Thyroid hormone induces the expression of 4-1BB and activation of caspases in a thyroid hormone receptor-dependent manner Toshiko Yamada-Okabe 1 , Yasuo Satoh 2, * and Hisafumi Yamada-Okabe 1,3 1 Department of Hygiene, 2 Department of Surgery, School of Medicine, Yokohama City University, Fukuura, Kanazawa, Yokohama; 3 Pharmaceutical Research Department 4, Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd, Kajiwara, Kamakura, Kanagawa, Japan Thyroid hormone has various effects on cell proliferation, growth and apoptosis. To gain more insight into the molecular dynamics caused by thyroid hormone, gene expression in HeLaTR cells that constitutively over- expressed the thyroid hormone receptor (TR) was analyzed. Gene expression profiling of the HeLaTR cells with an oligonucleotide microarray yielded 229 genes whose expression was significantly altered by T3. Among these genes, the expression of 4-1BB, which is known to initiate a signal cascade activating NF-jB, was significantly up-regu- lated by T3. Although treatment of the HeLaTR cells with T3 did not induce expression of NF-jB reporter luciferase, even in the presence of the 4-1BB-Ligand, it increased the caspase activities. An increase in the caspase activities was also observed in the HeLaTR cells transfected with 4-1BB cDNA, and the 4-1BB-Ligand further increased the caspase activities of the HeLaTR cells overexpressing the 4-1BB. Furthermore, up-regulation of 4-1BB and an increase in caspase activities also occurred in the rat FRTL cells that expressed only authentic TR. These results demonstrate that the expression of 4-1BB serves as the mediator of signals from T3 to activate caspases. Keywords: thyroid hormone; thyroid hormone receptor; 4-1BB gene expression; apoptosis; caspase. Thyroid hormone plays an important role in metabolism, growth and development in a wide variety of organisms. Thyroid hormone is involved in the regulation of the lifecycle of coelenterates [1] and the metamorphosis of amphibians [2]. In mammals, thyroid hormone is required for normal body growth and maturation including brain development, and its deficiency causes cretinism, the full-blown syndrome of congenital hypo- thyroidism [3]. T4 and T3 are synthesized in thyroid follicular cells under the control of thyroid stimulating hormones (TSH) and circuit the blood stream by binding to serum proteins such as transthyretin. At peripheral tissues, T4 is deiodinated by iodothyronine deiodinase to become active thyroid hor- mone, T3 [3] 1 . Biological functions of T3 are mediated by thyroid hormone receptors (TRs) that belong to the nuclear hormone receptor family. TRs are encoded by two distinct genes and are expressed as TRa,TRb1orTRb2 [4–6]. Each TR forms either a homo- or a hetero-dimer with a retinoid X receptor and binds to the thyroid hormone response element (TRE) that is present in the promoter/ enhancer of certain genes [7]. In the absence of T3, a corepressor such as N-CoR or SMRT is associated with the homo- or heterodimer of TR and represses the transcription mediated by TR. Upon binding of T3 to TR, the corepressor is displaced by a coactivator such as SRC1, TIF2 (GRIP1) or TRAM-1 (p/CIP, AIB1, ACTR, RAC3), making TR active in transcription [8,9]. Recently, a number of genes that were regulated by thyroid hormone were identified by cDNA microarray with hepatic RNA prepared from hypothyroid mice treated with T3 [10,11], and the expression of approximately 2.5% of the examined genes (55 of 2225 genes) appeared to be modulated by thyroid hormone treatment. Genes under the control of T3 included those involved in hepatocyte proliferation, cell survival (apoptosis), gluconeogenesis, lipogenesis, and insu- lin signaling [10]. As thyroid hormone causes growtharrest of thyrotropic tumors, genes involved in thyrotropic tumor growth were also explored by cDNA microarray. Among 1176 genes examined, 40 genes were down-regulated and seven were up-regulated by thyroid hormone [12]. However, because these studies were carried out with cells or tissues expressing only low levels of TR, cells might only weakly respond to T3, and therefore, cellular responses to T3 might not be clearly detected by DNA microarray. In this study, we examined the effects of T3 on gene expression in the HeLa cells that overexpressed TR by oligonucleotide microarray for approximately 11 000 Correspondence to H. Okabe, Pharmaceutical Research Department 4, Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd, 200 Kajiwara, Kamakura, Kanagawa, 247-8530, Japan. Fax: + 81 467 45 6782, Tel.: + 81 467 45 4382, E-mail: okabehsf@chugai-pharm.co.jp Abbreviations: TR, thyroid hormone receptor; TSH, thyroid stimulating hormone; TRE, thyroid hormone response element; FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle’s medium; CMV, cytomegalovirus; SV40, simian virus 40; PMA, phorbol 12-myristate 13-acetate (PMA); 4-1BB-L, 4-1BB-ligand. *Present address: Department of Surgery, Ito Shimin Hospital. (Received 22 March 2003, revised 10 May 2003, accepted 23 May 2003) Eur. J. Biochem. 270, 3064–3073 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03686.x human genes. Among the number of genes whose expres- sion was significantly altered by T3, the expression of 4-1BB 2 that encodes a TNF receptor superfamily protein was strongly induced by T3. Although 4-1BB can initiate a signal to activate NF-jB, T3 did not affect the NF-jB activity but increased caspase activity. Activation of the caspases occurred in the HeLa cells overexpressing the 4-1BB cDNA even without the addition of an extra 4-1BB- Ligand. Furthermore, up-regulation of 4-1BB expression, increase in the caspase activities, and impairment of cell proliferation were also observed in the rat FRTL cells that expressed only authentic TR. Thus, it appears that T3 up-regulates the expression of 4-1BB and that 4-1BB can activate caspases to induce apoptosis. Materials and methods Generation of cell lines that constitutively overexpress TR The entire open reading frame (ORF) of the human thyroid hormone receptor (hTRa1) cDNA was amplified by PCR using primers containing the sequences of the 5¢-and 3¢-coding regions of TR and ligated at the HindIII cleavage site of pCMV-Tag4 (Stratagene). The HeLa S3 cells were transfected with either the resulting plasmid or pCMV-Tag4 by lipofectamine reagent (Invitrogen) and selected with 1mgÆmL )1 of G418 for 2 weeks. G418-resistant clones were isolated and tested for expression levels of hTRa1. Cells from a single clone that expressed a high level of TR were designated HeLaTR and used for the study. HeLa S3 cells were cultured in Dulbecco’s modified Eagle’s medium (DME) supplemented with 10% fetal bovine serum (FBS). Exposure of the cells to T3 was carried out in DME supplemented with 10% FBS that had been treated with charcoal and dextran (HyClone). The nucleotide sequences of the primers used for amplifying the hTRa1 cDNA were: 5¢-CCCGGGAAGCTTCGGACCATGG AACAGAAGCCAAGCAAGGTG-3¢ and 5¢-CCCGGG GTCGACGACTTCCTGATCCTCAAAGACCTC-3¢.In order to overexpress 4-1BB and TRAF1 in the HeLaTR cells, the entire coding regions of the 4-1BB and TRAF1 cDNAs were amplified by RT-PCR and ligated at the HindIII site of pRC/CMV eukaryotic expression plasmid. The resulting plasmid DNA together with pSV2hph [13] was transfected into the HeLaTR cells and the cells were selected with 0.4 mgÆmL )1 of hygromycin B (Invitrogen). Cells derived from the single clone that overexpressed 4-1BB or TRAF1 were designated HeLaTR/4-1BB and HeaLaTR/ TRAF1, respectively. Primers for amplifying the 4-1BB and TRAF1 cDNAs were: 5¢-GAATTCAAGCTTATGGGA AACAGCTGTTACAACATA-3¢ and 5¢-GAATTCAAG CTTCACAGTTCACATCCTCCTTCTTCT-3¢ for 4-1BB, 5¢-CCCGGGATATCATGGCCTCCAGCTCAGGCAG CAGTC-3¢ and 5¢-CCCGGGATATCTAAGTGCTGG TCTCCACAATGCACT-3¢ for TRAF1. Isolation of RNA and RT-PCR Sub-confluent cells in 10-cm diameter dishes were lysed with Trizol reagent (Invitrogen), and total RNA was recovered in the aqueous phase by centrifugation 3 (15 000 g)andthen precipitated by isopropyl alcohol. Single-stranded cDNA was synthesized with 1 lg of the total RNA and was used as the template for RT-PCR. Amplification of cDNA was performed by 30 cycles of consecutive incubations at 94 °C for 30 s, 60 °C for 30 s and 72 °C for 90 s with primers for the indicated cDNAs and an RNA LA PCR kit (AMV) Ver 1.1 (Takara, Shiga, Japan). Immunoprecipitation Sub-confluent cells in 6-cm diameter dishes were incubated in DME containing 2% FBS that had been dialyzed against saline and 40 lCi of a mixture of [ 35 S]methionine and [ 35 S]cysteine (1000 Ci mmol )1 ;pro-mix L -[ 35 S] in vitro cell labelling mix, Amersham) for 2 h. Cell extracts were prepared by lysing the cells with RIPA buffer: [150 m M NaCl, 1% NP4O, 0.5% deoxycholate, 0.1% SDS, 50 m M Tris-HCl (pH 8.0)] 4 ,andthehTRa1 protein was immuno- precipitated from the cell extracts as described in a previous paper but with 2 lgÆmL )1 of the anti-(TR polyclonal Ig) (clone FL-408, Santa Cruz) [14]. The hTRa1proteinwas separated by 10% SDS/PAGE and visualized with the Fuji BAS 2000 system. Luciferase reporter assay To monitor the TR-mediated transcription, the reporter plasmid was constructed with the pGL2-promoter plasmid (Promega) that carries the firefly luciferase gene of Photinus pyralis linked to the simian virus-40 (SV-40) promoter. The DR4 oligonucleotide that contained the TRE sequences and control DR0 oligonucleotide were inserted between the SmaIandBglII cleavage sites of the pGL2-Promoter plasmid. The resulting plasmids, designated DR0-pGL2- luc and DR4-pGL2-luc, respectively, were transfected into the HeLaTR cells with lipofectamine reagent (Invitrogen). To determine the NF-jB activity, pTAL-luc (Clontech) that carries the firefly luciferase gene of Photinus pyralis linked to the TATA-like promoter region from the herpes simplex virus thymidine kinase promoter, and pNFjB-luc (Clon- tech), in which four copies of the NF-jB consensus sequence had been inserted into pTAL-luc, were transfected into HeLaTR cells; the resulting cells were cultured in the presence or absence of T3, 4-1BB-Ligand or TNFa for 48 h (T3), or 24 h (4-1BB-Ligand and TNFa). Thereafter, the cells were washed with NaCl/P i twice, harvested and suspended in the reporter lysis buffer (Promega). After lysing the cells, the cell extracts were recovered by centri- fugation at 15 000 g for 2 min at 4 °Cand10lL of the cell lysate were mixed with 50 lL of the luciferase assay reagent (Promega). The luciferase activities in the cell extracts were measured with Turner Designs Luminometer Model TD-20/20 (Promega). The nucleotide sequences of DR4 were 5¢-GGGAGGACAGATCAGGACAA-3¢ and 5¢-GA TCTTGTCCTGATCTGTCCTCCC-3¢, and those of the DR0 oligonucleotide were, 5¢-GGGAGGACAAGGAC AA-3¢ and 5¢-GATCTTGTCCTTGTCCTCCC-3¢. Electrophoretic mobility shift assay Nuclear extracts were prepared as described by Dignam et al. [15]. Two microliters of the oligonucleotide harboring Ó FEBS 2003 T3 induces 4-1BB in a TR-dependent manner (Eur. J. Biochem. 270) 3065 the NF-jB consensus sequence (Promega) that had been end-labelled with [c- 32 P]ATP (6000 CiÆmmol )1 ; Amersham Pharmacia) was incubated in a reaction mixture (10-lL final volume) containing 4% (w/v) Ficoll, 20 m M Hepes/KOH (pH 7.9), 50 m M KCl, 1 m M EDTA, 1 m M dithiothreitol, 2.5 lg of poly(dI-dC) (Roche Diagnostics), 6 m M MgCl 2 and 10-lg protein amounts of nuclear extract at room temperature for 10 min. Thereafter, 2 lL aliquot was electrophoresed on a 6% polyacrylamide gel and radio- labelled oligonucleotides were visualized with an image analyzer (Fuji BAS2000). In the control experiment, 100 times molar excess of nonradiolabelled NF-jB oligonucleo- tide was added to the reaction mixture. DNA microarray cDNA was synthesized from the total RNA with reverse transcriptase (SuperScript TM Choice System, Life Technologies) and an oligo-dT primer that contained the sequences for the T7 promoter. The resulting cDNA was extracted with phenol/chloroform and purified with Phase Lock Gel TM Light (Eppendorf). cRNA was synthesized with MEGAscript T7kit (Ambion) and the cDNA as a template. During the synthesis, Bio-11-CTP and Bio-16-UTP (Roche Biochemicals) were used to label the cRNA products. Thereafter, the cRNA was separated from mononucleotides and short oligonucleotides by column chromatography on CHROMA SPIN + STE-100 column (Clontech). HuU95A array (Affymetrix) was used for high-density oligonucleotide arrays. To hybridize with oligonucleotides on the chips, the cRNA was fragmented at 95 °Cfor35 min in a buffer containing 40 m M Tris/acetate (pH 8.1), 100 m M potassium acetate and 30 m M magnesium acetate. Hybrid- ization was carried out in 200 lL of a buffer containing 0.1 M Mes (pH 6.7), 1 M NaCl, 0.01% (v/v) Triton X-100, 20 lg herring sperm DNA, 100 lg acetylated bovine serum albumin, 10 lg of the fragmented cRNA and biotinylated- control oligonucleotides at 45 °C for 12 h. After the chips were washed with a buffer containing 0.01 M Mes (pH 6.7), 0.1 M NaCl and 0.001% (v/v) Triton X-100, they were further incubated with biotinylated anti-streptavidin Ig and stained with streptavidin R-Phycoerythrin (Molecular Probes) to amplify intensities. Each pixel level was collected by laser scanner (Affymetrix) and the levels of the expression of each cDNA and of the reliability (Present/Absent call) were calculated with software ( AFFYMETRIX MICROARRAY SUITE ver.4.0). For comparisons of the expression level of each gene, average differences below 20 were considered to be 20. Determination of the caspase activities One million cells of HeLa, HeLaTR, HeLaTR/4-1BB, HeLaTR/TRAF1 and FRTL were plated onto 10-cm diameter 5 dishes and incubated for 48 h in the presence or absence of 50 ngÆmL )1 T3. In some experiments, the 4-1BB- Ligand (PeproTech EC, UK) at a final concentration of 100 ngÆmL )1 or TNFa (Endogen) at a final concentration of 400 UÆmL )1 was added to the medium 24 h before harvesting the cells. In some of the experiments, Z-VAD- FMK (Promega), a caspase inhibitor, was added to the cells to give a final concentration of 75 l M . Cells were washed with NaCl/P i twice, harvested, suspended in 50 lLofthe Cell Lysis buffer (Promega), and lysed by twice repeated freezing and thawing. After centrifugation at 15 000 g for 20 min, the supernatants were collected and used as the cell extracts. Caspase activities were determined with a colori- metric caspase assay system (Promega) in 100 lLof reaction mixture containing 15 lL(100lg) cell extracts and acetyl-DEVD-p-nitroanilide as a substrate. After incu- bation at 37 °C overnight, absorbance at 405 nm was measured with a microplate reader (Bio-Rad). Cell proliferation assay One thousand cells of HeLaTR and FRTL were cultured in 100 lL of the medium in the presence or absence of T3 and Z-VAD-FMK at 37 °C for 48 h. At the end of the culture, 20 lL of CellTiter 96 AQ ueous One Solution Reagent (Promega) containing 5-(3-carboxymethoxyphenyl)-2-(4,5- dimenthylthiazoly)-3-(4-sulfophenyl) tetrazolium salt (MTS) was added to the medium, and the cells were incubated at 37 °C for 1–4 h. Absorbance at 490 nm, which represents a viable cell, was measured with a microplate reader. Detection of DNA fragmentation Both floating and adherent cells of HeLaTR and FRTL that were cultured in the presence or absence of T3 were collected and lysed with a lysis buffer containing 10 m M Tris/HCl (pH 8.0), 10 m M EDTA and 0.5% (v/v) Triton X-100. Cell lysates were treated with RNase A (0.1 mgÆmL )1 )at37°C for 1 h and then with proteinase K (1 mgÆmL )1 )at50°C for 2 h. DNA was extracted with phenol/chloroform mixture and precipitated by isopropa- nol. Five lg of DNA was fractionated by agarose gel electrophoresis. Results Generation of a cell line that overexpressed the TR In order to identify genes whose expression is regulated by thyroid hormone, we created a cell line that constitutively expressed the human TRa1 and thereby responded to T3. The hTRa1 cDNA connected to the cytomegalovirus (CMV) promoter was transfected to HeLa cells (clone S3) and cells that expressed a detectable level of hTRa1were selected. RT-PCR demonstrated that cells from one such clone, designated HeLaTR, expressed certain amounts of hTRa1 mRNA, whereas the vector-transfected HeLa cells contained undetectable levels of hTRa1 mRNA (Fig. 1A). Consistent with the results of RT-PCR, the detectable amounts of the hTRa1 protein was immunoprecipitated from the extracts of the HeLaTR cells but not from the vector-transfected HeLa cells (Fig. 1B). The TRb mRNA in HeLaTR and in the vector-transfected HeLa cells was below the level detectable by RT-PCR (data not shown). Next, we confirmed the functionality of hTRa1inthe HeLaTR cells. The HeLaTR cells were transfected with the plasmid of DR4-pGL2-luc or DR0-pGL2-luc that carried the firefly luciferase gene linked to the SV40 promoter. DR4 contains the sequence of TRE, but DR0 does not. Therefore, the transcription of the luciferase from the DR4-pGL2 enhancer/promoter should be dependent on the activation of TR and that from the DR0-pGL2 3066 T. Yamada-Okabe et al. (Eur. J. Biochem. 270) Ó FEBS 2003 enhancer/promoter would be independent of TR. As expected, T3 increased the luciferase activities derived from DR4-pGL2-luc by three- to fourfold but did not affect the activities from DR0-pGL2-luc (Fig. 1C). Optimal concen- trations of T3 to induce the luciferase activity in the HeLaTR cells ranged from 25 to 100 ngÆmL )1 ,whichis consistent with a previous report by Fondell et al.[16]. Furthermore, the increase in luciferase activities correlated with increase in the luciferase mRNA (not shown). No increase in the luciferase activity by T3 was observed in the vector-transfected HeLa cells. These results demonstrate that hTRa1wasactivatedbyT3intheHeLaTRcellsand stimulated the transcription of genes in a TRE-dependent manner and that the parental HeLa cells were TR-negative and thereby not responsive to T3. Genes whose expression is altered by T3 To identify the genes whose expression is regulated by T3, we performed oligonucleotide microarray for approxi- mately 11 000 human genes. Gene expression was com- pared between the HeLaTR cells and those treated with T3; genes whose mRNA levels showed a change of more than threefold between the two were selected. Among the 229 genes that matched this criterion, 113 genes were up-regulated and 116 genes were down-regulated by T3 (Fig. 2). The expression of these genes was further verified by RT-PCR. As conventional RT-PCR did not amplify detectable levels of the cDNA fragments from the mRNA, whose average difference in the DNA microarray was less than 400, we focused on the genes with average differences greater than 400 either in the HeLaTR cells or those treated with T3. RT-PCR confirmed that T3 up-regulated the expression of 4-1BB, pregnancy specific gene-7 (PSG7)andfmfc and down-regulated BMP-6 (Fig. 3A). 4-1BB encodes a protein of the TNF super- family that can activate NF-jB [17,18]. PSG7 is respon- sible for a protein of the carcinoembryonic antigen ( 6 CEA) family. Although the real function of PSG7 is unclear, PSG 7 protein families are thought to be essential for the maintenance of pregnancy, and their serum levels are increased in patients of choriocarcinoma and hydatidiform mole [19,20]. fmfc Codes for a four-kringle fragment of HGF that can antagonize NK4/HGF [21]. BMP-6 encodes a protein of the TGF-b superfamily that is induced during the osteoblast differentiation [22]. Fig. 1. Expression of the functional human TRa1inHeLacells.(A) Expression of the human TRa1 (TR) mRNA in HeLaTR cells. RNA was extracted from the subconfluent cultures of HeLaTR and vector-transfected HeLacellsandwasusedasthetemplatefor RT-PCR. RT-PCR was performed with the specific primer for hTRa1andGAPDH,and the PCR products after the indicated cycles of PCR were analysed by agarose gel electro- phoresis. (B) Expression of the human TRa1 protein in HeLaTR cells. Subconfluent cul- tures of HeLaTR and vector-transfected HeLa cells were labelled with [ 35 S]methionine/cys- teine, and the TRa1 protein was immunopre- cipitated from the cell extracts with an anti- human TRa Ig. The human TRa1proteinwas fractionated on a polyacrylamide gel and visualized by an image analyzer. The arrow indicates the band of human TRa1protein. (C) Reporter luciferase activities induced by T3. HeLaTR and vector-transfected HeLa cells were transfected with plasmid carrying DR0orDR4thatwaslinkedtotheluciferase gene and cultured in the presence or absence of T3 for 48 h. Cell extracts were prepared from the cells, and the luciferase activities in the cell extracts were determined. One relative luci- ferase unit (RLU) represents 1.66 light units examined by recombinant luciferase. Ó FEBS 2003 T3 induces 4-1BB in a TR-dependent manner (Eur. J. Biochem. 270) 3067 As shown in Fig. 3B, RT-PCR at different time points after the addition of T3 revealed that 4-1BB expression was induced as early as 1 h after the T3 treatment and its mRNA level continued to increase at least up to 48 h. The PSG7 mRNA reached a detectable level within 2 h and remained at a low level until 24 h. Its level, however, was elevated strongly between 24 and 48 h. Whereas the HeLaTR cells expressed a certain level of the fmfc mRNA even in the absence of T3, T3 further increased its level at 2–4 h. The BMP6 mRNA level started declining between 2 and 4 h and continued to decline up to 48 h after the addition of T3. Thus, it appears that the expression levels of 4-1BB, fmfc,andBMP6 are all regulated during the early response to T3, but that of PSG7 is modulated during both early and late responses. We also examined the expression of TRAF1 and TRAF2 because they interact and regulate the function of the 4-1BB proteins to induce NF-jB [18]. In the absence of T3, neither HeLa nor HeLaTR cells expressed a detectable level of the TRAF1 mRNA. The level of the TRAF1 mRNA was stronglyincreasedbyT3intheHeLaTRcellsandtoamuch lesser extent in the vector-transfected HeLa cells. Like 4-1BB mRNA, the expression of TRAF1 was induced as early as 1 h after the T3 treatment and the TRAF1 mRNA levels continued to increase up to 48 h (Fig. 3B). On the other hand, certain amounts of the TRAF2 mRNA were detected both in the HeLaTR and vector-transfected HeLa cells even in the absence of T3, and T3 did not affect the level of the TRAF2 mRNA (Fig. 3B). Modulation of the caspase activities by T3 Upon binding to the 4-1BB-L, the ligand for 4-1BB, 4-1BB can trigger signal cascade to activate NF-jB[18].We therefore asked whether T3 could affect the NF-jB activity in the HeLaTR cells. The activity of NF-jB was assessed by transfecting the HeLaTR cells with the NF-jBluciferase reporter plasmid NFjB-Luc. Whereas TNFa strongly induced NF-jB reporter luciferase activity in the HeLaTR cells, T3 did not significantly affect the activity and rather decreased it (Fig. 4). The expression of the reporter luciferase was indeed mediated by NF-jB because only a Fig. 2. Genes whose expression was modulated by T3. Expression of mRNAs for approximately 11 000 genes in HeLaTR cells cultured in the presence or absence of T3 for 48 h were analyzed by oligonucleotide microarray. Genes whose mRNA levels differed by more than fivefold between the T3-untreated and T3-treated HeLaTR cells were selected with AFFYMETRIX MICROARRAY SUITE ver.4.0 software and are shown. The number on each bar represents the -fold change of the mRNA level between the two. Genes up-regulated by T3 are shown in the upper half, and those down- regulated by T3 are listed in the lower half. Each gene is annotated with its GenBank accession number. Dotted bars, genes with an average difference greater than 400 after up-regulation or before down-regulation by T3. Filled bars, genes with an average difference less than 400 even after up-regulation or before down-regulation by T3. 3068 T. Yamada-Okabe et al. (Eur. J. Biochem. 270) Ó FEBS 2003 very low level of luciferase activity was observed in the cells transfected with plasmid carrying the luciferase gene with- out the NF-jB enhancer sequences (Fig. 4). Because 4-1BB has to be dimerized by binding to its ligand to initiate signal cascade, we also treated the HeLaTR cells with T3 and 4-1BB-L. However, even in the presence of 100 ngÆmL )1 4-1BB-L, a concentration 10 times higher than that of the ED 50 for IL-8 production in human peripheral blood mononuclear cells, the NF-jB reporter luciferase activity was not induced by T3 (Fig. 4). This was a rather unexpected result, so we next explored other possible effects of T3. As T3 is known to induce apoptosis in rat pancreatic beta-cell and in amphibian metamorphosis [2,23], we examined the effects of T3 on the caspase activities in the HeLaTR cells. As shown in Fig. 5, T3 increased the caspase activities in the HeLaTR cells to a level similar to that of TNFa (Fig. 5A). The activity detected in this assay was indeed caspase activity because a pan-caspase inhibitor, Z-VAD-FMK, completely inhibited the generation of p-nitroanilide from acetyl-DEVD-p-nitroanilide (Fig. 5B) 8 in HeLaTR cells. Proliferation of the cells was also impaired in the presence of T3, though the degree of the inhibition of proliferation by T3 was rather weak (Fig. 5C). According to the results of DNA microarray and RT-PCR, T3 most affected the expression of 4-1BB.This prompted us to examine whether the expression of 4-1BB alone would cause the activation of caspases. To address this possibility, we generated HeLaTR/4-1BB cells in which the 4-1BB cDNA was stably expressed under the control of the CMV promoter. RT-PCR revealed that the HeLaTR/ 4-1BB cells, but not the parental HeLaTR cells, contained a high level of 4-1BB mRNA (Fig. 5D). In the HeLaTR/ 4-1BB cells, the caspase activities significantly increased as compared to those of the parental HeLaTR cells and the caspase activity level in the HeLaTR/4-1BB cells was even higher than that of the T3-treated HeLaTR cells. Interest- ingly, treatment of the HeLaTR/4-1BB cells with 100 ngÆmL )1 4-1BB-L further elevated the caspase activities (Fig. 5A). The same results were obtained with other clones of HeLaTR/4-1BB cells. As TRAF1 expression was also strongly induced by T3, we created HeLaTR/TRAF1 cells that stably overexpressed TRAF1 under the control of the CMV promoter and examined the caspase activities. Although HeLaTR/TRAF1 expressed significant amounts of TRAF1 mRNA (Fig. 5C), the caspase activity in the HeLaTR/TRAF1 cells was almost the same as that in the parental HeLaTR cells, indicating that overexpression of TRAF1 did not significantly influence the caspase activities (Fig. 5A). We also asked whether the up-regulation of 4-1BB expression and activation of caspases by T3 occurred in other cells. Rat FRTL cells that originated from normal thyroid epithelial cells were used because they were not genetically manipulated and T3 bound to the FRTL cells [24]. RT-PCR demonstrated that the FRTL cells indeed expressed a certain level of TRa (Fig. 6A). As in the HeLaTR cells, T3 treatment of the FRTL cells significantly increased the level of the 4-1BB mRNA and decreased the level of the BMP6 mRNA. Furthermore, a marked increase in the Z-VAD-FMK-sensitive caspase activities was also observed in the FRTL cells that were cultured in the presence of T3 (Fig. 6B). Caspase activities in the FRTL cell extracts prepared from cells treated with T3 were signifi- cantly inhibited by caspase 3 inhibitor DEVD-FMK and also, to lesser extents, by caspase 1 inhibitor YVAD-CMK and by caspase 8 inhibitor IETD-FMK (not shown), indicating that several caspases were activated by T3. In addition, T3 impaired the proliferation of the cells and significantly increased the degree of DNA fragmentation in FRTL cells (Fig. 6C,D). Thus, it appears that induction of the expression of 4-1BB and subsequent activation of caspases by T3 leads to apoptosis in rat FRTL cells. Discussion Using oligonucleotide microarray and subsequent RT-PCR, we identified the genes whose expression is regulated by TR. Although previous papers also describe Fig. 3. Modulation of the mRNA expression by T3. (A) Expression of the mRNA for 4-1BB, PSG7, fmfc, BMP-6, TRAF1, TRAF2 and GAPDH. Total RNA was extracted from the subconfluent cultures of HeLaTR and from the vector-transfected HeLa cells that were cultured in the presence or absence of T3 for 48 h. RT-PCR was performed with the specific primer for the indicated genes and the total RNA as the template. The PCR products collected after 30 cycles of PCR were analyzed by agarose gel electrophoresis. (B) Total RNA used as the template for the RT-PCR was extracted from the subconfluent cultures of the HeLaTR cells that were cultured in the presence of T3 for the indicated time. RT-PCR was performed as in (A). Ó FEBS 2003 T3 induces 4-1BB in a TR-dependent manner (Eur. J. Biochem. 270) 3069 genes that are up- or down-regulated by TR using cDNA microarray [10,11], most of the genes identified in this study were not discovered in previous findings. One of the differences between our study and previous studies is that we used HeLaTR cells that constitutively overexpressed TR for the analysis. Because T3 significantly increased the activities of the reporter luciferase in the HeLaTR, the increased level of TR in the HeLaTR cells amplified cellular responses to T3, which allowed us to identify additional genes that are regulated by TR. Another possibility includes a difference in the type of DNA chips used for the study. Sensitivity may differ between oligonucleotide microarray and cDNA microarray. We identified 4-1BB, TRAF1, PSG7,andfmfc as genes that were highly up-regulated by T3 and BMP-6 as one significantly down-regulated by T3. Modulation of the expression levels of 4-1BB, TRAF1, PSG7, fmfc and BMP-6 occurred within 2–4 h after the addition of T3, indicating that they are all early response genes. Further- more, up-regulation or down-regulation of these genes continued to occur at least for 2 days after exposure to T3. This suggests that prolonged changes in the expres- sion levels of these genes cause profound effects on the cells. Among genes whose expression is modulated by T3, 4-1BB is responsible for a protein that can provide costimulatory signals to stimulate T-cell proliferation [25]. Expression of 4-1BB has been thought to be rather restricted, in the body, to activated T-cells 9 ; it is induced by the activation of T-cells from peptide-MHC complexes and mitotic stimulants such as phorbol 12-myristate 13-acetate (PMA) 10 [26]. Our results show that T3 induced the transcription of 4-1BB in the HeLa cells harboring the functional TR and also in rat FRTL cells, which suggests the involvement of 4-1BB not only in T-cell-mediated immune responses but also in other systems where functional TR is expressed. As well as treatment of the HeLaTR cells with T3, overexpression of 4-1BB in the HeLaTR cells led to the activation of caspases. Activation of caspases by the overexpression of 4-1BB was not an artefact of over- expressing any gene because caspase activities were not elevated in the HeLaTR cells overexpressing TRAF1.These results demonstrate that T3 induces the expression of 4-1BB, which in turn activates caspases. Activation of caspases in the HeLaTR cells overexpressing 4-1BB Fig. 4. Effects of T3 on the activity of NF-jB. (A) Reporter luciferase activities induced by T3. The cells of HeLaTR were transfected with pTAL-luc or pNFjB-luc and were cultured in the presence or absence of T3, 4-1BB-L, or TNFa for 48 h (T3) or 24 h (4-1BB-L and TNFa). Cell extracts were prepared and luciferase activities in the cell extracts were determined by luciferase assay. Open bars represent the relative luciferase activities in the cells transfected with pTAL-luc and filled bars indicate those of cells transfected with pNF-jB-luc. One relative luciferase unit (RLU) represents 1.66 light units examined by recom- binant luciferase. (B) Electrophoretic mobility shift assay. The oligo- nucleotide harboring the NF-jB consensus sequence that had been end-labelled with [c- 32 P]ATP was incubated in the absence (lanes 1 and 4) or presence of nuclear extracts prepared from HeLaTR cells (U, lanes 2 and 5) and T3-treated HeLaTR cells (T, lanes 3 and 6) at room temperature for 10 min. Thereafter, 2-lL aliquots of reaction mixture was electrophoresed on a 6% polyacrylamide gel and radio- labelled oligonucleotides were visualized by an image analyzer. As a control experiment, 100 times molar excess of nonradiolabelled NF-jB oligonucleotide was added to the reaction mixture (lanes 4, 5 and 6). 3070 T. Yamada-Okabe et al. (Eur. J. Biochem. 270) Ó FEBS 2003 occurred even without the addition of the 4-1BB-L. This implies that the activation of caspases by 4-1BB occurs independently of the 4-1BB-L. In fact, we observed that TKS-1, an anti-mouse 4-1BB-L monoclonal Ig that neut- ralizes 4-1BB-L, did not repress the T3-induced activation of caspases in HeLaTR cells or rat FRTL cells. As TKS-1 was raised against a peptide whose sequence was identical between mouse and rat 4-1BB-L [27], it should react with rat 4-1BB-L at the least. Nevertheless, we cannot rule out the possibility that a certain level of the 4-1BB-L was expressed and secreted from HeLaTR cells, because the addition of 4-1BB-L to the culture media of the HeLaTR/4-1BB cells further elevated the caspase activities. Further study is necessary to clarify the involvement of 4-1BB-L in the activation of caspases by T3. The biological significance of the modulation of PSG7, fmfc and BMP-6 expression by T3 also awaits additional studies. Up-regulation of 4-1BB and the subsequent activation of the caspases occurred not only in HeLaTR cells but also in rat FRTL cells that expressed only authentic TR, indicating that the up-regulation of 4-1BB and the subsequent activation of the caspases were not specific to the HeLaTR cells but rather common cellular responses to T3. Further- more, T3 induced DNA fragmentation and impaired the proliferation of rat FRTL cells. This implies that T3 induces apoptosis, which is mediated by 4-1BB, and subsequent activation of caspases in the tissues and organs in the body. Impairment of cell proliferation by T3 was more profound in FRTL cells than in HeLaTR cells. As HeLaTR cells are derived from tumor cells, it is possible that HeLaTR cells express anti-apoptotic factors thereby becoming more resistant to apoptotic stimuli. Nevertheless, both proapop- totic effects and anti-apoptotic effects by 4-1BB have been demonstrated; activation of 4-1BB abrogated the granulo- cyte-macrophage colony-stimulating factor-mediated anti- apoptosis in neutrophils and eosinophils [28,29], whereas it induced Bcl-x L and Bfl-1 expression and promoted the survival of CD8+ T lymphocytes [30]. Thus, proapoptotic and anti-apoptotic action of 4-1BB may depend on the cell and tissue types. As mentioned, 4-1BB can generate signals to activate NF-jB [18], but T3 did not activate NF-jBinthe HeLaTR cells even in the presence of the 4-1BB-L. The 4-1BB protein physically associates with TNF receptor- associated factor 1 and 2 (TRAF1 and TRAF2) through two TRAF binding sites present in its cytoplasmic domain [18]. The TRAF1 and TRAF2 proteins have similar affinities to the 4-1BB proteins, but 4-1BB complexed with TRAF1 and that with TRAF2 act differently with respect to the NF-jB activation. The 4-1BB/TRAF2 complexes dimerize and activate NF-jB in HEK293 cells, while expression of TRAF1 abrogate the NF-jB activation Fig. 5. Effects of T3 and 4-1BB on the caspase activities and proliferation of HeLaTR cells. Caspase activities in the cell extracts were determined by colorimetric assay with acetyl-DEVD-p-nitroanilide as a substrate. The cells of HeLaTR and HeLaTR/4-1BB were cultured in the presence or absence of T3, 4-1BB-L or TNFa for 48 h (T3) or 24 h (4-1BB-L and TNFa) (A). The HeLaTR cells were cultured in the presence or absence of T3 or Z-VAD-FMK for 48 h (B). Thereafter, the cells were harvested, and the cell extracts were prepared. Absorbance at 405 nm representing the activities of caspases is shown. (C) Effects of T3 on cell proliferation. Five hundred cells were plated on 96-well microplates and cultured in the presence or absence of T3 or Z-VAD-FMK. At 48 h, 20 lL of CellTiter 96 AQ ueous One Solution Reagent was added to the cells, and cells were further incubated for 2 h. Absorbance at 490 nm, which represents the number of viable cells, is shown. (D) Expression of 4–1BB and TRAF1 mRNA. Levels of 4-1BB and TRAF1 mRNA were determined by RT-PCR. Total RNA was extracted from the subconfluent cultures of the HeLaTR, HeLaTR/4-1BB, and HeLaTR/TRAF1 cells and was used as the template for RT-PCR. After 30 cycles of PCR, the PCR products were separated on a 1% agarose gel. Ó FEBS 2003 T3 induces 4-1BB in a TR-dependent manner (Eur. J. Biochem. 270) 3071 induced by TNFa, IL-1 or by the expression of TRAF2, TRAF6 or AITR [18]. Negative regulation of NF-jBby TRAF1 was also demonstrated by an inducible expression of TRAF1 in HEK293T cells and TRAF1-deficient mice [31,32]. On the other hand, in HeLa cells, it was reported that the expression of the full length TRAF1 potentiated the activation of NF-jB induced by TNFa or IL-1, but that of the C-terminal part of TRAF1 blocked it [33]. In this study, T3 did not activate NF-jB of the HeLaTR cells despite that the expression of both 4-1BB and TRAF1 were strongly induced by T3 and that of TRAF2 remained at a high level, being unaffected by T3. Because TRAF2A, a splice variant of TRAF2, was shown to inhibit the NF-jB activation [34], one possibility is that HeLaTR cells expressed TRAF2A. Nevertheless, the mechanism of how 4-1BB induces apoptosis remains elusive. Cannons et al. [35] reported that in T cell activation, 4-1BB recruits TRAF2 and activates apoptosis signal-regulating kinase-1 (ASK1), which leads to downstream activation of JNK/ SAPK activation [35]. Therefore, it is possible that 4-1BB activates ASK1 to induce apoptosis. Acknowledgements We thank T. Aono for technical assistance, K. 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(1999) Role of the stress kinase pathway in signaling via the T cell costimulatory receptor 4-1BB. J. Immunol. 163, 2990–2998. Ó FEBS 2003 T3 induces 4-1BB in a TR-dependent manner (Eur. J. Biochem. 270) 3073 . Kanazawa, Yokohama; 3 Pharmaceutical Research Department 4, Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd, Kajiwara, Kamakura, Kanagawa, Japan Thyroid. designated HeLaTR /4-1BB and HeaLaTR/ TRAF1, respectively. Primers for amplifying the 4-1BB and TRAF1 cDNAs were: 5¢-GAATTCAAGCTTATGGGA AACAGCTGTTACAACATA-3¢