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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 increased the TNF-α transcript and its release by MH-S cells 48 h post–radiation.(A) MH-S cells were left untreated (0 Gy) or irradiated (4 Gy) and culture supernatants were collected at the indicated time points. Released TNF-α levels were quantified using ELISA kits according to the manufacturer’s instruction. (B) Cells were treated as above, and RNA was isolated and quantified at the indicated time points as described in the materials and methods. (C) MH-S cells were either sham irradiated or irradiated with 4 Gy. 48 h post-irradiation TNF-α mRNA level, stability and synthesis was determined using BrU pulse-chase labeling experiment as described previously [28].
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pone-0057290-g001: Radiation increased the TNF-α transcript and its release by MH-S cells 48 h post–radiation.(A) MH-S cells were left untreated (0 Gy) or irradiated (4 Gy) and culture supernatants were collected at the indicated time points. Released TNF-α levels were quantified using ELISA kits according to the manufacturer’s instruction. (B) Cells were treated as above, and RNA was isolated and quantified at the indicated time points as described in the materials and methods. (C) MH-S cells were either sham irradiated or irradiated with 4 Gy. 48 h post-irradiation TNF-α mRNA level, stability and synthesis was determined using BrU pulse-chase labeling experiment as described previously [28].

Mentions: As lung macrophages are the major TNF-α producing cell upon radiation, we began by assessing the effect of radiation on TNF-α secretion by the mouse lung macrophage cell line, MH-S. We treated MH-S with different doses (2 and 4 Gy) of radiation and quantified TNF-α secretion into the culture supernatant using ELISA between 30 min till 48 h post-radiation. Two Gy had no major effects on TNF-α release until 48 h after radiation (data not shown); however 4 Gy consistently induced the secretion of the cytokine at 48 h post-irradiation (Fig. 1). To understand the time course of radiation-induced TNF-α secretion, we harvested the culture supernatants at various time points post-radiation (Fig. 1). We found that there was a 2.4 fold (control, 45±4.5 pg/ml; +RT, 110±5.2 pg/ml) increase in TNF-α secretion (Fig. 1A) and a 2.1±0.18 fold increase in TNF-α transcript (Fig. 1B) 48 h after radiation. To distinguish the involvements of mRNA synthesis versus alteration of mRNA stability, we performed BrU pulse-chase labeling assay (see methods). We found that radiation increased the stability of TNF-α transcript to 1.82±0.37 fold (n = 3), whereas no significant change in synthesis was noted at 48 h post-irradiation (Fig. 1C). Metabolic labeling of MH-S cells with 35S-methionine followed by immunoprecipitation of TNF-α using specific antibody showed that radiation did not change the rate of TNF-α translation up to 48 h post-irradiation (Fig. S1). From these analyses we concluded that irradiation of MH-S cells increases TNF-α transcript stability, which may be responsible for the increased secretion of the cytokine.


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 increased the TNF-α transcript and its release by MH-S cells 48 h post–radiation.(A) MH-S cells were left untreated (0 Gy) or irradiated (4 Gy) and culture supernatants were collected at the indicated time points. Released TNF-α levels were quantified using ELISA kits according to the manufacturer’s instruction. (B) Cells were treated as above, and RNA was isolated and quantified at the indicated time points as described in the materials and methods. (C) MH-S cells were either sham irradiated or irradiated with 4 Gy. 48 h post-irradiation TNF-α mRNA level, stability and synthesis was determined using BrU pulse-chase labeling experiment as described previously [28].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585360&req=5

pone-0057290-g001: Radiation increased the TNF-α transcript and its release by MH-S cells 48 h post–radiation.(A) MH-S cells were left untreated (0 Gy) or irradiated (4 Gy) and culture supernatants were collected at the indicated time points. Released TNF-α levels were quantified using ELISA kits according to the manufacturer’s instruction. (B) Cells were treated as above, and RNA was isolated and quantified at the indicated time points as described in the materials and methods. (C) MH-S cells were either sham irradiated or irradiated with 4 Gy. 48 h post-irradiation TNF-α mRNA level, stability and synthesis was determined using BrU pulse-chase labeling experiment as described previously [28].
Mentions: As lung macrophages are the major TNF-α producing cell upon radiation, we began by assessing the effect of radiation on TNF-α secretion by the mouse lung macrophage cell line, MH-S. We treated MH-S with different doses (2 and 4 Gy) of radiation and quantified TNF-α secretion into the culture supernatant using ELISA between 30 min till 48 h post-radiation. Two Gy had no major effects on TNF-α release until 48 h after radiation (data not shown); however 4 Gy consistently induced the secretion of the cytokine at 48 h post-irradiation (Fig. 1). To understand the time course of radiation-induced TNF-α secretion, we harvested the culture supernatants at various time points post-radiation (Fig. 1). We found that there was a 2.4 fold (control, 45±4.5 pg/ml; +RT, 110±5.2 pg/ml) increase in TNF-α secretion (Fig. 1A) and a 2.1±0.18 fold increase in TNF-α transcript (Fig. 1B) 48 h after radiation. To distinguish the involvements of mRNA synthesis versus alteration of mRNA stability, we performed BrU pulse-chase labeling assay (see methods). We found that radiation increased the stability of TNF-α transcript to 1.82±0.37 fold (n = 3), whereas no significant change in synthesis was noted at 48 h post-irradiation (Fig. 1C). Metabolic labeling of MH-S cells with 35S-methionine followed by immunoprecipitation of TNF-α using specific antibody showed that radiation did not change the rate of TNF-α translation up to 48 h post-irradiation (Fig. S1). From these analyses we concluded that irradiation of MH-S cells increases TNF-α transcript stability, which may be responsible for the increased secretion of the cytokine.

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