Low Dose Rate Radiation Induced Secretion of TGF-β3 Together With an Activator in Small Extracellular Vesicles Modifies Low Dose Hyper-Radiosensitivity Through ALK1 Binding

Abstract

Hyper-radiosensitivity (HRS) is the increased sensitivity to low doses of ionizing radiation observed in most cell lines. We previously demonstrated that HRS is permanently abolished in cells irradiated at a low dose rate (LDR), in a mechanism dependent on transforming growth factor β3 (TGF-β3). In this study, we aimed to elucidate the activation and receptor binding of TGF-β3 in this mechanism. T-47D cells were pre-treated with inhibitors of potential receptors and activators of TGF-β3, along with addition of small extracellular vesicles (sEVs) from LDR primed cells, before their radiosensitivity was assessed by the clonogenic assay. The protein content of sEVs from LDR primed cells was analyzed with mass spectrometry. Our results show that sEVs contain TGF-β3 regardless of priming status, but only sEVs from LDR primed cells remove HRS in reporter cells. Inhibition of the matrix metalloproteinase (MMP) family prevents removal of HRS, suggesting an MMP-dependent activation of TGF-β3 in the LDR primed cells. We demonstrate a functional interaction between TGF-β3 and activin receptor like kinase 1 (ALK1), by showing that TGF-β3 removes HRS through ALK1 binding, independent of ALK5 and TGF-βRII. These results are an important contribution to a more comprehensive understanding of the mechanism behind TGF-β3 mediated removal of HRS.

TGF-β3 increases the severity of radiation-induced oral mucositis and salivary gland fibrosis in a mouse model

“…Considering this, the endogenous TGF-β1 concentration and its change after RT, in addition to the administered TGF-β3 Furthermore, both isoforms have been shown to bind to and signal through TGF-β type I receptors activin-like kinase (ALK) 1 and ALK5, with downstream phosphorylation of Smad 1/5/8 and Smad 2/3, respectively, resulting in different functional effects (Lux et al 1999;Shi and Massagué 2003;De Kroon et al 2015). In a recent study, we discovered that ALK5 inhibition induced ALK1 binding by TGF-β3, indicating a competition between the two receptors, where ALK5 has the higher affinity (Hanson et al 2022). Thus the relative availability of different TGF-β receptors, in addition to the concentration of the cytokines, may influence the development of fibrosis mediated by TGF-β.…”