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Phosphorylation of the transcriptional coregulator alphaNAC by DNA-PKcs regulates osteoblastogenesis

ABSTRACT: In order to identify pathways affected by inhibition of the DNA-dependent protein kinase, catalytic subunit in osteoblasts, we performed a whole genome gene expression profiling using the Affymetrix mouse GeneChip system (MoGene-2.0 gene ST arrays). The osteoblastic cell line MC3T3-E1 was grown in osteogenic medium and treated with DMSO (vehicle) or NU7441, an inhibitor of DNA-dependent protein kinase, for 14 days. Six biological samples were used in this experiment, three replicates in the control group and three replicates in the treated group. Isolated RNA was subjected to microarray analysis.

ORGANISM(S): Mus musculus

PROVIDER: GSE151332 | GEO | 2020/05/28

REPOSITORIES: GEO

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

Project description: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.

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Genomic characterization of osteoblastic differentiation of MC-3T3E1 cells

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Caspase-9 is a positive regulator of osteoblastic cell migration identified by diaPASEF proteomics

Project description:Caspase-9 is traditionally considered as the initiator caspase of the intrinsic apoptotic pathway. In the last decade, however, other functions beyond initiation/execution of cell death have been described including cell type-dependent regulation of proliferation, differentiation/maturation, mitochondrial and endosomal/lysosomal homeostasis. As previous studies revealed non-apoptotic functions of caspases in osteogenesis and bone homeostasis, this study was performed to identify proteins and pathways deregulated by knockout of caspase-9 in mouse MC3T3-E1 osteoblasts. Data-independent acquisition – parallel accumulation serial fragmentation (diaPASEF) proteomics was used to compare protein profiles of control and caspase-9 knockout cells. A total of 7669 protein groups were quantified and 283 upregulated/141 downregulated protein groups were associated with the caspase-9 knockout phenotype. The deregulated proteins were mainly enriched for those associated with cell migration/motility and DNA replication/repair. Altered migration was confirmed in MC3T3-E1 cells with genetic and pharmacological inhibition of caspase-9. ABHD2, an established regulator of cell migration, was identified as a possible substrate of caspase-9. We conclude that caspase-9 acts as a modulator of osteoblastic MC3T3-E1 cell migration and therefore may be involved in bone remodeling and fracture repair.

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

Project description:Phosphorylation of the transcriptional coregulator aNAC by DNA-PKcs regulates osteoblastogenesis

| PRJNA594371 | ENA

Phosphorylation of the transcriptional coregulator alphaNAC by DNA-PKcs regulates osteoblastogenesis

Project description:Phosphorylation of the transcriptional coregulator alphaNAC by DNA-PKcs regulates osteoblastogenesis

| PRJNA635498 | ENA

Titanium with nanotopography attenuates the osteoclast-induced disruption of osteoblast differentiation by regulating histone methylation

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DNA-PKcs is a key transcriptional and splicing regulator of AR-Vs in advanced prostate cancer

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2023-09-11 | GSE242255 | GEO

Nfil3, a target of the NACA transcriptional coregulator, affects osteoblast and osteocyte gene expression differentially.

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