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* This work was supported by DGICYT Grant PM97-0065, CAM Grant 08.1/0025/97–99, and a grant from Fundación Salud 2000 (Spain).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Recipient of a fellowship from the Spanish Ministerio de Educación y Cultura.
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 G1/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·cyclin-dependent kinase 2 complexes and the phosphorylation of the retinoblastoma protein, two important steps in the transition from G1 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.
specific somatostatin receptor type 2
green fluorescent protein
cAMP-dependent protein kinase
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 cyclin·Cdk complexes is the retinoblastoma (Rb) gene product, which acts as a transcriptional repressor. During G1 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 D·Cdk4, cyclin D·Cdk6 in mid-G1 phase, and cyclin E·Cdk2 in the G1/S boundary (which completes the phosphorylation of Rb in additional sites) (for review see Refs.
). Another level of Cdk activity regulation results from the action of Cdk inhibitors that bind to cyclin·Cdk complexes and either inhibit their kinase activities or prevent their activation by Cdk-activating kinases (for review see Refs.
). 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 cyclin·Cdk 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 D·Cdk4 and Cdk6 complex formation (
). FRTL-5 cells provide an excellent system to study the mechanisms that govern progression from G1 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 (
); 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 (
We have studied how this hormone regulates two important mediators of the G1-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·Cdk2 association, cyclin E·Cdk2 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 TSH-induced intracellular cAMP levels.
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 (
) 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 (
), 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 (
). 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 (
), we did not study the participation of this kinase in TSH-induced 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. (
) demonstrated, in Wistar rat thyroid cells, that the mitogenic signals initiated by cAMP diverge to include PKA-dependent pathways, leading to p70s6kinase, and PKA-independent 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 p70s6 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 G1/S transition. The main regulators of this transition are the G1 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-G1 phase; thereafter, cyclin E·Cdk2 becomes active and completes this process by phosphorylating Rb on additional sites. Although we focused our study on the late G1 phase, mediated mainly by cyclin E·Cdk2 complexes, several pieces of evidence suggest that cyclin D·Cdk-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 (
), 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 G1-S progression control (
), 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 E·Cdk2 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 G1 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 G1/S progression in FRTL-5 cells (
) 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 (
). 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 (
). In addition, we show that overexpression of an interfering mutant (RhoA N19) or a specific inhibitor (TC3) induces G1 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 (
). 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.
We thank Drs. Isabel Barroso and Juan A. Velasco for the critical reading of this manuscript and Catherine Mark for linguistic assistance. We are indebted to Dr. Leonard Kohn (NHIDDK, National Institutes of Health, Bethesda, MD) for the HMG-CoA reductase cDNA, to Dr. Silvio Gutkind (NCI, NIH, Bethesda, MD) for the RhoA QL, RhoA N19, and TC3 expression vectors, and to Dr. Julian Downward (Imperial Cancer Research Foundation, London, UK) for the p110CAAX and pαΔ85iSH2-N expression vectors.