Abstract
Osteoarthritis is a global problem and the treatment of early disease is a clear area of unmet clinical need. Treatment strategies include cell therapies utilising chondrocytes e.g. autologous chondrocyte implantation and mesenchymal stem/stromal cells (MSCs) e.g. microfracture. The result of repair is often considered suboptimal as the goal of treatment is a more accurate regeneration of the tissue, hyaline cartilage, which requires a more detailed understanding of relevant biological signalling pathways. In this study, we describe a modulator of regulatory pathways common to both chondrocytes and MSCs. The chondrocytes thought to be cartilage progenitors are reported to reside in the superficial zone of articular cartilage and are considered to have the same developmental origin as MSCs present in the synovium. They are relevant to cartilage homeostasis and, like MSCs, are increasingly identified as candidates for joint repair and regenerative cell therapy. Both chondrocytes and MSCs can be regulated by the Wnt and TGFβ pathways. Dishevelled Binding Antagonist of Beta-Catenin (Dact) family of proteins is an important modulator of Wnt and TGFβ pathways. These pathways are key to MSC and chondrocyte function but, to our knowledge, the role of DACT protein has not been studied in these cells.
DACT1 and DACT2 were localised by immunohistochemistry in the developing joints of mouse embryos and in adult human cartilage obtained from knee replacement. RNAi of DACT1 and DACT2 was performed on isolated chondrocytes and MSCs from human bone marrow. Knockdown efficiency and cell morphology was confirmed by qPCR and immunofluorescence. To understand which pathways are affected by DACT1, we performed next-generation sequencing gene expression analysis (RNAseq) on cells where DACT1 had been reduced by RNAi. Top statistically significant (p < 0 .05) 200 up and downregulated genes were analysed with Ingenuity® Pathway Analysis software.
We observed DACT1 and DACT2 in chondrocytes throughout the osteoarthritic tissue, including in chondrocytes forming cell clusters. On the non-weight bearing and visually undamaged cartilage, DACT1 and DACT2 was localised to the articular surface. Furthermore, in mouse embryos (E.15.5), we observed DACT2 at the interzones, sites of developing synovial joints, suggesting that DACT2 has a role in cartilage progenitor cells. We subsequently analysed the expression of DACT1 and DACT2 in MSCs and found that both are expressed in synovial and bone marrow-derived MSCs. We then performed an RNAi knockdown experiment. DACT1 knockdown in both chondrocyte and MSCs caused the cells to undergo apoptosis within 24 hours. The RNA-seq study of DACT1 silenced bone marrow-derived MSCs, from 4 different human subjects, showed that loss of DACT1 has an effect on the expression of genes involved in both TGFβ and Wnt pathways and putative link to relevant cell regulatory pathways.
In summary, we describe for the first time, the presence and biological relevance of DACT1 and DACT2 in chondrocytes and MSCs. Loss of DACT1 induced cell death in both chondrocytes and MSCs, with RNA-seq analysis revealing a direct impact on transcript levels of genes involved in the Wnt and TFGβ signalling, key regulatory pathways in skeletal development and repair.
