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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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MEK-ERK1/2 signaling pathway is required for radiation-induced c-Jun phosphorylation.(A) Cell lysates collected from 10 Gy-irradiated BV2 cells were immunoblotted with anti-phosphorylated MEK1/2 (sc-7995R) antibody. (B) BV2 cells were transfected with HA-MEK1, HA-MEK1K97M (kinase-dead mutant), and vector control. Protein levels of p-c-Jun and p-ERK1/2 (upper and lower panels) are shown. (C) BV2 cells that were treated or untreated with 10 μM U0126 were irradiated with 10 Gy. p-c-Jun and p-ERK1/2 levels were analyzed by Western blot. (D) Two shRNAs (sh-MEK1-212 and sh-MEK1-495) were lentivirally delivered into BV2 cells to knockdown MEK1 protein. Knockdown efficiency and protein levels of p-c-Jun and p-ERK1/2 were detected with the indicated antibodies. The quantification results for (C) and (D) are shown in Figure S2.
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pone-0036739-g004: MEK-ERK1/2 signaling pathway is required for radiation-induced c-Jun phosphorylation.(A) Cell lysates collected from 10 Gy-irradiated BV2 cells were immunoblotted with anti-phosphorylated MEK1/2 (sc-7995R) antibody. (B) BV2 cells were transfected with HA-MEK1, HA-MEK1K97M (kinase-dead mutant), and vector control. Protein levels of p-c-Jun and p-ERK1/2 (upper and lower panels) are shown. (C) BV2 cells that were treated or untreated with 10 μM U0126 were irradiated with 10 Gy. p-c-Jun and p-ERK1/2 levels were analyzed by Western blot. (D) Two shRNAs (sh-MEK1-212 and sh-MEK1-495) were lentivirally delivered into BV2 cells to knockdown MEK1 protein. Knockdown efficiency and protein levels of p-c-Jun and p-ERK1/2 were detected with the indicated antibodies. The quantification results for (C) and (D) are shown in Figure S2.

Mentions: As the last downstream kinases in the Ras-Raf-MEK-ERK1/2 MAP kinase cascade, ERK1 and ERK2 are directly activated by MEK [21]. To confirm that radiation-induced c-Jun phosphorylation is dependent on the activation of this MAP kinase signaling pathway, we examined activation of MEK1 in irradiated BV2 cells. As shown in Figure 4A, radiation rapidly stimulated MEK1 phosphorylation. To validate the capability of MEK1 in modulating c-Jun phosphorylation, HA-tagged wild type (WT) MEK1 and its kinase-dead mutant MEK1K97M[24] were overexpressed in BV2 cells by transient transfection. As shown in Figure 4B, MEK1 (WT) dramatically induced phosphorylation of ERK1/2 as well as c-Jun, while the vector or MEK1K97M did not increase c-Jun phosphorylation. We also treated the BV2 cells with U0126, a MEK1 inhibitor, and observed markedly decreased phosphorylation of ERK1/2 and c-Jun in the irradiated cells (Figure 4C, and Figures S2A and S2B). To specifically disrupt MEK1 function, two lentiviral shRNAs constructs (sh-MEK1-212 and sh-MEK1-495) with different target sequences on MEK1 were generated. As illustrated in Figure 4D, and Figures S2C and S2D, sh-MEK1-212 efficiently depleted MEK1 protein level in BV2 cells and concurrently reduced radiation-stimulated phosphorylation of both ERK1/2 and c-Jun. By contrast, the sh-MEK1-495 construct, which marginally decreased MEK1, did not block radiation-increased phosphorylation of ERK1/2 and c-Jun. Collectively, these results indicate that MEK1 is indispensable for N-terminal c-Jun phosphorylation in response to radiation. Our data also suggest that the canonical MEK1-ERK1/2 signaling pathway contributes to radiation-induced c-Jun activation.


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)

MEK-ERK1/2 signaling pathway is required for radiation-induced c-Jun phosphorylation.(A) Cell lysates collected from 10 Gy-irradiated BV2 cells were immunoblotted with anti-phosphorylated MEK1/2 (sc-7995R) antibody. (B) BV2 cells were transfected with HA-MEK1, HA-MEK1K97M (kinase-dead mutant), and vector control. Protein levels of p-c-Jun and p-ERK1/2 (upper and lower panels) are shown. (C) BV2 cells that were treated or untreated with 10 μM U0126 were irradiated with 10 Gy. p-c-Jun and p-ERK1/2 levels were analyzed by Western blot. (D) Two shRNAs (sh-MEK1-212 and sh-MEK1-495) were lentivirally delivered into BV2 cells to knockdown MEK1 protein. Knockdown efficiency and protein levels of p-c-Jun and p-ERK1/2 were detected with the indicated antibodies. The quantification results for (C) and (D) are shown in Figure S2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351464&req=5

pone-0036739-g004: MEK-ERK1/2 signaling pathway is required for radiation-induced c-Jun phosphorylation.(A) Cell lysates collected from 10 Gy-irradiated BV2 cells were immunoblotted with anti-phosphorylated MEK1/2 (sc-7995R) antibody. (B) BV2 cells were transfected with HA-MEK1, HA-MEK1K97M (kinase-dead mutant), and vector control. Protein levels of p-c-Jun and p-ERK1/2 (upper and lower panels) are shown. (C) BV2 cells that were treated or untreated with 10 μM U0126 were irradiated with 10 Gy. p-c-Jun and p-ERK1/2 levels were analyzed by Western blot. (D) Two shRNAs (sh-MEK1-212 and sh-MEK1-495) were lentivirally delivered into BV2 cells to knockdown MEK1 protein. Knockdown efficiency and protein levels of p-c-Jun and p-ERK1/2 were detected with the indicated antibodies. The quantification results for (C) and (D) are shown in Figure S2.
Mentions: As the last downstream kinases in the Ras-Raf-MEK-ERK1/2 MAP kinase cascade, ERK1 and ERK2 are directly activated by MEK [21]. To confirm that radiation-induced c-Jun phosphorylation is dependent on the activation of this MAP kinase signaling pathway, we examined activation of MEK1 in irradiated BV2 cells. As shown in Figure 4A, radiation rapidly stimulated MEK1 phosphorylation. To validate the capability of MEK1 in modulating c-Jun phosphorylation, HA-tagged wild type (WT) MEK1 and its kinase-dead mutant MEK1K97M[24] were overexpressed in BV2 cells by transient transfection. As shown in Figure 4B, MEK1 (WT) dramatically induced phosphorylation of ERK1/2 as well as c-Jun, while the vector or MEK1K97M did not increase c-Jun phosphorylation. We also treated the BV2 cells with U0126, a MEK1 inhibitor, and observed markedly decreased phosphorylation of ERK1/2 and c-Jun in the irradiated cells (Figure 4C, and Figures S2A and S2B). To specifically disrupt MEK1 function, two lentiviral shRNAs constructs (sh-MEK1-212 and sh-MEK1-495) with different target sequences on MEK1 were generated. As illustrated in Figure 4D, and Figures S2C and S2D, sh-MEK1-212 efficiently depleted MEK1 protein level in BV2 cells and concurrently reduced radiation-stimulated phosphorylation of both ERK1/2 and c-Jun. By contrast, the sh-MEK1-495 construct, which marginally decreased MEK1, did not block radiation-increased phosphorylation of ERK1/2 and c-Jun. Collectively, these results indicate that MEK1 is indispensable for N-terminal c-Jun phosphorylation in response to radiation. Our data also suggest that the canonical MEK1-ERK1/2 signaling pathway contributes to radiation-induced c-Jun activation.

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