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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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Tristetraprolin negatively regulates radiation-induced TNF-α production.(A) MH-S cells were treated with either control (C) or TTP (T) siRNA and 24 h post-transfection cells were left un-irradiated or irradiated with 4 Gy. Cell lysates were prepared 48 h post-radiation and immunoblotted with the indicated antibodies. (B) TNF-α levels secreted in the culture supernatant were quantitated on the above mentioned samples and represented as fold change considering the sham irradiated control siRNA-treated sample as 1. (C) Bone marrow from ttp (+/+) and ttp (−/−) mice were isolated and differentiated into macrophages as described in the materials and methods. Left panel shows the PCR based genotyping of the mice used in the study and the right panel confirms macrophage differentiation using F4/80 immunofluorescence staining. (D) Bone marrow derived macrophages were either sham irradiated or radiated with 4 Gy. 12 h post-irradiation RNA samples were isolated and TNF-α transcript levels were quantitated as described in materials and methods. (E) MH-S cells were transfected with either vector control DNA or with various TTP constructs (WT, Ser52Ala, Ser178Ala, and Ser52/178Ala). 24 h post-transfection, cells were exposed to 4 Gy, and TNF-α secretion was quantified in culture supernatant 48 h post-radiation.
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pone-0057290-g002: Tristetraprolin negatively regulates radiation-induced TNF-α production.(A) MH-S cells were treated with either control (C) or TTP (T) siRNA and 24 h post-transfection cells were left un-irradiated or irradiated with 4 Gy. Cell lysates were prepared 48 h post-radiation and immunoblotted with the indicated antibodies. (B) TNF-α levels secreted in the culture supernatant were quantitated on the above mentioned samples and represented as fold change considering the sham irradiated control siRNA-treated sample as 1. (C) Bone marrow from ttp (+/+) and ttp (−/−) mice were isolated and differentiated into macrophages as described in the materials and methods. Left panel shows the PCR based genotyping of the mice used in the study and the right panel confirms macrophage differentiation using F4/80 immunofluorescence staining. (D) Bone marrow derived macrophages were either sham irradiated or radiated with 4 Gy. 12 h post-irradiation RNA samples were isolated and TNF-α transcript levels were quantitated as described in materials and methods. (E) MH-S cells were transfected with either vector control DNA or with various TTP constructs (WT, Ser52Ala, Ser178Ala, and Ser52/178Ala). 24 h post-transfection, cells were exposed to 4 Gy, and TNF-α secretion was quantified in culture supernatant 48 h post-radiation.

Mentions: Tristetraprolin (TTP) regulates mRNA stability of various inflammatory cytokines, most notably TNF-α (see Introduction). As the irradiation-induced TNF-α increase in MH-S cells was associated with increased TNF-α mRNA stability (Fig. 1C), we wished to characterize the involvement of TTP in radiation-induced TNF-α production. We used three major strategies: (i) siRNA-mediated gene silencing (ii) macrophages isolated from TTP-deficient mice and (iii) over-expression of super-active TTP mutants. TTP siRNA was able to down-regulate about 70% of the endogenous protein levels in MH-S cells (Fig. 2A, lane 2) which caused approximately a 2.5 fold increase in TNF-α secretion by macrophages even without radiation. Furthermore, in the control siRNA-treated group, 4 Gy caused a decrease of about 50% of endogenous TTP levels compared to the sham-irradiated group (Fig. 2A, lane 3), which was associated with a 2.5 fold increase in TNF-α secretion. This change was similar to that observed after TTP siRNA treatment without radiation. Interestingly, only about 5% of TTP remained after the combination of radiation and TTP siRNA (Fig. 2A, lane 4), which was associated with an additional 1.6 fold increase in TNF-α secretion (P<0.001; Fig. 2B). To better understand the role of TTP, we isolated bone marrow from ttp (+/+) and ttp (−/−) mice and differentiated them to macrophages as evidenced by the F4/80 positive staining (Fig. 2C). As shown in Fig. 2D, macrophages isolated from wild type mice showed a trend of increased TNF-α mRNA levels (1.48±0.17) upon 4 Gy of irradiation similar to irradiated MH-S cells. As reported earlier, the basal TNF-α transcript level was higher (2.13±0.01) in ttp- macrophages, which upon radiation increased to 2.53±0.02 fold 12 h post-irradiation (Fig. 2D). Conversely, transient over-expression of either wild type (wt) TTP or mutants of TTP that are unable to be phosphorylated [Ser52A (S52A), Ser178A (S178A), and SS52/178AA] and inactivated caused a significant inhibition of radiation-induced TNF-α secretion 48 h post-radiation compared to vector transfected cells (Fig. 2E). Vector control transfected MH-S cells showed a 2.2±0.3 fold increase in TNF-α secretion upon radiation as compared to non-irradiated cells; cells transfected with TTP-SS52/178AA showed only a modest increase of about 1.3±0.1 fold (P<0.0017). These experiments demonstrate TTP involvement in radiation-induced TNF-α secretion.


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)

Tristetraprolin negatively regulates radiation-induced TNF-α production.(A) MH-S cells were treated with either control (C) or TTP (T) siRNA and 24 h post-transfection cells were left un-irradiated or irradiated with 4 Gy. Cell lysates were prepared 48 h post-radiation and immunoblotted with the indicated antibodies. (B) TNF-α levels secreted in the culture supernatant were quantitated on the above mentioned samples and represented as fold change considering the sham irradiated control siRNA-treated sample as 1. (C) Bone marrow from ttp (+/+) and ttp (−/−) mice were isolated and differentiated into macrophages as described in the materials and methods. Left panel shows the PCR based genotyping of the mice used in the study and the right panel confirms macrophage differentiation using F4/80 immunofluorescence staining. (D) Bone marrow derived macrophages were either sham irradiated or radiated with 4 Gy. 12 h post-irradiation RNA samples were isolated and TNF-α transcript levels were quantitated as described in materials and methods. (E) MH-S cells were transfected with either vector control DNA or with various TTP constructs (WT, Ser52Ala, Ser178Ala, and Ser52/178Ala). 24 h post-transfection, cells were exposed to 4 Gy, and TNF-α secretion was quantified in culture supernatant 48 h post-radiation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585360&req=5

pone-0057290-g002: Tristetraprolin negatively regulates radiation-induced TNF-α production.(A) MH-S cells were treated with either control (C) or TTP (T) siRNA and 24 h post-transfection cells were left un-irradiated or irradiated with 4 Gy. Cell lysates were prepared 48 h post-radiation and immunoblotted with the indicated antibodies. (B) TNF-α levels secreted in the culture supernatant were quantitated on the above mentioned samples and represented as fold change considering the sham irradiated control siRNA-treated sample as 1. (C) Bone marrow from ttp (+/+) and ttp (−/−) mice were isolated and differentiated into macrophages as described in the materials and methods. Left panel shows the PCR based genotyping of the mice used in the study and the right panel confirms macrophage differentiation using F4/80 immunofluorescence staining. (D) Bone marrow derived macrophages were either sham irradiated or radiated with 4 Gy. 12 h post-irradiation RNA samples were isolated and TNF-α transcript levels were quantitated as described in materials and methods. (E) MH-S cells were transfected with either vector control DNA or with various TTP constructs (WT, Ser52Ala, Ser178Ala, and Ser52/178Ala). 24 h post-transfection, cells were exposed to 4 Gy, and TNF-α secretion was quantified in culture supernatant 48 h post-radiation.
Mentions: Tristetraprolin (TTP) regulates mRNA stability of various inflammatory cytokines, most notably TNF-α (see Introduction). As the irradiation-induced TNF-α increase in MH-S cells was associated with increased TNF-α mRNA stability (Fig. 1C), we wished to characterize the involvement of TTP in radiation-induced TNF-α production. We used three major strategies: (i) siRNA-mediated gene silencing (ii) macrophages isolated from TTP-deficient mice and (iii) over-expression of super-active TTP mutants. TTP siRNA was able to down-regulate about 70% of the endogenous protein levels in MH-S cells (Fig. 2A, lane 2) which caused approximately a 2.5 fold increase in TNF-α secretion by macrophages even without radiation. Furthermore, in the control siRNA-treated group, 4 Gy caused a decrease of about 50% of endogenous TTP levels compared to the sham-irradiated group (Fig. 2A, lane 3), which was associated with a 2.5 fold increase in TNF-α secretion. This change was similar to that observed after TTP siRNA treatment without radiation. Interestingly, only about 5% of TTP remained after the combination of radiation and TTP siRNA (Fig. 2A, lane 4), which was associated with an additional 1.6 fold increase in TNF-α secretion (P<0.001; Fig. 2B). To better understand the role of TTP, we isolated bone marrow from ttp (+/+) and ttp (−/−) mice and differentiated them to macrophages as evidenced by the F4/80 positive staining (Fig. 2C). As shown in Fig. 2D, macrophages isolated from wild type mice showed a trend of increased TNF-α mRNA levels (1.48±0.17) upon 4 Gy of irradiation similar to irradiated MH-S cells. As reported earlier, the basal TNF-α transcript level was higher (2.13±0.01) in ttp- macrophages, which upon radiation increased to 2.53±0.02 fold 12 h post-irradiation (Fig. 2D). Conversely, transient over-expression of either wild type (wt) TTP or mutants of TTP that are unable to be phosphorylated [Ser52A (S52A), Ser178A (S178A), and SS52/178AA] and inactivated caused a significant inhibition of radiation-induced TNF-α secretion 48 h post-radiation compared to vector transfected cells (Fig. 2E). Vector control transfected MH-S cells showed a 2.2±0.3 fold increase in TNF-α secretion upon radiation as compared to non-irradiated cells; cells transfected with TTP-SS52/178AA showed only a modest increase of about 1.3±0.1 fold (P<0.0017). These experiments demonstrate TTP involvement in radiation-induced TNF-α secretion.

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