title: O-GlcNAc modification of the runt-related transcription factor 2 (Runx2) links osteogenesis and nutrient metabolism in bone marrow mesenchymal stem cells.
abstract: 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.
authors.affiliation: From the ‡Department of Oral Health Sciences; Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina, 29425.
authors.forename: Alexis K
authors.initials: AK
authors.lastname: Nagel
authors.affiliation: From the ‡Department of Oral Health Sciences; Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina, 29425 ballle@musc.edu.
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Ball
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology; D055419:Bone Morphogenetic Protein 7 / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008745:Methylation; D051379:Mice; D008810:Mice, Inbred C57BL; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D015533:Transcriptional Activation / Q000187:drug effects
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C480020:Atrnl1 protein, mouse; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D055419:Bone Morphogenetic Protein 7; D050659:Core Binding Factor Alpha 1 Subunit; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; C493716:Runx2 protein, mouse; D001120:Arginine; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; D000117:Acetylglucosamine