Báo cáo khoa học: Thyroid hormone induces the expression of 4-1BB and activation of caspases in a thyroid hormone receptor-dependent manner pptx

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 auth

Thyroid hormone induces the expression of 4-1BB and activation

of caspases in a thyroid hormone receptor-dependent manner

Toshiko Yamada-Okabe1, Yasuo Satoh2,* and Hisafumi Yamada-Okabe1,3

1

Department of Hygiene,2Department of Surgery, School of Medicine, Yokohama City University, Fukuura, Kanazawa,

Yokohama;3Pharmaceutical 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 HeLaTRcells that constitutively

over-expressed the thyroid hormone receptor (TR) was analyzed

Gene expression profiling of the HeLaTRcells 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 HeLaTRcells 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 HeLaTRcells transfected with 4-1BB cDNA, and the 4-1BB-Ligand further increased the caspase activities of the HeLaTRcells 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; 4-1BBgene 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, TRb1 or TRb2 [4–6] Each TRforms 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-CoRor SMRT is associated with the homo- or heterodimer of TRand 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 TRactive 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 signainsu-ling [10] As thyroid hormone causes growth arrest 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 TRby 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)

human genes Among the number of genes whose

expres-sion was significantly altered by T3, the expresexpres-sion 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

4-1BBcDNA 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 TRand 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

1 mgÆmL)1of 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 TRwere

designated HeLaTRand 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 HeLaTRcells and the cells were

selected with 0.4 mgÆmL)1of 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

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 94C for 30 s, 60C 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 [35S]methionine and [35S]cysteine (1000 Ci mmol)1; pro-mixL-[35S] in vitro cell labelling mix, Amersham) for 2 h Cell extracts were prepared by lysing the cells with RIPA buffer: [150 mM NaCl, 1% NP4O, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 8.0)]

4 , and the hTRa1 protein was immuno-precipitated from the cell extracts as described in a previous paper but with 2 lgÆmL)1of the anti-(TRpolyclonal Ig) (clone FL-408, Santa Cruz) [14] The hTRa1 protein was 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 pyralislinked to the simian virus-40 (SV-40) promoter The DR4 oligonucleotide that contained the TRE sequences and control DR0 oligonucleotide were inserted between the SmaI and BglII cleavage sites of the pGL2-Promoter plasmid The resulting plasmids, designated DR0-pGL2-luc and DR4-pGL2-DR0-pGL2-luc, respectively, were transfected into the HeLaTRcells 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 HeLaTRcells; 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/Pi 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 4C and 10 lL 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

the NF-jB consensus sequence (Promega) that had been

end-labelled with [c-32P]ATP (6000 CiÆmmol)1; Amersham

Pharmacia) was incubated in a reaction mixture (10-lL final

volume) containing 4% (w/v) Ficoll, 20 mM Hepes/KOH

(pH 7.9), 50 mMKCl, 1 mMEDTA, 1 mMdithiothreitol,

2.5 lg of poly(dI-dC) (Roche Diagnostics), 6 mM MgCl2

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 (SuperScriptTM 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 GelTM 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 95C for 35 min

in a buffer containing 40 mMTris/acetate (pH 8.1), 100 mM

potassium acetate and 30 mMmagnesium acetate

Hybrid-ization was carried out in 200 lL of a buffer containing

0.1MMes (pH 6.7), 1MNaCl, 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 45C for 12 h After the chips

were washed with a buffer containing 0.01MMes (pH 6.7),

0.1M 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)1T3 In some experiments, the

4-1BB-Ligand (PeproTech EC, UK) at a final concentration of

100 ngÆmL)1or 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 lM Cells were washed

with NaCl/Pitwice, harvested, suspended in 50 lL of the

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 lL of reaction mixture containing 15 lL (100 lg) cell extracts and acetyl-DEVD-p-nitroanilide as a substrate After incu-bation at 37C overnight, absorbance at 405 nm was measured with a microplate reader (Bio-Rad)

Cell proliferation assay One thousand cells of HeLaTRand FRTL were cultured in

100 lL of the medium in the presence or absence of T3 and Z-VAD-FMK at 37C for 48 h At the end of the culture,

20 lL of CellTiter 96 AQueous 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 37C 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 HeLaTRand FRTL that were cultured in the presence or absence of T3 were collected and lysed with a lysis buffer containing 10 mM Tris/HCl (pH 8.0), 10 mM EDTA and 0.5% (v/v) Triton X-100 Cell lysates were treated with RNase A (0.1 mgÆmL)1) at 37C for 1 h and then with proteinase K (1 mgÆmL)1) at 50C 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 hTRa1 were 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 HeLaTRcells but not from the vector-transfected HeLa cells (Fig 1B) The TRb mRNA in HeLaTRand in the vector-transfected HeLa cells was below the level detectable by RT-PCR (data not shown) Next, we confirmed the functionality of hTRa1 in the HeLaTRcells The HeLaTRcells 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 TRand that from the DR0-pGL2

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

HeLaTRcells ranged from 25 to 100 ngÆmL)1, which is

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 hTRa1 was activated by T3 in the HeLaTR cells and

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 HeLaTRcells 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 HeLaTRcells

or those treated with T3 RT-PCR confirmed that T3 up-regulated the expression of 4-1BB, pregnancy specific gene-7 (PSG7) and fmfc 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 (

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

TRa1 in HeLa cells (A) Expression of the

human TRa1 (TR) mRNA in HeLaTR cells.

RNA was extracted from the subconfluent

cultures of HeLaTRand vector-transfected

HeLa cells and was used as the template for

RT-PCR RT-PCR was performed with the

specific primer for hTRa1 and GAPDH, and

the PCRproducts after the indicated cycles of

PCRwere analysed by agarose gel

electro-phoresis (B) Expression of the human TRa1

protein in HeLaTRcells Subconfluent

cul-tures of HeLaTRand 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 anti-human TRa1 protein was

fractionated on a polyacrylamide gel and

visualized by an image analyzer The arrow

indicates the band of human TRa1 protein.

(C) Reporter luciferase activities induced by

T3 HeLaTRand vector-transfected HeLa

cells were transfected with plasmid carrying

DR 0 or DR 4 that was linked to the luciferase

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.

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

HeLaTRcells 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, and BMP6 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 HeLaTRcells expressed a detectable level of

the TRAF1 mRNA The level of the TRAF1 mRNA was

strongly increased by T3 in the HeLaTR cells and to a much

lesser extent in the vector-transfected HeLa cells Like 4-1BBmRNA, 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 HeLaTRand 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 HeLaTRcells The activity of NF-jB was assessed by transfecting the HeLaTRcells with the NF-jB luciferase 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 HeLaTRcells 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.

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 HeLaTRcells 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

ED50 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

HeLaTRcells As shown in Fig 5, T3 increased the caspase

activities in the HeLaTRcells 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 HeLaTRcells 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 HeLaTRcells, 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 HeLaTRcells and the

caspase activity level in the HeLaTR/4-1BB cells was even

higher than that of the T3-treated HeLaTRcells

Interest-ingly, treatment of the HeLaTR/4-1BB cells with

100 ngÆmL)14-1BB-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 HeLaTRcells, indicating that overexpression of TRAF1did 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 HeLaTRcells, 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 PCRwere analyzed by agarose gel electrophoresis (B) Total RNA used as the template for the RT-PCRwas extracted from the subconfluent cultures of the HeLaTRcells that were cultured in the presence of T3 for the indicated time RT-PCRwas performed as in (A).

genes that are up- or down-regulated by TRusing 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 HeLaTRcells that constitutively overexpressed TR

for the analysis Because T3 significantly increased the

activities of the reporter luciferase in the HeLaTR, the increased level of TRin the HeLaTRcells 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, and fmfc 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

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 TRand 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 TRis expressed

As well as treatment of the HeLaTRcells with T3, overexpression of 4-1BB in the HeLaTRcells 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 HeLaTRcells overexpressing TRAF1 These results demonstrate that T3 induces the expression of 4-1BB, which in turn activates caspases Activation of caspases in the HeLaTRcells overexpressing 4-1BB

Fig 4 Effects of T3 on the activity of NF-jB (A) Reporter luciferase activities induced by T3 The cells of HeLaTRwere 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 HeLaTRcells (U, lanes 2 and 5) and T3-treated HeLaTRcells (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).

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 HeLaTRcells 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 HeLaTRcells, 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 HeLaTRcells 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 HeLaTRcells As HeLaTRcells are derived from tumor cells, it is possible that HeLaTRcells express anti-apoptotic factors thereby becoming more resistant to apoptotic stimuli Nevertheless, both proapop-totic effects and anti-apopproapop-totic 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-xLand 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-jB in the HeLaTRcells 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 HeLaTRand 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 HeLaTRcells 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.

induced by TNFa, IL-1 or by the expression of TRAF2,

TRAF6 or AITR [18] Negative regulation of NF-jB by

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 Okumura for anti-4-1BB-L monoclonal Ig TKS-1, and F Ford for proofreading the manuscript This study is supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan

to T Y.-O.

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