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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

Effect of 4-PBA on the levels of GRP78 and CHOP in ZEA-treated RAW 264.7 macrophages. RAW 264.7 macrophages were treated 24 h with 30 µM ZEA or 30 µM ZEA with 700 nM 4-PBA. (A,B) Confocal immunofluorescence photomicrography showed the expression of CHOP protein (green fluorescence) and GRP78 protein (red fluorescence) in the control (the upper panels), ZEA (the center panels) or ZEA + 4-PBA cells (the bottom panels). Bars = 20 µm; (C,D) Western blot analysis of GRP78 and CHOP in ZEA-treated RAW 264.7 macrophages. The analyses of the band intensities on 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-f004: Effect of 4-PBA on the levels of GRP78 and CHOP in ZEA-treated RAW 264.7 macrophages. RAW 264.7 macrophages were treated 24 h with 30 µM ZEA or 30 µM ZEA with 700 nM 4-PBA. (A,B) Confocal immunofluorescence photomicrography showed the expression of CHOP protein (green fluorescence) and GRP78 protein (red fluorescence) in the control (the upper panels), ZEA (the center panels) or ZEA + 4-PBA cells (the bottom panels). Bars = 20 µm; (C,D) Western blot analysis of GRP78 and CHOP in ZEA-treated RAW 264.7 macrophages. The analyses of the band intensities on 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: To confirm the role of ER stress in ZEA-induced apoptosis, RAW 264.7 macrophages were treated with ZEA in the presence or absence of the ER stress inhibitor 4-PBA. Similar results were observed using the MTT assay and flow cytometry. Treatment with 700 nM 4-PBA significantly promoted cell viability and prevented cell apoptosis caused by 30 µM ZEA at 24 h, respectively (Figure 3A–C, p < 0.05). Meanwhile, 4-PBA markedly reduced the immunofluorescence staining of GRP78 and CHOP produced by 30 µM ZEA in RAW 264.7 macrophages (Figure 4A,B). In addition, Western blot analyses also revealed that the protein levels of GRP78 and CHOP in the ZEA-induced RAW 264.7 macrophages were significantly decreased after treatment of 4-PBA (Figure 4C,D, p < 0.05).


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)

Effect of 4-PBA on the levels of GRP78 and CHOP in ZEA-treated RAW 264.7 macrophages. RAW 264.7 macrophages were treated 24 h with 30 µM ZEA or 30 µM ZEA with 700 nM 4-PBA. (A,B) Confocal immunofluorescence photomicrography showed the expression of CHOP protein (green fluorescence) and GRP78 protein (red fluorescence) in the control (the upper panels), ZEA (the center panels) or ZEA + 4-PBA cells (the bottom panels). Bars = 20 µm; (C,D) Western blot analysis of GRP78 and CHOP in ZEA-treated RAW 264.7 macrophages. The analyses of the band intensities on 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-f004: Effect of 4-PBA on the levels of GRP78 and CHOP in ZEA-treated RAW 264.7 macrophages. RAW 264.7 macrophages were treated 24 h with 30 µM ZEA or 30 µM ZEA with 700 nM 4-PBA. (A,B) Confocal immunofluorescence photomicrography showed the expression of CHOP protein (green fluorescence) and GRP78 protein (red fluorescence) in the control (the upper panels), ZEA (the center panels) or ZEA + 4-PBA cells (the bottom panels). Bars = 20 µm; (C,D) Western blot analysis of GRP78 and CHOP in ZEA-treated RAW 264.7 macrophages. The analyses of the band intensities on 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: To confirm the role of ER stress in ZEA-induced apoptosis, RAW 264.7 macrophages were treated with ZEA in the presence or absence of the ER stress inhibitor 4-PBA. Similar results were observed using the MTT assay and flow cytometry. Treatment with 700 nM 4-PBA significantly promoted cell viability and prevented cell apoptosis caused by 30 µM ZEA at 24 h, respectively (Figure 3A–C, p < 0.05). Meanwhile, 4-PBA markedly reduced the immunofluorescence staining of GRP78 and CHOP produced by 30 µM ZEA in RAW 264.7 macrophages (Figure 4A,B). In addition, Western blot analyses also revealed that the protein levels of GRP78 and CHOP in the ZEA-induced RAW 264.7 macrophages were significantly decreased after treatment of 4-PBA (Figure 4C,D, p < 0.05).

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