NOTCH2 promotes osteoclast maturation and metabolism and modulates the transcriptome profile during osteoclastogenesis
Ernesto Canalis, Lauren Schilling, Jungeun Yu, Emily Denker
Abstract
Notch signaling plays a key regulatory role in bone remodeling and NOTCH2 enhances osteoclastogenesis, an effect that is mostly mediated by its target gene Hes1 . In the present study, we explored mechanisms responsible for the enhanced osteoclastogenesis in bone marrow derived macrophages (BMM) from Notch2 tm1.1Ecan , harboring a NOTCH2 gain-of-function mutation, and control mice. Notch2 tm1.1Ecan mice are osteopenic and have enhanced osteoclastogenesis. Bulk RNA-Seq and gene set enrichment analysis (GSEA) of Notch2 tm1.1Ecan BMMs cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) revealed enrichment of genes associated with enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis. These pathways were not enhanced in the context of a Hes1 inactivation. Analysis of single cell RNA-Seq data of pooled control and Notch2 tm1.1Ecan BMMs treated with M-CSF or M-CSF and RANKL for 3 days identified 11 well-defined cellular clusters. Pseudotime trajectory analysis indicated a trajectory of clusters expressing genes associated with osteoclast progenitors, osteoclast precursors and mature cells. There was an increased number of cells expressing gene markers associated with the osteoclast and with an unknown, albeit related, cluster in Notch2 tm1.1Ecan than in control BMMs as well as enhanced expression of genes associated with osteoclast progenitors and precursors in Notch2 tm1.1Ecan cells. In conclusion, BMM cultures display cellular heterogeneity, and NOTCH2 enhances osteoclastogenesis, increases mitochondrial and metabolic activity of osteoclasts and affects cell cluster allocation in BMMs.