The aim of this study was to investigate the role of the TRB3 gene in macrophage apoptosis induced by oxidized low-density lipoprotein ox-LDL.. Furthermore, TRB3-silenced macrophages sho
Trang 1monocyte-derived macrophage apoptosis
Yuan-yuan Shang1,*, Zhi-hao Wang1,*, Li-ping Zhang2, Ming Zhong1, Yun Zhang1, Jing-ti Deng2 and Wei Zhang1
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] Macrophage apoptosis 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 macrophage apoptosis induced by oxidized low-density lipoprotein (ox-LDL) We found that, in human monocyte-derived macrophages, TRB3 is upregulated by 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 macrophage apoptosis induced by ox-LDL There-fore, TRB3 in part mediates the macrophage apoptosis 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.
Trang 2Signal 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 macrophage apoptosis remains to be elucidated
Tribbles, a Drosophila protein, slows progression
through the G2 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 macrophage apoptosis 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 apoptosis by 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 upregulated by ox-LDL
To examine whether TRB3 mRNA expression in
mac-rophages was regulated by ox-LDL, macmac-rophages 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
8 10
2 4 6
0
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.
Trang 3Macrophages 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)1ox-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 mediates macrophage apoptosis 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 macrophage apoptosis by 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–110 µg·mL–125 µg·mL–150 µg·mL–1
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 kDa
37 kDa
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.
Trang 4Treatment 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 by ox-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
TRB3
Vector TRB3
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.
Trang 5viability 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 macrophage apoptosis induced by ox-LDL Therefore, TRB3 was confirmed to mediate
in part the macrophage apoptosis 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 Human monocyte-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
**
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
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
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.
Trang 6In addition, TRB3 mRNA expression was upregulated
in macrophages in a dose- and time-dependent manner
upon stimulation with ox-LDL Moreover, TRB3
promoted macrophage apoptosis 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 macrophage apoptosis [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 macrophage apoptosis increased the size and
number of aortic atheromatous plaques and
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 upregulated by 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 G2 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.
Activated caspase-3
GAPDH
TRB3
ox-LDL siTRB3
19 kDa
17 kDa
Fig 8 Western blot analysis of expression of activated caspase-3 protein (A) Macrophages were treated with 50 lgÆmL)1ox-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.
Trang 7reduced 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
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 by ox-LDL, which further confirms that TRB3 mediates ox-LDL-induced macrophage apoptosis through caspase-3 Although TRB3 bridges the gap between macrophage apoptosis 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 mediates macrophage apoptosis via the MAPK pathway, which requires further study Macrophage apoptosis promotes vulnerable athero-sclerotic plaque progression As we found that TRB3
is implicated in macrophage apoptosis 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 macrophage apoptosis 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 upregulated by ox-LDL in a dose- and time-dependent manner, exceeds that of other TRBs in naturally differentiated human macrophages TRB3 in part mediates macrophage apoptosis 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 cgradi-entrifugation technique [29] Cells were plated in 12- or 6-well plates at 3· 105
cellsÆmL)1and cultured in complete culture medium [RPMI-1640 containing 5% human serum (Sigma), 100 IUÆmL)1 penicillin and 100 lgÆmL)1
Trang 8streptomycin (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 RNeasy 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 50C for
2 min, 95C for 10 min, then 40 cycles of 95 C for 15 min
followed by 60C for 1 min The relative changes in gene
expression were analyzed by the 2()DDCT) 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¢-GAAGTTATCAGTCGACAT
GCGAGCCACCCCTCTGGCT-3¢ (forward) and 5¢-AT
GGTCTAGAAAGCTTCCATACAGACCACTT-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 95C
for 15 min, followed by 35 amplification cycles of
denatur-ation at 92C for 15 s, annealing at 55 C for 30 s, and
extension at 72C for 1 min, with a final extension step at
72C 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 SalI 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 vecvec-tor) 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· 105 cell-sÆmL)1, and incubated at 37C in a 5% CO2 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,
Trang 95 mgÆmL)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 37C, 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 ApoStrand 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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