-EDTA buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA], and finally resuspended in 45 µl of the PI 3-kinase reaction buffer [20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5 mM EGTA]. PI 3-kinase assay was initiated by the addition of 5 µl of the mixture to give a final concentration of 20 mM MgCl2, 1 mM dithiothreitol, 30 µM ATP, 10 µCi [{gamma}-32P]ATP (3000 Ci/mmol; GE Healthcare UK Ltd.), and 20 µg bovine liver PI (Avanti Polar Lipids, Inc., Alabaster, AL). After the incubation at 25 C for 20 min, 100 µl chloroform-methanol-HCl (10:20:1) was added to the reaction mixture to stop a reaction. A lipid product was extracted, spotted onto a silica gel plate, and developed with a solvent containing chloroform-methanol-25% ammonia water-water (43:38:6:6). 32P radioactivity incorporated into PI was measured by autoradiography.

Statistics
Statistical analyses of data were performed by one-way factorial ANOVA using StatView software (Abacus Concepts, Inc., Berkeley, CA). Fisher’s projected least significant difference was performed to study the significance between different conditions. The results shown are the mean ± SEM. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Prolonged cAMP pretreatment enhances IGF-I-induced DNA synthesis and shortens the time of entry into the S phase
DNA synthesis was measured in FRTL-5 cells pretreated with or without Bt2cAMP for 24 h (cAMP pretreatment) and then stimulated with IGF-I for indicated times. As shown in Fig. 1Go, cAMP pretreatment enhanced DNA synthesis induced by IGF-I, which reflected an increase in the number of cells entered into the S phase. In addition, cAMP pretreatment caused a shift in the peak of DNA synthesis after IGF-I stimulation from 32–44 to 20–36 h, which indicated that cAMP pretreatment shortened the time for cells to enter the S phase in response to IGF-I.


Figure 1
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  		FIG. 1. DNA synthesis in FRTL-5 cells pretreated with Bt2cAMP and treated with IGF-I in the absence or presence of a PI 3-kinase inhibitor. Quiescent FRTL-5 cells were pretreated with or without 1 mM Bt2cAMP for 24 h (cAMP pretreatment). After washing to remove reagents, cells were treated with 100 ng/ml IGF-I for the indicated times. LY294002 (LY) (50 µM) was added into the medium during cAMP pretreatment (A) or during IGF-I treatment (B). [Methyl-3H]thymidine incorporation into DNA was measured during the last 4 h. The experiments were performed in triplicates, and the mean ± SEM was shown. In some points, SEM was too small to denote error bars. Similar results were obtained in two independent experiments.

 
Prolonged cAMP pretreatment enhances an increase in G1 cyclins and a decrease in a CKI induced by IGF-I
We then analyzed the amounts of G1 cyclins, including cyclin D1 and cyclin E, CDKs, including CDK2, CDK4, and CDK6, and CKIs p16Ink4, p21Cip1, and p27Kip1, in cells pretreated with or without Bt2cAMP for 24 h and then treated with IGF-I for indicated times (Fig. 2Go). Prolonged cAMP treatment by itself increased the amounts of cyclin D1, cyclin E, CDK4, and p21Cip1. IGF-I treatment by itself also increased cyclin D1, cyclin E, CDK4, and p21Cip1 (Fig. 2Go). In addition, cAMP pretreatment markedly potentiated IGF-I-dependent increases in cyclin D1 (at 3 h IGF-I treatment), cyclin E, and CDK2. It is notable that cyclin D1 increased gradually up to 24 h in response to IGF-I without cAMP pretreatment, whereas it reached a maximum level at 3 h and maintained this level at least up to 24 h in response to IGF-I after cAMP pretreatment. More intriguingly, we found that one of the CKIs, p27Kip1, slightly decreased after treatment with Bt2cAMP or IGF-I by itself but markedly decreased in cells pretreated with Bt2cAMP then treated with IGF-I. CDK6 and p16Ink4 were not detected.


Figure 2
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  		FIG. 2. Changes in G1 cyclins, CDKs and CKIs in FRTL-5 cells pretreated with Bt2cAMP and treated with IGF-I. After cAMP pretreatment, cells were treated with 100 ng/ml IGF-I for the indicated times. The cell lysates were subjected to IB with indicated antibodies. Densitometric analysis was performed, and the means ± SEM of the results of three independent experiments are shown in the lower graphs. *, P < 0.05. N.S., Not significant.

 
Prolonged cAMP pretreatment followed by IGF-I treatment causes a marked increase in G1 CDK activities
Next, we investigated the effect of cAMP pretreatment on IGF-I-dependent activation of G1 CDKs and the phosphorylation of Rb. Cyclin D-associated CDK activity was evaluated by measuring Rb Ser807/811 phosphorylation according to a previous report (46) (Fig. 3AGo, upper panel), and cyclin E-associated CDK activity was measured by a conventional kinase assay using histone H1 as a substrate (Fig. 3AGo, lower panel). The total phosphorylation levels of Rb were analyzed by mobility shift assay on SDS-PAGE (Fig. 3BGo). IGF-I stimulation by itself caused little activation of cyclin D- and cyclin E-associated CDKs or phosphorylation of Rb. In contrast, cAMP pretreatment enhanced cyclin D-associated CDK activity weakly at 3–6 h after IGF-I stimulation, which correlates with an increase in cyclin D1 shown in Fig. 2Go. The cAMP pretreatment potentiated more effectively IGF-I-dependent activation of cyclin D- and cyclin E-associated CDKs, as well as total Rb phosphorylation, at 12–24 h after commencement of IGF-I stimulation (Fig. 3Go). This was closely associated with a marked increase in cyclin E and a decrease in p27Kip1 (Fig. 2Go). The marked increase in CDK2 protein levels (Fig. 2Go) was observed only after augmentation of CDK activities. Considering these results together, we concluded that synergistic activation of cyclin D- and cyclin E-associated CDKs reflects, at least in a part, increases in cyclin D1 and cyclin E, and a decrease in p27Kip1.


Figure 3
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  		FIG. 3. CDK activation and Rb phosphorylation in FRTL-5 cells pretreated with Bt2cAMP and treated with IGF-I. After cAMP pretreatment, cells were treated with 100 ng/ml IGF-I for the indicated times. The cell lysates were subjected to IB with indicated antibodies (upper panel of A and B). Cyclin E-associated CDK activity was measured as histone H1 phosphorylation activity in the immunoprecipitates with anti-cyclin E antibody (lower panel of A). Representative blots of three independent experiments are shown.

 
Prolonged cAMP pretreatment enhances IGF-I-induced increases in cyclin D1 mRNA levels and translation
We next aimed to elucidate the molecular mechanisms underlying this regulation of cyclin D1 levels. Gene expression of cyclin D1 was first analyzed by NB (Fig. 4AGo). Treatment with IGF-I by itself increased mRNA levels to some extent; however, cAMP pretreatment markedly potentiated IGF-I-dependent increases in cyclin D1 mRNA at 3 h, suggesting that the enhanced increase in cyclin D1 protein is regulated at least in part at the mRNA level. Because recent reports suggested that the translation rate and the protein stability of cyclin D1 could be also modified by mitogenic stimuli (47, 48), we studied these regulations in cells pretreated with Bt2cAMP for 24 h and then treated with IGF-I (Fig. 4Go, B and C). The cAMP pretreatment enhanced the IGF-I-induced formation of the cyclin D1 mRNA-polysome complex, indicating that cAMP pretreatment also increases the rate of translation of the cyclin D1 mRNA induced by IGF-I (Fig. 4BGo). To evaluate the degradation rate of cyclin D1 in cells pretreated with or without Bt2cAMP for 24 h and treated with IGF-I for 12 h, we measured cyclin D1 levels at indicated times after addition of an inhibitor of protein synthesis, CHX (Fig. 4CGo). The degradation rate of cyclin D1 was not affected by cAMP pretreatment. These results indicated that the synergistic increase in cyclin D1 was controlled at both the mRNA and translation levels.


Figure 4
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  		FIG. 4. Gene expression, translation rate, and stability of cyclin D1 in FRTL-5 cells pretreated with Bt2cAMP and treated with IGF-I. After cAMP pretreatment, the cells were treated with 100 ng/ml IGF-I for the indicated times (A), 3 h (B) or 12 h (C). A, Total RNA was extracted and subjected to NB analysis using a random primed 32P-cDNA probe corresponding to cyclin D1. To confirm that equal amounts of RNA were loaded, the results of ethidium bromide staining of 18 S rRNA are shown. The right graph shows the mean ± SEM of the results of the densitometric analysis of three independent experiments. B, Cytoplasmic extracts were applied to a 15–60% sucrose gradient and separated by centrifugation. RNA was extracted from each fractionated sample and subjected to NB analysis using 32P-labeled cyclin D1 cDNA probe. The rRNA was detected mainly in no. 2–4 fractions, indicating that monosomes were separated into these fractions. The relative intensity of each band (amount of cyclin D1 mRNA in each fraction/amount of total cyclin D1 mRNA) was plotted in the lower graph. C, Twelve hours after the commencement of IGF-I treatment, CHX (10 µg/ml) was added into the medium to block de novo protein synthesis, and cell culture was continued for indicated times. The cell lysates were subjected to IB with anti-cyclin D1 antibody. The density of each band was plotted with a logarithmic scale on the ordinate in the right graph. The regression lines are shown. *, P < 0.05. N.S., Not significant.

 
Prolonged cAMP pretreatment enhances the IGF-I-induced proteasome-dependent degradation of p27Kip1
It was reported that, in the G1-S phase transition, p27Kip1 protein levels were regulated by its gene expression (49) and protein degradation through the ubiquitin-proteasome pathway (50, 51). A series of experiments was performed to determine if cAMP and IGF-I treatments result in similar changes in p27Kip1 mRNA levels and protein stability that could lead to CDK activation and DNA synthesis. Northern blot analysis revealed that, whereas p27Kip1 mRNA was decreased by Bt2cAMP treatment alone, it was also decreased by IGF-I treatment (Fig. 5AGo). After the commencement of IGF-I stimulation, p27Kip1 mRNA levels were continuously low either with or without cAMP pretreatment, suggesting that de novo synthesis of p27Kip1 was largely blocked under these conditions. To determine whether p27Kip1 protein degradation was affected by Bt2cAMP and/or IGF-I treatment, we pretreated cells with or without Bt2cAMP for 24 h, and measured the levels of p27Kip1 during IGF-I treatment with addition of a proteasome inhibitor (MG132). The decrease in p27Kip1 protein induced by IGF-I treatment was restored by addition of MG132, either with or without cAMP pretreatment (Fig. 5BGo). In addition, whereas normal molecular mass of p27Kip1 was 27 kDa, we detected a large-sized p27Kip1 (>100 kDa) when cells were pretreated with Bt2cAMP for 24 h and then treated with IGF-I in the presence of MG132 (Fig. 5CGo). IB using anti-ubiquitin antibody confirmed that this was polyubiquitinated p27Kip1 (Fig. 5DGo). We found that IGF-I induced and cAMP pretreatment further enhanced the accumulation of polyubiquitinated p27Kip1. These results showed that cAMP pretreatment enhances IGF-I-dependent p27Kip1 protein degradation through the ubiquitin-proteasome pathway.


Figure 5
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  		FIG. 5. Gene expression and degradation of p27Kip1 in FRTL-5 cells pretreated with Bt2cAMP and treated with IGF-I. A, After cAMP pretreatment, the cells were treated with 100 ng/ml IGF-I for the indicated times. Total RNA was extracted and subjected to NB analysis using 32P-labeled p27Kip1 cDNA probe. B, After cAMP pretreatment, the cells were treated with 100 ng/ml IGF-I in the presence or absence of 10 µM MG132 for 24 h. Cells were homogenized, and the lysates were subjected to IB with anti-p27Kip1 antibody. C and D, After cAMP pretreatment, the cells were treated with 100 ng/ml IGF-I in the presence of 10 µM MG132 for the indicated times (C) or 12 h (D). The lysates were subjected to IP with anti-p27Kip1 antibody. The immunoprecipitates were subjected to IB with indicated antibodies. The densities of the smears above 100 kDa in the IB using anti-ubiquitin antibody were measured, and the mean ± SEM of the results of six independent experiments is shown in the right graph (D). *, P < 0.05. N.S., Not significant.

 
PI 3-kinase is activated in distinct modes in response to cAMP or IGF-I stimulus
Because we have found that PI 3-kinase was activated in response to either cAMP or IGF-I stimuli in FRTL-5 cells, and activation by both was essential for cell proliferation (26, 27), we tested the effects of cAMP or IGF-I stimuli on the activity of different isoforms, p110{alpha}, β or {gamma}-catalytic subunits of PI 3-kinase. The basal activity of p110{alpha} was high, and it increased about 1.5-fold in response to IGF-I stimuli (Fig. 6AGo). In contrast, p110β activity was very low in the basal state, and IGF-I treatment increased its activity nearly 30-fold. Prolonged cAMP treatment did not affect these activities; however, the PI 3-kinase activity bound to its p85 regulatory subunit increased by 1.7-fold (data not shown) as described previously (26). We could detect little activity of p110{gamma} (data not shown).


Figure 6
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  		FIG. 6. Activity of PI 3-kinase p110{alpha} and p110β, and their association with phosphotyrosyl proteins in FRTL-5 cells pretreated with Bt2cAMP and treated with IGF-I. After cAMP pretreatment, the cells were treated with 100 ng/ml IGF-I for 5 min (A) or 1 min (B). Cells were homogenized, and the lysates were subjected to IP with anti-p110{alpha} antibody or anti-p110β antibody. A, PI 3-kinase activity was measured in the immunoprecipitates. The mean ± SEM of three replicate dishes is shown, along with the representative result of the autoradiography. PIP, Phosphatidylinositol phosphate. Ori, origin (sample spotting line). B, Total cell lysates and the immunoprecipitates were subjected to IB with indicated antibodies. Representative blots of two independent experiments are shown. *, P < 0.05. N.S., Not significant.

 
It is widely accepted that a p110{alpha}/β-subunit binds to a p85 regulatory subunit, and this heterodimer is recruited to the membrane through the association with phosphotyrosyl proteins, leading to production of phosphatidylinositol 3,4,5-triphosphate (PIP3), followed by activation of downstream signal pathway(s) (52). We then analyzed the association of p110{alpha}/β and phosphotyrosyl proteins in response to cAMP or IGF-I stimuli. Both p110{alpha} and p110β associated with 125-kDa phosphotyrosyl protein (p125) in response to prolonged cAMP treatment, and with 100- to 185-kDa phosphotyrosyl protein(s), which include IRS-2, in response to IGF-I treatment (Fig. 6BGo). Moreover, cAMP pretreatment potentiated the association of p110{alpha}/β with these 100- to 185-kDa phosphotyrosyl protein(s). We observed the smear pattern of IRS-2 in the immunoprecipitates with anti-p110{alpha}/β-antibody (Fig. 6BGo), but the reason remains unclear. Together, the results indicate that cAMP treatment caused association of active p110{alpha} with phosphotyrosyl p125, and IGF-I treatment activated p110{alpha}/β bound to phosphotyrosyl IRS-2, which was potentiated by cAMP pretreatment.

PI 3-kinase activated in response to cAMP or IGF-I stimulus plays different roles in cell-cycle progression
To determine the roles of each mode of activation of PI 3-kinase in response to cAMP or IGF-I in potentiation of DNA synthesis, we added a PI 3-kinase inhibitor (LY294002) during pretreatment with Bt2cAMP for 24 h or during treatment with IGF-I, and then measured DNA synthesis every 4 h during IGF-I treatment (Fig. 1Go, A and B). The inhibition of PI 3-kinase only during cAMP pretreatment abolished the potentiation of DNA synthesis without affecting IGF-I-induced DNA synthesis (Fig. 1AGo). In contrast, inhibition of PI 3-kinase during IGF-I treatment blocked the induction of DNA synthesis (Fig. 1BGo). These results clearly showed that both modes of activation of PI 3-kinase in response to cAMP or IGF-I are crucial for induction of synergistic DNA synthesis but that each mode of activation of PI 3-kinase has a unique role in cell-cycle progression from G1 to S phase.

PI 3-kinase activated in response to cAMP or IGF-I stimulus plays important roles in increasing the translation rate or mRNA levels of cyclin D1, respectively
We conducted studies to determine the roles of PI 3-kinase activation in response to cAMP or IGF-I in increases in cyclin D1 mRNA and translation rate of cyclin D1, using a PI 3-kinase inhibitor. We added LY294002 during pretreatment with Bt2cAMP for 24 h or treatment with IGF-I for the indicated times, and measured mRNA as well as protein levels of cyclin D1. We found that inhibition of PI 3-kinase during cAMP pretreatment suppressed the enhancement of the IGF-I-dependent increase in cyclin D1 protein (Fig. 7AGo), whereas it did not affect the increase in its mRNA (Fig. 7BGo). These data suggested that cAMP-dependent PI 3-kinase activation is necessary not for changes in cyclin D1 mRNA but for an increased rate of translation of its mRNA. In contrast, inhibition of PI 3-kinase during IGF-I treatment suppressed the increase in cyclin D1 protein as well as its mRNA (Fig. 7Go, C and D), indicating that IGF-I-dependent PI 3-kinase activation plays an important role in inducing the increase in cyclin D1 mRNA.


Figure 7
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  		FIG. 7. Effect of PI 3-kinase inhibitor addition during cAMP pretreatment or IGF-I treatment on mRNA and protein levels of cyclin D1, in FRTL-5 cells pretreated with Bt2cAMP and treated with IGF-I. After cAMP pretreatment, cells were treated with 100 ng/ml IGF-I. LY294002 (LY) (50 µM) was added into the medium during cAMP pretreatment (A and B) or IGF-I treatment (C and D). Cell culture was continued for the indicated times, and the lysates were subjected to IB with anti-cyclin D1 antibody (A and C). RNA was extracted from cells treated with IGF-I for 3 h and subjected to NB analysis using 32P-labeled cyclin D1 cDNA probe (B and D). Graphs show the mean ± SEM of the densitometric analysis from four (A and C) or three (B and D) independent experiments. *, P < 0.05. N.S., Not significant.

 
PI 3-kinase activated in response to IGF-I stimulus is a requisite for the decrease in p27Kip1
We analyzed the roles of PI 3-kinase activation in response to cAMP or IGF-I in the decrease in p27Kip1 (Fig. 8Go). Inhibition of PI 3-kinase during cAMP pretreatment did not affect the protein levels of p27Kip1 (Fig. 8AGo). In contrast, inhibition of PI 3-kinase during IGF-I treatment suppressed the decrease in p27Kip1 protein levels by cAMP pretreatment (Fig. 8BGo). These results showed that PI 3-kinase activated by IGF-I, but not by cAMP, plays a crucial role in the decrease in p27Kip1.


Figure 8
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  		FIG. 8. Effect of PI 3-kinase inhibitor addition during cAMP pretreatment or IGF-I treatment on protein levels of p27Kip1, in FRTL-5 cells pretreated with Bt2cAMP and treated with IGF-I. After cAMP pretreatment, cells were treated with 100 ng/ml IGF-I. LY294002 (LY) (50 µM) was added into the medium during cAMP pretreatment (A) or IGF-I treatment (B). Cell culture was continued for 24 h, and the lysates were subjected to IB with anti-p27Kip1 antibody. Graphs show the mean ± SEM of the densitometric analysis from five independent experiments. *, P < 0.05. N.S., Not significant.

 
Both modes of activation of PI 3-kinase in response to cAMP or IGF-I stimulus play important roles in increasing the mRNA levels of cyclin E
It is well known that activation of cyclin D-associated CDKs induces cyclin E gene expression (35). As shown in Fig. 3AGo, we observed obvious activation of cyclin D-associated CDKs in cells pretreated with Bt2cAMP for 24 h followed by treatment with IGF-I for 12 h. Therefore, we measured gene expression of cyclin E by NB (Fig. 9Go). The cAMP pretreatment remarkably potentiated IGF-I-dependent increases in cyclin E at 12 h after the commencement of IGF-I treatment, suggesting that the marked increase in cyclin E is regulated at the mRNA level, thus reflecting cyclin D-associated CDK activation. In addition, we found that inhibition of PI 3-kinase during cAMP pretreatment or during IGF-I treatment suppressed an increase in cyclin E mRNA levels as well as its protein levels (Fig. 10Go). These results indicate that both modes of activation of PI 3-kinase in response to cAMP or IGF-I stimulus are essential for the marked increase in cyclin E, which may be mediated by cyclin D-associated CDK activation.


Figure 9
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  		FIG. 9. Gene expression of cyclin E in FRTL-5 cells pretreated with Bt2cAMP and treated with IGF-I. After cAMP pretreatment, the cells were treated with 100 ng/ml IGF-I for the indicated times. Total RNA was extracted and subjected to NB analysis using 32P-labeled cyclin E cDNA probe. Graphs show the mean ± SEM of the densitometric analysis from four independent experiments. *, P < 0.05. N.S., Not significant.

 

Figure 10
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  		FIG. 10. Effect of PI 3-kinase inhibitor addition during cAMP pretreatment or IGF-I treatment on mRNA and protein levels of cyclin E, in FRTL-5 cells pretreated with Bt2cAMP and treated with IGF-I. After cAMP pretreatment, cells were treated with 100 ng/ml IGF-I. LY294002 (LY) (50 µM) was added into the medium during cAMP pretreatment (A and B) or IGF-I treatment (C and D). Cell culture was continued for indicated times, and the lysates were subjected to IB with anti-cyclin E antibody (A and C). RNA was extracted from cells treated with IGF-I for 18 h and subjected to NB analysis using 32P-labeled cyclin E cDNA probe (B and D). Graphs show the mean ± SEM of the densitometric analysis from four (A), five (C), or three (B and D) independent experiments. *, P < 0.05.

 
Both modes of activation of PI 3-kinases in response to cAMP or IGF-I are essential for increases in G1 CDK activities
Finally, we studied the effects on CDK activation of addition of a PI 3-kinase inhibitor during cAMP pretreatment or IGF-I treatment (Fig. 11Go). Inhibition of PI 3-kinase activity during cAMP pretreatment or IGF-I treatment suppressed cAMP- and IGF-I-induced Rb Ser807/811 phosphorylation, as well as cyclin E-associated CDK activation. These results clearly showed that both modes of activation of PI 3-kinase in response to cAMP or IGF-I are essential for marked increases in G1 CDK activities.


Figure 11
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  		FIG. 11. Effect of PI 3-kinase inhibitor addition during cAMP pretreatment or IGF-I treatment on CDK activation and Rb phosphorylation, in FRTL-5 cells pretreated with Bt2cAMP and treated with IGF-I. After cAMP pretreatment, the cells were treated with 100 ng/ml IGF-I for 24 h. LY294002 (LY) (50 µM) was added into the medium during cAMP pretreatment or IGF-I treatment. The cell lysates were subjected to IB with anti-phospho Rb (S807/811) antibody (upper panel). Cyclin E-associated CDK activity was measured as histone H1 phosphorylation activity in the immunoprecipitates with anti-cyclin E antibody (lower panel). Representative blots of three independent experiments are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study was undertaken to elucidate the mechanisms of cell-cycle progression in response to cAMP or IGF-I in thyroid FRTL-5 cells. A series of our experiments indicated that prolonged cAMP stimulus enhanced IGF-I-induced activation of cyclin D- and cyclin E-dependent kinases, which well explained the potentiation of DNA synthesis induced by IGF-I.

How are cell-cycle regulatory proteins regulated in FRTL-5 cells in response to cAMP and/or IGF-I stimulus? Previous studies by Yamamoto et al. (44) and others (38, 39, 40, 41, 42, 43) showed that cyclin D1, cyclin D3, cyclin E, CDK2, and CDK4 increased, and p27Kip1 decreased by the stimulation with TSH and/or IGF-I/insulin. We further studied detailed changes not previously reported in the amounts and activities of cell-cycle regulatory proteins in cells pretreated with cAMP followed by IGF-I treatment. Among the G1 cyclins, CDKs, and CKIs that we studied, cyclin D1, cyclin E, CDK4, and p21Cip1 increased in response to either cAMP or IGF-I alone (Fig. 2Go). Recently, cyclin D3 was shown to play important roles in cell-cycle progression of dog thyroid cells (19, 53), and we also observed cAMP- or IGF-I-dependent cyclin D3 increases in FRTL-5 cells (data not shown). However, these changes in cell-cycle regulatory proteins in response to cAMP or IGF-I alone appeared not to be sufficient to induce CDK activation (Fig. 3Go). In contrast, prolonged cAMP pretreatment followed by IGF-I treatment caused a marked increase in CDK activities (Fig. 3Go), and under this condition we found that cAMP pretreatment remarkably potentiated IGF-I-dependent increases in cyclin D1, cyclin E, and CDK2 (Fig. 2Go), but not cyclin D3 (data not shown). Especially in the case of cyclin D1 different from other proteins, the effect of cAMP pretreatment on potentiation of IGF-I-dependent increases was prominent until 12 h after IGF-I treatment, indicating that cAMP pretreatment shortened the time to stimulate its protein expression. We have found for the first time that p27Kip1 markedly decreased in response to cAMP pretreatment followed by IGF-I treatment (Fig. 2Go).

Comparing the time course of changes in these protein levels (Fig. 2Go) with CDK activities (Fig. 3Go), cyclin D1 increases before the marked CDK activation in response to cAMP and IGF-I, and cyclin E and p27Kip1 change simultaneously with the marked CDK activation, but an increase in CDK2 was observed after the CDK activation. These results suggested that changes in cyclin D1, cyclin E, and p27Kip1 play central roles in cyclin D- and cyclin E-associated CDK activation in this model. These changes of cell-cycle regulatory proteins in FRTL-5 cells were observed in other rat thyroid cell lines but not to be consistent with those in dog thyroid cells (19). For example, cyclin D1 decreases and p27Kip1 increases in response to TSH in dog thyroid cells. This inconsistency may be due to the differences of species, or between primary culture cells and subcultured cell lines.

We then studied the molecular mechanisms underlying the regulation of protein levels of cyclin D1 and p27Kip1 in response to cAMP and/or IGF-I stimulus.

It has been shown that the increases in cyclin D1 in response to mitogenic stimuli are mediated by different mechanisms, such as up-regulation of gene transcription (54), acceleration of translation (47, 55), and stabilization of its protein (48). In FRTL-5 cells, it has been reported that cyclin D1 mRNA increases by the simultaneous stimulation with TSH and IGF-I/insulin (39, 40, 56). We demonstrated that cAMP stimulus enhanced IGF-I-dependent increases in cyclin D1 mRNA (Fig. 4AGo) and translation rates of cyclin D1 (Fig. 4BGo) but did not affect its stability (Fig. 4CGo). Thus, our studies provide the first evidence that activation of the cAMP pathway plays important roles in the translational control of cyclins in thyroid cells as well as other types of cells.

In response to mitogenic stimuli, levels of p27Kip1 have been decreased via suppression of gene expression (49), and protein degradation through the ubiquitin-proteasome pathway (57). In FRTL-5 cells, it has been reported that p27Kip1 mRNA decreased by the simultaneous stimulation with TSH and IGF-I (40). We observed decreases of p27Kip1 mRNA in response to either cAMP or IGF-I alone (Fig. 5Go), but these decreases could not explain the marked reduction of p27Kip1 protein. Our studies first indicated that cAMP stimulus enhanced IGF-I-induced protein degradation of p27Kip1 through the ubiquitin-proteasome pathway (Fig. 5Go).

Recently, it was proposed that activation of PI 3-kinase and its downstream signals has multiple roles in the regulation of cell-cycle machinery (58). In thyroid FRTL-5 cells, either TSH stimulus via a cAMP-dependent signal transduction pathway or IGF-I stimulus via a tyrosine kinase-dependent signal transduction pathway induces activation of PI 3-kinase through distinct mechanisms (26, 27, 59, 60). We have furthered the understanding of this pathway by showing that each mode of activation of PI 3-kinase is indispensable for the synergistic cell-cycle progression in response to cAMP and IGF-I (26, 27).

We had reported that in FRTL-5 cells prolonged cAMP stimulus by TSH causes tyrosine phosphorylation of an unidentified 125-kDa protein (26). This phosphotyrosyl protein can associate with a p85 regulatory subunit of PI 3-kinase, leading to an increase in the PI 3-kinase activity in this complex. It is well established that the class I PI 3-kinases play a crucial role in growth factor signal transduction (52). Class I PI 3-kinases contain four isoforms of the catalytic subunit, known as p110{alpha}, β, {delta}, and {gamma}. The {alpha} and β-isoforms have a broad tissue distribution, although expression of the {delta} and {gamma}-isoforms is more restricted and predominantly detected in leukocytes (52). We observed PI 3-kinase activation bound to a p85 regulatory subunit, but not p110{alpha}/β-activation after prolonged cAMP treatment (Fig. 6AGo), indicating that only a small percentage of activated p110{alpha}/β is bound to p85. The present study demonstrated that p125 associates with p85-p110{alpha} as well as p85-p110β heterodimers in response to prolonged cAMP treatment, whereas we detected the high activity of only p110{alpha} when cells were in the basal state or treated with cAMP. These results suggested that p85-p110{alpha} PI 3-kinase bound to p125 mainly produces PIP3 around p125 during cAMP treatment (Fig. 6Go). Our studies showing that the majority of p125 is detected in the plasma membrane fraction (data not shown) predict that PIP3 is produced at that site. Our subsequent study using a PI 3-kinase inhibitor showed that cAMP-dependent activation of PI 3-kinase through this pathway is essential for potentiation of DNA synthesis and shortening of the time to enter the S phase (Fig. 1AGo).

In parallel with these observations, we have reported that in FRTL-5 cells, pretreatment with TSH or Bt2cAMP potentiated tyrosine phosphorylation of IRS-2 as well as its binding to p85 PI 3-kinase induced by IGF-I, and these changes were reflected in the markedly increased activation of PI 3-kinase (27). Our results indicated that IGF-I induced rapid tyrosine phosphorylation of IRS-2, leading to its association with p85-p110{alpha} as well as p85-p110β heterodimers, which are activated in response to IGF-I treatment, suggesting that both of these protein complexes possess high PI 3-kinase activity (Fig. 6Go). IRS-2 in FRTL-5 is detected around endosomes (Okajima, H. personal communication), implying that the sites of PIP3 production are different from these after prolonged cAMP treatment. The present study using a PI 3-kinase inhibitor indicated that IGF-I-dependent activation of PI 3-kinase through this pathway is essential for induction of DNA synthesis (Fig. 1BGo).

What are the roles of PI 3-kinase activated in response to each stimulus, cAMP or IGF-I in the regulation of cyclin D1, cyclin E, and p27Kip1?

Addition of a PI 3-kinase inhibitor during cAMP pretreatment suppressed a marked increase in cyclin D1 protein but not in cyclin D1 mRNA levels (Fig. 7Go, A and B), suggesting that cAMP-dependent PI 3-kinase activation plays an important role in the enhancement of cyclin D1 translation. As for the downstream signals of PI 3-kinase, previous reports indicated that TSH or cAMP analog activates S6 kinase in a PI 3-kinase-dependent manner (60, 61) and that, in breast cancer cells, S6 kinase up-regulates the translation of cyclin D1 (47). Similarly, we observed that the inhibition of S6 kinase by rapamycin during cAMP pretreatment abolished cAMP-dependent enhancement of the increase in cyclin D1 induced by IGF-I (data not shown). Considering these results together, we speculate that cAMP-dependent PI 3-kinase activation causes S6 kinase activation, resulting in an increase in the cyclin D1 translation rate.

In this study we demonstrated that cAMP pretreatment enhanced the IGF-I-dependent increase in the mRNA level of cyclin D1 and that IGF-I-dependent PI 3-kinase activation was necessary for this increase (Fig. 7Go, C and D). Because we have previously reported that cAMP pretreatment augmented the activation of PI 3-kinase bound to IRS-2 in FRTL-5 cells (27), we conclude that the enhanced increase in cyclin D1 mRNA is mainly mediated by IGF-I-dependent activation of PI 3-kinase bound to IRS-2, which is augmented by cAMP signals. Schmidt et al. (54) reported that the PI 3-kinase-Akt-FoxO pathway was important for an increase in cyclin D1 mRNA. However, we found that Akt activity did not reflect cAMP-dependent potentiation of PI 3-kinase activity bound to IRS-2 induced by IGF-I in FRTL-5 cells (data not shown), suggesting that the enhanced increase in cyclin D1 mRNA is caused via a novel mechanism.

We also showed that IGF-I-dependent PI 3-kinase activation was necessary for the induction of p27Kip1 degradation (Fig. 8BGo). It is consistent with the previous report that addition of a PI 3-kinase inhibitor suppressed insulin-dependent decrease in p27Kip1 protein levels in FRTL-5 cells (62). It is well established that mitogenic stimuli induce p27Kip1 phosphorylation by cyclin E-dependent kinase, and this triggers the ubiquitination of p27Kip1 by SCFSkp2 E3 ubiquitin ligase (50, 51, 63, 64). In this study we found that degradation of p27Kip1 might be induced independently of the phosphorylation of p27Kip1 by cyclin E-dependent kinase because p27Kip1 was degraded even though cyclin E-dependent kinase activity was completely inhibited by addition of a PI-3 kinase inhibitor during cAMP pretreatment (Figs. 8AGo and 11Go). Thus, the mechanism that mediates IGF-I-dependent PI 3-kinase activation leading to p27Kip1 ubiquitination is still unclear; however, p27Kip1 also seems to be under the control of IGF-I-dependent activation of PI 3-kinase bound to IRS-2.

Regarding cyclin E, we certified that cAMP stimulus enhanced IGF-I-dependent increases in cyclin E mRNA (Fig. 9Go). Cyclin E transcription is known to be up-regulated in an E2F-dependent manner, which is mainly triggered by the activation of cyclin D-associated CDKs (35). In our experiments, cyclin E mRNA increased in response to cAMP and IGF-I, coinciding with the activation of cyclin D-associated CDKs (Figs. 3AGo and 9Go). In addition, the increase in cyclin E mRNA was suppressed when the activation of cyclin D-associated CDKs was inhibited by the addition of PI 3-kinase inhibitor during cAMP pretreatment or IGF-I treatment (Fig. 10Go, B and D). Considering these results together, we conclude that the increase in cyclin E mRNA is due to up-regulation of its transcription by E2F, which is triggered by the activation of cyclin D-associated CDKs.

Finally, the increase in cyclin E and decrease in p27Kip1 well explained prominent activation of cyclin E-associated CDK followed by phosphorylation of Rb (Fig. 3Go), leading to synergistic DNA synthesis (Fig. 1Go). However, it cannot be excluded that other mechanisms such as cyclin-CDK assembly and phosphorylation of CDK may also be involved in the CDK activation induced by cAMP and IGF-I, as proposed using a dog thyroid cell culture system (65, 66).

In the present study, we have several novel findings. Long-term activation of the cAMP signaling pathway or short-term activation of the IGF-I signaling pathway potentiated by prolonged cAMP stimulus increases the PI 3-kinase activity in different manners and causes association of the p85-p110{alpha}/β-complex with different phosphotyrosyl proteins. In addition, distinct modes of activation of PI 3-kinase in response to cAMP or IGF-I differentially regulate gene expression, translation, or protein stability of the cell-cycle regulatory proteins. At last, all of these changes in concert converge in marked activation of CDKs, making it possible to promote cell cycle from G1 to S phase. Further analyses of the detailed mechanisms underlying the downstream signals of each PI 3-kinase control gene expression, translation, or protein stability of the target proteins are in progress at our laboratory.

In conclusion, the present study demonstrated the molecular mechanisms underlying the thyroid cell proliferation synergistically induced by cAMP and IGF-I. Our working hypothesis is shown in Fig. 12Go; prolonged cAMP stimulus enhances IGF-I-dependent activation of p110{alpha}/β PI 3-kinase bound to phosphorylated IRS-2, which leads to marked increases in cyclin D1 mRNA and p27Kip1 degradation. At the same time, these prolonged cAMP signals also up-regulate the translation rate of IGF-I-induced cyclin D1 mRNA mainly through activation of p110{alpha} PI 3-kinase bound to phosphorylated p125. These converging signals result in marked increases in G1 cyclins and decreases in p27Kip1, followed by prominent activation of CDKs and subsequent G1-S phase transition. Thus, distinct modes of activation of PI 3-kinase in response to cAMP or IGF-I play different but essential roles in this novel signaling cross talk that regulates cell-cycle machinery in FRTL-5 cells.


Figure 12
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  		FIG. 12. Working hypothesis of the roles of distinct modes of activation of PI 3-kinase in response to cAMP or IGF-I stimulus in CDK activation leading to synergistic cell proliferation in FRTL-5 cells. The prolonged cAMP stimulus activates PI 3-kinase bound to phosphotyrosyl protein p125, and this PI 3-kinase activation is necessary for enhancing the translation rate of cyclin D1 mRNA. On the other hand, prolonged cAMP stimulus enhances IGF-I-dependent activation of PI 3-kinase bound to IRS-2, and this strong PI 3-kinase activation induces a marked increase in cyclin D1 mRNA and degradation of p27Kip1. As a result of these signal convergences, cyclin D1 increases and p27Kip1 decreases synergistically, leading to cyclin E expression. These changes cause CDK activation, which promotes cell-cycle progression from G1 to S phase. Tyr P, Tyrosine phosphorylation.

 


   Acknowledgments
 
We thank Dr. Leonard Kohn (Edison Biotechnology Institute, Ohio University, Athens, OH) and Interthyr Research Foundation (Baltimore, MD) for the gift of FRTL-5 cells, the National Hormone and Pituitary Program (National Institute of Diabetes and Digestive and Kidney Diseases) for providing bovine TSH, and Dr. Jay Cross (University of Toronto, Toronto, Ontario, Canada) for the kind gift of mouse cyclin D1 cDNA and mouse cyclin E cDNA. We also thank Dr. Toshiaki Ohkuma (Fujisawa Pharmaceutical Co., Osaka, Japan) for the donation of recombinant human IGF-I, as well as support for this research. Finally, we are thankful for the helpful discussions with the late Dr. Van Wyk (University of North Carolina at Chapel Hill, Chapel Hill, NC), Dr. Marco Conti (Stanford University, Stanford, CA), Dr. Steven Boyages (Westmead Hospital, University of Sydney, Westmead, New South Wales, Australia), Dr. Susan Hall (University of North Carolina at Chapel Hill), Dr. Kazuhiro Chida (University of Tokyo, Tokyo, Japan), and Dr. Asako Takenaka (Meiji University, Kanagawa, Japan).


   Footnotes
 
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan [(B)(2 ) no. 11460126 and (A)(2 ) no. 16208028] (to S.-I.T.).

Present address for T.N.: TUBERO/Tohoku University Biomedical Engineering Research Organization, Tohoku University, Sendai 980-8575, Japan.

Present address for H.A.: Bristol-Myers K.K., Pharmacology Laboratory, Early Development, Shinjuku i-Land Tower 5-1, Nishi-Shinjuku 6-chome, Shinjuku-ku, Tokyo 163-1328, Japan.

Present address for M.A.: National Institute of Sensory Organs, National Hospital Organization Tokyo Medical Center, 2-5-1 Higashiga-oka, Meguro-ku, Tokyo 152-8902, Japan.

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 10, 2008

1 T.F. and T.N. contributed equally to this work. Back

Abbreviations: Bt2cAMP, Dibutyryl cAMP; CDK, cyclin-dependent kinase; CHX, cycloheximide; CKI, cyclin-dependent kinase inhibitor; HRP, horseradish peroxidase; IB, immunoblotting; IP, immunoprecipitation; IRS, insulin receptor substrate; NB, Northern blotting; PI, phosphatidylinositol; PIP3, phosphatidylinositol 3,4,5-triphosphate; Rb, retinoblastoma protein; SDS, sodium dodecyl sulfate.

Received October 23, 2007.

Accepted for publication April 3, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Liu JP, Baker J, Perkins AS, Robertson EJ, Efstratiadis A 1993 Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 75:59–72[Medline]
  2. Baker J, Liu JP, Robertson EJ, Efstratiadis A 1993 Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:73–82[CrossRef][Medline]
  3. Powell-Braxton L, Hollingshead P, Warburton C, Dowd M, Pitts-Meek S, Dalton D, Gillett N, Stewart TA 1993 IGF-I is required for normal embryonic growth in mice. Genes Dev 7:2609–2617[Abstract/Free Full Text]
  4. Jones JI, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–34[Abstract/Free Full Text]
  5. Stiles CD, Capone GT, Scher CD, Antoniades HN, Van Wyk JJ, Pledger WJ 1979 Dual control of cell growth by somatomedins and platelet-derived growth factor. Proc Natl Acad Sci USA 76:1279–1283[Abstract/Free Full Text]
  6. Leof EB, Wharton W, van Wyk JJ, Pledger WJ 1982 Epidermal growth factor (EGF) and somatomedin C regulate G1 progression in competent BALB/c-3T3 cells. Exp Cell Res 141:107–115[CrossRef][Medline]
  7. Stiles CD, Pledger WJ, Tucker RW, Martin RG, Scher CD 1980 Regulation of the Balb/c-3T3 cell cycle-effects of growth factors. J Supramol Struct 13:489–499[CrossRef][Medline]
  8. Bonassar LJ, Trippel SB 1997 Interaction of epidermal growth factor and insulin-like growth factor-I in the regulation of growth plate chondrocytes. Exp Cell Res 234:1–6[CrossRef][Medline]
  9. Ristow HJ 1996 Studies on stimulation of DNA synthesis with epidermal growth factor and insulin-like growth factor-I in cultured human keratinocytes. Growth Regul 6:96–109[Medline]
  10. Qureshi FG, Tchorzewski MT, Duncan MD, Harmon JW 1997 EGF and IGF-I synergistically stimulate proliferation of human esophageal epithelial cells. J Surg Res 69:354–358[CrossRef][Medline]
  11. Stull MA, Richert MM, Loladze AV, Wood TL 2002 Requirement for IGF-I in epidermal growth factor-mediated cell cycle progression of mammary epithelial cells. Endocrinology 143:1872–1879[Abstract/Free Full Text]
  12. Itagane Y, Inada H, Fujita K, Isshiki G 1991 Interactions between steroid hormones and insulin-like growth factor-I in rabbit chondrocytes. Endocrinology 128:1419–1424[Abstract/Free Full Text]
  13. Ghahary A, Chakrabarti S, Murphy LJ 1990 Localization of the sites of synthesis and action of insulin-like growth factor-I in the rat uterus. Mol Endocrinol 4:191–195[Abstract/Free Full Text]
  14. Stewart AJ, Johnson MD, May FE, Westley BR 1990 Role of insulin-like growth factors and the type I insulin-like growth factor receptor in the estrogen-stimulated proliferation of human breast cancer cells. J Biol Chem 265:21172–21178[Abstract/Free Full Text]
  15. Iwamura M, Sluss PM, Casamento JB, Cockett AT 1993 Insulin-like growth factor I: action and receptor characterization in human prostate cancer cell lines. Prostate 22:243–252[Medline]
  16. Van Wyk JJ, Conti M, Del Monte P, Takahashi SI 1990 The role of somatomedins in the growth and function of endocrine tissues. In: Sizonenko PC, Aubert ML, eds. Developmental endocrinology. New York: Raven Press; 127–140
  17. Tramontano D, Moses AC, Veneziani BM, Ingbar SH 1988 Adenosine 3',5'-monophosphate mediates both the mitogenic effect of thyrotropin and its ability to amplify the response to insulin-like growth factor I in FRTL5 cells. Endocrinology 122:127–132[Abstract/Free Full Text]
  18. Takahashi S, Conti M, Van Wyk JJ 1990 Thyrotropin potentiation of insulin-like growth factor-I dependent deoxyribonucleic acid synthesis in FRTL-5 cells: mediation by an autocrine amplification factor(s). Endocrinology 126:736–745[Abstract/Free Full Text]
  19. Kimura T, Van Keymeulen A, Golstein J, Fusco A, Dumont JE, Roger PP 2001 Regulation of thyroid cell proliferation by TSH and other factors: a critical evaluation of in vitro models. Endocr Rev 22:631–656[Abstract/Free Full Text]
  20. Thomas GA, Davies HG, Williams ED 1994 Site of production of IGF1 in the normal and stimulated mouse thyroid. J Pathol 173:355–360[CrossRef][Medline]
  21. Tode B, Serio M, Rotella CM, Galli G, Franceschelli F, Tanini A, Toccafondi R 1989 Insulin-like growth factor-I: autocrine secretion by human thyroid follicular cells in primary culture. J Clin Endocrinol Metab 69:639–647[Abstract/Free Full Text]
  22. Cheung NW, Lou JC, Boyages SC 1996 Growth hormone does not increase thyroid size in the absence of thyrotropin: a study in adults with hypopituitarism. J Clin Endocrinol Metab 81:1179–1183[Abstract]
  23. Miyakawa M, Saji M, Tsushima T, Wakai K, Shizume K 1988 Thyroid volume and serum thyroglobulin levels in patients with acromegaly: correlation with plasma insulin-like growth factor I levels. J Clin Endocrinol Metab 67:973–978[Abstract/Free Full Text]
  24. Cheung NW, Boyages SC 1997 The thyroid gland in acromegaly: an ultrasonographic study. Clin Endocrinol (Oxf) 46:545–549[CrossRef][Medline]
  25. Clement S, Refetoff S, Robaye B, Dumont JE, Schurmans S 2001 Low TSH requirement and goiter in transgenic mice overexpressing IGF-I and IGF-Ir receptor in the thyroid gland. Endocrinology 142:5131–5139[Abstract/Free Full Text]
  26. Nedachi T, Akahori M, Ariga M, Sakamoto H, Suzuki N, Umesaki K, Hakuno F, Takahashi SI 2000 Tyrosine kinase and phosphatidylinositol 3-kinase activation are required for cyclic adenosine 3',5'-monophosphate-dependent potentiation of deoxyribonucleic acid synthesis induced by insulin-like growth factor-I in FRTL-5 cells. Endocrinology 141:2429–2438[Abstract/Free Full Text]
  27. Ariga M, Nedachi T, Akahori M, Sakamoto H, Ito Y, Hakuno F, Takahashi S 2000 Signalling pathways of insulin-like growth factor-I that are augmented by cAMP in FRTL-5 cells. Biochem J 348(Pt 2):409–416
  28. Sherr CJ 1994 G1 phase progression: cycling on cue. Cell 79:551–555[CrossRef][Medline]
  29. Draetta GF 1994 Mammalian G1 cyclins. Curr Opin Cell Biol 6:842–846[CrossRef][Medline]
  30. Sherr CJ, Roberts JM 1995 Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 9:1149–1163[Free Full Text]
  31. Morgan DO 1995 Principles of CDK regulation. Nature 374:131–134[CrossRef][Medline]
  32. Hinds PW, Mittnacht S, Dulic V, Arnold A, Reed SI, Weinberg RA 1992 Regulation of retinoblastoma protein functions by ectopic expression of human cyclins. Cell 70:993–1006[CrossRef][Medline]
  33. Kato J, Matsushime H, Hiebert SW, Ewen ME, Sherr CJ 1993 Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4. Genes Dev 7:331–342[Free Full Text]
  34. Hiebert SW, Chellappan SP, Horowitz JM, Nevins JR 1992 The interaction of RB with E2F coincides with an inhibition of the transcriptional activity of E2F. Genes Dev 6:177–185[Abstract/Free Full Text]
  35. Ohtani K, DeGregori J, Nevins JR 1995 Regulation of the cyclin E gene by transcription factor E2F1. Proc Natl Acad Sci USA 92:12146–12150[Abstract/Free Full Text]
  36. Sala A, Nicolaides NC, Engelhard A, Bellon T, Lawe DC, Arnold A, Grana X, Giordano A, Calabretta B 1994 Correlation between E2F-1 requirement in the S phase and E2F-1 transactivation of cell cycle-related genes in human cells. Cancer Res 54:1402–1406[Abstract/Free Full Text]
  37. Sherr CJ, Roberts JM 1999 CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13:1501–1512[Free Full Text]
  38. Hirai A, Nakamura S, Noguchi Y, Yasuda T, Kitagawa M, Tatsuno I, Oeda T, Tahara K, Terano T, Narumiya S, Kohn LD, Saito Y 1997 Geranylgeranylated rho small GTPase(s) are essential for the degradation of p27Kip1 and facilitate the progression from G1 to S phase in growth-stimulated rat FRTL-5 cells. J Biol Chem 272:13–16[Abstract/Free Full Text]
  39. Lazzereschi D, Coppa A, Minicione G, Lavitrano M, Fragomele F, Colletta G 1997 The phosphatase inhibitor okadaic acid stimulates the TSH-induced G1-S phase transition in thyroid cells. Exp Cell Res 234:425–433[CrossRef][Medline]
  40. Carneiro C, Alvarez CV, Zalvide J, Vidal A, Dominguez F 1998 TGF-β1 actions on FRTL-5 cells provide a model for the physiological regulation of thyroid growth. Oncogene 16:1455–1465[CrossRef][Medline]
  41. Noguchi Y, Nakamura S, Yasuda T, Kitagawa M, Kohn LD, Saito Y, Hirai A 1998 Newly synthesized Rho A, not Ras, is isoprenylated and translocated to membranes coincident with progression of the G1 to S phase of growth-stimulated rat FRTL-5 cells. J Biol Chem 273:3649–3653[Abstract/Free Full Text]
  42. Takahashi SI, Nedachi T, Fukushima T, Umesaki K, Ito Y, Hakuno F, Van Wyk JJ, Conti M 2001 Long-term hormonal regulation of the cAMP-specific phosphodiesterases in cultured FRTL-5 thyroid cells. Biochim Biophys Acta 1540:68–81[Medline]
  43. Paternot S, Dumont JE, Roger PP 2006 Differential utilization of cyclin D1 and cyclin D3 in the distinct mitogenic stimulations by growth factors and TSH of human thyrocytes in primary culture. Mol Endocrinol 20:3279–3292[Abstract/Free Full Text]
  44. Yamamoto K, Hirai A, Ban T, Saito J, Tahara K, Terano T, Tamura Y, Saito Y, Kitagawa M 1996 Thyrotropin induces G1 cyclin expression and accelerates G1 phase after insulin-like growth factor I stimulation in FRTL-5 cells. Endocrinology 137:2036–2042[Abstract]
  45. Ambesi-Impiombato FS, Parks LAM, Coon HG 1980 Culture of hormone-dependent functional epithelial cells from rat thyroid. Proc Natl Acad Sci USA 77:3455–3459[Abstract/Free Full Text]
  46. Zarkowska T, Mittnacht S 1997 Differential phosphorylation of the retinoblastoma protein by G1/S cyclin-dependent kinases. J Biol Chem 272:12738–12746[Abstract/Free Full Text]
  47. Koziczak M, Hynes NE 2004 Cooperation between fibroblast growth factor receptor-4 and ErbB2 in regulation of cyclin D1 translation. J Biol Chem 279:50004–50011[Abstract/Free Full Text]
  48. Diehl JA, Cheng M, Roussel MF, Sherr CJ 1998 Glycogen synthase kinase-3β regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 12:3499–3511[Abstract/Free Full Text]
  49. Medema RH, Kops GJ, Bos JL, Burgering BM 2000 AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 404:782–787[CrossRef][Medline]
  50. Carrano AC, Eytan E, Hershko A, Pagano M 1999 SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat Cell Biol 1:193–199[CrossRef][Medline]
  51. Tsvetkov LM, Yeh KH, Lee SJ, Sun H, Zhang H 1999 p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27. Curr Biol 9:661–664[CrossRef][Medline]
  52. Hawkins PT, Anderson KE, Davidson K, Stephens LR 2006 Signalling through class I PI3Ks in mammalian cells. Biochem Soc Trans 34:647–662[CrossRef][Medline]
  53. Van Keymeulen A, Bartek J, Dumont JE, Roger PP 1999 Cyclin D3 accumulation and activity integrate and rank the comitogenic pathways of thyrotropin and insulin in thyrocytes in primary culture. Oncogene 18:7351–7359[CrossRef][Medline]
  54. Schmidt M, Fernandez de Mattos S, van der Horst A, Klompmaker R, Kops GJ, Lam EW, Burgering BM, Medema RH 2002 Cell cycle inhibition by FoxO forkhead transcription factors involves downregulation of cyclin D. Mol Cell Biol 22:7842–7852[Abstract/Free Full Text]
  55. Rosenwald IB, Kaspar R, Rousseau D, Gehrke L, Leboulch P, Chen JJ, Schmidt EV, Sonenberg N, London IM 1995 Eukaryotic translation initiation factor 4E regulates expression of cyclin D1 at transcriptional and post-transcriptional levels. J Biol Chem 270:21176–21180[Abstract/Free Full Text]
  56. Coppa A, Mincione G, Mammarella S, Ranieri A, Colletta G 1995 Epithelial rat thyroid cell clones, escaping from transforming growth factor β negative growth control, are still inhibited by this factor in the ability to trap iodide. Cell Growth Differ 6:281–290[Abstract]
  57. Pagano M, Tam SW, Theodoras AM, Beer-Romero P, Del Sal G, Chau V, Yew PR, Draetta GF, Rolfe M 1995 Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 269:682–685[Abstract/Free Full Text]
  58. Liang J, Slingerland JM 2003 Multiple roles of the PI3K/PKB (Akt) pathway in cell cycle progression. Cell Cycle 2:339–345[Medline]
  59. Ciullo I, Diez-Roux G, Di Domenico M, Migliaccio A, Avvedimento EV 2001 cAMP signaling selectively influences Ras effectors pathways. Oncogene 20:1186–1192[CrossRef][Medline]
  60. Suh JM, Song JH, Kim DW, Kim H, Chung HK, Hwang JH, Kim JM, Hwang ES, Chung J, Han JH, Cho BY, Ro HK, Shong M 2003 Regulation of the phosphatidylinositol 3-kinase, Akt/protein kinase B, FRAP/mammalian target of rapamycin, and ribosomal S6 kinase 1 signaling pathways by thyroid-stimulating hormone (TSH) and stimulating type TSH receptor antibodies in the thyroid gland. J Biol Chem 278:21960–21971[Abstract/Free Full Text]
  61. Cass LA, Meinkoth JL 1998 Differential effects of cyclic adenosine 3',5'-monophosphate on p70 ribosomal S6 kinase. Endocrinology 139:1991–1998[Abstract/Free Full Text]
  62. Saito J, Kohn AD, Roth RA, Noguchi Y, Tatsumo I, Hirai A, Suzuki K, Kohn LD, Saji M, Ringel MD 2001 Regulation of FRTL-5 thyroid cell growth by phosphatidylinositol (OH) 3 kinase-dependent Akt-mediated signaling. Thyroid 11:339–351[CrossRef][Medline]
  63. Montagnoli A, Fiore F, Eytan E, Carrano AC, Draetta GF, Hershko A, Pagano M 1999 Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation. Genes Dev 13:1181–1189[Abstract/Free Full Text]
  64. Sutterluty H, Chatelain E, Marti A, Wirbelauer C, Senften M, Muller U, Krek W 1999 p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells. Nat Cell Biol 1:207–214[CrossRef][Medline]
  65. Depoortere F, Van Keymeulen A, Lukas J, Costagliola S, Bartkova J, Dumont JE, Bartek J, Roger PP, Dremier S 1998 A requirement for cyclin D3-cyclin-dependent kinase (cdk)-4 assembly in the cyclic adenosine monophosphate-dependent proliferation of thyrocytes. J Cell Biol 140:1427–1439[Abstract/Free Full Text]
  66. Paternot S, Coulonval K, Dumont JE, Roger PP 2003 Cyclic AMP-dependent phosphorylation of cyclin D3-bound CDK4 determines the passage through the cell cycle restriction point in thyroid epithelial cells. J Biol Chem 278:26533–26540[Abstract/Free Full Text]




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