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.
Highlights
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
To confirm that OGA inhibition, and the concomitant rise in protein O-GlcNAc modification, augments Runx2-dependent transactivation, the activity of the 6OSE2luciferase reporter gene was measured in MC3T3E1 pre-osteoblasts cultured in the presence or absence of BMP2/7 and a selective OGA inhibitor, Thiamet G
We report for the first time novel sites of O-GlcNAc modification and methylation on human type II Runx2
Summary
Mesenchymal stem cells differentiated in the presence of the OGA inhibitor, demonstrating that direct or BMP2/7induced inhibition of OGA is associated with increased ALP activity. These findings link O-GlcNAc cycling to the Runx2-dependent regulation of the early ALP marker under osteoblast differentiation conditions. We have detected O-GlcNAcylation of proteins integral to the BMP2TAK1-MAPK signaling axis in pre-osteoblasts [44], the regulation and contribution of O-GlcNAc cycling enzymes during osteoblast differentiation and bone formation is virtually unexplored
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