Growth arrest of thyrotropic tumors by thyroid hormone is correlated with novel changes in Wnt-10A

Molecular and Cellular Endocrinology 238 (2005) 57–67

Growth arrest of thyrotropic tumors by thyroid hormone is correlated with novel changes in Wnt-10A Janice M. Kerr ∗ , David F. Gordon, Whitney W. Woodmansee, Virginia D. Sarapura, E. Chester Ridgway, William M. Wood University of Colorado Health Sciences Center, Department of Medicine, Division of Endocrinology, Metabolism, and Diabetes, MS8106, P.O. Box 6511 Denver, CO 80262, USA Received 7 December 2004; accepted 7 March 2005

Abstract The molecular mechanism underlying thyroid hormone inhibition of thyrotrope cell growth is poorly understood. A comprehensive screen for T3 -regulated genes involved in thyrotrope cell regulation was performed by Affymetrix MGU74A Genechip microarray analyses, which compared total RNA from hypothyroid versus 24 h T3 -treated TtT-97 tumors. Of the 13,000 genes screened, a number of novel, T3 -responsive candidate genes were identified. Within the Wnt family of growth factors, only Wnt-10A transcripts were abundantly expressed in hypothyroid TtT-97 tumors, and were down-regulated with T3 by 6 h of treatment. In addition, nuclear ␤-catenin, which is a downstream mediator of canonical Wnt signaling, was decreased at the protein and functional levels. TtT-97 growth suppression was associated with decreased cyclin A transcript levels. We conclude that treatment of thyrotropic TtT-97 tumors with T3 resulted in the decreased expression of Wnt-10A, and that thyroid hormone may inhibit growth via cyclin A regulation. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Wnt-10A; Thyroid hormone; Thyrotropic tumor

1. Introduction Among the multiple and diverse effects of thyroid hormones (T4 and T3 ), are its central role as a regulator of pituitary thyrotrope function. Thyroid hormones (TH) inhibit thyroid stimulating hormone (TSH) production and thyrotrope cell proliferation (Shupnik et al., 1994). Most of THs actions are mediated by its binding to nuclear thyroid receptors, which are ligand-inducible transcription factors of the steroid hormone/retinoic acid receptor superfamily (Lazar, 1993). The molecular basis of thyroid hormones inhibitory effects on TSH production is at the level of decreased transcription of the ␣- and TSH␤-subunit genes (Shupnik et al., 1985; Chin et al., 1985). The growth-inhibitory effect of TH on thyrotrope cell proliferation has also been well recognized. Protracted hypothyroidism can result in pituitary enlarge∗

Corresponding author. Tel.: +1 303 724 3935; fax: +1 303 724 3920. E-mail address: [email protected] (J.M. Kerr).

0303-7207/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2005.03.004

ment secondary to thyrotrope cell hyperplasia, sufficient to cause compressive symptomatology and panhypopituitarism (Beck-Peccoz et al., 1996). Treatment of hypothyroidism with TH rapidly reverses this pituitary enlargement, although the pathophysiology underlying this inhibitory effect is still poorly understood. As a durable model for thyrotrope hyperplasia, the mouse TtT-97 thyrotropic tumor has been used in our laboratory for several years. The TtT-97 tumor is a well-differentiated murine thyrotrope that secretes TSH and has a retained physiological response to TH (34). Physiological doses of TH in TtT-97 tumor-bearing mice inhibit cell proliferation and result in the gradual tumor involution (Sarapura et al., 1993). Previously, we demonstrated that treatment of TtT-97 tumors with TH is associated with the up-regulation of somatostatin types 1 and 5 receptors, which may mediate tumor involution (James et al., 1997; Woodmansee et al., 2000). Furthermore, utilizing a combination of cDNA microarray, which screened for 1176 transcripts, and Northern blot analyses, we identified several possible primary targets of thyroid

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hormone action (Wood et al., 2002). In particular, decreased expression of genes encoding receptors and ligands with mitogenic potential was observed, including: brain-derived neurotrophic factor, its receptor trkB, and thyrotropin releasing hormone receptor, subtype 1. Using microarrays to study the proliferative effects of TH on GC cells, a somatotrophic rat cell line, Cheng and colleagues identified 358 novel, TH-responsive genes from the 4400 genes screened (Miller et al., 2001). Interestingly, among the changed transcripts, were a paradoxical, down-regulation of six Wnt signaling components. The Wnt pathway is named for its most proximal ligand, Wnt, which activates diverse cellular processes including proliferation, differentiation, and morphogenesis (Cadigan and Nusse, 1997). The best understood Wnt signaling pathways is the “canonical” pathway, which classically regulates cell proliferation through the stabilization of nuclear ␤-catenin. During murine pituitary organogenesis, Wnt ligands act in concert with other extrinsic signals, including bone morphogenetic proteins (BMPs) and fibroblast growth factor (FGF) family members to direct the development of the pituitary anlage and specification of progenitor cell identity (Treier et al., 1998). Wnt 4, in particular, serves an important role in the differentiation or expansion of specific cell types, with Wnt 4−/− null mice showing a reduction in the number of TSH, GH, and glycoprotein ␣-subunit producing cells (Stark et al., 1994). In addition, Wnt 5a is essential for normal pituitary gland formation, as Wnt 5a−/− mice exhibited abnormal dorsal pituitary morphology (Cha et al., 2004). Support for the importance of the canonical Wnt pathway in pituitary ontogeny came from the elegant study by Camper and colleagues (Douglas et al., 2001) in which a partial expression profile of the developing pituitary gland identified the presence of several Wnt signaling pathway members. In this report, we further characterize changes in gene expression of TtT-97 tumors treated with T3 , using a more comprehensive microarray analyses that screened 13,000 genes. We identified the novel expression of Wnt-10A in TtT-97 tumor cells, and its down-regulation with T3 . We also established concordant changes in the down-stream mediator of the Wnt signaling pathway, ␤-catenin, as well as decreased cyclin A levels. Furthermore, we showed changes in the expression of other candidate genes, including: Bone Morphogenetic Protein 7 and GADD45␣.

2. Materials and methods 2.1. Propagation of TtT-97 tumors and treatment with T3 TtT-97 thyrotropic tumor propagation and maintenance in male LAF1 hypothyroid mice were performed as previously described (Wood et al., 1989, 1991). To analyze the effect of thyroid hormone on gene expression, mice were given a single intraperitoneal injection of either L-T3 at a

dose of 10 ␮g/100 g body weight) (+T3 ), or vehicle (10 nM NaOH, denoted – T3 ). Twenty-four hours later, the animals were anesthetized by methoxyflurane inhalation, followed by decapitation. The tumors were then removed, weighed, dissected free of connective tissue, and immediately processed for RNA, protein extraction, or transfection studies as detailed below. For the time course studies, animals were also sacrificed at 2, 6, 10, and 48 h following T3 injection. Blood samples collected at the time of decapitation were analyzed for T3 levels. All vehicle-treated mice were hypothyroid, with T3 levels <10 ng/dl, whereas those given T3 were hyperthyroid with T3 levels in excess of 170 ng/dl at all time points measured. 2.2. Microarray analysis/selection of T3 -responsive genes Sample labeling, microarray hybridization, washing, and scanning were performed according to the manufacturer’s protocols (Affymetrix Inc., Santa Clara, CA). As a preliminary screening for differentially expressed genes, replicate arrays were performed using two different pooled samples, each consisting of total RNA samples from three different TtT-97 tumors. Prior to pooling, the quantity and quality of each RNA sample was assessed by a spectrophotometric comparison of the 260 and 280 nm wavelengths and by visualization of intact 28S and 18S ribosomal RNA bands on agarose gel electrophoresis. Fifteen micrograms of total RNA from both the T3 -treated and vehicle-treated samples was reverse transcribed using a T7-(dT)24 oligomer and Superscript II Reverse Transcriptase (Invitrogen Corporation, Carlsbad, CA). Subsequently, biotin-labeled cRNA was generated from the double-stranded cDNA template by in vitro transcription using T7 RNA polymerase and a BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY). Biotinylated cRNA (20 ␮g) was fragmented to an average size of 35–200 bases with fragmentation buffer [40 mM Tris–acetate, pH 8.1, 100 mM KOAc, 30 mM MgOAc, for 35 min at 94 ◦ C]. The fragmented, biotinylated cRNA (10 ␮g) was then hybridized to the Affymetrix Murine MG-U74A GeneChip array. The arrays were hybridized for 16 h at 45 ◦ C. Following hybridization, the arrays were washed and stained according to the standard Antibody Amplification for Eukaryotic Targets Protocol (Affymetrix, Santa Clara, CA). The GeneChip arrays were subsequently scanned at 488 and 570 nm with a confocal G2500AGeneArray Scanner (Agilent Technologies, Palo Alto, CA). Following data acquisition, the scanned images were quantified according to algorithms of Microarray Suite 5.0 (MAS 5.0) software. The scans from each array were globally scaled by setting the average signal intensity to a target signal of 500. The Affymetrix MG-U74 chip contained 12,626 probe sets with some redundancy for certain genes or splice variants. Pair-wise comparison between the treated and untreated samples was performed, and the fold change derived from

J.M. Kerr et al. / Molecular and Cellular Endocrinology 238 (2005) 57–67

the signal log ratios were averaged for the replicated genes, which were deemed ‘present’ on the detection call. Routine Quality Control parameters including visual array inspection, scaling factor, background noise, 3 /5 Housekeeping Gene ratios, and percent “present” calls were evaluated for each microarray experiment. 2.3. Northern blot analysis Tumors were excised and total RNA extracted from vehicle- and T3 -treated TtT-97 animals using the guanidinium isothiocyanate/CsCl gradient method as previously described (10). Purification of poly(A+) RNA by oligodeoxythymidine (dT) cellulose chromatography and Northern blot analysis, using paired sets of 10 ␮g aliquots of poly(A+)RNA from vehicle and T3 -treated tumors, were performed as described (Wood et al., 2002). The various 32 Plabelled cDNA fragments were generated by nick translation using a commercially available kit (Life Technologies Inc.). The filters were subjected to autoradiography, and probed with a mouse ␤-actin radiolabeled cDNA fragment to assess loading uniformity. 2.4. RNase protection assays Quantitative mRNA expression for Wnt ligands and cellcycle genes was determined using a RiboQuantRPA Kit (PharMingen, San Diego, CA) according to the manufacturer’s instructions, and 15 ␮g of total RNA from vehicle and T3 -treated TtT-97 tumors. 2.5. Western blot analyses Nuclear extracts were prepared from the single-cell preparations of TtT-97 tumors by radioimmunoprecipitation (RIPA) buffer as previously reported (Brugge and Erikson, 1977), with the following final reagent concentrations: 50 mM Tris–Cl, pH 8.0, 0.5% SDS, 5 mM EGTA, pH 8.0, 2 mM EDTA, pH 5.0, 25 mM NaF, 40 mM betaglycerophosphate, pH 7.2, 2 mM Na orthovanadate, 1 mM PMSF, 3 mM benzamidine, 10 ␮g/␮l leupeptin, and 1 mM DTT. The nuclear extracts were diluted with an equal volume of 2× treatment buffer [0.125 M Tris–Cl (pH 6.8), 4% SDS, 20% glycerol and 10% 2-mercaptoethanol], heated at 90 ◦ C for 3 min, then placed on ice. Western blot analyses of the protein extracts were performed as previously detailed (Wood et al., 2002), using the following primary antibodies: ␤-catenin (1:500 dilution, Cell Signal Transduction Labs), Cyclin D1 (1:250 dilution, Santa Cruz Antibodies, Santa Cruz, CA), and Cyclin D2 (1:250 dilution, Santa Cruz Antibodies, Santa Cruz, CA). The primary antibodies were then conjugated to an anti-rabbit horseradish peroxidase secondary antibody (1:5000 dilution, Santa Cruz Antibodies, Santa Cruz, CA), and the proteins detected by a chemiluminescent assay (Amersham Pharmacia Biotech, Piscataway, NJ).

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2.6. Immunohistochemistry staining Immunohistochemistry staining (Forni and de Petris, 1984) was performed on paraffin-embedded, 5 ␮m slices of hypothyroid TtT-97 tumors. Antigen retrieval and deparaffinization was performed by slide immersion in Borg Decloaker (Biocare Medical, pH 9.5), and sample heating for 20 min at 120 ◦ C and 20–25 psi in a Biocare Medical Decloaker Pressure cooker. The slides were then stained with ␤-catenin primary antibody (1:100, Santa Cruz Antibodies, Santa Cruz, CA, sc-7199), and anti-rabbit IgG secondary antibody (Alexa Fluor® 488 goat, 1:200, Molecular Probes Inc., Eugene, OR). Mounting media, consisting of a 1:1 dilution of Vectashield mounting solution and 4 ,6-diamidino-2phenylindole (DAPI), was then applied and the fluorescence was visualized by confocal laser scanning microscopy at the UCHSC Light Microscopy Facility. 2.7. Transient–transfection assays TtT-97 tumors were excised from hypothyroid TtT-97 tumor-bearing mice and single-cell suspensions obtained as previously described (Wood et al., 2002). The cells were treated with 10 ml Trypsin and DNAse I (10 ␮l, 1000 U) at 37 ◦ C for 55 min, and then washed with 50 ml of icecold Hank’s Buffered Saline Solution [(HBSS), 55 mM KCl, 5.0 mM KH2 PO4 , 1.5 M NaCl, 50 mM NaHCO3 , 5 mM NaHPO4 , 50 mM glucose and phenol Red, pH 7.4] supplemented with 0.1% HEPES buffer, 10 U penicillin and 10 ␮g/ml of streptomycin]. The cell count was determined with a hemocytometer and cells were resuspended in HBSS at a density of 50 × 106 cells/ml. Viable cells were assayed by trypan blue exclusion with viability approaching 90%. The cells were then sedimented by centrifugation at 4 ◦ C for 5 min (1000 × g) and resuspended in 1–2 ml of Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% charcoal-stripped fetal calf serum, 0.1% HEPES Buffer, 10 U penicillin, and 10 ␮g/ml of streptomycin. Primary cell cultures of TtT-97, at a concentration of 106 cells/plate, were then transiently transfected, by electroporation, with a ␤-catenin responsive promoter plasmid termed “Super8xTopflash.” As a negative control, “Super8x-FopFlash,” was utilized. A total of 10 ␮g of the reporter constructs, and 10 ng of CMV-Renilla luciferase were transiently transfected into TtT-97, by electroporation, as previously detailed (Sarapura et al., 1990). The cells were incubated in media containing either 10 nM T3 or vehicle (10 nM NaOH) for 24 h. After this time, the cells were harvested in 10 mM potassium phosphate buffer containing 1.0 mM DTT, and lysed with 100 ␮l of Passive Lysis Buffer (Promega Corp., Madison, WI). Thirty microlitres of lysate were assayed for luciferase activity, in duplicates, using a Luciferase Assay System (Promega Corp.). Luminescence was measured as relative light units (RLU) using the Monolight 2010 Luminometer (BD Pharmingen, San Diego, CA), and the luciferase activity

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was normalized against the Renilla activity to adjust for transfection efficiency. 2.8. Statistical methods Statistically significant differences in Super8xTopflash activity (P < 0.05) were calculated by one-way analysis of variance (ANOVA) utilizing Statview 4.0 software (Abacus Concepts, Berkeley, CA). All transfections were performed in triplicate and data represent the mean ± S.D. of three individual experiments. 2.9. Animal treatment Studies on LAF1 mice bearing TtT-97 thyrotropic tumors were conducted with the highest standard of humane animal care in accordance with the NIH guide for the Care and Use of Laboratory Animals. The animal protocols were approved by the Committee on Animal Care and Use at the University of Colorado Health Sciences Center (Denver, CO).

3. Results 3.1. Microarray analysis identified genes responsive to T3 treatment in TtT-97 tumors Affymetrix cDNA microarrays were used to extensively analyze differences in gene expression levels in TtT-97 thyrotropic tumors treated with T3 . Using the murine GeneChip® Genome U74A arrays, a total of ∼6000 functionally characterized genes and ∼6000 EST clusters were studied. Total RNA was isolated from TtT-97 tumors grown in hypothyroid mice treated with T3 (10 nM) or vehicle (EtOH) for 24 h. Differential expression levels of individual genes were then examined by comparing the relative hybridization signal from the vehicle and T3 -treated samples. Using as a selection criteria only those genes deemed “present” on call detection, and exhibiting a ≥2-fold, positive or negative change, in two independent microarray studies, 111 genes were upregulated by T3 and 129 genes were down-regulated by T3 . These genes were classified by broad functional class into 10 groups, with each gene being assigned to a single group. The gene name, Genbank accession number, and fold-change in transcript expression, relative to the hypothyroid state, are shown in Table 1 and Supplement 1. Each value represents an averaged number from the two microarray studies, and included all values that met the original inclusion criteria. The quality of the microarrays was evaluated for each of the four pooled sample. The quality control parameters were excellent and similarly matched between the two microarrays, and included: a high percentage of genes present (>57% ± 1.00), a high actin 3 /5 ratio (1.20 ± 0.03), and a high 3 /5 GAPDH ratio (0.91 ± 0.09). In addition, the microarrays were characterized by similarly low background noise (67 units ± 3.9) and scaling factors (1.8 ± 0.3).

Among the previously reported T3 -responsive genes, TRH-R1, the major isoform that binds thyrotropin releasing hormone, was decreased three-fold with T3 . In addition, Neuronatin (NNAT) was increased seven-fold in response to T3 , as previously demonstrated by Wood et al. (2002). Furthermore, ornithine decarboxylase (ODC), which has been associated with thyroid-hormone responsiveness (Miller et al., 2001), and pituitary tumorigenesis (Evans et al., 2001), was decreased four-fold with T3 treatment in TtT-97 tumors. Within the categories of growth factors and receptors, a number of novel candidate genes were identified, including a four-fold decrease in Wnt-10A expression with T3 -treatment. In addition, there was a striking, but divergent response of various BMPs, including: a seven-fold increase of BMP-7, a two-fold increase of BMP-6, and a 2.5-fold decreased level of BMP-4. Within the classes of transduction molecules and transcription factors, Dexras1, a GTP-binding protein identified in corticotrophs (Kemppainen and Behrend, 1998), was induced six-fold with T3 , and Six5, a member of the Six gene family implicated in progenitor cell proliferation (Li et al., 2002), was decreased four-fold with T3 . Changes in multiple cell-cycle genes, including modulators of the G1/S and G2/M checkpoints, were also noted on the microarray (Table 1). Specifically, Cyclin A2 and cdk6, which are G1/S checkpoint regulators, were decreased significantly by T3 treatment. In addition, the G2/M checkpoint genes: cyclin B1 and cdk1(cdc2A), and multiple minichromosome maintenance (MCM) deficit genes, including: MCM-2,-3,-4, and -6 were decreased with T3 treatment. Within the family of growth suppressor genes, GADD45␣, which modulates the G1/S and G2/M checkpoints (Wang et al., 1999), and apoptosis (Harkin et al., 1999), was increased seven-fold in TtT-97. GADD45␣ (GADD45) is one of three isoforms, including: GADD45␤ and GADD45␥, which are members of the growth arrest and DNA-damage-inducible family of proteins (Fornace et al., 1988). Relevant to pituitary pathology, Klibanski and colleagues demonstrated that the GADD45␥ isoform was absent in a majority of nonfunctional pituitary adenomas (Zhang et al., 2002). The thyroid hormone responsiveness of GADD45 was demonstrated by Cheng and colleagues, who showed that GADD45 transcripts were rapidly induced by thyroid hormone in rat CG cells, which proliferate in response to TH (Miller et al., 2001). In addition, VEGF, which is a key regulator of tumor angiogenesis, was decreased two-fold in TH-treated TtT-97 tumors on the microarrays. VEGF over-expression has been described in several human neoplasms, including functioning and non-functioning pituitary adenomas (McCabe et al., 2002). Lastly, in keeping with thyroid hormones diverse action on cellular function, a number of genes involved in energy metabolism, cell activity, and transcriptional activation were regulated by T3 treatment (Supplement 1). Several of these genes are known to be TH-responsive including, but not limited to: malic enzyme, tubulin alpha, Na+/K+ ATPase beta subunit, fatty acid transport protein 3, bcl-3, ␤-galactoside

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Table 1 T3 -responsive genes in TtT-97 tumors using differential hybridization of Affymetrix Genechip (MGU74) microarrays Gene

GenBank ID

Description

Fold


Growth factors Wnt10a BMP7 BMP6 BMP4 Vegfc Gap43 Blcap

U61969 X56906 X80992 X56848 U73620 AI841303 AW121500

Wingless MMTV integration site-10a Bone morphogenetic protein-7; cell growth, differentiation, and/or maintenance Bone morphogentic protein-6 Bone morphogenetic protein-4 Regulation of angiogenesis Growth associated protein 43 Bladder cancer associated protein homolog

−4.3 7.0 2.0 −2.5 −2.7 −2.8 3.3

Receptors Nnat Sst2 Agpt2 Gpr56 Trhr Gabrg2 Vdr Trfr Ror2 Gfra2 Nr1h4 Nrc31 Folr1 Ptprm Erb b3 Thbd Crlf1 Gpc4

X83569 AF008914 AF004326 AI841654 M59811 M62374 D31969 X57349 AI596034 AF002701 U09416 X04435 M64782 AI847852 AI006228 X14432 AA270365 X83577

Neuronatin; putative calcium channel receptor Somatostatin receptor, subtype 2 Vascular endothelial growth factor receptor binding G-protein coupled receptor activity, unknown ligand Thyrotropin releasing hormone receptor Gamma-aminobutyric acid (GABA-A) receptor; subunit gamma 2 Vitamin D receptor Transferrin receptor Receptor tyrosie kinase-like orphan receptor 2 Glial cell line derived neurotrophic factor family receptor alpha 2 Ligand-dependent nuclear receptor sub-family 1 Ligand-dependent nuclear receptor sub-family 3 Folate receptor 1 Protein tyrosine phosphatase, receptor type, M v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 Thrombomodulin; anticoagulation activity Cytokine receptor-like factor 1 Glypican 4; transmembrane receptor

6.0 4.1 −2.5 −2.1 −3.7 −4.5 2.5 2.0 2.9 −5.1 2.1 2.0 3.3 −2.2 3.1 −2.1 −2.6 −2.6

Signal transduction/regulatory genes Rasd1 AF009246 Rapga1 AI853638 Arhu AW121294 Camk2b X63615 Mapk12 Y13439 Stk6 U80932 Stk18 L29480 Shc3 U46854 Calmbp1 AF062378 Zap70 AI386093 Nek2 AF013166

RAS, dexamethasone-induced 1 (Dexras) Rap1, GTPase-activating protein 1 Ras homolog gene family, member U, GTPase mediated signal transduction Calcium/calmodulin-dependent protein kinase II, beta Mitogen-activated protein kinase 12 Serine/threonine kinase 6 Serine/threonine kinase 18 Src homology 2 domain-containing transforming protein C3 Calmodulin binding protein 1 Zeta-chain (TCR) associated protein kinase NIMA (Never-in-mitosis gene A)-related expressed kinase 2

6.0 −3.2 2.0 2.4 −2.5 −2.9 −2.6 −6.7 −7.0 2.5 −2.6

Transcription factors Tcf712 Crem Elf3 Zfp467 Six5 Bhlhb2 Tcf19 Hmgb2 Lmo1 Fos Trp63 Solt Stac

Transcription factor 7-like 2, T-cell specific, HMG-box cAMP responsive element modulator E74-like factor 3 Zinc finger protein 467 Sine oculis-related homeobox 5 homolog Basic helix-loop-helx domain containing, class B2 Transcription factor 19 High mobility group box 2 LIM domain only 1 FBJ osteosarcom a oncogene Transformation-related protein 63 SoxLZ/Sox 6 leucine zipper binding protein Src homology three (SH3) and cysteine rich domain

−2.2 −2.5 2.7 2.5 −4.0 2.4 −2.2 −2.4 2.4 −3.7 −3.4 −2.9 −3.1

Mini Chromosome maintenance deficient 2; DNA replication Mini Chromosome maintenance deficient 4; DNA replication Cyclin A2; essential for regulation of G1/S and G2/M transitions Cyclin B1; essential for regulation at the G2/M checkpoint Mini Chromosome maintenancy deficient 3; DNA replication Mini Chromosome maintenance deficient 5; DNA replication Cell division cycle 6 homolog

−3.2 −2.6 −3.1 −3.1 −4.9 −6.4 −2.0

X65588 M60285 AF016294 AI551347 D83146 Y07836 AI049398 X67668 AW124311 V00727 AB010152 AA896295 D86639

Cell cycle/apoptosis genes Mcm2 D86725 Mcm4 D26089 Ccna2 X75483 Ccnb1 X64713 Mcm3 X62154 Mcm5 D26090 Cdc6 AJ223087

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Table 1 (Continued ) Gene Mad2I1 Gmnn Mk i67 Cdc2a Cks2 Gadd45a Chek1 Nusap1 Bcl2 Bcl211 Bcl3 Plal1 Reck Kif11 Pak3

GenBank ID

Description

Fold


U83902 AA681520 X82786 M38724 AA681998 U00937 AF016583 AA275196 L31532 AF032459 M90397 X95503 AB006960 AJ223293 U39738

MAD2 mitotic arrest deficient, homolog Geminin; cell cycle Mki67; antigen identified by monoclonal antibody Ki 67; cell proliferation marker Cell Division cycle 2 homolog; required for entry into S-phase and mitosis CDC28 protein kinase regulatory subunit 2 Growth arrest and DNA-damage inducible 45 alpha Checkpoint kinase 1 homolog; essential for G2/M transition Nucleolar and spindle-associated protein 1 B-cell leukemia/lymphoma 2 B-cell Leukemia/lymphoma 11 (apoptosis facilitator) B-cell leukemia/lymphoma 3 Pleiomorphic adenoma gene-like 1 Reversion-inducing-cysteine-rich protein; negative regulation of cell cycle Kinesin family member 11; centrosome separation p21 (CDKN1A) activated kinase 3

−2.4 −2.3 −3.3 −5.6 −3.1 7.5 −2.6 −3.9 −4.6 2.7 3.1 −3.2 3.2 −4.0 −2.2

␣-2,6-sialyltransferase, and FK506-binding proteins (Miller et al., 2001; Viguerie and Langin, 2003; Feng et al., 2000). 3.2. Effects of T3 on Wnt signaling components in TtT-97 tumors A more detailed analysis of Wnt gene transcripts was performed using RNAse protection assays which allowed for the simultaneous evaluation of 15 murine Wnt genes. Analyses

utilizing a Wnt multi-probe template showed the high expression of Wnt-10A in hypothyroid TtT-97 cells, and its downregulation with T3 , as observed on the microarray (Fig. 1A). In contrast, none of the other 14 Wnt mRNAs were significantly expressed, by the RNAse protection assay, suggesting a unique role for Wnt-10A in thyrotrope function the Wnt10A isoform. A time course for changes in the expression of Wnt-10A transcripts in TtT-97 tumors was then assessed by comparing

Fig. 1. (A) Identification of Wnt-10A transcripts in TtT-97 cells. RNase protection assays using 15 ␮g total RNA from hypothyroid (-T3 ) and 24 h T3 -treated (+T3 ) TtT-97 tumors simultaneously evaluated 15 Wnt ligands (Wnts #1-6, Left panel and Wnts #7a-15, Right panel). Shown in the left-most column of each radiogram is the undigested probe. A T3 non-regulated L32 transcript, provided in each kit, was used to evaluate loading uniformity. Autoradiographic exposure was performed at −80 ◦ C for 24 h. (B) Effect of time of T3 treatment on transcripts encoding Wnt-10A. A full-length coding region probe for mouse Wnt-10A was excised from plasmid provided by Dr. G. Shackelford, radiolabeled with 32 P, and analyzed by Northern blot analyses. Autoradiographic exposure was performed at −80◦ C for 24 h. The membrane was reprobed with a 32 P-labeled mouse ␤-actin fragment and subjected to autoradiography for 6 h. Radioactive bands corresponding to Wnt-10A mRNAs are shown with the approximate size in kb, derived using molecular weight standards loaded in a parallel lane.

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mRNA levels at 2, 6, and 24 h post-treatment. Northern blot analyses using 10 ␮g of poly(A+) mRNA showed that Wnt-10A levels were persistently decreased by 6 h after T3 treatment (Fig. 1B). This early change in mRNA expression suggests that Wnt-10A may be a primary transcriptional target for T3 in thyrotropes. Wnt signal transduction is mediated through a receptor complex comprised of Frizzled (Fzd) receptors (Bhanot et al., 1996). In order to characterize the Fzd receptor profile in our TtT-97 thyrotropes, we performed reverse transcriptionpolymerase chain reaction using hypothyroid TtT-97 RNA and primers specific for each frizzled receptor (Xu et al., 2001). Surprisingly, 6 of the possible 9 Fzd receptors were detected, including Frizzleds 1, 4, 5, 6, 7, and 9 (data not shown). Therefore, the specific Fzd receptor(s), mediating Wnt-10A signaling in TtT-97 cells is still unknown. Regarding changes in Fzd receptor expression with T3 treatment, the microarray data showed no appreciable change in the hybridization signal of several Fzd receptors, including: 1,4,6, and 7 (data not shown). It is possible that additional negative regulators of Fzd receptors, such as members of the secreted Frizzled-related proteins, may be important factors in determining differential receptor response. ␤-catenin is the important down-stream mediator of the canonical Wnt signaling pathway (Willert and Nusse, 1998), and was next evaluated in TtT-97 cells. To confirm the presence of ␤-catenin in TtT-97 cells, and determine its subcellular location, immunohistochemistry staining of hypothyroid samples was performed. Using a fluorescent primary anti-

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body against ␤-catenin and 4 ,6-diamidino-2-phenylindole (DAPI) staining to delineate the nucleus, nuclear ␤-catenin was not detected. Instead, an outer membranous predominance of ␤-catenin was observed, which is consistent with its known role in the cytoskeleton at adherens junctions (data not shown). To better assess levels of nuclear ␤-catenin, which is a mediator of transcriptional activation, Western blot analyses were performed using TtT-97 nuclear protein extracts. Comparison of hypothyroid animals versus 24 h T3 -treated animals demonstrated a significant decrease in ␤-catenin levels in the nucleus, consistent with down-regulation of the Wnt pathway (Fig. 2A). As a control, TtT-97 whole-cell lysates were evaluated, and total ␤-catenin levels were unchanged between the hypothyroid and T3 -treated animals (Fig. 2B). To further establish the functional significance of endogenous beta-catenin on gene activation, transient transfection studies were performed using primary cell cultures of TtT-97 tumors, and a well-established reporter assay for ␤-catenin/TCF activation (Super8xTopflash). This construct consists of eight TCF-4 binding elements fused upstream of a minimal promoter, which drives a luciferase reporter (Veeman et al., 2003). As a negative control, a vector with mutated TCF binding sites (Super8xFopflash), was used. The transfection studies of hypothyroid TtT-97 tumors demonstrated a 2.4-fold increase in the ␤-catenin/TCF response promoter, relative to the Super8xFopflash, and a subsequent 64% decreased activation with T3 treatment (P < 0.05) (Fig. 2B). These results are consistent with a T3 -mediated down-regulation of the canonical signaling pathway, presumably activated by Wnt-10A.

Fig. 2. (A) Decreased nuclear beta-catenin protein levels in TtT-97 thyrotrope cells following in vivo T3 treatment. Shown in the left panel is a Western blot analysis of 100 ␮g nuclear cell extracts from hypothyroid TtT-97 tumors (-T3 ) vs. 24 h T3 -treated (+T3 ) using an antibody against mouse beta-catenin. Loading uniformity was evaluated by comparison of actin levels. (B) Unchanged whole cell beta-catenin levels in TtT-97 thyrotrope cells following in vivo T3 treatment. Shown in the left panel is a Western blot analysis of 100 ␮g whole cell lysates from hypothyroid TtT-97 tumors (-T3 ) vs. 24 h T3 -treated (+T3 ) using an antibody against mouse beta-catenin. Loading uniformity was evaluated by comparison of actin levels. (C) Transient Transfection of TtT-97 tumors with the ␤-catenin/LEF reporter “Super8xTopFlash,” comparing hypothyroid (-T3 ) vs. T3 -treated for 24 h (+T3 ). Experiments were performed in triplicates, and the relative light units (RLU) are reported relative to the negative control “Super8xFopFlash.”

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3.3. Effect of T3 on PITX2 and cell-cycle genes in TtT-97 tumors Putative target genes of the ␤-catenin/TCF transcriptional complex were then studied to identify mediating factors of growth inhibition by T3 in TtT-97 tumors. Relevant to the pituitary gland, Rosenfeld and colleagues showed that PITX2, a transcription factor important for the development of thyrotropes, gonadotropes and somatotropes, was directly induced by the Wnt signaling pathway in ␣T3 -1 cells, a murine gonadotrophic cell line (Kioussi et al., 2002). Furthermore, they demonstrated that PITX2 activation resulted in the induction and/or stabilization of cyclin D1, cyclin D2, and c-

Myc transcripts (Kioussi et al., 2002; Briate et al., 2003). We subsequently studied PITX2 expression in TtT-97 tumors, in response to T3 , utilizing probes specific for the three isoforms, denoted a, b and c. Northern blot analyses comparing hypothyroid versus T3 -treated animals for 24 h, showed no significant change in any of the three PITX2 isoforms (Fig. 3A). As for cell-cycle genes, the microarray results showed a three-fold reduction of cyclin A2 and cyclin B1 transcripts, whereas cyclin D1, D2, and D3 levels were decreased less than two-fold. These changes in cell-cycle genes were confirmed by RNase protection assay (Fig. 3B). A concomitant decrease in cyclin A mRNA (Wood et al., 2002) and protein levels (unpublished data) after T3 treatment was observed. Conversely, both cyclins D1 and D2 protein levels were not significantly changed after 24 h in TtT-97 tumors (Fig. 3C).

4. Discussion

Fig. 3. (A) Effect of T3 treatment on transcripts encoding PITX2 Isoforms. Full length coding regions for the PITX2 isoform (a–c), provided by Dr. Sally Camper, were excised from plasmids, radiolabelled with 32 P, and analyzed by Northern blot analyses as described in Fig. 1B comparing hypothyroid (T3 ) vs. T3 -treated animals (+T3 ). (B) Decreased Cyclin A2, B1, D1, and D3 transcript levels in TtT-97 thyrotrope tumors following in vivo T3 treatment. Shown is the RNAse protection assay of 15 ␮g total RNA from TtT-97 tumors before (-T3 ) and after T3 treatment (+TH) for 24 h. (C) Unchanged cyclin D1 and D2 protein levels in TtT-97 thyrotrope tumors following in vivo T3 treatment. Shown is the Western blot analysis of 100 ␮g nuclear protein extracts from TtT-97 tumors before (-T3 ) and after T3 treatment (+T3 ) for 24 h.

The molecular mechanism underlying thyroid hormone inhibition of thyrotrope proliferation is poorly understood. Building upon our initial work of identifying T3 -regulated genes in TtT-97 tumors (Wood et al., 2002), we performed a more comprehensive microarray analysis utilizing the Affymetrix murine genechip. The microarrays identified changes in genes previously reported in association with thyroid-hormone responsiveness and/or abnormal pituitary cell growth. Among these genes were: GADD-45␣ (Miller et al., 2001), ornithine decarboxylase (Evans et al., 2001), and VEGF (McCabe et al., 2002). A novel candidate gene, Wnt-10A, was identified on the microarray, that may play a role in TtT-97 cell growth. Wnt10A transcripts are differentially expressed in embryonic and adult murine tissues, with very high levels of expression in the mature pituitary gland (Wang and Shackleford, 1996). The cell-type specificity of Wnt-10A and its exact role in the mature pituitary gland, however, are still unknown. We speculate that this ligand may play an important role in pituitary gland homeostasis, including thyrotrope hyperplasia, based on its high expression in hypothyroid TtT-97 tumors, its down-regulation with T3 , and the classical role of the canonical Wnt signaling pathway in cell proliferation. The signaling pathway transduced by Wnt-10A, in our TtT-97 tumor model, presumably involves the canonical Wnt pathway, based on our findings of decreased ␤-catenin levels at the mRNA and protein levels with T3 treatment. In addition, functional studies utilizing a ␤-catenin reporter showed decreased TCF transactivation, consistent with a down-regulation of nuclear ␤-catenin levels with T3 treatment. The role of ␤-catenin/TCF signaling in normal cell physiology is not well understood. Several downstream target genes of the ␤-catenin/TCF activation have been identified, mostly in human colon cancer cell lines, and include: cyclin D1 (Tetsu and McCormick, 1999), cyclin D2 (Mann et al.,

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1999), c-Myc (He et al., 1998), BMP-4 (Kim et al., 2002), FGF-4 (Klaus et al., 2002) and VEGF (Zhang et al., 2001). These genes are classically involved in normal growth, although over-expression from constitutive ␤-catenin activation has been demonstrated in tumorigenesis. Relevant to mature pituitary cells, Rosenfeld’s group demonstrated that activation of the canonical Wnt signaling pathway, in ␣T3 cells, resulted in the rapid induction of PITX2, which in turn directly activated and stabilized cyclins D1 and D2 transcripts, and induced c-Myc expression (Briate et al., 2003; Kioussi et al., 2002). It is unlikely that Wnt signaling is functioning in TtT-97 tumors by a similar mechanism, since PITX2 transcripts were unchanged with early T3 treatment. Furthermore, no significant changes in c-Myc transcripts (Wood et al., 2002), cyclin D1 and D2 mRNA (Wood et al., 2002) and cyclin D protein levels (Fig. 3C) were detected after T3 treatment. The factors that regulate Wnt ligands, in general, are also poorly characterized. To date, only one study of the effects of the steroid hormone superfamily members on Wnt-10A expression has been reported. Kirikoshi et al. demonstrated that Wnt-10A mRNA expression was unchanged by either retinoic acid treatment in NT2, a neuronal cell line, or by ␤-estradiol treatment in MCF-7 cells, a mammary cancer cell line (Kirikoshi et al., 2001). Relevant to our TtT-97 thyrotropic tumor model, there are five consensus thyroidhormone response element half-sites, defined by the sequence AGGTCA, within 1.5 kb of the Wnt-10A putative transcription start site. We speculate that some of these sites may function as negative regulators of Wnt-10A expression by T3 . As for regulation of Wnt signaling downstream of the Wnt ligand, there are multiple lines of evidence to support the role of nuclear hormone receptors in modulating ␤-catenin/TCF transcriptional activity. Nuclear hormone receptors including those for retinoic acid (Easwaran et al., 1999), vitamin D3 (Palmer et al., 2001), and androgen (Pawlowski et al., 2002) have been shown to repress ␤-catenin/TCF activation in a ligand-dependent manner. Relevant to thyroid hormone, Natsume et al. investigated the effect of T3 on the cyclin D1 promoter in 293T cells, a murine kidney cell line, and found that the transactivation by ␤-catenin/TCF/Lef-1 complex is inhibited by TR␤1 in a ligand- and dose-dependent manner (Natsume et al., 2003). It is possible, therefore, that crosssignaling with T3 and the canonical Wnt signaling pathway occurs at multiple levels, including the proximal ligand and the downstream TCF/LEF transcriptional complex. The finding of decreased cyclin A transcript levels in TtT97 tumors treated with T3 for 24 h, suggests that T3 -mediated growth arrest may occur partly through cyclin A regulation. In brief, cyclin A1 is implicated in cell cycle control in early mammalian embryogenesis (Sweeney et al., 1996), whereas cyclin A2 (or cyclin A) is expressed primarily in adult tissue. The cyclin A isoforms are the regulatory subunits of the cyclin-dependent kinases, cdk2 and cdk1, which are required for S phase progression and the G2/M transition, re-

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spectively (Tsai et al., 1991; Pagano et al., 1992). The microarray data demonstrated a three-fold reduction of cyclin A2 with T3 -treated TtT-97 cells, whereas cyclin A1 levels were not significantly changed. This finding was corroborated by an RNase protection assay, which evaluated cyclin A2 exclusively. Wood et al. previously reported a decreased expression, of both cyclin A1 and A2 transcripts, with a cyclin A2 predominance, and a corresponding decrease in cyclin A protein levels (unpublished data). We speculate that Wnt10A signaling may act in TtT-97 tumors through the novel mechanism of cyclin A regulation. This is supported by the observation that the murine cyclin A promoter contains two putative ␤-catenin/TCF consensus sequences, defined by the sequence 5 -A/TA/TCAAAG-3 , which are located approximately 2 kb upstream of the major transcriptional start site. Obviously, further studies are needed to establish the functional significance of Wnt-10A expression on cyclin A regulation in TtT-97 tumors. In conclusion, microarray analyses have identified the unique presence and high expression of Wnt-10A in TtT97, and its down-regulation with T3 . Additionally, the downstream mediator of the canonical Wnt signaling pathway, ␤catenin, was decreased at the transcript and protein levels. Furthermore, functional studies, using a TCF/LEF reporter, showed decreased ␤-catenin-mediated transactivation with T3 treatment in TtT-97 cells. Based on the presence of putative beta-catenin responsive sites in the cyclin A promoter, we speculate that Wnt-10A may be mediating growth in TtT-97 cells by this novel mechanism. These studies pave the way for further research to elucidate the mechanisms by which Wnt-10A regulates growth in thyrotrope cells. In addition, a number of other candidate genes from the family of growth factors and apoptotic genes were changed in response to T3 treatment in TtT-97 tumors, including: BMP-7 and GADD45␣, and warrant further investigation regarding their potential importance in regulating thyrotrope cell growth.

Acknowledgments We would like to thank Drs. Margaret Wierman and John Pawlowski at the University of Colorado Health Sciences Center for their helpful discussions. We are grateful to the following people for their generous provision of plasmids: Drs. Sally Camper (University of Michigan), Gregory Shackelford (Children’s Hospital Los Angeles Research Institute), Bert Vogelstein (John Hopkins University), Randall Moon (University of Washington), and Albert Fornace (National Cancer Institute). We would also like to thank Danielle Haakinson, Janet Dowding, and Elizabeth Tucker for their outstanding assistance. Furthermore, we acknowledge the excellent technical expertise of the Affymetrix Core Facility, the Light Microscopy Facility, and the DNA sequencing laboratory at UCHSC. This research was support by Public Health Service grants: CA47411, DK07446, and DK02813 from the

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National Institute of Health and the National Cancer Institute. Additional funding was provided by the Thorkildsen Research Fellowship Grant.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mce.2005. 03.004.

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