Low-dose IL-34 has no effect on osteoclastogenesis but promotes osteogenesis of hBMSCs partly via activation of the PI3K/AKT and ERK signaling pathways

Abstract

Background: Inflammatory microenvironment is significant to the differentiation and function of mesenchymal stem cells (MSCs). It evidentially influences the osteoblastogenesis of MSCs. IL-34, a newly discovered cytokine, playing a key role in metabolism. However, the research on its functional role in the osteogenesis of MSCs was rarely reported. Here, we described the regulatory effects of low-dose IL-34 on both osteoblastogenesis and osteoclastogenesis. Methods: We performed the osteogenic effects of hBMSCs by exogenous and overexpressed IL-34 in vitro, so were the osteoclastogenesis effects of mBMMs by extracellular IL-34. CCK-8 was used to assess the effect of IL-34 on the viability of hBMSCs and mBMMs. ALP, ARS, and TRAP staining was used to evaluate ALP activity, mineral deposition, and osteoclastogenesis, respectively. qRT-PCR and Western blotting analysis were performed to detect the expression of target genes and proteins. ELISA was used to evaluate the concentrations of IL-34. In vivo, a rat tibial osteotomy model and an OVX model were established. Radiographic analysis and histological evaluation were performed to confirm the therapeutic effects of IL-34 in fracture healing and osteoporosis. Statistical differences were evaluated by two-tailed Student’s t test, one-way ANOVA with Bonferroni’s post hoc test, and two-way ANOVA with Bonferroni multiple comparisons post hoc test in the comparison of 2 groups, more than 2 groups, and different time points of treated groups, respectively. Results: Promoted osteoblastogenesis of hBMSCs was observed after treated by exogenous or overexpressed IL-34 in vitro, confirmed by increased mineral deposits and ALP activity. Furthermore, exogenous or overexpressed IL-34 enhanced the expression of p-AKT and p-ERK. The specific AKT and ERK signaling pathway inhibitors suppressed the enhancement of osteoblastogenesis induced by IL-34. In a rat tibial osteotomy model, imaging and histological analyses testified the local injection of exogenous IL-34 improved bone healing. However, the additional IL-34 has no influence on both osteoclastogenesis of mBMMs in vitro and osteoporosis of OVX model of rat in vivo. Conclusions: Collectively, our study demonstrate that low-dose IL-34 regulates osteogenesis of hBMSCs partly via the PIK/AKT and ERK signaling pathway and enhances fracture healing, with neither promoting nor preventing osteoclastogenesis in vitro and osteoporosis in vivo.