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RUNX2 O‐GlcNAc modification links osteogenesis and nutrient metabolism in bone marrow mesenchymal stem cells (800.3)
Author(s) -
Nagel Alexis,
Ball Lauren
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.800.3
Subject(s) - runx2 , mesenchymal stem cell , chemistry , alkaline phosphatase , osteoblast , epigenetics , transcription factor , microbiology and biotechnology , progenitor cell , transcriptional regulation , biochemistry , stem cell , biology , enzyme , gene , in vitro
Decreased bone mineral density and increased risk of fracture in patients with Type I and II diabetes suggest that bone is a nutrient‐responsive tissue, however nutrient‐responsive mechanisms which modulate bone formation are not well understood. We previously identified multiple osteoblast proteins modified by O‐linked N‐acetylglucosamine (O‐GlcNAc), a post‐translational mechanism which modulates nutrient‐induced transcriptional and epigenetic programs. In this study we elucidate novel sites of O‐GlcNAc modification on runt‐related transcription factor 2 (RUNX2), the master osteogenic transcriptional regulator, using C18 nLC‐MS/MS and HexNAc product ion‐triggered ETD fragmentation. Sites of RUNX2 HexNAc modification reside within known PST‐ and N‐terminal activation domains. In addition to HexNAc modification, we observe acetylation and methylation of RUNX2 as well. O‐GlcNAc modification of endogenous RUNX2 was confirmed in bone marrow mesenchymal stem cells (BMMSCs) by click‐chemistry based chemoenzymatic labeling. Global elevation of protein O‐GlcNAc by Thiamet G (20 µM) inhibition of O‐GlcNAcase (OGA) significantly enhanced activity (65.8% over vehicle; n=3) and transcript expression (24.3% over vehicle; n=2) of the RUNX2‐target alkaline phosphatase (ALP) in osteogenic BMMSCs after 6 days. Addition of dexamethasone and rhBMP2/7 to the differentiation media resulted in decreased OGA activity (70.3% over vehicle; n=3) after 8 days. These data suggest that OGA and OGT modulate early osteogenic pathways in developing osteoblasts which may be partly mediated through glycosylation of RUNX2. Ongoing studies focus on determining the effect of RUNX2 O‐GlcNAcylation on transcriptional activity and protein interactions as they contribute to the regulation of osteogenesis. Grant Funding Source : RO1‐DE020925‐01A1