title: IL-6 Enhances Osteocyte-Mediated Osteoclastogenesis by Promoting JAK2 and RANKL Activity In Vitro.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.forename: Qing
authors.initials: Q
authors.lastname: Wu
authors.forename: Xiaokang
authors.initials: X
authors.lastname: Zhou
authors.forename: Danqing
authors.initials: D
authors.lastname: Huang
authors.forename: Yingchen
authors.initials: Y
authors.lastname: Ji
authors.forename: Feiwu
authors.initials: F
authors.lastname: Kang
mesh_terms: D000818:Animals; D002450:Cell Communication; D002460:Cell Line; D005260:Female; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D051379:Mice; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D010011:Osteocytes / Q000166:cytology / Q000378:metabolism*; D053245:RANK Ligand / Q000096:biosynthesis*; D051381:Rats; D050795:STAT2 Transcription Factor / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction; D020032:Tyrphostins / Q000494:pharmacology
chemical_list: C508600:IL6 protein, human; D015850:Interleukin-6; D053245:RANK Ligand; D050795:STAT2 Transcription Factor; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C494085:Stat2 protein, mouse; C505688:TNFSF11 protein, human; C505689:Tnfsf11 protein, mouse; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; C577686:interleukin-6, mouse; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2
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