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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 causes significant down-regulation of TTP in irradiated mouse lung.(A) C57BL/6 mice were either sham-irradiated or the whole lung was irradiated with a single dose of 15 Gy. Tissue lysates were prepared from three individual animals at each indicated time points post-radiation and immunoblotted using indicated antibodies. (B) Mouse lung cryo-sections from sham-radiated or 15 Gy single fraction radiated mice were isolated at indicated time points and immune-staining were performed as described in materials and methods.
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pone-0057290-g006: Radiation causes significant down-regulation of TTP in irradiated mouse lung.(A) C57BL/6 mice were either sham-irradiated or the whole lung was irradiated with a single dose of 15 Gy. Tissue lysates were prepared from three individual animals at each indicated time points post-radiation and immunoblotted using indicated antibodies. (B) Mouse lung cryo-sections from sham-radiated or 15 Gy single fraction radiated mice were isolated at indicated time points and immune-staining were performed as described in materials and methods.

Mentions: To determine if radiation induces p38 phosphorylation, TTP phosphorylation and TNF-α down-regulation in vivo, mice were irradiated, and the lungs were assessed at different time points post-radiation. As shown in Fig. 6A, there was a significant up-regulation of TTP (Ser178) phosphorylation and a down-regulation of steady state TTP levels between 72 h-2 weeks post-radiation. Furthermore, reprobing the membrane with Thr180 and Tyr182 phospho-specific p38 antibody showed increased phosphorylation as well. Finally, immunofluorescence staining of cryo-preserved lung sections confirmed that radiation caused an increase in TNF-α (Fig. 6B). These observations demonstrate that p38 phosphoryation (associated with activation), TTP phosphorylation and down regulation (associated with inactivation), and TNF-α induction occur in vivo as well as in vitro.


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 causes significant down-regulation of TTP in irradiated mouse lung.(A) C57BL/6 mice were either sham-irradiated or the whole lung was irradiated with a single dose of 15 Gy. Tissue lysates were prepared from three individual animals at each indicated time points post-radiation and immunoblotted using indicated antibodies. (B) Mouse lung cryo-sections from sham-radiated or 15 Gy single fraction radiated mice were isolated at indicated time points and immune-staining were performed as described in materials and methods.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057290-g006: Radiation causes significant down-regulation of TTP in irradiated mouse lung.(A) C57BL/6 mice were either sham-irradiated or the whole lung was irradiated with a single dose of 15 Gy. Tissue lysates were prepared from three individual animals at each indicated time points post-radiation and immunoblotted using indicated antibodies. (B) Mouse lung cryo-sections from sham-radiated or 15 Gy single fraction radiated mice were isolated at indicated time points and immune-staining were performed as described in materials and methods.
Mentions: To determine if radiation induces p38 phosphorylation, TTP phosphorylation and TNF-α down-regulation in vivo, mice were irradiated, and the lungs were assessed at different time points post-radiation. As shown in Fig. 6A, there was a significant up-regulation of TTP (Ser178) phosphorylation and a down-regulation of steady state TTP levels between 72 h-2 weeks post-radiation. Furthermore, reprobing the membrane with Thr180 and Tyr182 phospho-specific p38 antibody showed increased phosphorylation as well. Finally, immunofluorescence staining of cryo-preserved lung sections confirmed that radiation caused an increase in TNF-α (Fig. 6B). These observations demonstrate that p38 phosphoryation (associated with activation), TTP phosphorylation and down regulation (associated with inactivation), and TNF-α induction occur in vivo as well as in vitro.

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