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Endocrinology Vol. 142, No. 12 5261-5266
Copyright © 2001 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Identification of a Wnt/ß-Catenin Signaling Pathway in Human Thyroid Cells

K. Helmbrecht, A. Kispert, R. von Wasielewski and G. Brabant

Departments of Clinical Endocrinology (K.H., G.B.), Molecular Biology (A.K.), and Pathology (R.v.W.), Medizinische Hochschule Hannover, D-30625 Hannover, Germany

Address all correspondence and requests for reprints to: Dr. G. Brabant, Medizinische Hochschule Hannover, Klinische Endokrinologie, Carl Neuberg Strasse 1, D-30625 Hannover, Germany. E-mail: brabant.georg{at}mh-hannover.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ß-Catenin is a structural component of the adherens junctions. Outside the adherens junctions a complex consisting of glycogen synthase kinase 3ß, the tumor suppressor adenomatous polyposis coli, and axin constantly targets ß-Catenin for degradation to keep levels of free ß-Catenin low. Free ß-Catenin is able to bind to transcription factors of the T cell factor/lymphoid-enhancing factor family and to stimulate transcription of target genes. This signaling function of ß-Catenin is activated by extracellular Wnt factors that bind to Frizzled receptors and induce inhibition of ß-Catenin degradation.

By RT-PCR and subcloning, we observed the expression of five Wnt factors, three members of the Frizzled receptor family, and all known Disheveled isoforms in thyroid cells. Immunoprecipitation studies demonstrated the formation of the complex targeting ß-Catenin for degradation. Introduction of a degradation resistant ß-Catenin into the thyroid carcinoma cell line WRO induced appearance of monomeric ß-Catenin as shown by size fractionation and nuclear ß-Catenin immunostaining. Reporter gene assays demonstrated a stimulation of T cell factor/lymphoid-enhancing factor-mediated transcription in these cells. In ARO cells, a thyroid carcinoma cell line carrying a mutated adenomatous polyposis coli gene, monomeric ß-Catenin and nuclear immunostaining were observed. In summary, our data indicate that elements of the Wnt signaling pathway are expressed in thyroid cells and that this pathway is functionally active.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ß-CATENIN IS PART of the adherens junctions in epithelial cells that mediate Ca2+-dependent cell-cell adhesion. ß-Catenin binds to the intracellular domain of the transmembrane protein E-Cadherin and connects E-Cadherin via {alpha}-Catenin with actin filaments, a process essential for the formation and maintenance of epithelial tissues (reviewed in Ref. 1).

Free ß-Catenin is constantly targeted for degradation by a multiprotein complex consisting of Axin, the tumor suppressor adenomatous polyposis coli (APC), and the serine/threonine kinase glycogen synthase kinase 3ß (GSK3ß) (2). Axin and APC serve as scaffold proteins that bring GSK3ß and ß-Catenin into close contact, thereby facilitating phosphorylation of ß-Catenin by GSK3ß (3, 4). GSK3ß phosphorylates multiple conserved residues within the N-terminus of ß-Catenin (5). ß-TrCP, an F-box protein homologous to Drosophila slimb, was recently shown to bind to this regulatory domain of ß-Catenin in a phosphorylation-dependent manner, thereby recruiting ß-Catenin into the ubiquitin ligase complex (6). ß-Catenin becomes ubiquitinated and is then degraded in the proteasome resulting in low cytosolic ß-Catenin levels (7).

Recently, a new family of proteins, Wnt factors, has been shown to closely control levels of free ß-Catenin. Wnt factors are secreted glycoproteins that bind to the cell surface receptor Frizzled. Wnt binding to the Frizzled receptor induces the membrane recruitment and phosphorylation of the cytosolic protein Disheveled (8, 9). Wnt signaling antagonizes by a mechanism not fully elucidated the activity of GSK3ß. The multiprotein complex disintegrates (10). As a result, ß-Catenin is no longer targeted for degradation, but accumulates in the cytosol and enters the nucleus. There, ß-Catenin binds to members of the T cell factor/lymphoid-enhancing factor (TCF/LEF) family of transcription factors (reviewed in Ref. 11) and activates expression of target genes, including c-jun, c-myc, fra-1, and cyclin D1 (12, 13, 14).

The highly conserved Wnt signaling pathway regulates cell proliferation, differentiation, and cell fate decisions in species as divergent as nematodes, flies, frogs, and humans (reviewed in Ref. 15). Wnt signaling plays a central role in the development of the central nervous system, kidneys, placenta, reproductive tract, and limbs in vertebrates (16, 17, 18, 19), dorsoventral axis specification in Xenopus laevis (20, 21), tissue polarity in Drosophila melanogaster (reviewed in Ref. 22), and early embryonic cell fate decision in Caenorhabditis elegans (reviewed in Ref. 23).

An increased pool of ß-Catenin is known to have oncogenic potential due to constitutive signaling. Escape of ß-Catenin from degradation is caused by various genetic aberrations affecting components of the Wnt pathway. Originally, the murine Wnt-1 gene was characterized as a mammalian oncogene that becomes activated due to proviral insertion in murine breast cancer (24). Point mutations of the GSK3ß phosphorylation sites of ß-Catenin have been identified in a number of human carcinomas (reviewed in Refs. 25 and 26). Mutations of the tumor suppressor gene APC are detected in the majority of both sporadic and inherited colon cancers (reviewed in Ref. 27), and Axin gene mutations are reported in several human epithelial type carcinomas (28, 29).

An increase in free ß-Catenin might also result from a loss of E-Cadherin expression. This is characteristic of epithelial type carcinomas, including thyroid, and correlates to increased invasiveness of the tumors (30, 31).

Here, we demonstrate that members of the Wnt, Frizzled, and Disheveled gene families are expressed in human thyroid cells, and that the ß-Catenin degradation complex consisting of ß-Catenin, APC, and GSK3ß is formed in human thyroid cells. Free, monomeric ß-Catenin is detected in the thyroid carcinoma cell line WRO after transfection with a mutated degradation-resistant ß-Catenin and in the cell line ARO known to carry a mutation of the APC gene (32). In these cells nuclear localization of ß-Catenin is detected by immunocytochemical staining in contrast to a predominantly membranous localization in WRO cells transfected with an empty vector. TCF/LEF-mediated transcription is enhanced only in WRO cells transfected with a mutated ß-Catenin resistant to degradation. Our results are a first demonstration of a functional Wnt/ß-Catenin signaling pathway in human thyroid cells that might play an important role in proliferation and differentiation and, when dysregulated, in thyroid tumorigenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The cell lines WRO and ARO (33) were kept in RPMI 1640 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FCS (PAA Laboratories, Linz, Austria) in a humidified 5% CO2 atmosphere.

Tissues
Thyroid tissue (nodular goiter, normal tissue adjacent to a thyroid carcinoma) was obtained from patients who underwent surgery at the surgical department of our institution. Patients gave informed consent.

RT-PCR
RNA was extracted from cultured cells using RNA Easy (QIAGEN, Valencia, CA) and from tissues using the cesium gradient centrifugation procedure (34). For first strand cDNA synthesis reaction SuperScript II (Life Technologies, Inc.) was used. PCR was performed using the primers Wnt (forward, 5'-ggggaattctgyaartgygg; reverse, 5'-aaaatctagagcarcaccarwa) (35), Fz (forward, 5'-cagcgtcttgcccgaccagatcca; reverse, 5'-ctagcgccgctcttcgtgtacctg), Dvl (forward, 5'-ggcatctacattggctc; reverse, 5'-gagaaggtgatcttgttgac). To exclude the possibility of the presence of genomic DNA in the reaction, we performed control reactions without the RT step. The RT-PCR products were analyzed on agarose gels and subcloned using the TOPO TA cloning kit (Invitrogen, San Diego, CA). Positive clones were sequenced, and genes were identified by BLAST search.

Sequencing
For DNA sequencing, PCR products were purified using a commercial kit (QIAGEN), and sequencing was performed with the Thermosequenase radiolabeled terminator cycle sequencing kit (Amersham Pharmacia Biotech, Arlington Heights, IL).

Size-exclusion chromatography
Cells were lysed in 20 mM Tris (pH 8), 140 mM NaCl, 10% glycerol, 1 mM EGTA, 1 mM dithiothreitol, 1 mM Pefabloc, and 500 U/ml Trasylol, extracted for 30 min on ice, centrifuged for 10 min at 14,000 rpm, and applied on a Sephadex G-200 column (Amersham Pharmacia Biotech) equilibrated in buffer containing 20 mM Tris (pH 8), 150 mM NaCl, and 0.1% Triton X-100. Fractions of 1 ml were collected and submitted to Western blotting with a ß-Catenin antibody.

Immunoprecipitation
Cells were lysed for 20 min in PBS with 1% Tween 20, and supernatants were collected after centrifugation for 15 min at 15,000 rpm. Ten micrograms of primary antibodies against GSK3ß (Transduction Laboratories, Inc., Lexington, KY) or ß-Catenin (Transduction Laboratories, Inc.) were added, and the mixture was incubated for 1 h at 4 C. Fifty micrograms of antimouse IgG antibody bound to magnetic beads (DNA Research Instruments, Kent, UK) were added, and the mixture was incubated for another 1 h at 4 C. The immune complexes were washed three times in lysis buffer and resuspended in SDS-PAGE sample buffer. For control purposes an unrelated primary antibody was used.

Western blot
Fractions were submitted to SDS-PAGE (7.5%) (36), electrotransferred to nitrocellulose, and incubated with monoclonal antibodies against ß-Catenin (Transduction Laboratories, Inc.). Bound antibodies were visualized with a chemiluminescence detection method. For detection of GSK3ß, ß-Catenin and APC cells were lysed, and cell extracts were submitted to SDS-PAGE and Western blotting with a monoclonal antibody against GSK3ß (1 µg/ml; Transduction Laboratories), ß-Catenin (1 µg/ml; Transduction Laboratories), and APC (1 µg/ml; Oncogene Research, San Diego, CA).

Immunocytochemistry
Cells that were kept on chamber slides (Nunc, Copenhagen, Denmark) were fixed in methanol, incubated with a ß-Catenin antibody (Transduction Laboratories, Inc.), and subjected to the tyramine amplification technique. The cells were counterstained with hemalum.

Reporter gene analysis
WRO cells were transfected with 1 µg TOP Flash or FOP Flash plasmid (Upstate Biotechnology, Inc., Lake Placid, NY), 1 µg pSV ßgalactosidase vector (Promega Corp., Madison, WI), and 1 µg pCI neo containing mutant ß-Catenin (provided by Hans Clevers, Utrecht, The Netherlands) or the empty vector using Lipofectamine (Roche Molecular Biochemicals, Indianapolis, IN). The reporter constructs TOP Flash and FOP Flash contain three optimal copies of the TCF/LEF-binding site (TOP Flash) or mutated copies of the TCF/LEF-binding site (FOP Flash) upstream of a minimal thymidine kinase promoter directing transcription of a luciferase gene. Luciferase and ß-galactosidase activities were measured 48 h posttransfection using a Promega Corp. kit.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Detection of members of the Wnt gene family
We used an RT-PCR and TA cloning approach to detect expression of members of the Wnt gene family in thyroid cells. RNA was prepared from thyroid tissue and analyzed by RT-PCR with degenerated primers that bind to conserved sequences of all Wnt genes (35). The resulting products were cloned using the TA cloning procedure, and random clones were analyzed by sequencing. By BLAST search to confirm identity, the expressions of Wnt-2, Wnt-3, Wnt-4, Wnt-5A, and Wnt-10B were detected in thyroid tissue (Fig. 1Go).



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  		Figure 1. RT-PCR analysis for identification of Wnt, Frizzled, and Disheveled family members expressed in thyroid cells. Primers were chosen to detect multiple isoforms of Wnt, Frizzled, and Disheveled. RT-PCR analysis was performed on RNA from thyroid tissue. A portion of the PCR reaction mixture was cloned using the TA cloning technique, and random clones were sequenced to identify the encoded cDNAs. The data presented is a summary of the isoforms detected by this approach.

 
Detection of members of the Frizzled and Disheveled families
Using the same approach we analyzed expression of members of the Frizzled and Disheveled families. The Frizzled gene family in mammals comprises at least 10 members (15). By RT-PCR and subsequent TA cloning for sequence analysis of the PCR products the expression of three of them, Frizzled-1, Frizzled-2, and Frizzled-6, was detected in thyroid cells (Fig. 1Go). Additionally, expression of all three known Disheveled genes was observed (Fig. 1Go).

Detection of the ß-Catenin degradation targeting complex consisting of GSK3ß, ß-Catenin, and APC
To further determine the presence of intracellular effector molecules of the Wnt signaling pathway we performed immunoprecipitation experiments to detect the presence of the multiprotein complex consisting of GSK3ß, ß-Catenin, and APC. Cell lysates of the human thyroid carcinoma cell line WRO were incubated with antibodies directed against GSK3ß, ß-Catenin, or an unrelated protein for a control, and immune complexes were precipitated. Subsequent Western blot analysis demonstrated the presence of GSK3ß, ß-Catenin, and APC in both immune complexes, indicating formation of the degradation targeting complex in human thyroid cells (Fig. 2Go).



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  		Figure 2. Detection of the complex of GSK3ß, APC, and ß-Catenin by immunoprecipitation. Cell lysates were incubated with a GSK3ß antibody, a ß-Catenin antibody, or an unrelated antibody as a control, and immune complexes were precipitated and subjected to Western blot analysis with GSK3ß, APC, and ß-Catenin antibodies.

 
Regulation of the monomeric ß-Catenin pool
As accumulation of monomeric ß-Catenin is a critical step in Wnt signaling, we examined the pool of ß-Catenin in thyroid cells using size-exclusion chromatography. Free ß-Catenin with a molecular mass of 90 kDa can be easily separated from ß-Catenin that is part of a higher molecular mass complex. Lysates of thyroid carcinoma cell lines were subjected to size fractionation on a Sephadex 200 column, and fractions were processed in Western blot analysis with ß-Cateninspecific antibodies.

In lysates of WRO cells ß-Catenin immunoreactivity was observed in fractions that represent the void volume of the column (200 kDa) indicating that in these cells ß-Catenin is part of a higher molecular mass complex (Fig. 3Go). Disruption of protein complexes by incubation of WRO lysates with SDS was used for detection of free, monomeric ß-Catenin.



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  		Figure 3. Size fractionation of thyroid cell lysates. Size-exclusion chromatography was performed on lysates from WRO and ARO cells, and fractions were analyzed for ß-Catenin by immunoblotting. WRO cells were transiently transfected with an expression vector carrying the cDNA of a mutated ß-Catenin or with the empty vector (pCI neo). The molecular mass of the proteins eluted in the fractions is indicated below.

 
We introduced an expression vector carrying a mutant ß-Catenin cDNA where serine 33 is exchanged to tyrosine. Serine 33 is an essential GSK3ß phosphorylation site, and mutation of this residue prevents ß-Catenin from degradation (5). Size fractionation of a lysate of WRO cells transfected with mutant ß-Catenin revealed the appearance of monomeric ß-Catenin, indicating the accumulation of ß-Catenin not bound to protein complexes (Fig. 3Go).

Next, we tested whether a mutated APC gene induces an increase in the pool of monomeric ß-Catenin. The anaplastic thyroid carcinoma cell line ARO is known to carry a mutation in the APC gene (32). Size fractionation of ARO cell lysates revealed a pool of monomeric ß-Catenin compatible with an escape of ß-Catenin from degradation caused by a defect APC protein (Fig. 3Go).

Localization of ß-Catenin
Accumulation of monomeric ß-Catenin increases nuclear entry of ß-Catenin. There, ß-Catenin binds to transcription factors of the TCF/LEF family, providing a trans-activation domain for stimulation of transcription of a number of genes.

For studying intracellular ß-Catenin localization cells were cultured on coverslips and immunostained with a ß-Catenin antibody. Bound antibody is visualized by red color. In WRO cells a membranous localization of ß-Catenin is observed (Fig. 4Go). After transfection of a mutant Ser33->Tyr ß-Catenin a strong immunostaining of the cytoplasm and the nucleus is observed (Fig. 4Go). For better recognition the nucleus is specifically stained by the blue dye hemalum. Immunocytochemical analysis of ARO cells showed a comparable pattern with a cytoplasmic and nuclear ß-Catenin staining (Fig. 4Go).



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  		Figure 4. Immunocytochemical analysis of WRO and ARO cells with a ß-Catenin antibody. WRO cells were transiently transfected with an expression vector carrying the cDNA of a mutated ß-Catenin or with the empty vector (pCI neo).

 
Regulation of TCF/LEF transcriptional activation
Transactivation of gene expression by the ß-Catenin/TCF/LEF complex represents the nuclear target of the Wnt/ß-Catenin signaling pathway. To measure TCF/LEF-dependent transcriptional activation we performed reporter gene analysis with a vector containing three optimal copies of TCF/LEF sites (TOP Flash) upstream of a thymidine kinase minimal promoter and the firefly luciferase gene as reporter (Upstate Biotechnology, Inc.; Fig. 5Go). A vector with mutated copies of the TCF/LEF (FOP Flash) was used for measuring unspecific trans-activation (Upstate Biotechnology, Inc.). A ß-galactosidase vector was cotransfected for standardization. Cotransfection of TOP Flash with an expression vector containing the cDNA of Ser33->Tyr ß-Catenin induced an 8-fold induction of TCF/LEF-mediated reporter gene transcription compared with cotransfection of the empty vector.



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  		Figure 5. TCF/LEF-mediated transcription in thyroid cells. A reporter construct was transfected that contains three optimal copies of the TCF/LEF-binding site (TOP Flash) or mutated copies of the TCF/LEF-binding site (FOP Flash) upstream of a minimal TK promoter directing transcription of a luciferase gene. Cells were cotransfected with the pCI neo vector containing cDNA of mutated ß-Catenin or with the empty vector (pCI neo). A ß-galactosidase vector was cotransfected for standardization.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data present the first demonstration of the expression of components of the Wnt signaling pathway in human thyroid cells. By RT-PCR using primers binding to conserved regions of Wnt genes and subsequent subcloning for sequence analysis we observed that at least 5 different members of the Wnt family (Wnt-2, Wnt-3, Wnt-4, Wnt-5A, and Wnt-10B) are expressed in human thyroid tissue. The membranous receptors for Wnt ligands, Frizzled, are detectable by the same method, but the pattern of expression appears to be restricted to 3 of 10 currently known members (Frizzled-1, -2, and -6). We detected all members of the intracellular effector molecule of Frizzled, the cytosolic protein Disheveled, which acts on a degradation targeting complex for ß-Catenin. Immunoprecipitation studies indicated that this degradation complex consisting of APC, Axin, and GSK3ß is formed in thyroid cells.

In WRO cells, a thyroid carcinoma cell line expressing E-Cadherin, complexed ß-Catenin is the dominant form in the cytoplasm, and this complex may be dissociated by SDS. As shown in colon carcinoma cells mutations of APC disrupt the ß-Catenin degradation complex (37). Similarly, in ARO cells, an anaplastic thyroid carcinoma cell line carrying a mutation of APC, ß-Catenin is predominantly present in a monomeric, uncomplexed form. Moreover, when we introduced a mutant ß-Catenin where serine 33 is replaced by tyrosine in WRO cells to prevent ß-Catenin from degradation (5), a similar shift to monomeric ß-Catenin was induced. In both cell lines, ARO and Ser33->Tyr ß-Catenin transfected WRO cells, nuclear staining for ß-Catenin appears, indicating that comparable to other epithelial cells free ß-Catenin may be translocated to the nucleus and potentially interact with TCF/LEF transcription factors. Direct evidence for the latter mechanism was provided in studies using a reporter gene construct with optimal TCF/LEF-binding sites driving transcription of a luciferase gene. The activity of this reporter gene construct was approximately 8-fold increased upon cotransfection of a Ser33->Tyr ß-Catenin expression vector, suggesting the functional activity of the pathway.

Based on their biological activities in specific assays, vertebrate Wnts have been divided into two functional groups. Ectopic expression of one group of Wnt factors, including Wnt-1, Wnt-3A, Wnt-7A, and Wnt-8, induces a secondary axis in early Xenopus embryos (38) and transforms C57mg mammary epithelial cells (39). The other Wnt factors, including Wnt-4, Wnt-5A, and Wnt-11, are not axis inducing in Xenopus embryos, nor do they transform C57mg cells (38, 39). However, these Wnts are biologically active, because they alter cell movements and reduce cell adhesion when overexpressed in Xenopus (38, 40). These differences are explained by the existence of two different signaling pathways that are stimulated by Wnt factors. In the so-called canonical Wnt pathway, Wnt factors induce the stabilization of ß-Catenin and the activation of Wnt/ß-Catenin target genes, whereas an increase in intracellular Ca2+ and subsequent activation of PKC is stimulated by Wnt factors in the alternative pathway (41, 42). Among the Wnt and Frizzled families, Xenopus Wnt-5A, rat Frizzled-2, and mouse Frizzled-3, -4, and -6 stimulate Ca2+ release and/or activated PKC, but not Wnt/ß-Catenin targets (43, 44, 45). Although it appears that different Wnt proteins preferentially activate one of these two pathways, this distinction is not absolute, as the activity of Wnt proteins probably depends on the repertoire of receptors on the cell surface (41). Expression of Wnt-2, Wnt-3, Wnt-10B, and Frizzled-1 in human thyroid cells provides candidates for activation of ß-Catenin stabilization and stimulation of Wnt/ß-Catenin target gene expression in thyroid cells. On the other hand, the presence of Wnt-5A, Wnt-4, Frizzled-2, and Frizzled-6 in human thyroid cells suggests the simultaneous presence of alternative Wnt/Ca2+ signaling in human thyroid cells.

Wnt/ß-Catenin signaling is of growing interest for understanding pathological growth. Currently, little is known of effects on altered signaling via the Wnt/Ca2+ pathway, whereas activation of the canonical Wnt signaling pathway was observed in several epithelial type carcinomas (25, 27, 28, 29). In colon carcinomas mutations of components of the complex regulating ß-Catenin degradation initiate oncogenic transformation (46, 47). A role of ß-Catenin in the genesis of thyroid cancer is supported by recent observations of a high frequency of ß-Catenin mutations and nuclear ß-Catenin immunostaining in anaplastic thyroid carcinomas (48, 49). Additionally, papillary thyroid carcinomas associated with familial adenomatous polyposis, a hereditary disorder in which the APC gene is mutated, display nuclear ß-Catenin immunostaining (50). Together with our findings of the presence of all partners of the Wnt signaling pathway, these data suggest that Wnt/ß-Catenin may play an important role in the pathophysiology of thyroid growth and open a new field in our understanding of thyroid carcinogenesis.


   Acknowledgments
 
We thank H. Clevers (Department of Immunology, Utrecht University, Utrecht, The Netherlands) for kindly providing us with the human mutated ß-Catenin cDNA, and B. Bremer (Department of Clinical Endocrinology, Medizinische Hochschule Hannover, Hannover, Germany) for performing sequence analysis.


   Footnotes
 
This work was supported by Deutsche Krebshilfe under Grant 10927/Br4.

Abbreviations: APC, Adenomatous polyposis coli; GSK3ß, glycogen synthase kinase 3ß; TCF/LEF, T cell factor/lymphoid-enhancing factor.

Received April 10, 2001.

Accepted for publication August 17, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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