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 TTPSer178 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.
Highlights
Radiation-induced lung toxicity (RILT) limits the use of potentially curative high-dose-radiation for patients with thoracic malignancies [1] and occurs in up to 30% of treated patients [2,3]
As lung macrophages are the major tumor necrosis factor-alpha (TNF-a) producing cell upon radiation, we began by assessing the effect of radiation on TNFa secretion by the mouse lung macrophage cell line, MH-S
From these analyses we concluded that irradiation of MH-S cells increases TNF-a transcript stability, which may be responsible for the increased secretion of the cytokine
Summary
Radiation-induced lung toxicity (RILT) limits the use of potentially curative high-dose (chemo)-radiation for patients with thoracic malignancies [1] and occurs in up to 30% of treated patients [2,3]. TTP binds directly to the AU-rich element in the 39-UTR of TNF-a transcript leading to its destabilization and rapid degradation [19]. TTP knockout mice have high endogenous levels of TNF-a, due directly to the absence of TTP-mediated inhibition of TNF-a production [18]. In contrast to most proteins, phosphorylation of TTP leads to its inactivation ( producing effects similar to TTP knockdown). This is because, compared to the phosphorylated TTP, the unphosphorylated or de-phosphorylated form of TTP recruits more efficiently the deadenylase and mRNA decapping complexes to the AU-rich element containing TNF-a transcript to cause rapid degradation [20,21]. Absent or phosphorylated TTP is associated with increased TNF-a production, whereas unphosphorylated or de-phosphorylated TTP decreases TNF-a levels
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