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. 2025 Nov 1;14(11):927–940. doi: 10.1302/2046-3758.1411.BJR-2024-0518.R2

Therapeutic potential of IκB kinase epsilon inhibition in preventing meniscal degeneration of early osteoarthritis

Ryota Hirose 1, Yukio Akasaki 1,✉, Masanari Kuwahara 1, Taisuke Uchida 1, Yuki Hyodo 1, Mamiko Sakai 1, Takumi Kita 1, Ichiro Kurakazu 2, Martin K Lotz 2, Yasuharu Nakashima 1
PMCID: PMC12578550  PMID: 41173032

Abstract

Aims

Meniscal degeneration may precede or indicate early-stage osteoarthritis (OA); however, the pathogenetic involvement of the NF-κB pathway and its upstream IκB kinase (IKK) is unclear. This study investigated the functional role of IKK in the pathogenesis of meniscal degeneration, and the efficacy of IKKε inhibition as a therapeutic approach.

Methods

IKK expression in normal and OA human menisci was analyzed immunohistochemically. Gain- or loss-of-function experiments were performed in human meniscal cells. Additionally, meniscal degeneration was induced in wild-type mice and treated with intra-articular injection of the IKKε/TBK1 inhibitors amlexanox and BAY-985 every five days for four weeks. Meniscal degeneration was also induced in IKKε knockout mice. Mice were subsequently examined histologically.

Results

IKK protein expression was increased in human OA menisci. In vitro, the expression of meniscal degeneration-related factors was decreased after knockdown of each IKK, particularly IKKε, using small interfering RNA in human OA meniscal cells. Conversely, IKKε overexpression significantly increased the expression of these factors, and amlexanox and BAY-985 cancelled this effect. Western blot analysis showed that IKKε overexpression increased IκBα and p65 (RELA) phosphorylation. In vivo, both IKKε deletion and intra-articular injection of IKKε/TBK1 inhibitors protected mouse menisci against degeneration.

Conclusion

These results indicate that IKKs are involved in meniscal degeneration when it constitutes the preliminary or early stage of OA, with IKKε possibly playing a significant role. Furthermore, IKKε regulates meniscal degeneration through NF-κB signalling-mediated catabolism. Two IKKε/TBK1 inhibitors, amlexanox and BAY-985, are potential targets for the treatment of meniscal degeneration prior to OA.

Cite this article: Bone Joint Res 2025;14(11):927–940.

Keywords: Meniscus degeneration, IKKε, Inflammation, Osteoarthritis, NF-κB, Meniscal degeneration, early osteoarthritis, Meniscus, meniscal cells, osteoarthritis (OA), intra-articular injection, Western blot, RNA, pathogenesis, immunohistochemically

Article focus

  • We investigated the functional involvement of IκB kinase (IKK) isoforms in meniscal degeneration and the efficacy of IKKε inhibition.

Key messages

  • Inhibition of IKKε may be a promising therapeutic target for meniscal degeneration in osteoarthritis.

Strengths and limitations

  • The strengths of this study were that we characterized IKK isoform expression in human meniscus, the differential effects of each IKK isoform on the gene expression of meniscal degeneration-related factors, and the potential of IKKε inhibitors as novel therapeutic agents for meniscal degeneration.

  • One limitation is that the involvement of TANK-binding kinase 1, which is a target of amlexanox and BAY-985 as well as IKKε, has not been evaluated.

  • Other limitations of this study are the use of only male mice and lack of animal randomization.

Introduction

The meniscus is crucial for weight distribution, load bearing, shock absorption, and cartilage lubrication in the knee.1,2 Meniscal degeneration is closely associated with osteoarthritis (OA) onset and progression.3-5 Although early OA related to meniscal changes has gained attention because of its potential impact on prognosis,6-10 the mechanisms underlying meniscal degeneration remain unknown, and no pharmacological treatments exist to prevent or reverse these changes.

Meniscal degeneration results from mechanical instability, loading stress, and the production of inflammation-related proteins, including cytokines, chemokines, and matrix-degrading enzymes.11 Notably, OA menisci exhibit higher inflammatory factor levels than pre-OA menisci, and increased nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) expression in degenerated menisci suggests its role in degeneration.12

The IκB kinase (IKK) family, as upstream regulator of NF-κB, includes IKKα, IKKβ, IKKγ, and IKKε/TANK-binding kinase 1 (TBK1).13,14 When activated, IKK phosphorylates IκBα, leading to its degradation and releasing NF-κB dimers.15 While canonical IKKs (IKKα, IKKβ, and IKKγ) have been widely studied in cartilage pathology,16-19 recent studies implicate IKKε in OA pathogenesis through NF-κB activation, promoting chondrocyte catabolism and OA progression. Its inhibitors, Amlexanox and BAY-985, show potential in modifying OA.20,21 However, the role of IKK family, particularly IKKε in meniscal degeneration, remains unclear.

This study aimed to identify the predominant IKK involved in meniscal degradation by analyzing the expression and function in human tissues and cells. We also evaluated IKKε/TBK1 inhibitors and knockout mice to determine whether targeting IKKε can prevent meniscus degeneration prior to cartilage degeneration in OA mice model.

Methods

Clinical samples

Human knee joints from ten individuals aged 16 to 92 years were obtained at postmortem with the approval of the Institutional Review Board of the Scripps Research Institute, or obtained from patients undergoing total knee arthroplasty after the approval of the Ethics Committee of our institution. The meniscus tissues of normal knee joints were harvested from five healthy donors (mean age 31.2 years (16 to 46; SD 14.2)) with no history of joint disease. The meniscus tissues of human OA joints were obtained from five donors (mean age 76.2 years (67 to 92; SD 9.9)). Written informed consent was provided by all subjects.

Immunohistochemistry

Meniscus samples were fixed in 4% paraformaldehyde (Wako Pure Chemical Industries, Japan) for two days. After delipidation and decalcification, samples were embedded in paraffin and cut into 4 μm-thick sections. Antigen retrieval was performed by incubation overnight with 1 mM ethylenediaminetetraacetic acid (EDTA) at pH 8.0. Endogenous peroxidase activity was blocked by 3% hydrogen peroxidase in methanol for 30 minutes. For the blocking procedure, each specimen was placed in normal horse serum (Vectastain Universal Elite ABC kit; Vector Laboratories, USA) for 30 minutes and then incubated for one hour at room temperature with primary anti-IKKα antibody (Y463; Abcam, UK), primary anti-IKKβ antibody (SC8014; Santa Cruz Biotechnology, USA), primary anti-IKKγ antibody (MA5-32682; Thermo Fisher Scientific, USA), or primary anti-IKKε antibody (SC376114; Santa Cruz Biotechnology). Sections were incubated with biotinylated secondary antibodies for 30 minutes, followed by incubation with streptavidin–peroxidase complex (Vectastain Universal Elite ABC kit) for 30 minutes. Finally, the samples were counterstained with haematoxylin. The percentage of cells positive for IKKs in haematoxylin-stained sections was systematically determined on the basis of three images per section using BZ-II Analyzer software (Keyence, UK).

Cell isolation from human menisci and human meniscal cell culture

Human menisci were obtained aseptically from OA patients (mean age 69.1 years (59 to 77; SD 7.7)) as the OA sample and an osteosarcoma patient (seven-year-old female) who had undergone knee joint arthroplasty surgery as the normal sample. All patients provided informed consent, and the protocol was approved by the Ethics Committee of our institution. The inner menisci were minced and digested at 37°C with 2 mg/ml of collagenase for 12 hours. After digestion, meniscal cells were isolated and cultured in 10 cm dishes in Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12 with 10% fetal bovine serum (FBS), then used at the time of sub-confluence.20 To evaluate the gene expression of IKKs, cells were treated with 0.1 ng/ml interleukin-1β (IL-1β), 1 ng/ml tumour necrosis factor-α (TNFα), and 10 μg/ml lipopolysaccharide (LPS) for six hours.

Total RNA extraction and qRT-PCR

Total RNA was extracted from human meniscal cells using TRIzol reagent (Invitrogen, Thermo Fisher Scientific). Total RNA was reverse-transcribed to complementary DNA (cDNA) using the PrimeScript RT Reagent (Takara Bio, Japan). qRT-PCR was performed on a CFX Connect Real-Time System (Bio-Rad, USA) using TB Green Premix EX TaqII (Takara Bio). Data were normalized against the corresponding levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a housekeeping gene. The primers are summarized in Supplementary Table i.

Transfection of human meniscal cells with siRNA

Human OA meniscal cells were seeded in 12-well plates at a density of 0.75 × 105 cells/well with DMEM and 10% FBS. After one day, they were transfected with siRNAs (5 nM) targeting IKKα (siIKKα; Santa Cruz Biotechnology), IKKβ (siIKKβ; Santa Cruz Biotechnology), IKKε (siIKKε; Santa Cruz Biotechnology), or TBK1 (siTBK1; Santa Cruz Biotechnology) using RNAiMAX (Thermo Fisher Scientific). The control group was transfected with control siRNA (siCtrl; Santa Cruz Biotechnology). At 36 hours after transfection, cells were serum starved for 12 hours and then stimulated with IL-1β (0.1 ng/ml) for six hours.

Induction of IKKε overexpression and amlexanox or BAY-985 treatment of human meniscal cells

Recombinant adenoviral vectors encoding constitutively active IKKε (Ad-IKKε) or control green fluorescent protein (Ad-GFP) were purchased from SignaGen Laboratories (USA). Human meniscal cells were infected with adenovirus using Lipofectamine 3000 (Thermo Fisher Scientific) at 15 multiplicities of infection and cultured with or without 100 μM amlexanox (MedChem Express) or 10 μM BAY-985 (MedChem Express), both previously identified as IKKε inhibitors.22,23 At 36 hours after infection, cells were collected with or without IL-1β (0.1 ng/ml) stimulation during the final six hours of culture.

Western blotting of human meniscal cells

Whole-cell lysates were extracted from human meniscal cells using RIPA lysis buffer (Sigma-Aldrich, USA) with protease inhibitor (Sigma-Aldrich) and phosphatase inhibitor (Sigma-Aldrich). Cell lysates were electrophoresed in 4% to 12% gradient polyacrylamide gels (Thermo Fisher Scientific), and the resolved proteins were transferred to nitrocellulose membranes (Amersham Biosciences, USA). Membranes were blocked with blocking buffer (Takara Bio), washed in Tris-buffered saline with Tween (TBST), and incubated with primary antibodies (all from Cell Signaling Technology, USA) against IKKε (1:500; product no. 2905), phospho-p65 (p-p65, 1:500; product no. 3033), p65 (1:500; product no. 8242), phospho-IκBα (p-IκBα, 1:500; product no. 9246), IκBα (1:500; product no. 4814), and GAPDH (1:1,000; product no. 5174); all antibodies were diluted in Can Get Signal Immunoreaction Enhancer Solution 1 (TOYOBO, Japan). After washing in TBST, secondary anti-rabbit immunoglobulin G (IgG) antibodies (1:1,000) (product no. 7074; Cell Signaling Technology) or anti-mouse IgG antibodies (1:1,000) (sc-516102; Santa Cruz Biotechnology) were added. Immunoreactivity was detected with ECL Prime (Amersham Biosciences) and photographed using an Ez Capture MG (ATTO, Japan). Band densities were calculated using CS Analyzer version 6.0 (ATTO).24

Mice

All animal experiments were approved by the Animal Experiment Committee of our institution (project code: A23-281) and were performed according to the rules of our institution. Mice were housed in groups of three to five per cage at the Research Centre for Human Disease Modeling of our institution and were able to freely access food and water. The centre was maintained in specific pathogen-free conditions on a 12-hour light/dark cycle at all times.25 We have adhered to the ARRIVE guidelines and have included the ARRIVE checklist as Supplementary Material. Global IKKε knockout mice (IKKε−/−) and wild-type (WT) mice on a C57BL/6J background were used in all animal experiments. IKKε−/− mice were generated by Tom Maniatis (Columbia University College of Physicians and Surgeons). The sequences of the primers used for genotyping are shown in Supplementary Figure a. Male C57BL/6J mice were obtained from SLC Japan.

For histopathological assessment, knee sections in the medial sagittal plane were stained with Safranin O–fast green. Meniscal degeneration severity was quantified by Kwok’s meniscus scoring system on a scale of 0 to 24 for the anterior and posterior parts, respectively, with higher scores indicating greater severity of histopathological meniscus change,26 by two independent observers (YH, MS) in a blinded manner, with scores averaged to minimize observer bias. OA severity was similarly quantified by Osteoarthritis Research Society International (OARSI) histopathology grading on a scale of 0 to 6 for both the femur and tibia (total score of 0 to 12).27

DMM model in mice

DMM was achieved in mice by transection of the medial meniscotibial ligament (MMTL) and the medial collateral ligament (MCL) of eight-week-old mice, as previously described.28 As a control, sham surgery was performed in a separate group of mice using the same approach but without MMTL + MCL transection. The mice were euthanized four weeks after the operation, and knee joints were assessed for histological features of meniscal and cartilage degeneration. Mice were assigned to the two groups using a random number table. Each group contained seven mice, and group sizes were decided on the basis of a power analysis; at least seven mice per group were required to detect a minimum difference of 30% between mice postoperatively receiving amlexanox or BAY-985 versus saline in terms of meniscal degeneration based on the mean value derived using Kwok’s meniscus scoring system (score 0 to 24) (power = 0.8, α = 0.05), as determined in previous studies.20

Application of amlexanox or BAY-985 in the mouse DMM model

MMTL + MCL transection or sham operations were performed on the left knee joints of mice. The injection solution comprised 10 mM amlexanox or 10 mM BAY-985 stock solution (MedChemExpress, USA) diluted with saline. Ten microlitres of 100 μM amlexanox or 10 μM BAY-985, or saline as vehicle, was injected into the intra-articular space of each mouse knee joint, starting on the day of surgery and continuing every five days for four weeks. Mice were euthanized four weeks after the operation.

Immunofluorescence analysis of mouse knee joints

Knee joint sections were stained with a primary antibody against IKKε (SC376114; Santa Cruz Biotechnology), p-IκBα (NB100-81987; Novus Biologicals, USA), IL-6 (product no. 12912; Cell Signaling Technology), or MMP13 (ab39012; Abcam) at room temperature for one hour, then incubated with Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific). The numbers of cells positive for both IKKε and p-IκBα were quantified by counting immunopositive cells in sagittal sections of the knee joint at 200× magnification (IKKε, n = 5 mice per group; p-IκBα, n = 7 mice per group). The percentages of positively stained cells per section were counted using BZ-II Analyzer software.

Statistical analysis

All experiments were repeated at least five times. Data are presented as means (SD). The normality of the data was assessed by the Shapiro-Wilk test. When the distribution was normal, statistically significant differences between groups were determined by t-test (paired or independent-samples, as appropriate) or the Tukey-Kramer test. When the distribution was not normal, the Mann-Whitney U test (for independent samples) or the Wilcoxon signed-rank test (for paired samples), and the Steel–Dwass test was used as appropriate. All data analyses were performed using JMP statistical software version 16 (SAS Institute). P-values less than 0.05 were considered statistically significant.

Results

The expression characteristics of IKK isoforms in the inner and outer regions of human normal and OA menisci were investigated by immunohistochemical analysis. Regarding the protein expression of all IKK isoforms, the OA menisci exhibited a significantly higher percentage of immunostained cells in both the inner and outer regions compared with normal menisci (Figure 1). There was no consistent trend in the expression of IKK isoforms in normal meniscal tissue across ages ranging from 16 to 46 years (Supplementary Table ii).

Fig. 1.

Microscopy images and graphs comparing protein expression in normal and osteoarthritic cartilage, showing differences across inner and outer tissue regions. This figure presents a comparative analysis of protein expression in cartilage tissue from normal and osteoarthritic (OA) samples. The top section (labeled A) contains rows of microscopy images showing tissue sections stained for five proteins: IKKα, IKKβ, IKKγ, IKKe, and TBK1. Each row corresponds to one protein, and the columns represent inner and outer regions of cartilage under normal and OA conditions. Insets within each image provide magnified views of selected areas. The bottom section (labeled B) features scatter plots quantifying the percentage of cells positive for each protein in the inner and outer regions of both normal and OA cartilage. These plots show variation in protein expression between healthy and diseased tissue, with statistical indicators marking significant differences. The figure visually and quantitatively demonstrates how protein localization and abundance differ between cartilage regions and disease states.

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Expression of IκB kinase (IKK) isoforms is upregulated in both the inner and outer zones of meniscal tissue in osteoarthritis (OA) compared to normal tissue. a) Representative immunohistochemical staining images of IKK isoforms in the inner and outer zones of normal and OA human meniscal tissue (bar = 100 μm). The lower left panel shows a magnified view of the cells. The top row indicates the inner and outer zones of human meniscal tissue. b) Quantification of IKK isoform-positive cells in each group (n = 5 per group). All p-values in this figure were calculated using independent-samples t-test or Mann-Whitney U test. Symbols represent individual samples; thick horizontal lines with whiskers show the mean (SD). *p < 0.05; **p < 0.01; ***p < 0.001.

The expression characteristics of IKK isoforms in human OA and normal menisci were examined by gene expression analysis. First, we evaluated whether the cells isolated from the human meniscus exhibited characteristics of meniscus cells by comparing with chondrocytes obtained from the same donor using reverse transcription polymerase chain reaction (RT-PCR). It is known that aggrecan and type II collagen are highly expressed in cartilage,29 whereas type I collagen is expressed at higher levels in the meniscus compared to cartilage. Consistent with this, gene expression analysis of ACAN, COL2A1, and COL1A1, which encode aggrecan, type II collagen, and type I collagen, respectively, showed that meniscal cells had significantly lower expression of ACAN and COL2A1, and significantly higher expression of COL1A1 compared to chondrocytes (Supplementary Figure b). In human OA meniscal cells, the gene expressions of IKKα, IKKβ, IKKε, and TBK1 were significantly upregulated in the presence of IL-1β, TNFα, or LPS. Moreover, regarding the gene expressions of meniscus degeneration-related factors, IL-1β stimulation significantly increased the expression of IL-6, matrix metalloproteinase 1 (MMP1), MMP3, MMP13, A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), ADAMTS5, and p65, with the largest expression changes observed for IL-6, MMP1, MMP3, ADAMTS4, and ADAMTS5 under IL-1β stimulation (Figure 2a). In contrast, in human normal meniscal cells, only the gene expressions of IKKβ and IKKε were significantly increased in response to IL-1β, TNFα, or LPS stimulation, while TNFα stimulation also led to increased gene expressions of IKKα and IKKγ. Regarding the gene expressions of meniscus degeneration-related factors, IL-1β stimulation significantly increased the expression of IL-6, MMP1, MMP3, MMP13, ADAMTS4, ADAMTS5, and p65 (Supplementary Figure c).

Fig. 2.

Graphs comparing gene expression levels in osteoarthritic meniscal cells under different inflammatory conditions, showing significant differences for multiple genes. This figure presents a panel of bar graphs comparing the relative expression of multiple genes in meniscal cells from osteoarthritic (OA) tissue samples under four conditions: control, IL-1β, TNFα, and LPS stimulation. Genes analyzed include IKKα, IKKβ, IKKγ, IKKe, TBK1, IL6, MMP1, MMP3, MMP13, ADAMTS4, ADAMTS5, RUNX2, ENPP1, and p65. Individual data points are displayed with mean values and error bars. Statistically significant differences between conditions are indicated by asterisks. The figure highlights how inflammatory stimuli affect gene expression differently in OA versus normal meniscal cells, suggesting altered molecular responses in disease states.

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Differential responses of osteoarthritis (OA) human meniscal cells to inflammatory stimuli (interleukin-1β (IL-1β), tumour necrosis factor-α (TNFα), lipopolysaccharide (LPS)) in terms of IκB kinase (IKK) isoform and meniscal degeneration-related factor expression. Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of IKK isoform and meniscal degeneration–related factor messenger RNA (mRNA) levels in OA (n = 7) human meniscal cells left unstimulated (Ctrl) or stimulated with 0.1 ng/ml IL-1β, 1 ng/ml TNFα, or 10 μg/ml LPS for six hours. All p-values in this figure were calculated using paired t-test or Wilcoxon signed-rank test. Symbols represent individual samples; thick horizontal lines with whiskers show the mean (SD). *p < 0.05; **p < 0.01; ***p < 0.001.

To evaluate the effects of IKK knockdown, siRNA-transfected human OA meniscal cells stimulated with IL-1β were used. Figure 3a illustrates the impact of IKK knockdown on the gene expressions of other IKK isoforms. In particular, the gene expressions of IKKα and IKKβ were reciprocally suppressed, whereas IKKε knockdown did not affect the gene expressions of the other IKK isoforms. Moreover, IKKα knockdown significantly suppressed the gene expression of TBK1. Regarding the expressions of meniscal degeneration-related genes, IKKα knockdown significantly decreased the gene expressions of MMP1 and p65, while increasing those of ADAMTS4 and ADAMTS5. IKKβ knockdown resulted in decreased gene expressions of IL6, MMP3, RUNX2, and p65, along with increased gene expressions of MMP13, ADAMTS5, and ENPP1. Notably, IKKε knockdown significantly decreased the gene expressions of IL6, MMP1, MMP3, ADAMTS5, RUNX2, ENPP1, and p65 (Figure 3b).

Fig. 3.

Two-panel figure showing gene expression changes in meniscal cells under various conditions, including inflammatory stimulation and gene silencing, with significant differences across several genes. This figure consists of three panels labeled A, B, and C, each presenting scatter plots of gene expression data in meniscal cells under different experimental conditions. Panel A shows the expression of five genes related to the IKK family: IKKα, IKKβ, IKKγ, IKKε, and TBK1. Each plot compares control and stimulated conditions, with significant differences observed for all genes except TBK1. Panel B displays gene expression related to meniscus degeneration, including IL6, MMP1, MMP3, MMP13, ADAMTS4, ADAMTS5, RUNX2, ENPP1, and p65. Several genes show increased expression under inflammatory conditions, while RUNX2 shows a decrease. Changes in expression are evident for multiple genes, indicating the regulatory role of TBK1 and IKKε in meniscal degeneration pathways. Each graph includes individual data points, mean values, and statistical indicators of significance.

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Small interfering RNA (siRNA)-mediated knockdown of IκB kinase (IKK) isoforms in osteoarthritis (OA) human meniscal cells revealed that IKKε knockdown had the strongest effect on messenger RNA (mRNA) levels of meniscal degeneration–related factors. a) Effects of siRNA-mediated knockdown of IKK isoforms (siIKKα, siIKKβ, siIKKε) in 0.1 ng/ml IL-1β–stimulated OA meniscal cells on the mRNA levels of IKK isoforms (n = 7) and b) meniscal degeneration-related factors (n = 7). Nonactive siRNA (siCtrl) was used as a control. All p-values in this figure were calculated using Tukey-Kramer test or Steel-Dwass test. Symbols represent individual samples; thick horizontal lines with whiskers show the mean (SD). *p < 0.05; **p < 0.01; ***p < 0.001.

In human OA meniscal cells, TBK1 siRNA knockdown increases the gene expression of IKKε and several meniscal degeneration-related factors, and furthermore, IKKε knockdown in siTBK1-transfected cells reduces this increase.

To explore the functional role of TBK1, we analyzed the effect of TBK1 knockdown on the expression of inflammatory and catabolic genes in human OA meniscal cells stimulated with IL-1β. TBK1 siRNA knockdown significantly increased the expression of IKKε, MMP1, MMP3, MMP13, ADAMTS4, ADAMTS5, RUNX2, ENPP1, and p65 in human OA meniscal cells (Supplementary Figure d). To exclude the influence of increased IKKε expression, a double transfection with siTBK1 and siIKKε was performed. As a result, in meniscal cells receiving the siTBK1/siIKKε double transfection, the expression of MMP1, ADAMTS5, ENPP1, and p65 was significantly decreased compared to siTBK1-transfected cells. Moreover, compared to siCtrl, only MMP13 and ADAMTS4 showed a significant increase in expression (Supplementary Figure d).

Overexpression of IKKε notably promotes the expression of meniscal degeneration-related genes in human OA meniscal cells, and this effect is significantly suppressed by IKKε/TBK1 inhibitors (amlexanox and BAY-985).

The effects of IKKε overexpression and IKKε inhibitors on the expression of meniscal degeneration-related genes were evaluated using human OA meniscal cells transfected with an IKKε recombinant adenoviral vector. Transfection with the IKKε recombinant adenoviral vector increased IKKε protein expression (Figure 4a). In the absence of IL-1β, IKKε overexpression significantly elevated the gene expression of IL6, MMP1, MMP3, MMP13, ADAMTS4, ADAMTS5, ENPP1, and p65 (Figure 4b). In the presence of IL-1β, IKKε overexpression led to significant increases in the gene expression of IL6, MMP13, ADAMTS4, ADAMTS5, RUNX2, ENPP1, and p65, and these increases were significantly suppressed by treatment with either of the IKKε/TBK1 inhibitors (amlexanox or BAY-985) (Figure 4c). We confirmed that the inhibitor suppressed the gene expression of IL6 and MMP13 in a dose-dependent manner (Supplementary Figure e).

Fig. 4.

Three-panel figure showing IKKε protein levels and its impact on gene expression in meniscal cells under various experimental conditions, including IL-1β stimulation and inhibitor treatments. This figure consists of three panels labeled A, B, and C, illustrating the role of IKKε in regulating gene expression in meniscal cells. Panel A displays a Western blot comparing IKKε protein levels across three conditions: untreated (Ad-), GFP control, and IKKε overexpression. GAPDH is used as a loading control. A bar graph next to the blot quantifies the relative density of IKKε normalized to GAPDH, showing increased expression in the IKKε condition. Panel B contains scatter plots showing the relative expression of nine genes (IL6, MMP1, MMP3, MMP13, ADAMTS4, ADAMTS5, RUNX2, ENPP1, and p65) under two conditions: untreated and GFP with IL-1β stimulation. Panel C presents similar scatter plots for the same genes, comparing untreated cells with those treated with IL-1β plus either amlexanox or BAY-985. These plots reveal how IKKε modulation and pharmacological inhibition affect inflammatory and degenerative gene expression profiles. Each graph includes individual data points and statistical indicators of significance.

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IκB kinase (IKKε) overexpression significantly enhanced the expression of meniscal degeneration–related genes in osteoarthritis (OA) human meniscal cells, and this effect was inhibited by amlexanox and BAY-985. a) Western blot (left) and quantification (right) of IKKε protein levels in OA human meniscal cells (n = 5) after adenoviral vector-mediated overexpression of IKKε (Ad-IKKε) and stimulation with 0.1 ng/ml interleukin-1β (IL-1β). Green fluorescent protein adenoviral vector (Ad-GFP) was used as a vector control; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. b) Changes in meniscal degeneration-related gene expression in OA human meniscal cells with or without induced IKKε overexpression (unstimulated). c) Changes in 0.1 ng/ml IL-1β–induced meniscal degeneration-related gene expression in OA human meniscal cells with or without induced IKKε overexpression and with or without amlexanox (100 μM) or BAY-985 (10 μM) treatment. All p-values in this figure were calculated using paired t-test, Tukey-Kramer test, Wilcoxon signed-rank test, or Steel-Dwass test. Symbols represent individual samples; thick horizontal lines with whiskers show the mean (SD). *p < 0.05; **p < 0.01; ***p < 0.001.

To evaluate the role of IKKε in the NF-κB pathway, the phosphorylation levels of IκBα and p65 were analyzed in human OA meniscal cells overexpressing IKKε. IL-1β stimulation induced the phosphorylation of both IκBα and p65 (Figure 5a). In the absence of IL-1β, IKKε overexpression did not increase the phosphorylation level of IκBα compared to the Ad-GFP control. In contrast, in the presence of IL-1β, IKKε overexpression significantly increased IκBα phosphorylation, along with elevated levels of IκBα and phosphorylated p65 (p-p65). The increase in p-IκBα was significantly suppressed by treatment with either amlexanox or BAY-985, and the increase in p-p65 was significantly suppressed by BAY-985 treatment (Figure 5).

Fig. 5.

Western blot and bar graph analysis showing changes in IκBα and p65 protein levels in meniscal cells under IL-1β stimulation and different treatment conditions. This figure consists of two panels labeled A and B, illustrating the effects of IL-1β stimulation and various treatments on protein expression in meniscal cells. Panel A presents Western blot images for four proteins: IκBα, phosphorylated IκBα (p-IκBα), p65, and phosphorylated p65 (p-p65), with GAPDH used as a loading control. Samples are grouped by IL-1β treatment status and further divided into conditions including GFP control, IKKe overexpression, IKKe with Amlexanox, and IKKe with BAY-985. Panel B contains four bar graphs quantifying the relative density of each protein from the blots. Each graph shows fold changes in protein levels across the same treatment groups, with individual data points, mean values, and error bars. Statistically significant differences between conditions are marked with asterisks. The figure demonstrates how IKKe modulation and pharmacological inhibition influence NF-κB pathway activation in response to inflammatory stimulation.

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IκB kinase (IKKε) overexpression activated nuclear factor-κB (NF-κB) signalling in osteoarthritis (OA) human meniscal cells in the presence of interleukin-1β (IL-1β), and this effect was reversed by amlexanox and BAY-985 treatment. a) Western blot analysis and b) quantification of p-IκBα, IκBα, p-p65, and p65 levels in OA human meniscal cells after IKKε overexpression and treatment with amlexanox (100 μM) or BAY-985 (10 μM), either unstimulated or 20 minutes after IL-1β (0.1 ng/ml) stimulation. Green fluorescent protein adenoviral vector (Ad-GFP) was used as a vector control. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. All p-values in this figure were calculated using Tukey-Kramer test or Steel-Dwass test. Symbols represent individual samples; thick horizontal lines with whiskers show the mean (SD). *p < 0.05; **p < 0.01; ***p < 0.001.

The in vivo therapeutic effects of intra-articular injection of amlexanox or BAY-985 on meniscal degeneration were evaluated using a surgical OA mouse model. In this model, OA is induced by destabilization of the meniscus, resulting in abnormal mechanical loading-induced meniscal damage followed by subsequent cartilage damage. To detect early changes in meniscal degeneration before the progression of cartilage degeneration, mice were killed at four weeks after surgery. This model exhibited a significantly increased percentage of IKKε-positive cells in the meniscus compared with sham-operated mice (Figure 6a). Starting on the day of surgery and continuing every five days for four weeks, amlexanox solution, BAY-985 solution, or saline as a vehicle control was injected into the knee joint cavity. Consequently, the histopathological scores of both the anterior and posterior menisci were significantly reduced in the groups treated with amlexanox or BAY-985 compared with the saline group (Figures 6b and 6c). OARSI scores revealed only mild cartilage damage, with no significant changes observed due to the drug injections.

Fig. 6.

Three-panel figure showing IKKε expression and meniscus tissue scores in treated and untreated groups, with visual and quantitative comparisons across experimental conditions. This figure contains three panels labeled A, B, and C, illustrating the effects of surgical intervention and pharmacological treatments on IKKε expression and meniscus tissue integrity. Panel A includes immunofluorescence images of anterior and posterior meniscus sections from sham-operated and surgically operated groups, stained for IKKε and DAPI. A bar graph adjacent to the images quantifies IKKε expression, showing significantly higher levels in the operated group compared to sham. Panel B presents histological images of anterior and posterior meniscus sections treated with either saline or Amlexanox. Below these images, bar graphs display meniscus scores, indicating improved tissue condition in the Amlexanox-treated group. Panel C follows the same format, comparing saline-treated samples with those treated using BAY-985. The corresponding graphs show reduced meniscus scores in the BAY-985 group, suggesting therapeutic benefit. Statistical significance is indicated in all graphs, highlighting differences between treatment and control groups.

Open in a new tab

a) Representative immunofluorescence images (left) and quantification (right) of IκB kinase (IKKε) expression in the menisci of wild-type (WT) mice four weeks after destabilization of the medial meniscus (DMM) + medial collateral ligament (MCL) transection surgery (Ope) or sham surgery (n = 5 mice per group). Right panels show higher-magnification views of the boxed areas in the left panels (bar = 100 μm). b) and c) Representative Safranin O–fast green staining images of the menisci of WT mice four weeks after DMM + MCL transection and treatment with b) amlexanox, c) BAY-985, or saline as the vehicle control (top). Images were taken at ×100 magnification. Quantification of meniscal degeneration severity and articular cartilage histopathological changes in the indicated groups (bottom) (n = 7 mice per group). All p-values in this figure were calculated using independent-samples t-test or Mann-Whitney U test. Symbols represent individual samples; thick horizontal lines with whiskers show the mean (SD). *p < 0.05; **p < 0.01. DAPI, 4′,6-diamidino-2-phenylindole.

The expressions of p-IκBα, IL6, and MMP13 in mouse menisci were analyzed by immunofluorescence staining. At four weeks post-surgery, the percentage of p-IκBα-, IL6-, and MMP13-positive cells in the total meniscus was significantly reduced in the treatment groups with amlexanox or BAY-985 compared with the saline group (Supplementary Figure f).

Finally, to clarify the causal role of IKKε deletion in meniscal degeneration, an in vivo analysis was conducted using a surgical OA mouse model in IKKε knockout mice. The genotyping image of IKKε knockout mice is shown in Supplementary Figure a. No differences were observed in the whole-body phenotype between IKKε knockout mice and WT mice. Regarding body weight, no significant differences were noted between IKKε knockout and WT mice at two, four, eight, or 12 weeks of age. Furthermore, the development and structure of the meniscus and other joint tissues were normal in the knockout mice (Figure 7a). At four weeks post-surgery, the histopathological scores of the anterior and posterior menisci were significantly lower in IKKε knockout mice compared with WT mice, while no significant changes in early cartilage degeneration were observed based on the OARSI scores (Figure 7b). Additionally, when measuring the percentage of p-IκBα-, IL6-, and MMP13-positive cells in the entire meniscus at four weeks after surgery, IKKε knockout mice showed a significant decrease compared with WT mice (Figure 7c).

Fig. 7.

Comparative figure showing body weight, meniscus histology, and protein expression in wild-type and IKKε knockout mice, with visual and quantitative data across anterior and posterior regions. This figure presents a multi-panel comparison between wild-type (WT) and IKKε knockout (KO) mice, focusing on body weight progression, meniscus tissue integrity, and protein expression. Panel A includes a line graph tracking body weight over 12 weeks, accompanied by images of WT and KO mice. Histological sections of anterior and posterior meniscus tissue are shown for both groups, with bar graphs quantifying meniscus scores. Panel B expands on this with additional histological images and graphs comparing meniscus scores and OARSI scores between WT and KO mice. Panel C displays immunofluorescence staining for p-IκBα, Il6, and Mmp13 in anterior and posterior meniscus regions. Each staining image is paired with a scatter plot showing the percentage of positive cells, highlighting differences in inflammatory and degenerative marker expression between the two genotypes. The figure collectively illustrates the impact of IKKε deletion on joint tissue structure and molecular signaling.

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IκB kinase (IKKε) knockout attenuates the progression of meniscal degeneration in an early osteoarthritis (OA) mouse model. a) Left panel: representative images of wild-type (WT) and IKKε knockout (KO) mice showing their growth and development, as well as changes in body weight at the indicated ages. Middle panel: representative Safranin O–fast green staining images of the menisci of untreated WT and IKKε-KO mice at 12 weeks of age. Right panel: quantification of pathological changes in meniscal degeneration in each group. Values represent the mean (SD) (n = 5 mice per group). b) Representative Safranin O–fast green staining images of the menisci of WT and IKKε-KO mice four weeks after destabilization of the medial meniscus (DMM) + medial collateral ligament (MCL) transection surgery (top). Quantification of the severity of meniscal degeneration and articular cartilage histopathological changes in the indicated groups (bottom) (n = 7 mice per group). c) Representative immunofluorescence images (left) and quantification (right) of p-IκBα, interleukin-6 (IL-6), and matrix metalloproteinase-13 (MMP-13) expression in the menisci of WT and IKKε-KO mice four weeks after DMM + MCL transection surgery. Right panels show higher-magnification views of the boxed areas in the left panels (bar = 100 μm). All p-values in this figure were calculated using independent-samples t-test or Mann-Whitney U test. Symbols represent individual samples; thick horizontal lines with whiskers show the mean (SD). *p < 0.05; **p < 0.01.

Discussion

This study is the first to analyze the characteristics of IKK isoform expression in the human meniscus, the differences in the effects of IKK isoforms on the gene expressions of meniscal degeneration factors, and the potential of IKKε/TBK1 inhibitors as a pharmacological treatment for meniscal degeneration. IKK isoforms were highly expressed in the knee menisci of OA patients compared to healthy controls. The gene expressions of IKK isoforms in human OA meniscal cells were upregulated by inflammatory factors such as IL-1β, TNFα, and LPS, suggesting that IKK isoforms are deeply involved in the pathogenesis of human meniscal degeneration. In the knockdown experiments conducted in this study, IKKε knockdown had the most significant impact on the expression of meniscal degeneration-related factors. Gain-of-function experiments showed that IKKε overexpression increased the expression of meniscal degeneration-related factors, whereas the IKKε/TBK1 inhibitors (amlexanox and BAY-985) reversed this effect. Furthermore, in vivo, the IKKε/TBK1 inhibitors suppressed the progression of meniscal degeneration in the mouse DMM model. Additionally, the progression of meniscal degeneration was also suppressed in IKKε knockout mice, confirming the therapeutic effect.

IKK isoforms were highly expressed in OA meniscal tissue compared to normal meniscal tissue, and their expression, except for IKKγ, was upregulated in response to pro-inflammatory stimuli such as IL-1β, TNFα, and LPS. IKKγ is known as a regulatory subunit,14,30 and may have low responsiveness to pro-inflammatory stimuli. Furthermore, it is well known that the synovial fluid of OA patients contains high concentrations of inflammatory cytokines such as IL-1β and TNFα.31,32 Consistent with this, our study also confirmed an increase in IKK protein expression in degenerated menisci and an upregulation of IKK gene expression in meniscal cells in response to inflammatory cytokines. In normal meniscal cells, the upregulation of IKK isoforms in response to pro-inflammatory stimuli was limited compared to OA meniscal cells. However, in this study, we used meniscal cells from a seven-year-old donor, and it is possible that the stimulation time and concentration were insufficient. Additionally, a previous study reported that human non-OA chondrocyte released lower levels of inflammatory factors such as IL-6 compared to OA chondrocyte, suggesting that differences in responsiveness to stimuli may exist.33

IKKα is involved in both the canonical and non-canonical NF-κB pathways, while IKKβ is involved only in the canonical pathway. However, both had limited effects on the expression of meniscal degeneration factors in meniscal cells. In this study, the gene expressions of IKKα and IKKβ were reciprocally downregulated by siRNA. The IKK–NFκB pathway plays a crucial role in inflammation, immunity, cell proliferation, differentiation, and survival.34,35 Additionally, IKKα and IKKβ are known to have functions independent of NF-κB,36,37 which may also be relevant in the meniscus. Moreover, siIKKα influenced the gene expression of TBK1. IKK isoforms are known to be involved in common signalling pathways and interact with each other. These findings suggest that they may also possess regulatory functions in controlling each other’s expression.

Among the IKK isoforms, IKKε had the broadest impact on the expression of genes involved in OA pathogenesis, including IL6, MMPs, ADAMTSs, RUNX2, and ENPP1, in human OA meniscal cells. In this study, the expression of MMP13 and ADAMTS4 was not suppressed by siIKKε; however, previous research has shown that, unlike in normal meniscal cells, cytokine stimulation does not increase MMP13 production in OA meniscal cells,11 suggesting that inter-individual donor variability may be a contributing factor. Additionally, apart from inflammatory factors, meniscal hypertrophy contributes to calcification and fibrocartilage formation, ultimately leading to meniscal and cartilage degeneration.38,39 RUNX2 serves as a marker of meniscal hypertrophy,40 while ENPP1 overexpression has been observed in calcified meniscal areas and is thought to be involved as a precursor to cartilage calcification.41-43 In contrast to the effects of siIKKε, siTBK1 led to an increase in IKKε gene expression, which in turn upregulated MMPs, ADAMTSs, RUNX2, ENPP1, and p65. Subsequently, siIKKε significantly reduced the expression of MMP1, ADAMTS5, ENPP1, and p65 in siTBK1-transfected cells. Notably, TBK1 knockdown without IKKε upregulation (siTBK1 and siIKKε) only significantly increased the expression of MMP13 and ADAMTS4. Furthermore, the IKKε/TBK1 inhibitors, amlexanox and BAY-985, cancelled the increase in the expression of meniscal degeneration-related factors induced by IKKε overexpression in human OA meniscal cells. This suggests that the effects of these inhibitors are primarily mediated through IKKε inhibition. These results indicate that IKKε upregulation, rather than TBK1, plays a crucial role in the regulation of meniscal degeneration-related gene expression. There are conflicting reports on whether TBK1 consistently contributes to meniscal degeneration and OA pathogenesis.44,45 Further studies are necessary to elucidate the detailed mechanisms of IKKε/TBK1 signalling in meniscal degeneration.

The IKKε/TBK1 inhibitors, amlexanox and BAY-985, demonstrated therapeutic efficacy in delaying meniscal degeneration in the early stages of OA in the DMM mouse model. Amlexanox has already been used clinically for conditions such as oral ulcers and allergic diseases, and its safety profile has been well established. Additionally, as a GRK5 inhibitor, it has been shown to suppress cartilage degeneration progression eight weeks post-surgery in DMM model mice,21 suggesting that amlexanox could be rapidly and safely repurposed for therapeutic use. BAY-985 is a compound designed to selectively inhibit IKKε with high specificity.23 Due to its minimal side effects, it has the potential to be a safe therapeutic agent with fewer side effects and adverse events. Since it also suppresses cartilage degeneration progression eight weeks post-surgery in the DMM model mice,20 BAY-985 is also expected to be developed for clinical application as a therapeutic agent.

In the DMM mouse model, intra-articular administration of the IKKε/TBK1 inhibitors (amlexanox and BAY-985) suppressed IκBα phosphorylation, which was associated with reduced IL-6 and MMP13 protein expression in mouse meniscal tissue. These effects demonstrated a protective role against histopathological changes in the meniscus. Surprisingly, the suppression of meniscal degeneration was observed not only in the anterior meniscus, which was directly damaged, but also in the posterior meniscus. It is known that damaged menisci produce catabolic factors.11 However, our findings suggest that amlexanox and BAY-985 administration may have disrupted the vicious cycle of intra-articular inflammatory reactions by inhibiting catabolic factor production via NF-κB signalling at an early stage. In this study, histological changes were evaluated at four weeks post-surgery, focusing on the pre-onset phase of cartilage degeneration. A previous study using the DMM+ MCL mouse model reported minimal cartilage degeneration at four weeks post-surgery.28 In an experiment using rats, MMP13 expression increased in the meniscus as early as three days after ACLT surgery, whereas no such increase was observed in cartilage.46 These findings suggest that catabolic reactions in OA models of joint destabilization may initiate within the meniscal tissue. Furthermore, in IKKε knockout mice, at four weeks post-surgery, IκBα phosphorylation, IL-6, and MMP13 protein expression were significantly suppressed in meniscal tissue compared to WT mice, and histopathological changes in the meniscus were notably milder. These results suggest that IKKε plays a key causal role in meniscal degeneration and that even in the early OA stage – before cartilage degeneration occurs – IKKε could serve as a potential therapeutic target.

This study has several limitations. First, only cells isolated from the inner zone of the meniscus were used. Inner meniscus cells have been reported to respond more strongly to IL-1 compared to outer zone cells.47 Although both IKKε inhibitors demonstrated therapeutic effects in the inner zone cells, which are more susceptible to degeneration and have limited self-healing capacity, further investigation using cells from different meniscal regions is necessary to better understand the pathology of meniscus degeneration as a whole. Second, overexpression of IKKε did not consistently induce changes in RUNX2 gene expression in human OA meniscal cells. Additionally, although IKKε overexpression increased MMP1 and MMP3 gene expression in the presence of IL-1β, the increases were not statistically significant. Finally, although our findings suggest that IKKε plays a predominant role in regulating meniscus degeneration-related factors, the extent of TBK1 involvement remains unclear. Since both amlexanox and BAY-985 are ATP-competitive inhibitors, it was difficult to determine whether their effects were mediated through inhibition of IKKε, TBK1, or both. Further studies are needed to elucidate the role of TBK1 for future development and clinical application of these inhibitors.

In conclusion, the results of this study demonstrated that IKK isoforms are highly expressed in OA meniscal tissue, and that IKKε knockdown significantly suppresses catabolic responses mediated by NF-κB signalling. Furthermore, the IKKε/TBK1 inhibitors (amlexanox and BAY-985) were shown to have potential as therapeutic agents for meniscal degeneration and early OA.

Author contributions

R. Hirose: Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing

Y. Akasaki: Writing – review & editing, Conceptualization, Funding acquisition, Supervision

M. Kuwahara: Writing – review & editing, Funding acquisition, Methodology, Validation

T. Uchida: Conceptualization, Methodology, Writing – review & editing, Funding acquisition, Validation

Y. Hyodo: Investigation, Validation

M. Sakai: Investigation, Validation

T. Kita: Investigation, Validation

I. Kurakazu: Resources, Supervision

M. K. Lotz: Writing – review & editing, Supervision

Y. Nakashima: Writing – review & editing, Project administration, Supervision

Funding statement

The author(s) disclose receipt of the following financial or material support for the research, authorship, and/or publication of this article: this study was supported by JSPS KAKENHI (JP22K09306), a Grant-in-Aid for Early-Career Scientists (JP23K15717, JP24K19623) from the Japan Society for the Promotion of Science, the Ogata Science Promotion Foundation.

ICMJE COI statement

Y. Akasaki reports a grant from the Japanese Society for the Promotion of Science (JSPS KAKENHI (JP22K09306)) for this study. R. Hirose reports a grant from the Ogata Science Promotion Foundation for this study. M. Kuwahara reports a Grant-in-Aid for Early-Career Scientists (No. 23K07493) from the Japan Society for the Promotion of Science for this study. T. Uchida reports a Grant-in-Aid for Early-Career Scientists (No. 24K19623) from the Japan Society for the Promotion of Science for this study.

Data sharing

The data that support the findings for this study are available to other researchers from the corresponding author upon reasonable request.

Acknowledgements

The authors thank the anonymous peer reviewers of this manuscript for their constructive comments. This study was supported by the Research Support Center, Research Center for Human Disease Modeling, Kyushu University Graduate School of Medical Sciences. We also thank Dr Tom Maniatis (Columbia University College of Physicians and Surgeons) for generating and providing the IKKε−/− mice used in this study.

Ethical review statement

Human knee tissues were obtained with informed consent and with approval from the Institutional Review Board of the Scripps Research Institute and the Ethics Committee of Kyushu University. All animal experiments were approved by the Animal Experiment Committee of Kyushu University (approval ID: A23-281) and conducted according to institutional guidelines.

Open access funding

Open access funding was provided by the Ogata Science Promotion Foundation. No specific grant number is associated with this funding.

Supplementary material

Additional figures, tables, primer sequences, and extended experimental data supporting the findings of this study.

© 2025 Hirose et al. This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (CC BY-NC-ND 4.0) licence, which permits the copying and redistribution of the work only, and provided the original author and source are credited. See https://creativecommons.org/licenses/by-nc-nd/4.0/

Data Availability

The data that support the findings for this study are available to other researchers from the corresponding author upon reasonable request.

References

  • 1. Markes AR, Hodax JD, Ma CB. Meniscus form and function. Clin Sports Med. 2020;39(1):1–12. doi: 10.1016/j.csm.2019.08.007. [DOI] [PubMed] [Google Scholar]
  • 2. Sun H, Wen X, Li H, et al. Single-cell RNA-seq analysis identifies meniscus progenitors and reveals the progression of meniscus degeneration. Ann Rheum Dis. 2020;79(3):408–417. doi: 10.1136/annrheumdis-2019-215926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Sandell LJ, Aigner T. Articular cartilage and changes in arthritis. An introduction: cell biology of osteoarthritis. Arthritis Res. 2001;3(2):107–113. doi: 10.1186/ar148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Collins JE, Losina E, Nevitt MC, et al. Semiquantitative imaging biomarkers of knee osteoarthritis progression: data from the foundation for the National Institutes of Health Osteoarthritis Biomarkers Consortium. Arthritis Rheumatol. 2016;68(10):2422–2431. doi: 10.1002/art.39731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Erard J, Schmidt A, Batailler C, Shatrov J, Servien E, Lustig S. Higher knee survivorship in young patients with monocompartmental osteoarthritis and constitutional deformity treated by high tibial osteotomy then total knee arthroplasty compared to an early total knee arthroplasty: a comparative study at a minimum follow-up of ten years. Bone Jt Open. 2023;4(2):62–71. doi: 10.1302/2633-1462.42.BJO-2023-0002.R1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Bhattacharyya T, Gale D, Dewire P, et al. The clinical importance of meniscal tears demonstrated by magnetic resonance imaging in osteoarthritis of the knee. J Bone Joint Surg Am. 2003;85-A(1):4–9. doi: 10.2106/00004623-200301000-00002. [DOI] [PubMed] [Google Scholar]
  • 7. Englund M, Roemer FW, Hayashi D, Crema MD, Guermazi A. Meniscus pathology, osteoarthritis and the treatment controversy. Nat Rev Rheumatol. 2012;8(7):412–419. doi: 10.1038/nrrheum.2012.69. [DOI] [PubMed] [Google Scholar]
  • 8. Goebel L, Reinhard J, Madry H. Meniscal lesion. A pre-osteoarthritic condition of the knee joint. Orthopade. 2017;46(10):822–830. doi: 10.1007/s00132-017-3462-y. [DOI] [PubMed] [Google Scholar]
  • 9. Hada S, Kaneko H, Sadatsuki R, et al. The degeneration and destruction of femoral articular cartilage shows a greater degree of deterioration than that of the tibial and patellar articular cartilage in early stage knee osteoarthritis: a cross-sectional study. Osteoarthritis Cartilage. 2014;22(10):1583–1589. doi: 10.1016/j.joca.2014.07.021. [DOI] [PubMed] [Google Scholar]
  • 10. Castagno S, Gompels B, Strangmark E, et al. Understanding the role of machine learning in predicting progression of osteoarthritis. Bone Joint J. 2024;106-B(11):1216–1222. doi: 10.1302/0301-620X.106B11.BJJ-2024-0453.R1. [DOI] [PubMed] [Google Scholar]
  • 11. Stone AV, Loeser RF, Vanderman KS, Long DL, Clark SC, Ferguson CM. Pro-inflammatory stimulation of meniscus cells increases production of matrix metalloproteinases and additional catabolic factors involved in osteoarthritis pathogenesis. Osteoarthritis Cartilage. 2014;22(2):264–274. doi: 10.1016/j.joca.2013.11.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Monibi FA, Pannellini T, Otero M, Warren RF, Rodeo SA. Histologic and molecular features in pathologic human menisci from knees with and without osteoarthritis. J Orthop Res. 2022;40(2):504–512. doi: 10.1002/jor.25047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Yin M, Wang X, Lu J. Advances in IKBKE as a potential target for cancer therapy. Cancer Med. 2020;9(1):247–258. doi: 10.1002/cam4.2678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Shin CH, Choi DS. Essential roles for the non-canonical IκB kinases in linking inflammation to cancer, obesity, and diabetes. Cells. 2019;8(2):178. doi: 10.3390/cells8020178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Bhatt D, Ghosh S. Regulation of the NF-κB-mediated transcription of inflammatory genes. Front Immunol. 2014;5:71. doi: 10.3389/fimmu.2014.00071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Olivotto E, Otero M, Marcu KB, Goldring MB. Pathophysiology of osteoarthritis: canonical NF-κB/IKKβ-dependent and kinase-independent effects of IKKα in cartilage degradation and chondrocyte differentiation. RMD Open. 2015;1(Suppl 1):e000061. doi: 10.1136/rmdopen-2015-000061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Baumgartner T, Langenbach BP, Gianotti LRR, Müri RM, Knoch D. Frequency of everyday pro-environmental behaviour is explained by baseline activation in lateral prefrontal cortex. Sci Rep. 2019;9(1):9. doi: 10.1038/s41598-018-36956-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Grothe K, Flechsenhar K, Paehler T, et al. IκB kinase inhibition as a potential treatment of osteoarthritis – results of a clinical proof-of-concept study. Osteoarthritis Cartilage. 2017;25(1):46–52. doi: 10.1016/j.joca.2016.08.010. [DOI] [PubMed] [Google Scholar]
  • 19. Murahashi Y, Yano F, Kobayashi H, et al. Intra-articular administration of IκBα kinase inhibitor suppresses mouse knee osteoarthritis via downregulation of the NF-κB/HIF-2α axis. Sci Rep. 2018;8(1):16475. doi: 10.1038/s41598-018-34830-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Uchida T, Akasaki Y, Sueishi T, et al. Promotion of knee cartilage degradation by IκB kinase ε in the pathogenesis of osteoarthritis in human and murine models. Arthritis Rheumatol. 2023;75(6):937–949. doi: 10.1002/art.42421. [DOI] [PubMed] [Google Scholar]
  • 21. Sueishi T, Akasaki Y, Goto N, et al. GRK5 inhibition attenuates cartilage degradation via decreased NF-κB signaling. Arthritis Rheumatol. 2020;72(4):620–631. doi: 10.1002/art.41152. [DOI] [PubMed] [Google Scholar]
  • 22. Reilly SM, Chiang S-H, Decker SJ, et al. An inhibitor of the protein kinases TBK1 and IKK-ɛ improves obesity-related metabolic dysfunctions in mice. Nat Med. 2013;19(3):313–321. doi: 10.1038/nm.3082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Lefranc J, Schulze VK, Hillig RC, et al. Discovery of BAY-985, a highly selective TBK1/IKKε inhibitor. J Med Chem. 2020;63(2):601–612. doi: 10.1021/acs.jmedchem.9b01460. [DOI] [PubMed] [Google Scholar]
  • 24. Kurakazu I, Akasaki Y, Tsushima H, et al. TGFβ1 signaling protects chondrocytes against oxidative stress via FOXO1–autophagy axis. Osteoarthritis Cartilage. 2021;29(11):1600–1613. doi: 10.1016/j.joca.2021.07.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Kuwahara M, Akasaki Y, Kurakazu I, et al. C10orf10/DEPP activates mitochondrial autophagy and maintains chondrocyte viability in the pathogenesis of osteoarthritis. FASEB J. 2022;36(2):e22145. doi: 10.1096/fj.202100896R. [DOI] [PubMed] [Google Scholar]
  • 26. Kwok J, Onuma H, Olmer M, Lotz MK, Grogan SP, D’Lima DD. Histopathological analyses of murine menisci: implications for joint aging and osteoarthritis. Osteoarthritis Cartilage. 2016;24(4):709–718. doi: 10.1016/j.joca.2015.11.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Glasson SS, Chambers MG, Van Den Berg WB, Little CB. The OARSI histopathology initiative – recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthritis Cartilage. 2010;18:S17–S23. doi: 10.1016/j.joca.2010.05.025. [DOI] [PubMed] [Google Scholar]
  • 28. Kamekura S, Hoshi K, Shimoaka T, et al. Osteoarthritis development in novel experimental mouse models induced by knee joint instability. Osteoarthritis Cartilage. 2005;13(7):632–641. doi: 10.1016/j.joca.2005.03.004. [DOI] [PubMed] [Google Scholar]
  • 29. Fei K, Andress BD, Kelly AM, Chasse DAD, McNulty AL. Meniscus gene expression profiling of inner and outer zone meniscus tissue compared to cartilage and passaged monolayer meniscus cells. Sci Rep. 2024;14(1):27423. doi: 10.1038/s41598-024-78580-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Kravchenko VV, Mathison JC, Schwamborn K, Mercurio F, Ulevitch RJ. IKKi/IKKepsilon plays a key role in integrating signals induced by pro-inflammatory stimuli. J Biol Chem. 2003;278(29):26612–26619. doi: 10.1074/jbc.M303001200. [DOI] [PubMed] [Google Scholar]
  • 31. Yang L, Zhang J, Wang G. The effect of sodium hyaluronate treating knee osteoarthritis on synovial fluid interleukin -1β and clinical treatment mechanism. Pak J Pharm Sci. 2015;28(1 Suppl):407–410. [PubMed] [Google Scholar]
  • 32. Li ZM, Li M. Improvement in orthopedic outcome score and reduction in IL-1β, CXCL13, and TNF-α in synovial fluid of osteoarthritis patients following arthroscopic knee surgery. Genet Mol Res. 2017;16(3) doi: 10.4238/gmr16039487. [DOI] [PubMed] [Google Scholar]
  • 33. Eitner A, Sparing S, Kohler FC, et al. Osteoarthritis-induced metabolic alterations of human hip chondrocytes. Biomedicines. 2022;10(6):1349. doi: 10.3390/biomedicines10061349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Oeckinghaus A, Ghosh S. The NF-kappaB family of transcription factors and its regulation. Cold Spring Harb Perspect Biol. 2009;1(4):a000034. doi: 10.1101/cshperspect.a000034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Gilmore TD. Introduction to NF-κB: players, pathways, perspectives. Oncogene. 2006;25(51):6680–6684. doi: 10.1038/sj.onc.1209954. [DOI] [PubMed] [Google Scholar]
  • 36. Olivotto E, Otero M, Astolfi A, et al. IKKα/CHUK regulates extracellular matrix remodeling independent of its kinase activity to facilitate articular chondrocyte differentiation. PLoS One. 2013;8(9):e73024. doi: 10.1371/journal.pone.0073024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Jia C-H, Li M, Liu J, et al. IKK-β mediates hydrogen peroxide induced cell death through p85 S6K1. Cell Death Differ. 2013;20(2):248–258. doi: 10.1038/cdd.2012.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Behrendt P, Häfelein K, Preusse-Prange A, Bayer A, Seekamp A, Kurz B. IL-10 ameliorates TNF-α induced meniscus degeneration in mature meniscal tissue in vitro. BMC Musculoskelet Disord. 2017;18(1):197. doi: 10.1186/s12891-017-1561-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Du G, Zhan H, Ding D, et al. Abnormal mechanical loading induces cartilage degeneration by accelerating meniscus hypertrophy and mineralization after ACL injuries in vivo. Am J Sports Med. 2016;44(3):652–663. doi: 10.1177/0363546515621285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Lee KI, Gamini R, Olmer M, et al. Mohawk is a transcription factor that promotes meniscus cell phenotype and tissue repair and reduces osteoarthritis severity. Sci Transl Med. 2020;12(567):eaan7967. doi: 10.1126/scitranslmed.aan7967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Johnson K, Hashimoto S, Lotz M, Pritzker K, Goding J, Terkeltaub R. Up-regulated expression of the phosphodiesterase nucleotide pyrophosphatase family member PC-1 is a marker and pathogenic factor for knee meniscal cartilage matrix calcification. Arthritis Rheum. 2001;44(5):1071–1081. doi: 10.1002/1529-0131(200105)44:5&#x0003c;1071::AID-ANR187&#x0003e;3.0.CO;2-3. [DOI] [PubMed] [Google Scholar]
  • 42. Mitrovic DR, Stankovic A, Iriarte-Borda O, et al. The prevalence of chondrocalcinosis in the human knee joint. An autopsy survey. J Rheumatol. 1988;15(4):633–641. doi: 10.1136/ard.52.6.418. [DOI] [PubMed] [Google Scholar]
  • 43. Mitrovic D, Stankovic A, Morin J, et al. Anatomic incidence of meniscochondrocalcinosis of the knee. Rev Rhum Mal Osteoartic. 1982;49(7):495–499. [PubMed] [Google Scholar]
  • 44. Hu S, Mamun AA, Shaw J, et al. TBK1-medicated DRP1 phosphorylation orchestrates mitochondrial dynamics and autophagy activation in osteoarthritis. Acta Pharmacol Sin. 2023;44(3):610–621. doi: 10.1038/s41401-022-00967-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. Sun P, Xue Y. Silence of TANK-binding kinase 1 (TBK1) regulates extracellular matrix degradation of chondrocyte in osteoarthritis by janus kinase (JAK)-signal transducer of activators of transcription (STAT) signaling. Bioengineered. 2022;13(1):1872–1879. doi: 10.1080/21655979.2021.2018976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Tang Z, Yang L, Zhang J, et al. Coordinated expression of MMPs and TIMPs in rat knee intra-articular tissues after ACL injury. Connect Tissue Res. 2009;50(5):315–322. [PubMed] [Google Scholar]
  • 47. Fuller ES, Smith MM, Little CB, Melrose J. Zonal differences in meniscus matrix turnover and cytokine response. Osteoarthritis Cartilage. 2012;20(1):49–59. doi: 10.1016/j.joca.2011.10.002. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings for this study are available to other researchers from the corresponding author upon reasonable request.

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