TRB3,upregulatedbyox-LDL,mediates human
monocyte-derived macrophage apoptosis
Yuan-yuan Shang
1,
*, Zhi-hao Wang
1,
*, Li-ping Zhang
2
, Ming Zhong
1
, Yun Zhang
1
, Jing-ti Deng
2
and Wei Zhang
1
1 Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Public Health,
Department of Cardiology, Qilu Hospital of Shandong University, Ji’nan, China
2 Department of Anatomy, School of Medicine, Shandong University, Ji’nan, China
Acute coronary syndrome is the consequence of rupture
or erosion of pre-existing atherosclerotic plaques, with
subsequent formation of local thrombus, leading to crit-
ical occlusion of coronary arteries. The principal patho-
logical basis of acute coronary syndrome is vulnerable
plaques [1]. Macrophageapoptosis contributes signifi-
cantly to the development of vulnerable atherosclerotic
plaques [2–4]. A possible mechanism linking macro-
phage apoptosis to vulnerable plaque progression is that
the reduced level of macrophages fails to clear apoptotic
smooth muscle cells and macrophages, which leads to
secondary necrosis of these cells and facilitates forma-
tion of an atheromatous core within plaques. In addi-
tion, apoptotic macrophages can release cholesterol,
which results in accumulation of acicular cholesterol
crystals in the lipid core, thus injuring the fibrous cap of
plaques. In addition, apoptotic macrophages may be a
source of tissue factor, a procoagulant molecule that is
considered to play an important role in coagulation and
thrombosis associated with advanced plaques [3].
Keywords
apoptosis; atherosclerosis; macrophage;
ox-LDL; TRB3
Correspondence
W. Zhang, Department of Cardiology, Qilu
Hospital of Shandong University, Ji’nan
250012, China
Fax: +86 531 86169356
Tel: +86 531 82169339
E-mail: zhangweisdu@gmail.com
J t. Deng, Department of Anatomy, School
of Medicine of Shandong University, Ji’nan
250012, China
Fax: +86 531 86169356
Tel: +86 531 88382093
E-mail: jingtideng@hotmail.com
*These authors contributed equally to this
paper
(Received 16 January 2009, revised 4 March
2009, accepted 9 March 2009)
doi:10.1111/j.1742-4658.2009.06998.x
Tribble3 (TRB3), a mammalian homolog of Drosophila tribbles, slows cell-
cycle progression, and its expression is increased in response to various
stresses. The aim of this study was to investigate the role of the TRB3 gene
in macrophageapoptosis induced by oxidized low-density lipoprotein
(ox-LDL). We found that, in humanmonocyte-derived macrophages,
TRB3 is upregulatedby ox-LDL in a dose- and time-dependent manner.
The cell viability of TRB3-overexpressing macrophages was decreased, but
apoptosis was increased and the level of activated caspase-3 increased. Fac-
torial analyses revealed no significant interaction between TRB3 overex-
pression and ox-LDL stimulation with respect to macrophage apoptosis.
Furthermore, TRB3-silenced macrophages showed decreased apoptosis,
and TRB3-silenced cells treated with ox-LDL showed significantly
increased apoptosis. Silencing of TRB3 and ox-LDL stimulation showed
significant interaction for macrophage apoptosis, suggesting that TRB3
knockdown resisted the macrophageapoptosis induced by ox-LDL. There-
fore, TRB3 in part mediates the macrophageapoptosis induced by
ox-LDL, which suggests that TRB3 might be involved in vulnerable
atherosclerotic plaque progression.
Abbreviations
MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ox-LDL, oxidized low-density
lipoprotein; siRNA, small interfering RNA; SiTRB3, siRNA targeting TRB3; ssDNA, single-stranded DNA; TRB3, Tribble3.
2752 FEBS Journal 276 (2009) 2752–2761 ª 2009 The Authors Journal compilation ª 2009 FEBS
Signal transduction of apoptosis in macrophages
involves a complex network system. Various risk fac-
tors, such as an increased level of oxidized low-density
lipoprotein (ox-LDL), can induce macrophage apopto-
sis through multiple apoptotic signaling pathways, such
as Akt/protein kinase B (PKB) and mitogen-activated
protein kinase (MAPK) [5–7]. However, the mechanism
of macrophageapoptosis remains to be elucidated.
Tribbles, a Drosophila protein, slows progression
through the G
2
stage of the cell cycle [8]. Three mam-
malian orthologs, TRB1, TRB2 and TRB3, all contain
a consensus serine ⁄ threonine kinase catalytic core but
lack an ATP-binding pocket and so do not possess
kinase activity. Recently, TRBs have been shown to be
expressed in unstable regions of carotid plaques [9].
TRB3, also named neuronal cell death-inducible puta-
tive protein kinase, is expressed in the liver, thymus,
prostate and heart [10], and may have broad biological
activity. TRB3 has been reported to be an important
regulatory protein involved in signal pathways, and
works at least through CDC25 ⁄ String, Akt and
MAPK [11–13]. Activation of MAPK and inhibition
of Akt kinase activity result in macrophage apoptosis
[5–7], which is implicated in the development of vul-
nerable atherosclerotic plaques [2–4]. TRB3 may be
involved in macrophageapoptosis induced by ox-LDL,
and could play an essential role in the progression of
vulnerable plaques.
We investigated whether TRB3 is implicated in
ox-LDL-induced apoptosisby stimulating human
monocyte-derived macrophages with ox-LDL, then
transfecting them with a recombinant adenoviral
TRB3 construct or a small interfering RNA (siRNA)
targeting TRB3.
Results
TRBs mRNA expression in human macrophages
To determine whether the TRB genes TRB1, TRB2
and TRB3 were expressed in human monocyte-derived
macrophages, monocytes were first allowed to differen-
tiate naturally into macrophages. Quantitative real-
time PCR performed on days 1, 3, 5, 7, 9 and 11
showed that all three genes were expressed in mono-
cyte-derived macrophages. Macrophages predomi-
nantly expressed TRB3 (Fig. 1), and the level of TRB3
mRNA increased on day 3 and peaked on day 7.
TRB3 expression upregulatedby ox-LDL
To examine whether TRB3 mRNA expression in mac-
rophages was regulated byox-LDL, macrophages were
treated with various concentrations of ox-LDL or
LDL for 24 h. Quantitative real-time PCR showed
that expression of TRB3 mRNA was significantly
upregulated by ox-LDL but not by LDL (Fig. 2A).
The mRNA expression increased with increasing
ox-LDL concentration; a statistical difference between
ox-LDL and LDL treatments was observed at a con-
centration of 50 lgÆmL
)1
(4.72 ± 2.72 versus 1.04 ±
0.42, P < 0.01).
Fig. 1. TRBs mRNA expression in macrophages.
LDL
A
B
C
ox-LDL
LDL
ox-LDL
4
5
6
*
1
2
3
4
0
1
0 µg 5 µg 10 µg 25 µg 50 µg
0 h 4 h 8 h 12 h 24 h 48 h
Relative TRB3 mRNA level
8
10
2
4
6
0
Relative TRB3 mRNA level
LDL ox-LDL
TRB3
GAPDH
Fig. 2. TRB3 mRNA and protein expression are upregulated by
ox-LDL. (A) Quantitative real-time PCR analysis of macrophages in
serum-free medium treated with various concentrations of ox-LDL or
LDL for 24 h. (B) Quantitative real-time PCR analysis of macrophages
treated with 50 lgÆmL
)1
ox-LDL or LDL for various durations. (C)
Western blot analysis of macrophages treated with 50 lgÆmL
)1
ox-LDL or LDL for 24 h. For (A) and (B), expression was normalized
to that of GAPDH. *P < 0.05, **P < 0.01 versus LDL-treated cells.
Y y. Shang et al. TRB3 mediateshumanmacrophage apoptosis
FEBS Journal 276 (2009) 2752–2761 ª 2009 The Authors Journal compilation ª 2009 FEBS 2753
Macrophages treated with 50 lgÆmL
)1
ox-LDL or
LDL for various durations showed increased TRB3
mRNA expression with increasing time (Fig. 2B).
Expression of TRB3 mRNA was significantly higher
after a 24 h treatment with 50 lgÆmL
)1
ox-LDL than
with LDL treatment (3.32 ± 2.63 versus 1.14 ± 0.50,
P < 0.05) and was further increased after 48 h of
ox-LDL treatment (8.55 ± 4.78 versus 1.32 ± 0.46,
P < 0.01). The TRB3 protein level was also signifi-
cantly increased after treatment with 50 lgÆmL
)1
ox-LDL for 24 h (Fig. 2C).
Ox-LDL induces macrophage apoptosis
Macrophages were treated with various concentrations
of ox-LDL or LDL for 24 h, and then cell viability
was determined by MTT assay. Cell viability was sig-
nificantly reduced in cells treated with ox-LDL but not
those treated with LDL (Fig. 3A). Cell viability was
significantly lower with 50 lgÆmL
)1
ox-LDL treatment
than with 50 lgÆmL
)1
LDL treatment (42.5 ± 1.0%
versus 106.5 ± 16.3%, P < 0.05). However, at lower
concentrations, the reduction in cell viability with
ox-LDL was not significantly different from that with
LDL.
Macrophages were treated with 50 lgÆmL
)1
ox-LDL
or LDL for various durations. Incubation for 24 h
with ox-LDL resulted in a lower cell viability than
with LDL (42.5 ± 1.0% versus 89.6 ± 19.0%,
P < 0.01), and was further decreased after 48 h
treatment (16.8 ± 17.1% versus 106.5 ± 16.3%, P <
0.01) (Fig. 3B). Therefore, we selected 50 lgÆmL
)1
ox-LDL treatment for 24 h as the optimal stimulus in
subsequent experiments.
Macrophages treated with 50 lgÆmL
)1
ox-LDL for
24 h were subjected to western blot analysis to determine
the level of activated caspase-3 with 17 and 19 kDa, a
marker of apoptosis, and showed an increased level of
activated caspase-3 (see Fig. 8A below).
TRB3 mediatesmacrophageapoptosis induced
by ox-LDL
We were able to express the cloned TRB3 protein suc-
cessfully in mammalian cells using the adenoviral
expression system only, because of the lack of effec-
tiveness of other transfection techniques in this case.
The level of TRB3 protein was increased with increas-
ing multiplicities of infection with adenovirus (Fig. 4).
Endogenous TRB3 protein could not be detected in
nuclear extracts except when 100 lg total protein was
used for western blot analysis, which indicates that the
endogenous TRB3 protein is barely detectable and that
the protein is present at low abundance in human mac-
rophages.
As cell apoptosis increased with increased TRB3
expression, we investigated the role of TRB3 in
ox-LDL-induced macrophageapoptosisby determina-
tion of cell viability. Macrophages were transfected
with adeno-TRB3 or empty vector and incubated for
an additional 24 h with or without ox-LDL. MTT
assay results revealed a reduced cell viability of macro-
phages treated with 50 lgÆmL
)1
ox-LDL for 24 h
compared with vector controls (59.6 ± 8.5% versus
100 ± 0.2%, P < 0.01) (Fig. 5A), and in TRB3-over-
expressing macrophages compared with vector controls
(67.1 ± 18.2% versus 100 ± 0.2%, P < 0.01).
140
160
60
80
100
120
LDL
A
B
ox-LDL
LDL
ox-LDL
*
0
20
40
Control
2.5 µg·mL
–1
5 µg·mL
–1
10 µg·mL
–1
25 µg·mL
–1
50 µg·mL
–1
% cell viability% cell viability
120
140
40
60
80
100
*
0
20
40
0 h 4 h 8 h 12 h 24 h 48 h
*
Fig. 3. Effect of ox-LDL and LDL on macrophage cell viability by
MTT assay. (A) Macrophages were treated with various concentra-
tions of ox-LDL or LDL for 24 h. (B) Macrophages were treated
with 50 lgÆmL
)1
ox-LDL or LDL for various durations. *P < 0.05,
**P < 0.01 versus LDL-treated cells.
TRB3
GAPDH
Vector 50IFU 100IFU 200IFU
50 kD
a
37 kD
a
Fig. 4. Western blot analysis of TRB3 protein expression in vitro.
Macrophages were transfected with purified recombinant adeno-
TRB3 at multiplicities of infection of 50, 100 and 200 inclusion-
forming units (IFU) or with vector (control), and the TRB3 level was
analyzed 24 h later.
TRB3 mediateshumanmacrophageapoptosis Y y. Shang et al.
2754 FEBS Journal 276 (2009) 2752–2761 ª 2009 The Authors Journal compilation ª 2009 FEBS
Treatment of TRB3-overexpressing macrophages with
ox-LDL markedly reduced cell viability further com-
pared with vector control cells (46.0 ± 12.8% versus
100 ± 0.2%, P < 0.01) and with cells overexpressing
TRB3 alone (46.0 ± 12.8% versus 67.1 ± 18.2%,
P < 0.05), but no statistical difference was found
compared with ox-LDL-treated vector control cells
(46.0 ± 12.8% versus 59.6 ± 8.5%, P > 0.05).
Factorial analyses revealed a significantly lower cell
viability in macrophages treated with ox-LDL com-
pared to those that were not treated with ox-LDL
(50.4 ± 15.9% versus 69.0 ± 18.6%, P < 0.01), and
significantly lower cell viability in TRB3-overexpress-
ing cells than in non-TRB3-overexpressing cells
(46.9 ± 10.6% versus 73.5 ± 18.6%, P < 0.01)
(Table 1). However, overexpression of TRB3 and stim-
ulation with ox-LDL did not show a significant inter-
action for cell viability (P = 0.206). Thus,
overexpression of TRB3 compromises cell viability,
with further reduction caused by ox-LDL; however,
overexpression of TRB3 has no effect on cell viability
already reduced byox-LDL, which indicates that
TRB3 is involved in part in macrophage survival.
With regard to apoptosis, the single-stranded DNA
(ssDNA) absorbance of macrophages treated with
ox-LDL was significantly higher than that in vector
control cells (0.57 ± 0.02 versus 0.38 ± 0.04,
P < 0.01), and that of TRB3-overexpressing macro-
phages was also significantly increased (0.52 ± 0.12
versus 0.38 ± 0.04, P < 0.05) (Fig. 5B). In addition,
TRB3-overexpressing macrophages treated with
ox-LDL showed a significant increase in apoptosis
compared with vector control cells (0.77 ± 0.15 versus
0.38 ± 0.04, P < 0.01), ox-LDL-treated vector con-
trols (0.77 ± 0.15 versus 0.57 ± 0.02, P < 0.01) or
cells with TRB3 overexpression alone (0.77 ± 0.15
versus 0.52 ± 0.12, P < 0.01).
Factorial analyses revealed no significant interaction
between overexpressed TRB3 and ox-LDL stimulation
with respect to apoptosis (Table 1). Apoptosis was
significantly higher in TRB3-overexpressing than
non-TRB3-overexpressing cells (0.67 ± 0.14 versus
0.46 ± 0.10, P < 0.01) and higher in cells treated with
ox-LDL compared to those that were not treated with
ox-LDL (0.64 ± 0.18 versus 0.48 ± 0.10, P < 0.01).
Western blot analysis (see Fig. 8B below) showed an
increased activated caspase-3 protein level in TRB3-
overexpressing cells compared with vector control cells,
an increased level in ox-LDL-treated TRB3-overex-
pressing cells compared with ox-LDL-treated vector
controls, and an increased level compared with TRB3
overexpression alone.
To clarify the role of TRB3 in macrophage apopto-
sis, siTRB3 was transfected into macrophages to
silence TRB3 gene expression. The expression of TRB3
mRNA was significantly reduced after siTRB3 trans-
fection (Fig. 6). MTT assay results showed that the cell
1
0
20
40
60
80
100
120
140
160
A
B
0.6
0.8
Vector
P < 0.01
P < 0.05
P < 0.01
P < 0.05
P < 0.05
P < 0.01
P < 0.01
P < 0.01
0
0.2
0.4
Absorbance (405 nm) % cell viability
TRB3
Vector
TRB3
None ox-LDL
None ox-LDL
Fig. 5. Influence of TRB3 overexpression on macrophage apop-
tosis. Macrophages were transfected with adeno-TRB3 or empty
vector before incubation for an additional 24 h with or without
ox-LDL. (A) Cell viability evaluated by the MTT assay. (B) Apoptosis
detected by ELISA.
Table 1. Parameters of apoptosis for the various treatment groups comprising overexpression of TRB3 and ⁄ or stimulation with ox-LDL. Data
are means ± SD. TRB3, overexpression of TRB3; ox-LDL, oxidized low-density lipoprotein.
Group P-value
Control TRB3 ox-LDL TRB3 ⁄ ox-LDL TRB3 effect ox-LDL effect Interaction
Cell viability (%) 100 ± 0.2 67.1 ± 18.2 59.6 ± 8.5 46.0 ± 12.8 < 0.01 < 0.01 0.206
Apoptosis 0.38 ± 0.04 0.51 ± 0.12 0.57 ± 0.02 0.77 ± 0.15 < 0.01 < 0.01 0.520
Y y. Shang et al. TRB3 mediateshumanmacrophage apoptosis
FEBS Journal 276 (2009) 2752–2761 ª 2009 The Authors Journal compilation ª 2009 FEBS 2755
viability of macrophages transfected with siTRB3 was
higher than in those transfected with control siRNA
(100 ± 1.7% versus 78.8 ± 2.6%, P < 0.01), and the
cell viabilities of cells treated with ox-LDL
(47.8 ± 1.8% versus 78.8 ± 2.6%, P < 0.01) or
silenced TRB3 followed by treatment with ox-LDL
(57.5 ± 5.3% versus 78.8 ± 2.6%, P < 0.01) were
both markedly decreased compared to that of macro-
phages transfected with siTRB3. With ox-LDL treat-
ment, cell viability of TRB3-silenced cells was
significantly higher than for cells transfected with con-
trol siRNA (57.5 ± 5.3% versus 47.8 ± 1.8%,
P < 0.05) (Fig. 7A).
Factorial analyses revealed a significant interaction
between silenced TRB3 and ox-LDL stimulation with
respect to macrophage viability (P = 0.048). Cell
viability was significantly lower in macrophages treated
with ox-LDL compared to those that were not treated
with ox-LDL (52.2 ± 0.2% versus 89.4 ± 13.3%,
P < 0.01) and higher in TRB3-silenced cells than in
non-TRB3-silenced cells (73.5 ± 18.6 versus 46.9 ±
10.6, P < 0.01) (Table 2).
ELISA results showed that apoptosis of TRB3-
silenced macrophages was lower than that for control
siRNA-transfected cells (0.29 ± 0.01 versus
0.38 ± 0.05, P < 0.05) (Fig. 7B), but apoptosis of
ox-LDL-treated cells was significantly higher
(0.75 ± 0.09 versus 0.38 ± 0.05, P < 0.01) as was
that of TRB3-silenced cells (0.48 ± 0.02 versus
0.38 ± 0.05, P < 0.01). Apoptosis of TRB3-silenced
cells treated with ox-LDL was significantly lower than
that for control siRNA-transfected cells (0.48 ± 0.02
versus 0.75 ± 0.09, P < 0.01).
Factorial analyses showed that apoptosis of macro-
phages was significantly higher in cells treated with
ox-LDL compared to those that were not treated with
ox-LDL (0.56 ± 0.21 versus 0.39 ± 0.10, P < 0.01)
and was lower in TRB3-silenced cells than in
non-TRB3-silenced cells (0.33 ± 0.06 versus
0.62 ± 0.15, P < 0.01) (Table 2). The interaction
between silenced TRB3 and stimulation of ox-LDL
was significant with respect to macrophage apoptosis
(P = 0.001). Taken together, the results indicate that
TRB3 resists macrophageapoptosis induced by
ox-LDL. Therefore, TRB3 was confirmed to mediate
in part the macrophageapoptosis induced by ox-LDL.
The level of activated caspase-3 protein was
decreased upon siTRB3 transfection, but this reduction
was attenuated with subsequent ox-LDL treatment
(Fig. 8C). The level of activated caspase-3 in the
TRB3-silenced cells treated with ox-LDL was higher
than that for control siRNA-transfected cells treated
with ox-LDL.
Discussion
As regulatory proteins, TRBs play an important role
in signal regulation of apoptosis. As ox-LDL-induced
macrophage apoptosis is implicated in the formation
of vulnerable atherosclerotic plaques, we investigated
the role of the TRB3 gene in macrophage apoptosis
induced by ox-LDL. Humanmonocyte-derived macro-
phages expressed TRB1, TRB2 and especially TRB3.
1.6
0.8
1
1.2
1.4
0
0.2
0.4
0.6
Relative TRB3 mRNA level
**
Control siRNA siTRB3
Fig. 6. Expression of TRB3 mRNA after treatment with siRNA.
Macrophages were transfected with siTRB3 or control siRNA for
24 h, then quantitative real-time PCR was performed to analyze
TRB3 mRNA expression. **P < 0.01 versus control siRNA.
0
20
40
60
80
100
120
140
% cell viability
None ox-LDL
A
Control siRNA
siTRB3
Control siRNA
P < 0.01
P < 0.01
P < 0.01
P < 0.05
P < 0.01
P < 0.01
P < 0.01
P < 0.05
siTRB3
0.5
0.4
0.6
0.7
0.8
0.9
0
0.1
0.2
0.3
Absorbance (405 nm)
None ox-LDL
B
Fig. 7. Influence of TRB3 silencing on macrophage apoptosis. Mac-
rophages were transfected with siTRB3 or control siRNA for 24 h
before incubation for an additional 24 h with or without ox-LDL. (A)
Cell viability evaluated by MTT assay. (B) Apoptosis detected by
ELISA.
TRB3 mediateshumanmacrophageapoptosis Y y. Shang et al.
2756 FEBS Journal 276 (2009) 2752–2761 ª 2009 The Authors Journal compilation ª 2009 FEBS
In addition, TRB3 mRNA expression was upregulated
in macrophages in a dose- and time-dependent manner
upon stimulation with ox-LDL. Moreover, TRB3
promoted macrophageapoptosis and is involved in
ox-LDL-dependent macrophage apoptosis.
The ox-LDL level is considered a risk factor for ath-
erosclerosis. Ox-LDL is taken up by macrophages in a
rapid and uncontrolled manner, which accelerates the
formation of foam cells, the major cellular component
of fatty streaks. Ox-LDL may also mediate atherogen-
esis by inducing macrophageapoptosis [14,15].
Although a high concentration of ox-LDL is cytotoxic
for cells, a low concentration can protect cells and
attenuate apoptosis in monocytic cells [16]. We also
found that low concentrations of ox-LDL in human
monocyte-derived macrophages had no effect on apop-
tosis and high concentrations induced apoptosis.
Apoptosis was markedly increased in mouse peritoneal
macrophages after stimulation with ox-LDL, and
increased macrophageapoptosis increased the size and
number of aortic atheromatous plaques and macro-
phage infiltration of plaques [5], indicating that macro-
phage apoptosis promotes atherosclerosis progression.
TRBs, the regulation of which is cell type-specific
[17], are expressed in many types of cells, such as vas-
cular smooth muscle cells, human umbilical cord endo-
thelial cells, and HeLa and HepG2 cells. We found
that naturally differentiated macrophages expressed all
three TRB genes but predominantly TRB3. The
expression of TRB3 was significantly increased on
day 3 of differentiation into macrophages. We also
found that endogenous TRB3 protein was barely
detected in untreated macrophages as reported previ-
ously in untreated 293 cell [18].
TRB3 expression has been shown to be augmented
by multiple cellular stressors, including endoplasmic
reticulum stress, hypoxia, oxidative stress, high glucose
levels and advanced glycation end products [18–22],
but few reports exist of the regulation of TRB3 expres-
sion by ox-LDL in human primary macrophages. We
found that both mRNA and protein expression of
TRB3 was upregulatedby ox-LDL in human macro-
phages in a dose- and time-dependent manner, in
agreement with previous results [9]. Moreover, the
apoptosis of macrophages increased with increasing
expression of TRB3 mRNA and protein.
TRB3 inhibits cell mitosis and coordinates cell mor-
phogenesis and migration in Drosophila by regulating
String ⁄ CDC25 proteolysis and promoting the degrada-
tion of slbo [8,11]. As a feedback regulator of the
activating transcription factor 4– C ⁄ EBP homologous
protein (CHOP) pathway, TRB3 is involved in endo-
plasmic reticulum stress-induced apoptosis of HepG2
and COS-7 cells [19,23,24]. In addition, TRB3 expres-
sion in lymphocytes induces G
2
cell-cycle delay and
cellular depletion [25]. However, whether TRB3 is
involved in the apoptosis of human monocyte-derived
macrophages was unknown. Caspase-3, a key molecule
in the classical apoptotic pathway, plays an important
role in apoptosis induced by ox-LDL; ox-LDL induces
macrophage apoptosis through activation of caspase-3
[26], so expression of activated caspase-3 is used as the
primary measure of macrophage apoptosis. We found
that overexpression of TRB3 in human macrophages
Table 2. Parameters of apoptosis for the various treatment groups comprising silencing of the TRB3 gene and ⁄ or stimulation by ox-LDL.
Data are means ± SD. siTRB3, siRNA targeting TRB3; ox-LDL, oxidized low-density lipoprotein.
Group P-value
Control siTRB3 ox-LDL siTRB3 ⁄ ox-LDL siTRB3 effect ox-LDL effect Interaction
Cell viability (%) 78.8 ± 2.6 100 ± 1.7 47.8 ± 1.8 57.5 ± 5.3 < 0.01 < 0.01 0.048
Apoptosis 0.38 ± 0.05 0.29 ± 0.01 0.75 ± 0.09 0.48 ± 0.02 < 0.01 < 0.01 0.001
Activated caspase-3
ABC
GAPDH
ox-LDL – +
ox-LDL
TRB3
++––
+–+–
ox-LDL
siTRB3
––++
19 kD
a
17 kDa
–+–+
Fig. 8. Western blot analysis of expression of activated caspase-3 protein. (A) Macrophages were treated with 50 lgÆmL
)1
ox-LDL for 24 h.
(B) Macrophages were transfected with recombinant adeno-TRB3 or empty vector for 24 h, then incubated for an additional 24 h with or
without ox-LDL. (C) Macrophages were transfected with siTRB3 or control siRNA for 24 h, then incubated for an additional 24 h with or
without ox-LDL. Expression was normalized to that of GAPDH.
Y y. Shang et al. TRB3 mediateshumanmacrophage apoptosis
FEBS Journal 276 (2009) 2752–2761 ª 2009 The Authors Journal compilation ª 2009 FEBS 2757
reduced cell viability, increased apoptosis and aug-
mented activated caspase-3 expression, which suggests
a role in increased macrophage apoptosis. Apoptosis
was further increased in TRB3-overexpressing macro-
phages treated with ox-LDL. Thus, TRB3 is involved
in ox-LDL-dependent macrophage apoptosis, possibly
through caspase-3.
We found decreased cell viability of TRB3-over-
expressing macrophages treated with ox-LDL compared
with ox-LDL-treated control cells, but the differences
were not significant. This finding could be explained by
the higher level of TRB3 in macrophages treated with
ox-LDL than in cells treated by transfection alone,
which would compromise the effect of overexpressed
TRB3. In addition, our experiment required that macro-
phages with overexpressed TRB3 be incubated with
ox-LDL; as overexpression of TRB3 promoted macro-
phage apoptosis, many TRB3-overexpressing macro-
phages may have died before incubation with ox-LDL,
which would compromise the effect of ox-LDL. Fur-
thermore, the MTT assay and ELISA results differed.
ELISA showed higher apoptosis in TRB3-overexpress-
ing macrophages treated with ox-LDL than in control
cells treated with ox-LDL, but the MTT assay revealed
no significant difference. The ELISA results may have
been more accurate than MTT results in detecting cell
apoptosis, or many macrophages may have died, to indi-
cate higher apoptosis. Furthermore, the MTT assay
measures the net rate of apoptosis and proliferation,
and therefore TRB3 may influence macrophage prolifer-
ation rates just as TRB1 does [27].
Factorial analyses revealed that overexpression of
TRB3 and stimulation of ox-LDL can induce macro-
phage apoptosis, leading to reduced cell viability,
increased apoptosis and an increased level of activated
caspase-3. Ox-LDL aggravated the apoptosis of
TRB3-overexpressing macrophages, but overexpression
of TRB3 did not affect the apoptosis induced by
ox-LDL. The interaction of TRB3 overexpression and
stimulation by ox-LDL was not significant, indicating
that TRB3 is involved only in part in macrophage
apoptosis induced by ox-LDL.
To further clarify the function of TRB3, transfection
of siTRB3 into macrophages to silence TRB3 gene
expression resulted in increased macrophage viability,
decreased apoptosis and a reduced level of activated
caspase-3, which suggests decreased apoptosis of mac-
rophages. Combined with the results above, this dem-
onstrates that TRB3 alone promotes macrophage
apoptosis. However, ox-LDL treatment increased the
apoptosis of TRB3-silenced macrophages. Further
analysis showed that TRB3 knockdown and stimula-
tion with ox-LDL affect macrophage apoptosis, with
significant interaction between the treatments. In addi-
tion, TRB3 knockdown attenuated the macrophage
apoptosis, as indicated by the high level of activated
caspase-3, induced byox-LDL, which further confirms
that TRB3 mediates ox-LDL-induced macrophage
apoptosis through caspase-3. Although TRB3 bridges
the gap between macrophageapoptosis and stimula-
tion with ox-LDL, the specific cellular signal transduc-
tion mechanism is still unclear. Recently, TRB2 was
shown to regulate the inflammatory activation of
monocytes by the MAPK pathway [28]. Furthermore,
the TRB family has been reported to interact and
modify the activity of the MAPK system [24]. There-
fore, TRB3 may mediatesmacrophageapoptosis via
the MAPK pathway, which requires further study.
Macrophage apoptosis promotes vulnerable athero-
sclerotic plaque progression. As we found that TRB3
is implicated in macrophageapoptosis induced by
ox-LDL and that TRB3 knockdown can attenuate
ox-LDL-induced apoptosis, TRB3 may play a crucial
role in the development of vulnerable atherosclerotic
plaques by regulating apoptosis. However, more stud-
ies are necessary to elucidate the mechanism. Our pre-
liminary findings strongly suggest that TRB3
contributes to destabilization of atherosclerotic plaques
through its effect on macrophage apoptosis.
The signal regulation of macrophages involves a very
complex network system. ox-LDL has been found to
induce macrophageapoptosis through activation of mul-
tiple signaling pathways such as Akt and MAPK, and
now TRB3. These findings provide a basis for further
investigation of TRB3’s role in macrophage apoptosis
and formation of vulnerable atherosclerotic plaques.
In summary, expression of the regulatory protein
TRB3, which is upregulatedby ox-LDL in a dose- and
time-dependent manner, exceeds that of other TRBs in
naturally differentiated human macrophages. TRB3 in
part mediatesmacrophageapoptosis induced by
ox-LDL, which suggests that TRB3 might be involved
in vulnerable atherosclerotic plaque progression.
Experimental procedures
Isolation and culture of human monocyte-derived
macrophages
Peripheral blood mononuclear cells were isolated under
sterile conditions using endotoxin-free Histopaque-1077
medium (Sigma, St Louis, MO, USA) with a density gradi-
ent centrifugation technique [29]. Cells were plated in 12- or
6-well plates at 3 · 10
5
cellsÆmL
)1
and cultured in complete
culture medium [RPMI-1640 containing 5% human
serum (Sigma), 100 IUÆmL
)1
penicillin and 100 lgÆmL
)1
TRB3 mediateshumanmacrophageapoptosis Y y. Shang et al.
2758 FEBS Journal 276 (2009) 2752–2761 ª 2009 The Authors Journal compilation ª 2009 FEBS
streptomycin (both Gibco, Grand Island, NY, USA)] for
2 weeks for differentiation into macrophages. Macrophages
were treated or transfected after 2–4 weeks, and exposed for
4–48 h to various concentrations of ox-LDL (Intracel, Fred-
erick, MD, USA) in serum-free medium. Human natural
LDL was used as a negative control. The study protocol
was approved by the local ethics committee and conformed
to the principles outlined in the Declaration of Helsinki.
Quantitative real-time PCR
Total RNA was extracted from cells using an RNeasyÒ mini
kit (Qiagen, Hamburg, Germany). Single-stranded cDNA
was synthesized using hexamer primers and the Superscript
first-strand synthesis system (Invitrogen, Carlsbad, CA,
USA). Quantitative real-time PCR was performed using an
Applied Biosystems TaqMan 7900HT detection system
(Applied Biosystems, Hitchin, UK) with specific primers as
follows (gene symbols and Applied Biosystems primer set
numbers in parentheses): TRB1 (Hs00179769_m1); TRB2
(Hs00222224_m1); TRB3 (Hs00221754_m1). Reactions were
performed in a MicroAmp Optical 96-well reaction plate,
with each reaction mixture containing 1· Master Mix,
200 lm forward and reverse primers and 100 lm probe in a
total volume of 25 lL. PCR conditions were 50 °C for
2 min, 95 °C for 10 min, then 40 cycles of 95 °C for 15 min
followed by 60 °C for 1 min. The relative changes in gene
expression were analyzed by the 2()DDC
T
) method [30], and
normalized to the expression of GAPDH, as determined
using forward primer 5¢-GCCTTCCGTGTCCCCACT-3¢
and reverse primer 5¢-TGAGGGGGCCCTCCGACG-3¢.
cDNA cloning and construction of recombinant
adenoviral TRB3
Human TRB3 open reading frames (ORF) were amplified
by PCR using primers 5¢-GAAGTTATCA
GTCGACAT
GCGAGCCACCCCTCTGGCT-3¢ (forward) and 5¢-AT
GGTCTAG
AAAGCTTCCATACAGACCACTT-3¢ (reverse)
(restriction sites are underlined). The forward primer was
designed with a unique SalI site, and the reverse primer
with a unique HindIII site. PCR was performed using Pfu
Turbo DNA polymerase (Stratagene, La Jolla, CA, USA)
under the following conditions: initial denaturation at 95 °C
for 15 min, followed by 35 amplification cycles of denatur-
ation at 92 °C for 15 s, annealing at 55 °C for 30 s, and
extension at 72 °C for 1 min, with a final extension step at
72 °C for 10 min. A BD In-Fusion Dry-Down PCR cloning
kit (BD Biosciences, Franklin Lakes, NJ, USA) was used to
connect the TRB3 cDNA with a linearized pDNR-Dual
donor vector (BD Biosciences) at the unique Sal I and Hin-
dIII sites to construct the plasmid pDNR-Dual ⁄ TRB3,
which was transformed into Escherichia coli strain TOP10
(Invitrogen). After culturing of the transformed cells over-
night on LB-ampicillin medium (100 lg LB-agar mediumÆmL
ampicillin), the plasmid was amplified and isolated
from PCR-screened positive clones. DNA sequencing was
performed to verify the fidelity of the PCR amplification.
Adenoviral constructs were prepared using a BD Adeno-
X Expression System 2 (BD Biosciences) according to the
manufacturer’s protocol. Briefly, the TRB3 gene was trans-
ferred from the pDNR-Dual ⁄ TRB3 construct (donor vec-
tor) to pLP-Adeno-X viral DNA (acceptor vector). After
digestion with PacI, the recombinant adenoviral plasmid
was used to transfect HEK293 cells (Perkin Elmer,
Waltham, MA, USA). The adenovirus was isolated by the
freeze–thaw method, and purified by use of an Adeno-X
virus purification kit (BD Biosciences), and the virus titer
was determined using a BD Adeno-X Rapid Titer kit (BD
Biosciences).
Transfection of macrophages with recombinant
adenoviral TRB3 and siRNA
Macrophages were plated in six-well plates at 3 · 10
5
cell-
sÆmL
)1
, and incubated at 37 °C in a 5% CO
2
atmosphere.
Cells at 50–70% confluence were transfected with the puri-
fied recombinant adenoviral TRB3 construct (adeno-TRB3)
at multiplicities of infection of 50, 100 and 200 inclusion-
forming units using Lipofectamine 2000 reagent (Invitro-
gen). Cells were incubated for 24 h post-transfection before
treatment with ox-LDL or LDL.
Double-stranded RNA duplexes targeting human TRB3
(5¢-GGUGUACCCCGUCCAGGAA-3¢) and control
siRNA (purchased from Invitrogen) were transfected into
macrophages using Lipofectamine 2000 reagent.
Western blot analysis
Macrophages were lysed to prepare total cell extracts.
Nuclear and cytoplasmic extracts were prepared according to
the manufacturer’s instructions (Nuclear Extra Kit; Active
Motif, Carlsbad, CA, USA). Proteins were separated on
NuPAGE 4–12% Bis ⁄ Tris gels (Invitrogen), transferred to
nitrocellulose membranes, and incubated with antibody
against TRB3 (IMGENEX, San Diego, CA, USA), antibody
against caspase-3 or antibody against GAPDH (Abcam,
Cambridge, UK), then horseradish peroxidase-conjugated
secondary antibody (Abcam). Blots were developed using
Supersignal West Dura extended duration substrate (Perbio,
Tattenhall, UK). Images were captured using a Chemigenius
imaging system (Syngene, Cambridge, UK).
Detection of cell viability (MTT assay) and
apoptosis (ssDNA ELISA)
Macrophages cultured on 96-well plates were treated with
ox-LDL or LDL or transfected with adeno-TRB3 or
TRB3-targeting siRNA (siTRB3). At various time points,
Y y. Shang et al. TRB3 mediateshumanmacrophage apoptosis
FEBS Journal 276 (2009) 2752–2761 ª 2009 The Authors Journal compilation ª 2009 FEBS 2759
5mgÆmL
)1
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT) was added to each well to measure
cell viability [31]. After a 4 h incubation at 37 °C, 100 lL
of dimethylsulfoxide was added to the wells to dissolve
any precipitate. The absorbance was read at a wavelength
of 562 nm.
Detection of apoptotic macrophages was achieved using
an ApoStrandÔ ELISA apoptosis detection kit (BIOMOL
International, Plymouth Meeting, PA, USA), which mea-
sures ssDNA absorbance, according to the manufacturer’s
instructions.
Statistical analysis
All experiments were performed in triplicate and repeated
at least three times. Data are presented as means ± SD.
Comparisons among groups were performed using one-way
ANOVA. Interaction effects were tested by a general linear
model with a 2 · 2 factorial design. spss 16.0 (SPSS Inc.,
Chicago, IL, USA) was used for analysis. A P-value
< 0.05 was considered statistically significant.
Acknowledgements
This work was supported by the research grants from
the Key Technologies R & D Program of Shandong
Province (2006GG2202020), the National Natural Sci-
ence Foundation of China (30670874, 30570748 and
30871038) and the National Basic Research Program
of China (973 Program, grant number 2009CB521904).
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Y y. Shang et al. TRB3 mediateshumanmacrophage apoptosis
FEBS Journal 276 (2009) 2752–2761 ª 2009 The Authors Journal compilation ª 2009 FEBS 2761
. TRB3, upregulated by ox-LDL, mediates human
monocyte-derived macrophage apoptosis
Yuan-yuan Shang
1,
*, Zhi-hao. gene
in macrophage apoptosis induced by oxidized low-density lipoprotein
(ox-LDL). We found that, in human monocyte-derived macrophages,
TRB3 is upregulated by