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. 2022 Feb 2;61(2):856-864.
doi: 10.1093/rheumatology/keab440.

The role of TNFRSF11B in development of osteoarthritic cartilage

Alejandro Rodríguez Ruiz  1 , Margo Tuerlings  1 , Ankita Das  1 , Rodrigo Coutinho de Almeida  1 , H Eka D Suchiman  1 , Rob G H H Nelissen  2 , Yolande F M Ramos  1 , Ingrid Meulenbelt  1
Affiliations

The role of TNFRSF11B in development of osteoarthritic cartilage

Alejandro Rodríguez Ruiz et al. Rheumatology (Oxford). .

Abstract

Objectives: 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 3 D 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 eight changed significantly, with BMP6 showing the 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.

Keywords: TNFRSF11B; CCAL1; cartilage hypertrophy; chondrogenesis; matrix mineralization; osteoarthritis.

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Figures

<sc>Fig.</sc> 1
Fig. 1
Alcian blue staining of neo-cartilage (A) Representative images of 1-week neo-cartilage pellets as indicated (left: control chondrocytes; right: chondrocytes with TNFRSF11B overexpression). Scale bars: 50 μm. (B) Area of the pellets (n = 72), GAG-release in the medium (n = 27) and Alcian blue pixel intensity quantification (n = 18) for control and TNFRSF11B overexpressing chondrocytes.
<sc>Fig.</sc> 2
Fig. 2
Expression of matrix-related genes in neo-cartilage -ΔCt values for anabolic and catabolic genes in 1-week neo-cartilage pellets [control chondrocytes vs chondrocytes with TNFRSF11B overexpression (n = 18; *P-value <0.05; **P-value <10−3; *** P-value <10−6)].
<sc>Fig.</sc> 3
Fig. 3
COL1 and COL2 immunohistochemistry of neo-cartilage Representative images of 1-week neo-cartilage pellets as indicated (left: control chondrocytes; right: chondrocytes with TNFRSF11B overexpression). Scale bars: 50 µm.
<sc>Fig.</sc> 4
Fig. 4
Expression of osteogenic-related genes in neo-cartilage -ΔCt values for mineralization and bone formation genes in 1-week neo-cartilage pellets [control chondrocytes vs chondrocytes with TNFRSF11B overexpression (n = 18; *P-value <0.05; **P-value <10−3; ***P-value <10−6)].
<sc>Fig</sc>. 5
Fig. 5
Expression of TNFRSF11B-correlated genes in neo-cartilage -ΔCt values for TNFRSF11B-correlated genes in 1-week neo-cartilage pellets [control chondrocytes vs chondrocytes with TNFRSF11B overexpression (n = 18; *P-value <0.05; **P-value <10−3; ***P-value <10−6)].
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