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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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Radiation-induced phosphorylation of c-Jun and ERK1/2 in BV2 cells.(A) BV2 cells were irradiated with a single dose of 10 Gy, and sampled at the time points indicated. Cell lysates were sequentially immunoblotted with indicated antibodies in Western blot assays to detect phosphorylated and total proteins of c-Jun, ERK1/2, and JNK. β-actin was used as a loading control. Increased phosphorylation was found for c-Jun and ERK1/2 post irradiation, but not for JNKs. There was no obvious change for total c-Jun. The right panel shows the fold change for phosphorylation of c-Jun (Ser63 and Ser73) and ERK1/2 following irradiation. Data represent mean ± SD and the significant differences compared to control were indicated as *p<0.05 or **p<0.01. (B) BV2 cells were irradiated with different doses as shown and phosphorylation of c-Jun and ERK1/2 was detected at 0.5 and 1 h post-irradiation. Regardless of dose levels, radiation markedly stimulated phosphorylation of c-Jun and ERK1/2.
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pone-0036739-g001: Radiation-induced phosphorylation of c-Jun and ERK1/2 in BV2 cells.(A) BV2 cells were irradiated with a single dose of 10 Gy, and sampled at the time points indicated. Cell lysates were sequentially immunoblotted with indicated antibodies in Western blot assays to detect phosphorylated and total proteins of c-Jun, ERK1/2, and JNK. β-actin was used as a loading control. Increased phosphorylation was found for c-Jun and ERK1/2 post irradiation, but not for JNKs. There was no obvious change for total c-Jun. The right panel shows the fold change for phosphorylation of c-Jun (Ser63 and Ser73) and ERK1/2 following irradiation. Data represent mean ± SD and the significant differences compared to control were indicated as *p<0.05 or **p<0.01. (B) BV2 cells were irradiated with different doses as shown and phosphorylation of c-Jun and ERK1/2 was detected at 0.5 and 1 h post-irradiation. Regardless of dose levels, radiation markedly stimulated phosphorylation of c-Jun and ERK1/2.

Mentions: BV2 cells were irradiated with a single dose of 10 Gy γ-ray and cell lysates were collected for Western blot analyses. Radiation resulted in a prominent increase in c-Jun phosphorylation on Ser63 and Ser73 (Figure 1A), the critical phosphorylation sites correlating with enhanced transcriptional activity of c-Jun [23]. The induction of phosphorylation by radiation was rapid and sustained for several hours. Meanwhile, total c-Jun protein remained unchanged in the irradiated cells, indicating that the accumulation of phosphorylated c-Jun was not due to transcriptional upregulation. JNKs have been known to phosphorylate c-Jun on Ser63 and Ser73 residues. Unexpectedly, we did not observe concurrently activated JNKs following irradiation, indicated by their unchanged phosphorylation status on Thr183 and Tyr185 sites (Figure 1A). In contrast, radiation stimulated rapid phosphorylation of ERK1 and ERK2. Similar results were found in BV2 cells treated with different irradiation doses from 2.5 to 15 Gy. As shown in Figure 1B, phosphorylation was increased under each of the conditions for both c-Jun and ERK1/2. Nevertheless, induction patterns of the phosphorylation were somewhat different between low-dose and high-dose treated cells. For example, peak phosphorylation for c-Jun and ERK1/2 appeared at 0.5 h for lower doses of irradiation (2.5 and 5 Gy), but at 1 h for the highest dose (15 Gy). Moreover, there was an apparent correlation between post-irradiation phosphorylation levels of c-Jun and ERK1/2, suggesting that ERK1/2 mediate radiation-stimulated c-Jun phosphorylation.


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)

Radiation-induced phosphorylation of c-Jun and ERK1/2 in BV2 cells.(A) BV2 cells were irradiated with a single dose of 10 Gy, and sampled at the time points indicated. Cell lysates were sequentially immunoblotted with indicated antibodies in Western blot assays to detect phosphorylated and total proteins of c-Jun, ERK1/2, and JNK. β-actin was used as a loading control. Increased phosphorylation was found for c-Jun and ERK1/2 post irradiation, but not for JNKs. There was no obvious change for total c-Jun. The right panel shows the fold change for phosphorylation of c-Jun (Ser63 and Ser73) and ERK1/2 following irradiation. Data represent mean ± SD and the significant differences compared to control were indicated as *p<0.05 or **p<0.01. (B) BV2 cells were irradiated with different doses as shown and phosphorylation of c-Jun and ERK1/2 was detected at 0.5 and 1 h post-irradiation. Regardless of dose levels, radiation markedly stimulated phosphorylation of c-Jun and ERK1/2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351464&req=5

pone-0036739-g001: Radiation-induced phosphorylation of c-Jun and ERK1/2 in BV2 cells.(A) BV2 cells were irradiated with a single dose of 10 Gy, and sampled at the time points indicated. Cell lysates were sequentially immunoblotted with indicated antibodies in Western blot assays to detect phosphorylated and total proteins of c-Jun, ERK1/2, and JNK. β-actin was used as a loading control. Increased phosphorylation was found for c-Jun and ERK1/2 post irradiation, but not for JNKs. There was no obvious change for total c-Jun. The right panel shows the fold change for phosphorylation of c-Jun (Ser63 and Ser73) and ERK1/2 following irradiation. Data represent mean ± SD and the significant differences compared to control were indicated as *p<0.05 or **p<0.01. (B) BV2 cells were irradiated with different doses as shown and phosphorylation of c-Jun and ERK1/2 was detected at 0.5 and 1 h post-irradiation. Regardless of dose levels, radiation markedly stimulated phosphorylation of c-Jun and ERK1/2.
Mentions: BV2 cells were irradiated with a single dose of 10 Gy γ-ray and cell lysates were collected for Western blot analyses. Radiation resulted in a prominent increase in c-Jun phosphorylation on Ser63 and Ser73 (Figure 1A), the critical phosphorylation sites correlating with enhanced transcriptional activity of c-Jun [23]. The induction of phosphorylation by radiation was rapid and sustained for several hours. Meanwhile, total c-Jun protein remained unchanged in the irradiated cells, indicating that the accumulation of phosphorylated c-Jun was not due to transcriptional upregulation. JNKs have been known to phosphorylate c-Jun on Ser63 and Ser73 residues. Unexpectedly, we did not observe concurrently activated JNKs following irradiation, indicated by their unchanged phosphorylation status on Thr183 and Tyr185 sites (Figure 1A). In contrast, radiation stimulated rapid phosphorylation of ERK1 and ERK2. Similar results were found in BV2 cells treated with different irradiation doses from 2.5 to 15 Gy. As shown in Figure 1B, phosphorylation was increased under each of the conditions for both c-Jun and ERK1/2. Nevertheless, induction patterns of the phosphorylation were somewhat different between low-dose and high-dose treated cells. For example, peak phosphorylation for c-Jun and ERK1/2 appeared at 0.5 h for lower doses of irradiation (2.5 and 5 Gy), but at 1 h for the highest dose (15 Gy). Moreover, there was an apparent correlation between post-irradiation phosphorylation levels of c-Jun and ERK1/2, suggesting that ERK1/2 mediate radiation-stimulated c-Jun phosphorylation.

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