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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 11 5851-5861
Copyright © 2004 by The Endocrine Society

Decreased Expression of Retinoid X Receptor Isoforms in Human Thyroid Carcinomas

Yumi Takiyama, Naoyuki Miyokawa, Akira Sugawara, Shizuo Kato, Koichi Ito, Keisuke Sato, Kensuke Oikawa, Hiroya Kobayashi, Shoji Kimura and Masatoshi Tateno

The Second Department of Pathology (Y.T., K.S., K.O., H.K., M.T.), Surgical Pathology (N.M., S.Ka.), and School of Nursing (S.Ki.), School of Medicine, Asahikawa Medical College, Asahikawa 078-8510, Japan; Department of Comprehensive Medicine (A.S.), Tohoku University, Sendai 980-8578, Japan; and Ito Hospital (K.I.), Tokyo 150-8308, Japan

Address all correspondence and requests for reprints to: Yumi Takiyama, M.D., Ph.D., The Second Department of Internal Medicine, Asahikawa Medical College, Midorigaoka higashi 2-1-1-1, Asahikawa 078-8510, Japan. E-mail: taka0716{at}asahikawa-med.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Retinoid X receptors (RXRs) are ligand-inducible transcription factors that belong to the superfamily of nuclear hormone receptors. Because RXRs heterodimerize with thyroid hormone receptor, retinoic acid receptor, vitamin D3 receptor, and peroxisome proliferator-activated receptor, they play central roles in regulating a number of signaling pathways. To understand the roles of RXRs in human thyroid carcinogenesis, we have investigated the immunohistochemical expression of RXRs in normal and neoplastic thyroid tissues. Whereas nontumorous human thyroid cells exhibited distinct nuclear staining for the RXRs, thyroid carcinomas showed decreased nuclear expression of all three RXR isoforms. In particular, some thyroid carcinoma cells showed intense RXR-{alpha} cytoplasmic staining accompanied by decreased immunoreactivity in their nuclei. This subcellular localization of RXR-{alpha} was confirmed by Western blot analysis, which showed both lower nuclear expression levels of RXR-{alpha} and a cytosolic presence of RXR-related protein in neoplastic regions. We present here, for the first time, the histological distribution of each RXR protein ({alpha}, ß, and {gamma}) in human thyroid follicular cells. In addition, we found that the nuclear expression of RXRs was lower in thyroid carcinomas than in normal tissue. The differential expressions of these RXRs in thyroid carcinomas might be implicated in the pathogenesis of thyroid cancers.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RETINOIDS REGULATE A broad range of biological processes, including growth, differentiation, and development. They have been administered in anticancer and preventive cancer treatments in various clinical trials (1, 2, 3, 4, 5, 6). The biological actions of retinoic acid and its derivatives are mediated at the cellular level by two types of specific nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs) (7). RXRs are ligand-inducible transcription factors belonging to the superfamily of nuclear hormone receptors, and they heterodimerize with thyroid hormone receptor (TR), RAR, vitamin D3 receptor, and peroxisome proliferator-activated receptor (PPAR), and with other orphan receptors. Thus RXRs play central roles in regulating a number of signaling pathways (7). Recent studies have reported the involvement of nuclear hormone receptors in thyroid carcinogenesis (8, 9). Mutated TRs have been identified with significant frequency in papillary thyroid carcinomas (PTCs), suggesting that these TRs may be involved in the carcinogenesis of PTC (8). A PAX8-PPAR-{gamma} fusion gene was detected in 63% of follicular thyroid carcinomas (FTCs) but not in other thyroid tumors, indicating that this fusion may be useful in the diagnosis and treatment of FTCs (9). Because RXR is essential for the functioning of TRs and PPAR-{gamma}, it could also contribute to behaviors specific to PTCs or FTCs. Moreover, functional RXRs have been reported in the human thyroid carcinoma cell lines and tissue samples, and the specific decreased expression of RXR-ß mRNA was observed in anaplastic thyroid carcinoma (ATC) cells as well as in human thyroid carcinoma samples (10). In most epithelial malignant cell systems, retinoids have been found to inhibit DNA synthesis. Hence, the reduced expression of RXRs in human thyroid tumors could conceivably interfere with the retinoids’ inhibition of DNA synthesis and could thus contribute to the development of thyroid carcinomas.

Therefore, we hypothesize that abnormal expression of RXRs is also a critical step in human carcinogenesis and that the therapeutic effects of retinoids in thyroid cancer may be mediated through the normalization or enhancement of the biological functions of RXRs. The aims of the current study were to determine which RXR isoforms are present in thyroid carcinomas and to evaluate the expression of each RXR to determine whether or not differences in that expression in thyroid carcinomas could contribute to thyroid carcinogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue samples

Samples of pathologic human thyroid tissue were resected from patients whose thyroid carcinomas had been surgically removed. As a control, normal tissue counterparts of thyroid tumors were excised. The samples were immediately snap-frozen in liquid nitrogen after surgical removal and stored at –70 C until use. The surgical tissue samples were obtained with the informed consent of the patients according to the regulations of the local ethics committee.

RXR antibodies

Two polyclonal antibodies against human RXR-{alpha} (D-20 and {Delta}DN197) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The epitopes recognized by one of the anti-RXR-{alpha} antibodies (D-20) were residues 2–21, and those recognized by the other antibody ({Delta}DN197) were residues 198–462. Polyclonal antibodies against RXR-ß and RXR-{gamma} were raised against synthetic peptides containing the following mouse RXR amino acid residues: RXR-ß, 78–93; and RXR-{gamma}, 35–54 (11). The amino acid sequences of mouse RXRs used as immunogens to construct these antibodies bear 100% homology with the human RXR-{gamma} sequence and 83% homology (three amino acid differences) with the human RXR-ß isoform. The characterization of these RXR antibodies was confirmed by Western blotting and immunoprecipitation as described previously (11).

Immunohistochemistry

In this study, we examined RXR expression in 176 thyroid carcinomas [57 PTC, 40 FTC, 24 ATC, 28 medullary thyroid carcinomas (MTC), and 27 follicular adenomas (FA)]. Formalin-fixed sections of normal thyroid and carcinoma were deparaffinized and incubated with 1% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity and were then microwaved at 100 C for 15 min to retrieve antigenicity. Then the specimens were incubated overnight with the primary antibodies, rinsed in PBS, and subsequently incubated with ENVISION+ (Dako, Carpinteria, CA) for 30 min. The reactions were visualized with 3,3'-diaminobenzidine tetrahydrochloride after 2–3 min, revealing brown staining. The cell nuclei were then counterstained with hematoxylin. In the present study, we performed immunohistochemical preabsorption tests to confirm the specificity of the immunohistochemical reaction by preincubating the diluted antibodies with the corresponding antigen overnight at 4 C before the immunohistochemical procedure. The immunoreactivity of each RXR was evaluated independently by two of the authors (Y.T. and M.T). The intensity of staining was classified separately for the nucleus and the cytoplasm and was evaluated on a scale of 0–3 (0 and 3 corresponded to the absence and highest degree of staining, respectively). These independent assessments (between authors) did not differ by more than one scale level. Because normal follicular thyroid cells showed moderate (level = 2) or strong (level = 3) nuclear staining with absent (level = 0) or weak (level = 1) cytoplasmic staining, a tumor was considered to have a suppressed nuclear RXR expression when the nuclear intensity level of the tumor cell was less than 2 (moderate); if the cytoplasmic staining level was higher than 2, a tumor was considered to have increased cytoplasmic RXR expression.

Cell culture

Three carcinoma cell lines (thyroid papillary NPA, follicular WRO, and anaplastic ARO) were kindly donated by Dr. Yuji Nagayama (University of Nagasaki, Nagasaki, Japan). These cells were cultured in RPMI 1640 medium (Life Technologies, Inc. Oriental Co., Ltd., Tokyo, Japan) containing 10% heat-inactivated fetal calf serum (Life Technologies, Inc.) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) in 5% CO2 and at 95% humidity. The culture medium was changed every 3–4 d, and cells were passaged every 5–6 d.

RT-PCR

Total RNA was extracted from human thyroid whole tissue and thyroid cancer cell lines by homogenization using Sepasol-RNA I (Nacalai Tesque, Inc., Kyoto, Japan), according to the manufacturer’s protocol. The samples obtained were quantified by absorbance at 260 nm. cDNA was synthesized using an A-murine leukemia virus (A-MuLV) reverse transcriptase (AMV-RT) in a 20-µl reaction containing 1.6 µg oligo-p(dT)15 primer, 50 U RNase inhibitor, 1 mM of deoxynucleotide triphosphates, 2 µl of 10x reaction buffer, 5 mM MgCl2, 20 U AMV-RT (all from Roche Diagnostics Corp., Indianapolis, IN), and 2 µg total RNA. The reactions were incubated at 25 C for 10 min, 42 C for 60 min, 99 C for 55 min, and finally cooled to 4 C. cDNA was amplified using the Expand High Fidelity PCR System (Roche) in 25 µl of the final volume, which was one fifth of the reverse transcription products, 10 pmol each of the sense and antisense primers, and 1.75 U of Taq DNA polymerase. The oligonucleotide primers for RT-PCR were designed using the published sequence of each target gene. The primers and conditions used and the expected sizes from the reported cDNA sequences are listed in Table 1Go. To examine the presence of splicing variants, the sequences of the RXR-{alpha} primer pairs were as follows: forward primer, 5'-CAGACATGGACACCAAACAT-3' (nucleotide position: 64–83); and reverse primer 5'-CCGCAGGCCTAAGTCATTTG-3' (nucleotide position: 1465–1446). The numbers of PCR cycles were determined to amplify the products logarithmically. After the PCR products were visualized on 2% agarose gel stained with ethidium bromide, the amplified PCR products were excised from the gel and subcloned into TA cloning vectors (Invitrogen Co., Carlsbad, CA). Inserts were sequenced by an automatic DNA sequencer (Perkin-Elmer Cetus, Norwalk, CT).


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  		TABLE 1. Oligonucleotide sequences for RT-PCR amplification of mRNA species and nucleotide positions of oligonucleotides

 
Relative quantification of mRNAs of human RXR isoforms

The expression of each RXR isoform’s mRNA in tissues was analyzed by two-step real-time RT-PCR using LightCycler-DNA Master SYBR Green I (Roche) as described previously (12). ß-actin was used as the reference gene for the tissue examination. The same primers for RXR-{alpha}, RXR-ß, RXR-{gamma}, and ß-actin were used for the conservative RT-PCR (Table 1Go). For quantification, an external calibration curve was used. The RXR isoforms and the ß-actin PCR products amplified by each primer pair were cloned separately in pCR2.1 vector (Invitrogen). For each plasmid, a dilution containing from 103 to 109 double-stranded DNA molecules was used as a standard template for the production of an external calibration curve. The LightCycler instrument (Roche) was used to optimize the RT-PCR conditions for the MgCl2 concentrations, annealing temperatures, elongation time, and fluorescence acquisition temperature. PCR amplifications were performed in triplicate. The PCR efficiencies were calculated according to the following equation: E = 10–1/slope, where E is the PCR efficiency. Each mean slope of each external calibration curve was used as a representative slope value. The crossing point (CP) for each transcript was determined by the fit point method performed in the LightCycler software version 3.3 (Roche). The average triplicate CP value for each transcript was used as the representative CP value. The relative quantification ratio of each thyroid tissue sample was calculated according to the following equation: ratio = (Etarget){Delta}CPtarget(control – sample)/(Eref){Delta}CPref(control – sample). Etarget indicates the PCR efficiency when an RXR isoform’s primers and its standard template were used. Eref refers to the PCR efficiency when the reference gene (ß-actin) primer pairs and standard template were used. {Delta}CPtarget(control – sample) and {Delta}CPref(control – sample) refer to the CP deviation of each thyroid carcinoma vs. a control normal thyroid tissue (normal thyroid tissue paired with PTC 1) for each RXR isoform transcript and reference gene transcript, respectively (13).

Extract preparation

Protein extracts were prepared using a standard method described previously, with minor modifications (14). Briefly, human thyroid tissues were homogenized in 1 ml PBS with the Complete, Mini, EDTA-free protease inhibitor cocktail (Roche). Cultured cells were washed with medium without serum and twice with PBS and were collected using a cell scraper. The scraped cells or homogenates were then centrifuged at 1000 x g for 10 min at 4 C. The pellets were washed with PBS containing the protease inhibitors mentioned earlier and were resuspended in one to two volumes of prechilled hypotonic buffer A (10 mM HEPES, pH 7.9; 10 mM KCl; 1.5 mM MgCl2; 0.5 mM dithiothreitol; and protease inhibitor cocktail). After the suspension sat for 10 min on ice, 0.1 vol of 10% Nonidet P-40 was added to it, and the cells were lysed by vortexing. After centrifugation at 15,000 rpm for 30 sec, the supernatant obtained was collected as the cytoplasmic fraction. Soluble nuclear extracts (NEs) were prepared by resuspending the pellet in an equal volume of prechilled hypertonic buffer C (20 mM HEPES, pH 7.9; 0.42 M NaCl; 1.5 mM MgCl2; 0.2 mM EDTA; 25% glycerol; 0.5 mM dithiothreitol; and protease inhibitor cocktail), followed by incubation on ice for 30 min. After centrifugation at 13,000 rpm for 10 min, the supernatant collected constituted the NE.

Western blot analysis of RXR isoforms

The samples (standardized to 20 µg of total protein per lane) were mixed with the same volume of 2x loading buffer containing 2% sodium dodecyl sulfate and 10% ß-mercaptoethanol. They were then boiled for 5 min and analyzed on 4–12% NuPage bis-Tris SDS-PAGE gels (Invitrogen). After each gel was soaked in transfer buffer (glycine 33.3 mM, Tris 48 mM, sodium dodecyl sulfate 1.3 mM, and methanol 20% vol/vol), it was blotted onto an Immobilon-P membrane (Millipore, Bedford, MA) at 100 mA for 1 h. The blot was saturated overnight at 4 C in PBS and 0.1% Tween 20 (PBS-T buffer) containing 5% nonfat dry milk, washed twice in PBS-T for 10 min at room temperature, and then incubated with each of the rabbit antihuman RXR isoforms overnight. After the blot was washed three times in PBS-T (each wash lasted 10 min), the blot was stained with a 1:2000 dilution of peroxidase-conjugated antirabbit IgG antiserum (Amersham Pharmacia Biotech, Little Chalfont, UK) for 1 h at room temperature and was then washed again five times for 10 min each time in PBS-T. Bands were visualized by the enhanced chemiluminescence system according to the manufacturer’s instructions (Amersham).

Statistical analysis

The Friedman test was used to analyze differences in the expression levels of nuclear or cytoplasmic RXRs among different thyroid tumor histological types. The statistical differences in expression among the RXRs were compared in two groups of neoplasms using Tukey’s test post hoc. For comparative analysis, the two-tailed Fisher’s exact test was used to compare the differences in the expression of the RXR isoforms between the histological types. P < 0.05 was accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunohistochemical detection of RXR proteins in human thyroid follicular cells

To investigate a potential association between RXR expression and thyroid carcinogenesis, we first analyzed the expression of each RXR isoform in 57 PTC, 40 FTC, 24 ATC, 28 MTC, and 27 FA by immunohistochemistry. The upper half of Fig. 1Go demonstrates the results of this analysis. The lower half shows the results of Western blotting of nuclear or cytoplasmic protein preparations of a PTC and matched normal thyroid tissue. Each RXR isoform is represented by appropriately sized protein bands in the NEs from the normal thyroid tissues (Fig. 1Go, bottom, lane 1, arrow). The two immunoreactive bands in the NEs from the normal thyroid tissue are detected by the anti-RXR-{gamma} antibody (Fig. 1Go, right). The lower band (~50 kDa) corresponds to the form previously reported by Sugawara et al. (11), whereas the upper band (Fig. 1Go, bottom, arrowhead) has been considered to represent a different phosphorylation state, an alternate form of RXR-{gamma}, or a protein that cross-reacts with the RXR-{gamma} antiserum (15). The NEs from PTC, which showed no nuclear staining for any of the RXR isoforms in immunohistochemical analysis (Fig. 1Go, top, arrowhead), were negative in the Western blots (Fig. 1Go, lane 2). The cytoplasmic extract (CE) from PTC, which showed strong cytoplasmic immunostaining for RXR-{alpha} (Fig. 1Go, top left), reacted strongly at a molecular mass of approximately 44 kDa to the anti-RXR-{alpha} antibody (D-20; Fig. 1Go, asterisk).



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  		FIG. 1. Western blot analysis and immunohistochemistry of the PTC. Immunopositive cells appear brown as a result of 3,3'-diaminobenzidine tetrahydrochloride colorimetric reaction (top). Each anti-RXR isoform antibody detected the strong (level = 3) or moderate (level = 2) nuclear staining in normal (N) thyroid tissue (arrow) and absent (level = 0) nuclear staining in PTC (arrowhead). Normal thyroid is at left. Samples (standardized to 20 µg of total protein/lane) were size-separated on 4–12% NuPage bis-Tris SDS-PAGE gels and transferred to an Immobilon-P membrane. After blocking with 5% nonfat dry milk, the blot was incubated with the rabbit antihuman RXR isoforms and then stained with secondary antibody-linked horseradish peroxidase. Size standards are shown at the left. Lane 1, NE from normal thyroid tissue; lane 2, NE from PTC; lane 3, CE from normal thyroid tissue; and lane 4, CE from PTC. Appropriately sized protein bands in NEs from normal thyroid tissues were demonstrated for each RXR isoform (bottom, lane 1, arrow). The two immunoreactive bands in NE from normal thyroid tissue were detected by anti-RXR-{gamma} antibody (bottom right). The CE from PTC, which showed the strong cytoplasmic immunostaining for RXR-{alpha}, showed strong reaction at a molecular mass of approximately 44 kDa by anti-RXR-{alpha} antibody (D-20) (asterisk).

 
The RXR isoforms were readily detected in all thyroid tumors and surrounding normal thyroid tissues. These immunostainings were abolished when blocking peptides were added to the diluted antibodies for each RXR isoform (Fig. 2Go, B, D, and F). Normal follicular thyroid cells showed diffuse strong (level = 3) to moderate (level = 2) nuclear signals, whereas cytoplasmic staining was almost absent (Fig. 2Go, A, C, and E). Homogeneous staining in the nucleus was seen at a higher magnification (Fig. 2EGo, inset). The intensity of immunostaining by the RXR isoforms in the nuclei and cytoplasm of thyroid tumors was relatively uniform within each specimen. Due to differences in the affinities of the antibodies, it is not possible to draw conclusions about the relative expression levels of the three isoforms. However, these levels differed among the isoforms in normal thyroid tissues and thyroid carcinomas. We observed a tendency toward suppression of the nuclear expression of RXRs in thyroid tumor cells relative to that in adjacent normal thyroid follicular cells (Fig. 2Go, A and E). Unexpectedly, in addition to nuclear staining, a strong granular cytoplasmic staining was observed in some thyroid carcinomas by the anti-RXR-{alpha} antibody (D-20; Fig. 2AGo, inset); this staining was abolished by peptide preabsorption (Fig. 2BGo). In contrast with the PTCs, the FTCs showed higher levels of nuclear immunostaining of the RXR-{alpha} protein. Therefore, RXR-{alpha} indicated distinct nuclear staining without cytoplasmic staining in the FTCs (data not shown). Occasionally, the ATCs showed weak or absent nuclear staining of RXR-{alpha}, as evidenced by a mild granular cytoplasmic staining (Fig. 2GGo). Only the cytoplasmic distribution of RXR-{alpha} protein shown in the PTCs was observed in the MTCs; that is, neither RXR-ß nor RXR-{gamma} was detected in the nucleus or cytoplasm of the MTCs (Fig. 2EGo). Moreover, none of the RXR isoforms was detected in normal C cells by immunohistochemistry (data not shown). In contrast to the results obtained with thyroid carcinomas, the RXR-{alpha} (Fig. 2HGo) and RXR-ß nuclear staining of benign FA specimens was moderate and similar to that of normal follicular thyroid cells in the same section (data not shown).



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  		FIG. 2. Representative staining of RXR isoforms in thyroid carcinomas and normal (N) surrounding thyroid tissue. Immunopositive cells appear brown as a result of 3,3'-diaminobenzidine tetrahydrochloride colorimetric reaction. A, PTC. Normal thyroid is at lower right. RXR-{alpha} demonstrated strong (level = 3) to moderate (level = 2) nuclear staining in adjacent normal thyroid follicular cells. In contrast, moderate (level = 2) cytoplasmic staining was observed in PTC cells by anti-RXR-{alpha} antibody (D-20; original magnification, x200). The inset shows the higher magnification of PTC (original magnification, x400). The strong granular staining was seen in the cytoplasm in PTC. C, FTC cells protrude into a vessel in its capsule. Normal thyroid is at bottom. FTC demonstrated moderate (level = 2) nuclear staining with absent (level = 0) cytoplasmic staining of RXR-ß (magnification, x200). E, MTC. Normal thyroid is at left. MTC shows almost absent (level = 0) nuclear immunostaining of RXR-{gamma}, whereas surrounding normal thyroid follicular cells indicated strong (level = 3) nuclear staining (magnification, x200). Inset is the higher magnification of normal thyroid cells (original magnification, x400). G, Large-cell ATC with a lymphocytic reaction. ATC demonstrated absent (level = 0) nuclear staining with moderate (level = 2) and granular cytoplasmic staining of RXR-{alpha} (magnification, x200). H, FA had a thick capsule and displayed strong (level = 3) nuclear immunoreactivity for RXR-{alpha} (magnification, x200). B, D, and F, The positive staining appears specific by immunocytochemical criteria because each blocking peptide completely blocked the staining (magnification, x200).

 
Immunohistochemical evaluation of the expression patterns of RXR isoforms are summarized in Table 2Go. Most PTCs (42 of 57) showed decreased levels of nuclear expression, accompanied by moderate or strong cytoplasmic expression for RXR-{alpha}. However, as seen in Table 2Go, some PTCs (four of 57) indicated moderate or strong nuclear staining accompanied by moderate or strong intensities of cytoplasmic staining. Similarly, FTCs showing decreased levels of nuclear expression (21 of 40) were accompanied mostly by moderate or strong cytoplasmic staining (17 of 40) for RXR-{alpha}. Compared with PTCs, a greater proportion of FTCs (19 of 40) showed moderate or strong nuclear immunostaining for RXR-{alpha}. None of the ATCs indicated moderate or strong nuclear expression for RXR-{gamma}. In contrast to the carcinomas, FA showed distinct nuclear immunostaining for RXR-{alpha} and RXR-ß. The relationship between the expression of each RXR isoform and the tumor type was analyzed by the Friedman test. The nuclear and cytoplasmic expression values of the isoforms were significantly decreased in all four classes of thyroid carcinomas (P < 0.05), except for the cytoplasmic expression of RXR-{alpha} protein (P = 0.27). However, there was only a specific correlation between nuclear RXR-{gamma} expression and histological typing of thyroid tumors (P = 0.04), indicating a statistical difference between PTC and ATC in the nuclear expression of RXR-{gamma} (P < 0.05).


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  		TABLE 2. RXR isoform expressions in human thyroid carcinomas

 
Because normal follicular thyroid cells showed moderate (level = 2) or strong (level = 3) nuclear staining accompanied by absent (level = 0) or weak (level = 1) cytoplasmic staining, a tumor was considered to have a suppressed nuclear RXR expression when the nuclear intensity level of the tumor cell was lower than 2 (moderate); if the cytoplasmic staining level was higher than 2, a tumor was considered to have an increased cytoplasmic RXR expression. The intensity of staining was compared with that of normal thyroid follicular cells in the margin of each specimen. Figure 3Go summarizes the results of this investigation. The nuclear RXR-{alpha} immunostaining was substantially suppressed in PTCs (80.7%) and FTCs (52.5%; Fig. 3Go). Statistically, RXR-{alpha} nuclear suppression in PTCs was significantly greater than in FTCs (P = 0.004), whereas the cytoplasmic RXR-{alpha} immunostaining was profoundly greater in PTCs than in FTCs (P = 0.024; Fig. 3Go). Although all three RXR isotypes were suppressed in PTCs and FTCs, there was no statistically significant difference in RXR-ß or RXR-{gamma} expression between PTCs and FTCs. The percentage of positively stained (staining level of 2 or 3) FTC nuclei was higher than that of positively stained PTC nuclei in RXR-{alpha} (FTC vs. PTC, 47.5 vs. 19.3%) and in RXR-ß (FTC vs. PTC, 35 vs. 15.8%). However, the percentage of nuclei with positive RXR-{gamma} nuclear expression was divergent from the percentages for the other two RXR isoforms (FTC vs. PTC, 27.5 vs. 38.6%). A similarly low percentage of positively stained RXR-{gamma} nuclei was also obtained on benign FA. The only other statistically significant difference among expression levels of the RXR isoforms between FTC and FA was in nuclear RXR-ß staining. MTCs, which were derived from C cells and which did not show the positive immunostaining for each RXR isoforms, had significant suppression of nuclear expressions for RXR-{alpha}, RXR-ß, and RXR-{gamma} compared with other thyroid tumors (except ATC).



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  		FIG. 3. Distribution of RXR isoform immunostaining intensity in nucleus and cytoplasm in thyroid samples (57 PTCs, 40 FTCs, 24 ATCs, 28 MTCs, and 27 FAs). The intensity of staining of tumors was compared with that of normal thyroid follicular cells in the margin of each specimen. Because normal follicular thyroid cells showed moderate (level = 2) or strong (level = 3) nuclear staining accompanied by absent (level = 0) or weak (level = 1) cytoplasmic staining, tumors were considered to have a suppressed nuclear RXR expression when the nuclear intensity level of the tumor cell was less than 2 (moderate); if the cytoplasmic staining level was higher than 2, a tumor was considered to have the increased cytoplasmic RXR expression. The y-axis represents the percentage of cases in each histological category of tumor samples. Open bars show absent (level = 0) or weak (level = 1) immunostaining for each RXR isoform, and filled bars show moderate (level = 2) or strong (level = 3) staining for each RXR isoform. For comparative analysis, two-tailed Fisher’s exact test was used to compare the differences in the expression of the RXR isoforms between two histological types. P < 0.05 was accepted as statistically significant. **, P < 0.01; *, P < 0.05.

 
The nuclear RXR expression levels seemed to vary according to the extent of differentiation. The percentages of nuclei with suppressed nuclear expression of RXR isoforms were significantly higher in ATCs than in benign FA (P < 0.05). Furthermore, in general, the ATC specimens had much lower nuclear RXR-{alpha} or RXR-{gamma} expression levels than did specimens of highly differentiated thyroid carcinomas (PTCs and FTCs).

mRNA expression of RXR isoforms in PTC, FTC, MTC, and FA

Next, we examined the gene expression levels of the RXR isoforms in human thyroid tumors. First, we evaluated the possibility that PTCs showing cytoplasmic immunostaining for RXR-{alpha} protein would also express different sizes of mRNA; for this, we used primers for the complete mRNA of RXR-{alpha} (Fig. 4AGo). Identically sized fragments were obtained for both PTC and normal thyroid tissue (Fig. 4AGo). Then, we examined the quantitative measures of mRNA expression of the RXR isoforms in thyroid tumors and normal thyroid tissue using real-time RT-PCR. Before this step, though, to discriminate the amounts of contaminated DNA in the RNA samples, we performed conservative RT-PCR without AMV-RT, and then we confirmed the negative bands seen in the negative controls (data not shown). Using the LightCycler, we performed two-step real-time RT-PCR for six PTCs, one FTC, one MTC, and one FA (Fig. 4BGo). The RXR isoforms were differentially expressed in all nine normal thyroid tissues, but expression was lower in five (for RXR-{alpha} or RXR-ß) of six PTCs, in FTC, and in benign FA when compared with matched normal tissue. One tumor tissue (PTC 4, Fig. 4BGo) showed higher expression of all three RXR isoforms. Another tumor tissue (PTC 5, Fig. 4BGo) indicated higher expression of RXR-{gamma}. For all of the isoforms, there were no significant differences in gene expression between normal thyroid tissue and neoplastic lesions. The mRNAs of all the isoforms were detected in all specimens, even in the thyroid carcinomas in which nuclear immunostaining for RXR isoforms was absent (data not shown). Therefore, the reduction in the expression of RXR isoforms observed at the protein level in thyroid carcinomas was not associated with a reduction in all mRNAs encoding the three receptors.



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  		FIG. 4. A, RXR{alpha} transcripts detected by RT-PCR in paired normal thyroid tissue and PTC. To evaluate the possibility that PTC showing cytoplasmic immunostaining for RXR-{alpha} protein would also express different sizes of mRNA, we performed RT-PCR by using the mRNA extracted from the normal thyroid tissue and PTC. There was no different-sized band seen in both samples. Marker, 1-kb DNA ladder (Invitrogen). B, Relative quantification of RXR mRNA expression in representative PTCs, FTC, MTC, and FA. Real-time PCR was performed using a LightCycler system. The single cDNA obtained by RT-PCR from tissues was used as a template for real-time PCR. Each histogram indicates the relative expression ratio. The control tissue is surrounding normal thyroid tissue. ß-actin was used as a reference gene. Open bars are the matched normal thyroid tissues, and filled bars are the tumor tissues.

 
Western blotting of RXR-{alpha} in PTCs

To confirm the immunohistochemical cytoplasmic RXR protein expression in PTCs, we also performed Western blot analysis of PTCs. Identical results were obtained when CEs and NEs of PTCs were fractionated and subjected to Western blotting (Fig. 5BGo). The results in Fig. 5BGo show the presence of a 54-kDa protein species corresponding to the predicted size of RXR-{alpha} in NEs from the normal surrounding tissues. The NEs from tumor tissues, which showed absent or weak nuclear staining for RXR-{alpha} in immunohistochemical analysis, were weaker than those from normal tissues in Western blots (Fig. 5BGo). In CEs from all tumor specimens, anti-RXR-{alpha} antibody (D-20) stained proteins with molecular masses of approximately 47, 44, and 40 kDa, whereas marginal reactions were observed in CEs from normal thyroid cells (Fig. 5BGo).



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  		FIG. 5. Western blot analysis of RXR-{alpha} in PTC, FTC, and MTC. A, Structure of human RXR-{alpha} and epitopes of antibodies against RXR-{alpha}. RXR-{alpha} (A/B) (D-20): amino acids 2–21; RXR-{alpha} (E) ({Delta}DN197): amino acids 198–462 (A/B, transactivation domain; C, DNA-binding domain; D, hinge region; and E, ligand-binding domain). B, Western blot analysis of NEs and CEs from PTCs, which shows the moderate (level = 2) cytoplasmic immunostainings and the decreased nuclear immunostainings of RXR-{alpha} and surrounding normal thyroid tissues. N, Normal thyroid tissue; T, neoplastic lesions. Number indicates case number. C, Western blot analysis of NEs and CEs isolated from PTC (T) and surrounding normal tissue (N) using two specific RXR-{alpha} antibodies, antihuman RXR-{alpha} (A/B) (D-20) or antihuman RXR-{alpha} (E) ({Delta}DN197). D, Western blot analysis of MTCs and FTCs. Number indicates case number. An arrowhead indicates the mobility of 54 kDa, and an asterisk indicates the mobility of 44 kDa.

 
Western blot analysis using two specific RXR-{alpha} antibodies

Previous studies have reported that the RXR-{alpha} molecule gets truncated as a result of degradation (16, 17, 18). To evaluate the possible existence of a truncated RXR-{alpha} protein, we performed Western blotting of NEs and CEs from PTC using two specific RXR-{alpha} antibodies (Fig. 5AGo). An RXR-{alpha}-related protein with an apparent molecular mass in SDS-PAGE of approximately 44 kDa was observed by immunoblotting analysis using the specific RXR-{alpha} antibody raised against sequences in the NH2 terminus of human RXR-{alpha} (D-20; Fig. 5CGo, asterisk). However, Western blotting performed with an antibody raised against the complete ligand-binding domain (E domain) of human RXR-{alpha} ({Delta}DN197) failed to detect the 44-kDa protein (Fig. 5CGo). Furthermore, to examine the subcellular expression of RXR-{alpha} protein in FTC and MTC, we performed Western blot analysis of FTCs and MTCs with two different histological types, each showing absent (level = 0; FTC1 and MTC1, Fig. 5DGo) or moderate to strong (levels = 2 to 3; FTC2 and MTC2, Fig. 5DGo) cytoplasmic RXR-{alpha} expression. The immunohistochemical data were supported by Western blot analysis. The RXR-{alpha}-specific 54-kDa band in NE from normal thyroid tissues was darker than the band from MTCs (Fig. 5DGo, arrowhead). One FTC tumor with distinct nuclear RXR-{alpha} staining (FTC1, Fig. 5DGo) showed stronger immunoreactivity toward the 54-kDa protein than did the normal thyroid tissue. Immunoreactive protein was also detected at 44 kDa with the RXR-{alpha} (D-20) antibody, but it was not detected by another RXR-{alpha} antibody ({Delta}DN197), in the CE from two specimens (MTC2 and FTC2, Fig. 5DGo, asterisk).

Expression of RXR in thyroid carcinoma cell lines

Finally, we evaluated the expression levels of the RXR isoforms’ mRNAs in three human thyroid cancer cell lines, ARO, WRO, and NPA. Figure 6AGo shows that, although RXR-{alpha} and RXR-ß mRNAs were detected under basal conditions in all cell lines, RXR-{gamma} transcript was expressed in NPA and ARO but was undetectable in WRO, the FTC cell line. We also performed Western blot analysis of NE and CE protein extracts from human thyroid cancer cell lines. Figure 6BGo shows that the RXR-{alpha} protein (54 kDa) was present in NE, while approximately 44-kDa immunoreactive bands were seen in CE from all three cell lines. In contrast to the immunohistochemical findings for human thyroid carcinomas, nuclear RXR-{alpha} protein was expressed abundantly in ARO cells but only slightly in WRO cells.



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  		FIG. 6. Expression of RXR isoforms in human thyroid cancer cell lines. A, RXR isoform mRNA in thyroid carcinoma cell line. Total RNA extracted from the human thyroid cancer cell lines ARO, WRO, and NPA was reverse transcribed and amplified with specific primer, as described in Materials and Methods (Table 1Go). The size of each amplified cDNA is indicated. ß-actin gene served as the internal control. B, Expression of RXR-{alpha} protein in human thyroid cancer cell lines. NEs and CEs from ARO, WRO, and NPA cells were subjected to Western blot, and the membranes were incubated with antihuman RXR-{alpha} antibody (D-20). An arrow indicates the mobility of 54 kDa, and an asterisk indicates the mobility of 44 kDa.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we examined the distribution of RXR isoforms in the normal human thyroid and in thyroid carcinomas and identified a marked difference in receptor expression between these isoforms. Previous studies have shown that RXR-{alpha} and RXR-ß mRNAs are widely expressed (19, 20, 21, 22), whereas the expression of RXR-{gamma} mRNA is restricted to tissues such as those in skeletal muscle, heart, brain (21, 22), hypothalamus (23, 24), and pituitary (11, 15, 23, 25, 26, 27). Dolle et al. (23) first demonstrated the diffuse expression of RXR-{gamma} transcript in the murine thyroid gland. Very recently, two studies from separate laboratories demonstrated the localization of RXR-{gamma} transcripts in normal human thyroid tissues (28) and thyroid tumors (28, 29). Consistent with these two studies, our present data first confirmed the presence of RXR-{gamma} protein in normal and neoplastic human thyroid follicular cells (Fig. 1Go and Fig. 2Go, E and F). These findings were extended by demonstrating the expression of the RXR isoforms in vitro. The mRNA of both RXR-{alpha} and RXR-ß were found to be expressed in all three human thyroid cancer cell lines (ARO, WRO, and NPA; Fig. 6AGo). Interestingly, WRO cells derived from the FTC cell line did not express RXR-{gamma} mRNA (Fig. 6AGo). Our results agree with the recent data described by Haugen et al. (29), which indicated the complete absence of RXR-{gamma} protein expression in WRO cells. These cell lines also exhibited the cytoplasmic RXR-{alpha} protein shown in the human thyroid tissue samples (Fig. 6BGo). The differential RXR expression in these thyroid cancer cell lines may be a useful model with which to investigate the mechanisms underlying the translocation and degradation of RXR-{alpha} in human thyroid carcinomas, as well as the correlation between retinoic acid-induced response and expression of the RXRs.

This study reveals that the nuclear expression of RXR isoforms was decreased in human thyroid tumors (Fig. 3Go, Table 2Go). One previous report has shown a marked decreased or absent expression of RXR-ß mRNA in nine of 12 human thyroid carcinoma samples examined (three FTCs, three oncocytic carcinomas, and six PTCs), suggesting a possible role for RXR-ß in the pathophysiology of thyroid carcinomas (10). Recently, there have been two reports of the mRNA expression of RXR isoforms in human thyroid carcinomas and matched normal thyroid tissues (28, 29). Tang et al. (28) described a loss of mRNA expression observed in 18 cases for RAR-ß, six cases for RXR-{alpha}, and five cases for RXR-ß. In the same study, RAR-{gamma} and RXR-{gamma} were expressed in all 42 paired normal thyroid and PTC samples examined (28). Haugen et al. (29) found lower RAR-ß mRNA levels in seven of eight malignant thyroid tumors than in matched normal tissue. In contrast to Tang et al., Haugen et al. (29) observed that RXR-{gamma} mRNA is not expressed in normal thyroid tissue but is highly expressed in subsets of human thyroid carcinoma tissues and cell lines. We also observed the decreased expression of mRNA in five of the six PTCs for RXR-{alpha} and RXR-ß and in four of the six PTCs for RXR-{gamma} (Fig. 4BGo). These controversial results might be explained by the design of the primers, the efficiency of PCR amplification, or unknown methodological factors.

Cytoplasmic staining was unexpectedly observed in some thyroid carcinomas with the use of the anti-RXR-{alpha} antibody (Fig. 1Go and Fig. 2Go, A and G). Moreover, Western blot analysis showed that a novel RXR-{alpha}-related protein was expressed in the CEs from PTCs, MTCs, and FTCs (Figs. 1Go and Fig. 5Go, B–D). Some previous studies have identified RXR-{alpha}-related proteins similar in molecular size to those seen in our study (16, 17, 18, 30, 31, 32). Matsushima-Nishiwaki et al. (16) have reported that calpain cleaves RXR-{alpha} into two fragments with molecular masses of 47 and 44 kDa; each fragment was found to lack a portion of the N-terminal A/B domain in the NE of human hepatoma-derived HuH 7 cells. Furthermore, Nomura et al. (17) have indicated that cathepsin L-type protease in cytoplasm cleaves RXR-{alpha} into smaller fragments with molecular masses of 45, 43, and 31 kDa in two human-derived cell lines, HepG2 and JEG-3. They speculated that the 45- and 43-kDa fragments would lack the N-terminal A/B domain and that a fragment of 31 kDa would lack domains A through D of RXR-{alpha} (17). Our present findings were consistent with their results regarding the sizes of the RXR-{alpha}-related proteins. However, the results of Western blot analysis using two specific anti-RXR-{alpha} antibodies led us to speculate that the approximately 44-kDa protein lacked the C terminus of RXR-{alpha}, which might be a novel fragment of human RXR-{alpha} protein.

Previously, Blanco et al. (33) have shown that deletion of the C-terminal region converts RXR-{alpha} and RXR-ß to powerful dominant negative receptors that inhibit retinoic acid and vitamin D-dependent transcription by respective heterodimer partners. Similarly, homozygous mutant mice expressing a truncated RXR-{alpha} (RXR-{alpha} AF20) lacking C-terminal 18 amino acids, which includes the last helical {alpha} structure of the ligand-binding domain and the core of the activating domain of the activation function 2 of RXR{alpha} protein, exhibit a subset of abnormalities (34) previously observed in RXR-{alpha}–/– mutants (35). These results suggest that the truncated RXR-{alpha} protein could not possess transcriptional activity because, in addition to its cytoplasmic localization, it lacked part of (or changed the conformation of) the ligand-binding domain, including activation function, which cannot be recognized by the anti-RXR-{alpha} antibody ({Delta}DN197; Fig. 5Go, C and D). Because of the interesting effects of RXR-{alpha}, we wish to gain some additional understanding of the function of this novel truncated protein.

The proteolytic fragment of RXR-{alpha} in the cytosol was produced, resulting in the translocation of RXR-{alpha} from the nucleus to the cytoplasm. Katagiri et al. (36) have demonstrated that nerve growth factor induces the phosphorylation of Ser 105 of nerve growth factor-induced clone B (NGFI-B) in PC 12 pheochromocytoma cells, increasing the export of the NGFI-B-RXR heterodimer complex from the nucleus to the cytoplasm. Recently, a nuclear localization sequence (NLS) of RXR was identified in the DNA-binding domain of RXR-{alpha} (37). Mutations in this NLS caused predominant cytoplasmic localization of nlsYFP-RXR and prevented transcriptional activity (37). Future work will also clarify whether or not the phosphorylation of heterodimerizing nuclear hormone receptors (e.g. TR, RAR, PPAR-{gamma}, and vitamin D3 receptor) and/or the mutations in NLS of RXR-{alpha} regulate the subcellular distribution of RXR-{alpha} in thyroid carcinomas.

Retinoids have been administered as anticancer and preventive cancer treatments in various clinical trials, and previous studies of the effects of retinoids on thyroid cancer have demonstrated that they promote cell differentiation and regulate cellular proliferation (38, 39, 40, 41, 42, 43, 44, 45). Recently, two clinical studies evaluated the redifferentiation effects of retinoic acids on thyroid cancer (46, 47). Simon et al. (46) have reported observing increased iodide uptake in 21 patients (42%). On the other hand, Gruning et al. (47) have found some improvement in radioiodine uptake in five (20%) of 25 patients, concluding that the therapeutic effects of retinoic acid in thyroid cancer are less than previously reported. Reduced expression of RXRs in human thyroid tumors, as demonstrated in the present study, could conceivably interfere with the inhibitory actions of retinoids on DNA synthesis and may thereby contribute to the development of thyroid carcinomas. Our present data, in combination with these earlier results, lead us to propose the predictive parameters of successful redifferentiation therapy using retinoids on thyroid carcinomas.


   Acknowledgments
 
We are grateful to Mr. Toshimichi Fujisawa (Surgical Pathology, Ito Hospital, Tokyo, Japan) for help in preparation of the thyroid sections. We thank Mr. Masatoshi Sado (Surgical Pathology, Asahikawa Medical College, Asahikawa, Japan) and Ms. Rie Matsumoto (The Second Department of Pathology, Asahikawa Medical College) for technical assistance of immunohistochemistry. We also thank Mr. Shinichi Chiba (Asahikawa Medical College) for technical assistance of a LightCycler. Dr. Naoko Sanno (Nippon Medical School, Tokyo, Japan) is thanked for technical advice on immunohistochemistry. We particularly acknowledge Dr. Akira Takeshita (Toranomon Hospital, Tokyo, Japan) for stimulating the initiation and progress of this study.


   Footnotes
 
Abbreviations: A-MuLV, A-murine leukemia virus; AMV-RT, A-MuLV reverse transcriptase; ATC, anaplastic thyroid carcinoma; CE, cytoplasmic extract; CP, crossing point; FA, follicular adenoma; FTC, follicular thyroid carcinoma; MTC, medullary thyroid carcinoma; NE, nuclear extract; NLS, nuclear localization sequence; PPAR, peroxisome proliferator-activated receptor; PTC, papillary thyroid carcinoma; RAR, retinoic acid receptor; RXR, retinoid X receptor; TR, thyroid hormone receptor.

Received November 21, 2003.

Accepted July 25, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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