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Endoplasmic Reticulum Stress Cooperates in Zearalenone-Induced Cell Death of RAW 264.7 Macrophages.

Chen F, Li Q, Zhang Z, Lin P, Lei L, Wang A, Jin Y - Int J Mol Sci (2015)

Bottom Line: Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT) and flow cytometry assay.Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP), two ER stress-related marker genes.Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA) or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs), significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest Agriculture and Forestry University, Yangling 712100, Shaanxi, China. cfl0604114114@163.com.

ABSTRACT
Zearalenone (ZEA) is a fungal mycotoxin that causes cell apoptosis and necrosis. However, little is known about the molecular mechanisms of ZEA toxicity. The objective of this study was to explore the effects of ZEA on the proliferation and apoptosis of RAW 264.7 macrophages and to uncover the signaling pathway underlying the cytotoxicity of ZEA in RAW 264.7 macrophages. This study demonstrates that the endoplasmic reticulum (ER) stress pathway cooperated in ZEA-induced cell death of the RAW 264.7 macrophages. Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT) and flow cytometry assay. Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP), two ER stress-related marker genes. Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA) or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs), significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death. In summary, our results suggest that ZEA induces the apoptosis and necrosis of RAW 264.7 macrophages in a dose- and time-dependent manner via the ER stress pathway in which the activation of CHOP plays a critical role.

No MeSH data available.


Related in: MedlinePlus

ZEA induces the ER stress-related proteins GRP78 and CHOP in RAW 264.7 macrophages. (A,B) The expression of GRP78 and CHOP was analyzed by western blot. Cells were treated with different concentrations of ZEA (control, 10, 30 and 50 µM) for 12 h; (C,D) the expression of GRP78 and CHOP was analyzed by western blot. RAW264.7 macrophages were treated for different times (0, 6, 12 and 24 h) with 30 µM ZEA. Analyses of the band intensity on the films are presented as the relative ratio of GRP78 and CHOP to β-actin. Statistical analysis is shown in the bar graphs. Data are presented as the mean ± SEM of three independent experiments. Bars with different letters are significantly different (p < 0.05).
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ijms-16-19780-f002: ZEA induces the ER stress-related proteins GRP78 and CHOP in RAW 264.7 macrophages. (A,B) The expression of GRP78 and CHOP was analyzed by western blot. Cells were treated with different concentrations of ZEA (control, 10, 30 and 50 µM) for 12 h; (C,D) the expression of GRP78 and CHOP was analyzed by western blot. RAW264.7 macrophages were treated for different times (0, 6, 12 and 24 h) with 30 µM ZEA. Analyses of the band intensity on the films are presented as the relative ratio of GRP78 and CHOP to β-actin. Statistical analysis is shown in the bar graphs. Data are presented as the mean ± SEM of three independent experiments. Bars with different letters are significantly different (p < 0.05).

Mentions: The effect of ZEA on the ER stress-related gene (GRP78 and CHOP) expression was studied via western blot analysis. ZEA treatment significantly induced the expression of GRP78 compared with the control, although treatment with 10, 30 and 50 µM ZEA and 30 µM ZEA for 6, 12 and 24 h did not produce significantly different results (Figure 2). CHOP protein was weakly detected after treatment with 10 µM ZEA for 12 h and 30 µM for 6 h, while treatment with 30 µM ZEA for 12 h dramatically induced the expression of CHOP, while treatment with 50 µM ZEA for 12 h and 30 µM for 24 h decreased the level of CHOP (Figure 2).


Endoplasmic Reticulum Stress Cooperates in Zearalenone-Induced Cell Death of RAW 264.7 Macrophages.

Chen F, Li Q, Zhang Z, Lin P, Lei L, Wang A, Jin Y - Int J Mol Sci (2015)

ZEA induces the ER stress-related proteins GRP78 and CHOP in RAW 264.7 macrophages. (A,B) The expression of GRP78 and CHOP was analyzed by western blot. Cells were treated with different concentrations of ZEA (control, 10, 30 and 50 µM) for 12 h; (C,D) the expression of GRP78 and CHOP was analyzed by western blot. RAW264.7 macrophages were treated for different times (0, 6, 12 and 24 h) with 30 µM ZEA. Analyses of the band intensity on the films are presented as the relative ratio of GRP78 and CHOP to β-actin. Statistical analysis is shown in the bar graphs. Data are presented as the mean ± SEM of three independent experiments. Bars with different letters are significantly different (p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4581325&req=5

ijms-16-19780-f002: ZEA induces the ER stress-related proteins GRP78 and CHOP in RAW 264.7 macrophages. (A,B) The expression of GRP78 and CHOP was analyzed by western blot. Cells were treated with different concentrations of ZEA (control, 10, 30 and 50 µM) for 12 h; (C,D) the expression of GRP78 and CHOP was analyzed by western blot. RAW264.7 macrophages were treated for different times (0, 6, 12 and 24 h) with 30 µM ZEA. Analyses of the band intensity on the films are presented as the relative ratio of GRP78 and CHOP to β-actin. Statistical analysis is shown in the bar graphs. Data are presented as the mean ± SEM of three independent experiments. Bars with different letters are significantly different (p < 0.05).
Mentions: The effect of ZEA on the ER stress-related gene (GRP78 and CHOP) expression was studied via western blot analysis. ZEA treatment significantly induced the expression of GRP78 compared with the control, although treatment with 10, 30 and 50 µM ZEA and 30 µM ZEA for 6, 12 and 24 h did not produce significantly different results (Figure 2). CHOP protein was weakly detected after treatment with 10 µM ZEA for 12 h and 30 µM for 6 h, while treatment with 30 µM ZEA for 12 h dramatically induced the expression of CHOP, while treatment with 50 µM ZEA for 12 h and 30 µM for 24 h decreased the level of CHOP (Figure 2).

Bottom Line: Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT) and flow cytometry assay.Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP), two ER stress-related marker genes.Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA) or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs), significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest Agriculture and Forestry University, Yangling 712100, Shaanxi, China. cfl0604114114@163.com.

ABSTRACT
Zearalenone (ZEA) is a fungal mycotoxin that causes cell apoptosis and necrosis. However, little is known about the molecular mechanisms of ZEA toxicity. The objective of this study was to explore the effects of ZEA on the proliferation and apoptosis of RAW 264.7 macrophages and to uncover the signaling pathway underlying the cytotoxicity of ZEA in RAW 264.7 macrophages. This study demonstrates that the endoplasmic reticulum (ER) stress pathway cooperated in ZEA-induced cell death of the RAW 264.7 macrophages. Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT) and flow cytometry assay. Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP), two ER stress-related marker genes. Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA) or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs), significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death. In summary, our results suggest that ZEA induces the apoptosis and necrosis of RAW 264.7 macrophages in a dose- and time-dependent manner via the ER stress pathway in which the activation of CHOP plays a critical role.

No MeSH data available.


Related in: MedlinePlus