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Molecular Endocrinology 18 (9): 2321-2332
Copyright © 2004 by The Endocrine Society

Thyrotropin and Serum Regulate Thyroid Cell Proliferation through Differential Effects on p27 Expression and Localization

Aurélia E. Lewis, Aphrothiti J. Fikaris, Gregory V. Prendergast and Judy L. Meinkoth

Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Judy L. Meinkoth, Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104-6084. E-mail: Meinkoth{at}pharm.med.upenn.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyroid cell proliferation is regulated by the concerted action of TSH/cAMP and serum growth factors. The specific contributions of cAMP-dependent vs. -independent signals to cell cycle progression are not well understood. We examined the molecular basis for the synergistic effects of TSH and serum on G1/S phase cell cycle progression in rat thyroid cells. Although strictly required for thyroid cell proliferation, TSH failed to stimulate G1 phase cell cycle progression. Together with serum, TSH increased the number of cycling cells. TSH enhanced the effects of serum on retinoblastoma protein hyperphosphorylation, cyclin-dependent kinase 2 activity, and cyclin A expression. Most notably, TSH and serum elicited strikingly different effects on p27 localization. TSH stimulated the nuclear accumulation of p27, whereas serum induced its nuclear export. Unexpectedly, TSH enhanced the depletion of nuclear p27 in serum-treated cells. Furthermore, only combined treatment with TSH and serum led to rapamycin-sensitive p27 turnover. Together, TSH and serum stimulated p70S6K activity that remained high through S phase. These data suggest that TSH regulates cell cycle progression, in part, by increasing the number of cycling cells through p70S6K-mediated effects on the localization of p27.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE EFFECTS OF TSH on thyroid cell proliferation and function are mediated primarily through cAMP. Although TSH dependent, the proliferation of thyroid cells requires collaboration between TSH/cAMP and serum growth factors (1). The specific contributions of cAMP-dependent and -independent signals to cell cycle transit are not clear. This is important to understand given the critical role of abnormal thyroid proliferation in human disease, including goiter and thyroid cancer. Up to now, the most complete model of thyroid cell cycle progression has been generated from studies performed in primary canine thyroid cells. In these cells, insulin stimulates cyclin D3 expression (2), whereas TSH assembles and localizes cyclin D3/cyclin-dependent kinase (CDK)-4 complexes to the nucleus (2, 3). This results in the redistribution of p27 from cyclin E/CDK-2 to cyclin D3/CDK-4 complexes (4), presumably allowing CDK-2 activation, although direct measurements of CDK-2 activity in these cells have not been reported. Far less is known regarding G1/S phase cell cycle progression in rat thyroid cells. Moreover, there is considerable controversy regarding the effects of TSH in these cells. TSH has been reported to stimulate cell cycle progression in the absence of insulin in FRTL-5 cells (5). These authors reported that TSH decreased p27 expression (5, 6), an effect opposite to that seen in canine cells (7). However, Saito et al. (8) reported that TSH fails to decrease p27 expression in FRTL-5 cells. Although several reports have shown that TSH-containing growth medium decreases p27 expression in FRTL-5 cells (8, 9, 10), it is not clear that this is due to TSH.

One confounding factor in the reported studies concerns the protocol used to examine cell cycle progression. Canine thyroid cells are starved in the presence of insulin and then stimulated with TSH. When added to cells starved in the absence of insulin, insulin stimulates D cyclin expression and a modest assembly of cyclin D3/CDK-4 complexes (2). Therefore, starvation in the presence of insulin could mask its contribution to the acute effects of TSH. Many studies in rat thyroid cells employ a 24-h pretreatment with TSH, followed by exposure to insulin/IGF-I, potentially masking acute effects of TSH (11, 12, 13). When added to quiescent cells, TSH/cAMP elicits acute effects on signaling pathways that impinge upon protein kinase A, phosphatidylinositol 3kinase (PI3K), p70S6K, Ras, and Rap 1. Interference with these pathways impairs TSH-stimulated DNA synthesis (14, 15, 16, 17, 18).

We set out to examine the acute contributions of TSH and serum growth factors to cell cycle progression. Our studies were performed in Wistar rat thyroid (WRT) (19) and PC-Cl3 (20) cells, continuous lines of rat thyroid follicular cells that retain markers of thyroid differentiation including TSH-dependent proliferation and function (reviewed in Ref. 18). When added to cells starved in the absence of all growth factors, TSH failed to stimulate G1 phase cell cycle progression and cell proliferation, whereas serum was only a weak mitogen. Together, TSH and serum conferred rapid and sustained proliferation. Intriguingly, these studies revealed that cAMP-dependent and -independent signals converge at the level of CDK-2 activity through surprisingly different effects on the localization of p27.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Synergistic Effects of TSH and Serum on Thyroid Cell Proliferation
TSH acts synergistically with serum growth factors to regulate thyroid cell proliferation (reviewed in Refs. 18 and 21), although there are reports that TSH alone stimulates proliferation in rat thyroid FRTL-5 cells (5, 6). To identify the respective contributions of TSH and serum growth factors to thyroid cell proliferation, the effects of TSH, insulin, and calf serum, alone and in combination, on cell proliferation were investigated. As depicted in Fig. 1AGo, growth curve experiments revealed that treatment with TSH, insulin, or calf serum alone did not increase cell number (see legend), whereas TSH/insulin stimulated a very modest increase in cell number over 4 d. Together, TSH/calf serum (T/CS) reproduced the robust increase in cell number seen with fully supplemented thyroid cell growth medium or 3H (see Materials and Methods). Similar to the effects of TSH/CS, forskolin (f) and calf serum (f/CS) synergistically increased cell number. Similar results were obtained in PC-Cl3 cells (22), demonstrating that the synergistic effects of TSH/CS on cell proliferation are not unique to WRT cells (Fig. 1BGo).



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  		Fig. 1. Synergistic Effects of cAMP/CS on Cell Proliferation

Quiescent (A) WRT or (B) PC-Cl3 cells were stimulated with TSH (T) (solid circles), forskolin (f) (open squares), calf serum (CS) (solid triangles), insulin (ins) (broken line with x), TSH/ins (open inverted triangles), TSH/CS (open circles), f/CS (asterisks), or 3H (solid square) for 4 d and cell number was determined. Ni (cell number at day N) over No (cell number in unstimulated cells at day N) (mean ± SD) is shown. The values for treatment with single agents in WRT cells at d 4 were as follows: TSH, 1.6 ± 0.3; f, 2.2 ± 0.1; CS, 2.4 ± 0.4; ins, 2.2 ± 0.7. The values for combined treatments in WRT cells were as follows: TSH/ins, 3.8 ± 0.7; TSH/CS, 13 ± 1.7; f/CS, 16.5 ± 1.9; and 3H, 17.3 ± 2.0. For PC-Cl3 cells the values at d 4 were: TSH, 1.2 ± 0.1; f, 1.1 ± 0.1; CS, 1.5 ± 0.1; ins, 1.2 ± 0.3; TSH/ins, 1.4 ± 0.2; TSH/CS, 6.7 ± 0.9; f/CS, 8.3 ± 0.3; and 3H, 10.3 ± 0.8. C, Quiescent cells (basal, b) were stimulated with TSH (T), f, CS, f/CS, T/CS, f/CS, or 3H for 30 h, with BrdU added for 28–30 h. Data shown are mean ± SEM of two to six experiments per condition.

 
To determine that the increase in cell number stimulated by TSH/CS reflected an increase in the number of cycling cells, effects on cell cycle progression were analyzed. Time course experiments using 3H (data not shown) revealed that the maximal decrease in G1 phase cells and maximal increase in S phase cells were observed at 24 h after treatment. TSH/CS or forskolin/CS stimulated cell cycle progression with kinetics similar to 3H (Table 1Go). Calf serum alone stimulated cell cycle transit, but its effects were more modest than those of 3H. Alone, TSH failed to stimulate G1 phase cell cycle progression even over prolonged times (Table 2Go). Similar results were observed with forskolin (data not shown).


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  		Table 1. Effects of TSH, Forskolin, and CS on Cell Cycle Progression

 

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  		Table 2. TSH Does Not Stimulate G1 Phase Cell Cycle Progression

 
DNA synthesis experiments confirmed that TSH and forskolin failed to stimulate entry into S phase when added alone (Fig. 1CGo). Calf serum (CS) stimulated DNA synthesis, but to a lesser extent than 3H. Similar to their effects on cell number, TSH/CS and forskolin/CS reproduced the effects of 3H growth medium on DNA synthesis. Together, these findings demonstrate that cAMP and CS synergistically stimulate thyroid cell proliferation.

Characterization of G1 Phase Cell Cycle Progression in Response to 3H
To elucidate the molecular basis for the synergistic effects of TSH and CS on cell proliferation, we first characterized the regulation of G1 phase cell cycle progression by 3H. When added to quiescent cells, 3H stimulated cyclin A expression beginning at 15 h and maximally at 24 h (Fig. 2Go, row 1). The induction of cyclin A expression was preceded by increased expression of cyclin E (row 2). Expression of all three D cyclins was low in quiescent cells. 3H increased cyclin D1 (row 3) and D3 (row 4) expression, but had no effect on the expression of cyclin D2 (row 5). CDK-4, the catalytic partner of D cyclins, was detected in quiescent cells, and its expression was increased slightly by 3H (row 6). In contrast, CDK-2 expression was markedly stimulated by 3H (row 7).



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  		Fig. 2. Effects of 3H on the Expression of Cell Cycle-Regulatory Proteins

Quiescent WRT cells (b) were stimulated with 3H for the indicated times (hours), and the expression of cell cycle-regulatory proteins was assessed by Western immunoblotting. Equivalent protein loading and transfer were confirmed with ß-actin. At least three time course experiments were performed with similar results for each of the proteins analyzed.

 
The CDK inhibitors, p21 and p27, act as assembly factors and inhibitors of cyclin/CDK complexes (23, 24, 25). Mitogens typically increase p21 (26) and decrease p27 expression during G1 phase (27). Expression of p21 was undetectable in quiescent WRT cells, whereas p27 was abundantly expressed (rows 8 and 9). 3H modestly increased p21 expression (row 8) and decreased p27 levels (row 9). Decreased p27 expression coincided with the appearance of the faster migrating, activated form of CDK-2 (row 7) (28), suggesting that p27 is turned over by active CDK-2 in these cells as in other cells (29, 30, 31).

Effects of TSH and Serum on G1 Phase Cell Cycle Progression
To corroborate the negative effects of TSH on cell cycle progression (Table 2Go), its effects on important regulators of G1/S phase cell cycle progression were analyzed. Inactivation of the retinoblastoma protein (Rb) through hyperphosphorylation in late G1 is required for entry into S phase. Rb hyperphosphorylation enables the derepression of E2F (32), leading to E2F-dependent transcription of S phase genes such as cyclin A (33, 34). Figure 3AGo (row 1) demonstrates that TSH failed to stimulate Rb phosphorylation at sites phosphorylated by CDK-4 and CDK-2 (35) for up to 72 h. TSH also failed to stimulate Rb hyperphosphorylation (data not shown). In agreement with these findings, TSH did not increase cyclin A expression (Fig. 3AGo, row 3). These results differ from those reported in FRTL-5 cells in which TSH stimulated Rb hyperphosphorylation (6).



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  		Fig. 3. Effects of TSH and CS on Cyclin A Expression, Rb Phosphorylation, and CDK-2 Activity

Quiescent WRT cells (b) were stimulated for (A) 24–72 h with TSH (T) or (B) 15–30 h with serum (CS) or TSH/serum (T/CS). 3H stimulation for 20 h was used as a positive control in panel A. Rb phosphorylation on serines 807/811 (Rb-P) was assessed in the middle panel of panels A and B. The phosphorylation status of total Rb was determined using an antibody to Rb (Rb) (B, upper panel). Three independent experiments with similar results were performed using the Rb antibody and a single experiment using Rb-P. In panel B, the samples for CS vs. T/CS were analyzed on the same gels and exposed for the same times. The same blots were probed for ß-actin. C, Quiescent cells (b) were stimulated with TSH, CS, TSH/CS, or 3H, and CDK-2 activity was assessed. The autoradiograph shown was overexposed to illustrate the modest stimulation of CDK-2 activity by CS. Two experiments were performed with similar results for TSH, and three experiments were performed for all other treatments.

 
Calf serum alone stimulated Rb hyperphosphorylation (upshifted Rb species), but with delayed kinetics compared with TSH/CS (Fig. 3BGo, top) or 3H (data not shown). This was particularly evident at 20 h post treatment where more hyperphosphorylated Rb protein was observed in TSH/CS cells compared with cells treated with CS alone. TSH/CS stimulated Rb phosphorylation at the CDK-4 and CDK-2 sites, whereas the effects of CS alone on these sites were very modest (Fig. 3BGo, middle). Similar to Rb hyperphosphorylation, TSH/CS elicited synergistic effects on cyclin A expression (Fig. 3BGo, bottom).

To determine the mechanism through which TSH/CS collaboratively regulate Rb phosphorylation, their effects on CDK-2 activity were analyzed. Preliminary studies revealed that 3H stimulated CDK-2 activity by 15 h post treatment. TSH failed to stimulate CDK-2 activity (Fig. 3CGo). Calf serum stimulated CDK-2 activity, but its effects were far more modest than those of 3H. Together, TSH/CS elicited synergistic effects on CDK-2 activity. Therefore, the effects of TSH/CS on Rb phosphorylation can be explained, at least in part, by their effects on CDK-2 activity.

Regulation of p27 by TSH and Serum
CDK-2 activity is regulated in a complex manner that includes assembly with cyclins, phosphorylation, and complexation with CDK inhibitors. At high stoichiometry, binding of p27 to CDK-2 inhibits its activity (23, 36, 37). To elucidate the mechanistic basis for the synergistic effects of TSH/CS on CDK-2 activity, their effects on the expression and localization of p27 were investigated. TSH failed to decrease p27 expression (Fig. 4AGo, T). Calf serum alone did not decrease p27 expression (Fig. 4BGo, CS). Surprisingly, together TSH/CS decreased p27 expression with kinetics similar to those of 3H. Decreased expression was first detected at 24 h and continued over the next 6 h. The decrease in p27 was CDK 2 dependent (31, 38, 39) because treatment with the CDK-2 inhibitor roscovitine reversed its down-regulation (Fig. 4Go, C and D). Whereas decreased p27 expression could result in increased CDK-2 activity, TSH/CS stimulated CDK-2 activity at times (15 h) that preceded their effects on p27 expression (24 h). Therefore, the synergistic effects of TSH/CS on CDK-2 activity are not likely to be due to decreased p27 expression.



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  		Fig. 4. TSH and CS Synergistically Decrease p27 Expression

Quiescent WRT cells (b) were stimulated for (A) 24–72 h with TSH (T) or (B) 15–30 h with serum (CS), TSH/CS (T/CS), or 3H, and cell lysates subjected to Western blotting for p27 expression. In panel B, the blots for CS, T/CS, and 3H were exposed for the same times. Equivalent protein loading was confirmed using ß-actin. Three time course experiments were performed for each treatment. C, Quiescent WRT cells (b) were pretreated with roscovitine (30 µM) for 60 min before stimulation with CS, T/CS, or 3H for 30 h. D, Roscovitine did not affect basal levels of p27 expression in starved cells. Three experiments were performed with the same results.

 
p27 shuttles between the nucleus and cytoplasm (38, 39). Similar to a report in human thyroid cells (40), p27 is nuclear in quiescent rat thyroid cells (Fig. 5AGo, basal or b). Stimulation with 3H resulted in the rapid loss of nuclear p27. Nuclear p27 staining was reduced within 6 h and further decreased at 15 h and 24 h after treatment (Fig. 5AGo). Unlike 3H, TSH did not decrease nuclear p27 staining (Fig. 5BGo, T). Compared with starved cells, TSH increased the proportion of cells with nuclear p27 (Fig. 5Go, A and B). Calf serum (CS) decreased nuclear p27, but its effects were less robust than those of 3H. This was particularly evident at 15 h, when the majority of 3H-treated cells were devoid of nuclear p27 (Fig. 5AGo), whereas a significant proportion of CS-treated cells retained p27 in the nucleus (Fig. 5BGo). Strikingly, together TSH/CS decreased nuclear p27 levels with similar kinetics (data not shown) and to a similar extent as 3H (Fig. 5Go, A and B). Similar results were observed for forskolin/CS (data not shown). Therefore, although TSH fails to decrease nuclear p27 on its own, it enhances the ability of CS to decrease nuclear p27 expression.



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  		Fig. 5. Effects of TSH and CS on p27 Localization

A, Quiescent WRT cells (b) were stimulated with 3H for 6–24 h and subjected to immunostaining for p27. Top row shows p27, and bottom row shows DAPI staining of the same fields. B, p27 localization in quiescent cells stimulated with TSH (T), serum (CS), or TSH/serum (T/CS) for 15 h in the same experiment as panel A. DAPI staining of the same fields is shown (left panels). At least four random fields of cells (80–100 cells) were analyzed in each experiment, and four experiments were performed with similar results.

 
To determine whether the decrease in nuclear p27 was required for cell cycle progression, we examined whether cells with nuclear p27 entered S phase (Fig. 6Go). The majority of quiescent and forskolin-, and TSH-treated cells exhibited nuclear p27, and very few of these cells entered S phase (Table 3Go and Fig. 6Go). More serum-treated cells were bromodeoxyuridine (BrdU) positive, and these cells lacked nuclear p27. Very few TSH/CS- or forskolin/CS-treated cells exhibited nuclear p27 expression, whereas correspondingly more of these cells were BrdU positive. Irrespective of treatment, more than 95% of BrdU-positive cells lacked detectable nuclear p27 (Table 3Go). These results suggest that TSH/CS elicits synergistic effects on G1 phase cell cycle progression by increasing the number of cycling cells through effects on p27 localization.



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  		Fig. 6. Nuclear p27 Is Absent in Cells Undergoing DNA Synthesis

Quiescent WRT cells (b) were stimulated with TSH (T), serum (CS), or TSH/serum (T/CS) for 18 h, and BrdU was added for the last 2 h. Cells were stained for DAPI, BrdU, and p27. Cells outlined in white boxes depict BrdU-positive cells that are p27 negative. Data represent one of three experiments performed with similar results.

 

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  		Table 3. DNA Synthesis Is Inversely Correlated with Nuclear p27

 
Regulation of p27 Localization
To examine whether TSH/CS stimulated nuclear export of p27, the effects of leptomycin B, an inhibitor of nuclear export, were assessed. Leptomycin B prevented the loss of nuclear p27 in cells treated with CS, TSH/CS, or 3H (Fig. 7Go). These results demonstrate that CS, TSH/CS, and 3H stimulate nuclear export of p27. Interestingly, export of p27 (seen within 6–15 h; Fig. 5Go, A and B) preceded the decrease in p27 expression observed on Western blots (24–30 h; Fig. 4BGo). Decreased p27 expression was temporally correlated with increased CDK-2 activity, as assessed by the downshift in CDK-2 migration (Fig. 2Go) and direct measurements of CDK-2 activity (Fig. 3CGo). These data, together with the finding that roscovitine restored nuclear p27 (Fig. 4CGo), suggest that CDK-2 activity mediates the decrease in p27 expression, possibly through targeting p27 for proteasomal degradation (31, 38, 39). The earlier kinetics of p27 export vs. turnover might indicate that p27 is exported to the cytoplasm where it is degraded.



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  		Fig. 7. Serum, TSH/Serum, and 3H Stimulate Nuclear Export of p27

Quiescent WRT cells were pretreated for 60 min without (–) or with (+) leptomycin B (5 ng/ml) and then stimulated with serum (CS), TSH/serum (TSH/CS), or 3H for 24 h, and subjected to immunostaining for p27. The same fields of cells stained with DAPI (left panels) vs. p27 (right panels) are shown. The % cells with nuclear p27 in the absence vs. presence of leptomycin were: CS, 18% (385) vs. 61% (200); TSH/CS, 14% (354) vs. 46% (261); and 3H, 8% (359) vs. 41% (394). The number of cells scored for each treatment is shown in parentheses. Three independent experiments were performed for each treatment with similar results.

 
To examine how TSH and CS together deplete nuclear p27, we examined the effects of inhibition of PI3K and p70S6K activity, because TSH stimulates their activities (14, 15). Treatment with LY294002 (LY), an inhibitor of PI3K activity, or rapamycin, an inhibitor of mTOR, restored nuclear p27 levels (Fig. 8Go, B and C). Interestingly, they did so in very different ways. LY increased p27 expression regardless of the stimulus (Fig. 8AGo). Similarly, LY increased nuclear p27 in both CS- and TSH/CS-treated cells (Fig. 8BGo). In contrast, rapamycin selectively reversed the effects of TSH in cells treated with TSH/CS (Fig. 8Go, A and C). Rapamycin restored nuclear p27, but only to the level seen in cells treated with CS alone. These results suggest that the ability of TSH to enhance the nuclear depletion of p27 relies on p70S6K activity.



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  		Fig. 8. p27 Expression and Localization Are Differentially Affected by Rapamycin (Rap) and LY

A, Quiescent WRT cells pretreated for 60 min without (–) or with (+) LY (15 µM) or Rap (1 nM) were stimulated for 30 h with serum (CS), TSH (T), or TSH/serum (T/CS), and p27 expression was analyzed by Western blotting. All samples were analyzed on the same gel. The same membranes were probed with ß-actin. Three experiments were performed with similar results. B and C, Cells pretreated as in panel A were stimulated with serum (CS) and TSH/serum (T/CS) for 24 h in panel B or 16 h in panel C and subjected to p27 immunostaining. The same fields of cells stained with DAPI are shown. The percent of cells with nuclear p27 in the absence vs. presence of LY were: CS, 23% (488) vs. 69% (417); and TSH/CS, 13% (300) vs. 60% (387). The percent cells with nuclear p27 in the absence vs. presence of rapamycin were: CS, 18% (228) vs. 22% (325); and TSH/CS, 6% (163) vs. 23% (362). The number of cells scored is shown in parentheses. Data shown represent one of three similar experiments.

 
To further investigate the roles of PI3K and p70S6K in the regulation of p27, the effects of TSH and CS on Akt and p70S6k activity were assessed. Calf serum stimulated a transient increase in Akt activity as monitored by phosphorylation at serine 473 (Fig. 9AGo, CS). Akt activity returned to basal levels within 18 h of CS treatment. Together with TSH, CS stimulated higher levels of Akt phosphorylation, and they persisted for longer times. Similarly, together TSH and CS stimulated a sustained increase in p70S6k activity, compared with treatment with CS alone (Fig. 9BGo). Importantly, under these conditions, Akt and p70S6k activity remained high over the times required to deplete nuclear p27 (15 h).



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  		Fig. 9. TSH/CS Elicit Synergistic Effects on Akt and p70S6k Activity

Quiescent WRT cells (b) were stimulated with serum (CS) or TSH/serum (T/CS) for the times indicated and Akt and p70S6k activity was assessed by Western blotting with (A) Ser473 phospho-specific Akt (Akt-P) and with (B) phospho-S6 antibody (S6-P), a substrate of p70S6k. Four independent experiments were performed for Akt-P and five for S6-P. Blots in panels A and B were analyzed on the same gels and exposed for the same times. ß-Actin was used as a loading control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We set out to determine the molecular basis for the synergistic effects of TSH/cAMP and serum growth factors on thyroid cell proliferation. Our studies focused on TSH and CS as only combined treatment with these factors reproduced the effects of thyroid cell growth medium (3H) on cell proliferation. When used alone, TSH failed to stimulate cell cycle progression. Although CS alone stimulated cell cycle progression in some cells, a significant proportion of CS-stimulated cells failed to enter the cell cycle even over a prolonged time course (data not shown). Therefore, whereas TSH is required for cell proliferation, the mitogenic activity of TSH is strictly dependent on the presence of serum growth factors in rat thyroid cells as in canine cells (21).

To elucidate the molecular basis for their synergistic effects, the effects of TSH and CS, individually and in combination, on cell cycle regulation were investigated. Compared with 3H, CS elicited modest and delayed effects on cyclin A expression, Rb hyperphosphorylation, and CDK-2 activity. TSH enhanced the effects of serum on each of these markers of G1/S phase transit, restoring regulation similar to that seen with 3H. Unexpectedly, 3H, TSH, and CS exhibited striking differences in their effects on p27 expression and localization. 3H stimulated only a modest decrease in p27 levels during G1, indicating that cell cycle progression through G1 proceeds in the presence of p27 in thyroid cells. Individually, TSH and CS failed to decrease p27 expression. Nuclear p27 was high in quiescent cells and further increased by TSH. In contrast, 3H induced the rapid loss of p27 from the nucleus. Calf serum also depleted nuclear p27; however, its effects were less robust than those of 3H. Whereas 3H reduced nuclear p27 in most cells, a substantially higher proportion of CS-treated cells retained nuclear p27. Remarkably, when added together with CS, TSH enhanced the depletion of nuclear p27, reproducing the effects of 3H. An inverse correlation between nuclear p27 and DNA synthesis was revealed. 3H and TSH/CS decreased nuclear p27 and increased the number of S phase cells. TSH maintained nuclear p27 and failed to stimulate DNA synthesis. Colabeling experiments revealed that, irrespective of the stimulus, BrdU-positive cells lacked nuclear p27. These findings strongly suggest that p27 must exit the nucleus in order for thyroid cells to enter S phase.

Our data support the following model for G1/S phase cell cycle regulation in rat thyroid cells. Acting in concert, TSH and CS deplete nuclear stores of p27, allowing activation of nuclear CDK-2 and entry into S phase. Intriguingly, CS and TSH regulate p27 in very different ways. Calf serum stimulates the nuclear export of p27, evidenced by the finding that leptomycin B prevented nuclear export of p27 in CS-treated cells. Calf serum-stimulated cells devoid of nuclear p27 enter S phase and are likely capable of continued cell cycle progression. As they represent only a fraction of the population, this accounts for the modest effects of CS on cyclin A expression, cell cycle progression, and cell number compared with 3H. TSH increased the number of cycling cells by increasing the number of CS-stimulated cells devoid of nuclear p27. Because on its own TSH does not stimulate p27 export, we infer that TSH prevents the reentry of CS-exported p27 into the nucleus (see below), possibly through the enhanced turnover of p27. In support of this idea, p27 expression declined only after combined treatment with TSH and CS (or 3H). Moreover, export of p27 from the nucleus temporally preceded its degradation. The early modest decrease in p27 expression in 3H- or TSH/CS-treated cells coincided with increased cyclin E expression, and the later more robust decline of p27 coincided with increased cyclin A expression, suggesting that cyclin/CDK-2 complexes target p27 for proteasomal turnover (31, 38, 39). Indeed, treatment with the CDK-2 inhibitor roscovitine restored p27 levels in TSH/CS and 3H-treated cells.

p27 shuttles between the nucleus and cytoplasm (38, 39). Phosphorylation of p27 by MAPK, Akt, hKIS, and p90rsk has been reported to result in its cytoplasmic accumulation (27, 39, 41). Treatment with LY restored p27 expression and nuclear localization in CS- and TSH/CS-treated cells, suggesting that p27 expression and localization are under tonic control by PI3K-mediated signals. In contrast, rapamycin restored p27 expression and nuclear localization selectively in TSH/CS-treated cells. This suggests that p70S6K activity is required for the ability of TSH to localize p27 in the cytoplasm, thus facilitating its proteolysis. In support of this idea, individually TSH and CS stimulated transient p70S6k activity, whereas together they stimulated activation that was sustained throughout the G1 to S phase transition. Based on this, it is tempting to speculate that increased p70S6k activity during late G1 and S phase is required to limit access of p27 to the nucleus. A recent report demonstrated that p90rsk phosphorylates p27 and anchors it in the cytoplasm by promoting its association with 14–3-3 proteins (42). It is possible that p70S6k acts by a similar mechanism, thereby rendering p27 more susceptible to proteasomal turnover. A large number of thyroid tumors exhibit cytoplasmic p27 expression. In some cases, overexpression of D cyclins has been shown to sequester p27 in the cytoplasm (40), raising the possibility that p70S6k may also regulate D cyclin expression or assembly.

The inability of TSH to drive cell cycle progression was inconsistent with our previous data that TSH alone stimulates DNA synthesis (14). Our earlier studies were performed in cells arrested in basal medium containing 0.2% BSA. Inclusion of 0.2% BSA or 0.2% serum in basal medium restored the ability of TSH and forskolin to stimulate DNA synthesis (data not shown). However, even in the presence of 0.2% BSA or serum, TSH and forskolin failed to stimulate entry into S phase as monitored by fluorescence-activated cell sorting (FACS) analysis, Rb hyperphosphorylation, or cyclin A expression. The discordance between the effects of TSH/cAMP on BrdU incorporation and cell cycle progression (in the presence of BSA or CS) is not a consequence of the assay or cell line. Saito et al. (8) reported a similar discrepancy using [3H]thymidine incorporation in FRTL-5 cells, where TSH stimulated DNA synthesis, but not cell cycle progression.

Our findings provide an important new insight into the regulation of thyroid cell proliferation. These data unambiguously demonstrate that TSH fails to stimulate molecular markers of G1/S phase cell cycle progression, including stimulation of CDK-2 activity, Rb phosphorylation, and cyclin A expression in rat thyroid cells. These findings agree well with those reported in canine thyroid cells (2, 4, 43) but differ substantially from earlier reports in FRTL-5 cells (6). These data reaffirm the essential role played by serum growth factors in TSH-dependent proliferation (21) and indicate that TSH is not a true mitogen in either canine or rat thyroid cells. The inability of TSH to stimulate G1 phase cell cycle progression also provides an explanation for its unusual effects on p27. cAMP increases p27 expression in many cells, including canine thyroid cells (7). Typically, elevations in cAMP stimulate G1 arrest. Why would TSH, a factor required for proliferation, increase p27 expression? It is possible that p27 is required for the effects of TSH on differentiated function. Consistent with this idea, p27 expression is markedly reduced in Ras-transformed thyroid cells in which differentiated gene expression is abolished (44). Also, we observed increased p27 expression in WRT cells stably expressing activated Rap 1, cells in which differentiated gene expression is enhanced (45). cAMP stimulates p27 expression in most cell types, whereas its stimulatory effects on p70S6k may be restricted to cells in which cAMP stimulates proliferation (14). The unique ability of cAMP to stimulate p70S6k activity in these cells may provide a cell type-specific mechanism through which p27 expression can be down-regulated to promote cell cycle progression, and allow TSH to dually regulate proliferation and differentiated function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Insulin, transferrin, and Coon’s modified Ham’s F12 medium were from Sigma Chemical Co. (St. Louis, MO). Calf serum and protein A-agarose were from Life Technologies, Inc. (Gaithersburg, MD). Antibodies against cyclins D1 (sc-450), D2 (sc-593), D3 (sc-182), E (sc-481), and A (sc-596) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), as were antibodies to CDK-2 (sc-163 and sc-163G), CDK-4 (sc-260), CDK-6 (sc-177), p21 (sc-397), p27 (sc-527), and ß-actin (sc-1615). Rb antibody (clone MAb-1/Rb20B3) was from Zymed Laboratories, Inc. (South San Francisco, CA). Phospho-Akt (Ser473) antibody and phospho-Rb antibody (Ser 807/811) were from Cell Signaling Technology (Beverly, MA). Phospho-S6 antibody was a generous gift from Dr. Morris Birnbaum (University of Pennsylvania). The p27 (K25020) antibody used for immunostaining was from Transduction Laboratories, Inc. (Lexington, KY). Leptomycin B and roscovitine were from Sigma. LY was from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA), and rapamycin was from Calbiochem (La Jolla, CA).

Cell Culture
WRT and PC-Cl3 cells were propagated in Coon’s modified Ham’s F12 medium supplemented with 1 mU/ml TSH, 10 µg/ml insulin, 5 µg/ml transferrin, 5% donor calf serum, and antibiotics (3 hormone or 3H medium) as described previously (46). Subconfluent cells were rinsed and incubated in Coon’s modified Ham’s F12 medium, free of TSH, insulin, serum, and BSA (basal medium) for 48–72 h to render the cells quiescent. TSH was used at 1 mU/ml, insulin at 10 µg/ml, and calf serum at 5% in all experiments.

Growth Curve Assays
Cells (2.5 x 105) were plated overnight. The following morning, duplicate plates were counted to determine plating efficiency, and the remaining plates were washed three times and starved in basal medium for 72 h. At this time, duplicate plates were counted to determine the number of cells present, and the remaining plates were stimulated with TSH (T), forskolin (f), insulin (ins), 5% calf serum (CS), TSH/insulin, forskolin/insulin, TSH/serum, forskolin/serum, and 3H or held in basal medium (see Cell Culture above). Duplicate plates for each treatment were harvested at each time point and cell number determined by counting on a hemocytometer. Eight growth curve experiments were performed for 3H, six for TSH, three for insulin, TSH/serum, and TSH/insulin, and two for serum alone in WRT cells. A single growth curve was performed to confirm that forskolin and forskolin/serum elicited effects similar to TSH and TSH/serum. Two growth curve experiments were performed for each stimulation in PC-Cl3 cells.

DNA Synthesis
Cells were plated on 1 x 35 mm glass coverslips and allowed to grow for 3 d, at which time the cells were at most 50% confluent. After starvation in basal medium for 48 h, the cells were stimulated and pulse labeled with BrdU for the times indicated. Cells were fixed in 3.7% formaldehyde/PBS and stained with sheep anti-BrdU (BioDesign, Inc., Carmel, NY), fluorescein isothiocyanate-conjugated antisheep IgG, and 4',6-diamidino-2-phenylindole (DAPI) (to stain all nuclei). In some experiments (Fig. 6Go and Table 3Go), cells were also stained with p27 antibody (see below). Four fields (selected randomly) that together comprised more than 200 cells were scored blinded for each condition. The number of BrdU-positive cells is expressed as a percent of the total number of cells scored.

Cell Cycle Progression
Trypsinized adherent cells were collected by centrifugation, fixed in 70% ethanol in PBS at 4 C for 30 min, stained with propidium iodide (0.1 mg/ml) in 0.1% Triton X-100, 0.1 mM EDTA containing 100 U/ml RNAse A in PBS for 30 min, and subjected to FACS analysis. FACS analysis was performed by the Wistar Institute cytometry facility (Philadelphia, PA) on an EPICS XL flow cytometer (Coulter Corp., Hialeah, FL).

Western Immunoblotting
Cells were washed in ice-cold PBS and lysed, and protein determinations were made as described previously (45). Equal amounts of cellular protein (typically 40 µg) were resolved on 8% (Rb /phospho-Rb) or 10–12% (cyclins/CDKs/CDK inhibitors) SDS-PAGE and transferred to nitrocellulose. Membranes were probed with primary antibodies (0.2 µg/ml) overnight at 4 C or for 2 h at room temperature. Immunoblots were incubated for 1 h at room temperature with secondary antibodies conjugated to horseradish peroxidase (0.2 µg/ml) and antibody detection was performed using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ). To control for protein loading, the membranes were cut and probed for the protein in question and actin as a loading control.

CDK-2 Activity
Cells were washed with ice-cold PBS and lysates prepared in immunoprecipitation lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, freshly supplemented with 2.5 mM EGTA, 1 mM NaF, 10 µg/ml leupeptin, 20 µg/ml aprotinin, 1 mM sodium orthovanadate and 1 mM pefabloc]. Lysates were subjected to one freeze-thaw cycle and clarified by centrifugation. Cell extract (500 µg) was incubated with 3 µg CDK-2 antibody for 1 h at 4 C, and with 40 µl of 50% slurry of protein A-agarose for 1 h at 4 C. Immune complexes were washed four times in lysis buffer, two times in kinase buffer (50 mM HEPES, pH 7.5; 10 mM MgCl2; 1 mM dithiothreitol), and resuspended in 40 µl kinase buffer containing 10 µM ATP, 50 µg/ml histone H1, and 10 µCi of [{gamma}-32P]ATP. After incubation for 30 min at 30 C, the samples were boiled for 5 min with reducing buffer, separated by electrophoresis on a 12% gel, and transferred to nitrocellulose membrane. Membranes were subjected to autoradiography and Western blotted for CDK-2.

Immunostaining
Cells were fixed in 3.7% formaldehyde in PBS for 10 min at room temperature, washed twice with PBS, and incubated with p27 antibody diluted 1:100 in 0.5% Nonidet P-40, 1 mg/ml BSA in PBS for 1 h at 37 C, followed by incubation with Texas Red-conjugated antimouse antibody (1:200) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and DAPI. Digital images were collected using Zeiss-AxioVision 3.1 software (Carl Zeiss Vision, Munich, Germany). At least four fields of cells selected randomly (~80 cells per field) were imaged per condition. All images in an individual experiment were exposed for the same times.


   ACKNOWLEDGMENTS
 
We thank Dr. J. Alan Diehl for many helpful discussions and suggestions.


   FOOTNOTES
 
This work was supported by United States Public Health Service Grant DK45696 (to J.L.M.).

Present address for A.E.L: Department of Biomedicine, University of Bergen, 5009 Bergen, Norway.

A.E.L. and A.J.F. made equal contributions to this manuscript and should both be considered as first authors.

Abbreviations: BrdU, Bromodeoxyuridine; CDK, cyclindependent kinase; CS, calf serum, DAPI, 4',6-diamidino-2-phenylindole; f, forskolin; FACS, fluorescence-activated cell sorting; 3H, 3 hormone containing growth medium; LY, LY294002; PI3K, phosphatidylinositol 3-kinase; Rb, retinoblastoma protein; WRT, Wistar rat thyroid.

Received for publication March 10, 2004. Accepted for publication May 21, 2004.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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