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Tristetraprolin mediates radiation-induced TNF-α production in lung macrophages.

Ray D, Shukla S, Allam US, Helman A, Ramanand SG, Tran L, Bassetti M, Krishnamurthy PM, Rumschlag M, Paulsen M, Sun L, Shanley TP, Ljungman M, Nyati MK, Zhang M, Lawrence TS - PLoS ONE (2013)

Bottom Line: To study the in vivo relevance, mouse lungs were irradiated with a single dose (15 Gy) and assessed at varying times for TTP alterations.In conclusion, irradiation of lung macrophages causes TTP inactivation via p38-mediated phosphorylation and proteasome-mediated degradation, leading to TNF-α production.These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, United States of America. dipray@umich.edu

ABSTRACT
The efficacy of radiation therapy for lung cancer is limited by radiation-induced lung toxicity (RILT). Although tumor necrosis factor-alpha (TNF-α) signaling plays a critical role in RILT, the molecular regulators of radiation-induced TNF-α production remain unknown. We investigated the role of a major TNF-α regulator, Tristetraprolin (TTP), in radiation-induced TNF-α production by macrophages. For in vitro studies we irradiated (4 Gy) either a mouse lung macrophage cell line, MH-S or macrophages isolated from TTP knockout mice, and studied the effects of radiation on TTP and TNF-α levels. To study the in vivo relevance, mouse lungs were irradiated with a single dose (15 Gy) and assessed at varying times for TTP alterations. Irradiation of MH-S cells caused TTP to undergo an inhibitory phosphorylation at Ser-178 and proteasome-mediated degradation, which resulted in increased TNF-α mRNA stabilization and secretion. Similarly, MH-S cells treated with TTP siRNA or macrophages isolated from ttp (-/-) mice had higher basal levels of TNF-α, which was increased minimally after irradiation. Conversely, cells overexpressing TTP mutants defective in undergoing phosphorylation released significantly lower levels of TNF-α. Inhibition of p38, a known kinase for TTP, by either siRNA or a small molecule inhibitor abrogated radiation-induced TNF-α release by MH-S cells. Lung irradiation induced TTP(Ser178) phosphorylation and protein degradation and a simultaneous increase in TNF-α production in C57BL/6 mice starting 24 h post-radiation. In conclusion, irradiation of lung macrophages causes TTP inactivation via p38-mediated phosphorylation and proteasome-mediated degradation, leading to TNF-α production. These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT.

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Radiation-induced TTP degradation and TNF-α secretion is inhibited significantly by the proteasome inhibitor MG132.(A) MH-S cells were metabolically labeled with 35S-methionine, were either sham-irradiated or irradiated with 4 Gy, and chased with cold methionine for indicated time periods. After the chase period cell lysates were subjected to immunoprecipitation using TTP antibody, immunocomplexes were resolved by SDS-PAGE and autoradiography. (B) TTP protein’s half life in sham-irradiated (-RT) and irradiated (4 Gy) groups were determined by densitometric scanning of the autoradiographs followed by quantitation using Image J1.32j software (NIH, Bethesda, MD). Relative protein levels were determined in comparison to sample isolated immediately after the pulse labeling (0 h chase). (C) MH-S cells were either sham-irradiated or radiated with 4 Gy, and 44 h after radiation 2 µM of MG132 was added. Cell lysates were collected 4 h after MG132 addition and immunoblotted for TTP and TNF-α. GAPDH was used as loading control.
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pone-0057290-g004: Radiation-induced TTP degradation and TNF-α secretion is inhibited significantly by the proteasome inhibitor MG132.(A) MH-S cells were metabolically labeled with 35S-methionine, were either sham-irradiated or irradiated with 4 Gy, and chased with cold methionine for indicated time periods. After the chase period cell lysates were subjected to immunoprecipitation using TTP antibody, immunocomplexes were resolved by SDS-PAGE and autoradiography. (B) TTP protein’s half life in sham-irradiated (-RT) and irradiated (4 Gy) groups were determined by densitometric scanning of the autoradiographs followed by quantitation using Image J1.32j software (NIH, Bethesda, MD). Relative protein levels were determined in comparison to sample isolated immediately after the pulse labeling (0 h chase). (C) MH-S cells were either sham-irradiated or radiated with 4 Gy, and 44 h after radiation 2 µM of MG132 was added. Cell lysates were collected 4 h after MG132 addition and immunoblotted for TTP and TNF-α. GAPDH was used as loading control.

Mentions: To better understand the radiation-induced TTP down-regulation, endogenous TTP protein’s half-life in MH-S cells was determined using 35S-methionine pulse-chase assay. As shown in Fig. 4A and calculated in Fig. 4B, in sham-irradiated MH-S cells, TTP protein’s half life was calculated to be >12 h, whereas 4 Gy reduced the half-life to about 9.5 h.To determine if proteasomal degradation was responsible for radiation-induced TTP down-regulation, we treated MH-S cells with MG132 (a proteasomal inhibitor) for 4 h prior to harvest. MG132 protected against radiation-induced TTP down-regulation and reduced radiation-induced TNF-α release (Fig. 4C). These data suggest that radiation reduces the TTP protein’s half-life via proteasomal degradation and further confirm the role of TTP in suppressing TNF-α release.


Tristetraprolin mediates radiation-induced TNF-α production in lung macrophages.

Ray D, Shukla S, Allam US, Helman A, Ramanand SG, Tran L, Bassetti M, Krishnamurthy PM, Rumschlag M, Paulsen M, Sun L, Shanley TP, Ljungman M, Nyati MK, Zhang M, Lawrence TS - PLoS ONE (2013)

Radiation-induced TTP degradation and TNF-α secretion is inhibited significantly by the proteasome inhibitor MG132.(A) MH-S cells were metabolically labeled with 35S-methionine, were either sham-irradiated or irradiated with 4 Gy, and chased with cold methionine for indicated time periods. After the chase period cell lysates were subjected to immunoprecipitation using TTP antibody, immunocomplexes were resolved by SDS-PAGE and autoradiography. (B) TTP protein’s half life in sham-irradiated (-RT) and irradiated (4 Gy) groups were determined by densitometric scanning of the autoradiographs followed by quantitation using Image J1.32j software (NIH, Bethesda, MD). Relative protein levels were determined in comparison to sample isolated immediately after the pulse labeling (0 h chase). (C) MH-S cells were either sham-irradiated or radiated with 4 Gy, and 44 h after radiation 2 µM of MG132 was added. Cell lysates were collected 4 h after MG132 addition and immunoblotted for TTP and TNF-α. GAPDH was used as loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585360&req=5

pone-0057290-g004: Radiation-induced TTP degradation and TNF-α secretion is inhibited significantly by the proteasome inhibitor MG132.(A) MH-S cells were metabolically labeled with 35S-methionine, were either sham-irradiated or irradiated with 4 Gy, and chased with cold methionine for indicated time periods. After the chase period cell lysates were subjected to immunoprecipitation using TTP antibody, immunocomplexes were resolved by SDS-PAGE and autoradiography. (B) TTP protein’s half life in sham-irradiated (-RT) and irradiated (4 Gy) groups were determined by densitometric scanning of the autoradiographs followed by quantitation using Image J1.32j software (NIH, Bethesda, MD). Relative protein levels were determined in comparison to sample isolated immediately after the pulse labeling (0 h chase). (C) MH-S cells were either sham-irradiated or radiated with 4 Gy, and 44 h after radiation 2 µM of MG132 was added. Cell lysates were collected 4 h after MG132 addition and immunoblotted for TTP and TNF-α. GAPDH was used as loading control.
Mentions: To better understand the radiation-induced TTP down-regulation, endogenous TTP protein’s half-life in MH-S cells was determined using 35S-methionine pulse-chase assay. As shown in Fig. 4A and calculated in Fig. 4B, in sham-irradiated MH-S cells, TTP protein’s half life was calculated to be >12 h, whereas 4 Gy reduced the half-life to about 9.5 h.To determine if proteasomal degradation was responsible for radiation-induced TTP down-regulation, we treated MH-S cells with MG132 (a proteasomal inhibitor) for 4 h prior to harvest. MG132 protected against radiation-induced TTP down-regulation and reduced radiation-induced TNF-α release (Fig. 4C). These data suggest that radiation reduces the TTP protein’s half-life via proteasomal degradation and further confirm the role of TTP in suppressing TNF-α release.

Bottom Line: To study the in vivo relevance, mouse lungs were irradiated with a single dose (15 Gy) and assessed at varying times for TTP alterations.In conclusion, irradiation of lung macrophages causes TTP inactivation via p38-mediated phosphorylation and proteasome-mediated degradation, leading to TNF-α production.These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, United States of America. dipray@umich.edu

ABSTRACT
The efficacy of radiation therapy for lung cancer is limited by radiation-induced lung toxicity (RILT). Although tumor necrosis factor-alpha (TNF-α) signaling plays a critical role in RILT, the molecular regulators of radiation-induced TNF-α production remain unknown. We investigated the role of a major TNF-α regulator, Tristetraprolin (TTP), in radiation-induced TNF-α production by macrophages. For in vitro studies we irradiated (4 Gy) either a mouse lung macrophage cell line, MH-S or macrophages isolated from TTP knockout mice, and studied the effects of radiation on TTP and TNF-α levels. To study the in vivo relevance, mouse lungs were irradiated with a single dose (15 Gy) and assessed at varying times for TTP alterations. Irradiation of MH-S cells caused TTP to undergo an inhibitory phosphorylation at Ser-178 and proteasome-mediated degradation, which resulted in increased TNF-α mRNA stabilization and secretion. Similarly, MH-S cells treated with TTP siRNA or macrophages isolated from ttp (-/-) mice had higher basal levels of TNF-α, which was increased minimally after irradiation. Conversely, cells overexpressing TTP mutants defective in undergoing phosphorylation released significantly lower levels of TNF-α. Inhibition of p38, a known kinase for TTP, by either siRNA or a small molecule inhibitor abrogated radiation-induced TNF-α release by MH-S cells. Lung irradiation induced TTP(Ser178) phosphorylation and protein degradation and a simultaneous increase in TNF-α production in C57BL/6 mice starting 24 h post-radiation. In conclusion, irradiation of lung macrophages causes TTP inactivation via p38-mediated phosphorylation and proteasome-mediated degradation, leading to TNF-α production. These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT.

Show MeSH
Related in: MedlinePlus