| BackgroundAtherosclerosis (AS) and its complications caused by acute cardiovascular disease event have become the number one killer of mankind, its prevention and treatment have been a worldwide public health priorities and challenges. Coronary artery atherosclerosis is the principal cause of acute coronary syndrome (ACS) and is the single largest killer of both men and women. ACS is the consequence of rupture or erosion of preexisting atherosclerotic plaque, with subsequent formation of local thrombus leading to critical occlusion of coronary arteries. The principal pathological basis of ACS is vulnerable plaques.Macrophage apoptosis contributes much to the development of vulnerable atherosclerotic plaques. A possible mechanism linking macrophage apoptosis to vulnerable plaque progression is the reduced level of macrophages failing to clear the apoptotic smooth muscle cells and macrophages, which leads to secondary necrosis of these cells and facilitates the formation of an atheromatous core within plaques. As well, apoptotic macrophages can release cholesterol, which results in accumulation of acicular cholesterol crystal in the lipid core, thus injuring the fibrous cap of plaques. In addition, apoptotic macrophages can be a source of tissue factor, a procoagulant molecule considered to play an important role in coagulation and thrombosis associated with advanced plaques.Signal transduction of apoptosis in macrophages is a complex network system. Various risk factors such as increased level of oxidized low-density lipoprotein (ox-LDL) can induce macrophage apoptosis through multiple apoptotic signaling pathways, such as Akt, NF-kB and MAPKs. However, the mechanism of macrophage apoptosis remains to be elucidated.Tribbles (TRBs), a Drosophila protein, slows progression through the G2 stage of the cell cycle. 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. TRB3, also named neuronal cell death-inducible putative protein kinase (NIPK), is expressed in liver, thymus, prostate, pancreas and heart and may have wide biological activity. Recently, TRB3 has been shown to be a very important regulatory protein involved in signal pathways and works at least through CDC25/String, Akt and MAPKs. The activation of MAPKs and inhibition of Akt kinase activity result in macrophage apoptosis, which is implied in the development of vulnerable atherosclerotic plaques. TRB3 could be involved in macrophage apoptosis induced by ox-LDL and could play an essential role in the progression of vulnerable plaques.Therefore, we hypothesized that TRB3 may be implicated in ox-LDL-induced apoptosis by stimulating human monocyte-derived macrophages with ox-LDL, through MAPKs, Akt and NF-kB signaling pathways.Objective1. To investigate the expression of TRBs in human monocyte-derived macrophages.2. To observe the effect of ox-LDL on TRB3 in macrophages.3. To explore whether TRB3 affects macrophage apoptosis induced by ox-LDL and to study the possible signal transduction mechanism.MethodsWe took primary human monocyte-derived macrophages as the object of this research, then quantitative real time PCR, western blot, ELISA, etc. were used to observe:1. Monocytes were first allowed to differentiate into macrophages naturally. Quantitative real-time PCR performed on days 1,3,5,7,9 and 11 detected the mRNA expression of TRB1, TRB2, and TRB3 in monocyte-derived macrophages.2. Macrophages were treated with various concentrations of ox-LDL and LDL (0,5,10,25 and 50μg/ml) or various times (0,4,8,12,24, and 48h), then quantitative real time PCR and western blot analysis were used to detect TRB3 mRNA and protein level.3. Cloning of human TRB3 and constructing of TRB3 adenovirus vectorBased on human TRB3 sequence in Genebank, we designed one pair of primers. Using total DNA extracted from human whole blood and reverse transcript polymerase chain reaction, we cloned the CDS sequence of human TRB3. BD Adeno-XTM Expression System 2 was used to construct adenovirus containing human TRB3.293 cells were infected with the TRB3 plasmid. Recombinant adenovirus from a single plaque was expanded and purified, viral titer was determined by plaque assay.4. Design, synthesis and screening of TRB3 targeting small interfering RNA (siRNA).5. Macrophages were transfected with recombinant adeno viral TRB3 or TRB3 siRNA, and cell viability was examined by MTT assay, apoptosis by ELISA measurement of ssDNA absorbance, and caspase-3 level by western blot.6. After overexpressing or silencing TRB3 expression, the change of Akt, MAPKs and NF-kB in macrophages were measured.Results1. TRBs mRNA expression in human macrophagesAll three genes were expressed in monocyte-derived macrophages. Macrophages predominantly expressed TRB3; the TRB3 mRNA level was significantly increased on day 3 and peaked on day 7.2. TRB3 expression upregulated by ox-LDLOur data showed compared with LDL treatment, TRB3 mRNA and protein level in macrophages increased with increasing ox-LDL concentration and treatment time.3. Cloning of human TRB3 and constructing TRB3 adenovirusWe have successfully cloned human TRB3, and its sequence was identical to the human TRB3 sequence in Genebank. Using BD Adeno-XTM Expression System 2, we constructed human TRB3 adenovirus.4. TRB3 mediates macrophage apoptosis induced by ox-LDLCompared with control cells, cell viability of TRB3-overexpressing macrophages was decreased (P<0.01), but apoptosis was increased (P<0.05) and caspase-3 level increased. With ox-LDL treatment, TRB3-overexpressing macrophages showed increased apoptosis, as deduced by decreased cell viability and increased apoptosis (P<0.01) and caspase-3 level compared with controls. Factorial analyses revealed no significant interaction between overexpression of TRB3 and stimulation with ox-LDL on macrophage apoptosis.TRB3-silenced macrophages showed decreased apoptosis, as inferred by increased cell viability (P<0.01), and decreased apoptosis (P<0.05) and caspase-3 level compared with controls. TRB3-silenced cells treated with ox-LDL showed significantly reduced cell viability (P<0.01), accompanied by increased apoptosis (P<0.01) and caspase-3 level compared with controls. Silenced TRB3 and ox-LDL stimulation showed significant interaction for macrophage apoptosis (P=0.048, P=0.001), which suggests that TRB3 knockdown in part resisted macrophage apoptosis induced by ox-LDL.5. Effect of TRB3 on Akt in macrophagesCompared with control cells, the level of p-Akt of TRB3-overexpressing macrophages was decreased. With ox-LDL treatment, TRB3-overexpressing macrophages showed decreased p-Akt level compared with controls.TRB3-silenced macrophages showed increased p-Akt level compared with controls. TRB3-silenced cells treated with ox-LDL showed significantly increased p-Akt level compared with controls.6. Effect of TRB3 on MAPKs in macrophagesThe activity of c-Fos and c-Jun in TRB3-overexpressing macrophages with ox-LDL treatment were lower than control cells(P<0.05, P<0.05). TRB3-silenced cells treated with ox-LDL showed significantly increased AP-1 activity. c-Fos activity increased 1.3 folds and c-Jun activity increased 1.3 folds compared to control cells.TRB3-silenced macrophages showed increased p-p38 level compared with controls. TRB3-silenced cells treated with ox-LDL showed significantly increased p-p38 level compared with controls.7. Effect of TRB3 on NF-kB in macrophagesox-LDL decreased the activity of NF-kB in macrophages in a time-dependent manner compared to LDL treatment. After treatment with ox-LDL for 24h, the activity of p50 and p65 was significantly lower than that following treatment with LDL for 24h and was further decreased after 48h ox-LDL treatmet. However, overexpression of TRB3 has no effect on the activity of p50 and p65 reduced by ox-LDL.Conclusions1. Human monocyte-derived macrophages expressed TRB1, TRB2 and especially TRB3.2. TRB3 expression was upregulated in macrophages in a dose- and time-dependent manner on stimulation with ox-LDL.3. TRB3 in part mediates macrophage apoptosis induced by ox-LDL through Akt and MAPKs signaling pathway. BackgroundThe endoplasmic reticulum (ER) near the nucleus region in the cytoplasm is a central organelle of eukaryotic cells as the place of calcium storage, lipid synthesis, protein folding and protein maturation. A variety of factors such as hypoxia, hyperglycemia, chemical poisons and so on leading to depletion of endoplasmic reticulum Ca2+, inhibition of endoplasmic reticulum protein glycosylation, mismatched disulfide, accumulation of unfolded protein (unfolded protein response, UPR), excessive protein traffics and so on, can make the endoplasmic reticulum function change, collectively referred to endoplasmic reticulum stress (ER stress).Recently, ER stress has been shown to be involved in several diseases with ER stress-dependent cell death, such as diabetes and Alzheimer disease, especially ER stress plays an essential role in the progression of atherosclerosis.CCAAT/enhancer binding proteins homologus protein (CHOP, also known as growth arrest and DNA damage-inducible protein 153), consisting of a transcriptional activation domain and a leucine zipper for DNA binging, is a member of the C/EBP family transcription factor family that can regulate the expression of a large number of target genes during cell stress response. The expression level of CHOP is low under normal conditions but induced by ER stress. CHOP is considered to be a main mediating factor in ER stress and acts as an inducer of cell cycle arrest and apoptosis. CHOP-mediated apoptosis during ER stress has been shown to be involved in several diseases with ER stress-dependent cell death. High expression of CHOP was observed at all stages of atherosclerotic lesion development in apolipoprotein E-deficicent mice, and both in early lesion and late advanced lesion CHOP expression increaseed ER stress promoted atherosclerosis progression via acculation of free cholesterol. However, whether lipoprotein ox-LDL mediates atherogenesis by CHOP-dependent ER stress pathway remains unclear.Tribble3 (TRB3), a mammalian homolog of Drosophila Tribbles, is a recently recognized atypical inactibe kinase that inhibits mitosis and be implicated in cell cycle regulation and aopotosis. TRB3 is reported to participate in the signal pathway of ER stress, and induced by various forms ER stress, such as tunicamycin, cannabinoids and thapsigargin. Recent studies show that TRB3 acts as a target gene of CHOP during ER stress. CHOP with a novel dimerizing partner ATF4 activates TRB3 promoter[19]. We previously found that TRB3, upregulated by ox-LDL, mediates human monocyte-derived macrophage apoptosis.Taken together, we hypothesized that CHOP regulates ox-LDL-induced TRB3 expression and is involved in macrophages apoptosis through TRB3 pathway andObjective1. To investigate the mRNA expression of CHOP and ATF4 in human monocyte-derived macrophages.2. To observe the effect of ER stress on TRB3 expression in macrophages.3. To explore the interaction between TRB3 and CHOP.Methods1. Human monocyte-derived macrophages were treated with various concentrations of ox-LDL and LDL (0,2.5,5,10,25 and 50μg/ml))for various times (0,4,8,16,24, and 48h), then quantitative real time PCR and western blot analysis was used to detect CHOP and ATF4 mRNA and protein level.2. Macrophages treated with 2μg/ml tunicamycin for 0,2,4,6,8,24h, the mRNA level of TRB3 was measured by quantitative real time PCR.3. After overexpression CHOP by CHOP expression plasmid, macrophages were incubated with ox-LDL. The mRNA expression of TRB3 was measured.4. Macrophages were transfected with adenoviral TRB3 or TRB3 siRNA, and the mRNA expression of CHOP was dectected.Results1. CHOP and ATF4 mRNA expression upregulated by ox-LDLOur data showed that the expression of CHOP mRNA was significantly upregulated by ox-LDL but not LDL. This expression increased with increasing ox-LDL concentration. Furthermore, the expression of CHOP mRNA was significantly upregulated by ox-LDL at the concentration of 25 or 50μg/ml as compared to LDL treatment.Macrophages treated with 50μg/ml ox-LDL or LDL for various times showed increased expression of CHOP mRNA with increasing time. After treatment with 50μg/ml ox-LDL for 24 hr, the expression of CHOP mRNA was significantly higher than that with LDL treatment (P<0.05) and was further increased with 48-hr ox-LDL treatment (P<0.01).Also,50μg/ml ox-LDL increased the expression of ATF4 mRNA in a time-dependent manner. ATF4 mRNA expression peaked at 24 hr, but the peak level was less than CHOP (1.6 folds vs 3 folds). Ox-LDL induced TRB3 expression in a time-dependent manner, too. The induction of TRB3 mRNA was later than that of CHOP and ATF4 mRNA.2. ER stress induces mRNA expression of TRB3, CHOP and ATF4Macrophages treated with 2μg/ml tunicamycin for various times showed augmented TRB3 mRNA expression in a time-dependent manner. Compared with control cells, the significantly increase occurred at 6 hr and the peak level at 24 hr.3. CHOP overexpression promotes TRB3 expressionMacrophages were transfected with CHOP expression plasmid and incubated for an additional 24 hr with or without ox-LDL. TRB3 mRNA expression of CHOP-overexpressing macrophages was also found significantly increased as compared with controls. Moreover, treatment of CHOP-overexpressing macrophages with ox-LDL markedly increased TRB3 expression as compared with controls. 4. Effect of TRB3 on CHOP expressionTo better characterize the interaction between CHOP and TRB3, adenoviral TRB3 were transfected into macrophages to overexpress TRB3. CHOP mRNA expression of TRB3-overexpressing macrophages was significantly lower than that of control cells.The TRB3-targeting siRNA (siTRB3) was transfected into macrophages to silence TRB3 gene expression. CHOP mRNA expression of TRB3-silenced macrophages was higher than that of control cells but not significantly, whereas with ox-LDL treatment, CHOP mRNA expression of treated cells was significantly higher.Conclusion1. CHOP and ATF4 mRNA expression was upregulated in human monocyte-derived macrophages in a dose- and time-dependent manner on stimulation with ox-LDL.2. ox-LDL can induce ER stress in human macrophage.3. TRB3 mRNA expression was increased during ER stress.4. CHOP promoted TRB3 expression and TRB3 exerted a feedback inhibition on the expression of CHOP in macrophages, so ATF4/CHOP-TRB3 may be involved in ox-LDL-dependent macrophage apoptosis. |
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