WTAP-mediated m6A modification of FRZB triggers the inflammatory response via the Wnt signaling pathway in osteoarthritis

Abstract

Osteoarthritis (OA) is the most common form of arthritis. However, the exact pathogenesis remains unclear. Emerging evidence shows that N6-methyladenosine (m⁶A) modification may have an important role in OA pathogenesis. This study aimed to investigate the role of m⁶A writers and the underlying mechanisms in osteoarthritic cartilage. Among m⁶A methyltransferases, Wilms tumor 1-associated protein (WTAP) expression most significantly differed in clinical osteoarthritic cartilage. WTAP regulated extracellular matrix (ECM) degradation, inflammation and antioxidation in human chondrocytes. Mechanistically, the m⁶A modification and relative downstream targets in osteoarthritic cartilage were assessed by methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing, which indicated that the expression of frizzled-related protein (FRZB), a secreted Wnt antagonist, was abnormally decreased and accompanied by high m⁶A modification in osteoarthritic cartilage. In vitro dysregulated WTAP had positive effects on β-catenin expression by targeting FRZB, which finally contributed to the cartilage injury phenotype in chondrocytes. Intra-articular injection of adeno-associated virus-WTAP alleviated OA progression in a mouse model, while this protective effect could be reversed by the application of a Wnt/β-catenin activator. In summary, this study revealed that WTAP-dependent RNA m⁶A modification contributed to Wnt/β-catenin pathway activation and OA progression through post-transcriptional regulation of FRZB mRNA, thus providing a potentially effective therapeutic strategy for OA treatment.

Fig. 1. WTAP was upregulated in TNF-α-induced chondrocytes and osteoarthritic cartilage.

a Relative mRNA expression of m⁶A methylases (METLL3, METLL4, VIRMA, FTO, ALKBH5, and WTAP) measured by qRT‒PCR in osteoarthritic (n = 6) vs. uninjured cartilage tissues (n = 7). b Relative mRNA expression of ADAMTS4, ADAMTS5, MMP13, IL-6, IL-8, and iNOS measured by qRT‒PCR in human chondrocytes with or without TNF-α treatment (50 ng/mL). c, d Western blotting (c) and quantitative analysis (d) of ADAMTS4, ADAMTS5, and MMP13 in human chondrocytes with or without TNF-α treatment (50 ng/mL). e DCFH-DA staining for ROS levels in chondrocytes treated with TNF-α (50 ng/mL). Scale bar: 200 µm. f Relative mRNA expression levels of m⁶A methylases (METLL3, METLL4, VIRMA, FTO, ALKBH5, and WTAP) measured by qRT‒PCR in chondrocytes with or without TNF-α treatment (50 ng/mL). g, h Western blotting (g) and corresponding quantitative analysis (h) of WTAP protein expression in osteoarthritic cartilage. Data are presented as the means ± SEMs. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 2. WTAP overexpression aggravated the TNF-α-induced osteoarthritic chondrocyte phenotype.

a–d Overexpression efficiency of WTAP in human chondrocytes. Relative WTAP expression measured by qRT‒PCR (a), Western blotting (b), quantitative analysis (c), and IF staining (d) in chondrocytes after transfection with flag-WTAP. Scale bars: 100 µm. e The m⁶A level of total RNA in chondrocytes with or without WTAP overexpression. f Relative mRNA expression of ADAMTS4, ADAMTS5, MMP13, IL-6, IL-8, and iNOS was measured by qRT‒PCR in chondrocytes with or without WTAP overexpression. g, h Western blotting (g) and quantitative analysis (h) of ADAMTS4, ADAMTS5, and MMP13 expression in chondrocytes with or without WTAP overexpression. i, j Detection of ROS (i) and quantitative analysis (j) in chondrocytes with or without WTAP overexpression. Scale bars: 200 µm. Data are presented as the means ± SEMs. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 3. Knocking out WTAP alleviated the osteoarthritic chondrocyte phenotype.

a GFP identification in human chondrocytes transfected with WTAP gene knockout lentivirus (KO-WTAP). Scale bars: 200 µm. b–e Knockout efficiency of WTAP in human chondrocytes. Relative WTAP expression measured by qRT‒PCR (b), Western blotting (c), quantitative analysis (d), and IF staining (e) of chondrocytes after transfection with KO-WTAP. f The m⁶A level of total RNA in chondrocytes with or without KO-WTAP transfection. g Relative mRNA expression of ADAMTS4, ADAMTS5, MMP13, IL-6, IL-8, and iNOS measured by qRT‒PCR in chondrocytes with or without KO-WTAP transfection. h, i Western blotting (h) and quantitative analysis (i) of ADAMTS4, ADAMTS5 and MMP13.

Fig. 4. The Wnt pathway inhibitor FRZB abnormally elevated m⁶A methylation in OA.

a Statistical analysis of m⁶A peak gene number. b The distribution of different m⁶A peaks in different regions of the reference genome. c Volcano plot illustrating the distributions of differentially expressed m⁶A genes. d Heatmap of differential gene clustering. e Functional analysis of hypermethylated genes based on the STRING database (the genes marked in red are genes related to cartilage development, https://cn.string-db.org/). f Cartilage development-related genes corresponding to the methylation sequencing score, gene expression and P value score. g, h FRZB expression was assessed by qRT‒PCR (g) and Western blotting (h) in human chondrocytes with or without treatment with the m⁶A methylation inhibitor DAA (50 µM). i, j FRZB expression was assessed in human chondrocytes treated with TNF-α (50 ng/ml). Relative FRZB expression measured by qRT‒PCR (i), Western blotting and quantitative analysis (j) of FRZB protein in human osteoarthritic chondrocytes. k KEGG analysis of m⁶A difference genes in osteoarthritic (n = 3) vs. normal cartilage tissues (n = 2). Data are presented as the means ± SEMs. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 5. The FRZB-Wnt/β-catenin axis was regulated by WTAP in chondrocytes.

a–c Protein levels of FRZB and β-catenin were analyzed in chondrocytes with or without WTAP overexpression. d, e Relative mRNA expression levels of FRZB (d) and β-catenin (e) were measured by qRT‒PCR in chondrocytes with or without WTAP overexpression. f, g IF staining of intracellular FRZB (f) and β-catenin (g) in TNF-α-pretreated cells with or without WTAP overexpression. Scale bars: 100 µm. h–j Protein levels of FRZB and β-catenin were analyzed in TNF-α-pretreated chondrocytes with or without KO-WTAP transfection. k, l Relative mRNA expression levels of FRZB (k) and β-catenin (l) were measured by qRT‒PCR in TNF-α-pretreated cells with or without KO-WTAP transfection. m, n IF staining of intracellular FRZB (m) and β-catenin (n) expression in TNF-α-pretreated cells with or without KO-WTAP transfection. Scale bars: 100 µm. Data are presented as the means ± SEMs. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 6. WTAP regulated Wnt/β-catenin signaling activation and the osteoarthritic chondrocyte phenotype by m⁶A modification of FRZB.

a, b FRZB expression was analyzed in chondrocytes with WTAP overexpression after DAA (50 µM) treatment by qRT‒PCR (a) and IF staining (b). Scale bars: 100 µm. c, d β-catenin expression was analyzed in chondrocytes with WTAP overexpression after DAA (50 µM) treatment by qRT‒PCR (c) and IF staining (d). Scale bars: 100 µm. e Relative mRNA expression of ADAMTS4, ADAMTS5, MMP13, IL-6, IL-8, and iNOS measured by qRT‒PCR in chondrocytes with WTAP overexpression after DAA (50 µM) treatment. f, g Western blotting (f) and quantitative analysis (g) of ADAMTS4, ADAMTS5 and MMP13 expression in chondrocytes with WTAP overexpression after DAA (50 µM) treatment. h, i Detection of ROS (h) and quantitative analysis (i) in chondrocytes with WTAP overexpression after DAA (50 µM) treatment. Scale bars: 200 µm. Data are presented as the means ± SEMs. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 7. WTAP inhibition alleviated OA progression through Wnt/β-catenin signaling inactivation.

a Safranin O/fast green staining of the cartilage in the indicated groups at 8 weeks after DMM surgery. b OARSI scoring of knee OA was evaluated by Safranin O/fast green staining (n = 6). c, d IHC staining of COL2, ADAMTS5, MMP13, FRZB, and β-catenin in cartilage from the indicated groups at 8 weeks after DMM surgery. e Open field test in different groups. f The corresponding quantitative analysis of (e) (n = 6). g Footprint images in different groups and statistical analysis (n = 6). Data are presented as the means ± SEMs. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 8

Proposed model for the regulation of WTAP on OA progression through FRZB/β-catenin signaling activation.