TRB3, upregulated by ox-LDL, mediates human monocyte-derived macrophage apoptosis

Authors

  • Yuan-yuan Shang,

    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
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    • These authors contributed equally to this paper

  • Zhi-hao Wang,

    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
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    • These authors contributed equally to this paper

  • Li-ping Zhang,

    1.  Department of Anatomy, School of Medicine, Shandong University, Ji’nan, China
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  • Ming Zhong,

    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
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  • Yun Zhang,

    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
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  • Jing-ti Deng,

    1.  Department of Anatomy, School of Medicine, Shandong University, Ji’nan, China
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  • Wei Zhang

    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
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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

Abstract

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. Factorial analyses revealed no significant interaction between TRB3 overexpression 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. Therefore, 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

Acute coronary syndrome is the consequence of rupture or erosion of pre-existing atherosclerotic plaques, with subsequent formation of local thrombus, leading to critical occlusion of coronary arteries. The principal pathological basis of acute coronary syndrome is vulnerable plaques [1]. Macrophage apoptosis contributes significantly to the development of vulnerable atherosclerotic plaques [2–4]. A possible mechanism linking macrophage 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 formation of an atheromatous core within plaques. In addition, 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].

Signal transduction of apoptosis in macrophages involves a complex network system. Various risk factors, such as an increased level of oxidized low-density lipoprotein (ox-LDL), can induce macrophage apoptosis 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 mammalian 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 putative 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 vulnerable 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 differentiate 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 monocyte-derived macrophages. Macrophages predominantly expressed TRB3 (Fig. 1), and the level of TRB3 mRNA increased on day 3 and peaked on day 7.

Figure 1.

TRBs mRNA expression in macrophages.

TRB3 expression upregulated by ox-LDL

To examine whether TRB3 mRNA expression in macrophages was regulated by ox-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 concentration of 50 μg·mL−1 (4.72 ± 2.72 versus 1.04 ± 0.42, P < 0.01).

Figure 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 μg·mL−1 ox-LDL or LDL for various durations. (C) Western blot analysis of macrophages treated with 50 μg·mL−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.

Macrophages treated with 50 μg·mL−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 μg·mL−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 significantly increased after treatment with 50 μg·mL−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 significantly reduced in cells treated with ox-LDL but not those treated with LDL (Fig. 3A). Cell viability was significantly lower with 50 μg·mL−1 ox-LDL treatment than with 50 μg·mL−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.

Figure 3.

 Effect of ox-LDL and LDL on macrophage cell viability by MTT assay. (A) Macrophages were treated with various concentrations of ox-LDL or LDL for 24 h. (B) Macrophages were treated with 50 μg·mL−1 ox-LDL or LDL for various durations. *P < 0.05, **P < 0.01 versus LDL-treated cells.

Macrophages were treated with 50 μg·mL−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 μg·mL−1 ox-LDL treatment for 24 h as the optimal stimulus in subsequent experiments.

Macrophages treated with 50 μg·mL−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 successfully in mammalian cells using the adenoviral expression system only, because of the lack of effectiveness of other transfection techniques in this case. The level of TRB3 protein was increased with increasing multiplicities of infection with adenovirus (Fig. 4). Endogenous TRB3 protein could not be detected in nuclear extracts except when 100 μg 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 macrophages.

Figure 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.

As cell apoptosis increased with increased TRB3 expression, we investigated the role of TRB3 in ox-LDL-induced macrophage apoptosis by determination 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 macrophages treated with 50 μg·mL−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-overexpressing macrophages compared with vector controls (67.1 ± 18.2% versus 100 ± 0.2%, P < 0.01). Treatment of TRB3-overexpressing macrophages with ox-LDL markedly reduced cell viability further compared 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%, > 0.05).

Figure 5.

 Influence of TRB3 overexpression on macrophage apoptosis. 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.

Factorial analyses revealed a significantly lower cell viability in macrophages treated with ox-LDL compared 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-overexpressing 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 stimulation with ox-LDL did not show a significant interaction for cell viability (= 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.

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.
 GroupP-value
ControlTRB3ox-LDLTRB3/ox-LDLTRB3 effectox-LDL effectInteraction
Cell viability (%)100 ± 0.267.1 ± 18.259.6 ± 8.546.0 ± 12.8< 0.01< 0.010.206
Apoptosis0.38 ± 0.040.51 ± 0.120.57 ± 0.020.77 ± 0.15< 0.01< 0.010.520

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 macrophages 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 controls (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-overexpressing 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 apoptosis, siTRB3 was transfected into macrophages to silence TRB3 gene expression. The expression of TRB3 mRNA was significantly reduced after siTRB3 transfection (Fig. 6). MTT assay results showed that the cell 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 macrophages transfected with siTRB3. With ox-LDL treatment, cell viability of TRB3-silenced cells was significantly higher than for cells transfected with control siRNA (57.5 ± 5.3% versus 47.8 ± 1.8%, P < 0.05) (Fig. 7A).

Figure 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.

Figure 7.

 Influence of TRB3 silencing on macrophage apoptosis. Macrophages 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.

Factorial analyses revealed a significant interaction between silenced TRB3 and ox-LDL stimulation with respect to macrophage viability (= 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).

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.
 GroupP-value
ControlsiTRB3ox-LDLsiTRB3/ox-LDLsiTRB3 effectox-LDL effectInteraction
Cell viability (%)78.8 ± 2.6100 ± 1.747.8 ± 1.857.5 ± 5.3< 0.01< 0.010.048
Apoptosis0.38 ± 0.050.29 ± 0.010.75 ± 0.090.48 ± 0.02< 0.01< 0.010.001

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 macrophages 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 (= 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.

Figure 8.

 Western blot analysis of expression of activated caspase-3 protein. (A) Macrophages were treated with 50 μg·mL−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.

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 macrophages expressed TRB1, TRB2 and especially TRB3. In 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 atherosclerosis. 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 atherogenesis 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 apoptosis 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 macrophage infiltration of plaques [5], indicating that macrophage apoptosis promotes atherosclerosis progression.

TRBs, the regulation of which is cell type-specific [17], are expressed in many types of cells, such as vascular smooth muscle cells, human umbilical cord endothelial 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 previously 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 expression by ox-LDL in human primary macrophages. We found that both mRNA and protein expression of TRB3 was upregulated by ox-LDL in human macrophages 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 morphogenesis and migration in Drosophila by regulating String/CDC25 proteolysis and promoting the degradation of slbo [8,11]. As a feedback regulator of the activating transcription factor 4– C/EBP homologous protein (CHOP) pathway, TRB3 is involved in endoplasmic reticulum stress-induced apoptosis of HepG2 and COS-7 cells [19,23,24]. In addition, TRB3 expression 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 reduced cell viability, increased apoptosis and augmented activated caspase-3 expression, which suggests a role in increased macrophage apoptosis. Apoptosis was further increased in TRB3-overexpressing macrophages 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-overexpressing 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 macrophages with overexpressed TRB3 be incubated with ox-LDL; as overexpression of TRB3 promoted macrophage apoptosis, many TRB3-overexpressing macrophages may have died before incubation with ox-LDL, which would compromise the effect of ox-LDL. Furthermore, the MTT assay and ELISA results differed. ELISA showed higher apoptosis in TRB3-overexpressing 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 indicate higher apoptosis. Furthermore, the MTT assay measures the net rate of apoptosis and proliferation, and therefore TRB3 may influence macrophage proliferation rates just as TRB1 does [27].

Factorial analyses revealed that overexpression of TRB3 and stimulation of ox-LDL can induce macrophage 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 macrophages. Combined with the results above, this demonstrates that TRB3 alone promotes macrophage apoptosis. However, ox-LDL treatment increased the apoptosis of TRB3-silenced macrophages. Further analysis showed that TRB3 knockdown and stimulation with ox-LDL affect macrophage apoptosis, with significant interaction between the treatments. In addition, 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 stimulation with ox-LDL, the specific cellular signal transduction 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]. Therefore, TRB3 may mediates macrophage apoptosis via the MAPK pathway, which requires further study.

Macrophage apoptosis promotes vulnerable atherosclerotic 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 studies are necessary to elucidate the mechanism. Our preliminary 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 multiple 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 gradient centrifugation technique [29]. Cells were plated in 12- or 6-well plates at 3 × 105 cells·mL−1 and cultured in complete culture medium [RPMI-1640 containing 5% human serum (Sigma), 100 IU·mL−1 penicillin and 100 μg·mL−1 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, Frederick, 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 μm forward and reverse primers and 100 μm probe in a total volume of 25 μL. 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(−ΔΔCT) 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′-GAAGTTATCAGTCGACATGCGAGCCACCCCTCTGGCT-3′ (forward) and 5′-ATGGTCTAGAAAGCTTCCATACAGACCACTT-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 denaturation 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 SalI and HindIII sites to construct the plasmid pDNR-Dual/TRB3, which was transformed into Escherichia coli strain TOP10 (Invitrogen). After culturing of the transformed cells overnight on LB-ampicillin medium (100 μg LB-agar medium·mL 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 transferred from the pDNR-Dual/TRB3 construct (donor vector) 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 × 105 cells·mL−1, and incubated at 37 °C in a 5% CO2 atmosphere. Cells at 50–70% confluence were transfected with the purified recombinant adenoviral TRB3 construct (adeno-TRB3) at multiplicities of infection of 50, 100 and 200 inclusion-forming units using Lipofectamine 2000 reagent (Invitrogen). 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, 5 mg·mL−1 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well to measure cell viability [31]. After a 4 h incubation at 37 °C, 100 μL 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 measures 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 Science Foundation of China (30670874, 30570748 and 30871038) and the National Basic Research Program of China (973 Program, grant number 2009CB521904).

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