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

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.

Rapid Proteasomal Degradation of Posttranscriptional Regulators of the TIS11/Tristetraprolin Family Is Induced by an Intrinsically Unstructured Region Independently of Ubiquitination

“…The stability of TIS11/TTP proteins is known to be defined by their phosphorylation status, which in turn determines their degradation rate (20)(21)(22). Deregulation of this control can have dramatic effects on cell fate, leading to defects in the inflammatory response (23), increased radiation-induced lung toxicity (24), and tumorigenesis (25). Previous studies established the proteasome as the proteolytic complex responsible for the degradation of several TIS11/TTP proteins, as its inhibition stabilizes TTP (20) and BRF1 (21).…”

Involvement of p38-βTrCP-Tristetraprolin-TNFα axis in radiation pneumonitis

“…As Ttp knockout mice were found to be sensitive to radiation pneumonitis, and as we previously reported that radiation causes TTP protein inactivation primarily via enhanced inhibitory phosphorylation as well as via inducing protein degradation [ 10 ], we next focused our studies on improving the mechanistic understanding of TTP degradation machinery. To ascertain whether radiation-induced TTP protein degradation is mediated via ubiquitination-mediated proteasomal degradation, we utilized both overexpression and endogenous systems.…”

“…We previously identified an RNA binding anti-inflammatory protein, Tristetraprolin (TTP) as a major negative regulator of radiation-induced TNF-α synthesis by mouse lung macrophages [ 10 ]. We found radiation promotes TTP inactivation via phosphorylation and proteasomal degradation, which correlates with increased TNF-α synthesis and release.…”

“…Specific phosphorylation of TTP at serine 186 by p38 MAPK inactivates its mRNA-destabilizing ability [ 19 ]. We also found that radiation-induces activation of p38 leading to phosphorylation of TTP at Ser 186 [ 10 ]. While analyzing the amino acid sequence, we further noted that upon phosphorylation of Ser 186 , surrounding residues ( 186 S P FSGLPS 192 ) can potentially serve as a recognition motif (phospho-degron) for an F-box family ubiquitin ligase (E3), SCF β-TrCP (consensus D p SGxx p S, where x could be any amino acid) [ 20 , 21 ].…”

Mir-138 plays an important role in diabetic nephropathy through SIRT1-p38-TTP regulatory axis

“…Increased DN inflammation was associated with the downregulation of the RNA-binding protein tristetraprolin (TTP), also known as zinc finger protein 36 homolog (ZFP36). TTP has been reported to regulate the stability of multiple target mRNAs, including proinflammatory cytokines and chemokines such as TNFα, IL-6, and IL-8 [26,27]. TTPdeficient mice exhibit a severe inflammation syndrome characterized by cachexia, polyarthritis, and autoimmunity.…”