The Proliferative Status of Thyrotropes Is Dependent on Modulation of Specific Cell Cycle Regulators by Thyroid Hormone
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《内分泌学杂志》
Division of Endocrinology, Metabolism
Diabetes, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado 80045
Abstract
In this report we have examined changes in cell growth parameters, cell cycle effectors, and signaling pathways that accompany thyrotrope growth arrest by thyroid hormone (TH) and growth resumption after its withdrawal. Flow cytometry and immunohistochemistry of proliferation markers demonstrated that TH treatment of thyrotrope tumors resulted in a reduction in the fraction of cells in S-phase that is restored upon TH withdrawal. This is accompanied by dephosphorylation and rephosphorylation of retinoblastoma (Rb) protein. The expression levels of cyclin-dependent kinase 2 and cyclin A, as well as cyclin-dependent kinase 1 and cyclin B, were decreased by TH, and after withdrawal not only did these regulators of Rb phosphorylation and mitosis increase in their expression but so too did the D1 and D3 cyclins. We also noted a rapid induction and subsequent disappearance of the type 5 receptor for the growth inhibitor somatostatin with TH treatment and withdrawal, respectively. Because somatostatin can arrest growth by activating MAPK pathways, we examined these pathways in TtT-97 tumors and found that the ERK pathway and several of its upstream and downstream effectors, including cAMP response element binding protein, were activated with TH treatment and deactivated after its withdrawal. This led to the hypothesis that TH, acting through increased type 5 somatostatin receptor, could activate the ERK pathway leading to cAMP response element binding protein-dependent decreased expression of critical cell cycle proteins, specifically cyclin A, resulting in hypophosphorylation of Rb and its subsequent arrest of S-phase progression. These processes are reversed when TH is withdrawn, resulting in an increase in the fraction of S-phase cells.
Introduction
THE ANTERIOR PITUITARY is an endocrine gland that continuously regulates its capacity to produce hormones in response to differing physiological and pathological states. One example of this regulatory adjustment is the expansion or contraction of specific pituitary cell types to meet the demands of fluctuating blood levels of the target organ hormone. It has long been recognized that hypothyroidism in humans and in radiothyroidectomized rodents results in pituitary enlargement caused by thyrotrope cell hyperplasia and that treatment with thyroid hormone (TH) not only corrects the symptoms of hypothyroidism but also reverses the pituitary enlargement by reducing the thyrotrope population (1, 2, 3). More recent reports have correlated TH levels with changes in mitotic activity of thyrotropes (4, 5, 6). The precise mechanism underlying the antiproliferative effect of TH on thyrotrope cell growth is currently unknown, although mechanisms involving cross-talk between TH and TRH, somatostatin, estrogens, and epidermal growth factor signaling pathways have recently been reviewed (7). The mouse TtT-97 tumor is a model of a well differentiated thyrotrope hyperplasia that secretes TSH (8, 9). It is propagated by subscapular injection of dispersed tumor tissue into hypothyroid mice. When mice with palpable tumors are administered TH there is a measurable reduction in tumor mass that is reversed after withdrawal of the hormone (10). Therefore, TtT-97 tumors represent an ideal model system in which to study the mechanism underlying regulation of thyrotrope cell growth by TH.
It is generally accepted that growth is regulated by controlling entry into the cell cycle. An actively proliferating cell progresses through the various phases of the cycle including the DNA synthetic (S) phase followed by mitosis (M) ultimately leading to division resulting in two daughter cells. Whether a cell divides or not depends on its ability to reach a certain checkpoint after which it is irreversibly committed to traverse the S-phase. The critical gatekeeper of the pathway that leads to S-phase commitment is the product of the gene encoding retinoblastoma (Rb) protein. In its hypophosphorylated state, Rb binds to and inactivates members of the E2F family of transcription factors preventing them from activating certain key genes required for initiation and progression through the S-phase (11). Thus, phosphorylation of Rb is critical for regulation of the cell cycle and any process that interferes with it would result in growth arrest. Rb is phosphorylated by cyclin-dependent kinases (CDKs), which are complexes of catalytic (cdk) and regulatory (cyclin) subunits. Various activating cyclins associate with different CDKs to phosphorylate Rb at various stages of the cell cycle. For example, levels of the D cyclins within cells are dependent on sustained growth factor stimulation and interact with cdk4 and cdk6 to initiate phosphorylation of Rb (12), whereas E and A cyclins combine with cdk2 to maintain Rb in a hyperphosphorylated state even after growth factor influences have been withdrawn (13). A decrease in expression in thyrotropes of one or more of these activating influences by TH would preclude S-phase entry and lead to growth arrest. Conversely, induction of CDK inhibitors, which bind to and repress the phosphorylating capacity of cyclin/cdk complexes (14), would also lead to growth arrest. We previously published a report of early gene expression changes in the TtT-97 thyrotrope tumor model treated for 24 h with TH. The results of these studies showed a decrease in transcripts encoding cyclin A isoforms as well as those for cdk2 but not for other cyclins (E and D1, -2, and -3) or cdks (4, 5, and 6) (15). In the same study, we also showed that the only CDK inhibitor to increase its expression (of seven examined) was p15, which is frequently down-regulated in human pituitary adenomas (16). Increased p15 expression has also been associated with hypophosphorylation of Rb and cell cycle arrest in cultured pituitary cells (17).
Somatostatin is a neuropeptide widely distributed throughout the central and peripheral nervous systems as well as many other tissues. Since its initial isolation from the hypothalamus, somatostatin has been shown to be an important factor in the regulation of anterior pituitary function. Somatostatin analogs have been shown to have antiproliferative and antisecretory effects on a variety of neuroendocrine tumors (18), including pituitary adenomas (19). Furthermore, in humans with TSH-secreting tumors, the somatostatin analog, octreotide, induces tumor shrinkage (20). Somatostatin acts via a family of five G protein-coupled receptors that are expressed in numerous tissues (21). Our laboratory has previously shown that inhibition of TtT-97 tumor growth by TH treatment is associated with increased expression of the gene for type 5 somatostatin receptors (sst5) (15), resulting in the appearance of somatostatin binding sites within the tumor (22, 23).
Activation of the ERK1/2 MAPK pathway is generally related to the growth-promoting actions of ligands for G protein-coupled receptors (24). However, growth inhibition may also be a consequence of ERK1/2 activation as in the case of hepatocyte growth factor-induced HepG2 cell cycle arrest (25), which is also associated with Rb hypophosphorylation as a result of decreased cyclin A expression and down-regulation of cdk2 (26). In a report with relevance to the current study, Lahlou et al. (27) demonstrated that growth inhibition of CHO cells, stably expressing ssts, by a somatostatin analog, resulted in an activation of the ERK pathway.
In the studies presented here, we extend our previous findings (15) by demonstrating that the reversible regulation of thyrotrope tumor cell growth by the presence or absence of TH is achieved by altering the expression of only a few cell cycle genes that determine the number of cells occupying the DNA S-phase. We show that this is reflected in the phosphorylation state of Rb, which is probably dependent on the level of cyclin A protein. Furthermore, employing a shorter time course of sst5 mRNA expression, we showed that it is increased by TH as early as 6 h, suggesting that it could be a direct transcriptional target of TH. Finally, as was the case for CHO cell growth arrest (27), the ERK pathway was shown to be activated as a possible response to sst5 stimulation. We postulate that changes in cyclin A gene expression occur as a consequence of the somatostatin-activated ERK pathway via the downstream nuclear effectors p90RSK and phospho-cAMP response element binding protein (phospho-CREB).
Materials and Methods
Experimental design
LAF1 mice were rendered hypothyroid by radiothyroidectomy (150 μCi Na131I per mouse ip) 6 wk before injection of TtT-97 tumor tissue, as has been previously described (10). Approximately 28 wk later, hypothyroid animals bearing TtT-97 tumors were entered into the experimental design. Animals were divided into four experimental groups: hypothyroid mice treated for a short time with vehicle (A) or TH (B) or treated chronically with TH (C) and then withdrawn from hormone (D). Groups A and B received either an ip injection of normal saline vehicle or L-T3 (100 μg/kg body weight) and were killed and tumor tissue harvested 1 d later. Group C hypothyroid animals were treated for 17–23 d with L-T4 (1 mg/liter in 0.75% ethanol) in the drinking water before being killed (TH maintained). Group D hypothyroid animals were treated for 14–21 d with oral L-T4 as above and then killed 2 or 3 d after withdrawal from TH (TH withdrawn). The experimental protocol was carried out twice with at least three tumor-bearing mice in each treatment group. Tumor tissue was harvested and processed for flow cytometry and immunohistochemistry as well as RNA and protein analysis. Baseline animal weights and tumor sizes were recorded before starting any drug treatment, and final measurements were obtained at the time of killing. Tumor size was obtained by using calipers to take measurements in three perpendicular dimensions such that elliptical volume could be calculated [ellipsoid volume = 4/3(abc), where a, b, and c were the three dimensional radius values]. Blood was collected, and total serum TH was measured by RIA using the Coat-a-Count T4 kit according to the manufacturer’s instructions (Diagnostic Products Corp., Los Angeles, CA). Each TtT-97 tumor sample was subdivided at the time the animal was killed. A 3-g tumor specimen was immediately homogenized in 10 vol of 4 M guanidinium isothiocyanate supplemented with 5% -mercaptoethanol and stored at –20 C before RNA isolation. A second tumor specimen was stored in buffered formalin for subsequent histology and immunohistochemistry. The remainder of the tumor was immediately processed into a single-cell suspension for flow cytometry (see below) and whole-cell extracts (see below). After a single-cell suspension was generated from the tumor tissue, viable cells (stained with Trypan blue) were counted using a hemocytometer, resuspended in 1x calcium- and magnesium-free Dulbecco’s PBS (Life Technologies, Inc., Gaithersburg, MD) and divided for assay by either flow cytometry or Western blot analysis.
Flow cytometry
TtT-97 cells in a single-cell suspension were assayed by flow cytometry as follows. An aliquot of 0.5 x 106 cells was placed in 0.5 ml Krishan’s propidium iodine stain [0.224 g sodium citrate, 9.22 mg propidium iodide, 1 ml of 1% Nonidet P40, 2 ml boiled RNase A (1 mg/ml) in a total volume of 200 ml water]. Cells were analyzed for percentage of cells in each phase of the cell cycle using a Beckman Coulter FC500 flow cytometer. Doublets were excluded from the analysis using the peak vs. integral gating method. ModFit LT software (Verity Software House, Topsham, ME) was used for cell cycle analysis. All cell cycle analyses were performed by the University of Colorado Health Sciences Center Flow Cytometry Core Facility.
Immunohistochemistry
Immunohistochemistry was performed on tissue samples fixed in formalin and embedded in paraffin. Immunostaining for proliferating cell nuclear antigen (PCNA) was performed on TtT-97 tumor tissue (4 μm thick) mounted on slides using a PCNA detection kit according to the manufacturer’s instructions (Zymed Laboratories Inc., San Francisco, CA). The percentage of PCNA-positive stained cells was obtained by randomly counting 10 fields of TtT-97 tumor cells using the x100 power (oil immersion) objective of a Nikon Eclipse E600 microscope (Nikon, Boston, MA).
Northern blot analysis and RNase protection assays
Tumors were excised and total RNA extracted from vehicle- and TH-treated or -withdrawn TtT-97 tumor-bearing animals using the guanidinium isothiocyanate/CsCl gradient method as previously described (28). Purification of poly(A+) RNA by oligo(dT) cellulose chromatography and Northern blot analysis, using 10-μg aliquots of poly(A+) RNA from vehicle-treated, TH-treated, and TH-withdrawn TtT-97 tumors, were performed as described (15). The various 32P-labeled cDNA probes were generated by nick translation using a commercially available kit (Life Technologies). The sources of TSH, sst5, cdk2, and E2F1 probes were described previously (15, 29). Plasmids containing inserts for GADD45a and Dexras1 were kindly provided by Drs. Fornace (National Cancer Institute, Frederick, MD) and Kemppainen (Auburn University, Montgomery, AL), respectively. Filters were subjected to autoradiography and reprobed with a mouse -actin cDNA fragment to assess uniformity of loading.
Quantitative mRNA expression for cdk and cyclin genes was determined by RNase protection analyses employing RiboQuantRPA array kits (PharMingen, San Diego, CA) according to the manufacturer’s instructions, using 15 μg of total RNA from vehicle-treated, TH-treated, and TH-withdrawn TtT-97 tumors.
Western blot analysis
TtT-97 whole-cell extracts were obtained as previously described (15). Protein concentration was determined using Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. TtT-97 whole-cell extracts (30–100 μg) were separated on a 10% polyacrylamide-SDS gel and transferred to an Immobilon-P (polyvinylidene difluoride) membrane (Millipore, Bedford, MA) by overnight electroblotting at 100 mA (4 C) or 1.5 h at 100–120 V if using a Mini Protean 3 cell gel transfer system (Bio-Rad Laboratories, Hercules, CA). Nonspecific binding was blocked with 5% nonfat milk in Tris-buffered saline/Tween 20 (TBST) [10 mM Tris-Cl (pH 7.5), 137 mM NaCl, 0.2% Tween] for 2 h at room temperature. Filters were then incubated overnight at 4 C with a specific primary antibody followed by incubation with the appropriate secondary antibody, either goat antirabbit (sc2004; Santa Cruz Biotechnology, Santa Cruz, CA) or goat antimouse (sc2005; Santa Cruz Biotechnology) antibodies in 5% milk TBST for 2 h at room temperature. After three 10-min washes in TBST, protein was detected using an ECL chemiluminescent kit (Amersham Life Science, Arlington Heights, IL). Proteins were visualized and molecular weights estimated by comparison with size standards run in an adjacent lane. The antibodies, their catalog numbers, and the dilution used in these studies are outlined below (sc denotes antibodies from Santa Cruz Biotechnology, and cs denotes antibodies from Cell Signaling Technology, Beverly, MA): total Rb (1:1000, cs 9309), phospho-Rb 807/814 (1:500, cs 9308), cyclin A2 (1:1000, sc 596), E2F1 (1:1000, sc 251), phospho-MAPK p38 (1:1000, cs 9211), phospho-c-Jun N-terminal kinase (phospho-JNK) (1:1000, cs 9251), phospho-ERK 1/2 (1:750, cs 9101), phosho-MAPK kinase (phospho-MEK) 1/2 (1:1000, cs 9121), p90RSK (1:1000, cs 9341), phospho-CREB (1:1000, cs 9191), total CREB (1:1000 for cs 9192 or 1:5000 for sc 186X), and -actin (Sigma A5441, 1:10,000 to 1:50,000; Sigma Chemical Co., St. Louis, MO).
MEK kinase (MEKK) assays
Kinase activities of whole-cell extracts of TtT-97 tumors were assayed essentially as described (27) using c-Raf (sc-7267), B-Raf (sc-166), MEKK-1 (sc-437) or MEKK-3 (611102; Transduction Labs, Lexington, KY) immunoprecipitates, [-32P]ATP as a phosphate donor, and recombinant full-length MEK1 (sc-4025) as a substrate.
Statistics
All results were analyzed using a two-way ANOVA and post hoc Tukey paired comparisons. Significance was defined as P value < 0.05.
Animal care
All animals were treated in a humane manner in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and under protocols approved by the committee on animal care and use of the University of Colorado Health Sciences Center.
Results
TH treatment is associated with thyrotropic tumor size reduction
As we have previously shown, treatment with long-term physiological replacement of L-T4 caused a measurable reduction in tumor size (10). In the current studies, after approximately 21 d of L-T4 in the drinking water, tumor size was reduced by 40.15 ± 8.08% (n = 10 mice). We were unable to assess any change in tumor size in the animals that received TH for only 1 d or in those that were withdrawn for 2–3 d, because of the delayed nature of the gross changes in tumor size.
Thyrotropic tumors are responsive to withdrawal of TH as assessed by reactivation of TSH gene expression
To ensure that TH-withdrawn TtT-97 tumors retained their well-characterized response to a hypothyroid state by reexpressing the TSH gene, we measured steady-state TSH mRNA levels by Northern blot analysis. Figure 1 shows that 21-d TH-treated tumors that had high serum TH levels (T4 = 10.18 ± 0.74 μg/dl; n = 10) had a low level of TSH transcripts that dramatically increased after TH deprivation, demonstrating that withdrawal of TH that resulted in hypothyroid serum TH levels (T4 = 0.2 ± 0.17 μg/dl; n = 10) was already exerting an effect by 2 d of hormone withdrawal at the mRNA level presumably by reactivation of TSH gene transcription (30).
TH levels determine the percentage of TtT-97 tumor cells in S-phase
We next assessed tumor growth by quantifying the percentage of cells in the DNA synthetic phase, or S-phase, at each stage of TH treatment and withdrawal. Shown in Fig. 2 are the flow cytometry profiles of TtT-97 tumor cells dispersed from hypothyroid mice (A), mice treated for 1 d with TH (B), and mice TH-treated for 21 d (C) and similarly treated mice after subsequent withdrawal of TH for 2 d (D). It can be clearly seen that the area between the major peaks corresponding to the S-phase population, which is readily visible in the hypothyroid state (Fig. 2A), is noticeably reduced at 1 and 21 d of TH treatment and that this area dramatically reappears in mice withdrawn from TH. When the S-phase percentages were averaged for at least three mice in each thyroidal status group, the number of cells in S-phase significantly (P < 0.05) decreased from 4.2 ± 0.4% in the hypothyroid state to 1.6 ± 0.2% after 1 d of TH treatment. The population of cells within S-phase further declined to 1.2 ±0.6% after 21 d of TH when significant shrinkage of tumor mass was clearly evident. Subsequent TH withdrawal for 2 d resulted in 8 ± 0.9% of the cells occupying the S-phase (P < 0.05 compared with vehicle and TH-maintained mice). The TH-associated changes in growth were verified by staining sections of the tumors from each thyroidal state with an antibody against the proliferation marker PCNA (Fig. 2). Detection of PCNA demonstrated the presence of a sizeable number of positively staining cells in the hypothyroid state (quantitated by visual estimation to be 4.7 ± 1.0%), which then decreased to 2.4 ± 3% after 1 d of TH treatment. After 21 d of treatment, the percentage of PCNA-positive cells declined further to 1.0 ± 5% but then was restored to hypothyroid levels (10.3 ± 5.0%) after 2 d of withdrawal from TH. These changes in cell cycle parameters were also confirmed by visually counting mitotic indices, which also correlated with the TH-dependent growth changes (data not shown).
TH alters the phosphorylation state of Rb
The results of the flow cytometry and PCNA data suggested that TH regulates thyrotropic tumor growth by altering the fraction of cells that is actively synthesizing DNA presumably by blocking entry into S-phase, although the data do not rule out an increased exit from S-phase. To distinguish between these two possibilities, we investigated the effect of TH on the levels and specifically the phosphorylation state of Rb, which regulates the checkpoint of entry into S-phase. Figure 3A shows a Western blot probed with an anti-Rb antibody that revealed that total Rb levels remained relatively similar in the hypothyroid state and after 1 or 2 d of treatment with TH, although a slightly slower migrating band, representing the phosphorylated protein, decreased with TH treatment. Using an antibody raised against Rb activated by phosphorylation at serines 807 and 811 confirmed that phospho-Rb levels progressively diminish with TH treatment maintained for 21 d and that phosphorylation is restored after withdrawal of TH for 2 d (Fig. 3B). Thus, these data suggest that TH blocks entry of thyrotropic tumor cells into S-phase by reducing the amount of phosphorylated Rb that is subsequently rephosphorylated after TH withdrawal, resulting in increased S-phase occupancy.
TH regulates the expression levels of effectors of Rb phosphorylation
We previously showed that TH has profound effects on the transcript levels of certain genes involved in Rb phosphorylation (15). These included decreases in the mRNA levels of cdk2 and cyclin A as well as changes in specific CDK inhibitors. However, many other cell cycle genes including those for cdk4 and -6 and their activating D and E cyclins, as well as the other CDK inhibitors, were relatively unaffected by TH treatment and were therefore unlikely to play a role in the control of thyrotropic tumor cell growth by TH. To see whether the same effectors of Rb phosphorylation were involved in the resumption of growth after TH withdrawal, we employed multitemplate ribonuclease protection assays (RPAs) that allowed the simultaneous assessment of the transcript levels of several catalytic cdks and regulatory cyclins. Figure 4A shows that, as before (15), mRNAs for cdk4 and cdk5, as well as a series of other cdks not previously investigated, were unchanged either by treatment with or withdrawal from TH. Because cdk2 was not represented on the CDK RPA array and because its mRNA level was previously shown to be decreased by TH (15), we performed a comparative Northern blot analysis on poly(A+) RNA derived from a tumor maintained for 21 d on TH vs. a similarly treated one withdrawn from hormone for 2 d. The results in Fig. 4B show that the suppressed expression of cdk2 is reinstituted after TH withdrawal. The RPA analysis for the cyclins is shown in Fig. 5A. Cyclin A transcripts, as before (15), were dramatically down-regulated by TH treatment and subsequently reexpressed after TH withdrawal. In addition, transcripts for cyclins D1 and D3 were also increased, suggesting that restoration of thyrotropic tumor growth by withdrawal of TH, unlike its repression, involved additional pathways mediated by G1-phase cyclins (D1 and D3) that are usually associated with extracellular growth factor stimulation. Figure 5B demonstrates that the effect of TH on cyclin A expression was also exerted at the protein level as demonstrated by Western blot analysis. Interestingly expression of E2F1, a protein that phospho-Rb dissociates from, and that activates S-phase genes, is altered at the transcript level in response to TH (decreased with treatment and increased after withdrawal) (Fig. 6A) but is not affected at the protein level with TH treatment (Fig. 6B)
Expression of regulators of the G2/M transition was altered by TH
The response of other cdks and cyclins to TH, particularly those governing the transition from the G2 phase to mitosis (G2/M) was simultaneously evaluated by the RPA arrays. As is shown in Figs. 4A and 5A, transcripts for both cdk1 and cyclin B1, which phosphorylate substrates related to mitotic spindle formation to facilitate cell division (31, 32), were decreased by TH treatment. After TH withdrawal for 2 d, they returned to levels similar to those in the hypothyroid state and therefore could also be contributing to the cell cycle arrest and resumption of growth associated with exposure to TH and its subsequent withdrawal. Because complexes of cdk1 activated by cyclin B1 are disrupted by the protein GADD45a (33), we sought to determine whether TH also affected the expression of this cell cycle inhibitor. Figure 7 demonstrates that in contrast to the observed down-regulation of cdk1 and cyclin B1, GADD45a transcripts are increased with TH and decreased after its withdrawal. Alterations in GADD45a expression would result in modulation of cdk1 activity, which correlate with the TH-associated growth changes.
Specific MAPK pathways are activated by TH treatment of TtT-97 tumors
We sought to determine the mechanism by which TH regulates the levels of these cell cycle effectors. Those cell cycle regulators shown here to be affected by TH are ubiquitously expressed and are present in cells whose growth is not affected by TH and are therefore probably a secondary target of a pathway that is more restricted to thyrotrope cells. One possibility is that TH mediates its effect via a somatostatinergic pathway. We previously reported that prolonged treatment of TtT-97 tumors with TH was correlated with the appearance of somatostatin binding sites on the surface of these tumor cells and also with increased transcripts for sst5 (22). We also showed that only sst5 transcripts were increased after short-term (1-d) exposure to TH (15). We now show that expression of sst5 transcripts occurs very rapidly, within 6 h of TH administration (Fig. 8A), and could represent a primary transcriptional target of TH. Furthermore, sst5 mRNA decreased to undetectable levels after TH withdrawal (Fig. 8B). Lahlou et al. (27) reported that a somatostatin analog inhibits the growth of CHO cells that have been stably transfected with ssts and that it does this by activating specific MAPK pathways. We therefore wanted to determine whether MAPK pathways were being activated in thyrotrope cells in response to TH. When we examined the levels of activated, or phosphorylated, MAPKs we found that phospho-ERK1/2 and phospho-38MAPK, to a lesser extent, but not phospho-JNK, were increased after treatment with TH for 1 d and even more so after 2 d, whereas total ERK was unchanged (Fig. 9). Several downstream targets of activated ERK1/2, specifically activated p90RSK and phospho-CREB, were also increased by TH, whereas levels of total CREB remained unaffected (Fig. 10). When we investigated the potential upstream kinases that could be activating ERK1/2 we found that levels of phospho-MEK were also elevated in response to TH (Fig. 10). When Lahlou et al. (27) assayed the MEK activating potential of the CHO cell extracts they found that B-Raf kinase, but not c-Raf, was elevated with somatostatin treatment. However, when we assayed extracts of TtT-97 tumors for kinase activity of several of the most commonly reported MEK activators (MEKKs), including c-Raf, B-Raf, MEKK1, and MEKK3 (34, 35), we found that none of them had enhanced MEK-phosphorylating capability as a result of TH treatment (data not shown). When we examined the levels of ERK pathway proteins that were activated by TH treatment, specifically phospho-MEK, phospho-RK1/2, and phospho-CREB, after withdrawal from TH, they decreased to levels similar to or below those in the hypothyroid state (Fig. 11). Lahlou et al. (27) also showed that the Ras-related GTP binding protein Rap1 played an upstream role in the inhibition of growth by somatostatin. We previously reported that a glucocorticoid-responsive Ras family member, Dexras 1, was increased by TH treatment in a microarray analysis (36). Figure 12 confirms by Northern blot analysis that mRNA levels for Dexras 1, which was originally cloned from pituitary-derived cells and exhibits close sequence homology to Rap family members (37), are dramatically increased in TtT-97 tumors by 1 d of TH treatment and subsequently decrease after TH withdrawal.
Discussion
The molecular mechanism by which TH exerts its effects on cell growth remains largely unknown. The major goal of our studies is to understand how TH specifically regulates the growth of thyrotrope cells. Prolonged TH treatment of hypothyroid mice bearing TtT-97 tumors, an excellent thyrotrope model of TH regulation, results in a dramatic reduction in tumor size (10). We previously showed that relatively short-term exposure of thyrotropic tumors to TH for times up to 24 h resulted in changes in the mRNA levels encoding certain growth factors and their receptors as well as transcripts corresponding to key cell cycle mediators including cyclin A and cdk2 (15). In this report, we now demonstrate that modulation of the expression levels of these and other cell cycle regulators by the presence or absence of TH determines the growth state of thyrotropic tumor cells and that this is mediated through reversible changes in the percentage of cells occupying the S-phase of the cell cycle. We also show that this is accomplished by altering the phosphorylation state of Rb, the gatekeeper of the G1- to S-phase checkpoint (11). In thyrotropes, this appears to be achieved by modulating the expression levels of cyclin A and its catalytic partner cdk2, leading to the Rb phosphorylation changes observed. Finally, based on studies reported by Lahlou et al. (27), we postulate a relationship between the expression of sst5, which is rapidly up-regulated by and therefore a putative primary transcriptional target of TH, and somatostatin ligand-triggered activation of the ERK signaling pathway. We demonstrated that the activation state of members of this pathway, including MEK upstream and p90RSK and CREB downstream of ERK as well as the expression of the Ras homolog, Dexras 1, are dependent on the serum TH level sensed by the tumor cells.
Although activation of CREB is usually associated with growth stimulation and cyclin gene activation (38, 39), in some instances it has been shown to lead to inhibition of growth (40, 41) and also of cyclin gene expression (42, 43). The data presented here are consistent with the hypothesis that in thyrotropic tumor cells treated with TH, phosphorylation of CREB, as a result of sustained ERK pathway activation, leads to transcriptional suppression of cyclin A and cdk2 leading to hypophosphorylation of Rb and inhibition of the G1 to S progression. A reversal of this process occurs when TH is withdrawn; sst5 and Dexras 1 levels decrease and the ERK pathway is inactivated resulting in dephosphorylation of CREB and reexpression of the specific cell cycle regulators cyclinA and cdk2.
Interestingly, although we observed little alteration, if any, in D cyclin mRNA levels with TH treatment for 1 d, in a previous report by Northern analysis (15) and confirmed here by RPA arrays, we did see a dramatic increase in both cyclin D1 and D3 transcripts after TH withdrawal. This represented the only difference in the altered expression of several cell cycle mediators that was noted between TH treatment and its subsequent withdrawal and suggests that cyclin D1 and D3 may play a more important role in growth resumption induced by TH withdrawal. A possible explanation for this is that the D cyclins, which preferentially respond to extracellular growth factor stimulation, may be more influenced by a concerted reappearance of stimulatory growth factors and receptors such as the TRH receptor, brain-derived neurotrophic factor/tyrosine kinase B, Wnt10a, and bone morphogenetic protein 4, all of whose expression was shown previously to be repressed by TH (15, 36). In this regard, it has been reported that -catenin, which is stabilized by activation of Wnt pathways, can mediate transactivation of the cyclin D1 promoter in colon carcinoma cells and that this is negatively regulated by TH (44). In contrast, Cheng and coworkers (45) showed that Wnt signaling was silenced in response to TH-stimulated proliferation of pituitary-derived GC cells.
The use of the multitemplate RPA arrays uncovered a novel aspect of cell cycle control by TH of which we were previously unaware. Transcripts for cyclin B1 and cdk1, both of which are involved in facilitating the G2/M transition (46) and subsequent cell division, were decreased by TH and reexpressed after its withdrawal. Interestingly, expression of GADD45a, a p53-regulated stress protein that inhibits cell division by dissociating cyclinB1/cdk1 complexes (33), showed an opposite pattern of expression, being induced by TH and repressed by its withdrawal. Thus, the overall effect of TH on the levels of these key G2/M modulators would be to block progression from the G2 phase to mitosis and thus further contribute to thyrotropic tumor growth inhibition. There are no reports in the literature of TH affecting the expression of cyclin B1 or cdk1 and thus the G2/M transition. However, Cheng and coworkers (47) showed that TR1 could antagonize the function of p53 and, becauseGADD45a is a p53-regulated gene (48), TH could conceivably alter its expression. Although in this fashion GADD45a could represent a transcriptional target for TH, its promoter is also reported to be activated by MAPK (49). Interestingly, the gene for another GADD45 family member has been reported to be deleted in a high proportion of nonsecreting pituitary tumors (50).
Flow cytometry and PCNA immunostaining showed that the proportion of TtT-97 cells in S-phase is a function of thyroidal status, with the presence of TH decreasing the number of replicating cells. Although TH has been reported to stimulate growth, particularly in hepatocytes (51, 52, 53) and pituitary GC cells (45, 47), in other studies it has been shown to decrease the percentage of cells in S-phase. Inhibition of growth by TH has been reported for terminally differentiating oligodendrocytes (54, 55) and chondrocytes (56), murine and rat Sertoli cells (57, 58), and neuroblastoma cells stably overexpressing the TR1 isoform (59). In all of these reports where TH repressed growth, expression levels of cyclins or cdks were not reported to be affected, despite the fact that Rb was hypophosphorylated. Instead, Rb hypophosphorylation was attributed to increased levels of the CDK inhibitor p27. We previously reported no change in the expression of p27 mRNA levels after TH treatment of TtT-97 tumors (15), suggesting a different mechanism for thyrotrope growth inhibition by TH from what is described for these other cell types.
We were surprised to find that TH treatment of TtT-97 thyrotropes, which resulted in growth inhibition, led to sustained ERK activation because suppression of growth is usually associated with down-regulation of MAPK pathways (60, 61, 62). Furthermore, TH-stimulated growth is usually correlated with an activation of MAPK (53, 63). Other hormones, for example steroids, have been shown to stimulate growth and activate ERK pathways as was reported for androgens in LNCaP prostate cancer cells (64) and endometrial carcinomas (65), estrogens in breast cancer cells (66, 67), and aldosterone in rat cardiac fibroblasts (68). In contrast, glucocorticoids inhibited proliferation of smooth muscle cells with no effect on ERK activity or phosphorylation (69). Nevertheless, there have been several reports where growth inhibition or differentiation was associated with MAPK activation as in growth suppression of HL-60 myeloblastic leukemia cells by retinoids (70) and nerve growth factor or cAMP-induced differentiation of neuroendocrine PC12 cells (71, 72). Of particular relevance to the current studies was the finding of Lahlou et al. (27) that CHO cells stably transfected with ssts could be growth arrested by treatment with a somatostatin analog and that this resulted in activation of the ERK cascade. We had previously reported that prolonged treatment of TtT-97 tumors resulted in the up-regulation of sst5 transcripts and the appearance of somatostatin binding sites on the surface of the tumor cells (22). Somatostatin has long been known to possess antiproliferative properties (73) and has been used in the treatment of pituitary tumors (19), particularly those arising from thyrotropes (74, 75). Lahlou et al. (27) went on to show that the MAPK pathway members activated by somatostatin included Src, Ras, Rap1, B-Raf, MEK, and ERK. Here we also showed TH activation of MEK and ERK1/2 as well as the downstream targets p90RSK and CREB in thyrotropic tumors but were unable to identify the activator of MEK. Interestingly, we identified a small GTP-binding Rap1-related protein, Dexras 1, which is expressed in mouse pituitary cells (37, 76) and not previously known to be TH regulated. Furthermore, we demonstrated that Dexras 1 transcripts were increased in TtT-97 tumors by TH and decreased after withdrawal and speculate that Dexras 1 could possibly represent the as yet unknown MEKK activator, although this will have to await future studies.
In summary, we have shown that TH control of thyrotrope cell growth is caused by reversible changes in the number of replicating cells as a result of alterations in the expression of a limited cadre of key regulators that affect cell cycle progression. We also showed that proteins that represent possible primary gene targets of TH action, and that are present in the pituitary, could activate a putative somatostatinergic pathway that leads to activation of a MAPK signaling cascade and culminate in changes in critical G1/S and G2/M transition regulators. Based on studies describing the regulation of growth by other hormones, we believe that the mechanism of TH control of thyrotrope proliferation described here represents a unique mode of hormone-regulated growth.
Acknowledgments
We are grateful to Drs. A. J. Fornace Jr. (Bethesda, MD) and R. J. Kemppainen (Auburn, AL) for the generous provision of plasmids. We also thank Janet Dowding and Amina Gordon for excellent technical assistance and Dr. Andrew Bradford for help with MEK kinase assays.
Footnotes
Flow cytometry was carried out by the University of Colorado Cancer Center Flow Cytometry Core, supported by National Institutes of Health Grant 5 P30 CA 46934. This research was supported by National Institutes of Health Grants CA-47411 and DK-36843 (to E.C.R.) and DK-02813 (to W.W.W.).
First Published Online October 13, 2005
Abbreviations: CDK, Cyclin-dependent kinase; CREB, cAMP response element binding protein; JNK, c-Jun N-terminal kinase; MEK, MAPK kinase; MEKK, MEK kinase; PCNA, proliferating cell nuclear antigen; Rb, retinoblastoma; RPA, ribonuclease protection assay; sst5, type 5 somatostatin receptor; TBST, Tris-buffered saline/Tween 20; TH, thyroid hormone.
Accepted for publication October 3, 2005.
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Diabetes, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado 80045
Abstract
In this report we have examined changes in cell growth parameters, cell cycle effectors, and signaling pathways that accompany thyrotrope growth arrest by thyroid hormone (TH) and growth resumption after its withdrawal. Flow cytometry and immunohistochemistry of proliferation markers demonstrated that TH treatment of thyrotrope tumors resulted in a reduction in the fraction of cells in S-phase that is restored upon TH withdrawal. This is accompanied by dephosphorylation and rephosphorylation of retinoblastoma (Rb) protein. The expression levels of cyclin-dependent kinase 2 and cyclin A, as well as cyclin-dependent kinase 1 and cyclin B, were decreased by TH, and after withdrawal not only did these regulators of Rb phosphorylation and mitosis increase in their expression but so too did the D1 and D3 cyclins. We also noted a rapid induction and subsequent disappearance of the type 5 receptor for the growth inhibitor somatostatin with TH treatment and withdrawal, respectively. Because somatostatin can arrest growth by activating MAPK pathways, we examined these pathways in TtT-97 tumors and found that the ERK pathway and several of its upstream and downstream effectors, including cAMP response element binding protein, were activated with TH treatment and deactivated after its withdrawal. This led to the hypothesis that TH, acting through increased type 5 somatostatin receptor, could activate the ERK pathway leading to cAMP response element binding protein-dependent decreased expression of critical cell cycle proteins, specifically cyclin A, resulting in hypophosphorylation of Rb and its subsequent arrest of S-phase progression. These processes are reversed when TH is withdrawn, resulting in an increase in the fraction of S-phase cells.
Introduction
THE ANTERIOR PITUITARY is an endocrine gland that continuously regulates its capacity to produce hormones in response to differing physiological and pathological states. One example of this regulatory adjustment is the expansion or contraction of specific pituitary cell types to meet the demands of fluctuating blood levels of the target organ hormone. It has long been recognized that hypothyroidism in humans and in radiothyroidectomized rodents results in pituitary enlargement caused by thyrotrope cell hyperplasia and that treatment with thyroid hormone (TH) not only corrects the symptoms of hypothyroidism but also reverses the pituitary enlargement by reducing the thyrotrope population (1, 2, 3). More recent reports have correlated TH levels with changes in mitotic activity of thyrotropes (4, 5, 6). The precise mechanism underlying the antiproliferative effect of TH on thyrotrope cell growth is currently unknown, although mechanisms involving cross-talk between TH and TRH, somatostatin, estrogens, and epidermal growth factor signaling pathways have recently been reviewed (7). The mouse TtT-97 tumor is a model of a well differentiated thyrotrope hyperplasia that secretes TSH (8, 9). It is propagated by subscapular injection of dispersed tumor tissue into hypothyroid mice. When mice with palpable tumors are administered TH there is a measurable reduction in tumor mass that is reversed after withdrawal of the hormone (10). Therefore, TtT-97 tumors represent an ideal model system in which to study the mechanism underlying regulation of thyrotrope cell growth by TH.
It is generally accepted that growth is regulated by controlling entry into the cell cycle. An actively proliferating cell progresses through the various phases of the cycle including the DNA synthetic (S) phase followed by mitosis (M) ultimately leading to division resulting in two daughter cells. Whether a cell divides or not depends on its ability to reach a certain checkpoint after which it is irreversibly committed to traverse the S-phase. The critical gatekeeper of the pathway that leads to S-phase commitment is the product of the gene encoding retinoblastoma (Rb) protein. In its hypophosphorylated state, Rb binds to and inactivates members of the E2F family of transcription factors preventing them from activating certain key genes required for initiation and progression through the S-phase (11). Thus, phosphorylation of Rb is critical for regulation of the cell cycle and any process that interferes with it would result in growth arrest. Rb is phosphorylated by cyclin-dependent kinases (CDKs), which are complexes of catalytic (cdk) and regulatory (cyclin) subunits. Various activating cyclins associate with different CDKs to phosphorylate Rb at various stages of the cell cycle. For example, levels of the D cyclins within cells are dependent on sustained growth factor stimulation and interact with cdk4 and cdk6 to initiate phosphorylation of Rb (12), whereas E and A cyclins combine with cdk2 to maintain Rb in a hyperphosphorylated state even after growth factor influences have been withdrawn (13). A decrease in expression in thyrotropes of one or more of these activating influences by TH would preclude S-phase entry and lead to growth arrest. Conversely, induction of CDK inhibitors, which bind to and repress the phosphorylating capacity of cyclin/cdk complexes (14), would also lead to growth arrest. We previously published a report of early gene expression changes in the TtT-97 thyrotrope tumor model treated for 24 h with TH. The results of these studies showed a decrease in transcripts encoding cyclin A isoforms as well as those for cdk2 but not for other cyclins (E and D1, -2, and -3) or cdks (4, 5, and 6) (15). In the same study, we also showed that the only CDK inhibitor to increase its expression (of seven examined) was p15, which is frequently down-regulated in human pituitary adenomas (16). Increased p15 expression has also been associated with hypophosphorylation of Rb and cell cycle arrest in cultured pituitary cells (17).
Somatostatin is a neuropeptide widely distributed throughout the central and peripheral nervous systems as well as many other tissues. Since its initial isolation from the hypothalamus, somatostatin has been shown to be an important factor in the regulation of anterior pituitary function. Somatostatin analogs have been shown to have antiproliferative and antisecretory effects on a variety of neuroendocrine tumors (18), including pituitary adenomas (19). Furthermore, in humans with TSH-secreting tumors, the somatostatin analog, octreotide, induces tumor shrinkage (20). Somatostatin acts via a family of five G protein-coupled receptors that are expressed in numerous tissues (21). Our laboratory has previously shown that inhibition of TtT-97 tumor growth by TH treatment is associated with increased expression of the gene for type 5 somatostatin receptors (sst5) (15), resulting in the appearance of somatostatin binding sites within the tumor (22, 23).
Activation of the ERK1/2 MAPK pathway is generally related to the growth-promoting actions of ligands for G protein-coupled receptors (24). However, growth inhibition may also be a consequence of ERK1/2 activation as in the case of hepatocyte growth factor-induced HepG2 cell cycle arrest (25), which is also associated with Rb hypophosphorylation as a result of decreased cyclin A expression and down-regulation of cdk2 (26). In a report with relevance to the current study, Lahlou et al. (27) demonstrated that growth inhibition of CHO cells, stably expressing ssts, by a somatostatin analog, resulted in an activation of the ERK pathway.
In the studies presented here, we extend our previous findings (15) by demonstrating that the reversible regulation of thyrotrope tumor cell growth by the presence or absence of TH is achieved by altering the expression of only a few cell cycle genes that determine the number of cells occupying the DNA S-phase. We show that this is reflected in the phosphorylation state of Rb, which is probably dependent on the level of cyclin A protein. Furthermore, employing a shorter time course of sst5 mRNA expression, we showed that it is increased by TH as early as 6 h, suggesting that it could be a direct transcriptional target of TH. Finally, as was the case for CHO cell growth arrest (27), the ERK pathway was shown to be activated as a possible response to sst5 stimulation. We postulate that changes in cyclin A gene expression occur as a consequence of the somatostatin-activated ERK pathway via the downstream nuclear effectors p90RSK and phospho-cAMP response element binding protein (phospho-CREB).
Materials and Methods
Experimental design
LAF1 mice were rendered hypothyroid by radiothyroidectomy (150 μCi Na131I per mouse ip) 6 wk before injection of TtT-97 tumor tissue, as has been previously described (10). Approximately 28 wk later, hypothyroid animals bearing TtT-97 tumors were entered into the experimental design. Animals were divided into four experimental groups: hypothyroid mice treated for a short time with vehicle (A) or TH (B) or treated chronically with TH (C) and then withdrawn from hormone (D). Groups A and B received either an ip injection of normal saline vehicle or L-T3 (100 μg/kg body weight) and were killed and tumor tissue harvested 1 d later. Group C hypothyroid animals were treated for 17–23 d with L-T4 (1 mg/liter in 0.75% ethanol) in the drinking water before being killed (TH maintained). Group D hypothyroid animals were treated for 14–21 d with oral L-T4 as above and then killed 2 or 3 d after withdrawal from TH (TH withdrawn). The experimental protocol was carried out twice with at least three tumor-bearing mice in each treatment group. Tumor tissue was harvested and processed for flow cytometry and immunohistochemistry as well as RNA and protein analysis. Baseline animal weights and tumor sizes were recorded before starting any drug treatment, and final measurements were obtained at the time of killing. Tumor size was obtained by using calipers to take measurements in three perpendicular dimensions such that elliptical volume could be calculated [ellipsoid volume = 4/3(abc), where a, b, and c were the three dimensional radius values]. Blood was collected, and total serum TH was measured by RIA using the Coat-a-Count T4 kit according to the manufacturer’s instructions (Diagnostic Products Corp., Los Angeles, CA). Each TtT-97 tumor sample was subdivided at the time the animal was killed. A 3-g tumor specimen was immediately homogenized in 10 vol of 4 M guanidinium isothiocyanate supplemented with 5% -mercaptoethanol and stored at –20 C before RNA isolation. A second tumor specimen was stored in buffered formalin for subsequent histology and immunohistochemistry. The remainder of the tumor was immediately processed into a single-cell suspension for flow cytometry (see below) and whole-cell extracts (see below). After a single-cell suspension was generated from the tumor tissue, viable cells (stained with Trypan blue) were counted using a hemocytometer, resuspended in 1x calcium- and magnesium-free Dulbecco’s PBS (Life Technologies, Inc., Gaithersburg, MD) and divided for assay by either flow cytometry or Western blot analysis.
Flow cytometry
TtT-97 cells in a single-cell suspension were assayed by flow cytometry as follows. An aliquot of 0.5 x 106 cells was placed in 0.5 ml Krishan’s propidium iodine stain [0.224 g sodium citrate, 9.22 mg propidium iodide, 1 ml of 1% Nonidet P40, 2 ml boiled RNase A (1 mg/ml) in a total volume of 200 ml water]. Cells were analyzed for percentage of cells in each phase of the cell cycle using a Beckman Coulter FC500 flow cytometer. Doublets were excluded from the analysis using the peak vs. integral gating method. ModFit LT software (Verity Software House, Topsham, ME) was used for cell cycle analysis. All cell cycle analyses were performed by the University of Colorado Health Sciences Center Flow Cytometry Core Facility.
Immunohistochemistry
Immunohistochemistry was performed on tissue samples fixed in formalin and embedded in paraffin. Immunostaining for proliferating cell nuclear antigen (PCNA) was performed on TtT-97 tumor tissue (4 μm thick) mounted on slides using a PCNA detection kit according to the manufacturer’s instructions (Zymed Laboratories Inc., San Francisco, CA). The percentage of PCNA-positive stained cells was obtained by randomly counting 10 fields of TtT-97 tumor cells using the x100 power (oil immersion) objective of a Nikon Eclipse E600 microscope (Nikon, Boston, MA).
Northern blot analysis and RNase protection assays
Tumors were excised and total RNA extracted from vehicle- and TH-treated or -withdrawn TtT-97 tumor-bearing animals using the guanidinium isothiocyanate/CsCl gradient method as previously described (28). Purification of poly(A+) RNA by oligo(dT) cellulose chromatography and Northern blot analysis, using 10-μg aliquots of poly(A+) RNA from vehicle-treated, TH-treated, and TH-withdrawn TtT-97 tumors, were performed as described (15). The various 32P-labeled cDNA probes were generated by nick translation using a commercially available kit (Life Technologies). The sources of TSH, sst5, cdk2, and E2F1 probes were described previously (15, 29). Plasmids containing inserts for GADD45a and Dexras1 were kindly provided by Drs. Fornace (National Cancer Institute, Frederick, MD) and Kemppainen (Auburn University, Montgomery, AL), respectively. Filters were subjected to autoradiography and reprobed with a mouse -actin cDNA fragment to assess uniformity of loading.
Quantitative mRNA expression for cdk and cyclin genes was determined by RNase protection analyses employing RiboQuantRPA array kits (PharMingen, San Diego, CA) according to the manufacturer’s instructions, using 15 μg of total RNA from vehicle-treated, TH-treated, and TH-withdrawn TtT-97 tumors.
Western blot analysis
TtT-97 whole-cell extracts were obtained as previously described (15). Protein concentration was determined using Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. TtT-97 whole-cell extracts (30–100 μg) were separated on a 10% polyacrylamide-SDS gel and transferred to an Immobilon-P (polyvinylidene difluoride) membrane (Millipore, Bedford, MA) by overnight electroblotting at 100 mA (4 C) or 1.5 h at 100–120 V if using a Mini Protean 3 cell gel transfer system (Bio-Rad Laboratories, Hercules, CA). Nonspecific binding was blocked with 5% nonfat milk in Tris-buffered saline/Tween 20 (TBST) [10 mM Tris-Cl (pH 7.5), 137 mM NaCl, 0.2% Tween] for 2 h at room temperature. Filters were then incubated overnight at 4 C with a specific primary antibody followed by incubation with the appropriate secondary antibody, either goat antirabbit (sc2004; Santa Cruz Biotechnology, Santa Cruz, CA) or goat antimouse (sc2005; Santa Cruz Biotechnology) antibodies in 5% milk TBST for 2 h at room temperature. After three 10-min washes in TBST, protein was detected using an ECL chemiluminescent kit (Amersham Life Science, Arlington Heights, IL). Proteins were visualized and molecular weights estimated by comparison with size standards run in an adjacent lane. The antibodies, their catalog numbers, and the dilution used in these studies are outlined below (sc denotes antibodies from Santa Cruz Biotechnology, and cs denotes antibodies from Cell Signaling Technology, Beverly, MA): total Rb (1:1000, cs 9309), phospho-Rb 807/814 (1:500, cs 9308), cyclin A2 (1:1000, sc 596), E2F1 (1:1000, sc 251), phospho-MAPK p38 (1:1000, cs 9211), phospho-c-Jun N-terminal kinase (phospho-JNK) (1:1000, cs 9251), phospho-ERK 1/2 (1:750, cs 9101), phosho-MAPK kinase (phospho-MEK) 1/2 (1:1000, cs 9121), p90RSK (1:1000, cs 9341), phospho-CREB (1:1000, cs 9191), total CREB (1:1000 for cs 9192 or 1:5000 for sc 186X), and -actin (Sigma A5441, 1:10,000 to 1:50,000; Sigma Chemical Co., St. Louis, MO).
MEK kinase (MEKK) assays
Kinase activities of whole-cell extracts of TtT-97 tumors were assayed essentially as described (27) using c-Raf (sc-7267), B-Raf (sc-166), MEKK-1 (sc-437) or MEKK-3 (611102; Transduction Labs, Lexington, KY) immunoprecipitates, [-32P]ATP as a phosphate donor, and recombinant full-length MEK1 (sc-4025) as a substrate.
Statistics
All results were analyzed using a two-way ANOVA and post hoc Tukey paired comparisons. Significance was defined as P value < 0.05.
Animal care
All animals were treated in a humane manner in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and under protocols approved by the committee on animal care and use of the University of Colorado Health Sciences Center.
Results
TH treatment is associated with thyrotropic tumor size reduction
As we have previously shown, treatment with long-term physiological replacement of L-T4 caused a measurable reduction in tumor size (10). In the current studies, after approximately 21 d of L-T4 in the drinking water, tumor size was reduced by 40.15 ± 8.08% (n = 10 mice). We were unable to assess any change in tumor size in the animals that received TH for only 1 d or in those that were withdrawn for 2–3 d, because of the delayed nature of the gross changes in tumor size.
Thyrotropic tumors are responsive to withdrawal of TH as assessed by reactivation of TSH gene expression
To ensure that TH-withdrawn TtT-97 tumors retained their well-characterized response to a hypothyroid state by reexpressing the TSH gene, we measured steady-state TSH mRNA levels by Northern blot analysis. Figure 1 shows that 21-d TH-treated tumors that had high serum TH levels (T4 = 10.18 ± 0.74 μg/dl; n = 10) had a low level of TSH transcripts that dramatically increased after TH deprivation, demonstrating that withdrawal of TH that resulted in hypothyroid serum TH levels (T4 = 0.2 ± 0.17 μg/dl; n = 10) was already exerting an effect by 2 d of hormone withdrawal at the mRNA level presumably by reactivation of TSH gene transcription (30).
TH levels determine the percentage of TtT-97 tumor cells in S-phase
We next assessed tumor growth by quantifying the percentage of cells in the DNA synthetic phase, or S-phase, at each stage of TH treatment and withdrawal. Shown in Fig. 2 are the flow cytometry profiles of TtT-97 tumor cells dispersed from hypothyroid mice (A), mice treated for 1 d with TH (B), and mice TH-treated for 21 d (C) and similarly treated mice after subsequent withdrawal of TH for 2 d (D). It can be clearly seen that the area between the major peaks corresponding to the S-phase population, which is readily visible in the hypothyroid state (Fig. 2A), is noticeably reduced at 1 and 21 d of TH treatment and that this area dramatically reappears in mice withdrawn from TH. When the S-phase percentages were averaged for at least three mice in each thyroidal status group, the number of cells in S-phase significantly (P < 0.05) decreased from 4.2 ± 0.4% in the hypothyroid state to 1.6 ± 0.2% after 1 d of TH treatment. The population of cells within S-phase further declined to 1.2 ±0.6% after 21 d of TH when significant shrinkage of tumor mass was clearly evident. Subsequent TH withdrawal for 2 d resulted in 8 ± 0.9% of the cells occupying the S-phase (P < 0.05 compared with vehicle and TH-maintained mice). The TH-associated changes in growth were verified by staining sections of the tumors from each thyroidal state with an antibody against the proliferation marker PCNA (Fig. 2). Detection of PCNA demonstrated the presence of a sizeable number of positively staining cells in the hypothyroid state (quantitated by visual estimation to be 4.7 ± 1.0%), which then decreased to 2.4 ± 3% after 1 d of TH treatment. After 21 d of treatment, the percentage of PCNA-positive cells declined further to 1.0 ± 5% but then was restored to hypothyroid levels (10.3 ± 5.0%) after 2 d of withdrawal from TH. These changes in cell cycle parameters were also confirmed by visually counting mitotic indices, which also correlated with the TH-dependent growth changes (data not shown).
TH alters the phosphorylation state of Rb
The results of the flow cytometry and PCNA data suggested that TH regulates thyrotropic tumor growth by altering the fraction of cells that is actively synthesizing DNA presumably by blocking entry into S-phase, although the data do not rule out an increased exit from S-phase. To distinguish between these two possibilities, we investigated the effect of TH on the levels and specifically the phosphorylation state of Rb, which regulates the checkpoint of entry into S-phase. Figure 3A shows a Western blot probed with an anti-Rb antibody that revealed that total Rb levels remained relatively similar in the hypothyroid state and after 1 or 2 d of treatment with TH, although a slightly slower migrating band, representing the phosphorylated protein, decreased with TH treatment. Using an antibody raised against Rb activated by phosphorylation at serines 807 and 811 confirmed that phospho-Rb levels progressively diminish with TH treatment maintained for 21 d and that phosphorylation is restored after withdrawal of TH for 2 d (Fig. 3B). Thus, these data suggest that TH blocks entry of thyrotropic tumor cells into S-phase by reducing the amount of phosphorylated Rb that is subsequently rephosphorylated after TH withdrawal, resulting in increased S-phase occupancy.
TH regulates the expression levels of effectors of Rb phosphorylation
We previously showed that TH has profound effects on the transcript levels of certain genes involved in Rb phosphorylation (15). These included decreases in the mRNA levels of cdk2 and cyclin A as well as changes in specific CDK inhibitors. However, many other cell cycle genes including those for cdk4 and -6 and their activating D and E cyclins, as well as the other CDK inhibitors, were relatively unaffected by TH treatment and were therefore unlikely to play a role in the control of thyrotropic tumor cell growth by TH. To see whether the same effectors of Rb phosphorylation were involved in the resumption of growth after TH withdrawal, we employed multitemplate ribonuclease protection assays (RPAs) that allowed the simultaneous assessment of the transcript levels of several catalytic cdks and regulatory cyclins. Figure 4A shows that, as before (15), mRNAs for cdk4 and cdk5, as well as a series of other cdks not previously investigated, were unchanged either by treatment with or withdrawal from TH. Because cdk2 was not represented on the CDK RPA array and because its mRNA level was previously shown to be decreased by TH (15), we performed a comparative Northern blot analysis on poly(A+) RNA derived from a tumor maintained for 21 d on TH vs. a similarly treated one withdrawn from hormone for 2 d. The results in Fig. 4B show that the suppressed expression of cdk2 is reinstituted after TH withdrawal. The RPA analysis for the cyclins is shown in Fig. 5A. Cyclin A transcripts, as before (15), were dramatically down-regulated by TH treatment and subsequently reexpressed after TH withdrawal. In addition, transcripts for cyclins D1 and D3 were also increased, suggesting that restoration of thyrotropic tumor growth by withdrawal of TH, unlike its repression, involved additional pathways mediated by G1-phase cyclins (D1 and D3) that are usually associated with extracellular growth factor stimulation. Figure 5B demonstrates that the effect of TH on cyclin A expression was also exerted at the protein level as demonstrated by Western blot analysis. Interestingly expression of E2F1, a protein that phospho-Rb dissociates from, and that activates S-phase genes, is altered at the transcript level in response to TH (decreased with treatment and increased after withdrawal) (Fig. 6A) but is not affected at the protein level with TH treatment (Fig. 6B)
Expression of regulators of the G2/M transition was altered by TH
The response of other cdks and cyclins to TH, particularly those governing the transition from the G2 phase to mitosis (G2/M) was simultaneously evaluated by the RPA arrays. As is shown in Figs. 4A and 5A, transcripts for both cdk1 and cyclin B1, which phosphorylate substrates related to mitotic spindle formation to facilitate cell division (31, 32), were decreased by TH treatment. After TH withdrawal for 2 d, they returned to levels similar to those in the hypothyroid state and therefore could also be contributing to the cell cycle arrest and resumption of growth associated with exposure to TH and its subsequent withdrawal. Because complexes of cdk1 activated by cyclin B1 are disrupted by the protein GADD45a (33), we sought to determine whether TH also affected the expression of this cell cycle inhibitor. Figure 7 demonstrates that in contrast to the observed down-regulation of cdk1 and cyclin B1, GADD45a transcripts are increased with TH and decreased after its withdrawal. Alterations in GADD45a expression would result in modulation of cdk1 activity, which correlate with the TH-associated growth changes.
Specific MAPK pathways are activated by TH treatment of TtT-97 tumors
We sought to determine the mechanism by which TH regulates the levels of these cell cycle effectors. Those cell cycle regulators shown here to be affected by TH are ubiquitously expressed and are present in cells whose growth is not affected by TH and are therefore probably a secondary target of a pathway that is more restricted to thyrotrope cells. One possibility is that TH mediates its effect via a somatostatinergic pathway. We previously reported that prolonged treatment of TtT-97 tumors with TH was correlated with the appearance of somatostatin binding sites on the surface of these tumor cells and also with increased transcripts for sst5 (22). We also showed that only sst5 transcripts were increased after short-term (1-d) exposure to TH (15). We now show that expression of sst5 transcripts occurs very rapidly, within 6 h of TH administration (Fig. 8A), and could represent a primary transcriptional target of TH. Furthermore, sst5 mRNA decreased to undetectable levels after TH withdrawal (Fig. 8B). Lahlou et al. (27) reported that a somatostatin analog inhibits the growth of CHO cells that have been stably transfected with ssts and that it does this by activating specific MAPK pathways. We therefore wanted to determine whether MAPK pathways were being activated in thyrotrope cells in response to TH. When we examined the levels of activated, or phosphorylated, MAPKs we found that phospho-ERK1/2 and phospho-38MAPK, to a lesser extent, but not phospho-JNK, were increased after treatment with TH for 1 d and even more so after 2 d, whereas total ERK was unchanged (Fig. 9). Several downstream targets of activated ERK1/2, specifically activated p90RSK and phospho-CREB, were also increased by TH, whereas levels of total CREB remained unaffected (Fig. 10). When we investigated the potential upstream kinases that could be activating ERK1/2 we found that levels of phospho-MEK were also elevated in response to TH (Fig. 10). When Lahlou et al. (27) assayed the MEK activating potential of the CHO cell extracts they found that B-Raf kinase, but not c-Raf, was elevated with somatostatin treatment. However, when we assayed extracts of TtT-97 tumors for kinase activity of several of the most commonly reported MEK activators (MEKKs), including c-Raf, B-Raf, MEKK1, and MEKK3 (34, 35), we found that none of them had enhanced MEK-phosphorylating capability as a result of TH treatment (data not shown). When we examined the levels of ERK pathway proteins that were activated by TH treatment, specifically phospho-MEK, phospho-RK1/2, and phospho-CREB, after withdrawal from TH, they decreased to levels similar to or below those in the hypothyroid state (Fig. 11). Lahlou et al. (27) also showed that the Ras-related GTP binding protein Rap1 played an upstream role in the inhibition of growth by somatostatin. We previously reported that a glucocorticoid-responsive Ras family member, Dexras 1, was increased by TH treatment in a microarray analysis (36). Figure 12 confirms by Northern blot analysis that mRNA levels for Dexras 1, which was originally cloned from pituitary-derived cells and exhibits close sequence homology to Rap family members (37), are dramatically increased in TtT-97 tumors by 1 d of TH treatment and subsequently decrease after TH withdrawal.
Discussion
The molecular mechanism by which TH exerts its effects on cell growth remains largely unknown. The major goal of our studies is to understand how TH specifically regulates the growth of thyrotrope cells. Prolonged TH treatment of hypothyroid mice bearing TtT-97 tumors, an excellent thyrotrope model of TH regulation, results in a dramatic reduction in tumor size (10). We previously showed that relatively short-term exposure of thyrotropic tumors to TH for times up to 24 h resulted in changes in the mRNA levels encoding certain growth factors and their receptors as well as transcripts corresponding to key cell cycle mediators including cyclin A and cdk2 (15). In this report, we now demonstrate that modulation of the expression levels of these and other cell cycle regulators by the presence or absence of TH determines the growth state of thyrotropic tumor cells and that this is mediated through reversible changes in the percentage of cells occupying the S-phase of the cell cycle. We also show that this is accomplished by altering the phosphorylation state of Rb, the gatekeeper of the G1- to S-phase checkpoint (11). In thyrotropes, this appears to be achieved by modulating the expression levels of cyclin A and its catalytic partner cdk2, leading to the Rb phosphorylation changes observed. Finally, based on studies reported by Lahlou et al. (27), we postulate a relationship between the expression of sst5, which is rapidly up-regulated by and therefore a putative primary transcriptional target of TH, and somatostatin ligand-triggered activation of the ERK signaling pathway. We demonstrated that the activation state of members of this pathway, including MEK upstream and p90RSK and CREB downstream of ERK as well as the expression of the Ras homolog, Dexras 1, are dependent on the serum TH level sensed by the tumor cells.
Although activation of CREB is usually associated with growth stimulation and cyclin gene activation (38, 39), in some instances it has been shown to lead to inhibition of growth (40, 41) and also of cyclin gene expression (42, 43). The data presented here are consistent with the hypothesis that in thyrotropic tumor cells treated with TH, phosphorylation of CREB, as a result of sustained ERK pathway activation, leads to transcriptional suppression of cyclin A and cdk2 leading to hypophosphorylation of Rb and inhibition of the G1 to S progression. A reversal of this process occurs when TH is withdrawn; sst5 and Dexras 1 levels decrease and the ERK pathway is inactivated resulting in dephosphorylation of CREB and reexpression of the specific cell cycle regulators cyclinA and cdk2.
Interestingly, although we observed little alteration, if any, in D cyclin mRNA levels with TH treatment for 1 d, in a previous report by Northern analysis (15) and confirmed here by RPA arrays, we did see a dramatic increase in both cyclin D1 and D3 transcripts after TH withdrawal. This represented the only difference in the altered expression of several cell cycle mediators that was noted between TH treatment and its subsequent withdrawal and suggests that cyclin D1 and D3 may play a more important role in growth resumption induced by TH withdrawal. A possible explanation for this is that the D cyclins, which preferentially respond to extracellular growth factor stimulation, may be more influenced by a concerted reappearance of stimulatory growth factors and receptors such as the TRH receptor, brain-derived neurotrophic factor/tyrosine kinase B, Wnt10a, and bone morphogenetic protein 4, all of whose expression was shown previously to be repressed by TH (15, 36). In this regard, it has been reported that -catenin, which is stabilized by activation of Wnt pathways, can mediate transactivation of the cyclin D1 promoter in colon carcinoma cells and that this is negatively regulated by TH (44). In contrast, Cheng and coworkers (45) showed that Wnt signaling was silenced in response to TH-stimulated proliferation of pituitary-derived GC cells.
The use of the multitemplate RPA arrays uncovered a novel aspect of cell cycle control by TH of which we were previously unaware. Transcripts for cyclin B1 and cdk1, both of which are involved in facilitating the G2/M transition (46) and subsequent cell division, were decreased by TH and reexpressed after its withdrawal. Interestingly, expression of GADD45a, a p53-regulated stress protein that inhibits cell division by dissociating cyclinB1/cdk1 complexes (33), showed an opposite pattern of expression, being induced by TH and repressed by its withdrawal. Thus, the overall effect of TH on the levels of these key G2/M modulators would be to block progression from the G2 phase to mitosis and thus further contribute to thyrotropic tumor growth inhibition. There are no reports in the literature of TH affecting the expression of cyclin B1 or cdk1 and thus the G2/M transition. However, Cheng and coworkers (47) showed that TR1 could antagonize the function of p53 and, becauseGADD45a is a p53-regulated gene (48), TH could conceivably alter its expression. Although in this fashion GADD45a could represent a transcriptional target for TH, its promoter is also reported to be activated by MAPK (49). Interestingly, the gene for another GADD45 family member has been reported to be deleted in a high proportion of nonsecreting pituitary tumors (50).
Flow cytometry and PCNA immunostaining showed that the proportion of TtT-97 cells in S-phase is a function of thyroidal status, with the presence of TH decreasing the number of replicating cells. Although TH has been reported to stimulate growth, particularly in hepatocytes (51, 52, 53) and pituitary GC cells (45, 47), in other studies it has been shown to decrease the percentage of cells in S-phase. Inhibition of growth by TH has been reported for terminally differentiating oligodendrocytes (54, 55) and chondrocytes (56), murine and rat Sertoli cells (57, 58), and neuroblastoma cells stably overexpressing the TR1 isoform (59). In all of these reports where TH repressed growth, expression levels of cyclins or cdks were not reported to be affected, despite the fact that Rb was hypophosphorylated. Instead, Rb hypophosphorylation was attributed to increased levels of the CDK inhibitor p27. We previously reported no change in the expression of p27 mRNA levels after TH treatment of TtT-97 tumors (15), suggesting a different mechanism for thyrotrope growth inhibition by TH from what is described for these other cell types.
We were surprised to find that TH treatment of TtT-97 thyrotropes, which resulted in growth inhibition, led to sustained ERK activation because suppression of growth is usually associated with down-regulation of MAPK pathways (60, 61, 62). Furthermore, TH-stimulated growth is usually correlated with an activation of MAPK (53, 63). Other hormones, for example steroids, have been shown to stimulate growth and activate ERK pathways as was reported for androgens in LNCaP prostate cancer cells (64) and endometrial carcinomas (65), estrogens in breast cancer cells (66, 67), and aldosterone in rat cardiac fibroblasts (68). In contrast, glucocorticoids inhibited proliferation of smooth muscle cells with no effect on ERK activity or phosphorylation (69). Nevertheless, there have been several reports where growth inhibition or differentiation was associated with MAPK activation as in growth suppression of HL-60 myeloblastic leukemia cells by retinoids (70) and nerve growth factor or cAMP-induced differentiation of neuroendocrine PC12 cells (71, 72). Of particular relevance to the current studies was the finding of Lahlou et al. (27) that CHO cells stably transfected with ssts could be growth arrested by treatment with a somatostatin analog and that this resulted in activation of the ERK cascade. We had previously reported that prolonged treatment of TtT-97 tumors resulted in the up-regulation of sst5 transcripts and the appearance of somatostatin binding sites on the surface of the tumor cells (22). Somatostatin has long been known to possess antiproliferative properties (73) and has been used in the treatment of pituitary tumors (19), particularly those arising from thyrotropes (74, 75). Lahlou et al. (27) went on to show that the MAPK pathway members activated by somatostatin included Src, Ras, Rap1, B-Raf, MEK, and ERK. Here we also showed TH activation of MEK and ERK1/2 as well as the downstream targets p90RSK and CREB in thyrotropic tumors but were unable to identify the activator of MEK. Interestingly, we identified a small GTP-binding Rap1-related protein, Dexras 1, which is expressed in mouse pituitary cells (37, 76) and not previously known to be TH regulated. Furthermore, we demonstrated that Dexras 1 transcripts were increased in TtT-97 tumors by TH and decreased after withdrawal and speculate that Dexras 1 could possibly represent the as yet unknown MEKK activator, although this will have to await future studies.
In summary, we have shown that TH control of thyrotrope cell growth is caused by reversible changes in the number of replicating cells as a result of alterations in the expression of a limited cadre of key regulators that affect cell cycle progression. We also showed that proteins that represent possible primary gene targets of TH action, and that are present in the pituitary, could activate a putative somatostatinergic pathway that leads to activation of a MAPK signaling cascade and culminate in changes in critical G1/S and G2/M transition regulators. Based on studies describing the regulation of growth by other hormones, we believe that the mechanism of TH control of thyrotrope proliferation described here represents a unique mode of hormone-regulated growth.
Acknowledgments
We are grateful to Drs. A. J. Fornace Jr. (Bethesda, MD) and R. J. Kemppainen (Auburn, AL) for the generous provision of plasmids. We also thank Janet Dowding and Amina Gordon for excellent technical assistance and Dr. Andrew Bradford for help with MEK kinase assays.
Footnotes
Flow cytometry was carried out by the University of Colorado Cancer Center Flow Cytometry Core, supported by National Institutes of Health Grant 5 P30 CA 46934. This research was supported by National Institutes of Health Grants CA-47411 and DK-36843 (to E.C.R.) and DK-02813 (to W.W.W.).
First Published Online October 13, 2005
Abbreviations: CDK, Cyclin-dependent kinase; CREB, cAMP response element binding protein; JNK, c-Jun N-terminal kinase; MEK, MAPK kinase; MEKK, MEK kinase; PCNA, proliferating cell nuclear antigen; Rb, retinoblastoma; RPA, ribonuclease protection assay; sst5, type 5 somatostatin receptor; TBST, Tris-buffered saline/Tween 20; TH, thyroid hormone.
Accepted for publication October 3, 2005.
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