Nalesso G , Thomas BL , Sherwood JC , Yu J , Addimanda O , Eldridge SE , Thorup AS , Dale L , Schett G , Zwerina J , Eltawil N , Pitzalis C , Dell'Accio F .

MR/K013076/1 Medical Research Council , MRC_MR/K013076/1 Medical Research Council , 19654 Versus Arthritis, MR/N010973/1 Medical Research Council

             wound healing
             canonical Wnt signaling pathway

Xla Wt + wnt16 + wnt8a (G, H)
Xla Wt + wnt8a (G, H)

	Figure 5. WNT16 is a weak activator of the β-catenin-dependent pathway but also limits its activation by other more potent canonical WNTs. WNT16 caused a weak upregulation of the canonical WNT reporter gene Axin2 compared with WNT3a, in human articular chondrocytes (HAC) (N=3 patients, 4 replicates each) (A). Lubricin expression caused by WNT16 stimulation (ng/mL) in HAC (B), and mouse superficial zone cell (SZC) (C), was higher than that caused by WNT3a stimulation. Lubricin expression decreased in the presence of canonical inhibitor DKK1 (100 ng/mL) in mouse SZC (N=4) (C). All at 24 h. Following destabilisation of the medial meniscus (DMM) surgery, wnt16−/− mice had a higher expression of Axin2 mRNA in their joints (D). WNT16 reduced the capacity of WNT3a to activate the SUPER8XTOPFlash reporter assay in HEK293 cells treated with WNT3a, alone and in combination with WNT16 (ng/mL) (N=4) (E and F). wnt16 inhibited axis duplication caused by wnt8 in Xenopus laevis (G and H). Embryos were injected ventrally with 0.02 ng/µL wnt16 or 0.002 ng/µL wnt8 at the four-cell stage and left for 2 days to develop (n=36–47).
	Figure 1. WNT16 is upregulated in experimental osteoarthritis induced by destabilisation of the medial meniscus (DMM). Wild-type mice received DMM of the left knee and sham operation on the right and were killed at 2†days (C and D), 7†days (E and F) and 8†weeks (G) post surgery. (A) WNT16 mRNA was upregulated in the whole joints of DMM-operated mice compared with sham-operated controls, reaching statistical significance after 7†days. WNT16 protein was undetectable in non-operated knees (B), but was upregulated at 2†days following DMM (C) and, to a lesser extent, following sham operation (D). WNT16 expression was further increased in DMM-operated mice at 7†days (E) and to less of an extent following sham operation (F) (joint tissues labelled as follows: C, cartilage; B, subchondral bone; M, meniscus). After 8†weeks, (G) WNT16 was no longer detected in DMM-operated knees. (H) Isotype negative control at the 2-day time point. N=3 per condition.
	Figure 2. Wnt16−/− mice are more susceptible to instability-induced osteoarthritis. The 10-week-old mice (7 wild-type (WT) and 10 wnt16−/− (KO)) were subjected to destabilisation of the medial meniscus and the outcome was compared 8†weeks following surgery. (A) Safranin O staining of the medial compartment of representative sections. (B) OARSI histological score. (C) Intensity of the safranin O in the medial tibial plateau. LF, lateral femur; LT, lateral tibia; MF, medial femur; MT, medial tibia.
	Figure 3. Lubricin was upregulated following destabilisation of the medial meniscus (DMM). Wild-type (WT) and wnt16−/− mice were subjected to DMM and killed 2†days thereafter. (A) The joint surface, including the articular cartilage and the subchondral bone, was dissected and processed for gene expression analysis by real-time PCR. Lubricin was upregulated in WT mice but not in wnt16−/− mutants at mRNA level 2†days following DMM (N≥10). (B) At protein level, lubricin staining following DMM was detected in the superficial layer of the articular cartilage and in a region between the intermediate and the deep layer in WT mice. (C) The area of the articular cartilage positive for lubricin staining 2†days following DMM was significantly higher in WT mice (N=4) than in wnt16−/− mice (N=6). (D) WNT16 recombinant protein upregulated lubricin mRNA expression in bovine articular chondrocyte (BAC) at 24†h (N=3). KO, knockout. *p<0.05; **p<0.01; ***p<0.001.
	Figure 4. Disruption of WNT16 results in apoptosis of superficial cartilage cells and in the loss of superficial zone molecular markers. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of representative sections from the medial compartment of the knee of wild-type (WT) and wnt16−/− mice 2†days after destabilisation of the medial meniscus (DMM) (N=6) (A). The TUNEL staining (shown in red) was superimposed on the Nomarski image for anatomical orientation. Quantification of TUNEL-positive cells in the superficial and deep layers of the articular cartilage within the medial compartment (most affected in DMM) and lateral compartment (mildly or unaffected) (B). Gene expression analysis of superficial zone cells from wnt16−/− mice with or without rescue with exogenous 200†ng/mL recombinant WNT16 (N=4) (C). KO, knockout. *p<0.05.
	Figure 5. WNT16 is a weak activator of the β-catenin-dependent pathway but also limits its activation by other more potent canonical WNTs. WNT16 caused a weak upregulation of the canonical WNT reporter gene Axin2 compared with WNT3a, in human articular chondrocytes (HAC) (N=3 patients, 4 replicates each) (A). Lubricin expression caused by WNT16 stimulation (ng/mL) in HAC (B), and mouse superficial zone cell (SZC) (C), was higher than that caused by WNT3a stimulation. Lubricin expression decreased in the presence of canonical inhibitor DKK1 (100 ng/mL) in mouse SZC (N=4) (C). All at 24 h. Following destabilisation of the medial meniscus (DMM) surgery, wnt16−/− mice had a higher expression of Axin2 mRNA in their joints (D). WNT16 reduced the capacity of WNT3a to activate the SUPER8XTOPFlash reporter assay in HEK293 cells treated with WNT3a, alone and in combination with WNT16 (ng/mL) (N=4) (E and F). wnt16 inhibited axis duplication caused by wnt8 in Xenopus laevis (G and H). Embryos were injected ventrally with 0.02 ng/µL wnt16 or 0.002 ng/µL wnt8 at the four-cell stage and left for 2 days to develop (n=36–47).
	Figure 6. Cartilage injury causes activation of WNT signalling through downregulation of antagonists such as FRZB and upregulation of several agonists such as WNT16 and WNT8.14 18 WNT16 buffers the canonical WNT activation to homeostatic levels through its capacity to directly support a weak activation and preventing excessive activation induced by other ligands. Excessive canonical WNT activation causes cartilage breakdown by driving inappropriate maturation particularly within the superficial zone progenitor cells,8 18 whereas homeostatic levels of activation are necessary for supporting the superficial progenitor population and lubricin expression (this work and refs 5, 7).

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