Phosphorylation of the transcriptional coregulator aNAC by DNA-PKcs regulates osteoblastogenesisÂ

The αNAC (alpha chain of the Nascent polypeptide-Associated Complex) transcriptional coregulator is developmentally expressed in osteoblasts and regulates osteoblast differentiation in vitro and in vivo. αNAC can activate or repress gene transcription, a function that is dynamically regulated by post-translational modification. Phosphorylation of residue Ser132 stimulates the sumoylation of αNAC on Lys127 to repress gene expression. Using in vitro kinase assays, we show that Ser132 phosphorylation is mediated by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Pharmacological inhibition of DNA-PKcs kinase activity or gene silencing of Prkdc (encoding DNA-PKcs) in murine osteoblastic MC3T3-E1 cells and human adipose-derived mesenchymal stromal cells markedly enhanced osteogenesis and the expression of osteoblast differentiation marker genes. ChIP-seq identified Ezh2 as a target of the αNAC/DNA-PKcs signaling pathway. Mechanistically, inhibition of DNA-PKcs repressed Ezh2 expression, induced cell cycle block, and increased osteogenesis by significantly enhancing the bone morphogenetic protein 2 (BMP-2) response in osteoblasts and other mesenchymal cells. Importantly, in vivo inhibition of the kinase enhanced bone biomechanical properties, and bones from osteoblast-specific conditional Prkdc-knockout mice exhibited increased stiffness. In conclusion, DNA-PKcs is a negative regulator of osteoblast differentiation, and therefore DNA-PKcs inhibitors may have therapeutic potential for bone regeneration and metabolic bone diseases.