Tristetraprolin Mediates Radiation-Induced TNF-α Production in Lung Macrophages

The efficacy of radiation therapy for lung cancer is limited by radiation-induced lung toxicity (RILT). Although tumor necrosis factor-alpha (TNF-alpha) signaling plays a critical role in RILT, the molecular regulators of radiation-induced TNF-alpha production remain unknown. We investigated the role of a major TNF-alpha regulator, Tristetraprolin (TTP), in radiation-induced TNFa 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-alpha 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-alpha mRNA stabilization and secretion. Similarly, MH-S cells treated with TTP siRNA or macrophages isolated from ttp (2/2) mice had higher basal levels of TNF-alpha, which was increased minimally after irradiation. Conversely, cells overexpressing TTP mutants defective in undergoing phosphorylation released significantly lower levels of TNF-alpha. Inhibition of p38, a known kinase for TTP, by either siRNA or a small molecule inhibitor abrogated radiation-induced TNF-alpha release by MH-S cells. Lung irradiation induced TTP Ser178 phosphorylation and protein degradation and a simultaneous increase in TNF-alpha 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-alpha production. These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT.