TRIM24-RIP3 axis perturbation accelerates osteoarthritis pathogenesis

Objectives Recently, necroptosis has attracted increasing attention in arthritis research; however, it remains unclear whether its regulation is involved in osteoarthritis (OA) pathogenesis. Since receptor-interacting protein kinase-3 (RIP3) plays a pivotal role in necroptosis and its dysregulation is involved in various pathological processes, we investigated the role of the RIP3 axis in OA pathogenesis. Methods Experimental OA was induced in wild-type or Rip3 knockout mice by surgery to destabilise the medial meniscus (DMM) or the intra-articular injection of adenovirus carrying a target gene (Ad-Rip3 and Ad-Trim24 shRNA). RIP3 expression was examined in OA cartilage from human patients; Trim24, a negative regulator of RIP3, was identified by microarray and in silico analysis. Connectivity map (CMap) and in silico binding approaches were used to identify RIP3 inhibitors and to examine their direct regulation of RIP3 activation in OA pathogenesis. Results RIP3 expression was markedly higher in damaged cartilage from patients with OA than in undamaged cartilage. In the mouse model, adenoviral RIP3 overexpression accelerated cartilage disruption, whereas Rip3 depletion reduced DMM-induced OA pathogenesis. Additionally, TRIM24 knockdown upregulated RIP3 expression; its downregulation promoted OA pathogenesis in knee joint tissues. The CMap approach and in silico binding assay identified AZ-628 as a potent RIP3 inhibitor and demonstrated that it abolished RIP3-mediated OA pathogenesis by inhibiting RIP3 kinase activity. Conclusions TRIM24-RIP3 axis perturbation promotes OA chronicity by activating RIP3 kinase, suggesting that the therapeutic manipulation of this pathway could provide new avenues for treating OA.

protease inhibitors. Equal amounts of cell extracts were resolved by SDS-PAGE (6% stacking gels and 10% running gels) and analyzed by immunoblotting.

Reverse transcription (RT)-PCR and qPCR
Total RNA was extracted from articular chondrocytes using TRIzol reagent (Molecular Research Center (Cincinnati, OH, USA)), reverse transcribed into complementary DNA (cDNA) using ImProm-II™ Reverse Transcriptase (Promega (Madison, WI, USA)), and amplified by PCR or qPCR with primers as summarized in Supplementary Table S3. qPCR was performed using SYBR premix Ex Taq (TaKaRa Bio, Kusatsu, Shiga, Japan), with results normalized to Gapdh and expressed as fold-changes relative to the control.

Collagenase and aggrecanase activity assay
Chondrocytes were seeded in 6-well dishes (2 × 10 5 cells·well) and infected with Ad-C or Ad-Rip3. The cells were incubated for 36 h in DMEM without fetal bovine serum (FBS). The culture medium was collected; equal volumes were concentrated using Viva® Spin Columns (Sartorius Stedim Biotech, Göttingen, Germany) according to the manufacturer's protocol.
Concentrated samples were assayed for total collagenase activity using EnzCheck™ Gelatinase/Collagenase Assay kits (Molecular Probes, Eugene, OR, USA). Collagenase activity was measured as fluorescent signals using the SYNERGY H1 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at Ex/Em = 485/530 nm. Aggrecanase activity was assessed using an aggrecanase activity assay kit (Abnova, Taipei, Taiwan). The aggrecanase level was quantified in concentrated supernatant by measuring the absorbance at Human OA cartilage and mouse knee joints were fixed in 4 % paraformaldehyde and embedded in paraffin. Mouse knee joints were decalcified for 2 weeks in 0.5 M EDTA (pH 7.4). Paraffin-embedded samples were stained with Safranin-O or Alcian blue or immunostained. Cartilage destruction was assessed in the experimental OA mouse model by three observers blinded to the experimental groupings and scored according to the OARSI (Osteoarthritis Research Society International) grading system (grade 0-6). OARSI scores were presented as the mean maximum score for each mouse. Representative Safranin-O staining images were selected from the most advanced lesions in each section and osteophyte maturity quantified as described previously. 4 Subchondral bone sclerosis was determined by measuring subchondral bone plate thickness. Immunohistochemical staining was performed in human and mouse cartilage sections with MMP3, MMP13, and MLKL (Abcam), COX2 and TRIM24 (Proteintech), and RIP3 (Enzo Biochem) antibodies.

Microarray analyses
Mouse articular chondrocytes were infected with Ad-Rip3 or Ad-C (MOI, 800) for 36 h.

Gene set enrichment analysis (GSEA)
GSEA was performed using java GSEA software (ver. 4.0.3; Broad Institute, MIT). 8 Genes were ranked according to their expression; those up-or downregulated following RIP3 overexpression were selected as the RIP3-related gene set.

Statistical analysis
All experiments were performed independently at least four times. Two independent groups were compared using the Shapiro-Wilk normality test, Levene's homogeneity of variance test, and a two-tailed independent t-test. Multiple comparisons were made using the Shapiro-Wilk test, Levene's test, and one-way analysis of variance with Bonferroni's post-hoc test.
Data based on ordinal grading systems were analyzed using non-parametric Mann-Whitney U tests. P values < 0.05 were considered statistically significant.  Genes upregulated in osteoarthritic cartilage (150 genes) 3830406C13Rik Cdkn2b Genes downregulated in osteoarthritic cartilage (71 genes) Agtr2 Cmtm5 Statistical analyses were performed using a two-tailed t-test. Values are presented as the means ± SEM and assessed using one-way ANOVA with Bonferroni's test (A and D) or a two-tailed t-test (C).