A two phase regulation of bone regeneration: IL-17F mediates osteoblastogenesis via C/EBP-β in vitro

T lymphocytes and pro-inflammatory cytokines, specifically interleukin-17F (IL-17F) have been identified as important regulators in bone regeneration during fracture repair. To better understand the molecular mechanisms of IL-17F-mediated osteoblastogenesis, a mouse pre-osteoblast cell line (MC3T3-E1) was utilized to characterize the intracellular signal transduction of IL-17F. Comparisons to the established canonical Wnt signaling pathway were made using Wnt3a ligand. Our results demonstrated greater bone marker gene expression in IL-17F-treated cells, compared to cells treated with Wnt3a. Western blot analysis confirmed degradation of β-catenin and up-regulation of two key proteins in osteoblast differentiation, Runx2 and C/EBP-β, in response to IL-17F treatment. RNA silencing of IL-17F receptors, IL-17Ra and IL-17Rc via siRNA transfection resulted in decreased expression of Act2, Runx2, and C/EBP-β, demonstrating the direct ligand-receptor interaction between IL-17F and IL-17Ra/c as an activator of osteoblastogenesis. Our findings suggest that IL-17F promotes osteoblast differentiation independent of the canonical Wnt pathway and β-catenin signaling, presenting new insights on modulating the adaptive immune response in the inflammatory phase, temporally distinct from the reparative and remodeling phases of fracture healing.