The positive effects of secreting cytokines IL-17 and IFN-γ on the early-stage differentiation and negative effects on the calcification of primary osteoblasts in vitro

Abstract

Objective: Interleukin-17 (IL-17) and interferon-gamma (IFN-γ) are all pro-inflammatory cytokines produced by specific subsets of T-cells and are also considered crucial regulators in bone remodeling, but their effects on osteogenesis have not been carefully studied. So, this study aimed to investigate the effects of secreting cytokines IL-17 and IFN-γ on the osteogenesis of primary osteoblasts and to clarify the potential roles of the related Janus activated kinase 2 (JAK2) and downstream signal transducer and activator of transcription 3 (STAT3) signaling pathway in bone remodeling.

Methods: The proliferation of osteoblasts was evaluated by MTT assay. Osteogenic activity was tested by alkaline phosphatase (ALP) activity assay and alizarin red staining. The mRNA levels of ALP, osteocalcin, osteoprotegerin (OPG), Runt-related transcription factor 2 (Runx2) and receptor activator of nuclear factor-kappa B ligand (RANKL) were also measured by real-time quantitative PCR. The JAK2-STAT3 pathway was evaluated by Western blot.

Results: Osteoblasts showed no obvious proliferation when treated with IL-17 and/or IFN-γ, but higher ALP activities were observed in primary osteoblasts treated with IL-17 or IL-17 + IFN-γ in induction medium. We also found that IL-17 could promote the gene expression of Alp, Runx2, Osteocalcin, Opg, and Rankl, while IFN-γ might attenuate this effect. Nevertheless, IL-17 and IFN-γ exhibited an inhibitory effect on the calcification of primary osteoblasts. We also found that IL-17 could directly facilitate RANKL expressions by JAK2-STAT3 pathway.

Conclusion: The positive effects of IL-17 and IFN-γ on the early-stage differentiation and the negative effects on the calcification of murine calvarial osteoblasts contribute to our understanding of the role and interaction of inflammatory factors in the bone remodeling and as fundamental mechanisms involved in the destruction of alveolar bone.

Keywords: IFN-γ; IL-17; Osteoblast calcification; Osteoblast differentiation; Primary osteoblasts.