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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 CHOP on the ZEA-induced cell death of RAW 264.7 macrophages. RAW 264.7 macrophages were transduced with CHOP or non-targeting lentiviral-mediated shRNAs. (A) The constructs themselves could express GFP, and the proportion of cells that were transduced was 90% by flow cytometry (data not shown). Fluorescence images of RAW 264.7 macrophages transducted for 48 h with negative control shRNA (shNC) (a), shCHOP (b) and control (c) virus-containing supernatant; GFP expression was observed under light (top panels) or fluorescence microscopy (bottom panels). Bars = 50 µm; (B,C) The expression levels of CHOP were determined by western blot analysis. RAW 264.7 macrophages were transduced with lentiviruses expressing either shCHOP or shNC, and were then treated with 30 μM ZEA for 24 h. The analyses of the band intensity on the films are presented as the relative ratio of CHOP to β-actin. Statistical analysis is shown in the bar graphs; (D) Transduced RAW 264.7 macrophages with lentiviruses expressing either shCHOP or shNC were treated with 0, 30 and 50 μM ZEA and then cell viability was measured after 24 h by the MTT assay; (E,F) RAW 264.7 macrophages were tranduced with shNC or shCHOP to confirm the effects of shCHOP. To determine the apoptotic effect of ZEA on the RAW 264.7 macrophages, cells were transduced with shNC or shCHOP for 48 h and then incubated for 24 h in the absence or presence of 30 μM ZEA. Apoptosis was determined by Annexin V-PE/7-AAD staining. B1 is the part of cell death caused by mechanical damage, B2 is the part of late apoptotic or necrotic cells, B3 is the part of the normal cells, and B4 is the part of early apoptotic cells. 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-f005: Effect of CHOP on the ZEA-induced cell death of RAW 264.7 macrophages. RAW 264.7 macrophages were transduced with CHOP or non-targeting lentiviral-mediated shRNAs. (A) The constructs themselves could express GFP, and the proportion of cells that were transduced was 90% by flow cytometry (data not shown). Fluorescence images of RAW 264.7 macrophages transducted for 48 h with negative control shRNA (shNC) (a), shCHOP (b) and control (c) virus-containing supernatant; GFP expression was observed under light (top panels) or fluorescence microscopy (bottom panels). Bars = 50 µm; (B,C) The expression levels of CHOP were determined by western blot analysis. RAW 264.7 macrophages were transduced with lentiviruses expressing either shCHOP or shNC, and were then treated with 30 μM ZEA for 24 h. The analyses of the band intensity on the films are presented as the relative ratio of CHOP to β-actin. Statistical analysis is shown in the bar graphs; (D) Transduced RAW 264.7 macrophages with lentiviruses expressing either shCHOP or shNC were treated with 0, 30 and 50 μM ZEA and then cell viability was measured after 24 h by the MTT assay; (E,F) RAW 264.7 macrophages were tranduced with shNC or shCHOP to confirm the effects of shCHOP. To determine the apoptotic effect of ZEA on the RAW 264.7 macrophages, cells were transduced with shNC or shCHOP for 48 h and then incubated for 24 h in the absence or presence of 30 μM ZEA. Apoptosis was determined by Annexin V-PE/7-AAD staining. B1 is the part of cell death caused by mechanical damage, B2 is the part of late apoptotic or necrotic cells, B3 is the part of the normal cells, and B4 is the part of early apoptotic cells. 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 further examine the role of ER stress in ZEA-induced cellular apoptosis, we used lentiviral-transduced shRNA to down-regulate CHOP expression. With this strategy, we confirmed that the ER stress pathway is required for ZEA-induced apoptosis in RAW 264.7 macrophages (Figure 5A–C). The MTT assay showed that CHOP knockdown effectively increased the cell viability compared with the control after treatment with 30 µM ZEA for 24 h (Figure 5D, p < 0.05). Treatment with 30 µM ZEA for 24 h after transducing the shCHOP lentivirus significantly suppressed ZEA-induced cell apoptosis compared with the control (Figure 5E,F, 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 CHOP on the ZEA-induced cell death of RAW 264.7 macrophages. RAW 264.7 macrophages were transduced with CHOP or non-targeting lentiviral-mediated shRNAs. (A) The constructs themselves could express GFP, and the proportion of cells that were transduced was 90% by flow cytometry (data not shown). Fluorescence images of RAW 264.7 macrophages transducted for 48 h with negative control shRNA (shNC) (a), shCHOP (b) and control (c) virus-containing supernatant; GFP expression was observed under light (top panels) or fluorescence microscopy (bottom panels). Bars = 50 µm; (B,C) The expression levels of CHOP were determined by western blot analysis. RAW 264.7 macrophages were transduced with lentiviruses expressing either shCHOP or shNC, and were then treated with 30 μM ZEA for 24 h. The analyses of the band intensity on the films are presented as the relative ratio of CHOP to β-actin. Statistical analysis is shown in the bar graphs; (D) Transduced RAW 264.7 macrophages with lentiviruses expressing either shCHOP or shNC were treated with 0, 30 and 50 μM ZEA and then cell viability was measured after 24 h by the MTT assay; (E,F) RAW 264.7 macrophages were tranduced with shNC or shCHOP to confirm the effects of shCHOP. To determine the apoptotic effect of ZEA on the RAW 264.7 macrophages, cells were transduced with shNC or shCHOP for 48 h and then incubated for 24 h in the absence or presence of 30 μM ZEA. Apoptosis was determined by Annexin V-PE/7-AAD staining. B1 is the part of cell death caused by mechanical damage, B2 is the part of late apoptotic or necrotic cells, B3 is the part of the normal cells, and B4 is the part of early apoptotic cells. 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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Related In: Results  -  Collection

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ijms-16-19780-f005: Effect of CHOP on the ZEA-induced cell death of RAW 264.7 macrophages. RAW 264.7 macrophages were transduced with CHOP or non-targeting lentiviral-mediated shRNAs. (A) The constructs themselves could express GFP, and the proportion of cells that were transduced was 90% by flow cytometry (data not shown). Fluorescence images of RAW 264.7 macrophages transducted for 48 h with negative control shRNA (shNC) (a), shCHOP (b) and control (c) virus-containing supernatant; GFP expression was observed under light (top panels) or fluorescence microscopy (bottom panels). Bars = 50 µm; (B,C) The expression levels of CHOP were determined by western blot analysis. RAW 264.7 macrophages were transduced with lentiviruses expressing either shCHOP or shNC, and were then treated with 30 μM ZEA for 24 h. The analyses of the band intensity on the films are presented as the relative ratio of CHOP to β-actin. Statistical analysis is shown in the bar graphs; (D) Transduced RAW 264.7 macrophages with lentiviruses expressing either shCHOP or shNC were treated with 0, 30 and 50 μM ZEA and then cell viability was measured after 24 h by the MTT assay; (E,F) RAW 264.7 macrophages were tranduced with shNC or shCHOP to confirm the effects of shCHOP. To determine the apoptotic effect of ZEA on the RAW 264.7 macrophages, cells were transduced with shNC or shCHOP for 48 h and then incubated for 24 h in the absence or presence of 30 μM ZEA. Apoptosis was determined by Annexin V-PE/7-AAD staining. B1 is the part of cell death caused by mechanical damage, B2 is the part of late apoptotic or necrotic cells, B3 is the part of the normal cells, and B4 is the part of early apoptotic cells. 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 further examine the role of ER stress in ZEA-induced cellular apoptosis, we used lentiviral-transduced shRNA to down-regulate CHOP expression. With this strategy, we confirmed that the ER stress pathway is required for ZEA-induced apoptosis in RAW 264.7 macrophages (Figure 5A–C). The MTT assay showed that CHOP knockdown effectively increased the cell viability compared with the control after treatment with 30 µM ZEA for 24 h (Figure 5D, p < 0.05). Treatment with 30 µM ZEA for 24 h after transducing the shCHOP lentivirus significantly suppressed ZEA-induced cell apoptosis compared with the control (Figure 5E,F, 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