IL-6 Enhances Osteocyte-Mediated Osteoclastogenesis by Promoting JAK2 and RANKL Activity In Vitro

Cell Physiol Biochem. 2017;41(4):1360-1369. doi: 10.1159/000465455. Epub 2017 Mar 9.

Abstract

Background/aims: Evidence suggests that IL-6 affects bone mass by modulating osteocyte communication towards osteoclasts. However, the mechanism by which IL-6 enhances osteocyte-mediated osteoclastogenesis is unclear. We aimed to investigate the inflammatory factors in serum after orthodontic surgery and their relationship between osteocytes and osteoclasts.

Methods: Serum was obtained from 10 orthognathic surgery patients, and inflammatory factors were detected by ELISA. We treated the osteocyte-like cell line MLO-Y4 with recombinant mouse IL-6 and IL-6 receptor (IL-6R), and used quantitative RT-PCR and Western blotting to explore Receptor activator of nuclear factor-κB ligand (RANKL) expression at both the mRNA and protein level. MLO-Y4 cells were co-cultured with osteoclast precursor cells, and the formation of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining. To explore the role of JAK2 in the osteocyte-mediated osteoclastogenesis, AG490, a JAK2 inhibitor, was used to inhibit the JAK2-STAT3 pathway in osteocytes.

Results: In our study, we found that IL-6 and RANKL were stimulated in serum 3-7 days after orthognathic surgery. Therefore, IL-6 and IL-6 receptor enhanced the expression of RANKL at both the mRNA and protein level in MLO-Y4. Furthermore, when MLO-Y4 cells were co-cultured with osteoclast precursor cells, it significantly stimulated osteoclastogenesis. Our study indicated that osteocytes could promote osteoclastic differentiation and the formation of TRAP-positive multinucleated cells after stimulation with IL-6 and IL-6R. Our results also indicated that treatment with IL-6 and IL-6R increased RANKL mRNA expression and the RANKL/OPG expression ratio. Meanwhile, the phosphorylation of Janus kinase 2 (JAK2) and Signal transducer and activator of transcription (STAT3) also correlated with RANKL levels. Furthermore, we investigated the effects of a specific JAK2 inhibitor, AG490, on the expression of RANKL in osteocyte-like MLO-Y4 cells and osteocyte-mediated osteoclastogenesis. The results showed that AG490 inhibited (p)-JAK2 and RANKL expression. Osteoclastic differentiation was decreased after pretreatment in MLO-Y4 with mouse IL-6/IL-6R and AG490; therefore, we concluded that IL-6 increased osteocyte-mediated osteoclastic differentiation by activating JAK2 and RANKL.

Conclusion: The effects of IL-6/il-6R and AG490 on osteocyte-mediated osteoclastogenesis contribute to our understanding of the role of inflammatory factors in the interaction between osteocytes and osteoclast precursors. IL-6 and RANKL are key factors for bone remodelling after the orthodontic surgery, and their roles in bone remodelling may be fundamental mechanisms accelerating tooth movement by orthodontic surgery.

Keywords: AG490; IL-6 receptor; Interleukin-6; Janus kinase; Osteoclastogenesis; Osteocyte; Receptor activator of nuclear factor-κB ligand; Signal transducer and activator of transcription; Surgery first.

MeSH terms

  • Animals
  • Cell Communication*
  • Cell Line
  • Female
  • Humans
  • Interleukin-6 / metabolism*
  • Janus Kinase 2 / antagonists & inhibitors
  • Janus Kinase 2 / metabolism*
  • Male
  • Mice
  • Osteoclasts / cytology
  • Osteoclasts / metabolism*
  • Osteocytes / cytology
  • Osteocytes / metabolism*
  • RANK Ligand / biosynthesis*
  • Rats
  • STAT2 Transcription Factor / metabolism
  • STAT3 Transcription Factor / metabolism
  • Signal Transduction*
  • Tyrphostins / pharmacology

Substances

  • IL6 protein, human
  • Interleukin-6
  • RANK Ligand
  • STAT2 Transcription Factor
  • STAT3 Transcription Factor
  • STAT3 protein, human
  • Stat2 protein, mouse
  • TNFSF11 protein, human
  • Tnfsf11 protein, mouse
  • Tyrphostins
  • alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide
  • interleukin-6, mouse
  • JAK2 protein, human
  • Jak2 protein, mouse
  • Janus Kinase 2