Intracellular Calcium Overload Promotes NFATc1-ATF3 Activation and Induces the Senescence-Associated Phenotype in Irradiated Osteocytes

Although calcium overload dysregulation has been implicated in cellular senescence, its role in ionizing radiation (IR)-induced osteocyte senescence, a key pathogenic mechanism underlying radiotherapy-associated bone injury, remains poorly explored. This study investigated whether IR-induced osteocyte senescence is mediated through the Ca2+-NFATc1-ATF3 pathway. Exposure to 2 Gy X-rays impaired osteocyte homeostasis, manifesting as reduced viability and proliferation, G2/M phase arrest, and dendritic retraction. IR also induced persistent DNA damage response and senescence-associated phenotypes, including increased γ-H2AX foci, SA-β-gal activity, condensed punctate DAPI-dense nuclear foci, p16/p21 expression, and pro-inflammatory SASP profile. Intracellular Ca2+ levels surged within 6 h post-irradiation and remained elevated for at least 72 h in a dose-dependent manner. Pharmacological Ca2+ modulation with BAPTA-AM or verapamil attenuated IR-induced intracellular Ca2+ accumulation, G2/M arrest, SA-β-gal positivity, p21/p53 upregulation, and SASP secretion. Conditioned medium from irradiated osteocytes inhibited BMSC-mediated osteogenesis and enhanced BMM-driven osteoclastogenesis, whereas Ca2+ modulation partially mitigated these paracrine effects. Mechanistically, IR promoted NFATc1 nuclear translocation and ATF3 upregulation. Collectively, these findings support an important role for pathological intracellular Ca2+ elevation in IR-induced osteocyte senescence and suggest that the Ca2+-NFATc1-ATF3 axis may represent a potential therapeutic target for mitigating radiation-associated disruption of bone homeostasis.