O-GlcNAc modification of the runt-related transcription factor 2 (Runx2) links osteogenesis and nutrient metabolism in bone marrow mesenchymal stem cells

Mol Cell Proteomics. 2014 Dec;13(12):3381-95. doi: 10.1074/mcp.M114.040691. Epub 2014 Sep 3.


Runx2 is the master switch controlling osteoblast differentiation and formation of the mineralized skeleton. The post-translational modification of Runx2 by phosphorylation, ubiquitinylation, and acetylation modulates its activity, stability, and interactions with transcriptional co-regulators and chromatin remodeling proteins downstream of osteogenic signals. Characterization of Runx2 by electron transfer dissociation tandem mass spectrometry revealed sites of O-linked N-acetylglucosamine (O-GlcNAc) modification, a nutrient-responsive post-translational modification that modulates the action of numerous transcriptional effectors. O-GlcNAc modification occurs in close proximity to phosphorylated residues and novel sites of arginine methylation within regions known to regulate Runx2 transactivation. An interaction between Runx2 and the O-GlcNAcylated, O-GlcNAc transferase enzyme was also detected. Pharmacological inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc from Ser/Thr residues, enhanced basal (39.9%) and BMP2/7-induced (43.3%) Runx2 transcriptional activity in MC3T3-E1 pre-osteoblasts. In bone marrow-derived mesenchymal stem cells differentiated for 6 days in osteogenic media, inhibition of OGA resulted in elevated expression (24.3%) and activity (65.8%) of alkaline phosphatase (ALP) an early marker of bone formation and a transcriptional target of Runx2. Osteogenic differentiation of bone marrow-derived mesenchymal stem cells in the presence of BMP2/7 for 8 days culminated in decreased OGA activity (39.0%) and an increase in the abundance of O-GlcNAcylated Runx2, as compared with unstimulated cells. Furthermore, BMP2/7-induced ALP activity was enhanced by 35.6% in bone marrow-derived mesenchymal stem cells differentiated in the presence of the OGA inhibitor, demonstrating that direct or BMP2/7-induced inhibition of OGA is associated with increased ALP activity. Altogether, these findings link O-GlcNAc cycling to the Runx2-dependent regulation of the early ALP marker under osteoblast differentiation conditions.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acetylglucosamine / metabolism*
  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism
  • Animals
  • Arginine / metabolism
  • Bone Marrow Cells / cytology
  • Bone Marrow Cells / drug effects
  • Bone Marrow Cells / metabolism*
  • Bone Morphogenetic Protein 2 / pharmacology
  • Bone Morphogenetic Protein 7 / pharmacology
  • Cell Differentiation / drug effects
  • Cell Line
  • Core Binding Factor Alpha 1 Subunit / genetics
  • Core Binding Factor Alpha 1 Subunit / metabolism
  • Enzyme Inhibitors / pharmacology
  • HEK293 Cells
  • Humans
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Mesenchymal Stem Cells / cytology
  • Mesenchymal Stem Cells / drug effects
  • Mesenchymal Stem Cells / metabolism*
  • Methylation
  • Mice
  • Mice, Inbred C57BL
  • N-Acetylglucosaminyltransferases / genetics
  • N-Acetylglucosaminyltransferases / metabolism
  • Osteoblasts / cytology
  • Osteoblasts / drug effects
  • Osteoblasts / metabolism*
  • Osteogenesis / drug effects
  • Osteogenesis / genetics*
  • Protein Processing, Post-Translational*
  • Transcriptional Activation / drug effects


  • Adaptor Proteins, Signal Transducing
  • Atrnl1 protein, mouse
  • Bmp2 protein, mouse
  • Bone Morphogenetic Protein 2
  • Bone Morphogenetic Protein 7
  • Core Binding Factor Alpha 1 Subunit
  • Enzyme Inhibitors
  • Membrane Proteins
  • Runx2 protein, mouse
  • Arginine
  • N-Acetylglucosaminyltransferases
  • O-GlcNAc transferase
  • Acetylglucosamine