NOTCH2 disrupts the synovial fibroblast identity and the inflammatory response of epiphyseal chondrocytes.

Notch signaling plays a fundamental role in the inflammatory response and has been linked to the pathogenesis of osteoarthritis in murine models of the disease and in humans. To address how Notch signaling modifies transcriptomes and cell populations, we examined the effects of NOTCH2 in chondrocytes from mice harboring a NOTCH2 gain-of-function mutation (Notch2tm1.1Ecan) and a conditional NOTCH2 gain-of-function model expressing the NOTCH2 intracellular domain (NICD2) from the Rosa26 locus (R26-NICD2 mice). Bulk RNA-Sequencing (RNA-Seq) of primary epiphyseal cells from both gain-of-function models established increased expression of pathways associated with the phagosome, genes linked to osteoclast activity in rheumatoid arthritis signaling and pulmonary fibrosis signaling. Expression of genes linked to collagen degradation was enhanced in Notch2tm1.1Ecan cells, while genes related to osteoarthritis pathways were increased in NICD2-expressing cells. Single cell (sc)RNA-Seq of cultured Notch2tm1.1Ecan cells revealed clusters of cells related to limb mesenchyme, chondrogenic cells and fibroblasts including articular synovial fibroblasts. Pseudotime trajectory revealed close associations among clusters in control cultures, but the cluster of articular/synovial fibroblasts was disrupted in cells from Notch2tm1.1Ecan mice. ScRNA-Seq showed similarities in the cluster distributions and pseudotime trajectories of NICD2-expressing and control cells, except for altered progression in a cluster of NICD2-expressing cells. In conclusion, NOTCH2 enhances the activity of pathways associated with inflammation in epiphyseal chondrocytes and disrupts the transcriptome profile of articular/synovial fibroblasts.