Irisin reduces bone fracture by facilitating osteogenesis and antagonizing TGF-8/Smad signaling in a growing mouse model of osteogenesis imperfecta

Objective: Osteogenesis imperfecta (OI) is a congenital disorder characterized by muscle defect and skeletal fragility, and no cure is yet available. Crosstalk between bone and muscle has become a new coming focus of therapeutic strategy in OI. Irisin, a secreted myokine, was found to be involved in regulating bone metabolism, and may be beneficial for the treatment of OI. However, its effects in OI have yet to be determined. This study sought to determine whether Irisin therapy is capable of reducing fracture risk in OI and to investigate the po-tential mechanisms of action.Methods: Fibronectin type III domain containing 5 (FNDC5)/Irisin expression was assessed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining. In vivo, X-ray was used for fracture count-ing and micro-CT, dynamic histomorphometry analysis, immunohistochemistry, histomorphometry, and biome-chanical test were used to evaluate the effects of Irisin on fracture frequency and bone quality in OI mouse model, oim/oim mouse. In vitro, osteogenesis-related gene expressions were determined by quantitative real-time PCR (qRT-PCR), western blot, and osteoblastogenesis assay were assessed by alkaline phosphatase (ALP) staining and alizarin red S (ARS) staining. Mechanistically, cell immunofluorescence staining, co-immunoprecipitation (co-IP) molecular docking, western blot, luciferase reporter assay, and chromatin immunoprecipitation (ChIP) assay were used for elucidating the mechanisms of how Irisin antagonized transforming growth factor-8 (TGF-8)/Smad signaling in oim/oim osteoblasts and further attenuated the inhibitory effect of TGF-81 on osteogenic differentiation.Results: Musculoskeletal system-related FNDC5/Irisin was decreased in the serum, muscle, and bone in oim/oim mice. Irisin administration reduced bone fracture and attenuated bone abnormalities by improving bone mass and strength and facilitating the expression of osteogenic differentiation markers. In vivo study and in vitro experi-ments showed that Irisin antagonized TGF-8/Smad signaling by interfering with TGF-81-TGF-8 receptor II (T8RII) binding. In oim/oim osteoblasts, Irisin alleviated TGF-81-induced suppression of osteogenic differentiation through both integrin-dependent and integrin-independent mechanisms. Independent of integrin receptors, Irisin affected osteogenesis by activating ERK/p38 signaling and counteracting TGF-8/Smad2/3 signaling. In particular, Irisin alleviated TGF-81-induced inhibition of Runx2 function at the osteocalcin promoter through decreasing Smad2/3 signaling and inducing HADC4/5 degeneration.Conclusions: Collectively, Irisin could effectively reduce bone fracture in oim/oim mice through promoting osteogenesis and counteracting TGF-beta/Smad signaling. Translational potential statement: Findings from this study provided evidence for using Irisin as a potential ther-apeutic reagent to prevent the progression of OI.