Abstract
Objective: To investigate the role of IL-18 in the regulation of osteogenic differentiation in human bone marrow mesenchymal stem cells (hBMSCs). Methods: To assess whether IL-18 affects the osteogenic differentiation of hBMSCs through the c-MYC/SLC7A5 axis, IL-18 dose-response and time-course experiments were performed to evaluate its impact on osteogenic differentiation. To confirm osteogenic differentiation, alizarin red staining calcium measurement were performed. RT-qPCR and western blotting were used to determine the expression levels of bone-specific markers ALP, RUNX2, and BMP2, as well as those of SLC7A5 and c-MYC. Furthermore, SLC7A5 and c-MYC expression was evaluated via immunofluorescence. To elucidate the roles of SLC7A5 and c-MYC in osteoblast differentiation, cells were transfected with SLC7A5 or c-MYC siRNAs, or treated with the SLC7A5-specific inhibitor JPH203 and c-MYC-specific inhibitor 10058-F4, and the expression of SLC7A5, c-MYC, and bone-specific markers ALP, RUNX2, and BMP2 was assessed. Results: Our results demonstrated that IL-18 increased calcium deposition in hBMSCs, and upregulated the expression of SLC7A5, c-MYC, ALP, RUNX2, and BMP2. Silencing of SLC7A5 or c-MYC using siRNA reduced the expression of ALP, RUNX2, and BMP2, while IL-18 treatment partially reversed the inhibitory effect of siRNA. Similar results were obtained by treating hBMSCs with SLC7A5 and c-MYC specific inhibitors, leading to significant reduction of the osteogenesis effect of IL-18 on hBMSCs. Conclusion: In conclusion, our results indicate that IL-18 promotes the osteogenic differentiation of hBMSCs via the SLC7A5/c-MYC pathway and, therefore, may play an important role in fracture healing. These findings will provide new treatment strategies for delayed fracture healing after splenectomy.
Highlights
In a preliminary clinical study, we previously demonstrated that fracture healing was significantly delayed in patients undergoing splenectomy, while inflammatory factors were significantly reduced during the acute phase (Xiao et al, 2017; Xiao et al, 2018)
Alizarin red staining showed that the number of mineralized nodules increased significantly at higher Interleukin 18 (IL-18) concentrations, with the highest staining intensity being observed at 100 ng/ml IL-18 (Figure 1A)
According to the quantitative analysis of calcium, Compared with 0ng group human BMSCs (hBMSCs) cultured in osteogenic induction medium showed that the amount of calcium deposition increased with the concentration (Figure 1B)
Summary
In a preliminary clinical study, we previously demonstrated that fracture healing was significantly delayed in patients undergoing splenectomy, while inflammatory factors were significantly reduced during the acute phase (Xiao et al, 2017; Xiao et al, 2018). These results indicated that during certain stages of bone healing, the inflammatory immune response at the fracture site is indispensable (Claes et al, 2012). In response to a fracture, the inflammatory microenvironment formed around the edges of the fracture recruits bone marrow mesenchymal stem cells (BMSCs) to the site to participate in fracture repair (Reich et al, 2020). Mesenchymal stem cells (MSCs) and other progenitor cells proliferate and form granulation tissue, which is eventually converted to a cartilaginous callus to stabilize the fracture site
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