NOTCH3 disrupts the osteoblast transcriptome in femoral bone of 1-month-old male C57BL/6 mice.

Notch signaling plays a fundamental role in skeletal physiology, and activation of NOTCH3 in osteoblasts/osteocytes impairs corticalization, causes cortical osteopenia and suppresses cancellous bone remodeling. To address possible mechanisms responsible, we explored transcriptome profiles in osteocytes and femoral bone from 1-month-old male C57BL/6 mice following activation of NOTCH3 in Dmp1-expressing cells. We used a NOTCH3 gain-of-function mouse line expressing the NOTCH3 intracellular domain (N3ICD) from the Rosa26 locus (R26-N3ICD mice) following the deletion of a loxP flanked STOP cassette, which was accomplished by crossing R26-N3ICD mice with Dmp1-Cre transgenics. Bulk RNA-Sequencing (RNA-Seq) of osteocytes revealed that expression of the N3ICD enhanced pathways associated with rheumatoid arthritis signaling, osteoclast differentiation, collagen degradation and the immune response. Single cell (sc)RNA-Seq of femoral bone from 1-month-old mice revealed clusters of cells related to vascular and osteogenic cells. Trajectory analysis showed a close association among selected clusters in control and N3ICD-expressing femurs. There were modest differences between N3ICD-expressing and control cells in cluster allocation, except for a decrease in the osteogenic cluster in N3ICD-induced cells. Analysis of intron/exon sequences demonstrated that NOTCH3 altered the velocity of the osteogenic cluster. In conclusion, NOTCH3 enhances the activity of selected pathways including osteoclast differentiation and suppresses the transcription of osteogenic cells in femoral bone.