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Radiation-induced c-Jun activation depends on MEK1-ERK1/2 signaling pathway in microglial cells.

Deng Z, Sui G, Rosa PM, Zhao W - PLoS ONE (2012)

Bottom Line: ERK1/2 directly interact with c-Jun in vitro and in cells; meanwhile, the JNK binding domain on c-Jun is not required for its interaction with ERK kinases.Radiation-induced reactive oxygen species (ROS) potentially contribute to c-Jun phosphorylation through activating the ERK pathway.Pharmacologic blockade of the ERK signaling pathway interferes with c-Jun activity and inhibits radiation-stimulated expression of c-Jun target genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology and Brain Tumor Center of Excellence, Wake Forest University School of Medicine, Winston-Salem, North Carolina, United States of America.

ABSTRACT
Radiation-induced normal brain injury is associated with acute and/or chronic inflammatory responses, and has been a major concern in radiotherapy. Recent studies suggest that microglial activation is a potential contributor to chronic inflammatory responses following irradiation; however, the molecular mechanism underlying the response of microglia to radiation is poorly understood. c-Jun, a component of AP-1 transcription factors, potentially regulates neural cell death and neuroinflammation. We observed a rapid increase in phosphorylation of N-terminal c-Jun (on serine 63 and 73) and MAPK kinases ERK1/2, but not JNKs, in irradiated murine microglial BV2 cells. Radiation-induced c-Jun phosphorylation is dependent on the canonical MEK-ERK signaling pathway and required for both ERK1 and ERK2 function. ERK1/2 directly interact with c-Jun in vitro and in cells; meanwhile, the JNK binding domain on c-Jun is not required for its interaction with ERK kinases. Radiation-induced reactive oxygen species (ROS) potentially contribute to c-Jun phosphorylation through activating the ERK pathway. Radiation stimulates c-Jun transcriptional activity and upregulates c-Jun-regulated proinflammatory genes, such as tumor necrosis factor-α, interleukin-1β, and cyclooxygenase-2. Pharmacologic blockade of the ERK signaling pathway interferes with c-Jun activity and inhibits radiation-stimulated expression of c-Jun target genes. Overall, our study reveals that the MEK-ERK1/2 signaling pathway, but not the JNK pathway, contributes to the c-Jun-dependent microglial inflammatory response following irradiation.

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ROS is associated with c-Jun phosphorylation.(A) 100 μM exogenous hydrogen peroxide was added into culture medium of BV2 cells. p-c-Jun and p-ERK1/2 were detected at the time points indicated. (B) c-Jun phosphorylation was detected for BV2 cell lysates treated or untreated with 5 mM NAC at the indicated time points after radiation. (C) Inhibition of radiation-induced ROS in BV2 cells by 50 μM apocynin. Intracellular ROS level was determined using the 2′, 7′-Dichlorofluorescein diacetate (DCFA-DA) based method. Data represent mean ± SD, *p<0.05. (D) Apocynin was added into the BV2 cell culture medium and phosphorylation of c-Jun was analyzed in the apocynin-treated cell lysates.
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pone-0036739-g006: ROS is associated with c-Jun phosphorylation.(A) 100 μM exogenous hydrogen peroxide was added into culture medium of BV2 cells. p-c-Jun and p-ERK1/2 were detected at the time points indicated. (B) c-Jun phosphorylation was detected for BV2 cell lysates treated or untreated with 5 mM NAC at the indicated time points after radiation. (C) Inhibition of radiation-induced ROS in BV2 cells by 50 μM apocynin. Intracellular ROS level was determined using the 2′, 7′-Dichlorofluorescein diacetate (DCFA-DA) based method. Data represent mean ± SD, *p<0.05. (D) Apocynin was added into the BV2 cell culture medium and phosphorylation of c-Jun was analyzed in the apocynin-treated cell lysates.

Mentions: Cellular oxidative stress is one of primary consequences caused by irradiation. We and others have observed increased generation of reactive oxygen species (ROS) following radiation in various cells, including microglia [25], [26], [27]. To test whether c-Jun phosphorylation is dependent on increased generation of ROS by irradiation, we treated BV2 cells with 100 µM H2O2 and observed that phosphorylated ERK1/2 and c-Jun were increased in BV2 cells following H2O2 treatment (Figure 6A). Conversely, we found that the treatment with 2.5 mM NAC, an antioxidant, attenuated the c-Jun phosphorylation in a time-dependent manner in irradiated BV2 cells (Figure 6B), revealing a delayed effect associated with a gradual ROS decrease by this oxidant scavenger. It is known that electron transport chain and NADPH oxidases are generators of ROS following irradiation [2], [26]. We therefore tested several inhibitors to study the relationship between this type of ROS generation and c-Jun activation. However we only found that apocynin, an inhibitor of NADPH oxidases, could be used in this study, because other tested inhibitors, such as TTFA, rotenone, NaN3, and Dpi, could nonspecifically affect c-Jun phosphorylation in non-irradiated BV2 cells (data not shown). After preincubating BV2 cells with apocynin, we observed a marked reduction of radiation-induced ROS in these cells (Figure 6C). Importantly, apocynin treatment also decreased c-Jun phosphorylation in response to irradiation (Figure 6D), suggesting the importance of ROS on radiation-stimulated c-Jun function.


Radiation-induced c-Jun activation depends on MEK1-ERK1/2 signaling pathway in microglial cells.

Deng Z, Sui G, Rosa PM, Zhao W - PLoS ONE (2012)

ROS is associated with c-Jun phosphorylation.(A) 100 μM exogenous hydrogen peroxide was added into culture medium of BV2 cells. p-c-Jun and p-ERK1/2 were detected at the time points indicated. (B) c-Jun phosphorylation was detected for BV2 cell lysates treated or untreated with 5 mM NAC at the indicated time points after radiation. (C) Inhibition of radiation-induced ROS in BV2 cells by 50 μM apocynin. Intracellular ROS level was determined using the 2′, 7′-Dichlorofluorescein diacetate (DCFA-DA) based method. Data represent mean ± SD, *p<0.05. (D) Apocynin was added into the BV2 cell culture medium and phosphorylation of c-Jun was analyzed in the apocynin-treated cell lysates.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351464&req=5

pone-0036739-g006: ROS is associated with c-Jun phosphorylation.(A) 100 μM exogenous hydrogen peroxide was added into culture medium of BV2 cells. p-c-Jun and p-ERK1/2 were detected at the time points indicated. (B) c-Jun phosphorylation was detected for BV2 cell lysates treated or untreated with 5 mM NAC at the indicated time points after radiation. (C) Inhibition of radiation-induced ROS in BV2 cells by 50 μM apocynin. Intracellular ROS level was determined using the 2′, 7′-Dichlorofluorescein diacetate (DCFA-DA) based method. Data represent mean ± SD, *p<0.05. (D) Apocynin was added into the BV2 cell culture medium and phosphorylation of c-Jun was analyzed in the apocynin-treated cell lysates.
Mentions: Cellular oxidative stress is one of primary consequences caused by irradiation. We and others have observed increased generation of reactive oxygen species (ROS) following radiation in various cells, including microglia [25], [26], [27]. To test whether c-Jun phosphorylation is dependent on increased generation of ROS by irradiation, we treated BV2 cells with 100 µM H2O2 and observed that phosphorylated ERK1/2 and c-Jun were increased in BV2 cells following H2O2 treatment (Figure 6A). Conversely, we found that the treatment with 2.5 mM NAC, an antioxidant, attenuated the c-Jun phosphorylation in a time-dependent manner in irradiated BV2 cells (Figure 6B), revealing a delayed effect associated with a gradual ROS decrease by this oxidant scavenger. It is known that electron transport chain and NADPH oxidases are generators of ROS following irradiation [2], [26]. We therefore tested several inhibitors to study the relationship between this type of ROS generation and c-Jun activation. However we only found that apocynin, an inhibitor of NADPH oxidases, could be used in this study, because other tested inhibitors, such as TTFA, rotenone, NaN3, and Dpi, could nonspecifically affect c-Jun phosphorylation in non-irradiated BV2 cells (data not shown). After preincubating BV2 cells with apocynin, we observed a marked reduction of radiation-induced ROS in these cells (Figure 6C). Importantly, apocynin treatment also decreased c-Jun phosphorylation in response to irradiation (Figure 6D), suggesting the importance of ROS on radiation-stimulated c-Jun function.

Bottom Line: ERK1/2 directly interact with c-Jun in vitro and in cells; meanwhile, the JNK binding domain on c-Jun is not required for its interaction with ERK kinases.Radiation-induced reactive oxygen species (ROS) potentially contribute to c-Jun phosphorylation through activating the ERK pathway.Pharmacologic blockade of the ERK signaling pathway interferes with c-Jun activity and inhibits radiation-stimulated expression of c-Jun target genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology and Brain Tumor Center of Excellence, Wake Forest University School of Medicine, Winston-Salem, North Carolina, United States of America.

ABSTRACT
Radiation-induced normal brain injury is associated with acute and/or chronic inflammatory responses, and has been a major concern in radiotherapy. Recent studies suggest that microglial activation is a potential contributor to chronic inflammatory responses following irradiation; however, the molecular mechanism underlying the response of microglia to radiation is poorly understood. c-Jun, a component of AP-1 transcription factors, potentially regulates neural cell death and neuroinflammation. We observed a rapid increase in phosphorylation of N-terminal c-Jun (on serine 63 and 73) and MAPK kinases ERK1/2, but not JNKs, in irradiated murine microglial BV2 cells. Radiation-induced c-Jun phosphorylation is dependent on the canonical MEK-ERK signaling pathway and required for both ERK1 and ERK2 function. ERK1/2 directly interact with c-Jun in vitro and in cells; meanwhile, the JNK binding domain on c-Jun is not required for its interaction with ERK kinases. Radiation-induced reactive oxygen species (ROS) potentially contribute to c-Jun phosphorylation through activating the ERK pathway. Radiation stimulates c-Jun transcriptional activity and upregulates c-Jun-regulated proinflammatory genes, such as tumor necrosis factor-α, interleukin-1β, and cyclooxygenase-2. Pharmacologic blockade of the ERK signaling pathway interferes with c-Jun activity and inhibits radiation-stimulated expression of c-Jun target genes. Overall, our study reveals that the MEK-ERK1/2 signaling pathway, but not the JNK pathway, contributes to the c-Jun-dependent microglial inflammatory response following irradiation.

Show MeSH
Related in: MedlinePlus