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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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JNK function on c-Jun phosphorylation in BV2 cells.BV2 cells were individually treated with U0126 (10 μM) and the JNK inhibitor SP600125 (5 μM). Cells were irradiated (10 Gy) or sham-irradiated. Cell lysates were collected 1 h post-irradiation. (A) Levels of p-c-Jun and p-ERK1/2 in the treated cells. (B) Levels of c-Jun, ERK, p-JNKs, and JNKs in the treated cells.
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pone-0036739-g005: JNK function on c-Jun phosphorylation in BV2 cells.BV2 cells were individually treated with U0126 (10 μM) and the JNK inhibitor SP600125 (5 μM). Cells were irradiated (10 Gy) or sham-irradiated. Cell lysates were collected 1 h post-irradiation. (A) Levels of p-c-Jun and p-ERK1/2 in the treated cells. (B) Levels of c-Jun, ERK, p-JNKs, and JNKs in the treated cells.

Mentions: The members of JNK family are primary kinases responsible for N-terminal phosphorylation of c-Jun under different physiological conditions in multiple cell types [17], [22]. A recent study showed that the JNK inhibitor SP600125 could reduce c-Jun phosphorylation in BV2 cells post-irradiation [25]. However, we did not observe JNK activation in irradiated BV2 cells, suggesting that JNKs may not be involved in modulation of c-Jun phosphorylation following irradiation [25]. To reconcile these conflicting findings, we treated BV2 cells with SP600125 and used the MEK inhibitor U0126 as a control. We indeed detected the inhibitory effect of SP600125 on c-Jun phosphorylation induced by radiation (Figure 5A); however, the JNK inhibitor SP600125 also reduced ERK1/2 phosphorylation (Figure 5A). We concluded that this unexpected effect could contribute to the reduced c-Jun phosphorylation reported previously [25]. Between these two kinase inhibitors, U0126 exhibited much better inhibition on ERK1/2 phosphorylation, but SP600125 showed a greater effect in reducing c-Jun phosphorylation (Figure 5A). These results suggest that JNKs, rather than ERK1/2, are key factors for basal c-Jun phosphorylation. Due to its dual inhibitory roles in JNK and ERK1/2, SP600125 would likely reduce both basal and radiation-induced phosphorylation of c-Jun. Indeed, we found that SP600125 was more potent than U0126 in reducing c-Jun phosphorylation following irradiation (Figure 5A). Further studies validated that JNK phosphorylation was decreased by SP600125, but not U0126, in this inhibitor assay (Figure 5B). Overall, our observations strongly suggested that radiation stimulates JNK-independent c-Jun phosphorylation via an activated ERK1/2 pathway, while JNKs are critical for c-Jun phosphorylation in other physiological conditions.


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)

JNK function on c-Jun phosphorylation in BV2 cells.BV2 cells were individually treated with U0126 (10 μM) and the JNK inhibitor SP600125 (5 μM). Cells were irradiated (10 Gy) or sham-irradiated. Cell lysates were collected 1 h post-irradiation. (A) Levels of p-c-Jun and p-ERK1/2 in the treated cells. (B) Levels of c-Jun, ERK, p-JNKs, and JNKs in the treated cells.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351464&req=5

pone-0036739-g005: JNK function on c-Jun phosphorylation in BV2 cells.BV2 cells were individually treated with U0126 (10 μM) and the JNK inhibitor SP600125 (5 μM). Cells were irradiated (10 Gy) or sham-irradiated. Cell lysates were collected 1 h post-irradiation. (A) Levels of p-c-Jun and p-ERK1/2 in the treated cells. (B) Levels of c-Jun, ERK, p-JNKs, and JNKs in the treated cells.
Mentions: The members of JNK family are primary kinases responsible for N-terminal phosphorylation of c-Jun under different physiological conditions in multiple cell types [17], [22]. A recent study showed that the JNK inhibitor SP600125 could reduce c-Jun phosphorylation in BV2 cells post-irradiation [25]. However, we did not observe JNK activation in irradiated BV2 cells, suggesting that JNKs may not be involved in modulation of c-Jun phosphorylation following irradiation [25]. To reconcile these conflicting findings, we treated BV2 cells with SP600125 and used the MEK inhibitor U0126 as a control. We indeed detected the inhibitory effect of SP600125 on c-Jun phosphorylation induced by radiation (Figure 5A); however, the JNK inhibitor SP600125 also reduced ERK1/2 phosphorylation (Figure 5A). We concluded that this unexpected effect could contribute to the reduced c-Jun phosphorylation reported previously [25]. Between these two kinase inhibitors, U0126 exhibited much better inhibition on ERK1/2 phosphorylation, but SP600125 showed a greater effect in reducing c-Jun phosphorylation (Figure 5A). These results suggest that JNKs, rather than ERK1/2, are key factors for basal c-Jun phosphorylation. Due to its dual inhibitory roles in JNK and ERK1/2, SP600125 would likely reduce both basal and radiation-induced phosphorylation of c-Jun. Indeed, we found that SP600125 was more potent than U0126 in reducing c-Jun phosphorylation following irradiation (Figure 5A). Further studies validated that JNK phosphorylation was decreased by SP600125, but not U0126, in this inhibitor assay (Figure 5B). Overall, our observations strongly suggested that radiation stimulates JNK-independent c-Jun phosphorylation via an activated ERK1/2 pathway, while JNKs are critical for c-Jun phosphorylation in other physiological conditions.

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