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Background: The chondrogenic differentiation of mesenchymal stem cells (MSCs) to enhance cartilage repair and regeneration is a promising strategy to alleviate osteoarthritis (OA) progression. Method: The potency of JD-312 in inducing chondrogenic differentiation of MSCs was assessed and verified. The efficacy of JD-312-treated MSCs was evaluated using a Sprague–Dawley rat DMM model. Additionally, the capacity of JD-312 to successfully recruit bone marrow-derived mesenchymal stem cells (BMSCs) for the treatment of OA in vitro was confirmed via intra-articular injection. The repair status of the articular cartilage was analyzed in vivo through histological examination. Result: In this study, we identify JD-312 as a novel non-toxic small molecule that can promote chondrogenic differentiation in human umbilical cord-derived MSCs (hUCMSCs) and human bone marrow MSCS (hBMSCs) in vitro. We also show that transient differentiation of MSCs with JD-312 prior to in vivo administration remarkably improves the regeneration of cartilage and promotes Col2a1 and Acan expression in rat models of DMM, in comparison to kartogenin (KGN) pre-treatment or MSCs alone. Furthermore, direct intra-articular injection of JD-312 in murine model of OA showed reduced loss of articular cartilage and improved pain parameters. Lastly, we identified that the effects of JD-312 are at least in part mediated via upregulation of genes associated with the focal adhesion, PI3K-Akt signaling and the ECM-receptor interaction pathways, and specifically cartilage oligomeric matrix protein (COMP) may play a vital role. Conclusion: Our study demonstrated that JD-312 showed encouraging repair effects for OA in vivo. The translational potential of this article: Together, our findings demonstrate that JD-312 is a promising new therapeutic molecule for cartilage regeneration with clinical potential.

Osteoarthritis (OA) is one of the most common chronic diseases in the world. However, current treatment modalities mainly relieve pain and inhibit cartilage degradation, but do not promote cartilage regeneration. In this study, we show that G protein-coupled receptor class C group 5 member B (GPRC5B), an orphan G-protein-couple receptor, not only inhibits cartilage degradation, but also increases cartilage regeneration and thereby is protective against OA. We observed that Gprc5b deficient chondrocytes had an upregulation of cartilage catabolic gene expression, along with downregulation of anabolic genes in vitro. Furthermore, mice deficient in Gprc5b displayed a more severe OA phenotype in the destabilization of the medial meniscus (DMM) induced OA mouse model, with upregulation of cartilage catabolic factors and downregulation of anabolic factors, consistent with our in vitro findings. Overexpression of Gprc5b by lentiviral vectors alleviated the cartilage degeneration in DMM-induced OA mouse model by inhibiting cartilage degradation and promoting regeneration. We also assessed the molecular mechanisms downstream of Gprc5b that may mediate these observed effects and identify the role of protein kinase B (AKT)-mammalian target of rapamycin (mTOR)-autophagy signaling pathway. Thus, we demonstrate an integral role of GPRC5B in OA pathogenesis, and activation of GPRC5B has the potential in preventing the progression of OA.

Primary bone cancer (PBC) comprises several subtypes each underpinned by distinctive genetic drivers. This driver diversity produces novel morphological features and clinical behaviour that serendipitously makes PBC an excellent metastasis model. Here, we report that some transfer RNA-derived small RNAs termed tRNA fragments (tRFs) perform as a constitutive tumour suppressor mechanism by blunting a potential pro-metastatic protein-RNA interaction. This mechanism is reduced in PBC progression with a gradual loss of tRNAGlyTCC cleavage into 5′ end tRF-GlyTCC when comparing low-grade, intermediate-grade and high-grade patient tumours. We detected recurrent activation of miR-140 leading to upregulated RUNX2 expression in high-grade patient tumours. Both tRF-GlyTCC and RUNX2 share a sequence motif in their 3′ ends that matches the YBX1 recognition site known to stabilise pro-metastatic mRNAs. Investigating some aspects of this interaction network, gain- and loss-of-function experiments using small RNA mimics and antisense LNAs, respectively, showed that ectopic tRF-GlyTCC reduced RUNX2 expression and dispersed 3D micromass architecture in vitro. iCLIP sequencing revealed YBX1 physical binding to the 3′ UTR of RUNX2. The interaction between YBX1, tRF-GlyTCC and RUNX2 led to the development of the RUNX2 inhibitor CADD522 as a PBC treatment. CADD522 assessment in vitro revealed significant effects on PBC cell behaviour. In xenograft mouse models, CADD522 as a single agent without surgery significantly reduced tumour volume, increased overall and metastasis-free survival and reduced cancer-induced bone disease. Our results provide insight into PBC molecular abnormalities that have led to the identification of new targets and a new therapeutic.

Background: Osteosarcoma (OS) is the most common type of primary bone cancer affecting children and adolescents. OS has a high propensity to spread meaning the disease is often incurable and fatal. There have been no improvements in survival rates for decades. This highlights an urgent need for the development of novel therapeutic strategies. Here, we report in vitro and in vivo data that demonstrates the role of purinergic signalling, specifically, the B isoform of the purinergic receptor P2RX7 (P2RX7B), in OS progression and metastasis. Methods: TE85 and MNNG-HOS OS cells were transfected with P2RX7B. These cell lines were then characterised and assessed for proliferation, cell adhesion, migration and invasion in vitro. We used these cells to perform both paratibial and tail vein injected mouse studies where the primary tumour, bone and lungs were analysed. We used RNA-seq to identify responsive pathways relating to P2RX7B. Results: Our data shows that P2RX7B expression confers a survival advantage in TE85 + P2RX7B and MNNG-HOS + P2RX7B human OS cell lines in vitro that is minimised following treatment with A740003, a specific P2RX7 antagonist. P2RX7B expression reduced cell adhesion and P2RX7B activation promoted invasion and migration in vitro, demonstrating a metastatic phenotype. Using an in vivo OS xenograft model, MNNG-HOS + P2RX7B tumours exhibited cancer-associated ectopic bone formation that was abrogated with A740003 treatment. A pro-metastatic phenotype was further demonstrated in vivo as expression of P2RX7B in primary tumour cells increased the propensity of tumour cells to metastasise to the lungs. RNA-seq identified a novel gene axis, FN1/LOX/PDGFB/IGFBP3/BMP4, downregulated in response to A740003 treatment. Conclusion: Our data illustrates a role for P2RX7B in OS tumour growth, progression and metastasis. We show that P2RX7B is a future therapeutic target in human OS.