Abstract
The T-lymphocyte secreted pro-inflammatory cytokine, interleukin-17F (IL-17F), was found to be a key mediator in the cellular response of the immune system in the early phase of fracture repair but its intracellular signaling processes are currently not known in osteoblasts. The objective of this study was to identify the signaling proteins and crucial gene targets involved in osteoblast activation via IL-17F. It was hypothesised that IL-17F stimulated osteoblast maturation through a novel GSK3beta / beta-catenin independent pathway.
Mouse pre-osteoblast cell line (MC3T3-E1) was used for IL-17F or Wnt3a treatment. Desired proteins were detected using western blot analysis (antibodies: Phospho-GSK-3beta (Tyr 216), Phospho-GSK-3beta (Ser9), Runx2/cbfa1, TRAF6, Act1, p-ERK2, p-JNK and p-MAPK, C/EBP-beta and & delta). Gene-specific siRNAs of mouse IL-17Ra, IL-17Rc and a non-targeting siRNA (control) were utilised. MC3T3-E1 were transfected with IL-17Ra, IL-17Rc or Negative Control and treated with IL-17F. Chromatin Immunoprecipitation (ChIP-qPCR) was used to evaluate the mouse Runx2 P1 promoter region.
IL-17F increased expression of Col1, BSP, Runx2/cbfa1 and osteocalcin in MC3T3-E1 cells. Western blot analysis confirmed expression of known Wnt signaling proteins TRAF6, Act1, p-ERK2, p-JNK and p-MAPK in both IL-17F and Wnt3a treated cultures, including up-regulation of Runx2/cbfa1, a key transcription factor associated with osteoblast differentiation. IL-17F up-regulation of Runx2/cbfa1 appears independent of the Wnt/beta-catenin pathway as phosphorylated GSK-3beta at the Ser9 site was not detected with IL-17F treatment. Despite this, IL-17F treatment still increased expression of Runx2/cbfa1 downstream, lending evidence for a GSK3beta/beta-catenin independent manner of IL-17F stimulated osteogenesis. While IL-17F and Wnt3a both induced expression of C/EBP-delta, only IL-17F treatment induced expression of C/EBP-beta, an upstream transcription factor of Runx2/cbfa1. Further, siRNA knock down of the IL-17 receptors directly decreased Act1, C/EBP-beta and Runx2/cfba1 expression. By ChIP analysis, IL-17F was shown to upregulate C/EBP-beta expression and stimulated its binding to the P1 Promoter of the Runx2/cbfa1 gene.
The C/EBP-beta transcription factor was shown to be a key regulator of early osteogenesis. C/EBP-beta up-regulates Runx2/cbfa1 expression by directly binding to the Runx2/cbfa1 P1 promoter in osteoblasts. C/EBP-beta was activated in the osteoblast by IL-17F but not by Wnt3a adding further support to a novel GSK3beta/beta-catenin independent pathway. Our data shows that IL-17F, a cytokine secreted by T-lymphocytes, stimulates osteoblast maturation through a novel GSK3beta/beta-catenin independent pathway and reveals a crucial interaction between C/EBP-beta and the Runx2/cbfa1 P1 promoter not previously been shown in osteogenesis signaling further.
