The ubiquitination of IL-6 by FBXO7 mitigates osteoarthritis through JAK1/STAT3 pathway modulation

The ubiquitination of IL-6 by FBXO7 mitigates osteoarthritis through JAK1/STAT3 pathway modulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The ubiquitination of IL-6 by FBXO7 mitigates osteoarthritis through JAK1/STAT3 pathway modulation ZhuSong Huang, Huiling Guo, XuChao Lin, JinFu Lan, WenHan Zhao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5012728/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Osteoarthritis (OA) is a chronic degenerative disease marked by cartilage destruction and subchondral bone remodeling which results in functional disability and pain. FBXO7 has been implicated in various inflammatory conditions, however, very little research has been done to establish its functional and molecular participation in the development and progression of OA. Here we show that the downregulation of FBXO7 may cause the progression and severity of OA. Articular cartilage obtained from OA patients and cartilage excised from DMM-induced OA from mice showed that FBXO7 was downregulated when compared to the controls. Mechanistically, we determined that FBXO7 interacts with the JAK1/STAT3 signaling pathway through IL-6. Immunoprecipitation assay revealed high-affinity physical interactions between FBXO7 and IL-6. When FBXO7 is downregulated in OA, the expression levels of IL-6 are elevated, which increases the activation of the JAK1/STAT3 signaling pathway. This process results in the elevation of MMP13 and suppression of type II collagen, two components crucial in the maintenance of articular cartilage homeostasis. However, overexpression of FBXO7 alleviated cartilage degradation by mediating the ubiquitination of IL-6 and degrading it, which led to the elevated expression of type II collagen, and reversed progression of OA. Therefore, targeting FBXO7 in the treatment of OA presents a promising avenue of therapy. Osteoarthritis FBOX7 IL-6 ubiquitination JAK1/STAT3 pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Osteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage destruction, osteophyte formation, and subchondral bone remodeling resulting in functional disability and joint pain( 1 , 2 ). OA is caused by aging, hard work, lifting heavy weights, and inflammation( 2 ). The bones in the human body are regularly regenerated via bone remodeling, which involves the removal of mature bone tissue from the skeleton (bone resorption) and the production of new bone (bone formation). Aging, hard work, heavy weight lifting, and inflammation can pathologically alter the properties of the bone remodeling process( 3 ). These pathological changes in the subchondral bone may result in abnormal bone remodeling( 4 ). Osteomorphs have been proven to participate in bone resorption and remodeling. Osteomorphs are derived from osteoclast fission and are able to rapidly mobilize to move to resorption sites and fuse into mature resorbing osteoclasts. Osteomorphs are implicated in the etiology of several bone diseases characterized by abnormal osteoclast physiology( 5 ). In the early stages of osteoarthritis (OA), osteoclasts induce an increase in abnormal bone remodeling in the subchondral bone. Furthermore, osteoclasts have been strongly associated with osteoarthritis through bone resorption. Although articular cartilage degradation has long been thought to be the cause of osteoarthritis, new research indicates that subchondral bone remodeling plays a crucial role in both initiating and driving the disease's progression( 6 ). FBXO7 has been reported to be overexpressed in osteomorphs, especially at the site of osteomorph division, and is regarded as an osteomorph marker( 5 , 7 ). Loss of mutations in FBXO7 was observed to produce bone phenotypes in mice( 8 ). This information implicates FBXO7 in bone development and resorption and can therefore be targeted for therapy. The FBXO7 protein belongs to the SKP7-Cullin-F-box type E3 ubiquitin ligases and plays important roles in targeting substrates for ubiquitination( 9 ). FBXO7 is implicated in inflammatory responses, and its inhibition suppresses inflammation by disrupting the ubiquitin machinery. A study by Li Yukun found that the high expression of FBXO7 was enriched with IL-6-JAK-STAT3 signaling( 10 ). Another study also observed that the overexpression of FBXO7 caused the increased release of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in lung inflammation( 11 ). These studies reveal that there is a regulatory relationship between FBXO7 and IL-6-JAK-STAT3 signaling. Most people with osteoarthritis (OA) have inflammation of the joints, and pro-inflammatory mediators like IL-6 play a crucial role in the disease's progression. The incidence and severity of OA patients' condition are correlated with higher levels of IL-6 in their serum or synovial fluid( 12 ). IL-6 plays a pivotal role in the development of cartilage pathology by activating the JAK1/STA3 pathways( 13 ). Studies show that IL-6 activation also suppresses collagen type II neo-synthesis in rabbit chondrocytes( 14 , 15 ) and induces matrix metalloproteinase MMP3 and MMP13, which mediate cartilage degradation( 12 ). The activation of the JAK1/STAT3 pathway plays an instrumental role in many inflammatory diseases including the osteoarticular system, particularly in the progression of OA( 16 ). In this study, we assess and determine the role of FBOX7 in the ubiquitination of IL-6 and JAK1/STAT3 pathway activation in the development and progression of osteoarthritis. Materials and Methods Human samples collection Human OA cartilage samples were collected from OA patients who were undergoing knee arthroplasty and healthy non-OA cartilage was excised from the knee joints of donors of trauma patients. The collection of the specimen was approved by the Ethics Committee Board of Fuzhou Second General Hospital (Approval No. 2022140). Written informed consent was obtained from all subjects before knee surgery. The cartilage samples were used for further evaluations using immunohistochemistry and histology. Animals Animal experiments were conducted on male C57BL/6J provided by Beijing Vital River Laboratory Animal Technology Company. All mice were housed in pathogen-free barrier facilities at 5 or less per cage and access to food and water. They were maintained at room temperature, with humidity ranging from 30–60%, and a 12-hour light/dark cycle. The mice were allocated to different experimental groups randomly. Animal welfare monitoring and euthanasia practices were implemented throughout the animal research. All animal experiments were approved by the Ethics Committee Board of Fujian Medical University (Approval No. IACUC FJMU 2023 − 0347) and followed the National Research Council and ARRIVE guidelines. OA mice model construction For the OA experiment model, the mice were placed under anesthesia using sodium pentobarbital (100 mg/kg) administered intraperitoneally by injection. Then the mice were traversed by the medial collateral ligament and destabilized by the medial meniscus (DMM) in the right knee. The mice were injected with replication-deficient lentivirus (Lenti) alone, Lenti-FBXO7, or saline via intra-articular administration 3 days, 7 days, 14 days, and 21 days after the DMM surgery. Eight weeks after the operation, the mice were sacrificed by the cervical dislocation method, and the knee joints were harvested. Cell culture and transfection Femoral articular cartilage was harvested from OA patients and non-OA donors. After conducting the OA model, on the 8th week, the knees of the mice were microdissected by surgically dissecting the OA cartilage under a surgical microscope. The tissue was digested with 0.2% type II collagenase for 40 minutes at 37°C. The isolated cells were washed with D-Hanks solution and suspended in DMEM/F12 (Gibco, USA) containing 10% FBS, 100U/mL penicillin (Gibco, USA), and 100mg/mL streptomycin (Gibco, USA). The chondrocytes were cultured in a high-humidity incubator (37°C; 5% CO 2 ). The media were changed every 2 days until the chondrocytes had grown into sheets and were over 85% confluent( 17 ). After 2 to 3 passages, the chondrocytes were used for future investigations. The FBXO7 overexpression was achieved through pcDNA3.1-FBXO7 transfection, with an empty pcDNA3.1 vector used as a control (Invitrogen, Life Technologies, Carlsbad, CA, USA). Negative control siRNA to knockdown FBXO7 (FBOX7 siRNA) (Sigma-Aldrich, St. Louis, MO, USA) was transfected into chondrocytes at a concentration of 50 nM Lipofectamine® 3000 Transfection Reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Senescence-associated β-galactosidase (SA-β-Gal) assay Using an SA-β-Gal staining kit (#9860, Cell Signaling Technology)as described( 18 ), SA-β-Gal staining was performed. Human chondrocytes were placed into 6-well plates at a density of 1.0×10 5 cells per well and cultured for about 48 hours at 37°C in an incubator. After culturing the cells for 48 hours, pcDNA3.1-FBXO, pcDNA3.1, FBXO7siRNA, and control siRNA were transfected by Lipofectamine® 3000 Transfection Reagent. Thereafter, cytochemical staining for SA-β-Gal was performed at pH6, and the positive cells were counted. Edu Assay Human OA chondrocytes transfected pcDNA3.1-FBXO7, pcDNA3.1, FBXO7siRNA, and control siRNA were used to perform Edu assay to analyze the relationship between FBXO7 expression and OA progression. EdU staining was performed according to the manufacturer’s instructions using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 555 (Beyotime Biotechnology, cat. no. C0075S). Briefly, the cells were incubated for 2 hours at 37°C after EdU was added to the culture media at a final concentration of 10 µM. After labeling, the cells were fixed for 15 minutes with 4% PFA and permeabilized for 10 minutes with 0.2% Triton X-100. The cells were then stained in the dark for 30 minutes with click reaction solution before being counterstained with Hoechst 33342. Under fluorescence microscopy (Olympus, Tokyo, Japan), EdU-positive cells were counted and the percentage of EdU-positive cells was reported. Flow cytometry Assay Cells were treated with trypsin and 200 µL Annexin V-FITC, then incubated for 10 minutes in the dark. Cells were rinsed with 200 µL PBS and 10 µL PI added. Cell apoptosis was identified by flow cytometry (Beckman Coulter). Cell immunofluorescence Chondrocytes from human donors and mice (after OA completion) were cultured on glass coverslips in 24-well plates at the density of 1×10 4 cells per well and transfected with pcDNA3.1-FBXO7, pcDNA3.1, FBXO7siRNA, and control siRNA. After 24-hour transfection, the cells on the coverslips were washed twice with PBS, cells were treated with 10ng/mL PA in the absence and presence of Xn for 24 hours for collagen II and MMP13 stain. The glass-seeded chondrocyte monolayers were fixed with 4% paraformaldehyde for 15 minutes at room temperature after they were washed with PBS three times. Then the cells were permeabilized for 10 minutes at room temperature with 0.3% Triton X-100 (in PBS). Then the cells were blocked using 5% BSA for 30 minutes. The chondrocytes were incubated with collagen II and MMP13 primary antibodies overnight at 4°C. The cells were then rinsed three times with PBS before being treated with Alexa Fluor® 647-conjugated goat anti-rabbit IgG (1:500) for 1 hour in the dark at room temperature. Finally, the cells were stained with DAPI to reveal their nuclei. Images were acquired using a fluorescence microscope. Western blotting analysis To determine certain protein expressions in transfected human chondrocytes and human SW-1353 chondrosarcoma cells stimulated with IL-1β, human chondrocytes were first transfected with pcDNA3.1-FBXO7, pcDNA3.1, FBXO7siRNA, and control siRNA. The cells were then lysed with RIPA buffer to extract the whole proteins. The total protein was quantified with a bicinchoninic acid (BCA) protein assay. After being separated and transferred onto polyvinylidenefluoride (PVDF) membranes, the membranes were blocked with 5% skim milk/BSA and incubated with primary antibodies against MMP13 (Cat# Ab39012; Abcam; 1:1000), COL2a1 (Cat# sc52655; Santa Cruz Biotechnology; 1:1000), anti-Fbxo7 antibody (Santa Cruz, sc-271763; 1:1000), JAK1 (Cat# 3332; Cell Signaling Technology; 1:1000), STAT3 (Cat# 9131, Cell Signaling Technology; 1:1000), anti-pSTAT3 (Cat# 9145; Cell Signaling Technology), and anti-IL-6 (Cat# 21865-1-AP; Rosemont, IL, Proteintech). Histology and immunohistochemistry analyses of OA Human OA-affected cartilage (OA) and healthy non-OA-affected cartilage (NC), and DMM-induced OA cartilage from mice (transfected with Lenti-FBXO7, Lenti-control, and saline) were fixed with 4% neutral-buffered paraformaldehyde (PFA; Solarbo, Beijing, China), embedded in paraffin, and sectioned into 6 µm thick slices. The cartilage sections were stained with Hematoxylin and eosin, Safranin O using the Safranin O kit (ICH World, Woodstock, NY, USA), and 0.05% Fast green FCF (f7258; Sigma-Aldrich). For immunohistochemical staining, the paraffin-embedded human articular cartilage sections from OA and NC patients were incubated with 3% H 2 O 2 for 10 minutes to block the endogenous peroxidase activity. This was followed by incubation with 10% goat serum for 1 hour at 21°C. Then the sections were incubated with primary antibodies against FBXO7 (ARP43128_P050; AVIVA, San Diego, USA). Next, the sections were incubated with horseradish peroxidase-linked secondary antibody (1:1, Immunologic, Duiven, The Netherlands) for 1 hour at room temperature. The integrated optical density (IOD) value of positive staining was evaluated using ImageJ software (National Institutes of Health, MD, USA). RNA-sequencing analysis The pathway enrichment (KEGG) and GO analyses, including biological process, from different gene lists were carried out by using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) ( http://david.abcc.ncifcrf.gov/ ). For GO classification and KEGG, the cutoff conditions were set as FDR = 0.05. The GO graphs of all enriched GO terms and volcano plots were drawn by ggplot2 package in R environment. Heatmaps were generated in R package ‘pheatmap’. Mass spectrometry analysis Chondrocytes were transfected with pcDNA3.1-FBXO7, pcDNA3.1, FBXO7siRNA, and control siRNA, then 48 hours later treated with IL-6, and the nuclear fraction was prepared by using a kit (Beyotime, P0028). The nuclear protein was immunoprecipitated with an anti-acetylation antibody and Protein A/G PLUS-agarose (sc-2003, Santa Cruz Biotechnology) overnight at 4°C. immunoprecipitated protein was resolved by 10% SDS-PAGE gels for Coomassie blue staining. The purified protein bands were cut out and digested with trypsin. LC-MS/MS analysis was performed on an EASY-nLC 1000 HPLC system (Thermo Scientific), which was directly interfaced with a Q Exactive mass spectrometer (Thermo Scientific). The analytical column was an AcclaimR PepMap RSLC column (50 µm ID, 15 cm length, C18, 2µm, 100 Å) (Thermo Scientific). The Q Extractive mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 2.2 SPI software and there was a single full-scan mass spectrum in the orbitrap (300–2000 m/z, 70,000 resolution) followed by 20 data-dependent MS/MS scans at 27% normalized collision energy (HCD). The MS/MS spectra from each LC-MS/MS rub were searched against the fasta files using Sequnet HT and phosphoRS 3.0 modules in the proteome Discoverer Software (Version PDL4, Thermo Scientific, USA). Co-immunoprecipitation For the co-immunoprecipitation assay, chondrocytes were transfected with Flag-FBXO7 alone, HA-IL-6 alone, or Flag-FBXO7 and HA-IL-6. The cells were then lysed with a cold RIPA lysis buffer. Lysates were centrifuged at 12,000g for 10 minutes, and the supernatants containing proteins were then incubated with 1 µg anti-FBXO7 and anti-HA-IL-6 antibodies at night at 4°C. Protein lysates were subsequently incubated with 20 µL pre-washed protein A/G PLUS-Agarose beads (sc-2003; Santa Cruz Biotechnology, USA) with gentle rotation for 3 hours at 4°C. The immunoprecipitates were analyzed by western blotting as described above using primary antibodies against FBXO7 to detect IL-6 protein or primary antibodies against IL-6 to detect FBXO7 protein. Ubiquitination Assay Chondrocytes were transfected with Flag-FBXO7, IL-1β, HA-FBXO7, Myc-k63-ub, and Flag-IL-6 K55R/K82R/K114R/K178R. Following 36 hours of transfection, the cells were treated with 20 µM MG132 for 8 hours and then lysed in RIPA (Low) Lysis Buffer (Meilunbio, China, Cat No: MA0153). For immunoprecipitation, the cell lysates were incubated with anti-FLAG M2 agarose beads (Sigma, USA) or anti-IL-6 Protein A/G immunoprecipitated magnetic beads (Bimake, China, Cat No: B23201) for 4 hours at 4°C. The bound beads were then washed four times with BC100 buffer (20 mM Tris-Cl, pH 7.9, 100 mM NaCl, 0.2 mM EDTA, 20% glycerol) containing 0.2% Triton X-100. The protein was eluted with FLAG peptide for 4 hours at 4°C. The ubiquitinated form of IL-6 was detected by western blotting with an anti-HA antibody. Statistical analysis In this investigation, one-way ANOVA and independent t-tests were employed when the data fit the normal distribution. GraphPad Prism 7.0 (GraphPad Software Inc., La Jolla, CA, USA) and SPSS 20.0 (SPSS, Inc., Chicago, IL, USA) were utilized for statistical analysis. The data were reported as a mean ± standard deviation (SD). P < 0.05 was determined to be statistically significant. Results The expression levels of FBXO7 in osteoarthritic cartilage and chondrocytes of patients Using cartilage from patients with OA (OA group) and healthy non-OA cartilage (NC group), we sought to investigate the participation of FBXO7 in OA progression. The results from the HE and Safranin O/fast green staining showed that the morphology of the cartilage from the OA group had severe cartilage erosion, development of osteophytes, and the thickening of the subchondral bone plate, whereas the cartilage from the NC group did not show any these signs (Fig. 1 ). The expression levels of FBXO7 in the human articular cartilage from OA and NC patients were evaluated using immunohistochemistry, the results showed that FBXO7 levels were downregulated in the cartilage of the OA group when compared to the NC group as depicted by the western blot (Fig. 2 a, b). Furthermore, the heat map and volcano plot demonstrated the differentially expressed genes in human OA chondrocytes and NC patients, it was observed that FBXO7 was significantly downregulated (Fig. 2 c). This implicates FBXO7 in the OA pathogenesis. FBXO7 is essential for the proliferation of chondrocytes To further clarify the relationship between FBXO7 expression and OA, the human chondrocytes were transfected with pcDNA3.1-FBXO7, pcDNA3.1, FBXO7siRNA, and control siRNA. The results from the western blot (Fig. 3 a) indicated that the expression level of FBXO7 was elevated in the chondrocytes transfected with pcDNA3.1-FBXO7 compared to the control group (pcDNA3.1), whereas when FBXO7 was silenced (FBXO7siRNA), the expression level of FBXO7 was decreased compared to the control group (control siRNA). This showed successful transfection. To determine the cell viability of chondrocytes transfected with different groups, flow cytometry was conducted. The level of cell apoptosis in the chondrocytes transfected with FBXO7 overexpression (pcDNA3.1-FBXO7) was substantially decreased compared to the control group. In comparison, the level of cell apoptosis was increased when FBXO7 was silenced compared to the control group (control siRNA) (Fig. 3 b). Furthermore, the Edu assay illustrated that the proliferation of chondrocytes was elevated in the group transfected with pcDNA3.1-FBXO7 compared to the control group but when FBXO7 was silenced, the level of cell proliferation was decreased compared to the control group (Fig. 3 c). Figure 3 d revealed that cells characterized by positive SA-β-gal were enhanced in chondrocytes that were transfected with FBXO7siRNA, indicating the loss of cell proliferative capacity when compared to the control group and the cells transfected with FBXO7 overexpression of FBXO7. These results show that FBXO7 promoted the proliferation of chondrocytes. FBXO7 regulates the expression of molecules correlated with cartilage homeostasis Collagen type II (Col II) is the main constituent of the extracellular matrix (ECM) of cartilage and is crucial for chondrocyte differentiation and hypertrophy in both normal cartilage formation and the pathophysiology of osteoarthritis. Collagenase-3 (MMP13) is another component of the chondrocyte ECM responsible for maintaining the homeostasis of chondrocytes, however, its expression is highly elevated in OA( 19 ). Using immunofluorescence to analyze the expression of these molecules in OA chondrocytes, it was observed that Col II was substantially increased in the chondrocytes when FBXO7 was overexpressed but significantly reduced when it was silenced (Fig. 4 a). Conversely, MMP13 was elevated in the chondrocytes when FBXO7 was silenced and downregulated when FBXO7 was overexpressed (Fig. 4 b). The activation of MMP13 and suppression of CoL II are promoted by IL-6. IL-6 is an important inflammatory cytokine secreted during the pathogenesis of AO. The results from the western blot show that IL-6 expression was elevated when FBXO7 was silenced but significantly reduced when FBXO7 was overexpressed (Fig. 4 c). FBXO7 is an ubiquitin ligase, it mediates the ubiquitination of IL-6 and degrades it. The degradation of IL-6 results in decreased levels of MMP13 and increased levels of Col II. This indicates that FBXO7 participates in the regulation of these molecules and can aid in the production of Col II and suppression of MMP 13 to mitigate OA. Activation of the JAK1/STAT3 pathway and its role in OA Research shows that IL-6 inflammatory cytokine is implicated in the development of OA through the activation of the JAK1/STAT3 signaling pathway. The JAK1/STAT3 pathway plays a crucial role in OA by promoting the secretion of inflammatory cytokines and MMP13 leading to chondrocyte inflammation and the degradation of cartilage( 20 ). When FBXO7 was overexpressed in OA chondrocytes, the levels of p-STAT3 and p-JAK1 expression were reduced as well as the expression of IL-6. However, when FBXO7 was silenced, the expression levels of p-JAK1 and p-STAT3 were elevated together with IL-6 levels (Fig. 5 a). This shows that the JAK1/STAT3 pathway system is involved in OA through FBXO7 and IL-6 participation. The downregulation of FBXO7 expression in OA may induce the elevation of IL-6 inflammatory cytokine and the activation of the JAK1/STAT3 pathway. Moreover, gene ontology revealed that the JAK1/STAT3 signaling pathway was enriched in the regulation of programmed cell death, collagen-containing extracellular matrix, and extracellular matrix structural components which were referred to as the biological process (BP), cellular components (CC), and molecular function (MF), respectively. GSEA and KEGG analysis demonstrated that the JAK1/STAT3 signaling pathway was upregulated in OA patients (Fig. 5 b). The JAK1/STAT3 signaling pathway may regulate the structural components of the extracellular matrix in the cartilage. Identification of FBXO7 downstream molecular targets The interaction between FBXO7 and IL-6 was determined using mass spectrometry analysis, where FBXO7 was used to pull down IL-6 (Fig. 6 a). The interaction between FBXO7 and IL-6 was further verified using coimmunoprecipitation assay and ubiquitination assay. The immunoprecipitation assay revealed high-affinity physical interactions between FBXO7 and IL-6 (Fig. 6 b) and the ubiquitination assay showed that K114 was the ubiquitinated lysine in human IL-6 (Fig. 6 c, d). Enhanced expression of FBXO7 attenuates surgically induced OA progression in mice To investigate the effects of FBXO7 in OA, mice were injected with Lenti-FBXO7 on 3, 7, 14, and 21 days after DMM surgery and sacrificed on week 8 (Fig. 6 a). The expression levels of Col II were upregulated in the cartilage tissue of mice overexpressing FBXO7 when compared to the control and saline groups. However, the expression levels of MMP13 were downregulated in the cartilage tissues overexpressing FBXO7 when compared to the control and saline groups (Fig. 6 b). Furthermore, in the mice injected with Lenti-FBXO, there was reduced articular erosion and the cartilage was greatly ameliorated (Fig. 6 c). This shows that FBXO7 is crucial in the maintenance of healthy cartilage, and can reverse the progression of OA. Discussion Osteoarthritis is a chronic degenerative disorder of multifactorial etiology marked by the loss of articular cartilage( 21 ). Though the risk factors of osteoarthritis have been well-studied( 22 ), the pathophysiological mechanism still remains unclear. In this study, we investigated FBXO7, which was downregulated in OA patients' cartilage. This investigation establishes the protective function of FBXO7 on articular cartilage against osteoarthritis. We demonstrate that FBXO7 was significantly downregulated in OA patients' cartilage tissues and chondrocytes and that there was a clear correlation between its level and the deterioration of cartilage. The results from the histological and immunohistochemical analyses showed that the cartilage from the OA patients was degraded and eroded compared to that of the non-OA patients with the expression levels of FBXO7 decreased in OA patients when compared to non-OA patients. From this, we can surmise that the decrease in the levels of FBXO7 may aggravate cartilage degradation and eventually lead to the onset or progression of OA. To confirm these findings, chondrocytes obtained from OA and non-OA patients were treated with the FBXO7 overexpression or FBXO7 knockdown and their respective controls. It was observed that FBXO7 knockdown elevated the level of chondrocyte apoptosis compared to the control, while its overexpression markedly decreased the level of chondrocyte apoptosis compared to the control. Congruently, the level of chondrocyte proliferation was substantially reduced with the knockdown of FBXO7 but increased when FBXO7 was overexpressed. Since the proliferation of chondrocytes is limited in OA( 23 ), the presence of FBXO7 in OA may be able to halt its progression by reverting the proliferative activity of chondrocytes. Over 90% of the collagen in adult articular cartilage is type II collagen( 24 ), which also acts as an anchor for chondrocytes. It is also believed to be responsible for regulating the proliferation of chondrocytes. Its degradation and reduction are usually observed in the pathological changes leading to osteoarthritis( 25 ). Under normal physiological conditions, the production of MMP13, a collagenase, in chondrocytes and cartilage is low and strictly controlled( 26 ). Its role is to aid in the proper resorption of hypertrophic cartilage and normal bone resorption( 27 , 28 ). However, during the pathophysiology of osteoarthritis, MMP13 causes erosion and destruction of articular cartilage by degrading type II collagen( 29 ). In our study, when FBXO7 was silenced, the expression levels of MMP13 were upregulated while those of type II collagen were downregulated. However, when FBXO7 was overexpressed, the reverse was true, MMP13 expression levels were downregulated while the expression levels of type II collagen were upregulated. This discovery is very significant in establishing the mechanism of osteoarthritis onset or progression, as well as its possible therapy. The results reveal that FBXO7 possibly modulates the expression levels of these crucial factors in osteoarthritis. Furthermore, we constructed a DMM-induced OA in mice by injecting lenti-FBXO7, lenti-control, and saline into the knee joints of the mice after the operation. We then studied the cartilage morphology from the three groups, the cartilage from the lenti-FBXO7 was less eroded and degraded compared to the lenti-control saline group. Moreover, the expression levels of MMP13 were markedly downregulated while the levels of type II collagen were upregulated in the lenti-FBXO7 group compared to the lenti-control and saline group. These findings further confirm that FBXO7 regulates the expression of MMP13 and type II collagen, and its presence in OA cartilage can restore the integrity of the cartilage thereby alleviating osteoarthritis. From our findings, we can deduce that FBXO7 regulates the expression levels of type II collagen and MMP13, the two factors that have been established to play crucial roles in the restoration or degradation of cartilage, respectively, and subsequently in the development of osteoarthritis. Establishing the molecular mechanism through which FBXO7 regulated these factors is vital as this elucidates the molecules and pathways responsible for initiating this pathological process. Using this information, we can curate effective therapeutic strategies for the treatment of osteoarthritis. To establish the molecular mechanism, RNA-seq analysis was performed, and downstream molecular targets of FBXO7. First, we established that FBXO7 was differentially expressed and downregulated in the chondrocytes from OA patients when compared to non-OA patients. According to the results of the Gene set enrichment analysis, the JAK/STAT pathway had significant changes. To correlate this pathway to FBXO7, we studied the expression levels of STAT and JAK when FBXO7 was silenced or overexpressed. It was observed that the expression levels of phosphorylated JAK1 and STAT3 were both upregulated when FBXO7 was silenced but downregulated when FBXO7 was overexpressed. A similar trend was also observed for the expression levels of IL-6. The phosphorylated forms of JAK1 and STAT3 were upregulated as JAK1 and STAT3 are activated by phosphrylation( 11 , 30 ). During inflammation in osteoarthritis, the secretion of IL-6 phosphorylates and activates JAK1 which then recruits STAT3, moreover, IL-6 can also activate STAT3( 31 ). IL-6 is implicated in the development of osteoarthritis( 32 , 33 ). Alone, IL-6 induces MMP13 which promotes cartilage degradation and suppresses type II collagen which promotes chondrocyte proliferation and cartilage regeneration( 34 ), however, the JAK1/STAT3 pathway activated by IL-6 also activates MMP13( 35 , 36 ) as well as suppressing type II collagen ( 16 ). FBXO7 can regulate the activation of the JAK1/STAT3 pathway through IL-6, thereby controlling osteoarthritis development. Since FBXO7 is an ubiquitin ligase, we surmise that it mediates the ubiquitination of IL-6 and degrades. When IL-6 is degraded, its expression levels are decreased and the JAK1/STAT3 pathway can not be activated. However, when FBXO7 is silenced, our results show that IL-6 is upregulated, this activates the JAK1/STAT3 pathway, which leads to the induction of MMP13 and suppression of type II collagen. Thus, by targeting FBXO7, therapy that actually targets chondrocyte proliferation and cartilage restoration can be developed. Conclusion We found that in DMM-induced OA mice, intra-articular injection of FBXO7 reduces cartilage breakdown, enhances type II collagen, and inhibits the expression of metallopeptidase 13 (MMP13). FBXO7 archives this by mediating the ubiquitination of IL-6 and degrading it, the decreased levels of IL-6 means that the JAK1/STAT3 signaling pathway is not activated. Consequently, this reduces the expression of MMP13 and increases the expression of Col II, thereby mitigating cartilage degradation and OA progression. FBXO7 has emerged as a viable target for osteoarthritis treatment and represents a major step forward in reducing or perhaps stopping the disease's progression. Declarations Ethical Approval and Consent to participate The collection of the specimen was approved by the Ethics Committee Board of Fuzhou Second General Hospital (Approval No. 2022140). All animal experiments were approved by the Ethics Committee Board of Fujian Medical University (Approval No. IACUC FJMU 2023 − 0347). Consent for publication: All authors have approved the manuscript and given their consent for submission and publication. Competing interests: The authors declare no competing interests. Fuding This work was supported by the Fujian Provincial Clinical Medical Research Center for First Aid and Rehabilitation in Orthopaedic Trauma (2020Y2014). Author Contribution All authors were involved in drafting or critically revising the manuscript for important intellectual content, and all authors approved the final version for publication. Z.S.H., M.C. and H.L.G. conceived the ideas and designed the experiments. Z.S.H., H.L.G., X.C.L., J.F.L, and W.H.Z. conducted experiments and analyzed the data. Z.S.H., H.L.G., and M.C. interpreted the data and wrote the manuscript. Acknowledgements: This work was supported by the Fujian Provincial Clinical Medical Research Center for First Aid and Rehabilitation in Orthopaedic Trauma (2020Y2014). The funders had no role in study design, data collection, data analysis, interpretation, or writing of this report. Availability of data and materials: The data that support the findings of this study are available on request from the corresponding author. References Kang D, Lee J. (2022) Selenophosphate synthetase 1 deficiency exacerbates osteoarthritis by dysregulating redox homeostasis 13: 779. Nagata K, et al. Runx2 and Runx3 differentially regulate articular chondrocytes during surgically induced osteoarthritis development. Nat Commun. 2022;13:6187. Zhu X, Chan YT, Yung PSH, Tuan RS, Jiang Y. Subchondral Bone Remodeling: A Therapeutic Target for Osteoarthritis. Front cell Dev biology. 2020;8:607764. Zhao X, et al. Osteoclasts secrete leukemia inhibitory factor to promote abnormal bone remodeling of subchondral bone in osteoarthritis. BMC Musculoskelet Disord. 2022;23:87. McDonald MM, et al. Osteoclasts recycle via osteomorphs during RANKL-stimulated bone resorption. Cell. 2021;184:1330–e13471313. Löfvall H, et al. Osteoclasts degrade bone and cartilage knee joint compartments through different resorption processes. Arthritis Res therapy. 2018;20:67. Huang T, et al. Effect of mitophagy in the formation of osteomorphs derived from osteoclasts. iScience. 2023;26:106682. Iqbal J, Zaidi M. Bone resorption goes green. Cell. 2021;184:1137–9. Deng H, Liang H, Jankovic J. F-box only protein 7 gene in parkinsonian-pyramidal disease. JAMA Neurol. 2013;70:20–4. Li Y, Yu J, Li R, Zhou H, Chang X. New insights into the role of mitochondrial metabolic dysregulation and immune infiltration in septic cardiomyopathy by integrated bioinformatics analysis and experimental validation. Cell Mol Biol Lett. 2024;29:21. Xin P, et al. The role of JAK/STAT signaling pathway and its inhibitors in diseases. Int Immunopharmacol. 2020;80:106210. Wiegertjes R, van de Loo FAJ, Blaney Davidson EN. A roadmap to target interleukin-6 in osteoarthritis. Rheumatology (Oxford). 2020;59:2681–94. Zeng R, et al. FOXM1 activates JAK1/STAT3 pathway in human osteoarthritis cartilage cell inflammatory reaction. Experimental biology Med (Maywood N J). 2021;246:644–53. Porée B, et al. Interleukin-6 (IL-6) and/or Soluble IL-6 Receptor Down-regulation of Human Type II Collagen Gene Expression in Articular Chondrocytes Requires a Decrease of Sp1·Sp3 Ratio and of the Binding Activity of Both Factors to the COL2A1 Promoter*. J Biol Chem. 2008;283:4850–65. Jikko A, et al. Effects of interleukin-6 on proliferation and proteoglycan metabolism in articular chondrocyte cultures. Cell Biol Int. 1998;22:615–21. Zhou Q et al. (2022) The potential roles of JAK/STAT signaling in the progression of osteoarthritis. Front Endocrinol 13. Wei L, Sun XJ, Wang Z, Chen Q. CD95-induced osteoarthritic chondrocyte apoptosis and necrosis: dependency on p38 mitogen-activated protein kinase. Arthritis Res therapy. 2006;8:R37. Debacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O. Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protoc. 2009;4:1798–806. Chan CM, et al. Cytokine-induced MMP13 Expression in Human Chondrocytes Is Dependent on Activating Transcription Factor 3 (ATF3) Regulation*. J Biol Chem. 2017;292:1625–36. Zhou Q, et al. The potential roles of JAK/STAT signaling in the progression of osteoarthritis. Front Endocrinol. 2022;13:1069057. Mahajan A, Verma S, Tandon V. Osteoarthritis. J Assoc Phys India. 2005;53:634–41. Spector TD, MacGregor AJ. (2004) Risk factors for osteoarthritis: genetics. Osteoarthritis and cartilage 12 Suppl A: S39-44. Li Y, Nie J, Deng C, Li H. P-15 promotes chondrocyte proliferation in osteoarthritis by regulating SFPQ to target the Akt-RUNX2 axis. J Orthop Surg Res. 2023;18:199. Roberts S, Menage J, Sandell LJ, Evans EH, Richardson JB. Immunohistochemical study of collagen types I and II and procollagen IIA in human cartilage repair tissue following autologous chondrocyte implantation. Knee. 2009;16:398–404. Lian C, et al. Collagen type II suppresses articular chondrocyte hypertrophy and osteoarthritis progression by promoting integrin β1 – SMAD1 interaction. Bone Res. 2019;7:8. Li S, Pritchard DM, Yu L-G. Regulation and Function of Matrix Metalloproteinase-13 in Cancer Progression and Metastasis. Cancers. 2022;14:3263. Behonick DJ, et al. Role of matrix metalloproteinase 13 in both endochondral and intramembranous ossification during skeletal regeneration. PLoS ONE. 2007;2:e1150. Duncan HF et al. (2022) The Critical Role of MMP13 in Regulating Tooth Development and Reactionary Dentinogenesis Repair Through the Wnt Signaling Pathway. Front Cell Dev Biology 10. Hu Q, Ecker M. (2021) Overview of MMP-13 as a Promising Target for the Treatment of Osteoarthritis. Int J Mol Sci 22. Apos, et al. The JAK-STAT Pathway: Impact on Human Disease and Therapeutic Intervention*. Annu Rev Med. 2015;66:311–28. Gao Q, et al. JAK/STAT Signal Transduction: Promising Attractive Targets for Immune, Inflammatory and Hematopoietic Diseases. Curr Drug Targets. 2018;19:487–500. Séguin CA, Bernier SM. TNFα suppresses link protein and type II collagen expression in chondrocytes: Role of MEK1/2 and NF-κB signaling pathways. J Cell Physiol. 2003;197:356–69. Namba A, et al. Effects of IL-6 and Soluble IL-6 Receptor on the Expression of Cartilage Matrix Proteins in Human Chondrocytes. Connect Tissue Res. 2007;48:263–70. Wiegertjes R, van de Loo FAJ, Blaney Davidson EN. A roadmap to target interleukin-6 in osteoarthritis. Rheumatology. 2020;59:2681–94. Legendre F, Dudhia J, Pujol J-P, Bogdanowicz P. JAK/STAT but Not ERK1/ERK2 Pathway Mediates Interleukin (IL)-6/Soluble IL-6R Down-regulation of Type II Collagen, Aggrecan Core, and Link Protein Transcription in Articular Chondrocytes: ASSOCIATION WITH A DOWN-REGULATION OF SOX9 EXPRESSION *. J Biol Chem. 2003;278:2903–12. Aida Y, et al. IL-6 and soluble IL-6 receptor stimulate the production of MMPs and their inhibitors via JAK—STAT and ERK—MAPK signalling in human chondrocytes. Cell Biol Int. 2012;36:367–76. Additional Declarations No competing interests reported. Supplementary Files Fulllength.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5012728","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":352049072,"identity":"8b7c6763-492e-4cd7-bf7f-1c9c115eb1fa","order_by":0,"name":"ZhuSong Huang","email":"","orcid":"","institution":"Fujian Medical University Union Hospital","correspondingAuthor":false,"prefix":"","firstName":"ZhuSong","middleName":"","lastName":"Huang","suffix":""},{"id":352049073,"identity":"af68ff23-2a37-41bc-afde-f3edd315e7d7","order_by":1,"name":"Huiling Guo","email":"","orcid":"","institution":"Fujian Medical University Union Hospital","correspondingAuthor":false,"prefix":"","firstName":"Huiling","middleName":"","lastName":"Guo","suffix":""},{"id":352049074,"identity":"14d48469-d22e-4e7b-8101-137e9fbfde00","order_by":2,"name":"XuChao Lin","email":"","orcid":"","institution":"Fujian Medical University Union Hospital","correspondingAuthor":false,"prefix":"","firstName":"XuChao","middleName":"","lastName":"Lin","suffix":""},{"id":352049075,"identity":"2ffab521-16da-4d1e-9c00-895c2e60d77a","order_by":3,"name":"JinFu Lan","email":"","orcid":"","institution":"Fuzhou Second General Hospital","correspondingAuthor":false,"prefix":"","firstName":"JinFu","middleName":"","lastName":"Lan","suffix":""},{"id":352049076,"identity":"6b5ba5ed-fd7e-42bd-8fdc-82f23b649235","order_by":4,"name":"WenHan Zhao","email":"","orcid":"","institution":"Fuzhou Second General Hospital","correspondingAuthor":false,"prefix":"","firstName":"WenHan","middleName":"","lastName":"Zhao","suffix":""},{"id":352049077,"identity":"538073d0-3704-4a1d-9e12-dfdcda4c66fe","order_by":5,"name":"Min Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYDACCRBhwMDP2MDAcCChQkJOnlgtko0NDIwPHpyxMDZsIEoLA4MkUCGz4cO2ikSGAwR08M9uPvboRsEdCeZpZ8wkEudJJDA2MD98dAOfJXeOpRvnGDyTYJydA9SyTSKPnYHN2DgHjxYDiRwz6RyDw3UwLcWMDTxs0vi15H8DaYHaMkciseEAQS05bDAtxgaJDURokbiRZgbVklb4IOGYhLFhMwG/8M9Ifiad8+ewhOHs5A0Hf9TUycmzNz98jE8LHCBikJkY5SBAOJ2MglEwCkbBiAUAXRNJql0TZ1sAAAAASUVORK5CYII=","orcid":"","institution":"Fujian Medical University Union Hospital","correspondingAuthor":true,"prefix":"","firstName":"Min","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-09-01 11:51:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5012728/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5012728/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":67094454,"identity":"dc01754f-1b34-4d16-87ec-2f9f1081a3aa","added_by":"auto","created_at":"2024-10-21 07:20:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3241547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of FBXO7 disrupts cartilage homeostasis and induces OA. \u003c/strong\u003eRepresentative gross appearance of the cartilage of OA patients and controls, H\u0026amp;E, and Safranin O/fast green staining. n = 6 independent biological replicates per group.\u003c/p\u003e","description":"","filename":"OnlineFIG1.png","url":"https://assets-eu.researchsquare.com/files/rs-5012728/v1/27776b9371a4dd2ed81f717f.png"},{"id":67094457,"identity":"14658ac3-3922-4633-9d71-4a1e227a91e0","added_by":"auto","created_at":"2024-10-21 07:20:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3741104,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of FBXO7 in OA. \u003c/strong\u003e\u0026nbsp;(a) Immunohistochemistry staining illustrating the expression of FBXO7 in articular cartilage of NC and OA human cartilage. n = 3 independent biological replicates per group. (b) Western blot images illustrating the expression of FBXO7 in human chondrocytes or Human SW-1353 chondrosarcoma cells stimulated with IL-1β. n = 3 independent biological replicates per group. (c) Heat map and Volcano plot demonstrating differentially expressed genes (fold change \u0026gt;2 or \u0026lt;0.5, Benjamini–Hochberg-corrected p) in OA chondrocytes vs controls.\u003c/p\u003e","description":"","filename":"OnlineFIG2.png","url":"https://assets-eu.researchsquare.com/files/rs-5012728/v1/7a0cc457fe039705bf953c8c.png"},{"id":67094455,"identity":"d77571b8-e236-4e24-a561-f430e0378c87","added_by":"auto","created_at":"2024-10-21 07:20:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7799114,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe overexpression of FBXO7 is associated with the proliferation of chondrocytes. \u003c/strong\u003e(a) Western blot images showing FBXO7 expression levels in human chondrocytes that were transfected by pcDNA3.1- FBXO7, FBXO7 siRNA, or their corresponding controls. (b) Chondrocyte apoptosis was assayed by flow cytometry in human chondrocytes that were transfected with pcDNA3.1- FBXO7 = 6 independent biological replicates per group. (c) Cell proliferation analysis in human chondrocytes transfected with pcDNA3.1- FBXO7, FBXO7siRNA, or their corresponding controls via EdU assay. n = 6 independent biological replicates per group. (d) SA-β-Gal positivity staining in chondrocytes transfected with pcDNA3.1- FBXO7, FBXO7siRNA, or their corresponding controls. The increased activity of SA-β-Gal could be observed in the FBXO7 siRNA group. Scale bar = 50 μm.\u003c/p\u003e","description":"","filename":"OnlineFIG3.png","url":"https://assets-eu.researchsquare.com/files/rs-5012728/v1/759f1b178b0d483381cb8263.png"},{"id":67094456,"identity":"0fa69d1a-a226-4edb-8c19-470ed4376c4e","added_by":"auto","created_at":"2024-10-21 07:20:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4427241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe overexpression of FBXO7 elevates the structural component of cartilage Col II and downregulates pro-OA inflammatory factor IL-6 and MMP13. \u003c/strong\u003e(a) Immunofluorescence staining of chondrocytes transfected with pcDNA3.1- FBXO7, FBXO7siRNA, or their corresponding controls to illustrate the expression of Col II and (b) MMP13. (c) Rescue assay of western blot illustrating that IL-6 overexpression effectively reversed the effects of FBXO7 overexpression.\u003c/p\u003e","description":"","filename":"OnlineFIG4.png","url":"https://assets-eu.researchsquare.com/files/rs-5012728/v1/af5a7cc4eb3669b1dd7d254a.png"},{"id":67094452,"identity":"f23f73cd-1d86-4d43-925e-8309fa688c8d","added_by":"auto","created_at":"2024-10-21 07:20:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2193584,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFBXO7 inhibits OA by regulating JAK1/STAT3 pathway via IL-6. \u003c/strong\u003e(a) The protein expressions of phosphorylated JAK1 and STAT3 in primary human chondrocytes transfected with pcDNA3.1- FBXO7, FBXO7 siRNA, or their corresponding controls. (b) Gene set enrichment analysis (GSEA) and GO analyses demonstrating JAK/STAT signaling pathway enriched in OA.\u003c/p\u003e","description":"","filename":"OnlineFIG5.png","url":"https://assets-eu.researchsquare.com/files/rs-5012728/v1/e8e6bca6a81382464e996e9a.png"},{"id":67094460,"identity":"78ba9b56-7903-47e1-b3a9-698a94ed30dd","added_by":"auto","created_at":"2024-10-21 07:20:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1421386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFBXO7 regulates the ubiquitination of IL-6 by targeting K114. \u003c/strong\u003e(a) Mass spectrometry was used to illustrate the pulling down of IL-6 by FBXO7. (b) Coimmunoprecipitation assay revealed high-affinity physical interactions between FBXO7 and IL-6. (c) Ubiquitination assay illustrates that IL-6 was targeted by FBXO7 for ubiquitination, and (d) K114 was identified as a ubiquitinated lysine in human IL-6.\u003c/p\u003e","description":"","filename":"OnlineFIG6.png","url":"https://assets-eu.researchsquare.com/files/rs-5012728/v1/b33baa1fe6889d3e2997a1be.png"},{"id":67094459,"identity":"e4510329-51ed-446f-9147-b5aa47e65299","added_by":"auto","created_at":"2024-10-21 07:20:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6847045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFBXO7 attenuates the progression of OA in DMM-induced OA mice. \u003c/strong\u003e(a) Scheme of OA treatment with intraocular injections of saline, Lenti-control, or Lenti-FBXO7 administered 3, 7, 14, and 21 days after DMM surgery. (b) Immunofluorescence analysis showing the expression of Col II and MMP13 in the cartilage tissues from OA mice after saline, Lenti-control, or Lenti-FBXO7 administration. (c) Representative images of H\u0026amp;E and Safranin O staining of cartilage tissues from OA mice 8 weeks after treatment.\u003c/p\u003e","description":"","filename":"OnlineFIG7.png","url":"https://assets-eu.researchsquare.com/files/rs-5012728/v1/db2e5e9dcbd99d789d8a41d7.png"},{"id":93947986,"identity":"c36c54a5-b838-42ed-beb6-93787472c21a","added_by":"auto","created_at":"2025-10-20 14:35:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3591492,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5012728/v1/607cc956-be1c-4a09-bd84-e2f0ed7fcaf6.pdf"},{"id":67095924,"identity":"0a28ae40-8ef6-447f-831e-68df7cb252e7","added_by":"auto","created_at":"2024-10-21 07:28:08","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4891825,"visible":true,"origin":"","legend":"","description":"","filename":"Fulllength.pptx","url":"https://assets-eu.researchsquare.com/files/rs-5012728/v1/db417e7f5c41d18728f55616.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The ubiquitination of IL-6 by FBXO7 mitigates osteoarthritis through JAK1/STAT3 pathway modulation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage destruction, osteophyte formation, and subchondral bone remodeling resulting in functional disability and joint pain(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). OA is caused by aging, hard work, lifting heavy weights, and inflammation(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The bones in the human body are regularly regenerated via bone remodeling, which involves the removal of mature bone tissue from the skeleton (bone resorption) and the production of new bone (bone formation). Aging, hard work, heavy weight lifting, and inflammation can pathologically alter the properties of the bone remodeling process(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). These pathological changes in the subchondral bone may result in abnormal bone remodeling(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOsteomorphs have been proven to participate in bone resorption and remodeling. Osteomorphs are derived from osteoclast fission and are able to rapidly mobilize to move to resorption sites and fuse into mature resorbing osteoclasts. Osteomorphs are implicated in the etiology of several bone diseases characterized by abnormal osteoclast physiology(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). In the early stages of osteoarthritis (OA), osteoclasts induce an increase in abnormal bone remodeling in the subchondral bone. Furthermore, osteoclasts have been strongly associated with osteoarthritis through bone resorption. Although articular cartilage degradation has long been thought to be the cause of osteoarthritis, new research indicates that subchondral bone remodeling plays a crucial role in both initiating and driving the disease's progression(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFBXO7 has been reported to be overexpressed in osteomorphs, especially at the site of osteomorph division, and is regarded as an osteomorph marker(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Loss of mutations in FBXO7 was observed to produce bone phenotypes in mice(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). This information implicates FBXO7 in bone development and resorption and can therefore be targeted for therapy. The FBXO7 protein belongs to the SKP7-Cullin-F-box type E3 ubiquitin ligases and plays important roles in targeting substrates for ubiquitination(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). FBXO7 is implicated in inflammatory responses, and its inhibition suppresses inflammation by disrupting the ubiquitin machinery. A study by Li Yukun found that the high expression of FBXO7 was enriched with IL-6-JAK-STAT3 signaling(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Another study also observed that the overexpression of FBXO7 caused the increased release of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in lung inflammation(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). These studies reveal that there is a regulatory relationship between FBXO7 and IL-6-JAK-STAT3 signaling.\u003c/p\u003e \u003cp\u003eMost people with osteoarthritis (OA) have inflammation of the joints, and pro-inflammatory mediators like IL-6 play a crucial role in the disease's progression. The incidence and severity of OA patients' condition are correlated with higher levels of IL-6 in their serum or synovial fluid(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). IL-6 plays a pivotal role in the development of cartilage pathology by activating the JAK1/STA3 pathways(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Studies show that IL-6 activation also suppresses collagen type II neo-synthesis in rabbit chondrocytes(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) and induces matrix metalloproteinase MMP3 and MMP13, which mediate cartilage degradation(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The activation of the JAK1/STAT3 pathway plays an instrumental role in many inflammatory diseases including the osteoarticular system, particularly in the progression of OA(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we assess and determine the role of FBOX7 in the ubiquitination of IL-6 and JAK1/STAT3 pathway activation in the development and progression of osteoarthritis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHuman samples collection\u003c/h2\u003e \u003cp\u003eHuman OA cartilage samples were collected from OA patients who were undergoing knee arthroplasty and healthy non-OA cartilage was excised from the knee joints of donors of trauma patients. The collection of the specimen was approved by the Ethics Committee Board of Fuzhou Second General Hospital (Approval No. 2022140). Written informed consent was obtained from all subjects before knee surgery. The cartilage samples were used for further evaluations using immunohistochemistry and histology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eAnimal experiments were conducted on male C57BL/6J provided by Beijing Vital River Laboratory Animal Technology Company. All mice were housed in pathogen-free barrier facilities at 5 or less per cage and access to food and water. They were maintained at room temperature, with humidity ranging from 30\u0026ndash;60%, and a 12-hour light/dark cycle. The mice were allocated to different experimental groups randomly. Animal welfare monitoring and euthanasia practices were implemented throughout the animal research. All animal experiments were approved by the Ethics Committee Board of Fujian Medical University (Approval No. IACUC FJMU 2023\u0026thinsp;\u0026minus;\u0026thinsp;0347) and followed the National Research Council and ARRIVE guidelines.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eOA mice model construction\u003c/h2\u003e \u003cp\u003eFor the OA experiment model, the mice were placed under anesthesia using sodium pentobarbital (100 mg/kg) administered intraperitoneally by injection. Then the mice were traversed by the medial collateral ligament and destabilized by the medial meniscus (DMM) in the right knee. The mice were injected with replication-deficient lentivirus (Lenti) alone, Lenti-FBXO7, or saline via intra-articular administration 3 days, 7 days, 14 days, and 21 days after the DMM surgery. Eight weeks after the operation, the mice were sacrificed by the cervical dislocation method, and the knee joints were harvested.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eCell culture and transfection\u003c/h2\u003e \u003cp\u003eFemoral articular cartilage was harvested from OA patients and non-OA donors. After conducting the OA model, on the 8th week, the knees of the mice were microdissected by surgically dissecting the OA cartilage under a surgical microscope. The tissue was digested with 0.2% type II collagenase for 40 minutes at 37\u0026deg;C. The isolated cells were washed with D-Hanks solution and suspended in DMEM/F12 (Gibco, USA) containing 10% FBS, 100U/mL penicillin (Gibco, USA), and 100mg/mL streptomycin (Gibco, USA). The chondrocytes were cultured in a high-humidity incubator (37\u0026deg;C; 5% CO\u003csub\u003e2\u003c/sub\u003e). The media were changed every 2 days until the chondrocytes had grown into sheets and were over 85% confluent(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). After 2 to 3 passages, the chondrocytes were used for future investigations. The FBXO7 overexpression was achieved through pcDNA3.1-FBXO7 transfection, with an empty pcDNA3.1 vector used as a control (Invitrogen, Life Technologies, Carlsbad, CA, USA). Negative control siRNA to knockdown FBXO7 (FBOX7 siRNA) (Sigma-Aldrich, St. Louis, MO, USA) was transfected into chondrocytes at a concentration of 50 nM Lipofectamine\u0026reg; 3000 Transfection Reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSenescence-associated β-galactosidase (SA-β-Gal) assay\u003c/h2\u003e \u003cp\u003eUsing an SA-β-Gal staining kit (#9860, Cell Signaling Technology)as described(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), SA-β-Gal staining was performed. Human chondrocytes were placed into 6-well plates at a density of 1.0\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well and cultured for about 48 hours at 37\u0026deg;C in an incubator. After culturing the cells for 48 hours, pcDNA3.1-FBXO, pcDNA3.1, FBXO7siRNA, and control siRNA were transfected by Lipofectamine\u0026reg; 3000 Transfection Reagent. Thereafter, cytochemical staining for SA-β-Gal was performed at pH6, and the positive cells were counted.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eEdu Assay\u003c/h2\u003e \u003cp\u003eHuman OA chondrocytes transfected pcDNA3.1-FBXO7, pcDNA3.1, FBXO7siRNA, and control siRNA were used to perform Edu assay to analyze the relationship between FBXO7 expression and OA progression. EdU staining was performed according to the manufacturer\u0026rsquo;s instructions using the BeyoClick\u0026trade; EdU Cell Proliferation Kit with Alexa Fluor 555 (Beyotime Biotechnology, cat. no. C0075S). Briefly, the cells were incubated for 2 hours at 37\u0026deg;C after EdU was added to the culture media at a final concentration of 10 \u0026micro;M. After labeling, the cells were fixed for 15 minutes with 4% PFA and permeabilized for 10 minutes with 0.2% Triton X-100. The cells were then stained in the dark for 30 minutes with click reaction solution before being counterstained with Hoechst 33342. Under fluorescence microscopy (Olympus, Tokyo, Japan), EdU-positive cells were counted and the percentage of EdU-positive cells was reported.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eFlow cytometry Assay\u003c/h2\u003e \u003cp\u003eCells were treated with trypsin and 200 \u0026micro;L Annexin V-FITC, then incubated for 10 minutes in the dark. Cells were rinsed with 200 \u0026micro;L PBS and 10 \u0026micro;L PI added. Cell apoptosis was identified by flow cytometry (Beckman Coulter).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell immunofluorescence\u003c/h2\u003e \u003cp\u003eChondrocytes from human donors and mice (after OA completion) were cultured on glass coverslips in 24-well plates at the density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well and transfected with pcDNA3.1-FBXO7, pcDNA3.1, FBXO7siRNA, and control siRNA. After 24-hour transfection, the cells on the coverslips were washed twice with PBS, cells were treated with 10ng/mL PA in the absence and presence of Xn for 24 hours for collagen II and MMP13 stain. The glass-seeded chondrocyte monolayers were fixed with 4% paraformaldehyde for 15 minutes at room temperature after they were washed with PBS three times. Then the cells were permeabilized for 10 minutes at room temperature with 0.3% Triton X-100 (in PBS). Then the cells were blocked using 5% BSA for 30 minutes. The chondrocytes were incubated with collagen II and MMP13 primary antibodies overnight at 4\u0026deg;C. The cells were then rinsed three times with PBS before being treated with Alexa Fluor\u0026reg; 647-conjugated goat anti-rabbit IgG (1:500) for 1 hour in the dark at room temperature. Finally, the cells were stained with DAPI to reveal their nuclei. Images were acquired using a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting analysis\u003c/h2\u003e \u003cp\u003eTo determine certain protein expressions in transfected human chondrocytes and human SW-1353 chondrosarcoma cells stimulated with IL-1β, human chondrocytes were first transfected with pcDNA3.1-FBXO7, pcDNA3.1, FBXO7siRNA, and control siRNA. The cells were then lysed with RIPA buffer to extract the whole proteins. The total protein was quantified with a bicinchoninic acid (BCA) protein assay. After being separated and transferred onto polyvinylidenefluoride (PVDF) membranes, the membranes were blocked with 5% skim milk/BSA and incubated with primary antibodies against MMP13 (Cat# Ab39012; Abcam; 1:1000), COL2a1 (Cat# sc52655; Santa Cruz Biotechnology; 1:1000), anti-Fbxo7 antibody (Santa Cruz, sc-271763; 1:1000), JAK1 (Cat# 3332; Cell Signaling Technology; 1:1000), STAT3 (Cat# 9131, Cell Signaling Technology; 1:1000), anti-pSTAT3 (Cat# 9145; Cell Signaling Technology), and anti-IL-6 (Cat# 21865-1-AP; Rosemont, IL, Proteintech).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHistology and immunohistochemistry analyses of OA\u003c/h2\u003e \u003cp\u003eHuman OA-affected cartilage (OA) and healthy non-OA-affected cartilage (NC), and DMM-induced OA cartilage from mice (transfected with Lenti-FBXO7, Lenti-control, and saline) were fixed with 4% neutral-buffered paraformaldehyde (PFA; Solarbo, Beijing, China), embedded in paraffin, and sectioned into 6 \u0026micro;m thick slices. The cartilage sections were stained with Hematoxylin and eosin, Safranin O using the Safranin O kit (ICH World, Woodstock, NY, USA), and 0.05% Fast green FCF (f7258; Sigma-Aldrich).\u003c/p\u003e \u003cp\u003eFor immunohistochemical staining, the paraffin-embedded human articular cartilage sections from OA and NC patients were incubated with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 10 minutes to block the endogenous peroxidase activity. This was followed by incubation with 10% goat serum for 1 hour at 21\u0026deg;C. Then the sections were incubated with primary antibodies against FBXO7 (ARP43128_P050; AVIVA, San Diego, USA). Next, the sections were incubated with horseradish peroxidase-linked secondary antibody (1:1, Immunologic, Duiven, The Netherlands) for 1 hour at room temperature. The integrated optical density (IOD) value of positive staining was evaluated using ImageJ software (National Institutes of Health, MD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRNA-sequencing analysis\u003c/h2\u003e \u003cp\u003eThe pathway enrichment (KEGG) and GO analyses, including biological process, from different gene lists were carried out by using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://david.abcc.ncifcrf.gov/\u003c/span\u003e\u003cspan address=\"http://david.abcc.ncifcrf.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). For GO classification and KEGG, the cutoff conditions were set as FDR\u0026thinsp;=\u0026thinsp;0.05. The GO graphs of all enriched GO terms and volcano plots were drawn by ggplot2 package in R environment. Heatmaps were generated in R package \u0026lsquo;pheatmap\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMass spectrometry analysis\u003c/h2\u003e \u003cp\u003eChondrocytes were transfected with pcDNA3.1-FBXO7, pcDNA3.1, FBXO7siRNA, and control siRNA, then 48 hours later treated with IL-6, and the nuclear fraction was prepared by using a kit (Beyotime, P0028). The nuclear protein was immunoprecipitated with an anti-acetylation antibody and Protein A/G PLUS-agarose (sc-2003, Santa Cruz Biotechnology) overnight at 4\u0026deg;C. immunoprecipitated protein was resolved by 10% SDS-PAGE gels for Coomassie blue staining. The purified protein bands were cut out and digested with trypsin. LC-MS/MS analysis was performed on an EASY-nLC 1000 HPLC system (Thermo Scientific), which was directly interfaced with a Q Exactive mass spectrometer (Thermo Scientific). The analytical column was an AcclaimR PepMap RSLC column (50 \u0026micro;m ID, 15 cm length, C18, 2\u0026micro;m, 100 \u0026Aring;) (Thermo Scientific). The Q Extractive mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 2.2 SPI software and there was a single full-scan mass spectrum in the orbitrap (300\u0026ndash;2000 m/z, 70,000 resolution) followed by 20 data-dependent MS/MS scans at 27% normalized collision energy (HCD). The MS/MS spectra from each LC-MS/MS rub were searched against the fasta files using Sequnet HT and phosphoRS 3.0 modules in the proteome Discoverer Software (Version PDL4, Thermo Scientific, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation\u003c/h2\u003e \u003cp\u003eFor the co-immunoprecipitation assay, chondrocytes were transfected with Flag-FBXO7 alone, HA-IL-6 alone, or Flag-FBXO7 and HA-IL-6. The cells were then lysed with a cold RIPA lysis buffer. Lysates were centrifuged at 12,000g for 10 minutes, and the supernatants containing proteins were then incubated with 1 \u0026micro;g anti-FBXO7 and anti-HA-IL-6 antibodies at night at 4\u0026deg;C. Protein lysates were subsequently incubated with 20 \u0026micro;L pre-washed protein A/G PLUS-Agarose beads (sc-2003; Santa Cruz Biotechnology, USA) with gentle rotation for 3 hours at 4\u0026deg;C. The immunoprecipitates were analyzed by western blotting as described above using primary antibodies against FBXO7 to detect IL-6 protein or primary antibodies against IL-6 to detect FBXO7 protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eUbiquitination Assay\u003c/h2\u003e \u003cp\u003eChondrocytes were transfected with Flag-FBXO7, IL-1β, HA-FBXO7, Myc-k63-ub, and Flag-IL-6 K55R/K82R/K114R/K178R. Following 36 hours of transfection, the cells were treated with 20 \u0026micro;M MG132 for 8 hours and then lysed in RIPA (Low) Lysis Buffer (Meilunbio, China, Cat No: MA0153). For immunoprecipitation, the cell lysates were incubated with anti-FLAG M2 agarose beads (Sigma, USA) or anti-IL-6 Protein A/G immunoprecipitated magnetic beads (Bimake, China, Cat No: B23201) for 4 hours at 4\u0026deg;C. The bound beads were then washed four times with BC100 buffer (20 mM Tris-Cl, pH 7.9, 100 mM NaCl, 0.2 mM EDTA, 20% glycerol) containing 0.2% Triton X-100. The protein was eluted with FLAG peptide for 4 hours at 4\u0026deg;C. The ubiquitinated form of IL-6 was detected by western blotting with an anti-HA antibody.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eIn this investigation, one-way ANOVA and independent t-tests were employed when the data fit the normal distribution. GraphPad Prism 7.0 (GraphPad Software Inc., La Jolla, CA, USA) and SPSS 20.0 (SPSS, Inc., Chicago, IL, USA) were utilized for statistical analysis. The data were reported as a mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was determined to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eThe expression levels of FBXO7 in osteoarthritic cartilage and chondrocytes of patients\u003c/h2\u003e \u003cp\u003eUsing cartilage from patients with OA (OA group) and healthy non-OA cartilage (NC group), we sought to investigate the participation of FBXO7 in OA progression. The results from the HE and Safranin O/fast green staining showed that the morphology of the cartilage from the OA group had severe cartilage erosion, development of osteophytes, and the thickening of the subchondral bone plate, whereas the cartilage from the NC group did not show any these signs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The expression levels of FBXO7 in the human articular cartilage from OA and NC patients were evaluated using immunohistochemistry, the results showed that FBXO7 levels were downregulated in the cartilage of the OA group when compared to the NC group as depicted by the western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). Furthermore, the heat map and volcano plot demonstrated the differentially expressed genes in human OA chondrocytes and NC patients, it was observed that FBXO7 was significantly downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). This implicates FBXO7 in the OA pathogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eFBXO7 is essential for the proliferation of chondrocytes\u003c/h2\u003e \u003cp\u003eTo further clarify the relationship between FBXO7 expression and OA, the human chondrocytes were transfected with pcDNA3.1-FBXO7, pcDNA3.1, FBXO7siRNA, and control siRNA. The results from the western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) indicated that the expression level of FBXO7 was elevated in the chondrocytes transfected with pcDNA3.1-FBXO7 compared to the control group (pcDNA3.1), whereas when FBXO7 was silenced (FBXO7siRNA), the expression level of FBXO7 was decreased compared to the control group (control siRNA). This showed successful transfection. To determine the cell viability of chondrocytes transfected with different groups, flow cytometry was conducted. The level of cell apoptosis in the chondrocytes transfected with FBXO7 overexpression (pcDNA3.1-FBXO7) was substantially decreased compared to the control group. In comparison, the level of cell apoptosis was increased when FBXO7 was silenced compared to the control group (control siRNA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Furthermore, the Edu assay illustrated that the proliferation of chondrocytes was elevated in the group transfected with pcDNA3.1-FBXO7 compared to the control group but when FBXO7 was silenced, the level of cell proliferation was decreased compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed revealed that cells characterized by positive SA-β-gal were enhanced in chondrocytes that were transfected with FBXO7siRNA, indicating the loss of cell proliferative capacity when compared to the control group and the cells transfected with FBXO7 overexpression of FBXO7. These results show that FBXO7 promoted the proliferation of chondrocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eFBXO7 regulates the expression of molecules correlated with cartilage homeostasis\u003c/h2\u003e \u003cp\u003eCollagen type II (Col II) is the main constituent of the extracellular matrix (ECM) of cartilage and is crucial for chondrocyte differentiation and hypertrophy in both normal cartilage formation and the pathophysiology of osteoarthritis. Collagenase-3 (MMP13) is another component of the chondrocyte ECM responsible for maintaining the homeostasis of chondrocytes, however, its expression is highly elevated in OA(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Using immunofluorescence to analyze the expression of these molecules in OA chondrocytes, it was observed that Col II was substantially increased in the chondrocytes when FBXO7 was overexpressed but significantly reduced when it was silenced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Conversely, MMP13 was elevated in the chondrocytes when FBXO7 was silenced and downregulated when FBXO7 was overexpressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The activation of MMP13 and suppression of CoL II are promoted by IL-6. IL-6 is an important inflammatory cytokine secreted during the pathogenesis of AO. The results from the western blot show that IL-6 expression was elevated when FBXO7 was silenced but significantly reduced when FBXO7 was overexpressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). FBXO7 is an ubiquitin ligase, it mediates the ubiquitination of IL-6 and degrades it. The degradation of IL-6 results in decreased levels of MMP13 and increased levels of Col II. This indicates that FBXO7 participates in the regulation of these molecules and can aid in the production of Col II and suppression of MMP 13 to mitigate OA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eActivation of the JAK1/STAT3 pathway and its role in OA\u003c/h2\u003e \u003cp\u003eResearch shows that IL-6 inflammatory cytokine is implicated in the development of OA through the activation of the JAK1/STAT3 signaling pathway. The JAK1/STAT3 pathway plays a crucial role in OA by promoting the secretion of inflammatory cytokines and MMP13 leading to chondrocyte inflammation and the degradation of cartilage(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). When FBXO7 was overexpressed in OA chondrocytes, the levels of p-STAT3 and p-JAK1 expression were reduced as well as the expression of IL-6. However, when FBXO7 was silenced, the expression levels of p-JAK1 and p-STAT3 were elevated together with IL-6 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). This shows that the JAK1/STAT3 pathway system is involved in OA through FBXO7 and IL-6 participation. The downregulation of FBXO7 expression in OA may induce the elevation of IL-6 inflammatory cytokine and the activation of the JAK1/STAT3 pathway. Moreover, gene ontology revealed that the JAK1/STAT3 signaling pathway was enriched in the regulation of programmed cell death, collagen-containing extracellular matrix, and extracellular matrix structural components which were referred to as the biological process (BP), cellular components (CC), and molecular function (MF), respectively. GSEA and KEGG analysis demonstrated that the JAK1/STAT3 signaling pathway was upregulated in OA patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The JAK1/STAT3 signaling pathway may regulate the structural components of the extracellular matrix in the cartilage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eIdentification of FBXO7 downstream molecular targets\u003c/h2\u003e \u003cp\u003eThe interaction between FBXO7 and IL-6 was determined using mass spectrometry analysis, where FBXO7 was used to pull down IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The interaction between FBXO7 and IL-6 was further verified using coimmunoprecipitation assay and ubiquitination assay. The immunoprecipitation assay revealed high-affinity physical interactions between FBXO7 and IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) and the ubiquitination assay showed that K114 was the ubiquitinated lysine in human IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eEnhanced expression of FBXO7 attenuates surgically induced OA progression in mice\u003c/h2\u003e \u003cp\u003eTo investigate the effects of FBXO7 in OA, mice were injected with Lenti-FBXO7 on 3, 7, 14, and 21 days after DMM surgery and sacrificed on week 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The expression levels of Col II were upregulated in the cartilage tissue of mice overexpressing FBXO7 when compared to the control and saline groups. However, the expression levels of MMP13 were downregulated in the cartilage tissues overexpressing FBXO7 when compared to the control and saline groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Furthermore, in the mice injected with Lenti-FBXO, there was reduced articular erosion and the cartilage was greatly ameliorated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). This shows that FBXO7 is crucial in the maintenance of healthy cartilage, and can reverse the progression of OA.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOsteoarthritis is a chronic degenerative disorder of multifactorial etiology marked by the loss of articular cartilage(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Though the risk factors of osteoarthritis have been well-studied(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), the pathophysiological mechanism still remains unclear. In this study, we investigated FBXO7, which was downregulated in OA patients' cartilage. This investigation establishes the protective function of FBXO7 on articular cartilage against osteoarthritis. We demonstrate that FBXO7 was significantly downregulated in OA patients' cartilage tissues and chondrocytes and that there was a clear correlation between its level and the deterioration of cartilage.\u003c/p\u003e \u003cp\u003eThe results from the histological and immunohistochemical analyses showed that the cartilage from the OA patients was degraded and eroded compared to that of the non-OA patients with the expression levels of FBXO7 decreased in OA patients when compared to non-OA patients. From this, we can surmise that the decrease in the levels of FBXO7 may aggravate cartilage degradation and eventually lead to the onset or progression of OA. To confirm these findings, chondrocytes obtained from OA and non-OA patients were treated with the FBXO7 overexpression or FBXO7 knockdown and their respective controls. It was observed that FBXO7 knockdown elevated the level of chondrocyte apoptosis compared to the control, while its overexpression markedly decreased the level of chondrocyte apoptosis compared to the control. Congruently, the level of chondrocyte proliferation was substantially reduced with the knockdown of FBXO7 but increased when FBXO7 was overexpressed. Since the proliferation of chondrocytes is limited in OA(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), the presence of FBXO7 in OA may be able to halt its progression by reverting the proliferative activity of chondrocytes.\u003c/p\u003e \u003cp\u003eOver 90% of the collagen in adult articular cartilage is type II collagen(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), which also acts as an anchor for chondrocytes. It is also believed to be responsible for regulating the proliferation of chondrocytes. Its degradation and reduction are usually observed in the pathological changes leading to osteoarthritis(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Under normal physiological conditions, the production of MMP13, a collagenase, in chondrocytes and cartilage is low and strictly controlled(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Its role is to aid in the proper resorption of hypertrophic cartilage and normal bone resorption(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). However, during the pathophysiology of osteoarthritis, MMP13 causes erosion and destruction of articular cartilage by degrading type II collagen(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In our study, when FBXO7 was silenced, the expression levels of MMP13 were upregulated while those of type II collagen were downregulated. However, when FBXO7 was overexpressed, the reverse was true, MMP13 expression levels were downregulated while the expression levels of type II collagen were upregulated. This discovery is very significant in establishing the mechanism of osteoarthritis onset or progression, as well as its possible therapy. The results reveal that FBXO7 possibly modulates the expression levels of these crucial factors in osteoarthritis. Furthermore, we constructed a DMM-induced OA in mice by injecting lenti-FBXO7, lenti-control, and saline into the knee joints of the mice after the operation. We then studied the cartilage morphology from the three groups, the cartilage from the lenti-FBXO7 was less eroded and degraded compared to the lenti-control saline group. Moreover, the expression levels of MMP13 were markedly downregulated while the levels of type II collagen were upregulated in the lenti-FBXO7 group compared to the lenti-control and saline group. These findings further confirm that FBXO7 regulates the expression of MMP13 and type II collagen, and its presence in OA cartilage can restore the integrity of the cartilage thereby alleviating osteoarthritis.\u003c/p\u003e \u003cp\u003eFrom our findings, we can deduce that FBXO7 regulates the expression levels of type II collagen and MMP13, the two factors that have been established to play crucial roles in the restoration or degradation of cartilage, respectively, and subsequently in the development of osteoarthritis. Establishing the molecular mechanism through which FBXO7 regulated these factors is vital as this elucidates the molecules and pathways responsible for initiating this pathological process. Using this information, we can curate effective therapeutic strategies for the treatment of osteoarthritis. To establish the molecular mechanism, RNA-seq analysis was performed, and downstream molecular targets of FBXO7. First, we established that FBXO7 was differentially expressed and downregulated in the chondrocytes from OA patients when compared to non-OA patients. According to the results of the Gene set enrichment analysis, the JAK/STAT pathway had significant changes. To correlate this pathway to FBXO7, we studied the expression levels of STAT and JAK when FBXO7 was silenced or overexpressed. It was observed that the expression levels of phosphorylated JAK1 and STAT3 were both upregulated when FBXO7 was silenced but downregulated when FBXO7 was overexpressed. A similar trend was also observed for the expression levels of IL-6. The phosphorylated forms of JAK1 and STAT3 were upregulated as JAK1 and STAT3 are activated by phosphrylation(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). During inflammation in osteoarthritis, the secretion of IL-6 phosphorylates and activates JAK1 which then recruits STAT3, moreover, IL-6 can also activate STAT3(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). IL-6 is implicated in the development of osteoarthritis(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Alone, IL-6 induces MMP13 which promotes cartilage degradation and suppresses type II collagen which promotes chondrocyte proliferation and cartilage regeneration(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), however, the JAK1/STAT3 pathway activated by IL-6 also activates MMP13(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) as well as suppressing type II collagen (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). FBXO7 can regulate the activation of the JAK1/STAT3 pathway through IL-6, thereby controlling osteoarthritis development. Since FBXO7 is an ubiquitin ligase, we surmise that it mediates the ubiquitination of IL-6 and degrades. When IL-6 is degraded, its expression levels are decreased and the JAK1/STAT3 pathway can not be activated. However, when FBXO7 is silenced, our results show that IL-6 is upregulated, this activates the JAK1/STAT3 pathway, which leads to the induction of MMP13 and suppression of type II collagen. Thus, by targeting FBXO7, therapy that actually targets chondrocyte proliferation and cartilage restoration can be developed.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe found that in DMM-induced OA mice, intra-articular injection of FBXO7 reduces cartilage breakdown, enhances type II collagen, and inhibits the expression of metallopeptidase 13 (MMP13). FBXO7 archives this by mediating the ubiquitination of IL-6 and degrading it, the decreased levels of IL-6 means that the JAK1/STAT3 signaling pathway is not activated. Consequently, this reduces the expression of MMP13 and increases the expression of Col II, thereby mitigating cartilage degradation and OA progression. FBXO7 has emerged as a viable target for osteoarthritis treatment and represents a major step forward in reducing or perhaps stopping the disease's progression.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eEthical Approval and Consent to participate\u003c/h2\u003e \u003cp\u003eThe collection of the specimen was approved by the Ethics Committee Board of Fuzhou Second General Hospital (Approval No. 2022140). All animal experiments were approved by the Ethics Committee Board of Fujian Medical University (Approval No. IACUC FJMU 2023\u0026thinsp;\u0026minus;\u0026thinsp;0347).\u003c/p\u003e \u003ch2\u003eConsent for publication:\u003c/h2\u003e \u003cp\u003e All authors have approved the manuscript and given their consent for submission and publication.\u003c/p\u003e \u003ch2\u003eCompeting interests:\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003ch2\u003eFuding\u003c/strong\u003e \u003cp\u003eThis work was supported by the Fujian Provincial Clinical Medical Research Center for First Aid and Rehabilitation in Orthopaedic Trauma (2020Y2014).\u003c/p\u003e \u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors were involved in drafting or critically revising the manuscript for important intellectual content, and all authors approved the final version for publication. Z.S.H., M.C. and H.L.G. conceived the ideas and designed the experiments. Z.S.H., H.L.G., X.C.L., J.F.L, and W.H.Z. conducted experiments and analyzed the data. Z.S.H., H.L.G., and M.C. interpreted the data and wrote the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eThis work was supported by the Fujian Provincial Clinical Medical Research Center for First Aid and Rehabilitation in Orthopaedic Trauma (2020Y2014). The funders had no role in study design, data collection, data analysis, interpretation, or writing of this report.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials:\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available on request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKang D, Lee J. 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Cell Biol Int. 2012;36:367\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Osteoarthritis, FBOX7, IL-6, ubiquitination, JAK1/STAT3 pathway","lastPublishedDoi":"10.21203/rs.3.rs-5012728/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5012728/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOsteoarthritis (OA) is a chronic degenerative disease marked by cartilage destruction and subchondral bone remodeling which results in functional disability and pain. FBXO7 has been implicated in various inflammatory conditions, however, very little research has been done to establish its functional and molecular participation in the development and progression of OA. Here we show that the downregulation of FBXO7 may cause the progression and severity of OA. Articular cartilage obtained from OA patients and cartilage excised from DMM-induced OA from mice showed that FBXO7 was downregulated when compared to the controls. Mechanistically, we determined that FBXO7 interacts with the JAK1/STAT3 signaling pathway through IL-6. Immunoprecipitation assay revealed high-affinity physical interactions between FBXO7 and IL-6. When FBXO7 is downregulated in OA, the expression levels of IL-6 are elevated, which increases the activation of the JAK1/STAT3 signaling pathway. This process results in the elevation of MMP13 and suppression of type II collagen, two components crucial in the maintenance of articular cartilage homeostasis. However, overexpression of FBXO7 alleviated cartilage degradation by mediating the ubiquitination of IL-6 and degrading it, which led to the elevated expression of type II collagen, and reversed progression of OA. Therefore, targeting FBXO7 in the treatment of OA presents a promising avenue of therapy.\u003c/p\u003e","manuscriptTitle":"The ubiquitination of IL-6 by FBXO7 mitigates osteoarthritis through JAK1/STAT3 pathway modulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-21 07:20:03","doi":"10.21203/rs.3.rs-5012728/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ad44d13b-bd1e-409b-9533-b282127ba5ce","owner":[],"postedDate":"October 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-20T14:26:52+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-21 07:20:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5012728","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5012728","identity":"rs-5012728","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}