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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 caused increased phosphorylation and down-regulation of TTP in MH-S cells.(A) MH-S cells were either sham irradiated or treated with 4 Gy. Cells were harvested at indicated time points and immunoblotted using indicated antibodies. (B) CHO cells overexpressing mouse TTP were treated with 4 Gy, harvested at different time points, and immunoblotted using indicated antibodies. (C) U2OS cells overexpressing human TTP were irradiated with either 4 or 8 Gy and immunoblotted using indicated antibodies.
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pone-0057290-g003: Radiation caused increased phosphorylation and down-regulation of TTP in MH-S cells.(A) MH-S cells were either sham irradiated or treated with 4 Gy. Cells were harvested at indicated time points and immunoblotted using indicated antibodies. (B) CHO cells overexpressing mouse TTP were treated with 4 Gy, harvested at different time points, and immunoblotted using indicated antibodies. (C) U2OS cells overexpressing human TTP were irradiated with either 4 or 8 Gy and immunoblotted using indicated antibodies.

Mentions: We have used two systems (endogenous and over-expressed) to study the effect of radiation on post-translational modifications of TTP. To determine if the effect of radiation was mediated by TTP phosphorylation, cell lysates were prepared from both non-irradiated and irradiated MH-S cells at various times starting at 10 min after radiation. Within 10 minutes after radiation, there was a significant increase in Ser178 phosphorylation of TTP, which gradually declined with time (Fig. 3A). A similar trend in TTP phosphorylation kinetics was detected when TTP over-expressing CHO cells (which express undetectable levels of TTP) were irradiated (Fig. 3B). Interestingly, at later time points (between 24–48 h post-radiation), we found a significant down-regulation of TTP protein levels in MH-S cells (Fig. 3A). There was a similar dose-dependent radiation-induced down-regulation of overexpressed TTP in U2OS cells (an osteosarcoma cell line having undetectable endogenous TTP) at 24 h after 4 and 8 Gy (Fig. 3C). These observations show that radiation causes an early enhancement of TTP Ser178 phosphorylation and late total TTP down-regulation.


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 caused increased phosphorylation and down-regulation of TTP in MH-S cells.(A) MH-S cells were either sham irradiated or treated with 4 Gy. Cells were harvested at indicated time points and immunoblotted using indicated antibodies. (B) CHO cells overexpressing mouse TTP were treated with 4 Gy, harvested at different time points, and immunoblotted using indicated antibodies. (C) U2OS cells overexpressing human TTP were irradiated with either 4 or 8 Gy and immunoblotted using indicated antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585360&req=5

pone-0057290-g003: Radiation caused increased phosphorylation and down-regulation of TTP in MH-S cells.(A) MH-S cells were either sham irradiated or treated with 4 Gy. Cells were harvested at indicated time points and immunoblotted using indicated antibodies. (B) CHO cells overexpressing mouse TTP were treated with 4 Gy, harvested at different time points, and immunoblotted using indicated antibodies. (C) U2OS cells overexpressing human TTP were irradiated with either 4 or 8 Gy and immunoblotted using indicated antibodies.
Mentions: We have used two systems (endogenous and over-expressed) to study the effect of radiation on post-translational modifications of TTP. To determine if the effect of radiation was mediated by TTP phosphorylation, cell lysates were prepared from both non-irradiated and irradiated MH-S cells at various times starting at 10 min after radiation. Within 10 minutes after radiation, there was a significant increase in Ser178 phosphorylation of TTP, which gradually declined with time (Fig. 3A). A similar trend in TTP phosphorylation kinetics was detected when TTP over-expressing CHO cells (which express undetectable levels of TTP) were irradiated (Fig. 3B). Interestingly, at later time points (between 24–48 h post-radiation), we found a significant down-regulation of TTP protein levels in MH-S cells (Fig. 3A). There was a similar dose-dependent radiation-induced down-regulation of overexpressed TTP in U2OS cells (an osteosarcoma cell line having undetectable endogenous TTP) at 24 h after 4 and 8 Gy (Fig. 3C). These observations show that radiation causes an early enhancement of TTP Ser178 phosphorylation and late total TTP down-regulation.

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