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Paraquat Induces Apoptosis through Cytochrome C Release and ERK Activation.

Seo HJ, Choi SJ, Lee JH - Biomol Ther (Seoul) (2014)

Bottom Line: Paraquat treatment resulted in activation of ERK, and U0126, inhibitors of the MEK/ERK signaling pathway, prevented apoptosis.Moreover, paraquat-induced apoptosis was associated with cytochrome C release, which could be prevented by treatment with the MEK inhibitors.Taken together, our findings suggest that ERK activation plays an active role in mediating paraquat-induced apoptosis of NIH3T3 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Thoracic and Cardiovascular Surgery, Chosun University School of Medicine, Gwangju 501-759, Republic of Korea.

ABSTRACT
Paraquat has been suggested to induce apoptosis by generation of reactive oxygen species (ROS). However, little is known about the mechanism of paraquat-induced apoptosis. Here, we demonstrate that extracellular signal-regulated protein kinase (ERK) is required for paraquat-induced apoptosis in NIH3T3 cells. Paraquat treatment resulted in activation of ERK, and U0126, inhibitors of the MEK/ERK signaling pathway, prevented apoptosis. Moreover, paraquat-induced apoptosis was associated with cytochrome C release, which could be prevented by treatment with the MEK inhibitors. Taken together, our findings suggest that ERK activation plays an active role in mediating paraquat-induced apoptosis of NIH3T3 cells.

No MeSH data available.


The effect of paraquat on activation of ERK1/2 MAPKs in NIH3T3 cells. (A) Cells were cotransfected with GAL4-Elk/firefly luciferase vector and control renilla luciferase vector (pRL-Luc). Different dose of paraquat were then treated for 12 h. The cells were lysed, and luciferase activity was measured. Firefly luciferase reading was normalized to that or the control renilla luciferase. Results are shown as means ± S.D. (n=3). *<0.05. (B) Upper panel, cells were treated with the different dose of paraquat for 12 h. Lower panel, cells were treated with 1 mM paraquat for indicated time. Activation of ERK1/2 was determined by an immune complex kinase assay using Elk-1 fusion protein as substrate. ERK1/2-induced phosphorylation of Elk-1 was measures by immunoblotting with phosphor-Elk-1(Ser383) antibody. The ERK1/2 protein levels were shown using immunoblotting of ERK1/2 as control of immunocomplex.
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f2-bt-22-503: The effect of paraquat on activation of ERK1/2 MAPKs in NIH3T3 cells. (A) Cells were cotransfected with GAL4-Elk/firefly luciferase vector and control renilla luciferase vector (pRL-Luc). Different dose of paraquat were then treated for 12 h. The cells were lysed, and luciferase activity was measured. Firefly luciferase reading was normalized to that or the control renilla luciferase. Results are shown as means ± S.D. (n=3). *<0.05. (B) Upper panel, cells were treated with the different dose of paraquat for 12 h. Lower panel, cells were treated with 1 mM paraquat for indicated time. Activation of ERK1/2 was determined by an immune complex kinase assay using Elk-1 fusion protein as substrate. ERK1/2-induced phosphorylation of Elk-1 was measures by immunoblotting with phosphor-Elk-1(Ser383) antibody. The ERK1/2 protein levels were shown using immunoblotting of ERK1/2 as control of immunocomplex.

Mentions: It is well established that MAPK signaling pathway mediates stress induced apoptosis (Kumar et al., 2014; Wang et al., 2014b). Therefore, we further analyzed the relation of MAPK signal in paraquat-induced apoptosis. We first investigated whether paraquat treatment led to ERK activation. NIH3T3 cells were cotransfected with Gal4-Elk1-firefly luciferase vector, which contains the Gal4 DNA binding domain fused to the Elk-1 carboxyl-terminal transactivation domain, and pRL-Luc, which contains the renilla luciferase gene, and then fresh medium containing different dose of paraquat was then added to cells. The cells were harvested 12 h later, and the luciferase activities were determined using a luminometer. Elk1 is a transcriptional factor that is activated in response to activation of mitogen activated protein kinase (MAPK) and the renilla luciferase plasmid (pRL-Luc) was used to normalize the transfection efficiency. As shown in Fig. 2A, luciferase activity was elevated by treatment of 0.1 mM paraquat and increased in a dose-dependent manner. To confirm the phosphorylation of ERK by paraquat, NIH3T3 cells were exposed to different dose of paraquat for various lengths of time, and ERK activity was the assessed using immunocomplex kinase assay, that is measurement of its phosphorylation by Western blot analysis with anti-phospho Elk-1 antibody. Total ERK protein levels were monitored using antibody capable of recognizing unphosphorylated forms of the proteins. As shown in Fig. 2B, 1 mM paraquat, which resulted in significant apoptosis, led to strong activation of ERK. Activation was apparent at about 30 min following treatment with 1 mM paraquat and persisted for at least 24 h.


Paraquat Induces Apoptosis through Cytochrome C Release and ERK Activation.

Seo HJ, Choi SJ, Lee JH - Biomol Ther (Seoul) (2014)

The effect of paraquat on activation of ERK1/2 MAPKs in NIH3T3 cells. (A) Cells were cotransfected with GAL4-Elk/firefly luciferase vector and control renilla luciferase vector (pRL-Luc). Different dose of paraquat were then treated for 12 h. The cells were lysed, and luciferase activity was measured. Firefly luciferase reading was normalized to that or the control renilla luciferase. Results are shown as means ± S.D. (n=3). *<0.05. (B) Upper panel, cells were treated with the different dose of paraquat for 12 h. Lower panel, cells were treated with 1 mM paraquat for indicated time. Activation of ERK1/2 was determined by an immune complex kinase assay using Elk-1 fusion protein as substrate. ERK1/2-induced phosphorylation of Elk-1 was measures by immunoblotting with phosphor-Elk-1(Ser383) antibody. The ERK1/2 protein levels were shown using immunoblotting of ERK1/2 as control of immunocomplex.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4256029&req=5

f2-bt-22-503: The effect of paraquat on activation of ERK1/2 MAPKs in NIH3T3 cells. (A) Cells were cotransfected with GAL4-Elk/firefly luciferase vector and control renilla luciferase vector (pRL-Luc). Different dose of paraquat were then treated for 12 h. The cells were lysed, and luciferase activity was measured. Firefly luciferase reading was normalized to that or the control renilla luciferase. Results are shown as means ± S.D. (n=3). *<0.05. (B) Upper panel, cells were treated with the different dose of paraquat for 12 h. Lower panel, cells were treated with 1 mM paraquat for indicated time. Activation of ERK1/2 was determined by an immune complex kinase assay using Elk-1 fusion protein as substrate. ERK1/2-induced phosphorylation of Elk-1 was measures by immunoblotting with phosphor-Elk-1(Ser383) antibody. The ERK1/2 protein levels were shown using immunoblotting of ERK1/2 as control of immunocomplex.
Mentions: It is well established that MAPK signaling pathway mediates stress induced apoptosis (Kumar et al., 2014; Wang et al., 2014b). Therefore, we further analyzed the relation of MAPK signal in paraquat-induced apoptosis. We first investigated whether paraquat treatment led to ERK activation. NIH3T3 cells were cotransfected with Gal4-Elk1-firefly luciferase vector, which contains the Gal4 DNA binding domain fused to the Elk-1 carboxyl-terminal transactivation domain, and pRL-Luc, which contains the renilla luciferase gene, and then fresh medium containing different dose of paraquat was then added to cells. The cells were harvested 12 h later, and the luciferase activities were determined using a luminometer. Elk1 is a transcriptional factor that is activated in response to activation of mitogen activated protein kinase (MAPK) and the renilla luciferase plasmid (pRL-Luc) was used to normalize the transfection efficiency. As shown in Fig. 2A, luciferase activity was elevated by treatment of 0.1 mM paraquat and increased in a dose-dependent manner. To confirm the phosphorylation of ERK by paraquat, NIH3T3 cells were exposed to different dose of paraquat for various lengths of time, and ERK activity was the assessed using immunocomplex kinase assay, that is measurement of its phosphorylation by Western blot analysis with anti-phospho Elk-1 antibody. Total ERK protein levels were monitored using antibody capable of recognizing unphosphorylated forms of the proteins. As shown in Fig. 2B, 1 mM paraquat, which resulted in significant apoptosis, led to strong activation of ERK. Activation was apparent at about 30 min following treatment with 1 mM paraquat and persisted for at least 24 h.

Bottom Line: Paraquat treatment resulted in activation of ERK, and U0126, inhibitors of the MEK/ERK signaling pathway, prevented apoptosis.Moreover, paraquat-induced apoptosis was associated with cytochrome C release, which could be prevented by treatment with the MEK inhibitors.Taken together, our findings suggest that ERK activation plays an active role in mediating paraquat-induced apoptosis of NIH3T3 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Thoracic and Cardiovascular Surgery, Chosun University School of Medicine, Gwangju 501-759, Republic of Korea.

ABSTRACT
Paraquat has been suggested to induce apoptosis by generation of reactive oxygen species (ROS). However, little is known about the mechanism of paraquat-induced apoptosis. Here, we demonstrate that extracellular signal-regulated protein kinase (ERK) is required for paraquat-induced apoptosis in NIH3T3 cells. Paraquat treatment resulted in activation of ERK, and U0126, inhibitors of the MEK/ERK signaling pathway, prevented apoptosis. Moreover, paraquat-induced apoptosis was associated with cytochrome C release, which could be prevented by treatment with the MEK inhibitors. Taken together, our findings suggest that ERK activation plays an active role in mediating paraquat-induced apoptosis of NIH3T3 cells.

No MeSH data available.