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

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:
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. 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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