cAMP inhibition of Akt is mediated by activated and phosphorylated Rap1b

Rap1b has been implicated in the transduction of the cAMP mitogenic signal. Rap1b is phosphorylated and activated by cAMP, and its expression in cells where cAMP is mitogenic leads to an increase in G(1)/S phase entry and tumor formation. The PCCL3 thyroid follicular cells represent a differentiated and physiologically relevant system that requires thyrotropin (TSH), acting via cAMP, for a full mitogenic response. In this model system, cAMP stimulation of DNA synthesis requires activation and phosphorylation of Rap1b by the cAMP-dependent protein kinase A (PKA). This scenario presents the challenge of identifying biochemical processes involved in the phosphorylation-dependent Rap1b mitogenic action. In thyroid cells, Akt has been implicated in the stimulation of cell proliferation by TSH and cAMP. However, the mechanism(s) by which cAMP regulates Akt activity remains unclear. In this study we show that in PCCL3 cells 1) TSH inhibits Akt activity via cAMP and PKA; 2) Rap1b is required for cAMP inhibition of Akt; and 3) transduction of the cAMP signal into Akt requires activation as well as phosphorylation of Rap1b by PKA.     

Phosphorylation of the Three Rb Protein Family Members Is a Common Step of the cAMP-, the Growth Factor, and the Phorbol Ester-Mitogenic Cascades but Is Not Necessary for the Hypertrophy Induced by Insulin

Thyrotropin (TSH) through the cAMP cascade and in the presence of insulin induces the proliferation of dog thyroid cells. In this work, it is shown that TSH via cAMP causes the phosphorylation of the three members of the pRb family, pRb, p107, and p130, with the same kinetics as those observed when these cells are stimulated by mitogens acting through a tyrosine kinase receptor or through activation of kinase C. It is the first described point of convergence of cAMP-dependent and -independent mitogenic pathways in dog thyrocytes and suggests that the phosphorylation of the three proteins may be involved in the initiation of DNA synthesis in these cells. We also show that insulin, which induces hypertrophy and is permissive for the TSH mitogenic action, does not provoke the phosphorylation of any pRb family member, suggesting that none of these phosphorylations is required for this effect.     

Protein synthesis during induction of DNA replication in thyroid epithelial cells: evidence for late markers of distinct mitogenic pathways

The synthesis of specific protein has been investigated in primary cultures of dog thyroid epithelial cells, which can be induced to progress into G1 phase, in the presence of insulin, by different types of mitogens: thyrotropin (TSH) acting through cyclic adenosine monophosphate (cAMP), epidermal growth factor (EGF), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or 10% serum. EGF, TPA, or serum specifically induce [35S] methionine labeling of protein 1 (Mr approximately 80,000). The effect of EGF on protein 1 labeling and DNA replication is dependent on insulin. The level of protein 1 labeling as well as that of DNA synthesis is higher when TSH or TSH + serum are added together with EGF. It peaks in mid-G1. TSH alone, in the presence of insulin, stimulates DNA replication without inducing protein 1 synthesis, which thus represents a cell-cycle-dependent event that is not obligatory in mitogenic activation through cyclic AMP. Among the eight proteins whose synthesis is stimulated by TSH, only the labeling of protein 7, molecular weight ratio (Mr approximately 38,000), correlates with the DNA synthetic activity of the cells. The present authors identified protein 7 as cyclin/proliferating cell nuclear antigen (PCNA), the auxiliary protein of DNA polymerase-delta. The effect of TSH on cyclin synthesis is already detectable when most of the cells are in late G1, but its stimulation by EGF or EGF + serum is delayed and detected only after extending the labeling period to the S-phase. These data support the view that the cAMP-mediated mitogenic pathway remains partly distinct from the better known pathways induced by growth factors and tumor promoters, even at late stages of the G1-phase.     

Impairment of the TSH signal transduction system in human thyroid carcinoma cells

In order to further evaluate the role of TSH in the proliferation and the differentiation of human thyroid carcinoma cells, we have analyzed the function of the TSH receptor in the established thyroid carcinoma cell lines NPA and WRO. The TSH signal transduction system in the carcinoma cells was also compared with that in normal thyroid cells. Although unresponsiveness to bovine and human TSH was demonstrated by measurement of cAMP production and [3H]thymidine incorporation after treatment of TSH, cAMP production was induced after stimulation of these cells by forskolin, cholera toxin, and isoproterenol. Specific binding to 125I-TSH was demonstrated in both NPA and WRO cells in addition to the existence of a TSH receptor mRNA and thyroglobulin mRNA species, although thyroid-specific gene expression in these cells was not regulated by TSH. These findings suggest that the unresponsiveness to TSH in these cells may be due to an abnormality of TSH receptor-G protein coupling rather than to a decreased level of TSH-receptor expression or a Gs protein abnormality.     

Effect of TSH in human thyroid cells: evidence for both mitogenic and antimitogenic effects.

The well-known mitogenic effects of TSH observed in vivo on the thyroid are not always reproducible of human thyroid cells in vitro where conflicting results have been obtained. In order to clarify this issue, we have used primary cultures of human thyroid cells obtained from normal tissue and maintained in serum-free medium for several days. In this in vitro model we have studied the effect of TSH on growth by measuring three different parameters: [3H]-thymidine incorporation, cell counts, and DNA measurement. Monolayer cultures were plated at both low and high cell density (2 x 10(4) and 8 x 10(4) cells/25 mm well, respectively). Although at either cell density cultures were equally able to functionally respond to TSH in terms of cAMP accumulation a significant growth response to TSH was observed only in low density cultures. In high density cultures TSH had an antimitogenic effect. Moreover, TSH potentiated the mitogenic effect of insulin only in low density cultures. In contrast to TSH, FCS induced a similar proliferative response at both high and low cell density. Following TSH stimulation, cAMP content was always increased, paralleling the effect of growth in low density but not in high density cultures. The cAMP analogues dibutyryl-cAMP and 8-bromo-cAMP, as well as cholera toxin and forskolin, did not mimic the mitogenic effect of TSH but had an antiproliferative effect. In addition, these agents blunted the proliferative effect of insulin. These data suggest that in thyroid cells TSH is able to elicit both a mitogenic and an antimitogenic effect depending on the environmental conditions such as cell density.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP regulation of Gs protein. Thyrotropin and forskolin increase the quantity of stimulatory guanine nucleotide-binding proteins in cultured thyroid follicles

This study was carried out to clarify the way in which thyrotropin (TSH) and forskolin regulate the adenylylcyclase complex in thyroid follicle cells. We examined the effects of chronic treatment of pig thyroid follicles with TSH or forskolin on the state of G proteins by (a) assaying adenylylcyclase activity, (b) analyzing the ADP-ribosylation of stimulatory G protein (Gs) by cholera toxin, and (c) quantifying the Gs subunits by Western blotting with antipeptide antibodies. Chronic exposure (18 h) of thyroid follicles to a low concentration of TSH (0.01-0.1 milliunit/ml) enhanced the subsequent response of adenylylcyclase to TSH. Higher concentration of TSH (1 milliunit/ml) induced a homologous desensitization of this response. In cells pretreated with forskolin, the TSH-stimulated adenylylcyclase activity was higher than in control cells. The forskolin-or guanosine 5'-(beta, gamma-imido) triphosphate (Gpp(NH)p)-stimulated adenylylcyclase activity was always significantly increased after chronic treatment of cells with TSH or forskolin. Treatment of cultured thyroid follicle membranes with [32P]NAD and cholera toxin resulted in labeling of the Gs alpha (45-52-kDa) component. Culturing follicles with TSH (0.001-1 milliunit/ml) or forskolin (0.01-10 microM) greatly affected the cholera toxin-mediated ADP-ribosylation of the Gs alpha subunit. Gs alpha labeling increased progressively to level off at 1 milliunit/ml TSH or 1 microM forskolin (150-200%). Gs alpha immunoreactivity was increased in parallel (200-300%). The immunoreactivity of G beta subunits in cells cultured with TSH or forskolin was also increased compared with control cells. Cycloheximide abolished the effects of TSH and forskolin on the follicles, suggesting that new protein synthesis is required. These results indicate that Gs protein subunits are up-regulated by TSH and forskolin and suggest that their synthesis in thyroid cells is mediated, at least in part, by a cyclic AMP-dependent mechanism.     

Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase.

Microinjection of a dominant interfering mutant of Ras (N17 Ras) caused a significant reduction in thyrotropin (thyroid-stimulating hormone [TSH])-stimulated DNA synthesis in rat thyroid cells. A similar reduction was observed following injection of the heat-stable protein kinase inhibitor of the cyclic AMP-dependent protein kinase. Coinjection of both inhibitors almost completely abolished TSH-induced DNA synthesis. In contrast to TSH, overexpression of cellular Ras protein did not stimulate the expression of a cyclic AMP response element-regulated reporter gene. Similarly, injection of N17 Ras had no effect on TSH-stimulated reporter gene expression. Moreover, overexpression of cellular Ras protein stimulated similar levels of DNA synthesis in the presence or absence of the heat-stable protein kinase inhibitor. Together, these results suggest that in Wistar rat thyroid cells, a full mitogenic response to TSH requires both Ras and cyclic APK-dependent protein kinase.     

Reciprocal modulation of thyrotropin actions by P1-purinergic agonists in FRTL-5 thyroid cells. Inhibition of cAMP pathway and stimulation of phospholipase C-Ca2+ pathway.

In FRTL-5 thyroid cells, thyrotropin (TSH) stimulates I- efflux in association with phospholipase C activation and Ca2+ mobilization. TSH also stimulates DNA synthesis, accompanied by cAMP accumulation. Significant activation of the phospholipase C-Ca2+ pathway requires 10-100 nM TSH a concentration 10(3) to 10(4) times higher than necessary to stimulate the cAMP pathway. When the P1-purinergic agonist, phenylisopropyladenosine (PIA) is added to the reaction medium, the former pathway is markedly enhanced, whereas the latter pathway is inhibited. As a result, in the presence of PIA, both TSH-induced pathways are activated at similar TSH concentrations. These PIA actions are completely reversed by a prior treatment of cells with islet-activating protein (IAP); pertussis toxin. When adenosine deaminase is added to the reaction medium, TSH-induced cAMP accumulation is significantly enhanced, suggesting an autocrine action of adenosine. In IAP-treated cells, the level of TSH-induced cAMP accumulation reaches that of deaminase-treated control cells, and no further increase is observed when adenosine deaminase is added. We conclude that in the thyroid, either an neural or autocrine adenosine signal, mediated by an IAP-sensitive G-protein, switches TSH signal transduction from the cAMP pathway to the phospholipase C-Ca2+ pathway.     

A mechanism generating heterogeneity in thyroid epithelial cells: suppression of the thyrotropin/cAMP-dependent mitogenic pathway after cell division induced by cAMP-independent factors

The mechanisms that generate the intercellular heterogeneity of functional and proliferation responses in a tissue are generally unknown. In the thyroid gland, this heterogeneity is peculiarly marked and it has been proposed that it could result from the coexistence of genetically different subpopulations of thyrocytes. To evaluate the heterogeneity of proliferative responses in primary culture of dog thyrocytes, we asked whether the progeny of cells having incorporated 3H thymidine in a first period of the culture could have a distinct proliferative fate during a second labeling period (incorporation of bromodeoxyuridine revealed by immunofluorescence staining combined with autoradiography of 3H thymidine). No growth-prone subpopulations were detected and the great majority of cells were found to response to either EGF or thyrotropin (TSH) through cAMP. However, only a fraction of cells replicated DNA at one given period and a clustered distribution of labeled cells within the monolayer, which was different for thymidine- or bromodeoxyuridine-labeled cells, indicates some local and temporal synchrony of neighboring cells. The TSH/cAMP-dependent division of thyrocytes preserved their responsiveness to both TSH and EGF mitogenic pathways. By contrast, cells that had divided during a momentary treatment with EGF lost the mitogenic sensitivity to TSH and cAMP (forskolin) but retained the sensitivity to EGF. Since cells that had not divided kept responsiveness to both TSH and EGF, this generated two subpopulations differing in mitogen responsiveness. The extinction of the TSH/cAMP-dependent mitogenic pathway was delayed (1-2 d) but stable. Cell fusion experiments suggest it was due to the induction of a diffusible intracellular inhibitor of the cAMP-dependent growth pathway. These findings provide a useful model of the generation of a qualitative heterogeneity in the cell sensitivity to various mitogens, which presents analogies with other epigenetic processes, such as differentiation and senescence. They shed a new light on the significance of the coexistence of different modes of cell cycle controls in thyroid epithelial cells.     

TSH induces insulin receptors that mediate insulin costimulation of growth in normal human thyroid cells

The mitogenic/goitrogenic effects of thyrotropin (TSH) on human thyrocytes in vitro and in vivo depend on permissive comitogenic effects of insulin-like growth factors (IGFs), which are mimicked in vitro by the low-affinity binding of high supraphysiological concentrations of insulin to IGF-I receptors. Contrary to general assumption, we show here that very low concentrations of insulin, acting through insulin receptors but not IGF-I receptors, can also support the stimulation of DNA synthesis by TSH in primary cultures of normal human thyrocytes. Moreover, TSH through cAMP increases the content of insulin receptors demonstrated by Western blotting and the cells' responsiveness to low insulin concentrations. These observations provide the first in vitro evidence in normal human thyroid cells of a functional interaction between TSH and insulin acting through its own receptor.     

Phosphatidylinositol 3-kinase-binding protein, PI3KAP/XB130, is required for cAMP-induced amplification of IGF mitogenic activity in FRTL-5 thyroid cells

We previously demonstrated that long-term pretreatment of rat FRTL-5 thyroid cells with TSH or cAMP-generating reagents potentiated IGF-I-dependent DNA synthesis. Under these conditions, cAMP treatment increased tyrosine phosphorylation of a 125-kDa protein (p125) and its association with a p85 regulatory subunit of phosphatidylinositol 3-kinase (p85 PI3K), which were suggested to mediate potentiation of DNA synthesis. This study was undertaken to identify p125 and to elucidate its roles in potentiation of DNA synthesis induced by IGF-I. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis revealed p125 to be a rat ortholog of human XB130, which we named PI3K-associated protein (PI3KAP). cAMP treatment elevated PI3KAP/XB130 mRNA and protein levels as well as tyrosine phosphorylation and interaction with p85 PI3K leading to increased PI3K activities associated with PI3KAP/XB130, supporting the role of PI3KAP/XB130 in DNA synthesis potentiation. Importantly, PI3KAP/XB130 knockdown attenuated cAMP-dependent potentiation of IGF-I-induced DNA synthesis. Furthermore, c-Src was associated with PI3KAP/XB130 and was activated in response to cAMP. Addition of Src family kinase inhibitors, PP1 or PP2, during cAMP treatment abolished tyrosine phosphorylation of PI3KAP/XB130 and its interaction with p85 PI3K. Finally, introduction of PI3KAP/XB130 into NIH3T3 fibroblasts lacking endogenous PI3KAP/XB130 enhanced IGF-I-induced DNA synthesis; however, a mutant Y72F incapable of binding to p85 PI3K did not show this response. Together, these data indicate that cAMP-dependent induction of PI3KAP/XB130, which is associated with PI3K, is required for enhancement of IGF mitogenic activities.     

Induction of DNA synthesis in dog thyrocytes in primary culture: synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin

We have investigated the growth effects of thyrotropin (TSH) (mimicked by forskolin and acting through cyclic AMP), epidermal growth factor (EGF), serum (10%) and insulin on quiescent dog thyroid epithelial cells in primary culture in a serum-free defined medium. These cells were previously shown to retain the capacity to express major thyroid differentiation markers. In the presence of insulin and after a similar prereplicative phase of 18 +/- 2h, TSH, EGF, and serum promoted DNA synthesis in such quiescent cells only a minority of which had proliferated in vitro before stimulation. The combination of these factors induced more than 90% of the cells to enter S phase within 48 h and near exponetial proliferation. Analysis of the cell cycle parameters of the stimulated cells revealed that the G1 period duration was similar to the length of the prereplicative phase of quiescent thyroid cells; this might indicate that they were in fact in an early G1 stage rather than in G0 prior to stimulation. TSH and EGF action depended on or was potentiated by insulin. Strikingly, nanomolar concentrations of insulin were sufficient to support stimulation of DNA synthesis by TSH, while micromolar concentrations of insulin were required for the action of EGF. This suggests that insulin supported the action of TSH by acting on its own high affinity receptors, whereas its effect on EGF action would be related to its somatomedinlike effects at high supraphysiological concentrations. Insulin stimulated the progression in the prereplicative phase initiated by TSH or forskolin. In addition, in some primary cultures TSH must act together with insulin to stimulate early events of the prereplicative phase. In the presence of insulin, EGF, and forskolin, an adenylate cyclase activator, markedly synergized to induce DNA synthesis. Addition of forskolin 24 h after EGF or EGF 24 h after forskolin also resulted in amplification of the growth response but with a lag equal to the prereplicative period observed with the single compound. This indicates that events induced by the second factor can no longer be integrated during the prereplicative phase set by the first factor. These findings demonstrate the importance of synergistic cooperation between hormones and growth factors for the induction of DNA synthesis in epithelial thyroid cells and support the proposal that essentially different mitogenic pathways--cyclic AMP-dependent or independent--may coexist in one cell.     

Induction of refractoriness to thyrotropin stimulation in cultured thyroid cells. Dependence on new protein synthesis.

Cultured dog thyroid cells were used to investigate the mechanism by which previous exposure to thyrotropin (TSH) induces refractoriness to further TSH stimulation of cellular adenosine 3'-5'-monophosphate (cAMP). Refractoriness of the cAMP response to TSH could not be overcome by exposure of the cells to supramaximal stimulatory concentrations of TSH. Although an unknown factor present in human and fetal calf serum was found to inhibit the thyroid cell cAMP response to TSH, this factor could not account for refractoriness because refractoriness could be induced in the absence of serum. Induction of thyroid refractoriness did not appear to be related to cellular concentrations of cyclic AMP, because equal refractoriness was produced by TSH alone or TSH plus the phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine. In addition, preincubation of thyroid cells in 10(-4) M cAMP did not result in subsequent refractoriness. Recovery from the refractory process required almost 24 h. Short term (15 min) stimulation with TSH did not produce thyroid cell refractoriness, and reversal of the stimulation was obtained by thorough washing of the cells. Long term TSH stimulation (16 h), however, resulted in both supramaximal cAMP response to TSH, and inclusion of TSH together with cycloheximide did not produce refractoriness. Cyclic AMP phosphodiesterase activity in thyroid cell homogenate was unaltered by TSH or dibutyryl cyclic AMP pretreatment of the cells for up to 24 h, or cycloheximide for up to 4 h. In contrast, TSH-stimulated, but not F--stimulated, adenylate cyclase activity was reduced in thyroid cell homogenates after preincubation of the cells in TSH. Refractoriness to TSH stimulation was not associated with an alteration in the binding of 125I-TSH to cultured thyroid cells. These studies suggest that the thyroid cAMP response to TSH is modulated by an inhibitory mechanism dependent upon new protein synthesis. TSH stimulation itself increases the degree of this inhibition through a mechanism not involving cAMP.     

Thyrotropin and insulin-like growth factor I regulation of tyrosine phosphorylation in FRTL-5 cells. Interaction between cAMP-dependent and growth factor-dependent signal transduction.

Pretreatment of rat FRTL-5 thyroid cells with thyrotropin (TSH) markedly potentiated the mitogenic response to insulin-like growth factor I (IGF-I) (Tramontano, D., Moses, A. C., Veneziani, B. M., and Ingbar, S. H. (1988) Endocrinology 122, 127-132; Takahashi, S.-I., Conti, M., and Van Wyk, J. J. (1990) Endocrinology 126, 736-745). The present study was undertaken to determine whether this synergism between TSH and IGF-I in FRTL-5 cells was correlated with changes in tyrosine phosphorylation of intracellular proteins. Tyrosine phosphorylation in intact cells was determined by gel electrophoresis and immunoblotting using monospecific anti-phosphotyrosine antibodies. Cells were preincubated for up to 24 h with TSH, dibutyryl cAMP, forskolin, or cholera toxin and then incubated for an additional 1 min in the absence or presence of IGF-I. As reported by others, IGF-I rapidly increased tyrosine phosphorylation of a 175-kDa protein as well as a less intense band of 90-100 kDa. Pretreatment for 6-12 h with either TSH or other agents that elevate intracellular cAMP potentiated the IGF-I-dependent tyrosine phosphorylation of the 175-kDa substrate by 3-5-fold. Since TSH did not increase IGF receptor number of kinase activity, the effect of TSH is assumed to be exerted at a step distal to IGF receptor tyrosine kinase. Surprisingly, IGF-I-independent tyrosine phosphorylation was also increased by pretreatment with TSH. When intact cells were analyzed TSH produced a time- and concentration-dependent increase in tyrosine phosphorylation of a prominent 120-125-kDa substrate and less prominent 100- and 80-kDa substrates. Assays using Triton X-100-soluble extracts incubated with MgCl2, ATP, and orthovanadate demonstrated that TSH pretreatment increased tyrosine phosphorylation over that observed in untreated cells. In this cell-free assay, TSH pretreatment enhanced the phosphorylation of multiple substrates. These studies suggest that a cAMP stimulus that initiates a trophic effect can be propagated indirectly through multiple pathways including enhancement of tyrosine phosphorylation.     

Endothelin-1 stimulates c-fos mRNA expression and acts as a modulator on cell proliferation of rat FRTL5 thyroid cells.

In FRTL5 thyroid cells, endothelin (ET)-1 alone had no effect on DNA synthesis but caused a transient increase in c-fos mRNA levels and stimulated IGF-I induced DNA synthesis and cell proliferation. By contrast, ET-1 inhibited the stimulatory effects of TSH actions on DNA synthesis, cell proliferation and c-AMP production. 8-Bromo-cAMP-induced DNA synthesis was also inhibited by ET-1, suggesting that ET-1 exerts its inhibitory effects at step(s) involving cAMP production and post cAMP pathway. ET-1-induced suppression of TSH actions were reversed by a C-kinase inhibitor, H-7. These results suggest that the effect of ET on functions of FRTL5 cells is, at least, in part mediated by C-kinase dependent pathway.     

Thyrotropin-induced mitogenesis is Ras dependent but appears to bypass the Raf-dependent cytoplasmic kinase cascade.

Cellular growth control requires the coordination and integration of multiple signaling pathways which are likely to be activated concomitantly. Mitogenic signaling initiated by thyrotropin (TSH) in thyroid cells seems to require two distinct signaling pathways, a cyclic AMP (cAMP)-dependent signaling pathway and a Ras-dependent pathway. This is a paradox, since activated cAMP-dependent protein kinase disrupts Ras-dependent signaling induced by growth factors such as epidermal growth factor and platelet-derived growth factor. This inhibition may occur by preventing Raf-1 protein kinase from binding to Ras, an event thought to be necessary for the activation of Raf-1 and the subsequent activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) and MAP kinase (MAPK)/ERKs. Here we report that serum-stimulated hyperphosphorylation of Raf-1 was inhibited by TSH treatment of Wistar rat thyroid cells, indicating that in this cell line, as in other cell types, increases in intracellular cAMP levels inhibit activation of downstream kinases targeted by Ras. Ras-stimulated expression of genes containing AP-1 promoter elements was similarly inhibited by TSH. On the other hand, stimulation of thyroid cells with TSH resulted in stimulation of DNA synthesis which was Ras dependent but both Raf-1 and MEK independent. We also show that Ras-stimulated DNA synthesis required the use of this kinase cascade in untreated quiescent cells but not in TSH-treated cells. These data suggest that in TSH-treated thyroid cells, Ras might be able to signal through effectors other than the well-studied cytoplasmic kinase cascade.     

Thyrotropin and dibutyryl cyclic AMP increase levels of c-myc and c-fos mRNAs in cultured rat thyroid cells

We have studied the effects of thyrotropin (TSH) on the growth and on the levels of the mRNAs of the cellular proto-oncogenes, c-myc, and c-fos, in the specific target of TSH action, the thyroid follicular cell. FRTL5 cells, a cloned line from normal rat thyroid gland that depends upon TSH for its replication, were maintained in a quiescent state for 5 days by keeping them in a medium devoid of serum or TSH. The addition of bovine TSH (bTSH, 1 nM) increased DNA synthesis and stimulated cell proliferation after a lag period of 24 h. This growth response was anteceded by prompt, but transient, increases in the levels of c-myc and c-fos mRNAs, with peak responses at 60 and 30 min, respectively. The minimally and maximally effective concentrations of bTSH were 0.01 mM and 1.0 nM, respectively. Dibutyryl cAMP (Bt2cAMP) stimulated cell growth and increased the level of c-myc mRNA in a concentration-dependent manner, with maximum effects at a Bt2cAMP concentration of 1 mM. At the single concentration tested (1 mM), Bt2cAMP also increased the level of c-fos mRNA. Hence, bTSH-stimulated mitogenesis in quiescent FRTL5 cells is associated with rapid, but short-lived, increases in the levels of the mRNAs of the proto-oncogenes, c-myc and c-fos. Since bTSH is known to stimulate adenylate cyclase in these cells, and since the effect of TSH on c-myc and c-fos mRNAs is mimicked by Bt2cAMP, it is possible that these responses to bTSH are mediated, at least in part, by cAMP.