Regulation of cytokine and chemokine expression by histone lysine methyltransferase MLL1 in rheumatoid arthritis synovial fibroblasts.

Histone lysine methylation is thought to play a role in the pathogenesis of rheumatoid arthritis (RA). We previously reported aberrant expression of the gene encoding mixed-lineage leukemia 1 (MLL1), which catalyzes methylation of histone H3 lysine 4 (H3K4), in RA synovial fibroblasts (SFs). The aim of this study was to elucidate the involvement of MLL1 in the activated phenotype of RASFs. SFs were isolated from synovial tissues obtained from patients with RA or osteoarthritis (OA) during total knee joint replacement. MLL1 mRNA and protein levels were determined after stimulation with tumor necrosis factor α (TNFα). We also examined changes in trimethylation of H3K4 (H3K4me3) levels in the promoters of RA-associated genes (matrix-degrading enzymes, cytokines, and chemokines) and the mRNA levels upon small interfering RNA-mediated depletion of MLL1 in RASFs. We then determined the levels of H3K4me3 and mRNAs following treatment with the WD repeat domain 5 (WDR5)/MLL1 inhibitor MM-102. H3K4me3 levels in the gene promoters were also compared between RASFs and OASFs. After TNFα stimulation, MLL1 mRNA and protein levels were higher in RASFs than OASFs. Silencing of MLL1 significantly reduced H3K4me3 levels in the promoters of several cytokine (interleukin-6 [IL-6], IL-15) and chemokine (C-C motif chemokine ligand 2 [CCL2], CCL5, C-X-C motif chemokine ligand 9 [CXCL9], CXCL10, CXCL11, and C-X3-C motif chemokine ligand 1 [CX3CL1]) genes in RASFs. Correspondingly, the mRNA levels of these genes were significantly decreased. MM-102 significantly reduced the promoter H3K4me3 and mRNA levels of the CCL5, CXCL9, CXCL10, and CXCL11 genes in RASFs. In addition, H3K4me3 levels in the promoters of the IL-6, IL-15, CCL2, CCL5, CXCL9, CXCL10, CXCL11, and CX3CL1 genes were significantly higher in RASFs than OASFs. Our findings suggest that MLL1 regulates the expression of particular cytokines and chemokines in RASFs and is associated with the pathogenesis of RA. These results could lead to new therapies for RA.

Association between omega-3 polyunsaturated fatty acids and osteoarthritis: results from the NHANES 2003-2016 and Mendelian randomization study.

BACKGROUND: Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) exhibit potential as therapeutics for a variety of diseases. This observational and Mendelian randomization (MR) study aims to explore the relationship between omega-3 PUFAs and osteoarthritis (OA). METHODS: Excluding individuals under 20 years old and those with missing data on relevant variables in the National Health and Nutrition Examination Survey (NHANES) spanning from 2003 to 2016, a total of 22 834 participants were included in this cross-sectional study. Weighted multivariable-adjusted logistic regression was used to estimate the association between omega-3 PUFAs and OA in adults. Moreover, restricted cubic splines were utilized to examine the dose-response relationship between omega-3 PUFAs and OA. To further investigate the potential causal relationship between omega-3 PUFAs and OA risk, a two-sample MR study was conducted. Furthermore, the robustness of the findings was assessed using various methods. RESULTS: Omega-3 PUFAs intake were inversely associated with OA in adults aged 40 ∼ 59 after multivariable adjustment [Formula: see text], with a nonlinear relationship observed between omega-3 PUFAs intake and OA [Formula: see text]. The IVW results showed there was no evidence to suggest a causal relationship between omega-3 PUFAs and OA risk [Formula: see text]. CONCLUSIONS: Omega-3 PUFAs were inversely associated with OA in adults aged 40 ∼ 59. However, MR studies did not confirm a causal relationship between the two.

TPX2 upregulates MMP13 to promote the progression of lipopolysaccharide-induced osteoarthritis.

Purpose: This study seeks to identify potential clinical biomarkers for osteoarthritis (OA) using bioinformatics and investigate OA mechanisms through cellular assays. Methods: Differentially Expressed Genes (DEGs) from GSE52042 (four OA samples, four control samples) were screened and analyzed with protein-protein interaction (PPI) analysis. Overlapping genes in GSE52042 and GSE206848 (seven OA samples, and seven control samples) were identified and evaluated using Gene Set Enrichment Analysis (GSEA) and clinical diagnostic value analysis to determine the hub gene. Finally, whether and how the hub gene impacts LPS-induced OA progression was explored by in vitro experiments, including Western blotting (WB), co-immunoprecipitation (Co-IP), flow cytometry, etc. Result: Bioinformatics analysis of DEGs (142 up-regulated and 171 down-regulated) in GSE52042 identified two overlapping genes (U2AF2, TPX2) that exhibit significant clinical diagnostic value. These genes are up-regulated in OA samples from both GSE52042 and GSE206848 datasets. Notably, TPX2, which AUC = 0.873 was identified as the hub gene. In vitro experiments have demonstrated that silencing TPX2 can alleviate damage to chondrocytes induced by lipopolysaccharide (LPS). Furthermore, there is a protein interaction between TPX2 and MMP13 in OA. Excessive MMP13 can attenuate the effects of TPX2 knockdown on LPS-induced changes in OA protein expression, cell growth, and apoptosis. Conclusion: In conclusion, our findings shed light on the molecular mechanisms of OA and suggested TPX2 as a potential therapeutic target. TPX2 could promote the progression of LPS-induced OA by up-regulating the expression of MMP13, which provides some implications for clinical research.

Inhibition of miR-199b-5p reduces pathological alterations in osteoarthritis by potentially targeting Fzd6 and Gcnt2.

Osteoarthritis (OA) is a degenerative disease with a high prevalence in the elderly population, but our understanding of its mechanisms remains incomplete. Analysis of serum exosomal small RNA sequencing data from clinical patients and gene expression data from OA patient serum and cartilage obtained from the GEO database revealed a common dysregulated miRNA, miR-199b-5p. In vitro cell experiments demonstrated that miR-199b-5p inhibits chondrocyte vitality and promotes extracellular matrix degradation. Conversely, inhibition of miR-199b-5p under inflammatory conditions exhibited protective effects against damage. Local viral injection of miR-199b-5p into mice induced a decrease in pain threshold and OA-like changes. In an OA model, inhibition of miR-199b-5p alleviated the pathological progression of OA. Furthermore, bioinformatics analysis and experimental validation identified Gcnt2 and Fzd6 as potential target genes of MiR-199b-5p. Thus, these results indicated that MiR-199b-5p/Gcnt2 and Fzd6 axis might be a novel therapeutic target for the treatment of OA.

Analysis of common differential gene expression in synovial cells of osteoarthritis and rheumatoid arthritis.

OBJECTIVE: To elucidate potential molecular mechanisms differentiating osteoarthritis (OA) and rheumatoid arthritis (RA) through a bioinformatics analysis of differentially expressed genes (DEGs) in patient synovial cells, aiming to provide new insights for clinical treatment strategies. MATERIALS AND METHODS: Gene expression datasets GSE1919, GSE82107, and GSE77298 were downloaded from the Gene Expression Omnibus (GEO) database to serve as the training groups, with GSE55235 being used as the validation dataset. The OA and RA data from the GSE1919 dataset were merged with the standardized data from GSE82107 and GSE77298, followed by batch effect removal to obtain the merged datasets of differential expressed genes (DEGs) for OA and RA. Intersection analysis was conducted on the DEGs between the two conditions to identify commonly upregulated and downregulated DEGs. Enrichment analysis was then performed on these common co-expressed DEGs, and a protein-protein interaction (PPI) network was constructed to identify hub genes. These hub genes were further analyzed using the GENEMANIA online platform and subjected to enrichment analysis. Subsequent validation analysis was conducted using the GSE55235 dataset. RESULTS: The analysis of differentially expressed genes in the synovial cells from patients with Osteoarthritis (OA) and Rheumatoid Arthritis (RA), compared to a control group (individuals without OA or RA), revealed significant changes in gene expression patterns. Specifically, the genes APOD, FASN, and SCD were observed to have lower expression levels in the synovial cells of both OA and RA patients, indicating downregulation within the pathological context of these diseases. In contrast, the SDC1 gene was found to be upregulated, displaying higher expression levels in the synovial cells of OA and RA patients compared to normal controls.Additionally, a noteworthy observation was the downregulation of the transcription factor PPARG in the synovial cells of patients with OA and RA. The decrease in expression levels of PPARG further validates the alteration in lipid metabolism and inflammatory processes associated with the pathogenesis of OA and RA. These findings underscore the significance of these genes and the transcription factor not only as biomarkers for differential diagnosis between OA and RA but also as potential targets for therapeutic interventions aimed at modulating their expression to counteract disease progression. CONCLUSION: The outcomes of this investigation reveal the existence of potentially shared molecular mechanisms within Osteoarthritis (OA) and Rheumatoid Arthritis (RA). The identification of APOD, FASN, SDC1, TNFSF11 as key target genes, along with their downstream transcription factor PPARG, highlights common potential factors implicated in both diseases. A deeper examination and exploration of these findings could pave the way for new candidate targets and directions in therapeutic research aimed at treating both OA and RA. This study underscores the significance of leveraging bioinformatics approaches to unravel complex disease mechanisms, offering a promising avenue for the development of more effective and targeted treatments.

GPX7 reduces chondrocyte inflammation and extracellular matrix degradation triggered by IL­1ß, via a mechanism mediated by ferroptosis.

During osteoarthritis (OA), chondrocytes become highly active, with increased matrix synthesis and inflammatory cytokine­induced catabolic pathways. Early intervention strategies targeting pathological changes may attenuate or halt disease progression. The present study aimed to reveal the role of glutathione peroxidase (GPX)7 in OA. For this purpose, a research model was established by inducing C28/I2 human chondrocytes with interleukin (IL)­1ß, and the expression level of GPX7 was determined. To explore its roles, C28/I2 cells were transfected to gain GPX7 overexpression. The effects of GPX7 overexpression on intracellular inflammation, extracellular matrix (ECM) degradation, apoptosis and ferroptosis were then evaluated. In addition, the cells were treated with the ferroptosis inducer, erastin, and its effects on the aforementioned phenotypes were assessed. The level of GPX7 was decreased in response to IL­1ß treatment, and GPX7 overexpression suppressed cellular inflammation, ECM degradation and apoptosis. Moreover, the reduction of lipid peroxidation, ferrous ions and transferrin indicated that GPX7 overexpression inhibited ferroptosis. Subsequently, inflammation, ECM degradation and apoptosis were found to be promoted in the cells upon treatment with erastin. These findings suggested that the regulatory role of GPX7 may be mediated by a pathway involving ferroptosis. On the whole, the present study revealed that GPX7 reduces IL­1ß­induced chondrocyte inflammation, apoptosis and ECM degradation partially through a mechanism involving ferroptosis. The results of the present study lay a theoretical foundation for subsequent OA­related research and may enable the development of translational strategies for the treatment of OA.

Knocking-down long non-coding RNA LINC01094 prohibits chondrocyte apoptosis via regulating microRNA-577/metal-regulatory transcription factor 1 axis.

PURPOSE: The abnormal function and survival of chondrocytes result in articular cartilage failure, which may accelerate the onset and development of osteoarthritis (OA). This study is aimed to investigate the role of LINC01094 in chondrocyte apoptosis. METHODS: The viability and apoptosis of lipopolysaccharide (LPS)-induced chondrocytes were evaluated through CCK-8 assay and flow cytometry analysis, respectively. The expression levels of LINC01094, miR-577 and MTF1 were detected by qRT-PCR. Dual luciferase reporter tests were implemented for the verification of targeted relationships among them. Western blotting was employed to measure the levels of pro-apoptotic proteins (Caspase3 and Caspase9). RESULTS: The viability of LPS-induced chondrocytes was overtly promoted by loss of LINC01094 or miR-577 upregulation, but could be repressed via MTF1 overexpression. The opposite results were observed in apoptosis rate and the levels of Caspase3 and Caspase9. LINC01094 directly bound to miR-577, while MTF1 was verified to be modulated by miR-577. Both LINC01094 and MTF1 were at high levels, whereas miR-577 was at low level in OA synovial fluid and LPS-induced chondrocytes. Furthermore, the highly expressed miR-577 abolished the influences of MTF1 overexpression on LPS-induced chondrocytes. CONCLUSIONS: Silencing of LINC01094 represses the apoptosis of chondrocytes through upregulating miR-577 expression and downregulating MTF1 levels, providing a preliminary insight for the treatment of OA in the future.

Long-term haplodeficency of DSPP causes temporomandibular joint osteoarthritis in mice.

BACKGROUND: Extracellular matrix (ECM) protein malfunction or defect may lead to temporomandibular joint osteoarthritis (TMJ OA). Dentin sialophophoprotein (DSPP) is a mandibular condylar cartilage ECM protein, and its deletion impacted cell proliferation and other extracellular matrix alterations of postnatal condylar cartilage. However, it remains unclear if long-term loss of function of DSPP leads to TMJ OA. The study aimed to test the hypothesis that long-term haploinsufficiency of DSPP causes TMJ OA. MATERIALS AND METHODS: To determine whether Dspp+/- mice exhibit TMJ OA but no severe tooth defects, mandibles of wild-type (WT), Dspp+/-, and Dspp homozygous (Dspp-/-) mice were analyzed by Micro-computed tomography (micro-CT). To characterize the progression and possible mechanisms of osteoarthritic degeneration over time in Dspp+/- mice over time, condyles of Dspp+/- and WT mice were analyzed radiologically, histologically, and immunohistochemically. RESULTS: Micro-CT and histomorphometric analyses revealed that Dspp+/- and Dspp-/- mice had significantly lower subchondral bone mass, bone volume fraction, bone mineral density, and trabecular thickness compared to WT mice at 12 months. Interestingly, in contrast to Dspp-/- mice which exhibited tooth loss, Dspp+/- mice had minor tooth defects. RNA sequencing data showed that haplodeficency of DSPP affects the biological process of ossification and osteoclast differentiation. Additionally, histological analysis showed that Dspp+/- mice had condylar cartilage fissures, reduced cartilage thickness, decreased articular cell numbers and severe subchondral bone cavities, and with signs that were exaggerated with age. Radiographic data showed an increase in subchondral osteoporosis up to 18 months and osteophyte formation at 21 months. Moreover, Dspp+/- mice showed increased distribution of osteoclasts in the subchondral bone and increased expression of MMP2, IL-6, FN-1, and TLR4 in the mandibular condylar cartilage. CONCLUSIONS: Dspp+/- mice exhibit TMJ OA in a time-dependent manner, with lesions in the mandibular condyle attributed to hypomineralization of subchondral bone and breakdown of the mandibular condylar cartilage, accompanied by upregulation of inflammatory markers.

Hyper-physiologic mechanical cues, as an osteoarthritis disease-relevant environmental perturbation, cause a critical shift in set points of methylation at transcriptionally active CpG sites in neo-cartilage organoids.

BACKGROUND: Osteoarthritis (OA) is a complex, age-related multifactorial degenerative disease of diarthrodial joints marked by impaired mobility, joint stiffness, pain, and a significant decrease in quality of life. Among other risk factors, such as genetics and age, hyper-physiological mechanical cues are known to play a critical role in the onset and progression of the disease (Guilak in Best Pract Res Clin Rheumatol 25:815-823, 2011). It has been shown that post-mitotic cells, such as articular chondrocytes, heavily rely on methylation at CpG sites to adapt to environmental cues and maintain phenotypic plasticity. However, these long-lasting adaptations may eventually have a negative impact on cellular performance. We hypothesize that hyper-physiologic mechanical loading leads to the accumulation of altered epigenetic markers in articular chondrocytes, resulting in a loss of the tightly regulated balance of gene expression that leads to a dysregulated state characteristic of the OA disease state. RESULTS: We showed that hyper-physiological loading evokes consistent changes in CpGs associated with expression changes (ML-tCpGs) in ITGA5, CAV1, and CD44, among other genes, which together act in pathways such as anatomical structure morphogenesis (GO:0009653) and response to wound healing (GO:0042060). Moreover, by comparing the ML-tCpGs and their associated pathways to tCpGs in OA pathophysiology (OA-tCpGs), we observed a modest but particular interconnected overlap with notable genes such as CD44 and ITGA5. These genes could indeed represent lasting detrimental changes to the phenotypic state of chondrocytes due to mechanical perturbations that occurred earlier in life. The latter is further suggested by the association between methylation levels of ML-tCpGs mapped to CD44 and OA severity. CONCLUSION: Our findings confirm that hyper-physiological mechanical cues evoke changes to the methylome-wide landscape of chondrocytes, concomitant with detrimental changes in positional gene expression levels (ML-tCpGs). Since CAV1, ITGA5, and CD44 are subject to such changes and are central and overlapping with OA-tCpGs of primary chondrocytes, we propose that accumulation of hyper-physiological mechanical cues can evoke long-lasting, detrimental changes in set points of gene expression that influence the phenotypic healthy state of chondrocytes. Future studies are necessary to confirm this hypothesis.

Associations between immune cells signatures and osteoarthritis: An integrated analysis of bidirectional Mendelian randomization and Bayesian colocalization.

BACKGROUND: Previous investigations have explored the associations between immune cell signatures and osteoarthritis (OA); however, causality remains unclear. This study employs an integrated analysis, combining bidirectional Mendelian randomization (MR) and Bayesian colocalization (Coloc), to investigate causal relationships between 731 immune cells signatures and OA, identifying shared causal variants. METHODS: Utilizing publicly available summary data, this study primarily employs inverse variance weighting (IVW). Supplementary methods include MR-Egger regression, weighted median, weight mode, and simple mode. Various sensitivity tests, including Cochran's Q test, MR pleiotropy Residual Sum and Outlier, and leave-one-out tests, were conducted to assess the robustness of the analysis results. Coloc was employed to identify shared causal genetic variants among potential associations. RESULTS: IVW analysis revealed 196 immune cell signatures potentially linked to OA across diverse subtypes. Reverse MR analyses indicated the causal impact of OA on the levels of 140 immune cell signatures, with subtype-specific variations. Notably, several specific associations, including CD64 on CD14-CD16 + monocyte for Hip OA (OR = 1.0593, 95 % CI: 1.0260-1.0938, P = 0.0004), HLA-DR on CD14 + CD16- monocyte (OR = 0.9664, 95 % CI: 0.9497-0.9834, P = 0.0001), HLA-DR on CD14 + monocyte (OR = 0.9680, 95 % CI: 0.9509-0.9853, P = 0.0003) in the Knee or Hip OA, PDL-1 on CD14-CD16 + monocyte by All OA (OR = 1.7091, 95 %CI:1.2494-2.3378, P = 0.0008), and herpesvirus entry mediator on effector memory CD4 + T cell by Spine OA (OR = 0.5200, 95 %CI:0.3577-0.7561, P = 0.0006) remained significant post-Bonferroni correction. Sensitivity tests validated the credibility of the IVW analysis. Additionally, Coloc revealed several potential associations among shared genetic variants, including rs115328872, rs1800973, and rs317667. CONCLUSIONS: Our findings provide evidence for the potential involvement of immune cell signatures in OA development, revealing avenues for early prevention and innovative therapeutic strategies.

Emerging technology has a brilliant future: the CRISPR-Cas system for senescence, inflammation, and cartilage repair in osteoarthritis.

Osteoarthritis (OA), known as one of the most common types of aseptic inflammation of the musculoskeletal system, is characterized by chronic pain and whole-joint lesions. With cellular and molecular changes including senescence, inflammatory alterations, and subsequent cartilage defects, OA eventually leads to a series of adverse outcomes such as pain and disability. CRISPR-Cas-related technology has been proposed and explored as a gene therapy, offering potential gene-editing tools that are in the spotlight. Considering the genetic and multigene regulatory mechanisms of OA, we systematically review current studies on CRISPR-Cas technology for improving OA in terms of senescence, inflammation, and cartilage damage and summarize various strategies for delivering CRISPR products, hoping to provide a new perspective for the treatment of OA by taking advantage of CRISPR technology.

SKP2-mediated ubiquitination and degradation of KLF11 promotes osteoarthritis via modulation of JMJD3/NOTCH1 pathway.

Osteoarthritis (OA) is the main cause of cartilage damage and disability. This study explored the biological function of S-phase kinase-associated protein 2 (SKP2) and Kruppel-like factor 11 (KLF11) in OA progression and its underlying mechanisms. C28/I2 chondrocytes were stimulated with IL-1ß to mimic OA in vitro. We found that SKP2, Jumonji domain-containing protein D3 (JMJD3), and Notch receptor 1 (NOTCH1) were upregulated, while KLF11 was downregulated in IL-1ß-stimulated chondrocytes. SKP2/JMJD3 silencing or KLF11 overexpression repressed apoptosis and extracellular matrix (ECM) degradation in chondrocytes. Mechanistically, SKP2 triggered the ubiquitination and degradation of KLF11 to transcriptionally activate JMJD3, which resulted in activation of NOTCH1 through inhibiting H3K27me3. What's more, the in vivo study found that KLF11 overexpression delayed OA development in rats via restraining apoptosis and maintaining the balance of ECM metabolism. Taken together, ubiquitination and degradation of KLF11 regulated by SKP2 contributed to OA progression by activation of JMJD3/NOTCH1 pathway. Our findings provide promising therapeutic targets for OA.

No genetic causal association between circulating alpha-tocopherol levels and osteoarthritis, a two-sample Mendelian randomization analysis.

The causal association between vitamin E status and osteoarthritis (OA) remains controversial in previous epidemiological studies. We employed a Mendelian randomization (MR) analysis to explore the causal relationship between circulating alpha-tocopherol levels (main forms of vitamin E in our body) and OA. The instrumental variables (IVs) of circulating alpha-tocopherol levels were obtained from a Genome-wide association study (GWAS) dataset of 7781 individuals of European descent. The outcome of OA was derived from the UK biobank. Two-sample MR analysis was used to estimate the causal relationship between circulating alpha-tocopherol levels and OA. The inverse-variance weighted (IVW) method was the primary analysis in this analysis. We used the MR-Egger method to determine horizontal pleiotropic in this work. The heterogeneity effect of instrumental IVs was detected by MR-Egger and IVW analyses. Sensitivity analysis was performed by removing single nucleotide polymorphism (SNP) one by one. Three SNPs (rs964184, rs2108622, and rs11057830) (P < 5E-8) strongly associated with circulating alpha-tocopherol levels were used in this analysis. The IVW-random effect indicated no causal relationship between circulating alpha-tocopherol levels and clinically diagnosed OA (OR = 0.880, 95% CI 0.626, 1.236, P = 0.461). Similarly, IVW analysis showed no causal association between circulating alpha-tocopherol levels and self-reported OA (OR = 0.980, 95% CI 0.954, 1.006, P = 0.139). Other methods of MR analyses and sensitivity analyses revealed consistent findings. MR-Egger and IVW methods indicated no significant heterogeneity between IVs. The MR-Egger intercept showed no horizontal pleiotropic. The results of this linear Mendelian randomization study indicate no causal association between genetically predicted alpha-tocopherol levels and the progression of OA. Alpha-tocopherol may not provide beneficial and more favorable outcomes for the progression of OA. Further MR analysis based on updated GWASs with more IVs is required to verify the results of our study.

Integration of metabolomics and transcriptomics provides insights into the molecular mechanism of temporomandibular joint osteoarthritis.

The deficiency of clinically specific biomarkers has made it difficult to achieve an accurate diagnosis of temporomandibular joint osteoarthritis (TMJ-OA) and the insufficient comprehension of the pathogenesis of the pathogenesis of TMJ-OA has posed challenges in advancing therapeutic measures. The combined use of metabolomics and transcriptomics technologies presents a highly effective method for identifying vital metabolic pathways and key genes in TMJ-OA patients. In this study, an analysis of synovial fluid untargeted metabolomics of 6 TMJ-OA groups and 6 temporomandibular joint reducible anterior disc displacement (TMJ-DD) groups was conducted using liquid and gas chromatography mass spectrometry (LC/GC-MS). The differential metabolites (DMs) between TMJ-OA and TMJ-DD groups were analyzed through multivariate analysis. Meanwhile, a transcriptomic dataset (GSE205389) was obtained from the GEO database to analyze the differential metabolism-related genes (DE-MTGs) between TMJ-OA and TMJ-DD groups. Finally, an integrated analysis of DMs and DE-MTGs was carried out to investigate the molecular mechanisms associated with TMJ-OA. The analysis revealed significant differences in the levels of 46 DMs between TMJ-OA and TMJ-DD groups, of which 3 metabolites (L-carnitine, taurine, and adenosine) were identified as potential biomarkers for TMJ-OA. Collectively, differential expression analysis identified 20 DE-MTGs. Furthermore, the integration of metabolomics and transcriptomics analysis revealed that the tricarboxylic acid (TCA) cycle, alanine, aspartate and glutamate metabolism, ferroptosis were significantly enriched. This study provides valuable insights into the metabolic abnormalities and associated pathogenic mechanisms, improving our understanding of TMJOA etiopathogenesis and facilitating potential target screening for therapeutic intervention.

DDX5 inhibits hyaline cartilage fibrosis and degradation in osteoarthritis via alternative splicing and G-quadruplex unwinding.

Hyaline cartilage fibrosis is typically considered an end-stage pathology of osteoarthritis (OA), which results in changes to the extracellular matrix. However, the mechanism behind this is largely unclear. Here, we found that the RNA helicase DDX5 was dramatically downregulated during the progression of OA. DDX5 deficiency increased fibrosis phenotype by upregulating COL1 expression and downregulating COL2 expression. In addition, loss of DDX5 aggravated cartilage degradation by inducing the production of cartilage-degrading enzymes. Chondrocyte-specific deletion of Ddx5 led to more severe cartilage lesions in the mouse OA model. Mechanistically, weakened DDX5 resulted in abundance of the Fn1-AS-WT and Plod2-AS-WT transcripts, which promoted expression of fibrosis-related genes (Col1, Acta2) and extracellular matrix degradation genes (Mmp13, Nos2 and so on), respectively. Additionally, loss of DDX5 prevented the unfolding Col2 promoter G-quadruplex, thereby reducing COL2 production. Together, our data suggest that strategies aimed at the upregulation of DDX5 hold significant potential for the treatment of cartilage fibrosis and degradation in OA.

Identification and verification of a novel signature that combines cuproptosis-related genes with ferroptosis-related genes in osteoarthritis using bioinformatics analysis and experimental validation.

BACKGROUND: Exploring the pathogenesis of osteoarthritis (OA) is important for its prevention, diagnosis, and treatment. Therefore, we aimed to construct novel signature genes (c-FRGs) combining cuproptosis-related genes (CRGs) with ferroptosis-related genes (FRGs) to explore the pathogenesis of OA and aid in its treatment. MATERIALS AND METHODS: Differentially expressed c-FRGs (c-FDEGs) were obtained using R software. Enrichment analysis was performed and a protein-protein interaction (PPI) network was constructed based on these c-FDEGs. Then, seven hub genes were screened. Three machine learning methods and verification experiments were used to identify four signature biomarkers from c-FDEGs, after which gene set enrichment analysis, gene set variation analysis, single-sample gene set enrichment analysis, immune function analysis, drug prediction, and ceRNA network analysis were performed based on these signature biomarkers. Subsequently, a disease model of OA was constructed using these biomarkers and validated on the GSE82107 dataset. Finally, we analyzed the distribution of the expression of these c-FDEGs in various cell populations. RESULTS: A total of 63 FRGs were found to be closely associated with 11 CRGs, and 40 c-FDEGs were identified. Bioenrichment analysis showed that they were mainly associated with inflammation, external cellular stimulation, and autophagy. CDKN1A, FZD7, GABARAPL2, and SLC39A14 were identified as OA signature biomarkers, and their corresponding miRNAs and lncRNAs were predicted. Finally, scRNA-seq data analysis showed that the differentially expressed c-FRGs had significantly different expression distributions across the cell populations. CONCLUSION: Four genes, namely CDKN1A, FZD7, GABARAPL2, and SLC39A14, are excellent biomarkers and prospective therapeutic targets for OA.

Specific isoforms of the ubiquitin ligase gene WWP2 are targets of osteoarthritis genetic risk via a differentially methylated DNA sequence.

BACKGROUND: Transitioning from a genetic association signal to an effector gene and a targetable molecular mechanism requires the application of functional fine-mapping tools such as reporter assays and genome editing. In this report, we undertook such studies on the osteoarthritis (OA) risk that is marked by single nucleotide polymorphism (SNP) rs34195470 (A > G). The OA risk-conferring G allele of this SNP associates with increased DNA methylation (DNAm) at two CpG dinucleotides within WWP2. This gene encodes a ubiquitin ligase and is the host gene of microRNA-140 (miR-140). WWP2 and miR-140 are both regulators of TGFß signaling. METHODS: Nucleic acids were extracted from adult OA (arthroplasty) and foetal cartilage. Samples were genotyped and DNAm quantified by pyrosequencing at the two CpGs plus 14 flanking CpGs. CpGs were tested for transcriptional regulatory effects using a chondrocyte cell line and reporter gene assay. DNAm was altered using epigenetic editing, with the impact on gene expression determined using RT-qPCR. In silico analysis complemented laboratory experiments. RESULTS: rs34195470 genotype associates with differential methylation at 14 of the 16 CpGs in OA cartilage, forming a methylation quantitative trait locus (mQTL). The mQTL is less pronounced in foetal cartilage (5/16 CpGs). The reporter assay revealed that the CpGs reside within a transcriptional regulator. Epigenetic editing to increase their DNAm resulted in altered expression of the full-length and N-terminal transcript isoforms of WWP2. No changes in expression were observed for the C-terminal isoform of WWP2 or for miR-140. CONCLUSIONS: As far as we are aware, this is the first experimental demonstration of an OA association signal targeting specific transcript isoforms of a gene. The WWP2 isoforms encode proteins with varying substrate specificities for the components of the TGFß signaling pathway. Future analysis should focus on the substrates regulated by the two WWP2 isoforms that are the targets of this genetic risk.

Data-driven identification of predictive risk biomarkers for subgroups of osteoarthritis using interpretable machine learning.

Osteoarthritis (OA) is increasing in prevalence and has a severe impact on patients' lives. However, our understanding of biomarkers driving OA risk remains limited. We developed a model predicting the five-year risk of OA diagnosis, integrating retrospective clinical, lifestyle and biomarker data from the UK Biobank (19,120 patients with OA, ROC-AUC: 0.72, 95%CI (0.71-0.73)). Higher age, BMI and prescription of non-steroidal anti-inflammatory drugs contributed most to increased OA risk prediction ahead of diagnosis. We identified 14 subgroups of OA risk profiles. These subgroups were validated in an independent set of patients evaluating the 11-year OA risk, with 88% of patients being uniquely assigned to one of the 14 subgroups. Individual OA risk profiles were characterised by personalised biomarkers. Omics integration demonstrated the predictive importance of key OA genes and pathways (e.g., GDF5 and TGF-ß signalling) and OA-specific biomarkers (e.g., CRTAC1 and COL9A1). In summary, this work identifies opportunities for personalised OA prevention and insights into its underlying pathogenesis.

Intra-articular injection of miRNA-1 agomir, a novel chemically modified miRNA agonists alleviates osteoarthritis (OA) progression by downregulating Indian hedgehog in rats.

Our objective in this study is to determine whether intra-articular injection of miRNA-1 can attenuate the progression of OA in rats by down regulating Ihh. Knee chondrocytes were isolated from male Sprague-Dawley rats aged 2-3 days. Second-generation chondrocytes were transfected with miR-1 mimic and empty vector with lipo3000 for 6 h and then stimulated with 10 ng/mL IL-1ß for 24 h. OA-related and cartilage matrix genes were quantified using real-time quantitative polymerase chain reaction (RT-qPCR). Two-month-old male Sprague-Dawley rats were divided into three groups (n = 30?): sham operation group + 50 µL saline, anterior cruciate ligament transection (ACLT) group + 50 µL miR-1 agomir (concentration), and control group ACLT + 50 µL miR-1 agomir. Treatment was started one week after the operation. All animals were euthanized eight weeks after the operation. X-rays and micro-CT were used to detect imaging changes in the knee joints. FMT was used to monitor joint inflammation in vivo. Safranin O staining was used to detect morphological changes in articular cartilage. Immunohistochemistry was used to detect Col2, Col10, metalloproteinase-13 (MMP-13). RT-qPCR was used to detect gene changes includingmiR-1, Col2, Col10, MMP-13, Ihh, Smo, Gli1, Gli2, and Gli3. Overexpression of miR-1 in IL-1ß-stimulated chondrocytes reduced the levels of Ihh, MMP-13, and Col10 but increased the levels of Col2 and aggrecan. Intra-articular injection of miR-1 agomir reduced osteophyte formation, inflammation, and prevented cartilage damage. RT-qPCR results indicated that the miR-1 agomir increased articular cartilage anabolism and inhibited cartilage catabonism. miR-1 can attenuate the progression of OA by downregulating Ihh.

FTH1 protects against osteoarthritis by MAPK pathway inhibition of extracellular matrix degradation.

OBJECTIVE: Ferritin heavy chain 1 (FTH1) is an important subunit of ferro-storing proteins and is indispensable for iron metabolism. Though it has been extensively studied in numerous organs and diseases, the relationship between FTH1 and osteoarthritis (OA) is unclear. DESIGN: Primary murine chondrocytes and cartilage explants were treated with FTH1 siRNA for 72 h. Mice were injected with adenovirus expressing FTH1 after destabilized medial meniscus (DMM) surgery. These approaches were used to determine the effect of FTH1 expression on the pathophysiology of OA. RESULTS: FTH1 expression was down regulated in OA patients and mice after DMM surgery. Knock down of FTH1 induced articular cartilage damage and extracellular matrix degradation in cartilage explants. Further, over expression of FTH1 reduced the susceptibility of chondrocytes to ferroptosis and reversed decrements in SOX9 and aggrecan after DMM surgery. Moreover, FTH1 relieved OA by inhibition of the chondrocyte MAPK pathway. CONCLUSION: This study found FTH1 to play an essential role in extracellular matrix degradation, ferroptosis, and chondrocytes senescence during OA progression. Further, injection of adenovirus expressing FTH1 may be a potential strategy for OA prevention and therapy.