BackgroundBoth loss- and gain-of-function of Wnt/β-catenin signaling in chondrocytes result in exacerbation of osteoarthritis (OA). Here, we examined the activity and roles of Wnt/β-catenin signaling in the superficial zone (SFZ) of articular cartilage.MethodsWnt/β-catenin signaling activity was analyzed using TOPGAL mice. We generated Prg4-CreERT2;Ctnnb1fl/fl and Prg4-CreERT2;Ctnnb1-ex3fl/wt mice for loss- and gain-of-function, respectively, of Wnt/β-catenin signaling in the SFZ. Regulation of Prg4 expression by Wnt/β-catenin signaling was examined in vitro, as were upstream and downstream factors of Wnt/β-catenin signaling in SFZ cells.ResultsWnt/β-catenin signaling activity, as determined by the TOPGAL reporter, was high specifically in the SFZ of mouse adult articular cartilage, where Prg4 is abundantly expressed. In SFZ-specific β-catenin-knockout mice, OA development was significantly accelerated, which was accompanied by decreased Prg4 expression and SFZ destruction. In contrast, Prg4 expression was enhanced and cartilage degeneration was suppressed in SFZ-specific β-catenin-stabilized mice. In primary SFZ cells, Prg4 expression was downregulated by β-catenin knockout, while it was upregulated by β-catenin stabilization by exon 3 deletion or treatment with CHIR99021. Among Wnt ligands, Wnt5a, Wnt5b, and Wnt9a were highly expressed in SFZ cells, and recombinant human WNT5A and WNT5B stimulated Prg4 expression. Mechanical loading upregulated expression of these ligands and further promoted Prg4 transcription. Moreover, mechanical loading and Wnt/β-catenin signaling activation increased mRNA levels of Creb1, a potent transcription factor for Prg4.ConclusionsWe demonstrated that Wnt/β-catenin signaling regulates Prg4 expression in the SFZ of mouse adult articular cartilage, which plays essential roles in the homeostasis of articular cartilage.

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Lubricin encoded by the proteoglycan 4 (Prg4) gene is produced from superficial zone (SFZ) cells of articular cartilage and synoviocytes, which is indispensable for lubrication of joint surfaces. Loss‐of‐function of human and mouse Prg4 results in early‐onset arthropathy accompanied by lost SFZ cells and hyperplastic synovium. Here, we focused on increases in the thickness of articular cartilage in Prg4‐knockout joints and analyzed the underlying mechanisms. In the late stage of articular cartilage development, the articular cartilage was thickened at 2 to 4 weeks and the SFZ disappeared at 8 weeks in Prg4‐knockout mice. Similar changes were observed in cultured Prg4‐knockout femoral heads. Cell tracking showed that Prg4‐knockout SFZ cells at 1 week of age expanded to deep layers after 1 week. In in vitro experiments, overexpression of Prg4 lacking a mucin‐like domain suppressed differentiation of ATDC5 cells markedly, whereas pellets of Prg4‐knockout SFZ cells showed enhanced differentiation. RNA sequencing identified matrix metalloproteinase 9 (Mmp9) as the top upregulated gene by Prg4 knockout. Mmp9 expressed in the SFZ was further induced in Prg4‐knockout mice. The increased expression of Mmp9 by Prg4 knockout was canceled by IκB kinase (IKK) inhibitor treatment. Phosphorylation of Smad2 was also enhanced in Prg4‐knockout cell pellets, which was canceled by the IKK inhibitor. Expression of Mmp9 and phosphorylated Smad2 during articular cartilage development was enhanced in Prg4‐knockout joints. Lubricin contributes to homeostasis of articular cartilage by suppressing differentiation of SFZ cells, and the nuclear factor‐kappa B‐Mmp9‐TGF‐β pathway is probably responsible for the downstream action of lubricin. © 2020 American Society for Bone and Mineral Research (ASBMR).

Objective. Transient receptor potential vanilloid channel 2 (TRPV2) is a Ca 2+ -permeable channel and plays a role in mediating intracellular Ca 2+ current via mechanical stimuli. This study was undertaken to examine the expression and role of TRPV2 in adult articular cartilage and the development of osteoarthritis (OA).Methods. We examined TRPV2 expression in mouse and human articular cartilage. We analyzed the development of OA in Col2a1-Cre ERt2 ;Trpv2 fl/fl mice and Trpv2 fl/fl littermates in the resection of the medial meniscus and medial collateral ligament model (n = 5 each), the destabilization of the medial meniscus model (n = 5 each), and the aging mouse model (n = 8-9 each). We examined marker protein expression in these joints, Ca 2+ influx by mechanical stimuli, and downstream pathways in vitro.Results. TRPV2 was expressed in mouse and human articular cartilage and ectopic ossification lesions. In all mouse models of OA examined, Col2a1-Cre ERt2 ;Trpv2 fl/fl mice were observed to have enhanced degradation of articular cartilage accompanied by decreased expression of lubricin/Prg4, and marked formation of periarticular ectopic ossification. Mechanical stress-induced Ca 2+ influx was decreased by Trpv2 knockout (KO). Prg4 induction by fluidflow shear stress was diminished in Trpv2-KO mouse chondrocytes, and this was mediated by the Ca 2+ /calmodulindependent protein kinase kinase-cyclic AMP response element binding protein axis. Hypertrophic differentiation was enhanced in Trpv2-KO mouse chondrocytes. Increased activity of calcineurin and nuclear translocation of nuclear factor in activated T cells 1 induced by fluid-flow shear stress or TRP agonist treatment was reversed by Trpv2 knockout.Conclusion. Our findings demonstrate regulation of articular cartilage by TRPV2 through Prg4 induction and suppression of ectopic ossification.

Ectopic endochondral ossification in the tendon/ligament is caused by repetitive mechanical overload or inflammation. Tendon stem/progenitor cells (TSPCs) contribute to tissue repair, and some express lubricin [proteoglycan 4 (PRG4)]. However, the mechanisms of ectopic ossification and association of TSPCs are not yet known. Here, we investigated the characteristics of Prg4-positive ( + ) cells and identified that R-spondin 2 (RSPO2), a WNT activator, is specifically expressed in a distinct Prg4 + TSPC cluster. The Rspo2 + cluster was characterized as mostly undifferentiated, and RSPO2 overexpression suppressed ectopic ossification in a mouse Achilles tendon puncture model via chondrogenic differentiation suppression. RSPO2 expression levels in patients with ossification of the posterior longitudinal ligament were lower than those in spondylosis patients, and RSPO2 protein suppressed chondrogenic differentiation of human ligament cells. RSPO2 was induced by inflammatory stimulation and mechanical loading via nuclear factor κB. Rspo2 + cells may contribute to tendon/ligament homeostasis under pathogenic conditions.