Primary cilium restricts TGF-β/SMAD signaling induced RIBEs in the co-culture model

Transforming growth factor β (TGF-β) is the predominant cytokine responding to ionizing radiation and participates in radiation induced bystander effects (RIBEs). Primary cilia (PC) coordinates with multiple signaling pathways, exhibit specialized functions in TGF-β signal transduction. Our previous studies using a medium transfer model revealed that PC modulates RIBEs by restricting TGF-β signaling. To further investigate PC's mechanistic role in RIBEs, proton microbeam radiation (MR) and partial radiation (PR) were used to arrange the co-culture bystander system. Key methodologies included siRNA-mediated PC formation modulation, inhibition of DNA damage response kinases ataxia telangiectasia-mutated gene (ATM), Ataxia telangiectasia and Rad3-related protein (ATR) or DNA-dependent protein kinase (DNApk), and quantitative analysis of γH2AX foci, cell proliferation, and reactive oxygen/nitrogen species (ROS/NO). The results showed that, firstly, PC inhibition in both PR and MR models significantly increased γH2AX foci formation and protein levels within 12 h while suppressing cell proliferation, which were reversed by TGF-β signaling inhibition. Secondly, although inhibition of ATM, ATR, or DNApk reduced γH2AX foci but resulted in decrease of proliferation rate. Scavenging ROS/NO similarly attenuated DNA damage but enhanced cell survival. Third, SMAD2/3 complex inhibition in PC-deficient bystander cells downregulated ATM/ATR expression and reduced intracellular NO/ROS levels. These results demonstrate that TGF-β1 drives RIBEs-associated DNA damage through canonical p-SMAD2/3 signaling, which amplifies ROS/NO production and hyperactivates ATM, ATR or DNApk. Crucially, PC acts as a regulatory role in DNA damages of RIBEs progression.