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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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ERK1 and ERK2 are indispensable for radiation-induced c-Jun phosphorylation in BV2 cells.(A) BV2 cells were transiently cotransfected with ERK1 and ERK2 expression vectors in doses of 75, 150 and 300 ng for each plasmid. The total DNA amounts in transfection were compensated with an empty vector. The transfected ERK1 and ERK2 led to a dose-dependent increase of phosphorylated ERK1/2 and c-Jun. (B) BV2 cells were treated with 10 µM ERK kinase inhibitor (Calbiochem Cat #328006) and DMSO as a control. Inhibitor-treated or untreated cells were collected at 1 h after 10 Gy irradiation. Western blots showed that ERK kinase inhibitor abolished radiation-stimulated phosphorylation of ERK1/2 and c-Jun. (C) To detect knockdown effects for ERK1 and ERK2 by lentiviral shRNA constructs in BV2 cells, two lentivirual shRNAs were prepared for each ERK kinase, respectively, and used for infecting BV2 cells. The cells were collected 48 h after lentiviral infection and subjected for Western blot analysis using the antibody against ERK1 (K32, sc-94), which also can recognize ERK2. Efficient depletion was found in the cell lysates infected with sh-ERK1-974, sh-ERK2-321, and sh-ERK2-772, but not sh-ERK1-536. (D) BV2 cells were individually infected with scrambled control, ERK1, and ERK2 shRNA lentiviruses, and subsequently irradiated with 10 Gy 48 h post infection and sampled at 1 h following radiation. There was no effect on radiation-induced c-Jun phosphorylation by individual depletion of ERK kinases. (E) BV2 cells were infected with combined lentiviruses (sh-ERK1-974/sh-ERK2-321 and sh-ERK1-974/sh-ERK2-772) and irradiated (10 Gy). Both shRNA combinations reduced radiation-stimulated phosphorylation of ERK1 and ERK2 to low levels, and decreased phosphorylated c-Jun after radiation. Bar graph depicts quantification (means ± SD) of relative c-Jun phosphorylation level, **p<0.01(#3 vs. #2; #4 vs. #2). (F) JNK levels in ERK1/2 knockdown BV2 cells.
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pone-0036739-g002: ERK1 and ERK2 are indispensable for radiation-induced c-Jun phosphorylation in BV2 cells.(A) BV2 cells were transiently cotransfected with ERK1 and ERK2 expression vectors in doses of 75, 150 and 300 ng for each plasmid. The total DNA amounts in transfection were compensated with an empty vector. The transfected ERK1 and ERK2 led to a dose-dependent increase of phosphorylated ERK1/2 and c-Jun. (B) BV2 cells were treated with 10 µM ERK kinase inhibitor (Calbiochem Cat #328006) and DMSO as a control. Inhibitor-treated or untreated cells were collected at 1 h after 10 Gy irradiation. Western blots showed that ERK kinase inhibitor abolished radiation-stimulated phosphorylation of ERK1/2 and c-Jun. (C) To detect knockdown effects for ERK1 and ERK2 by lentiviral shRNA constructs in BV2 cells, two lentivirual shRNAs were prepared for each ERK kinase, respectively, and used for infecting BV2 cells. The cells were collected 48 h after lentiviral infection and subjected for Western blot analysis using the antibody against ERK1 (K32, sc-94), which also can recognize ERK2. Efficient depletion was found in the cell lysates infected with sh-ERK1-974, sh-ERK2-321, and sh-ERK2-772, but not sh-ERK1-536. (D) BV2 cells were individually infected with scrambled control, ERK1, and ERK2 shRNA lentiviruses, and subsequently irradiated with 10 Gy 48 h post infection and sampled at 1 h following radiation. There was no effect on radiation-induced c-Jun phosphorylation by individual depletion of ERK kinases. (E) BV2 cells were infected with combined lentiviruses (sh-ERK1-974/sh-ERK2-321 and sh-ERK1-974/sh-ERK2-772) and irradiated (10 Gy). Both shRNA combinations reduced radiation-stimulated phosphorylation of ERK1 and ERK2 to low levels, and decreased phosphorylated c-Jun after radiation. Bar graph depicts quantification (means ± SD) of relative c-Jun phosphorylation level, **p<0.01(#3 vs. #2; #4 vs. #2). (F) JNK levels in ERK1/2 knockdown BV2 cells.

Mentions: To determine whether ERK1 and ERK2 regulate c-Jun phosphorylation in BV2 cells, ERK1 and ERK2 expression vectors were cotransfected into BV2 cells in increasing amounts to overexpress these two proteins. Forty-eight hours after transfection, c-Jun phosphorylation on Ser63 showed a dose-dependent increase, indicating the capability of ERK1/2 to stimulate c-Jun phosphorylation (Figure 2A). To further substantiate the role of ERK1/2 in radiation-induced c-Jun phosphorylation, an inhibitory assay was performed. The ERK inhibitor was found to attenuate phosphorylation of ERK1/2 but not JNK kinases and accordingly markedly reduced radiation-mediated phosphorylation of Ser63 on c-Jun (Figure 2B).


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)

ERK1 and ERK2 are indispensable for radiation-induced c-Jun phosphorylation in BV2 cells.(A) BV2 cells were transiently cotransfected with ERK1 and ERK2 expression vectors in doses of 75, 150 and 300 ng for each plasmid. The total DNA amounts in transfection were compensated with an empty vector. The transfected ERK1 and ERK2 led to a dose-dependent increase of phosphorylated ERK1/2 and c-Jun. (B) BV2 cells were treated with 10 µM ERK kinase inhibitor (Calbiochem Cat #328006) and DMSO as a control. Inhibitor-treated or untreated cells were collected at 1 h after 10 Gy irradiation. Western blots showed that ERK kinase inhibitor abolished radiation-stimulated phosphorylation of ERK1/2 and c-Jun. (C) To detect knockdown effects for ERK1 and ERK2 by lentiviral shRNA constructs in BV2 cells, two lentivirual shRNAs were prepared for each ERK kinase, respectively, and used for infecting BV2 cells. The cells were collected 48 h after lentiviral infection and subjected for Western blot analysis using the antibody against ERK1 (K32, sc-94), which also can recognize ERK2. Efficient depletion was found in the cell lysates infected with sh-ERK1-974, sh-ERK2-321, and sh-ERK2-772, but not sh-ERK1-536. (D) BV2 cells were individually infected with scrambled control, ERK1, and ERK2 shRNA lentiviruses, and subsequently irradiated with 10 Gy 48 h post infection and sampled at 1 h following radiation. There was no effect on radiation-induced c-Jun phosphorylation by individual depletion of ERK kinases. (E) BV2 cells were infected with combined lentiviruses (sh-ERK1-974/sh-ERK2-321 and sh-ERK1-974/sh-ERK2-772) and irradiated (10 Gy). Both shRNA combinations reduced radiation-stimulated phosphorylation of ERK1 and ERK2 to low levels, and decreased phosphorylated c-Jun after radiation. Bar graph depicts quantification (means ± SD) of relative c-Jun phosphorylation level, **p<0.01(#3 vs. #2; #4 vs. #2). (F) JNK levels in ERK1/2 knockdown BV2 cells.
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getmorefigures.php?uid=PMC3351464&req=5

pone-0036739-g002: ERK1 and ERK2 are indispensable for radiation-induced c-Jun phosphorylation in BV2 cells.(A) BV2 cells were transiently cotransfected with ERK1 and ERK2 expression vectors in doses of 75, 150 and 300 ng for each plasmid. The total DNA amounts in transfection were compensated with an empty vector. The transfected ERK1 and ERK2 led to a dose-dependent increase of phosphorylated ERK1/2 and c-Jun. (B) BV2 cells were treated with 10 µM ERK kinase inhibitor (Calbiochem Cat #328006) and DMSO as a control. Inhibitor-treated or untreated cells were collected at 1 h after 10 Gy irradiation. Western blots showed that ERK kinase inhibitor abolished radiation-stimulated phosphorylation of ERK1/2 and c-Jun. (C) To detect knockdown effects for ERK1 and ERK2 by lentiviral shRNA constructs in BV2 cells, two lentivirual shRNAs were prepared for each ERK kinase, respectively, and used for infecting BV2 cells. The cells were collected 48 h after lentiviral infection and subjected for Western blot analysis using the antibody against ERK1 (K32, sc-94), which also can recognize ERK2. Efficient depletion was found in the cell lysates infected with sh-ERK1-974, sh-ERK2-321, and sh-ERK2-772, but not sh-ERK1-536. (D) BV2 cells were individually infected with scrambled control, ERK1, and ERK2 shRNA lentiviruses, and subsequently irradiated with 10 Gy 48 h post infection and sampled at 1 h following radiation. There was no effect on radiation-induced c-Jun phosphorylation by individual depletion of ERK kinases. (E) BV2 cells were infected with combined lentiviruses (sh-ERK1-974/sh-ERK2-321 and sh-ERK1-974/sh-ERK2-772) and irradiated (10 Gy). Both shRNA combinations reduced radiation-stimulated phosphorylation of ERK1 and ERK2 to low levels, and decreased phosphorylated c-Jun after radiation. Bar graph depicts quantification (means ± SD) of relative c-Jun phosphorylation level, **p<0.01(#3 vs. #2; #4 vs. #2). (F) JNK levels in ERK1/2 knockdown BV2 cells.
Mentions: To determine whether ERK1 and ERK2 regulate c-Jun phosphorylation in BV2 cells, ERK1 and ERK2 expression vectors were cotransfected into BV2 cells in increasing amounts to overexpress these two proteins. Forty-eight hours after transfection, c-Jun phosphorylation on Ser63 showed a dose-dependent increase, indicating the capability of ERK1/2 to stimulate c-Jun phosphorylation (Figure 2A). To further substantiate the role of ERK1/2 in radiation-induced c-Jun phosphorylation, an inhibitory assay was performed. The ERK inhibitor was found to attenuate phosphorylation of ERK1/2 but not JNK kinases and accordingly markedly reduced radiation-mediated phosphorylation of Ser63 on c-Jun (Figure 2B).

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