The role of TNFRSF11B in development of osteoarthritic cartilage.
OBJECTIVES
Osteoarthritis (OA) is a complex genetic disease with different risk factors contributing to its development. One of the genes, TNFRSF11B, previously identified with gain-of-function mutation in a family with early-onset OA with chondrocalcinosis, is among the highest upregulated genes in lesioned OA cartilage (RAAK-study). Here, we determined the role of TNFRSF11B overexpression in development of OA.


METHODS
Human primary articular chondrocytes (9 donors RAAK study) were transduced using lentiviral particles with or without TNFRSF11B. Cells were cultured for 1 week in a 3D in-vitro chondrogenic model . TNFRSF11B overexpression was confirmed by RT-qPCR, immunohistochemistry and ELISA. Effects of TNFRSF11B overexpression on cartilage matrix deposition, matrix mineralization, and genes highly correlated to TNFRSF11B in RNA-sequencing dataset (r>|0.75|) were determined by RT-qPCR. Additionally, glycosaminoglycans and collagen deposition were visualized with Alcian blue staining and immunohistochemistry (COL1 and COL2).


RESULTS
Overexpression of TNFRSF11B resulted in strong upregulation of MMP13, COL2A1 and COL1A1. Likewise, mineralization and osteoblast characteristic markers RUNX2, ASPN and OGN showed a consistent increase. Among 30 genes highly correlated to TNFRSF11B, expression of only 8 changed significantly, with BMP6 showing highest increase (9-fold) while expression of RANK and RANKL remained unchanged indicating previously unknown downstream pathways of TNFRSF11B in cartilage.


CONCLUSION
Results of our 3D in vitro chondrogenesis model indicate that upregulation of TNFRSF11B in lesioned OA cartilage may act as a direct driving factor for chondrocyte to osteoblast transition observed in OA pathophysiology. This transition does not appear to act via the OPG/RANK/RANKL triad common in bone remodeling.


ETHICS APPROVAL AND CONSENT TO PARTICIPATE
The Medical Ethics Committee of the LUMC gave approval for the RAAK study (P08.239). Written informed consent was obtained from all donors.
['Ruiz|Alejandro Rodríguez|AR|', 'Tuerlings|Margo|M|', 'Das|Ankita|A|', 'de Almeida|Rodrigo Coutinho|RC|', 'Eka Suchiman|H|H|', 'Nelissen|Rob G H H|RGHH|', 'Ramos|Yolande F M|YFM|', 'Meulenbelt|Ingrid|I|']
TNFRSF11B osteoarthritis matrix mineralization chondrogenesis CCAL1 cartilage hypertrophy