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, but the mechanisms are poorly understood. We examined the effects of NOTCH2 in chondrocytes from Notch2tm1.1Ecan mice harboring a NOTCH2 gain-of-function mutation and from R26-NICD2 mice, a conditional gain-of-NOTCH2 function model, expressing the NOTCH2 intracellular domain (NICD2) from the Rosa26 locus. Bulk RNA-Sequencing (Seq) revealed increased expression of pathways associated with the phagosome, the role of osteoclasts in rheumatoid arthritis signaling and pulmonary fibrosis signaling in Notch2tm1.1Ecan and NICD2-expressing cultures. Collagen degradation was enhanced in Notch2tm1.1Ecan chondrocytes and the osteoarthritis pathway in NICD2-expressing cells. Single cell (sc)RNA-Seq analyzed 7,845 control and 7,141 Notch2tm1.1Ecan cells and used to define cell clusters. The more prevalent ones were constituted by limb mesenchyme, chondrogenic cells and fibroblasts including articular synovial fibroblasts. Pseudotime trajectory finding revealed a good association among clusters in control cultures, but this was disrupted in the articular/synovial cluster from Notch2tm1.1Ecan mice. scRNA-Seq of R26-NICD2 mice analyzed 9,957 cells in control and 9,924 in NICD2-expressing cultures. Neither the cluster distribution nor the pseudotime trajectory analysis revealed substantial differences between NIDC2-expressing and control cells except for an altered progression in an undefined cluster in 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. Overall design: Chondrocyte-enriched cells were isolated from the epiphyses of long bones of the hind and fore limbs from 3- to 4-day-old C57BL/6 mice.