Somatostatin interferes with thyrotropin-induced G1-S transition mediated by cAMP-dependent protein kinase and phosphatidylinositol 3-kinase. Involvement of RhoA and cyclin E x cyclin-dependent kinase 2 complexes.

cAMP-mediated cell proliferation is a complex process that involves multiple pathways. Using a cAMP-dependent cell system, FRTL-5 thyroid cells, we have previously demonstrated the existence of a precise autocrine loop in the control of cell proliferation that involves the positive effector thyrotropin (TSH) and the general inhibitor somatostatin. In search of the regulatory mechanisms responsible for the TSH and somatostatin control of cell proliferation, we analyzed the cell cycle regulatory proteins and the cellular pathways involved in the action of both signals. The results show that specific inhibition of cAMP-dependent protein kinase (PKA) and phosphatidylinositol (PI) 3-kinase blocks independently TSH-induced FRTL-5 cell proliferation and that somatostatin interferes with both signals. Each pathway activates different proteins required for G(1)/S progression. Thus, PKA is responsible for the TSH-induction of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA levels, RhoA activation, and down-regulation of p27(kip1). These correlated events are necessary for FRTL-5 cell proliferation after TSH stimulation. Moreover, TSH through PKA pathway increases cyclin-dependent kinase 2 levels, whereas PI 3-kinase signaling increases cyclin E levels. Together, both pathways finally converge, increasing the formation and activation of cyclin E x cyclin-dependent kinase 2 complexes and the phosphorylation of the retinoblastoma protein, two important steps in the transition from G(1) to S phase in growth-stimulated cells. Somatostatin exerts its antiproliferative effect inhibiting more upstream the TSH stimulation of PKA and PI 3-kinase, interfering with the TSH-mediated increases of intracellular cAMP levels by inactivation of adenylyl cyclase activity. Together, these results suggest the existence of a PKA-dependent pathway and a new PKA-independent PI 3-kinase pathway in the TSH/cAMP-mediated proliferation of FRTL-5 thyroid cells.

Cell cycle progression in mammalian cells requires the coordinated action of several classes of cyclin-dependent kinases (Cdk) 1 and cyclin complexes. One of the critical targets of cyclin⅐Cdk complexes is the retinoblastoma (Rb) gene product, which acts as a transcriptional repressor. During G 1 phase, Rb is hypophosphorylated and binds to E2F, a family of cell cycle transcription factors, inhibiting its activity. Rb is inactivated by a coordinated, sequential phosphorylation by cyclin D⅐Cdk4, cyclin D⅐Cdk6 in mid-G 1 phase, and cyclin E⅐Cdk2 in the G 1 /S boundary (which completes the phosphorylation of Rb in additional sites) (for review see Refs. [1][2][3]. This process leads to Rb dissociation from E2F, with the corresponding activation of genes containing E2F-binding sites in their promoters and implicated in G 1 /S transition (for review see Refs. 5 and 6). Another level of Cdk activity regulation results from the action of Cdk inhibitors that bind to cyclin⅐Cdk complexes and either inhibit their kinase activities or prevent their activation by Cdk-activating kinases (for review see Refs. 5 and 6). The Cdk inhibitors comprise two classes of proteins. The first includes the Ink4 family, so named for their ability to inhibit specifically the catalytic subunit of Cdk4 and Cdk6. The second class includes the Cip/Kip family, which can interact with many different cyclin⅐Cdk complexes. This family was initially described to interfere with the activity of cyclin D-, E-, and A-dependent kinases. More recent work revealed that although the Cip/Kip proteins are potent inhibitors of cyclin E-and A-dependent Cdk2, they can act as positive regulators of cyclin D⅐Cdk4 and Cdk6 complex formation (4,7). In this last Cdk inhibitor family, p27 kip1 is a widely distributed Cdk inhibitor that has an important role in regulating entry into and exit from the cell cycle.
We have recently demonstrated that p27 kip1 expression is down-regulated by thyrotropin (TSH) in FRTL-5 thyroid cells and that somatostatin prevents this TSH-induced down-regulation (8). FRTL-5 cells provide an excellent system to study the mechanisms that govern progression from G 1 to S phase, because in this cell type the transition from quiescent to proliferative cells requires the action of hormones and growth factors such as TSH, insulin, and insulin-like growth factor-I (9 -12). Studies in FRTL-5 thyroid cells, although performed in different hormonal backgrounds, show that TSH increases the expression of G 1 cyclins such as cyclin D1, D3, and E (13,14) as well as its partners Cdk2 and Cdk4 (13,15). Moreover, these effects correlate with down-regulation of p27 kip1 protein levels (8,13) and with an increase in the phosphorylation state of Rb (13), leading to the activation of cyclin⅐Cdk complexes and the progression of the cells through the cell cycle. TSH cell cycle induction is counteracted by cytostatic signals such as TGF-␤1 (13) and somatostatin (8,16,17). TGF-␤1 interference with TSH action has been studied in FRTL-5 cells (13); however, the mechanism of interference between somatostatin and TSH is unknown. It has recently been demonstrated, in another system, that somatostatin interferes with the insulin-mediated induction of cell cycle proteins by activating p27 kip1 (18). This action takes place through the specific somatostatin receptor type 2 (SSTR2) expressed in many cell types, including FRTL-5 cells (8).
The critical role of p27 kip1 in the cytostatic effects elicited by somatostatin (8,18) is the focus of our present study. Among other functions, p27 kip1 has been implicated in G 1 arrest induced by inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (19,20). These inhibitors interfere with cell cycle progression by suppressing the isoprenylation of proteins (21)(22)(23). A class of isoprenylated small GTP-binding proteins, termed Rho small GTPases, is proposed to be involved in G 1 /S transition in mouse fibroblasts (24) and also in FRTL-5 thyroid cells (15). RhoA, a member of a subgroup of the Ras superfamily, regulates a wide spectrum of cell functions such as cell growth, membrane trafficking, and transcription (for review see Ref. 25). RhoA expression promotes p27 kip1 degradation in FRTL-5 cells, leading to progress from G 1 to S phases (15). In addition, p27 kip1 is involved in G 1 arrest by cAMP, in cells in which this second messenger is an inhibitor of cell proliferation (26,27); conversely, cAMP signaling in FRTL-5 cells is the main mediator of thyroid cell proliferation in response to TSH (28).
We have studied how this hormone regulates two important mediators of the G 1 -S transition (Cdk2 and cyclin E associated with p27 kip1 ), as well as the role played by somatostatin in each control point of the TSH effect. Our work focused on the effect of TSH alone. The results obtained are due to TSH and not to the combined action of TSH with other hormones and growth factors, such as insulin or serum. The main signal pathways involved in TSH and somatostatin control of cell growth have been also studied. The results show that TSH regulates cell cycle proteins through at least two independent pathways that involve PKA and PI 3-kinase. These pathways induce different sets of cell cycle proteins that finally converge in Rb phosphorylation. Somatostatin prevents TSH modulation of p27 kip1 ⅐Cdk2 association, cyclin E⅐Cdk2 kinase activity, and the phosphorylation of Rb. Moreover, somatostatin also blocks the TSH-mediated induction of HMG-CoA-reductase mRNA levels as well as RhoA activation, two of the decisive events in FRTL-5 cells growth, which we show are mediated by PKA but not by PI 3-kinase. Finally, the mechanism by which somatostatin interferes with TSH effects involves inhibition of the adenylyl cyclase activity and the consecutive decrease in TSHinduced intracellular cAMP levels.

EXPERIMENTAL PROCEDURES
Materials-Tissue culture medium, bovine TSH, somatostatin, and bovine insulin were purchased from Sigma. H89, PD98059, wortmannin, and anti-Rb antibody were from Calbiochem. Octeotride (sandostatin) was from Sandoz Pharma Ltd. The Luminol kit, streptavidinhorseradish peroxidase conjugate, anti-Cdk2, anti-cyclin E, and anti-p27 antibodies were from Santa Cruz Biotechnology. Histone H1 was from Roche Molecular Biochemicals. Pravastatin was a gift from Bristol-Myers Squibb. Donor calf serum was from Life Technologies, Inc., and Nytran and nitrocellulose filters were from Schleicher & Schuell. The Biotrak cAMP enzyme immunoassay system (dual range) prestained protein marker, [␣-32 P]ATP, [␥-32 P]ATP, and [␣-32 P]dCTP were from Amersham Pharmacia Biotech. The IMMUNOcatcher kit was from CytoSignal Research Products.
Cell Culture-FRTL-5 thyroid cells (29) were cultured in Coon's modified Ham's F-12 medium supplemented with 5% donor calf serum and a hormone mixture (6H medium) including glycyl-L-histidyl-L-lysine acetate (10 ng/ml), hydrocortisone (10 nM), transferrin (5 g/ml), somatostatin (10 ng/ml), insulin (10 g/ml), and TSH (1 nM). Fresh medium was added every 2 or 3 days, and cultures were divided every 7 days. Before treatment, cell cultures were maintained for 3 days (otherwise indicated) in medium depleted of TSH, insulin, somatostatin, and 0.2% serum (3H medium) and were then maintained with each treatment at the concentration and for the time indicated below. Cells maintained in 3H medium (8) are completely viable. 2 To perform growth curve profiles FRTL-5 cells (4 ϫ 10 4 ) were maintained in basal medium (3H) for 3 days and then treated with TSH (1 nM), somatostatin (1 M), octeotride (100 ng/ml), H89 (10 M), PD98059 (50 M), wortmannin (25 nM), or pravastatin (100 g/ml) in the combinations indicated for each experiment. To avoid wortmannin degradation, this inhibitor was added every 6 h to cell culture medium. The number of viable cells was determined by cell counting at 36 h (otherwise indicated), and the means Ϯ S.D. of three independent experiments are represented.
Northern Blot Analysis-Total RNA was isolated by the guanidinium-isothiocyanate-phenol procedure (30). Samples of total RNA were electrophoresed in 1% agarose gels containing formaldehyde. RNA was transferred to Nytran membranes, and RNA integrity was revealed by methylene blue staining of the blots. Hybridization and washing were performed with a probe specific for HMG-CoA reductase (31) and labeled with [␣-32 P]dCTP by random priming.
Transient Transfections and Flow Cytometric Analysis-FRTL-5 cells grown to 80% confluence in 6H medium were transiently transfected (8) with 10 g of different expression vectors. The involvement of PI 3-kinase pathway was studied with the constitutively activated catalytic subunit (p110CAAX) or the dominant negative (p␣⌬85iSH2-N) that contains a deletion of the regulatory subunit of PI 3-kinase (32). The role of RhoA was analyzed with a dominant positive RhoA QL (33), dominant negative RhoA N19 (34), or the botulinum C3 exoenzyme, TC3 (35). In all cases cells were cotransfected with 1 g of expression vector encoding green fluorescent protein (GFP) (36). A group of cells was transfected with the corresponding empty vector as control. After transfection different hormonal treatments were used, as detailed in results. Then cells were harvested and the cell cycle distribution of propidium iodine-stained samples was performed as described previously (37), using a FACScan flow cytometer (Becton Dickinson Co.). At least 10,000 events were collected and analyzed. Data were integrated and plotted with the CellQuest software. GFP expression was used to normalize transfection efficiency.
Western Blots and Immunoprecipitation Assays-Total protein extracts (50 or 90 g for Rb detection) were subjected to 7.5, 10, and 12% SDS-polyacrylamide gel electrophoresis, for Rb, cyclin E and Cdk2, and p27 kip1 and RhoA respectively, and the proteins were transferred to nitrocellulose membranes. Membranes were blocked for 1-2 h at room temperature in TBS-T buffer (20 mM Tris-HCl, 137 mM NaCl, 0.1% Tween-20, pH 7.5) containing 5% non-fat milk. After incubation with the corresponding antibodies (1 g/ml, 1 h, room temperature) in TBS-T containing 5% non-fat milk, membranes were washed three times with TBS-T and then incubated with a 1:5000 dilution of streptavidin-horseradish peroxidase conjugate, followed by three washes of 5 min/each with TBS-T buffer. Immunoreactive bands were visualized using the Luminol kit as described by the manufacturer. RhoA levels were quantitated after densitometric scanning of the blots using the MacBas software.
For immunoprecipitation assays we used the IMMUNOcatcher kit. Briefly, cells were lysed as recommended by the manufacturer, and 500 g of total protein extracts were incubated with the primary antibody (1 h, room temperature). The samples were then incubated for 30 min at room temperature with 20 l of protein A/G-Sepharose. After centrifugation, the pellets were washed three times in lysis buffer, resuspended in 40 l of protein sample buffer, separated in SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes.
cAMP Assays-The Biotrak cAMP competitive enzyme immunoassay system was used, following the manufacturer's instructions. Briefly, FRTL-5 cells were grown in 96-well microtiter plates (10 4 -10 6 cells/ml), maintained in 3H medium for 3 days, and then treated with 1 nM TSH, 1 M somatostatin, 100 ng/ml octeotride, or the appropriate combination for 0.5, 1, 2.5, or 4 h. Cells were then lysed, moved to a donkey anti-rabbit Ig precoated microtiter plate, and incubated with anti-cAMP antiserum for 2 h at 4°C, after which samples were incubated with a cAMP-peroxidase-conjugated antibody (1 h, 4°C) and washed four times with washing buffer. The enzyme substrate was added immediately afterward to all wells and incubated (1 h, room temperature). Prior to optical density determination a plate reader at 450 nm, the reaction was terminated by adding 1 M sulfuric acid to each well. In parallel, we prepared a standard curve with concentrations cAMP from 12.5-3200 fmol/well. Each value represents the mean Ϯ S.D. of three different experiments.
Adenylyl Cyclase Assays-Cells in 10 mM Tris-HCl, pH 7.5, and 0.1 mM EDTA were homogenized at 0 -4°C by nitrogen cavitation using a 75 ml of Parr bomb; cells were equilibrated for 15 min at 750 p.s.i. N 2 , followed by slow release into a glass test tube. The homogenates were centrifuged (500 ϫ g, 10 min) to remove nuclei and unbroken cells. Membranes were collected by centrifugation of the supernatant fluid (18,000 ϫ g, 10 min), washed in 10 volumes of the same buffer, recentrifuged, resuspended in the same buffer (1-2 mg of protein/ml), and stored in aliquots at Ϫ80°C until use. As described previously (38)

PKA and PI 3-kinase Are Involved in TSH-induced FRTL-5
Thyroid Cell Proliferation-We previously described the existence of an autocrine loop in the control of FRTL-5 thyroid cell proliferation that involves TSH, the main regulator of thyroid cell proliferation, and somatostatin, a cytostatic agent (8). To elucidate the signal transduction pathways mediating TSH proliferation and the level at which somatostatin elicited its negative control, FRTL-5 cells were maintained for 3 days in a medium containing 0.2% serum and depleted of TSH, insulin, and somatostatin (3H medium). The cells were then treated for 36 h with TSH alone or combined with specific inhibitors of the most common transduction signal pathways or with somatostatin as described under "Experimental Procedures." Growth curve profiles (Fig. 1A) and cell cycle distribution after flow cytometric analysis (Fig. 1B) were determined. TSH increased cell number about 1.7-fold, and when added together with somatostatin, a detectable inhibition of cell number was observed. The most drastic inhibition of TSH induction of cell growth was obtained with the PKA inhibitor H89. A PI 3-kinase inhibitor, wortmannin, also inhibited TSH cell growth induction, although to a lesser extent than H89. The inhibitor of MEK (the upstream regulator of mitogen-activated protein kinase) PD98059 had no effect on TSH induction of cell growth in 3H cells. The results of G 1 /S ratio analysis are summarized in Fig. 1B. Cells maintained in 3H medium have an increase in G 1 over the S phase that indicates cell quiescence, with a virtual absence of DNA synthesis. TSH treatment of quiescent 3H cells clearly promotes G 1 transition to S phase, and the G 1 /S ratio decreases 75%. When the cells were treated with TSH plus somatostatin, H89, or wortmannin, however, accumulation was detected in G 1 phase, with a G 1 /S ratio similar to that observed in quiescent 3H cells. Again, PD98059 treatment had no effect on cell cycle distribution.
The involvement of PI 3-kinase in the TSH induction of FRTL-5 cells growth is very interesting and has not been previously reported in this cell line. Because of the relevance of these data, we confirm our observation using another experimental approach. FRTL-5 cells maintained in 6H medium were transiently transfected with an expression vector encoding a dominant negative of PI 3-kinase (32). Control cells were transfected with the empty vector. After transfection, cells were maintained for 36 h in 3H medium and then treated with TSH for other 36 h. In all transfection experiments, a plasmid containing GFP was cotransfected to normalize transfection efficiency, and then cytometric analysis was performed. The results show that control cells respond to TSH and have a cell cycle profile indicative of cells progressing through the G 1 /S phase (G 1 /S: 7.1), whereas cells expressing the dominant negative of PI 3-kinase do not respond to TSH and have a dramatic decrease in the S phase (G 1 /S: 12.9) (Fig. 1C). When an expression vector of the constitutively activated catalytic subunit of PI 3-kinase (32) was transfected in the same conditions as above, the cells respond to TSH and have a curve profile similar to control cells (data not shown).
Together all these data confirm two previous observations demonstrating that (i) TSH alone, in the absence of growth factors (0.2% serum) and insulin (3H medium), can induce thyroid cell growth (8,40), and (ii) somatostatin elicits a cytostatic effect (8). These data also demonstrate that TSH regulation of cell growth involves at least two cascades, one via PKA and another via PI 3-kinase.
TSH Regulates Cell Cycle Proteins Required to G 1 /S Transition-Once two of the main signal transduction pathways mediating TSH induction of FRTL-5 cell growth had been determined, we analyzed the cell cycle proteins involved in this response and, more precisely, those at the G 1 -S boundary. Cells maintained for 3 days in 3H medium (0 h) were treated with TSH for 12, 24, and 36 h, and Western blots for Cdk2, cyclin E, and p27 kip1 proteins were performed. Cdk2 and cyclin E levels increased with TSH in a time-dependent manner ( Fig. 2A), whereas the levels of the Cdk-dependent kinase inhibitor p27 kip1 decreased in parallel. Following the same experimental approach, TSH was added for 36 h to quiescent 3H cells, alone or together with somatostatin, H89, or wortmannin. Somatostatin decreased the TSH-mediated up-regulation of both Cdk2 and cyclin E proteins, and, as we have described previously (8), it also decreases TSH-mediated down-regulation of p27 kip1 (Fig. 2B, lane 3). We observe some differences when comparing the effects of PKA and PI 3-kinase inhibitors. H89 decreases TSH up-regulation of Cdk2, inhibits TSH down-regulation of p27 kip1 , and has no effect on cyclin E levels (Fig. 2B,  lane 4). Conversely, wortmannin only has an effect on cyclin E, decreasing its TSH up-regulation, but does not modify the TSH-regulated Cdk2 and p27 kip1 levels (Fig. 2B, lane 5). These results indicate that the two main pathways that mediate TSH effect on FRTL-5 cell proliferation, PKA and PI 3-kinase, di-verge in the regulation of these cell cycle proteins. PKA thus mediates the regulation of Cdk2 and p27 kip1 , whereas PI 3-kinase is involved in cyclin E regulation. Furthermore, somatostatin interferes with all of these effects, suggesting that its mechanism of action lies further upstream.
Somatostatin Decreases TSH-mediated Activation of Cyclin E⅐Cdk2 Complexes and Retinoblastoma Phosphorylation-The kinase activity of the cyclin E⅐Cdk2 complexes was then analyzed. We first studied the formation of cyclin E⅐Cdk2 complexes, and as predicted, the results demonstrate that TSH increases total Cdk2 protein levels as well as the cyclin E bound to it (Fig. 3A, lane 2). Somatostatin affects the complex formation inhibiting the TSH-dependent increase of Cdk2 and cyclin E (Fig. 3A, lane 3; see also Fig. 2B, lane 3). The specific inhibitors of PKA and PI 3-kinase affect complex formation in a different way. Thus, H89 decreases the Cdk2 protein immu-

FIG. 2. Regulation of G 1 cell cycle proteins in FRTL-5 cells.
A, time-dependent changes in the expression of G 1 cyclins and related molecules in TSH-stimulated FRTL-5 cells. B, effects of TSH, somatostatin and specific inhibitors of PKA and PI 3-kinase on cell cycle proteins required to G 1 /S transition. FRTL-5 cells were maintained in basal 3H medium and then treated with TSH alone or in combination with somatostatin (SS), H89, and wortmannin (Wort) for the indicated times and concentration described under "Experimental Procedures." Total extracts (50 g) were analyzed by immunoblotting with antibodies against cyclin E, Cdk2, and p27 kip1 .
FIG. 3. Effects of TSH treatment alone or combined with somatostatin, H89, or wortmannin on the cyclin E⅐Cdk2-p27 kip1 complexes and retinoblastoma protein levels. A, 500 g of total cell lysates were immunoprecipitated with anti-Cdk2 antibody and immunoblotted against Cdk2 and cyclin E. Cyclin E⅐Cdk2 complex activity were assayed for associated kinase activity using histone H1 as substrate. B, 500 g of total cell lysates were immunoprecipitated with anti-p27 antibody and immunoblotted against Cdk2 and p27 kip1 . C, total cell extract (90 g) were analyzed by immunoblotting with antibodies against Rb proteins. Both phosphorylated forms of Rb are shown. Cell treatment was as described in legend to Fig. 2B. Wb, Western blot. noprecipitated (Fig. 3A, lane 4), whereas wortmannin only affects the complex by inhibiting the cyclin E protein levels (Fig. 3A, lane 5; see also Fig. 2B, lane 5). We also determined the activity of these complexes using the H1 kinase assay. Cdk2 activity is maximal in the TSH-treated group (Fig. 3A,  bottom panel); no activity is detected in the other groups, except for wortmannin-treated cells, in which residual activity is observed. This effect is probably due to a small amount of p27 kip1 bound to Cdk2 in this group (Fig. 3B, lane 5). As seen in this panel, when p27 kip1 was immunoprecipitated and probed with p27-specific antibodies, we observed that p27 kip1 was almost undetectable in TSH-treated cells (lane 2). In the rest of the groups, p27 kip1 levels are very similar, except for the cells treated with wortmannin. These results indicate that most p27 kip1 is degraded in the group treated with TSH, so that cyclin E⅐Cdk2 should be free to phosphorylate its substrates, as shown above in the H1 kinase assay.
We next performed Western blot assays with a specific anti-Rb antibody. This antibody detects the active hyperphosphorylated form of the protein (Fig. 3C, upper band) as well as the inactive, more slowly migrating hypophosphorylated form (lower band). In quiescent 3H cells, Rb is expressed at very low levels (Fig. 3C, lane 1). TSH increased Rb protein levels as well as its phosphorylation state, seen as an increase in the upper phosphorylated band (Fig. 3C, lane 2). Conversely, somatostatin, H89, and wortmannin inhibited the TSH-induced phosphorylation of Rb protein (Fig. 3C, lanes 3-5). These data suggest that Rb could be the main inhibition mechanism of G 1 /S transition in our study of FRTL-5 thyroid cell growth.
Somatostatin Inhibits the TSH Induction of HMG-CoA Reductase-To study the signaling circuits involved in the TSH and somatostatin regulation of p27 kip1 levels, we tested the regulation of HMG-CoA reductase, because p27 kip1 has been implicated in the G 1 arrest induced by an inhibitor of this enzyme (19,20). Using Northern blot assays (Fig. 4A), we show that, in cells maintained in 3H medium, HMG-CoA reductase mRNA levels are undetectable (lane 1). TSH can increase these levels (lane 2), as has been previously demonstrated (31). Somatostatin (lane 3) and H89 (lane 4) block this TSH increase, as does the inhibitor of HMG-CoA reductase, pravastatin (lane 5). Wortmannin had no effect on TSH induction of HMG-CoA reductase, indicating that TSH induces the expression of this enzyme mainly via the PKA pathway. When pravastatin was added alone, in the absence of TSH induction, HMG-CoA-reductase mRNA levels were undetectable as observed in 3H group (data not shown). To confirm the importance of HMG-CoA reductase in TSH-mediated proliferation, growth curves and cytometric assays were performed in parallel studies. Pravastatin decreases the TSH induction of cell number (Fig.  4B) as well as the TSH decrease of G 1 /S ratio (Fig. 4C). These two experimental approaches clearly confirm that inhibition of isoprenoid synthesis promotes G 1 arrest in FRTL-5 cells and further demonstrate that TSH regulation of HMG-CoA reductase is mediated by PKA and not by the PI 3-kinase pathway.
RhoA Is Required for TSH-induced G 1 Progression-Because HMG-CoA reductase activity is necessary to isoprenylate small GTPases and RhoA is reported to be essential for the degradation of p27 kip1 , facilitating G 1 /S progression in FRTL-5 cells (15), we studied the role of this small GTPase in TSH-induced G 1 progression. We first determined whether RhoA is activated by TSH alone and/or this activation is inhibited by somatostatin, H89, wortmannin, or pravastatin. For this purpose, Western blot was performed with a specific anti-RhoA antibody, using fractionated proteins derived from the above experimental groups (Fig. 5A). After quantitation of the RhoA levels in membrane and cytosolic fractions (Fig. 5B), we show that RhoA is activated by TSH, because this hormone induces the translocation of RhoA from the cytoplasm to the membrane. Somatostatin, pravastatin, and the PKA inhibitor, but not the PI 3-kinase inhibitor, blocked TSH induction of RhoA activation.
To confirm the role of RhoA in FRTL-5 cell cycle, transfection experiments were performed with expression vectors encoding the cDNA of a dominant positive RhoA QL (32), the botulinum C3 exoenzyme TC3 (34), which specifically inactivates Rho proteins (41), or the dominant negative RhoA N19 (33). Control cells were transfected with the empty vector. In all transfection experiments, a plasmid containing GFP was cotransfected to normalize transfection efficiency, and transfected cells were analyzed cytometrically. The results (Fig. 5C) show that cells overexpressing RhoA have a cell cycle profile similar to FRTL-5 control cells progressing through the G 1 /S phase. When RhoA was inactivated with TC3, cells suffered arrest in G 1 , similar to that found when the dominant negative RhoA N19 was overexpressed. These data demonstrate that inactivation of RhoA is sufficient to arrest FRTL-5 cells in G 1 phase, revealing its

FIG. 4. Somatostatin and H89 inhibit TSH-induction of HMG-CoA reductase mRNA levels in FRTL-5 cells.
Total RNA was extracted from cells maintained 3 days in 3H medium and then treated for 12 h with TSH alone or combined with somatostatin (SS), H89, wortmannin (Wort), or pravastatin (Prav). A shows a representative Northern blot hybridized with HMG-CoA reductase probe. For loading control, the 18 S ribosomal RNA after methylene blue staining is shown. The lower panels show the effect of pravastatin treatment on cells number (B) and cell cycle distribution (represented as G 1 /S ratio) (C). Samples were collected after 36 h of treatments for FACS analysis and represented with CellQuest software. Values are arbitrary units considering 3H-maintained FRTL-5 cells as maximal G 1 /S ratio. The data are the means Ϯ S.D. of three independent experiments. importance in cell cycle control. Moreover, we demonstrate that TSH, probably through PKA and by activation of HMG-CoA reductase, induces RhoA translocation to the plasma membrane. Finally, we show that somatostatin is again able to counteract this effect of TSH.
Somatostatin Inhibits TSH Induction of FRTL-5 Cell Proliferation by Interfering with Adenylyl Cyclase Activity-The results presented thus far show that somatostatin is able to inhibit most TSH-induced effects on FRTL-5 cell proliferation. This suggests that somatostatin inhibition would involve a mechanism upstream of the signal transduction pathways stimulated by TSH. As we previously reported (8), somatostatin action on FRTL-5 cell growth is mediated through its specific receptor, SSTR2. Previous work has demonstrated that this receptor is coupled to adenylyl cyclase, inhibiting cAMP production (42). TSH, after binding to its own receptor, increases the intracellular levels of cAMP activating the PKA (28). This evidence suggests a plausible mechanism of somatostatin action through its SSTR2 receptor by inhibiting TSH induction of intracellular cAMP levels and the consecutive inhibition of PKA and PI 3-kinase pathways. To test this hypothesis, we used two different approaches. We determined the intracellular cAMP levels and the adenylyl cyclase activity in FRTL-5 cells maintained in basal 3H medium or treated with TSH alone or together with somatostatin for different periods of time and at various concentrations. The results demonstrate that TSH stimulates intracellular cAMP levels with respect to those observed in cells maintained in 3H medium (Fig. 6A). This stimulation occurs in a time-dependent manner (not shown), with maximum stimulation after 2 h of treatment. In parallel, TSH stimulates adenylyl cyclase activity (Fig. 6B); conversely, somatostatin abolished TSH induction of cAMP  (Fig. 6A). This effect was mimicked by a somatostatin analog, octeotride, that also acts through the SSTR2. Interestingly, somatostatin inhibits TSH induction of the adenylyl cyclase in a dose-dependent manner (Fig. 6B). These data demonstrate that somatostatin interference with TSH action already occurs in the first step of TSH signaling. DISCUSSION The control of cell proliferation requires a balance between positive effectors and cytostatic signals. In the case of FRTL-5 thyroid cells, we have recently demonstrated (8) that this control is elicited by an autocrine loop that involves TSH, as a positive effector, and somatostatin, as a cytostatic agent. Although it is widely accepted that cAMP is the main mediator of TSH stimulation of thyroid cell growth (28,43), increasing evidence suggests that the mechanism of TSH action involves more than the PKA classical signal transduction pathway (44). In different thyroid cell systems and with different hormonal backgrounds, TSH is able to activate PKA (28), protein kinase C (45,46), and mitogen-activated protein kinase (47), inducing distinct thyroid cell functions. Most the studies were performed in FRTL-5 cells, and in most of them, TSH action has been analyzed in the presence of permissive factors such as insulin/ insulin-like growth factor-I or serum growth factors (48). Insulin per se thus promotes moderate increases in protein and DNA synthesis in FRTL-5 and PC13 thyroid cells (49), as well as increases in G 1 cyclins, D1 and E (14). Moreover, in many studies, TSH effects on proliferation are masked by the presence of somatostatin in the culture medium (8). For this reason, we focused on the signal transduction pathways and the cell cycle progression mediated by TSH alone, in the absence of any other ligand, as well as the negative role elicited by somatostatin on TSH action. Our results show that specific inhibition of PKA and PI 3-kinase independently blocks TSH-induced FRTL-5 cell proliferation and that somatostatin interferes with both signals. Our data inhibiting MEK, the upstream regulator of mitogen-activated protein kinase, confirm previous results in dog thyrocytes (50) and Wistar rat thyroid cells (47), showing that the mitogen-activated protein kinase pathway is not involved in TSH-mediated proliferation. Although it has been described that TSH induces protein kinase C activation (45,46), we did not study the participation of this kinase in TSHinduced FRTL-5 proliferation, because such activation has been observed under unusual culture conditions, with high doses of TSH, insulin, and serum in the medium. In summary, our results suggest that the proliferative response of TSH in FRTL-5 cells involves at least two independent pathways, PKA and PI 3-kinase. During the preparation of this manuscript, Cass et al. (44) demonstrated, in Wistar rat thyroid cells, that the mitogenic signals initiated by cAMP diverge to include PKA-dependent pathways, leading to p70 s6 kinase, and PKAindependent pathways that regulate Akt and Rac1 via PI 3-kinase. These observations corroborate our results on the implication of the PI 3-kinase pathway in TSH-induced cell growth, suggesting TSH/cAMP-dependent induction of the PI 3-kinase signal. We have also observed that TSH increases p70 s6 kinase protein levels in FRTL-5 and that this activation is PKA-dependent and PI 3-kinase-independent. 2 To link the proliferative TSH-stimulated pathways with the cell cycle machinery, we determined the effects of PKA and PI 3-kinase inhibition on the cell cycle proteins necessary for G 1 /S transition. The main regulators of this transition are the G 1 cyclins (D and E) and their partner cyclin-dependent kinases (Cdk-2, Cdk-4, and Cdk-6). The most recognized function of cyclin D-dependent kinase is phosphorylation of Rb, which initiates in mid-G 1 phase; thereafter, cyclin E⅐Cdk2 becomes active and completes this process by phosphorylating Rb on additional sites. Although we focused our study on the late G 1 phase, mediated mainly by cyclin E⅐Cdk2 complexes, several pieces of evidence suggest that cyclin D⅐Cdk-4 and Cdk-6 must be also active in TSH-stimulated FRTL-5 cells: first, our previous results indicated TSH up-regulation of cyclin D1 (8), and second, the TSH-mediated increase in Rb phosphorylation observed here. Our results furthermore demonstrate that TSH increases cyclin E and Cdk-2 as well as down-regulates p27 kip1 protein levels. These effects are differentially mediated by PKA, which is responsible for the TSH increases of Cdk-2 and p27 kip1 down-regulation, and PI 3-kinase, which is necessary for TSH increases of cyclin E. TSH also increases Rb protein levels and its phosphorylated state, whereas PKA and PI 3-kinase inhibitors revert this effect, indicating that both pathways converge in the nucleus, leading finally to the Rb phosphorylation. These results indicate that PI 3-kinase-dependent increases in cyclin E might be explained in light of the recent view of G 1 -S progression control (4). PI 3-kinase is thus involved in the turnover of D-type cyclins. Inhibition of this pathway increases cyclin D1 degradation (51), with the consequent inhibition of Rb phosphorylation and E2F-dependent cyclin E transcription. Conversely, somatostatin inhibits both the TSH increases of cyclin E and Cdk-2 as well as TSH down-regulation of p27 kip1 . As expected, it also inhibits cyclin E⅐Cdk2 complexes, decreasing the amount of Cdk-2 immunoprecipitated and increasing the p27 kip1 bound to the complex and the TSH phosphorylation of Rb. p27 kip1 promotes G 1 arrest induced by inhibitors of HMG-CoA reductase, a rate-limiting enzyme in the synthesis of isoprenoids. A class of isoprenylated small GTPase, RhoA, is involved in G 1 /S progression in FRTL-5 cells (15). Our results confirm that TSH increases HMG-CoA reductase mRNA (31) and also demonstrate that somatostatin inhibits this up-regulation. In addition, the inhibition of HMG-CoA reductase activity promotes cell cycle arrest, confirming the important role of this enzyme in TSH-induced proliferation. The activity of this enzyme is PKA-dependent and PI 3-kinase-independent. The latter results correlate with an increase in RhoA levels in the membrane fraction of TSH-stimulated FRTL-5 cells; somatostatin, H89, and pravastatin, but not wortmannin, inhibit this effect, suggesting that PKA may activate RhoA in FRTL-5 cells. The mammalian Rho family of GTPases, including RhoA, Rac1, and Cdc42, plays a pivotal role in controlling many cellular functions including cell polarity, motility, proliferation, apoptosis, and cytokinesis (25). In other cell types, such as neural cells and lymphocytes, RhoA can be specifically phosphorylated at Ser-188 by PKA. This decreases the binding of RhoA to downstream effectors, suggesting that RhoA and cAMP have antagonistic regulatory roles in these cells (52). The role of cAMP is completely different in the thyroid, however; this second messenger thus activates a proliferative response in the majority of thyroid cell systems (11,53,54). In FRTL-5 thyroid cells, RhoA activation is essential for p27 kip1 degradation and the consequent G 1 /S progression; this activation is HMG-CoA reductase-dependent (15). In addition, we show that overexpression of an interfering mutant (RhoA N19) or a specific inhibitor (TC3) induces G 1 arrest, whereas overexpression of a dominant positive (RhoA QL) shows a cell cycle distribution similar to the control. Together, these data suggest that TSH stimulates RhoA in a PKA/HMG-CoA Red-dependent manner and that this activation may be responsible for TSH down-regulation of p27 kip1 , although further study is needed to demonstrate this hypothesis, currently under investigation.
We show that the specific inhibition of PKA and PI 3-kinase blocks different TSH effects, indicating their independence, whereas somatostatin inhibits all TSH effects studied. This suggests a mechanism for somatostatin action upstream of PKA and PI 3-kinase and indicates the inhibitory potential of somatostatin, which is able to counteract PKA-dependent and -independent proliferative effects. FRTL-5 thyroid cells express the somatostatin receptor SSTR2 (8), and this receptor can be coupled to several downstream effectors such as adenylyl cyclase or protein-tyrosine phosphatases (55,56). Moreover, TSH increases intracellular cAMP levels (28,43). We thus tested a possible mechanism of somatostatin inhibition by interference with TSH-mediated cAMP production. The results clearly show that TSH increases cAMP intracellular levels by activation of adenylyl cyclase, whereas somatostatin inhibits cAMP production by inactivation of this enzyme. The fact that somatostatin inhibits both PKA and PI 3-kinase pathways after TSH stimulation, by interfering with cAMP production, reinforces the idea of cAMP-dependent stimulation of both kinases.
Finally, we present a model for the action of TSH and somatostatin in FRTL-5 thyroid cell proliferation (Fig. 7). In this model, TSH/cAMP can activate both PKA and PI 3-kinases. Via PKA, TSH thus increases Cdk2 protein levels and RhoA activation in a HMG-CoA reductase-dependent manner, whereas via PI 3-kinase, TSH increases cyclin-E protein levels. Subsequent to these effects, each converges in the phosphorylation of Rb, leading to progression through the cell cycle. Conversely, somatostatin inhibits TSH-induced proliferation, decreasing cAMP levels by inhibition of the adenylyl cyclase activity.