Identification of Nedd9 as a TGF-β-Smad2/3 Target Gene Involved in RANKL-Induced Osteoclastogenesis by Comprehensive Analysis

PLoS One. 2016 Jun 23;11(6):e0157992. doi: 10.1371/journal.pone.0157992. eCollection 2016.

Abstract

TGF-ß is a multifunctional cytokine that is involved in cell proliferation, differentiation and function. We previously reported an essential role of the TGF-ß -Smad2/3 pathways in RANKL-induced osteoclastogenesis. Using chromatin immunoprecipitation followed by sequencing, we comprehensively identified Smad2/3 target genes in bone marrow macrophages. These genes were enriched in the gene population upregulated by TGF-ß and downregulated by RANKL. Recent studies have revealed that histone modifications, such as trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3), critically regulate key developmental steps. We identified Nedd9 as a Smad2/3 target gene whose histone modification pattern was converted from H3K4me3(+)/H3K4me27(+) to H3K4me3(+)/H3K4me27(-) by TGF-ß. Nedd9 expression was increased by TGF-ß and suppressed by RANKL. Overexpression of Nedd9 partially rescued an inhibitory effect of a TGF-ß inhibitor, while gene silencing of Nedd9 suppressed RANKL-induced osteoclastogenesis. RANKL-induced osteoclastogenesis were reduced and stimulatory effects of TGF-ß on RANKL-induced osteoclastogenesis were partially abrogated in cells from Nedd9-deficient mice although knockout mice did not show abnormal skeletal phenotypes. These results suggest that Nedd9 is a Smad2/3 target gene implicated in RANKL-induced osteoclastogenesis.

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics*
  • Animals
  • Bone Resorption / drug therapy
  • Bone Resorption / genetics*
  • Cell Differentiation / drug effects
  • Cell Differentiation / genetics
  • Epigenesis, Genetic
  • Gene Expression Profiling
  • Gene Expression Regulation*
  • Histones / metabolism
  • Male
  • Mice
  • Mice, Knockout
  • Osteoclasts / drug effects
  • Osteoclasts / metabolism*
  • Protein Binding
  • RANK Ligand / metabolism*
  • RANK Ligand / pharmacology
  • Smad2 Protein / metabolism*
  • Smad3 Protein / metabolism*
  • Transforming Growth Factor beta / metabolism*

Substances

  • Adaptor Proteins, Signal Transducing
  • Histones
  • NEDD9 protein, mouse
  • RANK Ligand
  • Smad2 Protein
  • Smad3 Protein
  • Transforming Growth Factor beta

Grants and funding

This work was supported by a Grant-in-aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology(#26253075). The URL is as follows; https://www.jsps.go.jp/j-grantsinai/index.html. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.