Acute activation of autophagy enables growth plate regeneration following radiation-induced injury.

Abstract

Purpose Radiation injury to growth plates commonly leads to skeletal late complications including short stature, limb length-discrepancy, and scoliosis/kyphosis in pediatric oncology patients. We aimed to understand the acute responses of direct growth plate irradiation that result in skeletal late complications. Materials and methods We first established an in vivo model of focal growth plate irradiation that recapitulates the clinical development of skeletal late complications and used it to explore the responses of growth plate chondrocytes within the first 72 hours of radiation exposure. To monitor acute effects of radiation exposure on human chondrocytes, rare human growth plate biopsies were exposed to ionizing radiation ex vivo. Using these approaches, we applied clonal genetic tracing and immunofluorescence to monitor changes at the cellular and molecular levels. Functional in vivo perturbations were conducted with clinically-relevant autophagy inhibitor, hydroxychloroquine. Results Growth plate irradiation disrupted the continuous production of chondrocytes required for bone elongation and was associated with DNA damage throughout the growth plate. Indicators of growth plate activity, SOX9 and the phosphorylated form of ribosomal protein S6, decreased during a 6- and 24-hour post-irradiation window but returned to normal levels 72 hours after irradiation. We identified a surge in autophagic flux throughout the growth plate during this window, based on temporal SQSTM1 and LAMP1 protein levels. The earliest stages of these response mechanisms are conserved between species and relevant to humans. Hydroxychloroquine treatment immediately after radiation injury in mice impaired growth plate regeneration, resulting in more severe late complications. Conclusion Our findings demonstrate that autophagy is an important acute response to irradiation in growth plate chondrocytes, revealing a novel potential therapeutic target for preventing radiation-induced skeletal late complications.