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Endocrinology, doi:10.1210/en.2006-0923
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Endocrinology Vol. 148, No. 3 1306-1312
Copyright © 2007 by The Endocrine Society

Gelsolin: A Novel Thyroid Hormone Receptor-ß Interacting Protein that Modulates Tumor Progression in a Mouse Model of Follicular Thyroid Cancer

Caroline S. Kim, Fumihiko Furuya, Hao Ying, Yasuhito Kato, John A. Hanover and Sheue-yann Cheng

Laboratory of Molecular Biology (C.S.K., F.F., H.Y., Y.K., S.C.), Center for Cancer Research, National Cancer Institute; and Laboratory of Cellular Biochemistry and Biology (J.A.H.), National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892-4264

Address all correspondence and requests for reprints to: Dr. Sheue-yann Cheng, National Cancer Institute, 37 Convent Drive, Room 5128, Bethesda, Maryland 20892-4264. E-mail: chengs{at}mail.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Follicular thyroid cancer (FTC) is known to metastasize to distant sites via hematogenous spread; however, the underlying pathways that contribute to metastasis remain unknown. Recent creation of a knockin mutant mouse that expresses a mutant thyroid hormone receptor-ß (TRßPV/PV mouse) that spontaneously develops thyroid cancer with metastasis similar to humans has provided new opportunities to study contributors to FTC metastasis. This study evaluates the role of gelsolin, an actin-regulatory protein, in modulating the metastatic potential of FTC. Gelsolin was previously found by cDNA microarray analysis to be down-regulated in TRßPV/PV mice as compared with wild-type mice. This study found an age-dependent reduction of gelsolin protein abundance in TRßPV/PV mice as tumorigenesis progressed. Knockdown of gelsolin by small interfering RNA resulted in increased tumor cell motility and increased gelsolin expression by histone deacetylase inhibitor (trichostatin A) led to decreased cell motility. Additional biochemical analyses demonstrated that gelsolin physically interacted with TRß1 or PV in vivo and in vitro. The interaction regions were mapped to the C terminus of gelsolin and the DNA binding domain of TR. The physical interaction of gelsolin with PV reduced its binding to actin, leading to disarrayed cytoskeletal architectures. These results suggest that PV-induced alteration of the actin/gelsolin cytoskeleton contributes to increased cell motility. Thus, the present study uncovered a novel PV-mediated oncogenic pathway that could contribute to the local tumor progression and metastatic potential of thyroid carcinogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID CANCER IS the most common endocrine malignancy and its incidence is growing. Differentiated thyroid cancer arises from follicular epithelial cells and can be classified into several subtypes, with the most common being either papillary or follicular thyroid cancer. Follicular thyroid carcinoma (FTC) differs from papillary in its predisposition for hematogenous vs. lymphatic spread, with its preferred metastatic sites being the lungs and bone (1). Although the majority of thyroid cancer cases follow a benign course, a small subset develop metastatic disease for which there are limited treatment options. To date, no reliable markers can predict the metastatic potential of FTC, and the biology of FTC metastasis remains an open field of investigation.

Progress in understanding the molecular basis of thyroid tumorigenesis has been advanced through studies of the TRßPV/PV mouse model (2). The TRßPV/PV mouse expresses a dominant-negative mutation in the thyroid hormone receptor (TR)-ß, denoted as PV. The PV mutation was identified in a patient with resistance to thyroid hormone (RTH). RTH is caused by mutations of the TRß gene and manifests symptoms as a result of decreased sensitivity to the thyroid hormone (T3) in target tissues (3). PV has a C insertion at codon 448 that produces a frame shift in the carboxyl-terminal 14 amino acids of TRß1 (4). PV has completely lost T3 binding and exhibits potent dominant-negative activity (5). As TRßPV/PV mice age, they spontaneously develop follicular thyroid carcinoma similar to human thyroid cancer with pathological progression from hyperplasia to vascular invasion, capsular invasion, anaplasia, and eventually metastasis (2, 6, 7).

The similarities in FTC progression between this model and humans provide unique opportunities to evaluate the changes during tumor progression and metastasis. Our previous microarray analysis comparing the thyroids of TRßPV/PV and wild-type mice identified genes differentially expressed in the mutant mice (6). Functional classification of these candidate genes suggested that the actin-regulatory protein gelsolin could play a role in thyroid carcinogenesis as indicated by a 5-fold down-regulation in the thyroid of the TRßPV/PV mouse (6). Gelsolin is an actin-binding protein, and in the presence of calcium, it is able to sever actin and cap the growing end of the released filament (8). It is involved in controlling cell morphology, motility, growth, and apoptosis (9). Phosphoinositides can bind to gelsolin and regulate its interaction with actin (9). Its important role in phospholipid signaling pathways was recently demonstrated by the observation that gelsolin-induced epithelial cell invasion is dependent on phosphatidylinositol 3-kinase-Rac pathways (10).

Expression of gelsolin is frequently reduced in many cancers including breast (11), stomach (12), colon (13), lung (14), bladder (15), prostate (16), and kidney (17) in both cancer cell lines and human tumors. These reports suggest the critical role of gelsolin in tumor progression. Whereas Aldred et al. (18) reported that gelsolin was down-regulated more than 2-fold in over 87% of pairwise comparisons in human FTC with normal thyroid tissue by microarray analysis, no studies have yet focused specifically on gelsolin’s role in thyroid cancer. Therefore, the role of gelsolin in thyroid carcinogenesis remains unknown.

The observation that the expression of gelsolin is significantly reduced during thyroid carcinogenesis of TRßPV/PV mice provided us with an opportunity to investigate its role in thyroid cancer. We discovered that PV-induced reduction of gelsolin expression increased tumor cell motility. Furthermore, we found that gelsolin physically interacted with TRß1 via its C-terminal region. The association of gelsolin with PV interfered with the interaction of gelsolin with actin, resulting in altered cytoskeletal architectures that affected cell motility. Thus, the present study uncovered not only an important role of gelsolin in local progression and metastatic potential of tumor cells but also a novel PV-mediated pathway contributing to thyroid carcinogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse strains and treatment
All aspects of the care and handling of the animals used in this study were approved by the National Cancer Institute Animal Care and Use Committee. TRßPV mice were generated via homologous recombination, as previously described (19). Genotyping of mice was performed using the primers and method previously reported (19).

Western blot analysis
Immunoblotting was performed as previously described with adjustments as described below (20). Thyroid lysate was prepared from age- and gender-matched mice using a final concentration of 50 mM Tris, 100 mM NaCl, 0.1% Triton X-100, protease inhibitors (1 mM phenylmethylsulfonylfluoride, 0.3 µM aprotinin, and 0.4 mM leupeptin = Complete Mini tab; Roche, Mannheim, Germany). A total of 50 µg of protein was loaded for each sample. Experiments were repeated three times using different groups of mice.

Lysate from primary thyroid cells was prepared as previously described (21). For each sample, 50 µg of protein were loaded. Antibodies that were used included gelsolin (graciously provided by Dr. Hisakazu Fujita, Hokkaido University, Sapporo, Japan, used at 1:20,000 dilution), protein disulfide isomerase at 0.5 µg/ml (no. 3632), and {alpha}-tubulin (T6199, used at 1:3000 dilution; Sigma, St. Louis, MO). Secondary antibodies included horseradish peroxidase-conjugated goat antimouse or antirabbit IgG (Amersham Biosciences, Piscataway, NJ) and were visualized with the Western Lightning chemiluminescence reagent plus system (PerkinElmer Life Sciences, Boston, MA). The blots were stripped with Re-Blot Plus (Chemicon, Temecula, CA) according to the manufacturer’s instructions and normalized to protein disulfide isomerase (PDI).

Primary cell culture and trichostatin A (TSA) treatment
Primary thyroid tumor cell lines from TRßPV mice were prepared and cultured at 37 C, 5% CO2 atmosphere in primary cell media as detailed previously (21). Cells were then exposed to TSA (Sigma) at a concentration of 500 ng/ml or an equivalent volume of dimethylsulfoxide (Sigma). Afterward, the primary thyroid tumor cells were either used for Western blot analysis or cell motility experiments as described below.

Cell motility
A modified Boyden chamber assay using serum as chemoattractant was performed as previously described (21). The assays were performed in 8-µm-pore transwells (6.5 mm; Costar, Corning, NY) in triplicate. Primary thyroid tumor cells were incubated at 37 C for 5 h. The wells were then decanted and the cells fixed in 1% glutaraldehyde in PBS (Sigma), stained with 0.1% crystal violet (Sigma) in water for 30 min. The wells were destained by rinsing with deionized water. Using separate cotton-tipped applicators soaked in 0.2% Triton X-100 (Sigma), both the nonmigrating cells on the upper side of the well and the migrating cells on the lower side were removed and placed in 1 ml of 0.2% Triton X-100. These samples were kept at 4 C overnight. The solution of each sample was measured at A590 using a spectrophotometer. Two independent experiments were performed in triplicate. Fold of increase in motility was calculated by dividing experimental percent motility to control percent motility.

Gelsolin small interfering (si) RNA
Mouse Stealth gelsolin siRNA was designed using the BLOCK-IT RNAi Designer Web site (Invitrogen, Carlsbad, CA). The siRNA gelsolin (siRNA gelsolin 703) sequences used were as follows: sense: 5'-GGC UCU GCC AGC AAC AAA UUU GAA A, antisense: 5'-UUU CAA AUU UGU UGC UGC CAG AGC C. Primary thyroid cells, whose preparation is described above, were grown in 60-mm dishes until approximately 30–50% confluent. Cells were then transfected with 100 nM siRNA using Lipofectamine 2000 (Invitrogen) per the manufacturer’s instructions. Cells were incubated for 4 d and then transfected again with gelsolin siRNA and incubated for 3 additional days before use in experiments. Control cells were transfected with Lipofectamine 2000 (Invitrogen) alone. Afterward, the primary thyroid cells were used for either Western blot or cell motility experiments as described above.

GST-binding assay
Binding of 35S-labeled TRß1 or PV to GST-gelsolin (GST-GSN) or truncated GST-gelsolin (GST-GSNc) was carried out as described previously with modifications (22). The plasmids GST-GSN and GST-GSNc were a kind gift from Dr. Chawnshang Chang (University of Rochester Medical Center, Rochester, NY). GST-GSN represents full-length human gelsolin, whereas GST-GSNc contains the carboxyl-terminal of gelsolin (23). A TNT kit (Promega, Madison, WI) was used to synthesize in vitro-translated 35S-labeled TRß1 and PV, which were later incubated with GST-GSN or GST-GSNc at 4 C for 24 h with constant shaking. Truncated TR constructs were previously described (20). The beads were then washed five times and the bound proteins were analyzed by SDS-PAGE; 20% input was loaded.

Coimmunoprecipitation
To analyze the association of TRß with gelsolin in cells, CV-1 cells were transfected with pFlag(f)-TRß1 in either the presence or absence of T3 in a manner similar to that described previously (20). Cell lysates (1.5 mg) were immunoprecipitated with 8 µg anti-Flag antibody or 8 µg antigelsolin antibody (kindly provided by Dr. Hisakazu Fujita, Hokkaido, Japan), followed by Western blot analysis using antigelsolin antibody or anti-Flag antibody, respectively. Rabbit IgG and mouse IgG (MOPC, Sigma M9269) were used for negative control samples.

Coimmunoprecipitation assays were also used to demonstrate the association of endogenous gelsolin with TRß1 or PV. Cells were transfected with 8 µg of pFlag(f)-TRß1 or pFlag(f)-PV with lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. Cells were cultured in medium with or without T3 (100 nM) and incubated for an additional 48 h. To determine the TR binding with gelsolin in TRß1 or PV-transfected PC cells, 500 µg of protein were treated with anti-FLAG M2 affinity gel (20 µl; gel suspension concentration 50%) (Sigma A2220) for 3 h according to manufacturer’s instructions. Western blot analysis was carried out as described above.

To analyze the association of gelsolin with ß-actin, thyroids from TRßPV/PV mice and wild-type mice were dissected. The gelsolin protein abundance of the thyroid extracts from wild-type and TRßPV/PV mice was first quantified by Western blot analysis. Thyroid extracts (1000, 750, 500, 250, and 125 µg) of wild-type mice and the corresponding TRßPV/PV thyroid extracts containing an equal amount of gelsolin at each extract concentration were immunoprecipitated with 4 µg antigelsolin followed by Western blot analysis using anti-ß-actin antibody. Similarly extracts containing equal amounts of actin in thyroid extracts of TRßPV/PV mice and wild-type mice were immunoprecipitated with antiactin antibody followed by Western blot analysis using antigelsolin antibody. Band intensities were measured by a National Institutes of Health software package (Image J 1.34.), and data were plotted.

Fluorescence confocal microscopy
Subcellular localization of actin and gelsolin was visualized by fluorescence confocal microscopy. Primary thyroid cultured cells derived from wild-type mice and tumor cells from TRßPV/PV mice were washed twice with PBS, fixed with 4% paraformaldehyde (10 min at room temperature), and permeabilized with 0.5% Triton X-100 in PBS (10 min at room temperature). Nonspecific binding of the antibodies was blocked with 3% BSA before incubation with the antiactin and antigelsolin antibodies at 4 C overnight to detect the endogenous actin and gelsolin. The cells were subsequently incubated with 1.0 µg/ml Alexa Fluor 488 fragment of goat antimouse IgG (Invitrogen; no. A11017) or tetramethyl rhodamine goat antirabbit IgG (Invitrogen; no. T2769). Nuclei were also stained with 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). Laser confocal scanning images were captured by using an Ultraview confocal head (PerkinElmer) on a TV200 inverted microscope (Zeiss, Thornwood, NY).

Statistical analysis
All data are expressed as mean ± SEM. Data were analyzed by unpaired t test with StatView 5.0 (Abacus Concepts, Inc., San Diego, CA). Differences with P <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Down-regulation of gelsolin in thyroids of TRßPV/PV mice
Prior microarray data set analysis comparing differentially expressed genes in thyroids from age- and gender-matched TRßPV/PV and wild-type mice found a greater than 6-fold reduction of gelsolin mRNA (6). To understand whether the gelsolin protein levels were reduced during thyroid carcinogenesis, we carried out Western blot analysis of thyroid tumors as mice aged. Figure 1AGo shows that, compared with wild-type mice, the protein levels of gelsolin were decreased in an age-dependent manner. After normalization using the {alpha}-tubulin loading controls, the quantitative comparison is shown in Fig. 1BGo. At 3–4 and 5–6 months, compared with wild-type mice, a significant reduction of gelsolin protein abundance in TRßPV/PV was observed (80 and 85%, respectively). At 10–12 months, whereas gelsolin protein levels were also reduced in the wild-type mice, gelsolin in the tumors of TRßPV/PV mice was further reduced (Fig. 1BGo). The temporal association of decreased gelsolin protein levels suggests an important role of gelsolin in tumor progression in this mouse model.


Figure 1
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  		FIG. 1. Age-dependent reduced expression of gelsolin in the thyroids of gender- and age-matched wild-type (WT) and TRßPV/PV mice. A, Thyroid tissue extracts were prepared as described in Materials and Methods. Shown are representative data comparing two mice from each genotype that were age and gender matched. Three separate experiments were performed. Thyroid extract (50 µg) was separated by gel electrophoresis, and Western blot analysis was carried out against gelsolin. {alpha}-Tubulin as a control for loading was also determined by Western blot analysis using anti-{alpha}-tubulin antibody. B, Densitometric analysis after normalization with {alpha}-tubulin loading controls. The reduction in gelsolin protein abundance in TRßPV/PV mice as compared with wild-type mice is significant (P < 0.05).

 
Regulation of tumor cell motility of TRßPV/PV mice by gelsolin
Gelsolin is known to play an important role in the modulation of the actin network and affects cellular functions including cell motility (8). To assess the functional roles of reduced expression of gelsolin in thyroid carcinogenesis, we first adopted the loss-of-function approach by performing siRNA experiments to further decrease gelsolin in primary thyroid tumor cells derived from TRßPV/PV mice. As shown by Western blot analysis, gelsolin was substantially reduced using siRNA in the primary thyroid tumor cells (compare lanes 1 and 2; Fig. 2A-aGo). Cell motility assays indicate that the primary thyroid tumor cells with lower gelsolin levels were able to migrate significantly faster (about 2.5-fold increase in migration; Fig. 2A-bGo). These data suggest that the reduced abundance of gelsolin may be involved in tumor progression through regulation of cell motility.


Figure 2
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  		FIG. 2. A, Knockdown of gelsolin increases primary thyroid tumor cell motility. a, TRßPV/PV mouse thyroids were removed and prepared into a single-cell suspension and gelsolin siRNA was transfected using Lipofectamine 2000 as described in Materials and Methods. Western blot analysis shows a reduction of gelsolin in cells exposed to siRNA, whereas PDI was used as a loading control. Cell motility experiments were performed as described in Materials and Methods. Two independent experiments from two different cell lines were performed in triplicate. b, The percentage of cells that were motile was calculated for each cell line and normalized to fold increase in motility, compared with control, defined as 1. Data presented as the mean ± SEM (n = 6), P < 0.05. B, HDAC inhibition increases gelsolin expression and retards primary thyroid tumor cell motility. TSA was administered to primary thyroid cells derived from TRßPV/PV mice. a, Cells were incubated in TSA or dimethylsulfoxide (control) for 24 h. After incubation, 50 µg of cellular lysate were used for Western blot analysis of gelsolin expression, whereas PDI was used as a control. Western blot confirms the increase in gelsolin in TSA-treated primary thyroid cells. b, Primary thyroid tumor cells (control and TSA treated) were used in a motility assay as described in Materials and Methods. Representative data from two independent experiments, performed in triplicate, presented as the mean ± SEM (n = 6), P < 0.05.

 
The above results would predict that an up-regulation in gelsolin expression would result in a decrease in motility. Whereas the regulators of gelsolin transcription have not been fully identified, it is known that histone deacetylase (HDAC) inhibitors can induce gelsolin expression in certain tumor cell lines (24, 25), although the effect in thyroid tumors is unknown. Therefore, we treated primary thyroid cells from TRßPV/PV mice with the HDAC inhibitor, TSA. As can be seen in Fig. 2B-aGo, gelsolin levels increased in the TSA-treated cells (compare lane 2 with lane 1), and this increase was accompanied by a significant decrease in cell motility (~2-fold; Fig. 2B-bGo). Taken together, these data indicate that gelsolin regulates tumor cell motility of TRßPV/PV mice. However, at present, we could not exclude the possibility that TSA could also affect the expression of other cellular proteins, thereby affecting cell motility.

Interaction of gelsolin with TRß1 or PV in vivo and in vitro
That gelsolin regulates thyroid tumor cell motility of TRßPV/PV mice prompted us to probe the pathways by which PV could mediate tumor cell motility via gelsolin. Nishimura et al. (23) found that gelsolin could bind to the DNA-binding domain of the androgen receptor. This finding led us to hypothesize that the cellular functions of gelsolin could be regulated via association with TRß1 or PV. We first assessed whether gelsolin could associate with TRß1 or PV in vivo by carrying out immunoprecipitation assays using thyroid extracts from wild-type and TRßPV/PV mice (Fig. 3Go). Lanes 2 and 4 show that the endogenous gelsolin was associated with TRß1 or PV, respectively, first by immunoprecipitation with anti-TRß1 (C4, lane 2; Fig. 3Go) or PV (no. 302, lane 4, Fig. 3Go) and then by Western blot using antigelsolin antibody. Lane 1 was a negative control, and lanes 3 and 5 were positive controls, showing the presence of endogenous gelsolin in the thyroids of wild-type and TRßPV/PV mice, respectively, by direct Western blot analysis. These results demonstrate that in vivo gelsolin is associated with TRß1 or PV.


Figure 3
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  		FIG. 3. Association of endogenous gelsolin with TRß1 or PV in thyroid of wild-type (WT) or TRßPV/PV mice, respectively. The association of gelsolin with TRß1 or PV was evaluated by coimmunoprecipitation. Thyroid extracts (400 µg) of wild-type mice (lane 2) or TRßPV/PV mice (lane 4) were immunoprecipitated with 5 µg of anti-TRß1 (C4) or anti-PV (no. 302) antibodies, respectively. Thyroid extracts (400 µg) of wild-type mice (lane 1) were also immunoprecipitated (IP) with an irrelevant monoclonal antibody (MOPC) as control. The immunoprecipitated proteins were analyzed by Western blot analysis with antigelsolin antibody. Lanes 3 and 5 show input by direct Western blot analysis (40 µg of lysate).

 
To assess whether the interaction of gelsolin with TRß1 or PV is regulated by T3, we transfected Flag-tagged TRß1 or Flag-tagged PV to a thyroid cell line (PC cells). Lanes 3 and 4 (Fig. 4AGo) show that the endogenous gelsolin also interacted with TRß1, and this interaction was increased by T3 (lane 4 vs. lane 3). However, because PV does not bind T3, no change in the extent of interaction was observed (lane 5 vs. lane 6). Similar findings were also observed in CV1 cells (Fig. 4BGo). A comparison of the band intensities in lanes 4 and 3 (Fig. 4B-aGo) revealed an increase in the association of the endogenous gelsolin with the transfected TRß1 by T3 when antigelsolin was used in the immunoprecipitation followed by Western blot analysis using anti-Flag antibody. This T3 effect was further confirmed first by immunoprecipitation with anti-Flag antibody and then by Western blot analysis using antigelsolin antibody (compare lane 2 with lane 1, Fig. 4B-bGo).


Figure 4
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  		FIG. 4. T3-dependent association of endogenous gelsolin with TRß1 or PV in a thyroid cell line (PC cells). A, PC cells were transfected with expression plasmids TRß1 [8 µg of pFlag(f)-TRß1; lanes 3, 4, 7, and 8] or PV [8 µg pFlag(f)-PV; lanes 5, 6, 9, and 10] in the absence or presence of T3 (100 nM). For coimmunoprecipitation, 500 µg of cell lysate was immunoprecipitated with anti-FLAG M2 affinity gel followed by Western blot analysis with polyclonal rabbit antigelsolin antibody. Lanes 7–10 show input by direct Western blot analysis (40 µg of tissue lysate). B, T3-dependent association of endogenous gelsolin with TRß1 in CV1 cells. CV-1 cells were transfected with expression plasmids TRß1 [5 µg of pFlag(f)-TRß1] in the presence or absence of T3 as described in Materials and Methods. Cell lysates (1.5 mg) were immunoprecipitated (IP) with 8 µg anti-gelsolin antibody (a) or 8 µg anti-Flag antibody (b) followed by Western blot analysis using anti-Flag antibody (a) or anti-gelsolin antibody (b). Rabbit IgG (lanes 1 and 2; a) or an irrelevant antibody, MOPC (lanes 3 and 4, b) were used for negative control.

 
A glutathione-S-transferase (GST)-pulldown assay was also used to map out the regions of TRß1 that interacted with gelsolin. Figure 5A-aGo shows the schematic representation of the full-length and truncated TRß1 used in the analysis. Lanes 1–4 show the input of 35S-labeled full-length and truncated TRß1, and lanes 5 and 6 (Fig. 5A-bGo) indicate the deletion of the A/B domains had no effect in the binding of TRß1 to GST-gelsolin. However, when the DNA binding domain was deleted (MD32, Fig. 5A-aGo), no binding to gelsolin was observed (Fig. 5A-bGo, lane 7), thus suggesting that the DNA binding domain is critical in the interaction of TRß1 with gelsolin. A truncated GST-gelsolin (GST-GSNc) containing only the C-terminal amino acids 281–755 still retained the ability to interact with TRß1 (Fig. 5BGo). Taken together, these results indicate that the DNA binding domain of TRß1 and the C-terminal region of gelsolin were critical in the interaction of these two proteins.


Figure 5
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  		FIG. 5. Mapping of interaction region of TRß1 bound to gelsolin by GST-pulldown assay. A-a, Schematic representation of full-length and truncated TRß1 proteins. A-b, Gelsolin interacts with the DNA-binding region of TRß1. The protein GST or fusion protein GST-gelsolin was incubated with 35S-labeled in vitro-translated full-length TRß1 and analyzed by SDS-PAGE as described in Materials and Methods. Twenty percent input was used. B, The C-terminal gelsolin interacted with TRß1. Full-length (lane 3) and C-terminal gelsolin (lane 4; GST-GSNc) fused to GST were incubated with 35S-labeled in vitro-translated full-length TRß1. Lane 1 shows the 20% input, and lane 2 shows the negative control of using GST alone.

 
Differential effects of TRß1 and PV on the interaction of gelsolin with actin
Recent studies have shown that gelsolin is an actin binding protein that severs, caps, and in some cases nucleates and bundles actin filaments (8, 26). The actin binding sites are located in amino terminal regions of gelsolin. Thus, gelsolin, in association with actin plays a critical role in maintaining cytoskeletal structure of cells to regulate cellular functions including cell morphology, motility, signaling, and apoptosis (8, 10, 26). We postulated that the interaction of gelsolin with TRß1 or PV could alter its interaction with actin differently and thereby alter gelsolin/actin-mediated cellular functions. To test this hypothesis, we analyzed the interaction of gelsolin with actin by coimmunoprecipitation in the thyroid extracts of wild-type and TRßPV/PV mice. With equal input of gelsolin amounts at a given extract concentration, immunoprecipitation of thyroid extracts by antigelsolin antibody followed by Western blot analysis with antiactin antibody showed a significant reduction in the recruitment of actin by gelsolin in the thyroid of TRßPV/PV mice (Fig. 6AGo; compare lanes 1–5 with lanes 6–10). Quantitative analysis shown in Fig. 6BGo indicates that PV significantly weakened the interaction of gelsolin with actin in the thyroid of TRßPV/PV mice as compared with TRß1 in wild-type mice. A similar reduction in the interaction of actin with gelsolin was also observed when coimmunoprecipitation was carried out by first immunoprecipitating actin followed by Western blot analysis using antigelsolin antibody (data not shown).


Figure 6
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  		FIG. 6. Reduced recruitment of actin by gelsolin in thyroid tumor cells of TRßPV/PV mice. Increasing concentrations of thyroid extracts from wild-type (WT) mice (1000, 750, 500, 250, and 125 µg in lanes 1, 2, 3, 4, and 5, respectively) were each immunoprecipitated (IP) with 4 µg antigelsolin antibody followed by Western blot analysis anti-ß-actin antibody. Using increasing thyroid tumor extracts of TRßPV/PV mice that were adjusted to have equal amounts of gelsolin, a similar analysis was carried out to determine the gelsolin bound actin (lanes 6–10). B, Band intensities were quantified by Image J 1.34. (National Institutes of Health image analysis software) and graphed.

 
To assess whether the weakening of the interaction of gelsolin with actin by PV is associated with perturbed cytoskeletal architectures, we compared the patterns of subcellular distribution of actin and gelsolin in cultured thyrocytes from wild-type mice and tumor cells from TRßPV/PV mice (Fig. 7Go). In cultured thyrocytes from wild-type mice, the endogenous gelsolin (Fig. 7aGo) and actin (Fig. 7bGo) displayed strong characteristic actin filament patterns. In the merged images (Fig. 7cGo), the colocalization of these two proteins was particularly evident in the cortical regions. However, in cultured tumor cells in which PV is expressed (Fig. 7dGo), consistent with the Western blot analysis shown in Fig. 1Go, the intensity of gelsolin signals was clearly lower with less prominent filament patterns (Fig. 7dGo). The characteristic cortical actin filament pattern was also less apparent in tumor cells (Fig. 7eGo). Furthermore, the appearance of intracellular actin bundles was altered. The morphological features of the merged image of actin/gelsolin in tumor cells (Fig. 7fGo) clearly differed from those shown for wild-type thyrocytes (Fig. 7cGo). These observations further support the notion that interaction of PV with gelsolin disrupts the normal actin/gelsolin cytoskeletal properties.


Figure 7
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  		FIG. 7. Altered actin/gelsolin cytoskeletal architectures in thyroid tumor cells of TRßPV/PV mice. Endogenous gelsolin and actin in primary thyroid cultured cells of wild-type mice and thyroid tumor cells of TRßPV/PV mice were visualized by confocal fluorescence microscopy as described in Materials and Methods. Gelsolin (a and d; red) and actin (b and e; green) were stained with antigelsolin and antiactin antibody followed by second antibodies for visualization as described in Materials and Methods. Panels c and f show the merged images of gelsolin and actin for wild-type cells and tumor cells, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Whereas an increasing number of studies have demonstrated the key role of gelsolin in tumor progression in many cancers, little is known about the part it plays in thyroid carcinogenesis. Using a TRßPV/PV mouse model, the present study found that reduced expression of gelsolin is closely associated with progression of FTC. Our results indicate that one of the functional consequences of reduced gelsolin is increased tumor cell motility (Fig. 2Go). Whereas the mechanisms by which the expression of gelsolin is reduced in the thyroid of the TRßPV/PV mouse have not been elucidated, our data indicate that its expression can be induced by T3 in wild-type mice (Kim, C. S., and S. Cheng, unpublished results), suggesting that gelsolin could be positively regulated by T3 via TRs. It is well documented that the expression of T3 positively regulated target genes such as the malic enzyme, S14, and deiodinase type 1 genes is repressed in the liver by the dominant-negative action of PV (19). Therefore, it is likely that the repression of the expression of gelsolin in the thyroids of TRßPV/PV mice is, at least in part, via the dominant-negative action of PV. But the precise mechanisms by which T3 induces gelsolin expression will require future studies.

However, the present study discovered that gelsolin is a novel TR-interacting protein. We found that TRß1 as well as PV interacted with gelsolin in cultured cells (Fig. 4Go) and the thyroid extracts (Fig. 3Go). Compared with TRß1, the interaction of PV with gelsolin significantly reduced the recruitment of actin by gelsolin (Fig. 6Go), resulting in an altered cytoskeleton (Fig. 7Go). The C-terminal region of gelsolin was shown to be critical in its interaction with TRß1 or PV. It is known that the C-terminal region of gelsolin also is critical in the binding to actin (26). Whereas the DNA binding domain of TR was shown to be critical in the binding to gelsolin, the differential effect of TRß1 and PV on gelsolin/actin interaction is less clear. It is entirely possible that the mutated C-terminal 16 amino acids (in helix 12) of PV, when bound to gelsolin, could deter the binding of gelsolin to actin.

In addition to regulating cytoskeletal structures, gelsolin has been shown to play a role in nucleus-initiated transcription activity (27). One notable example is the increase of the androgen-dependent transcriptional activity by gelsolin (23). Consistent with the reports by others (27), we also detected the colocolization of gelsolin with TRß1 in the nuclei of thyroid cells (data not shown). This nuclear colocalization suggests that gelsolin could affect the transcription activity of TRs. Indeed, using reporter assays, we found that gelsolin increased the T3-dependent transcription activity of TRß1 in a gelsolin concentration-dependent manner (Kim, C. S., and S. Cheng, unpublished results). These findings suggest that the cellular actions of gelsolin in the thyroid are not limited to its direct effects on the actin cytoskeletal organization but also could also be involved with the regulation of TRß1 target genes. This hypothesis awaits validation in future studies.

Follicular thyroid cancer is recognized for its ability to spread hematogenously. Our results show that alterations of the gelsolin/actin cytoskeleton are one of the mechanisms that could underlie the metastatic spread. Thus, this study demonstrates the utility of this mouse model in dissecting the molecular genetic changes during thyroid carcinogenesis. Because FTC human tissue samples are scarce, the TRßPV/PV mouse model provides many opportunities to investigate the factors that contribute to FTC progression and devise better therapeutic options.


   Acknowledgments
 
We thank Dr. Hisakazu Fujita for providing us with the gelsolin antibody and Dr. Chawnshang Chang for his GST-gelsolin plasmids.


   Footnotes
 
This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.

The authors have nothing to disclose.

First Published Online December 14, 2006

Abbreviations: FTC, Follicular thyroid carcinoma; GST, glutathione-S-transferase; GST-GSN, GST-gelsolin; GST-GSNc, GST-gelsolin with carboxyl terminal; HDAC, histone deacetylase; PDI, protein disulfide isomerase; RTH, resistance to thyroid hormone; si, small interfering; TR, thyroid hormone receptor; TSA, trichostatin A.

Received July 10, 2006.

Accepted for publication December 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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