PAX8-Peroxisome Proliferator-Activated Receptor (PPAR) Disrupts Normal PAX8 or PPAR Transcriptional Function and Stimulates Folli
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《内分泌学杂志》
Cancer Genetics Unit (A.Y.M.A., C.M., B.S., L.C., M.M., J.W., V.T., D.L., B.G.R., R.J.C.-B.), Kolling Institute of Medical Research, University of Sydney
Department of Endocrinology (D.L., B.G.R., R.J.C.-B.), Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia
Division of Metabolism (K.G.W., R.J.K.), Endocrinology and Diabetes, University of Michigan Medical Center, Ann Arbor, Michigan 48109
Abstract
Follicular thyroid carcinomas are associated with a chromosomal translocation that fuses the thyroid-specific transcription factor paired box gene 8 (PAX8) with the nuclear receptor peroxisome proliferator-activated receptor (PPAR). This study investigated the transcriptional mechanisms by which PAX8-PPAR regulates follicular thyroid cells. In HeLa cells, rat follicular thyroid (FRTL-5) cells, or immortalized human thyroid cells, PAX8-PPAR stimulated transcription from PAX8-responsive thyroperoxidase and sodium-iodide symporter promoters in a manner at least comparable with wild-type PAX8. In contrast, PAX8-PPAR failed to stimulate transcription from the thyroglobulin promoter and blocked the synergistic stimulation of this promoter by wild-type PAX8 and thyroid transcription factor-1. Unexpectedly, PAX8-PPAR transcriptional function on a PPAR-responsive promoter was cell-type dependent; in HeLa cells, PAX8-PPAR dominantly inhibited expression of the PPAR-responsive promoter, whereas in FRTL-5 and immortalized human thyroid cells PAX8-PPAR stimulated this promoter. In gel shift analyses, PAX8-PPAR bound a PPAR-response element suggesting that its transcriptional function is mediated via direct DNA contact. A biological model of PAX8-PPAR function in follicular thyroid cells was generated via constitutive expression of the fusion protein in FRTL-5 cells. In this model, PAX8-PPAR expression was associated with enhanced growth as assessed by soft agar assays and thymidine uptake. Therefore, PAX8-PPAR disrupts normal transcriptional regulation by stimulating some genes and inhibiting others, the net effect of which may mediate follicular thyroid cell growth and loss of differentiation that ultimately leads to carcinogenesis.
Introduction
PRIMARY THYROID CANCER may be classified according to its histological appearance as medullary, papillary, follicular, or anaplastic (1). Distinct molecular changes have now been described for each of these differing phenotypes. Papillary thyroid carcinoma is associated with chromosomal translocations fusing the RET (rearranged during transfection)-tyrosine kinase with papillary-specific genes (2), whereas medullary carcinoma is associated with activating mutations within RET (3). In contrast, follicular thyroid carcinoma was recently associated with a novel chromosomal translocation t(2;3)(q13;p25) (4). The genetic consequence of this translocation was to fuse the thyroid transcription factor paired box gene 8 (PAX8) with a nuclear receptor, peroxisome proliferator-activated receptor (PPAR). PAX8 is a critical regulator of thyroid differentiation and growth (5), whereas PPAR is a ligand-dependent transcription factor that is activated by the thiazolidinedione class of antidiabetic drugs (6, 7, 8) but is not known to have a thyroid-specific role. Therefore, the fusion protein is expressed in thyroid cells under the control of upstream PAX8 promoter elements.
Several studies have now confirmed this initial observation (9, 10, 11, 12, 13, 14). Overall, PAX8-PPAR has been detected in about 50% of follicular thyroid carcinomas by a combination of methods (RT-PCR, fluorescent in situ hybridization, and immunohistochemistry) (9, 10, 11, 12, 13). Contrary to the original report, however, PAX8-PPAR has now also been reported to occur in follicular adenomas, albeit less frequently (9, 10, 11, 12, 13). Our group noted that follicular adenomas containing the fusion protein were more likely to have a distinct histological phenotype (thick capsule, microfollicular architecture) (13) and two other studies also suggested that PAX8-PPAR was more likely to occur with a more aggressive phenotype (9, 12). Therefore, it is possible that PAX8-PPAR occurs early in follicular thyroid tumorigenesis or that benign tumors containing PAX8-PPAR possess other mechanisms to suppress carcinogenesis. However, given the histological difficulty in distinguishing follicular thyroid adenomas from carcinomas, a more provocative explanation is that apparently benign PAX8-PPAR-containing tumors are in fact noninvasive carcinomas.
In this regard, determining the mechanisms by which PAX8-PPAR causes follicular thyroid neoplasia is clearly important. At first it seemed probable that the fusion protein would retain at least some functional properties of its individual components, as has been noted for other fusion proteins involving transcription factors (15, 16). On the one hand, PPAR mediates transcriptional activation of target genes by recruiting transcriptional coactivators to cognate DNA-binding sites in a ligand-dependant manner (5, 6). Therefore, it was perhaps surprising that not only was PAX8-PPAR transcriptionally inactive on a PPAR-response element (PPRE), but the fusion protein also dominantly inhibited thiazolidinedione-induced gene transactivation by wild-type PPAR (4). It was not known whether the addition of PAX8 to the N terminus of PPAR in some way prevented its binding either with ligand or DNA. However, it is notable that in some circumstances, PPAR may interact with transcriptional corepressors to mediate gene silencing (17, 18, 19, 20), and that, in other situations, aberrant recruitment of corepressors has been associated with dominant-negative function of mutant nuclear receptors (21, 22, 23). Whereas PPAR was previously thought not to be expressed in normal thyroid, recent evidence suggests that it is expressed in certain malignant thyroid cell lines. Indeed, thiazolidinediones have been shown to inhibit growth and promote apoptosis in papillary thyroid cancer cells in vitro (24, 25). PPAR has also been implicated as a potential therapeutic target for several other cancer types including liposarcoma, breast, prostate, and colon (26, 27).
PAX8, on the other hand, is a member of a family of transcription factors containing the paired box DNA binding domain (located in the amino terminus of PAX8) (28). During development, PAX8 is expressed in thyroid, kidney, and neural tissue, but adult expression is restricted to thyroid tissue (29, 30). PAX8 targets include the thyroid-specific genes thyroglobulin (Tg), thyroperoxidase (TPO), and sodium-iodide symporter (NIS) (31). On the rat Tg promoter, Pax8 functions synergistically with another thyroid-specific transcription factor, thyroid transcription factor-1 (TITF1; also called TTF-1 and Nkx2–1), mediated via direct interaction on overlapping DNA binding sites (32, 33, 34).
To investigate the role of PAX8-PPAR in follicular thyroid tumorigenesis, we have studied the transcriptional function of PAX8-PPAR on thyroid- and PPAR-specific gene promoters and the effect of PAX8-PPAR on thyroid cell growth. We found that PAX8-PPAR has mixed transcriptional function, stimulating transcription of some thyroid-specific genes and inhibiting others. We also found that PAX8-PPAR transcriptional function was cell type specific, showing no PPAR-dependent function in heterologous cells but stimulating transcription from a PPAR-responsive promoter in thyroid cells. PAX8-PPAR stimulated proliferation and anchorage-independent growth of FRTL-5 cells. Overall these data suggest that PAX8-PPAR promotes neoplasia by directly interfering with normal transcriptional programming in follicular thyroid cells.
Materials and Methods
Cloning of expression plasmids
The human TPO promoter containing a PAX8 response element (–367 to +51 bp relative to the transcription start site) was amplified from human genomic DNA by forward and reverse primers 5'-ggggtaccgagctgcacccaacccaat-3' and 5'-ctagctagcagtaattttcacggctgt-3', respectively, and cloned into the pGL3-basic vector (Promega, Madison, WI) as a 419-bp KpnI/NheI fragment (35). The NISTK-Luc reporter construct contains a herpes simplex virus thymidine kinase promoter amplified from a TK-CAT plasmid by PCR using the forward and reverse primers 5'-tgtaaagatctggatccggccccgcccagcg-3' and 5'-tgtaaagatctatgccattgggatatatcaa-3', respectively, then cloned into the BglII site in pGL3-basic vector. The rat NIS upstream enhancer (rNUE) that harbors two PAX8 response elements (–2495 to –2264 bp relative to the translation start site) (36) was amplified by PCR using forward and reverse primers 5'-cggggtaccagattgcagctg-3' and 5'-ctagctagctctagaagaagg-3', respectively, and then cloned into the pGL3-TK plasmid as a 231-bp KpnI/NheI fragment such that the rNUE is upstream of the thymidine kinase promoter. The full-length NIS-Luc (flNIS-Luc) was a kind gift from Prof. S. Jhiang (The Ohio State University, Columbus, OH) and contains the rNUE from –2495 to –2264 joined to the proximal 2-kb rat NIS promoter from –1949 to –4 bp relative to the translation start site (37). The Tg-Luc reporter construct contains the human Tg promoter (–372 to +1 bp relative to the translation start site) cloned upstream of the luciferase gene. PPRETK-Luc contains three copies of the acyl-CoA oxidase PPAR response element (underlined: GTCGACAGGGGACCAGGACAAAGGTCACGTTCGGGAGTCGAC, three copies) cloned upstream of the TK-Luc reporter (38). Plasmids for Tg-Luc, PPRETK-Luc, TITF1, PAX8, PPAR, and PPAR mutant were kindly provided by Prof. V. K. K. Chatterjee (University of Cambridge, Cambridge, UK). The PAX8-PPAR plasmid was kindly provided by Dr. T. G. Kroll (University of Chicago, Chicago, IL).
Cell culture
HeLa and HEK293 cells were grown in DMEM (Life Technologies, Inc. Invitrogen, Grand Island, NY) supplemented with 10% fetal calf serum (Trace Biosciences, Castle Hill, Australia). Diploid FRTL-5 cells were grown in F12 Coon’s modification with L-glutamine and zinc sulfate medium (Sigma-Aldrich, St. Louis, MO) supplemented with 5% fetal calf serum, a six hormone combination (1 mIU/ml bovine TSH, 4 ng/ml insulin, 10 ng/ml somatostatin, 5 μg/ml transferrin, 4 mg/ml hydrocortisone, and 10 ng/ml glycyl-L-histidyl-L-lycine acetate; Sigma-Aldrich) and 50 mg/ml penicillin/streptomycin (Invitrogen Life Technologies). Nthy-ori cells were grown in RPMI 1640 supplemented with 10% fetal calf serum. Cells were grown in 37 C humidified conditions with 5% CO2.
Transient transfection experiments
HeLa and Nthy-ori cells were plated at 1 x 105 cells per well and FRTL-5 cells were plated at 2 x 105 cells per well and grown to approximately 80% confluence in 24-well plates. Media was changed to 2% charcoal-stripped calf serum in Opti-MEM (Invitrogen Life Technologies) before transfection. Transient transfections were performed using DMRIE-C (Invitrogen Life Technologies) with 1 μg of transcription factor expression plasmid (PAX8, PPAR, PAX8-PPAR, TITF1, or pcDNA3 empty vector), 1 μg of luciferase promoter (Tg-Luc, NISTK-Luc, flNIS-Luc, PPRE-Luc, or 8 μg TPO-Luc), and 1 μg of -galactosidase plasmid for HeLa cell transfections and 100 ng of transcription factor expression plasmid (PAX8, PPAR, PAX8-PPAR, or pcDNA3 empty vector), 100 ng of luciferase promoter (Tg-Luc, NISTK-Luc, PPRE-Luc, or 800 ng TPO-Luc), and 100 ng of -galactosidase plasmid for FRTL-5 and Nthy-ori cell transfections. Cells were incubated for 16–18 h and replenished with 5% charcoal-stripped calf serum in normal growing media for 24 h together with 10 μM ciglitizone (Sigma-Aldrich) or equivalent volume of solvent control as indicated. Cells were then lysed and assayed for luciferase activity (Promega) and -galactosidase activity was used for normalization of reporter activity. Transcriptional response was expressed relative to that seen with empty vector in the absence of ciglitizone, and data shown represent mean ± SEM of at least three experiments, each performed in triplicate wells.
Transfections in dog thyrocytes
Whole thyroid glands were removed from dogs that had been previously anesthetized and exsanguinated as part of an unrelated, institutionally approved study. Thyroid glands were removed within 10 min of exsanguination. Glands were trimmed and minced, and primary cultures of thyrocytes were obtained following the method of Uyttersprot et al. (39). Briefly, minced thyroid tissue was subjected to collagenase digestion, serially rinsed in RPMI, and plated in a serum-free media consisting of DMEM-F12 Ham-MCDB 105 in a 2:1:1 ratio, with additional growth factors including bovine TSH at 15 mIU/ml.
For transfection, primary thyrocytes were plated into 24-well plates in culture media without additives. Transfections were performed with lipofectamine and Plus reagents according to the manufacturer’s protocol (Invitrogen), and included 100 ng of firefly luciferase reporter plasmid, 100 ng of transcription factor expression plasmid (PAX8, PPAR, PAX8-PPAR, or empty vector), and an internal control Renilla luciferase plasmid (0.1 ng CMV-Rluc, 0.5–1 ng SV40-Rluc, or 5 ng tk-Rluc). After 3 h of transfection, equal volumes of culture media containing 20% charcoal-stripped calf serum and penicillin/streptomycin were added to the wells. Thirty hours after initial transfection, the culture media were replaced with media containing either 10 μM ciglitizone or vehicle for an additional 24 h. Thyrocytes were then rinsed, lysed, and analyzed for firefly and Renilla luciferase activities using the Promega dual luciferase reagents and protocol.
Stable FRTL-5 cell lines
FRTL-5 cells were plated at a density of 6 x 105 cells per 60-mm diameter tissue culture dish, 48 h before transfection. Transfections were performed using FuGENE6 (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s protocols with either 1 μg of pcDNA3 empty vector or PAX8-PPAR expression vector (containing the neomycin resistance gene). Eighteen hours after transfection, selection of transfected FRTL-5 cells was performed by replenishing cells with normal growing medium supplemented with 100 μg/ml Geneticin (Invitrogen Life Technologies) for 10 d. Fresh medium containing Geneticin was replaced twice a week until no cells were remaining in the control plate. Stable transfectants were pooled and maintained in media containing 100 μg/ml Geneticin.
Gel shift analyses
Gel shift experiments were performed using a PPRE oligonucleotide DR+1 made by annealing 5'-aaggggccaactaggtcaaaggtcaccggt-3' and 5'-aaggggaccggtgacctttgacctagttgg-3' primers and labeling with 32P-dCTP. Five to 10 μl of in vitro-translated products were incubated for 30 min at room temperature in a binding buffer composed of 20 mM HEPES (pH 7.8), 1 mM dithiothreitol, 0.1% Nonidet P-40, 50 mM KCl, 20% glycerol, and approximately 20,000 cpm of the radiolabeled probe in a final volume of 45 μl. In supershift studies, in vitro-translated products were preincubated for 30 min with 200 ng of anti-PPAR E8 antibody (sc-7273; Santa Cruz Biotechnology, Santa Cruz, CA) before addition of radiolabeled probe. Bound products were separated on a 6% PAGE in 0.5x Tris-borate-EDTA. The gel was fixed in 30% methanol and 10% acetic acid for 20 min, vacuum dried for 2 h at 80 C, and exposed to film at –80 C.
RNA isolation and RT-PCR
RNA was isolated using TRI Reagent (Sigma-Aldrich) according to the manufacturer’s protocol, and 1 μg RNA was reverse transcribed using Superscript II (Invitrogen Life Technologies) and oligo-dT primers (Invitrogen Life Technologies) as per protocol. The cDNAs were amplified by PCR using a forward primer derived from exon 6 of PAX8, 5'-cgcggatccgcattgactcacagagca-3' and an antisense primer from exon 1 of PPAR1, 5'-ccggaattcgaagtcaacagtagtgaa-3' for detection of the PAX8-PPAR fusion gene. The 2-microglobulin housekeeping gene was amplified using a forward primer derived from exon 2, 5'-acccccactgaaaaagatga-3' and an antisense primer from exon 4, 5'-atcttcaaacctccatgatg-3'.
Tritiated thymidine uptake assay
FRTL-5 cells were seeded into 24-well plates at a density of 1 x 105 cells per well and grown for 48 h. Each well was pulsed with 37 MBq of [methyl-3H]thymidine (PerkinElmer Cetus, Norwalk, CT) for 4 h at 37 C. Ciglitizone at 10 μM or equal volume of solvent control was added 24 h before the experiment. Cells were washed twice with ice cold PBS (Invitrogen Life Technologies) and once with cold 0.9% NaCl. Ice-cold methanol-acetic acid (3:1) was used to fix the cells for 2 h at 4 C. The methanol-acetic acid was removed and 0.5 M NaOH was added, and the cells were left overnight to solubilize. Lysate (500 μl) was mixed with 4 ml of liquid scintillant (Optima Gold) and counted for 2 min on a liquid scintillation counter (Packard 1500).
Anchorage-independent cell growth assay
A total of 1 x 104 cells from each stable cell line were trypsinized, resuspended in 1 ml of 0.5% agarose (SeaPlaque; Edwards Instrument Co., New South Wales, Australia), and layered on top of a solidified bottom layer of 0.5% agarose in a 6-well plate. One milliliter of normal growing media was added on top of the gel and changed twice a week. The number of colonies in each well was counted after 20 d by staining with 1 mg/ml p-iodonitrotetrazolium for 24 h. Ciglitizone was added for 48 h before staining.
Statistics
The statistical software package for Microsoft Excel was used to analyze the data. Pairwise comparisons using t tests were performed between groups as indicated. P 0.05 was considered significant.
Results
Transcriptional function of PAX8-PPAR on PAX8-responsive promoters in heterologous cells
We first examined the transcriptional function of PAX8-PPAR on reporter genes containing PAX8 response elements from within the NIS, TPO, and Tg genes, in heterologous HeLa cells that have previously been used for study of these promoters (32, 33, 34, 36, 40, 41). In these transient transfection experiments, wild-type PAX8 stimulated transcription more strongly from NISTK-Luc (2.4 ± 0.2-fold; Fig. 1A) than Tg-Luc (1.7 ± 0.3-fold; Fig. 1D), and we were unable to show significant PAX8 induction of full-length flNIS-Luc (1.3 ± 0.65-fold; Fig. 1B) or TPO-Luc constructs (1.2 ± 0.1-fold; Fig. 1C). In contrast, PAX8-PPAR stimulated NISTK-Luc (3.0 ± 0.4-fold; Fig. 1A), flNIS-Luc (4.5 ± 0.2-fold; Fig. 1B), and TPO-Luc (1.8 ± 0.2-fold; Fig. 1C). Furthermore, the addition of the PPAR agonist ciglitizone significantly enhanced PAX8-PPAR-mediated transcription of NISTK-Luc and TPO-Luc (Fig. 1, A and C), suggesting that the PPAR C terminus is functional within the fusion protein in some promoter contexts. Nevertheless, transcriptional activation by PAX8-PPAR on NISTK-Luc was at least an order of magnitude less sensitive to thiazolidinedione treatment than that observed with PPAR alone on the PPAR-responsive promoter (Fig. 1E).
However, on the PAX8-responsive Tg promoter, PAX8-PPAR not only failed to stimulate but indeed significantly repressed transcription relative to basal levels (0.73 ± 0.1-fold, P = 0.02; Fig. 1D). The addition of ciglitizone did not relieve the repressive effect of PAX8-PPAR on this promoter. Therefore, PAX8-PPAR appeared to be a ligand-dependent positive regulator of NIS and TPO promoter transcription, and a negative regulator of Tg promoter transcription in heterologous cells.
Because the Tg promoter is coordinately regulated by PAX8 and TITF1 together, we next sought to investigate PAX8-PPAR transcriptional function in the presence of TITF1. As expected, TITF1 stimulated Tg promoter transcription more strongly than PAX8 alone (9.5 ± 0.8-fold vs. 1.6 ± 0.1-fold, respectively, P = 0.04; Fig. 2). However, the addition of PAX8-PPAR inhibited TITF1-mediated transcription from the Tg promoter in a dominant-negative manner (0.7 ± 0.1-fold repression, P = 0.01; Fig. 2). In contrast, as previously described (31, 32, 33), the addition of PAX8 and TITF1 together synergistically activated transcription from the Tg promoter (17.3 ± 4.1-fold, P = 0.04 compared with TITF1 alone; Fig. 2). Nevertheless, PAX8/TITF1-mediated transcription was also dominantly inhibited by the presence of PAX8-PPAR (0.6 ± 0.04-fold repression, P = 0.05; Fig. 2). The addition of ciglitizone did not relieve this repressive effect (data not shown). Although PAX8-PPAR did not significantly inhibit wild-type PAX8 function alone on the Tg promoter (0.8 ± 0.2-fold repression, P = 0.17; Fig. 2), the relatively weak activation of this promoter by PAX8 may have limited our ability to detect this.
Transcriptional function of PAX8-PPAR on PAX8-responsive promoters in thyroid cells
We next wished to examine PAX8-PPAR function in cell lines relevant to its proposed role in thyroid neoplasia. We chose the well-differentiated Fischer rat thyroid (FRTL-5) cell line and human Nthy-ori cells that are commonly used in follicular thyroid cell models (42, 43) and that do not contain PAX8-PPAR (data not shown). Overall, we found that PAX8-dependent transcription by PAX8-PPAR was similar in thyroid cells compared with heterologous cells (Fig. 3). Although we expected that FRTL-5 and Nthy-ori cells contain endogenous PAX8, addition of transiently transfected PAX8 further stimulated expression from NISTK-Luc in both cell lines (Fig. 3, A and B). As in HeLa cells, PAX8-PPAR also stimulated expression of NISTK-Luc and TPO-Luc in either thyroid cell line (Fig. 3, A–D). Results of reporter gene expression in the presence of ciglitazone were intriguing, insomuch as there appeared to be a general augmentation of transcription independent of the addition of either PAX8 or PAX8-PPAR (Fig. 3, A–F), although these promoters lack a PPRE consensus sequence (data not shown). PAX8-PPAR-mediated transcription was not specifically activated by the addition of ciglitizone on any promoter in thyroid cells (Fig. 3, A–F). Importantly, the lack of PAX8-PPAR transcriptional activity on the Tg promoter was also observed in both thyroid cell lines (Fig. 3, E and F) indicating that PAX8-PPAR-dependent inactivity on this promoter is not due to lack of thyroid-specific transcriptional cofactors in heterologous cells.
Transcriptional function of PAX8-PPAR on PPAR-responsive promoters
We then examined PAX8-PPAR function on a PPAR-responsive promoter (38). In HeLa cells, wild-type PPAR stimulated basal transcription from PPRETK-Luc 4.4 ± 0.8-fold (P < 0.01) and responded to thiazolidinedione as expected (further stimulation by 2.1 ± 0.1-fold, P = 0.02; Fig. 4A). PAX8-PPAR in contrast failed to activate this promoter either in the absence or presence of PPAR agonist, but repression of this promoter was not observed (1.1 ± 0.1-fold change; Fig. 3A). Conversely, a synthetic PPAR dominant-negative mutant that has previously been shown to recruit corepressors (18) did repress basal transcription of PPRETK-Luc 0.7 ± 0.1-fold (P = 0.03), although this effect was partly reversed by the addition of thiazolidinedione (Fig. 4A). We also found that PAX8-PPAR is inactive and dominantly inhibits PPAR-mediated transcription from PPRETK-Luc in HEK293 cells (data not shown). Overall, our data in heterologous cells was comparable to a previous report that studied PAX8-PPAR on the PPAR-responsive aP2 enhancer (4).
In FRTL-5 and Nthy-ori cells, however, PAX8-PPAR stimulated transcription from PPRETK-Luc (Fig. 4, B and C). PAX8-PPAR-mediated basal transcription of this promoter was comparable to that seen with wild-type PPAR (3.0 ± 0.5-fold vs. 3.0 ± 0.2-fold, respectively, in FRTL-5 cells and 4.7 ± 0.3-fold vs. 4.2 ± 1.2-fold, respectively, in Nthy-ori cells). PAX8-PPAR-dependent transcription was significantly stimulated by 10 μM ciglitizone in Nthy-ori cells (Fig. 4C), but no significant ligand response was seen in FRTL-5 cells (Fig. 4B). The addition of ciglitizone stimulated PPAR-dependent transcription 2.8 ± 0.5-fold in FRTL-5 cells (Fig. 4B) and 15.5 ± 4.7-fold in Nthy-ori cells (Fig. 4C). By comparison, the dominant-negative PPAR mutant neither repressed nor stimulated this promoter in FRTL-5 cells (1.1 ± 0.1-fold change).
To address potential concerns that available thyroid cell lines may differ substantially from true thyrocytes, PPRETK-Luc was also transfected into primary cultures of dog thyrocytes (Fig. 4D). Luciferase was induced 8.1 ± 2.0-fold by PPAR in the absence of ciglitizone and 30.6 ± 6.3-fold in the presence of ciglitizone. Inductions by PAX8-PPAR were 7.2 ± 1.8-fold and 17 ± 3.0-fold in the absence and presence of ciglitizone, respectively. These data are similar to the findings in FRTL-5 and Nthy-ori cells.
PAX8-PPAR binds to a PPAR consensus DNA recognition sequence
Transcription factors mediate gene transactivation through binding to specific cognate response elements located in promoter regions. PPAR binds a consensus recognition sequence containing direct repeats of a hexamer spaced by one nucleotide (DR+1) (44). Therefore, we examined the ability of PAX8-PPAR to bind this cognate DNA element in gel shift analyses. PPAR preferentially interacts with this DNA sequence as a heterodimer with the retinoid X receptor (RXR) (Fig. 5, lane 3) although RXR homodimers also form a complex, albeit less efficiently (Fig. 5, lanes 1 and 2). The addition of an anti-PPAR monoclonal antibody results in a supershifted complex (Fig. 5, lane 4), confirming the identity of the DNA-bound PPAR-RXR heterodimer. PAX8-PPAR also bound this DNA sequence as a heterodimer with RXR (Fig. 5, lane 5) with a size difference consistent with the presence of PAX8 compared with PPAR-RXR alone. Translation from an internal start site in PAX8-PPAR produced a DNA-bound complex with RXR similar in mobility to the PPAR-RXR band. Once again, the addition of the anti-PPAR antibody confirmed the identity of the PAX8-PPAR-RXR heterodimer-DNA complex (Fig. 5, lane 6). No PPAR or PAX8-PPAR complexes were seen in the absence of RXR, nor did the addition of ciglitizone alter the mobility of either PPAR-RXR or PAX8-PPAR-RXR DNA-bound complexes (data not shown). We have not as yet been able to demonstrate PAX8-PPAR binding to PAX8 response elements in gel mobility shift experiments (data not shown), although it is possible that we are lacking some critical cofactor required for binding.
PAX8-PPAR increases proliferation of rat follicular thyroid cells
We then examined the effect of PAX8-PPAR expression on follicular thyroid cell growth. A recent report demonstrated that PAX8-PPAR expression increased growth and decreased apoptosis in human Nthy-ori cells (43). In contrast, we chose to express PAX8-PPAR in the better-differentiated FRTL-5 cell model (45). FRTL-5 cells were stably transfected with pcDNA3-PAX8-PPAR constitutively expressing the fusion protein under control of the cytomegalovirus promoter. We confirmed PAX8-PPAR expression by RT-PCR (Fig. 6A). PAX8-PPAR expression was associated with increased FRTL-5 cell growth as assessed by thymidine uptake (Fig. 6B) and by proliferation in semisolid media (46) (Fig. 6C). The addition of thiazolidinedione did not affect thymidine uptake (Fig. 6B) or soft-agar proliferation (data not shown) by PAX8-PPAR-expressing FRTL-5 cells.
Discussion
The chromosomal translocation t(2;3)(q13;p25) that produces the PAX8-PPAR fusion gene has been associated with a subset of follicular thyroid carcinomas and possibly adenomas, although the transcriptional mechanisms by which PAX8-PPAR stimulates thyroid neoplasia have not previously been studied in detail. A prevailing hypothesis is that PAX8-PPAR inhibits PPAR function in thyroid cells. In support of this, PAX8-PPAR was initially shown to disrupt PPAR-mediated transcription in a dominant-negative manner (4). A subsequent study also found that PAX8-PPAR dominantly inhibited PPAR-mediated transcription in human Nthy-ori cells and proposed that PAX8-PPAR-stimulated growth of these cells depended at least in part on inhibition of PPAR function (43). Finally, PPAR expression was shown to be down-regulated in follicular thyroid carcinomas that do not contain the PAX8-PPAR translocation (47, 48). In combination, therefore, these previous results suggested that follicular thyroid neoplasia may be associated with net loss of PPAR activity either through lack of endogenous PPAR expression or by PAX8-PPAR-mediated dominant inhibition of wild-type PPAR transcriptional function.
Our study provides combined evidence for two alternate hypotheses, namely that PAX8-PPAR affects PAX8 function on the one hand, and is capable of inducing PPAR-dependent transcription on the other. However the pattern of transcriptional regulation by PAX8-PPAR is complex, such that some PAX8-responsive promoters were up-regulated (TPO, NIS), whereas another (Tg) was inhibited in a dominant-negative manner. PAX8-PPAR function on a PPRE was similarly complex, capable of stimulating transcription from this promoter when in the appropriate cellular environment. These results are strongly supported by a recent report by Lacroix et al. (61) that found specific up-regulation of several PPAR target genes in tumors containing PAX8-PPAR, but not in those tumors lacking the translocation. Taken together, these data suggest that inappropriate induction of some PPAR-responsive genes, rather than their repression, may contribute to the pathogenesis of follicular thyroid cancer, a hypothesis that is all the more plausible considering the extremely low level of endogenous PPAR expression in normal thyrocytes. Therefore, the differences between PAX8-PPAR-mediated regulation of the acyl-CoA oxidase PPRE used in our study and the aP2 enhancer PPRE used by Powell et al. (43) are likely to be promoter and/or cofactor dependent. We are now studying PAX8-PPAR transcriptional function on those PPAR-target genes including angiopoietin-like 4 and aquaporin 7 that were found to be up-regulated in microarray analyses of PAX8-PPAR-positive tumors (61).
PAX8-PPAR responsiveness to PPAR agonist also appeared to be both promoter and cell type dependent. On the NIS promoter, transcriptional activation by PAX8-PPAR in response to thiazolidinedione treatment was comparatively weak and at least an order of magnitude less sensitive than that observed with PPAR alone on a PPAR-responsive promoter (Fig. 1E). Although stronger agonist responses were seen on a PPRE in thyroid cells, PAX8-PPAR transcription was still not augmented by ciglitizone to the same extent as for wild-type PPAR despite comparable basal activities (Fig. 4, C and D). Whether this relates to altered ligand affinity in the fusion protein, or to altered cofactor recruitment is not yet clear. We are also now examining whether PAX8-PPAR may be more responsive to tyrosine-based PPAR agonists, as has been previously reported with PPAR mutants that otherwise respond poorly to thiazolidinediones both in vitro and in vivo (18, 59).
Thus, our studies suggest that PAX8-PPAR has mixed function that can either replicate the transcriptional effects of either PAX8 or PPAR alone when the appropriate promoter- or cell-type-specific conditions are present; or inhibit gene expression when these necessary promoter- or cell-specific conditions are lacking. We have demonstrated that growth of rat FRTL-5 thyroid cells was stimulated by PAX8-PPAR expression, as has previously been shown for less well-differentiated human thyroid cells (43). In combination, our results suggest that PAX8-PPAR disrupts normal transcriptional pathways in thyroid cells by means of a complex mix of activation of some genes and inhibition of others, the net effect of which is to stimulate follicular thyroid neoplasia.
Differing histological phenotypes of thyroid carcinoma have all now been associated with specific molecular abnormalities. Papillary carcinoma is associated with a chromosomal translocation that leads to activation of the RET proto-oncogene (2, 49) or an activating mutation of the braf kinase (50). Medullary carcinoma is associated with RET mutations that lead to its constitutive activity within C-cells (3). Our finding that PAX8-PPAR stimulates PAX8-specific gene expression is consonant with its specific association with follicular thyroid cell neoplasms, and the known role of PAX8 in thyroid follicular cell differentiation (8). Conversely, the mechanism by which PAX8-PPAR inhibits transcription from the PAX8-responsive Tg promoter is unclear. It is possible that PAX8-PPAR binds the Tg promoter but cannot interact with TITF1 or other cofactors necessary for Tg transcription, and we found that PAX8-PPAR dominantly inhibited TITF1 mediated Tg-Luc transcription in a manner that might be consistent with steric hindrance (Fig. 2). Alternatively, PAX8-PPAR might recruit transcriptional corepressors via PPAR. Indeed, specific mutations in the extreme C terminus of PPAR have been previously shown to cause corepressor recruitment and dominant-negative transcriptional function by PPAR (18, 19). Furthermore, naturally occurring PPAR mutations associated with a syndrome of insulin resistance and lipodystrophy have also been shown to be associated with recruitment of corepressors to target genes (20). However, when we compared PAX8-PPAR with a corepressor-binding mutant PPAR (18), although both dominantly inhibited transcription from PPRETK-Luc in heterologous cells (Fig. 4A and data not shown), PAX8-PPAR clearly differed from the mutant PPAR in its transcriptional function in thyroid cells (Fig. 4, B–D). We speculate that this may be due to PAX8-mediated recruitment of thyroid-specific coactivators to the DNA-bound fusion protein (Fig. 5).
Dysregulated recruitment of transcriptional corepressors has also been implicated in other clinical syndromes, including thyroid hormone resistance syndrome associated with mutant thyroid hormone receptors (TR) (21) and acute promyelocytic leukemia associated with translocations fusing the myeloid-specific PML or PLZF genes with the retinoic acid receptor gene (22, 23, 51). Corepressor-mediated recruitment of histone deacetylases has been proposed to be a key pathogenic feature in acute promyelocytic leukemia (22, 23, 51). Similarly, recent work has also suggested that histone deacetylases may be important in thyroid carcinogenesis (52, 53, 54, 55, 56, 57, 58). Histone deacetylase inhibitors promote reexpression of NIS (52, 53, 54, 55, 56) and increase apoptosis (57, 58) in poorly differentiated thyroid cancer cell lines. We are now investigating whether these compounds specifically derepress PAX8-PPAR transcription.
Although PAX8-PPAR stimulates NIS and TPO promoter expression in our cell culture models, these genes may be down-regulated in follicular thyroid carcinomas (60). Indeed, the recent report by Lacroix et al. (61) did not find that Tg, TPO, or NIS genes were differentially expressed in follicular tumors harboring or not harboring the PAX8-PPAR translocation. However, only a few PAX8-PPAR tumors were analyzed (four in microarray, five in quantitative PCR experiments) and since multiple transcription factors regulate these promoters it may be too simplistic to expect PAX8-PPAR to have discernible effects in a small sample size. It is also possible that PAX8-mediated transcriptional function is dependent upon as yet unknown cofactors that are lost as thyroid cells become progressively undifferentiated. Even if basal NIS expression is not altered, it will be very interesting to determine whether PPAR agonists might up-regulate NIS expression in follicular carcinomas harboring the PAX8-PPAR translocation, which would have practical therapeutic relevance in enhancing tumor cell ablation by radioactive iodine.
Acknowledgments
We are indebted to Prof. V. K. K. Chatterjee (University of Cambridge) for providing expression vectors for TITF1, PAX8, PPAR, PPAR mutant, Tg-Luc, and PPRETK-Luc; Prof. S. Jhiang (The Ohio State University) for the full-length flNIS-Luc; and to Dr. T. G. Kroll (University of Chicago) for the PAX8-PPAR expression plasmid.
Footnotes
This work was supported by the Cure Cancer Australia Foundation, University of Sydney Cancer Research Fund (to B.G.R and R.J.C.-B.) and National Institutes of Health Grant DK44155 (to R.J.K.).
First Published Online September 22, 2005
1 A.Y.M.A. and C.M. contributed equally to this work.
Abbreviations: NIS, Sodium-iodide symporter; PAX8, paired box gene 8; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR-response element; rNUE, rat NIS upstream enhancer; RET, rearranged during transfection; RXR, retinoid X receptor ; Tg, thyroglobulin; TITF1, thyroid transcription factor-1; TPO, thyroperoxidase.
Accepted for publication September 13, 2005.
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Department of Endocrinology (D.L., B.G.R., R.J.C.-B.), Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia
Division of Metabolism (K.G.W., R.J.K.), Endocrinology and Diabetes, University of Michigan Medical Center, Ann Arbor, Michigan 48109
Abstract
Follicular thyroid carcinomas are associated with a chromosomal translocation that fuses the thyroid-specific transcription factor paired box gene 8 (PAX8) with the nuclear receptor peroxisome proliferator-activated receptor (PPAR). This study investigated the transcriptional mechanisms by which PAX8-PPAR regulates follicular thyroid cells. In HeLa cells, rat follicular thyroid (FRTL-5) cells, or immortalized human thyroid cells, PAX8-PPAR stimulated transcription from PAX8-responsive thyroperoxidase and sodium-iodide symporter promoters in a manner at least comparable with wild-type PAX8. In contrast, PAX8-PPAR failed to stimulate transcription from the thyroglobulin promoter and blocked the synergistic stimulation of this promoter by wild-type PAX8 and thyroid transcription factor-1. Unexpectedly, PAX8-PPAR transcriptional function on a PPAR-responsive promoter was cell-type dependent; in HeLa cells, PAX8-PPAR dominantly inhibited expression of the PPAR-responsive promoter, whereas in FRTL-5 and immortalized human thyroid cells PAX8-PPAR stimulated this promoter. In gel shift analyses, PAX8-PPAR bound a PPAR-response element suggesting that its transcriptional function is mediated via direct DNA contact. A biological model of PAX8-PPAR function in follicular thyroid cells was generated via constitutive expression of the fusion protein in FRTL-5 cells. In this model, PAX8-PPAR expression was associated with enhanced growth as assessed by soft agar assays and thymidine uptake. Therefore, PAX8-PPAR disrupts normal transcriptional regulation by stimulating some genes and inhibiting others, the net effect of which may mediate follicular thyroid cell growth and loss of differentiation that ultimately leads to carcinogenesis.
Introduction
PRIMARY THYROID CANCER may be classified according to its histological appearance as medullary, papillary, follicular, or anaplastic (1). Distinct molecular changes have now been described for each of these differing phenotypes. Papillary thyroid carcinoma is associated with chromosomal translocations fusing the RET (rearranged during transfection)-tyrosine kinase with papillary-specific genes (2), whereas medullary carcinoma is associated with activating mutations within RET (3). In contrast, follicular thyroid carcinoma was recently associated with a novel chromosomal translocation t(2;3)(q13;p25) (4). The genetic consequence of this translocation was to fuse the thyroid transcription factor paired box gene 8 (PAX8) with a nuclear receptor, peroxisome proliferator-activated receptor (PPAR). PAX8 is a critical regulator of thyroid differentiation and growth (5), whereas PPAR is a ligand-dependent transcription factor that is activated by the thiazolidinedione class of antidiabetic drugs (6, 7, 8) but is not known to have a thyroid-specific role. Therefore, the fusion protein is expressed in thyroid cells under the control of upstream PAX8 promoter elements.
Several studies have now confirmed this initial observation (9, 10, 11, 12, 13, 14). Overall, PAX8-PPAR has been detected in about 50% of follicular thyroid carcinomas by a combination of methods (RT-PCR, fluorescent in situ hybridization, and immunohistochemistry) (9, 10, 11, 12, 13). Contrary to the original report, however, PAX8-PPAR has now also been reported to occur in follicular adenomas, albeit less frequently (9, 10, 11, 12, 13). Our group noted that follicular adenomas containing the fusion protein were more likely to have a distinct histological phenotype (thick capsule, microfollicular architecture) (13) and two other studies also suggested that PAX8-PPAR was more likely to occur with a more aggressive phenotype (9, 12). Therefore, it is possible that PAX8-PPAR occurs early in follicular thyroid tumorigenesis or that benign tumors containing PAX8-PPAR possess other mechanisms to suppress carcinogenesis. However, given the histological difficulty in distinguishing follicular thyroid adenomas from carcinomas, a more provocative explanation is that apparently benign PAX8-PPAR-containing tumors are in fact noninvasive carcinomas.
In this regard, determining the mechanisms by which PAX8-PPAR causes follicular thyroid neoplasia is clearly important. At first it seemed probable that the fusion protein would retain at least some functional properties of its individual components, as has been noted for other fusion proteins involving transcription factors (15, 16). On the one hand, PPAR mediates transcriptional activation of target genes by recruiting transcriptional coactivators to cognate DNA-binding sites in a ligand-dependant manner (5, 6). Therefore, it was perhaps surprising that not only was PAX8-PPAR transcriptionally inactive on a PPAR-response element (PPRE), but the fusion protein also dominantly inhibited thiazolidinedione-induced gene transactivation by wild-type PPAR (4). It was not known whether the addition of PAX8 to the N terminus of PPAR in some way prevented its binding either with ligand or DNA. However, it is notable that in some circumstances, PPAR may interact with transcriptional corepressors to mediate gene silencing (17, 18, 19, 20), and that, in other situations, aberrant recruitment of corepressors has been associated with dominant-negative function of mutant nuclear receptors (21, 22, 23). Whereas PPAR was previously thought not to be expressed in normal thyroid, recent evidence suggests that it is expressed in certain malignant thyroid cell lines. Indeed, thiazolidinediones have been shown to inhibit growth and promote apoptosis in papillary thyroid cancer cells in vitro (24, 25). PPAR has also been implicated as a potential therapeutic target for several other cancer types including liposarcoma, breast, prostate, and colon (26, 27).
PAX8, on the other hand, is a member of a family of transcription factors containing the paired box DNA binding domain (located in the amino terminus of PAX8) (28). During development, PAX8 is expressed in thyroid, kidney, and neural tissue, but adult expression is restricted to thyroid tissue (29, 30). PAX8 targets include the thyroid-specific genes thyroglobulin (Tg), thyroperoxidase (TPO), and sodium-iodide symporter (NIS) (31). On the rat Tg promoter, Pax8 functions synergistically with another thyroid-specific transcription factor, thyroid transcription factor-1 (TITF1; also called TTF-1 and Nkx2–1), mediated via direct interaction on overlapping DNA binding sites (32, 33, 34).
To investigate the role of PAX8-PPAR in follicular thyroid tumorigenesis, we have studied the transcriptional function of PAX8-PPAR on thyroid- and PPAR-specific gene promoters and the effect of PAX8-PPAR on thyroid cell growth. We found that PAX8-PPAR has mixed transcriptional function, stimulating transcription of some thyroid-specific genes and inhibiting others. We also found that PAX8-PPAR transcriptional function was cell type specific, showing no PPAR-dependent function in heterologous cells but stimulating transcription from a PPAR-responsive promoter in thyroid cells. PAX8-PPAR stimulated proliferation and anchorage-independent growth of FRTL-5 cells. Overall these data suggest that PAX8-PPAR promotes neoplasia by directly interfering with normal transcriptional programming in follicular thyroid cells.
Materials and Methods
Cloning of expression plasmids
The human TPO promoter containing a PAX8 response element (–367 to +51 bp relative to the transcription start site) was amplified from human genomic DNA by forward and reverse primers 5'-ggggtaccgagctgcacccaacccaat-3' and 5'-ctagctagcagtaattttcacggctgt-3', respectively, and cloned into the pGL3-basic vector (Promega, Madison, WI) as a 419-bp KpnI/NheI fragment (35). The NISTK-Luc reporter construct contains a herpes simplex virus thymidine kinase promoter amplified from a TK-CAT plasmid by PCR using the forward and reverse primers 5'-tgtaaagatctggatccggccccgcccagcg-3' and 5'-tgtaaagatctatgccattgggatatatcaa-3', respectively, then cloned into the BglII site in pGL3-basic vector. The rat NIS upstream enhancer (rNUE) that harbors two PAX8 response elements (–2495 to –2264 bp relative to the translation start site) (36) was amplified by PCR using forward and reverse primers 5'-cggggtaccagattgcagctg-3' and 5'-ctagctagctctagaagaagg-3', respectively, and then cloned into the pGL3-TK plasmid as a 231-bp KpnI/NheI fragment such that the rNUE is upstream of the thymidine kinase promoter. The full-length NIS-Luc (flNIS-Luc) was a kind gift from Prof. S. Jhiang (The Ohio State University, Columbus, OH) and contains the rNUE from –2495 to –2264 joined to the proximal 2-kb rat NIS promoter from –1949 to –4 bp relative to the translation start site (37). The Tg-Luc reporter construct contains the human Tg promoter (–372 to +1 bp relative to the translation start site) cloned upstream of the luciferase gene. PPRETK-Luc contains three copies of the acyl-CoA oxidase PPAR response element (underlined: GTCGACAGGGGACCAGGACAAAGGTCACGTTCGGGAGTCGAC, three copies) cloned upstream of the TK-Luc reporter (38). Plasmids for Tg-Luc, PPRETK-Luc, TITF1, PAX8, PPAR, and PPAR mutant were kindly provided by Prof. V. K. K. Chatterjee (University of Cambridge, Cambridge, UK). The PAX8-PPAR plasmid was kindly provided by Dr. T. G. Kroll (University of Chicago, Chicago, IL).
Cell culture
HeLa and HEK293 cells were grown in DMEM (Life Technologies, Inc. Invitrogen, Grand Island, NY) supplemented with 10% fetal calf serum (Trace Biosciences, Castle Hill, Australia). Diploid FRTL-5 cells were grown in F12 Coon’s modification with L-glutamine and zinc sulfate medium (Sigma-Aldrich, St. Louis, MO) supplemented with 5% fetal calf serum, a six hormone combination (1 mIU/ml bovine TSH, 4 ng/ml insulin, 10 ng/ml somatostatin, 5 μg/ml transferrin, 4 mg/ml hydrocortisone, and 10 ng/ml glycyl-L-histidyl-L-lycine acetate; Sigma-Aldrich) and 50 mg/ml penicillin/streptomycin (Invitrogen Life Technologies). Nthy-ori cells were grown in RPMI 1640 supplemented with 10% fetal calf serum. Cells were grown in 37 C humidified conditions with 5% CO2.
Transient transfection experiments
HeLa and Nthy-ori cells were plated at 1 x 105 cells per well and FRTL-5 cells were plated at 2 x 105 cells per well and grown to approximately 80% confluence in 24-well plates. Media was changed to 2% charcoal-stripped calf serum in Opti-MEM (Invitrogen Life Technologies) before transfection. Transient transfections were performed using DMRIE-C (Invitrogen Life Technologies) with 1 μg of transcription factor expression plasmid (PAX8, PPAR, PAX8-PPAR, TITF1, or pcDNA3 empty vector), 1 μg of luciferase promoter (Tg-Luc, NISTK-Luc, flNIS-Luc, PPRE-Luc, or 8 μg TPO-Luc), and 1 μg of -galactosidase plasmid for HeLa cell transfections and 100 ng of transcription factor expression plasmid (PAX8, PPAR, PAX8-PPAR, or pcDNA3 empty vector), 100 ng of luciferase promoter (Tg-Luc, NISTK-Luc, PPRE-Luc, or 800 ng TPO-Luc), and 100 ng of -galactosidase plasmid for FRTL-5 and Nthy-ori cell transfections. Cells were incubated for 16–18 h and replenished with 5% charcoal-stripped calf serum in normal growing media for 24 h together with 10 μM ciglitizone (Sigma-Aldrich) or equivalent volume of solvent control as indicated. Cells were then lysed and assayed for luciferase activity (Promega) and -galactosidase activity was used for normalization of reporter activity. Transcriptional response was expressed relative to that seen with empty vector in the absence of ciglitizone, and data shown represent mean ± SEM of at least three experiments, each performed in triplicate wells.
Transfections in dog thyrocytes
Whole thyroid glands were removed from dogs that had been previously anesthetized and exsanguinated as part of an unrelated, institutionally approved study. Thyroid glands were removed within 10 min of exsanguination. Glands were trimmed and minced, and primary cultures of thyrocytes were obtained following the method of Uyttersprot et al. (39). Briefly, minced thyroid tissue was subjected to collagenase digestion, serially rinsed in RPMI, and plated in a serum-free media consisting of DMEM-F12 Ham-MCDB 105 in a 2:1:1 ratio, with additional growth factors including bovine TSH at 15 mIU/ml.
For transfection, primary thyrocytes were plated into 24-well plates in culture media without additives. Transfections were performed with lipofectamine and Plus reagents according to the manufacturer’s protocol (Invitrogen), and included 100 ng of firefly luciferase reporter plasmid, 100 ng of transcription factor expression plasmid (PAX8, PPAR, PAX8-PPAR, or empty vector), and an internal control Renilla luciferase plasmid (0.1 ng CMV-Rluc, 0.5–1 ng SV40-Rluc, or 5 ng tk-Rluc). After 3 h of transfection, equal volumes of culture media containing 20% charcoal-stripped calf serum and penicillin/streptomycin were added to the wells. Thirty hours after initial transfection, the culture media were replaced with media containing either 10 μM ciglitizone or vehicle for an additional 24 h. Thyrocytes were then rinsed, lysed, and analyzed for firefly and Renilla luciferase activities using the Promega dual luciferase reagents and protocol.
Stable FRTL-5 cell lines
FRTL-5 cells were plated at a density of 6 x 105 cells per 60-mm diameter tissue culture dish, 48 h before transfection. Transfections were performed using FuGENE6 (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s protocols with either 1 μg of pcDNA3 empty vector or PAX8-PPAR expression vector (containing the neomycin resistance gene). Eighteen hours after transfection, selection of transfected FRTL-5 cells was performed by replenishing cells with normal growing medium supplemented with 100 μg/ml Geneticin (Invitrogen Life Technologies) for 10 d. Fresh medium containing Geneticin was replaced twice a week until no cells were remaining in the control plate. Stable transfectants were pooled and maintained in media containing 100 μg/ml Geneticin.
Gel shift analyses
Gel shift experiments were performed using a PPRE oligonucleotide DR+1 made by annealing 5'-aaggggccaactaggtcaaaggtcaccggt-3' and 5'-aaggggaccggtgacctttgacctagttgg-3' primers and labeling with 32P-dCTP. Five to 10 μl of in vitro-translated products were incubated for 30 min at room temperature in a binding buffer composed of 20 mM HEPES (pH 7.8), 1 mM dithiothreitol, 0.1% Nonidet P-40, 50 mM KCl, 20% glycerol, and approximately 20,000 cpm of the radiolabeled probe in a final volume of 45 μl. In supershift studies, in vitro-translated products were preincubated for 30 min with 200 ng of anti-PPAR E8 antibody (sc-7273; Santa Cruz Biotechnology, Santa Cruz, CA) before addition of radiolabeled probe. Bound products were separated on a 6% PAGE in 0.5x Tris-borate-EDTA. The gel was fixed in 30% methanol and 10% acetic acid for 20 min, vacuum dried for 2 h at 80 C, and exposed to film at –80 C.
RNA isolation and RT-PCR
RNA was isolated using TRI Reagent (Sigma-Aldrich) according to the manufacturer’s protocol, and 1 μg RNA was reverse transcribed using Superscript II (Invitrogen Life Technologies) and oligo-dT primers (Invitrogen Life Technologies) as per protocol. The cDNAs were amplified by PCR using a forward primer derived from exon 6 of PAX8, 5'-cgcggatccgcattgactcacagagca-3' and an antisense primer from exon 1 of PPAR1, 5'-ccggaattcgaagtcaacagtagtgaa-3' for detection of the PAX8-PPAR fusion gene. The 2-microglobulin housekeeping gene was amplified using a forward primer derived from exon 2, 5'-acccccactgaaaaagatga-3' and an antisense primer from exon 4, 5'-atcttcaaacctccatgatg-3'.
Tritiated thymidine uptake assay
FRTL-5 cells were seeded into 24-well plates at a density of 1 x 105 cells per well and grown for 48 h. Each well was pulsed with 37 MBq of [methyl-3H]thymidine (PerkinElmer Cetus, Norwalk, CT) for 4 h at 37 C. Ciglitizone at 10 μM or equal volume of solvent control was added 24 h before the experiment. Cells were washed twice with ice cold PBS (Invitrogen Life Technologies) and once with cold 0.9% NaCl. Ice-cold methanol-acetic acid (3:1) was used to fix the cells for 2 h at 4 C. The methanol-acetic acid was removed and 0.5 M NaOH was added, and the cells were left overnight to solubilize. Lysate (500 μl) was mixed with 4 ml of liquid scintillant (Optima Gold) and counted for 2 min on a liquid scintillation counter (Packard 1500).
Anchorage-independent cell growth assay
A total of 1 x 104 cells from each stable cell line were trypsinized, resuspended in 1 ml of 0.5% agarose (SeaPlaque; Edwards Instrument Co., New South Wales, Australia), and layered on top of a solidified bottom layer of 0.5% agarose in a 6-well plate. One milliliter of normal growing media was added on top of the gel and changed twice a week. The number of colonies in each well was counted after 20 d by staining with 1 mg/ml p-iodonitrotetrazolium for 24 h. Ciglitizone was added for 48 h before staining.
Statistics
The statistical software package for Microsoft Excel was used to analyze the data. Pairwise comparisons using t tests were performed between groups as indicated. P 0.05 was considered significant.
Results
Transcriptional function of PAX8-PPAR on PAX8-responsive promoters in heterologous cells
We first examined the transcriptional function of PAX8-PPAR on reporter genes containing PAX8 response elements from within the NIS, TPO, and Tg genes, in heterologous HeLa cells that have previously been used for study of these promoters (32, 33, 34, 36, 40, 41). In these transient transfection experiments, wild-type PAX8 stimulated transcription more strongly from NISTK-Luc (2.4 ± 0.2-fold; Fig. 1A) than Tg-Luc (1.7 ± 0.3-fold; Fig. 1D), and we were unable to show significant PAX8 induction of full-length flNIS-Luc (1.3 ± 0.65-fold; Fig. 1B) or TPO-Luc constructs (1.2 ± 0.1-fold; Fig. 1C). In contrast, PAX8-PPAR stimulated NISTK-Luc (3.0 ± 0.4-fold; Fig. 1A), flNIS-Luc (4.5 ± 0.2-fold; Fig. 1B), and TPO-Luc (1.8 ± 0.2-fold; Fig. 1C). Furthermore, the addition of the PPAR agonist ciglitizone significantly enhanced PAX8-PPAR-mediated transcription of NISTK-Luc and TPO-Luc (Fig. 1, A and C), suggesting that the PPAR C terminus is functional within the fusion protein in some promoter contexts. Nevertheless, transcriptional activation by PAX8-PPAR on NISTK-Luc was at least an order of magnitude less sensitive to thiazolidinedione treatment than that observed with PPAR alone on the PPAR-responsive promoter (Fig. 1E).
However, on the PAX8-responsive Tg promoter, PAX8-PPAR not only failed to stimulate but indeed significantly repressed transcription relative to basal levels (0.73 ± 0.1-fold, P = 0.02; Fig. 1D). The addition of ciglitizone did not relieve the repressive effect of PAX8-PPAR on this promoter. Therefore, PAX8-PPAR appeared to be a ligand-dependent positive regulator of NIS and TPO promoter transcription, and a negative regulator of Tg promoter transcription in heterologous cells.
Because the Tg promoter is coordinately regulated by PAX8 and TITF1 together, we next sought to investigate PAX8-PPAR transcriptional function in the presence of TITF1. As expected, TITF1 stimulated Tg promoter transcription more strongly than PAX8 alone (9.5 ± 0.8-fold vs. 1.6 ± 0.1-fold, respectively, P = 0.04; Fig. 2). However, the addition of PAX8-PPAR inhibited TITF1-mediated transcription from the Tg promoter in a dominant-negative manner (0.7 ± 0.1-fold repression, P = 0.01; Fig. 2). In contrast, as previously described (31, 32, 33), the addition of PAX8 and TITF1 together synergistically activated transcription from the Tg promoter (17.3 ± 4.1-fold, P = 0.04 compared with TITF1 alone; Fig. 2). Nevertheless, PAX8/TITF1-mediated transcription was also dominantly inhibited by the presence of PAX8-PPAR (0.6 ± 0.04-fold repression, P = 0.05; Fig. 2). The addition of ciglitizone did not relieve this repressive effect (data not shown). Although PAX8-PPAR did not significantly inhibit wild-type PAX8 function alone on the Tg promoter (0.8 ± 0.2-fold repression, P = 0.17; Fig. 2), the relatively weak activation of this promoter by PAX8 may have limited our ability to detect this.
Transcriptional function of PAX8-PPAR on PAX8-responsive promoters in thyroid cells
We next wished to examine PAX8-PPAR function in cell lines relevant to its proposed role in thyroid neoplasia. We chose the well-differentiated Fischer rat thyroid (FRTL-5) cell line and human Nthy-ori cells that are commonly used in follicular thyroid cell models (42, 43) and that do not contain PAX8-PPAR (data not shown). Overall, we found that PAX8-dependent transcription by PAX8-PPAR was similar in thyroid cells compared with heterologous cells (Fig. 3). Although we expected that FRTL-5 and Nthy-ori cells contain endogenous PAX8, addition of transiently transfected PAX8 further stimulated expression from NISTK-Luc in both cell lines (Fig. 3, A and B). As in HeLa cells, PAX8-PPAR also stimulated expression of NISTK-Luc and TPO-Luc in either thyroid cell line (Fig. 3, A–D). Results of reporter gene expression in the presence of ciglitazone were intriguing, insomuch as there appeared to be a general augmentation of transcription independent of the addition of either PAX8 or PAX8-PPAR (Fig. 3, A–F), although these promoters lack a PPRE consensus sequence (data not shown). PAX8-PPAR-mediated transcription was not specifically activated by the addition of ciglitizone on any promoter in thyroid cells (Fig. 3, A–F). Importantly, the lack of PAX8-PPAR transcriptional activity on the Tg promoter was also observed in both thyroid cell lines (Fig. 3, E and F) indicating that PAX8-PPAR-dependent inactivity on this promoter is not due to lack of thyroid-specific transcriptional cofactors in heterologous cells.
Transcriptional function of PAX8-PPAR on PPAR-responsive promoters
We then examined PAX8-PPAR function on a PPAR-responsive promoter (38). In HeLa cells, wild-type PPAR stimulated basal transcription from PPRETK-Luc 4.4 ± 0.8-fold (P < 0.01) and responded to thiazolidinedione as expected (further stimulation by 2.1 ± 0.1-fold, P = 0.02; Fig. 4A). PAX8-PPAR in contrast failed to activate this promoter either in the absence or presence of PPAR agonist, but repression of this promoter was not observed (1.1 ± 0.1-fold change; Fig. 3A). Conversely, a synthetic PPAR dominant-negative mutant that has previously been shown to recruit corepressors (18) did repress basal transcription of PPRETK-Luc 0.7 ± 0.1-fold (P = 0.03), although this effect was partly reversed by the addition of thiazolidinedione (Fig. 4A). We also found that PAX8-PPAR is inactive and dominantly inhibits PPAR-mediated transcription from PPRETK-Luc in HEK293 cells (data not shown). Overall, our data in heterologous cells was comparable to a previous report that studied PAX8-PPAR on the PPAR-responsive aP2 enhancer (4).
In FRTL-5 and Nthy-ori cells, however, PAX8-PPAR stimulated transcription from PPRETK-Luc (Fig. 4, B and C). PAX8-PPAR-mediated basal transcription of this promoter was comparable to that seen with wild-type PPAR (3.0 ± 0.5-fold vs. 3.0 ± 0.2-fold, respectively, in FRTL-5 cells and 4.7 ± 0.3-fold vs. 4.2 ± 1.2-fold, respectively, in Nthy-ori cells). PAX8-PPAR-dependent transcription was significantly stimulated by 10 μM ciglitizone in Nthy-ori cells (Fig. 4C), but no significant ligand response was seen in FRTL-5 cells (Fig. 4B). The addition of ciglitizone stimulated PPAR-dependent transcription 2.8 ± 0.5-fold in FRTL-5 cells (Fig. 4B) and 15.5 ± 4.7-fold in Nthy-ori cells (Fig. 4C). By comparison, the dominant-negative PPAR mutant neither repressed nor stimulated this promoter in FRTL-5 cells (1.1 ± 0.1-fold change).
To address potential concerns that available thyroid cell lines may differ substantially from true thyrocytes, PPRETK-Luc was also transfected into primary cultures of dog thyrocytes (Fig. 4D). Luciferase was induced 8.1 ± 2.0-fold by PPAR in the absence of ciglitizone and 30.6 ± 6.3-fold in the presence of ciglitizone. Inductions by PAX8-PPAR were 7.2 ± 1.8-fold and 17 ± 3.0-fold in the absence and presence of ciglitizone, respectively. These data are similar to the findings in FRTL-5 and Nthy-ori cells.
PAX8-PPAR binds to a PPAR consensus DNA recognition sequence
Transcription factors mediate gene transactivation through binding to specific cognate response elements located in promoter regions. PPAR binds a consensus recognition sequence containing direct repeats of a hexamer spaced by one nucleotide (DR+1) (44). Therefore, we examined the ability of PAX8-PPAR to bind this cognate DNA element in gel shift analyses. PPAR preferentially interacts with this DNA sequence as a heterodimer with the retinoid X receptor (RXR) (Fig. 5, lane 3) although RXR homodimers also form a complex, albeit less efficiently (Fig. 5, lanes 1 and 2). The addition of an anti-PPAR monoclonal antibody results in a supershifted complex (Fig. 5, lane 4), confirming the identity of the DNA-bound PPAR-RXR heterodimer. PAX8-PPAR also bound this DNA sequence as a heterodimer with RXR (Fig. 5, lane 5) with a size difference consistent with the presence of PAX8 compared with PPAR-RXR alone. Translation from an internal start site in PAX8-PPAR produced a DNA-bound complex with RXR similar in mobility to the PPAR-RXR band. Once again, the addition of the anti-PPAR antibody confirmed the identity of the PAX8-PPAR-RXR heterodimer-DNA complex (Fig. 5, lane 6). No PPAR or PAX8-PPAR complexes were seen in the absence of RXR, nor did the addition of ciglitizone alter the mobility of either PPAR-RXR or PAX8-PPAR-RXR DNA-bound complexes (data not shown). We have not as yet been able to demonstrate PAX8-PPAR binding to PAX8 response elements in gel mobility shift experiments (data not shown), although it is possible that we are lacking some critical cofactor required for binding.
PAX8-PPAR increases proliferation of rat follicular thyroid cells
We then examined the effect of PAX8-PPAR expression on follicular thyroid cell growth. A recent report demonstrated that PAX8-PPAR expression increased growth and decreased apoptosis in human Nthy-ori cells (43). In contrast, we chose to express PAX8-PPAR in the better-differentiated FRTL-5 cell model (45). FRTL-5 cells were stably transfected with pcDNA3-PAX8-PPAR constitutively expressing the fusion protein under control of the cytomegalovirus promoter. We confirmed PAX8-PPAR expression by RT-PCR (Fig. 6A). PAX8-PPAR expression was associated with increased FRTL-5 cell growth as assessed by thymidine uptake (Fig. 6B) and by proliferation in semisolid media (46) (Fig. 6C). The addition of thiazolidinedione did not affect thymidine uptake (Fig. 6B) or soft-agar proliferation (data not shown) by PAX8-PPAR-expressing FRTL-5 cells.
Discussion
The chromosomal translocation t(2;3)(q13;p25) that produces the PAX8-PPAR fusion gene has been associated with a subset of follicular thyroid carcinomas and possibly adenomas, although the transcriptional mechanisms by which PAX8-PPAR stimulates thyroid neoplasia have not previously been studied in detail. A prevailing hypothesis is that PAX8-PPAR inhibits PPAR function in thyroid cells. In support of this, PAX8-PPAR was initially shown to disrupt PPAR-mediated transcription in a dominant-negative manner (4). A subsequent study also found that PAX8-PPAR dominantly inhibited PPAR-mediated transcription in human Nthy-ori cells and proposed that PAX8-PPAR-stimulated growth of these cells depended at least in part on inhibition of PPAR function (43). Finally, PPAR expression was shown to be down-regulated in follicular thyroid carcinomas that do not contain the PAX8-PPAR translocation (47, 48). In combination, therefore, these previous results suggested that follicular thyroid neoplasia may be associated with net loss of PPAR activity either through lack of endogenous PPAR expression or by PAX8-PPAR-mediated dominant inhibition of wild-type PPAR transcriptional function.
Our study provides combined evidence for two alternate hypotheses, namely that PAX8-PPAR affects PAX8 function on the one hand, and is capable of inducing PPAR-dependent transcription on the other. However the pattern of transcriptional regulation by PAX8-PPAR is complex, such that some PAX8-responsive promoters were up-regulated (TPO, NIS), whereas another (Tg) was inhibited in a dominant-negative manner. PAX8-PPAR function on a PPRE was similarly complex, capable of stimulating transcription from this promoter when in the appropriate cellular environment. These results are strongly supported by a recent report by Lacroix et al. (61) that found specific up-regulation of several PPAR target genes in tumors containing PAX8-PPAR, but not in those tumors lacking the translocation. Taken together, these data suggest that inappropriate induction of some PPAR-responsive genes, rather than their repression, may contribute to the pathogenesis of follicular thyroid cancer, a hypothesis that is all the more plausible considering the extremely low level of endogenous PPAR expression in normal thyrocytes. Therefore, the differences between PAX8-PPAR-mediated regulation of the acyl-CoA oxidase PPRE used in our study and the aP2 enhancer PPRE used by Powell et al. (43) are likely to be promoter and/or cofactor dependent. We are now studying PAX8-PPAR transcriptional function on those PPAR-target genes including angiopoietin-like 4 and aquaporin 7 that were found to be up-regulated in microarray analyses of PAX8-PPAR-positive tumors (61).
PAX8-PPAR responsiveness to PPAR agonist also appeared to be both promoter and cell type dependent. On the NIS promoter, transcriptional activation by PAX8-PPAR in response to thiazolidinedione treatment was comparatively weak and at least an order of magnitude less sensitive than that observed with PPAR alone on a PPAR-responsive promoter (Fig. 1E). Although stronger agonist responses were seen on a PPRE in thyroid cells, PAX8-PPAR transcription was still not augmented by ciglitizone to the same extent as for wild-type PPAR despite comparable basal activities (Fig. 4, C and D). Whether this relates to altered ligand affinity in the fusion protein, or to altered cofactor recruitment is not yet clear. We are also now examining whether PAX8-PPAR may be more responsive to tyrosine-based PPAR agonists, as has been previously reported with PPAR mutants that otherwise respond poorly to thiazolidinediones both in vitro and in vivo (18, 59).
Thus, our studies suggest that PAX8-PPAR has mixed function that can either replicate the transcriptional effects of either PAX8 or PPAR alone when the appropriate promoter- or cell-type-specific conditions are present; or inhibit gene expression when these necessary promoter- or cell-specific conditions are lacking. We have demonstrated that growth of rat FRTL-5 thyroid cells was stimulated by PAX8-PPAR expression, as has previously been shown for less well-differentiated human thyroid cells (43). In combination, our results suggest that PAX8-PPAR disrupts normal transcriptional pathways in thyroid cells by means of a complex mix of activation of some genes and inhibition of others, the net effect of which is to stimulate follicular thyroid neoplasia.
Differing histological phenotypes of thyroid carcinoma have all now been associated with specific molecular abnormalities. Papillary carcinoma is associated with a chromosomal translocation that leads to activation of the RET proto-oncogene (2, 49) or an activating mutation of the braf kinase (50). Medullary carcinoma is associated with RET mutations that lead to its constitutive activity within C-cells (3). Our finding that PAX8-PPAR stimulates PAX8-specific gene expression is consonant with its specific association with follicular thyroid cell neoplasms, and the known role of PAX8 in thyroid follicular cell differentiation (8). Conversely, the mechanism by which PAX8-PPAR inhibits transcription from the PAX8-responsive Tg promoter is unclear. It is possible that PAX8-PPAR binds the Tg promoter but cannot interact with TITF1 or other cofactors necessary for Tg transcription, and we found that PAX8-PPAR dominantly inhibited TITF1 mediated Tg-Luc transcription in a manner that might be consistent with steric hindrance (Fig. 2). Alternatively, PAX8-PPAR might recruit transcriptional corepressors via PPAR. Indeed, specific mutations in the extreme C terminus of PPAR have been previously shown to cause corepressor recruitment and dominant-negative transcriptional function by PPAR (18, 19). Furthermore, naturally occurring PPAR mutations associated with a syndrome of insulin resistance and lipodystrophy have also been shown to be associated with recruitment of corepressors to target genes (20). However, when we compared PAX8-PPAR with a corepressor-binding mutant PPAR (18), although both dominantly inhibited transcription from PPRETK-Luc in heterologous cells (Fig. 4A and data not shown), PAX8-PPAR clearly differed from the mutant PPAR in its transcriptional function in thyroid cells (Fig. 4, B–D). We speculate that this may be due to PAX8-mediated recruitment of thyroid-specific coactivators to the DNA-bound fusion protein (Fig. 5).
Dysregulated recruitment of transcriptional corepressors has also been implicated in other clinical syndromes, including thyroid hormone resistance syndrome associated with mutant thyroid hormone receptors (TR) (21) and acute promyelocytic leukemia associated with translocations fusing the myeloid-specific PML or PLZF genes with the retinoic acid receptor gene (22, 23, 51). Corepressor-mediated recruitment of histone deacetylases has been proposed to be a key pathogenic feature in acute promyelocytic leukemia (22, 23, 51). Similarly, recent work has also suggested that histone deacetylases may be important in thyroid carcinogenesis (52, 53, 54, 55, 56, 57, 58). Histone deacetylase inhibitors promote reexpression of NIS (52, 53, 54, 55, 56) and increase apoptosis (57, 58) in poorly differentiated thyroid cancer cell lines. We are now investigating whether these compounds specifically derepress PAX8-PPAR transcription.
Although PAX8-PPAR stimulates NIS and TPO promoter expression in our cell culture models, these genes may be down-regulated in follicular thyroid carcinomas (60). Indeed, the recent report by Lacroix et al. (61) did not find that Tg, TPO, or NIS genes were differentially expressed in follicular tumors harboring or not harboring the PAX8-PPAR translocation. However, only a few PAX8-PPAR tumors were analyzed (four in microarray, five in quantitative PCR experiments) and since multiple transcription factors regulate these promoters it may be too simplistic to expect PAX8-PPAR to have discernible effects in a small sample size. It is also possible that PAX8-mediated transcriptional function is dependent upon as yet unknown cofactors that are lost as thyroid cells become progressively undifferentiated. Even if basal NIS expression is not altered, it will be very interesting to determine whether PPAR agonists might up-regulate NIS expression in follicular carcinomas harboring the PAX8-PPAR translocation, which would have practical therapeutic relevance in enhancing tumor cell ablation by radioactive iodine.
Acknowledgments
We are indebted to Prof. V. K. K. Chatterjee (University of Cambridge) for providing expression vectors for TITF1, PAX8, PPAR, PPAR mutant, Tg-Luc, and PPRETK-Luc; Prof. S. Jhiang (The Ohio State University) for the full-length flNIS-Luc; and to Dr. T. G. Kroll (University of Chicago) for the PAX8-PPAR expression plasmid.
Footnotes
This work was supported by the Cure Cancer Australia Foundation, University of Sydney Cancer Research Fund (to B.G.R and R.J.C.-B.) and National Institutes of Health Grant DK44155 (to R.J.K.).
First Published Online September 22, 2005
1 A.Y.M.A. and C.M. contributed equally to this work.
Abbreviations: NIS, Sodium-iodide symporter; PAX8, paired box gene 8; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR-response element; rNUE, rat NIS upstream enhancer; RET, rearranged during transfection; RXR, retinoid X receptor ; Tg, thyroglobulin; TITF1, thyroid transcription factor-1; TPO, thyroperoxidase.
Accepted for publication September 13, 2005.
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