Neuromedin B is a new player in cartilage formation

Abstract

Purpose: In view of the importance of the TGFβ pathway in cartilage homeostasis and its deregulation in rheumatic diseases, we aimed at identifying genes modulating by TGFβ3 in the early phases of mesenchymal stromal cells (MSC) differentiation into chondrocytes and deciphering their role in maintaining cartilage phenotype. Methods: Transcriptomic analysis was performed on human bone marrow MSC, induced to differentiate into chondrocytes using micropellet culture with/without TGFβ3 and stopped at day 0.5/1/2/3 as compared to d0. Expression of chondrocyte markers and novel genes was quantified by RT-qPCR and western-blotting. Gain- and loss-of-function experiments were performed using recombinant neuromedin B (NMB) and siRNA transfection on MSC induced to chondrogenesis and in mature OA chondrocytes. Results: Transcriptomic data analysis identified genes significantly modulated in TGFβ3-induced micropellets. We selected NMB as one of the most highly up-regulated gene and as a secreted factor. Expression was increased by a 40-fold factor at d1, stabilized at d2-d3 and returned to basal expression at d21 of chondrogenesis. Enhanced expression of NMB was specific to chondrogenesis. NMB basal expression in MSC positively and significantly correlated with SOX9, COL2B, aggrecan and COL10 expression at d21. Down-regulation of NMB expression in MSCs resulted in partial inhibition of chondrogenesis while addition of rNMB did not impact differentiation. Expression of NMB and NMBR was also detected in chondrocytes. Addition of rNMB did not impact chondrogenic (COL2B, ACAN), hypertrophic (MMP13, AP) and fibrotic (COL1, COL3) markers but NMB inhibition induced these markers. Finally, we showed that NMB expression is associated with intracellular Ca2+ influx and calcineurin activity required for Sox9-induced chondrogenesis. Conclusions: NMB induction in the early stages of chondrogenesis is required for MSC differentiation. NMB expression needs to be down-regulated in late stages of chondrogenesis and finely regulated in mature chondrocytes to maintain their phenotype. High expression of NMB in MSC might be a predictive marker of chondrogenic potential.