| Objectives: Osteoarthritis(OA)is a considerably heterogeneous disease that is associated with age,gender,obesity,and joint injury.OA affects approximately 240 million people worldwide,50% of people over 65 years old are OA patients.With the accelerated progress of our country's population aging,the incidence of OA is still on the rise,resulting in a great economic burden to the society.OA is mainly described by subchondral bone remodeling,joint inflammation,osteophyte formation,and cartilage degeneration that is influenced by cell stress and cartilage matrix metabolism.OA pathogenesis involves elevated apoptosis and increased catabolism and decreased anabolism.Autophagy depends on the progression of OA.However,to date,there remains no disease-modifying treatment of OA.Therefore,it is of utmost importance to elucidate its pathogenesis,which can yield effective therapeutic strategies.It has been reported that the F-box protein family members,including FBXO32 and FBXO6,are related to OA.FBXO21,a member of the F-box protein family,is a subunit of the Skp1-cullin-F-box(SCF)ubiquitin E3 ligases,which acts on phosphorylationdependent ubiquitination degradation.Normally,FBXO21 degrades the P-glycoprotein and EID1 by ubiquitination.Dramatically,FBXO21 activates apoptosis signalregulating kinase 1(ASK1),instead of degrading,resulting in the wake of JNK and p38 pathways during antiviral innate response.However,its biological role in OA remains unknown and has not been studied.ERK is a serine/threonine protein kinase that can be activated by cytokines in OA.ERK is related to cartilage calcification and osteophyte formation but has little effect on cartilage matrix metabolism.It is mainly involved in regulating cell proliferation,apoptosis,and autophagy.Autophagy is a mechanism of cell self-protection that acts as an intracellular scavenger to maintain intracellular homeostasis and widely occurs in eukaryotic cells.Autophagy is a double-edged sword that may be beneficial and detrimental.In OA,autophagy increases in the early stage but decreases in the late stage.After we showed that ERK interacts with FBXO21 in a preliminary experiment,we hypothesized that FBXO21 may function by targeting ERK and autophagy.JUNB is a transcription factor(TF)containing a basic leucine zipper and belongs to the JUN family that includes JUND and c-JUN.JUNB is elevated in human chondrocytes and cartilage with OA and mice cartilage with OA.In addition,JUNB can bind to the promoter of the matrix metalloproteinase13(MMP13)that promotes the expression of MMP13 and reduces that of type II collagen(COL2A1)after the activation by cytokines such as interleukin(IL)-1?.We have previously found binding sites of JUNB in the FBXO21(Homo sapiens)promoter using the JASPAR database;thus,we hypothesized that JUNB functions by targeting FBXO21.In this study,the main objective was to evaluate the potential effects and mechanism of FBXO21 in OA degeneration.We firstly demonstrated FBXO21 accumulated in articular cartilage of patients with knee OA,articular cartilage of Sprague-Dawley(SD)rats with OA of aging and monosodium iodoacetate(MIA)model,and chondrocytes with OA.Then,we investigated underlying mechanism that could regulate cartilage degeneration in OA.Subsequently,we identified the involvement of the JUNBFBXO21-ERK axis in regulating apoptosis,anabolism,and catabolism of OA by inhibiting autophagy.To the best of our knowledge,this study is the first to explore the biological role of FBXO21 in OA,which sought to fill the gap in literature.Taken together,these results indicate that the novel functions of FBXO21 might provide a new therapeutic avenue for OA.Methods:1.The cartilage tissue and related clinicopathological characteristics of 24 patients with knee OA who underwent total knee arthroplasty were collected.According to the International Cartilage Repair Society(ICRS)grade,the cartilage was divided into undamaged and damaged cartilage.Western blot method was used to detect the expression of FBXO21 and other related molecules,and the cartilage was stained and scored histologically.2.MIA was injected into the knee joint cavity of rats to prepare OA rat models of different weeks,and aging OA rat models of different months were collected.X-ray,magnetic resonance and gross imaging were used for model evaluation,and Western blot and histochemistry were used to detect the expression of FBXO21 and other related molecules.3.Rat primary chondrocytes and SW1353 cell lines were treated with IL-1?,TNF-? and LPS respectively to simulate three different in vitro OA models,and Western blot and immunofluorescence were used to detect the expression of FBXO21 and other related molecules.4.FBXO21 adenovirus was injected into the knee joint cavity three times a week,and then MIA was injected to create a rat model of OA to study the biological effects of FBXO21 in OA in vivo.X-ray,magnetic resonance,gross imaging and histological staining were used to observe the degree of cartilage destruction,and Western blot and q PCR were used to detect the expression changes of related phenotypic molecules.5.Rat primary chondrocytes were infected with FBXO21 adenovirus first,and then treated with IL-1? to simulate an in vitro OA model to study the in vitro biological effects of FBXO21 in OA.Western blot and q PCR were used to detect the expression of related phenotypic molecules,and transmission electron microscopy,autophagy double-labeled adenovirus and flow cytometry were used to detect changes in autophagy and apoptosis.6.The primary rat chondrocytes were treated with IL-1?,followed by immunoprecipitation and mass spectrometry,and then co-immunoprecipitation to verify the potential downstream interaction proteins of FBXO21.7.The primary rat chondrocytes were infected with FBXO21 knockdown adenovirus,and then treated with IL-1? and the ERK activator Honokiol or autophagy inhibitor 3-MA at the same time to investigate whether FBXO21 could inhibit autophagy by activating ERK or promote degeneration by inhibiting autophagy.Western blot,autophagy double-labeled adenovirus and flow cytometry were used to detect changes in autophagy,apoptosis,anabolism and catabolism.8.IL-1? was added to the SW1353 cell line to simulate an in vitro OA model,and chromatin immunoprecipitation was used to prove that the transcription factor JUNB can bind to the promoter of FBXO21.9.Add JUNB knockdown adenovirus or plasmid and FBXO21 overexpression adenovirus or plasmid to rat primary chondrocytes or SW1353 cell lines at the same time,and finally add IL-1? treatment to study whether JUNB can promote degeneration by activating the expression of FBXO21.Western blot,autophagy double-labeled adenovirus and flow cytometry were used to detect changes in autophagy,apoptosis,anabolism and catabolism.Result:1.The expression of FBXO21 in the articular cartilage of the injured area of knee OA patients is elevated and is related to clinicopathological characteristics(1)X-ray and MRI images of 24 patients with knee OA: It was observed that the joint space of knee OA patients was significantly narrow,the cartilage surface was not smooth,and the osteophyte proliferation was obvious.(2)Cartilage histological staining score: The cartilage was stained with Alician Blue,Safranin O,Toluidine Blue and the International Osteoarthritis Research Association(OARSI)score,and the articular cartilage was divided into undamaged and damaged cartilage.The surface of undamaged cartilage is smooth and complete,and chondrocytes are arranged in a regular and orderly manner.On the contrary,cracks and cracks appeared on the surface of damaged cartilage,the surface was rough and the chondrocytes were arranged disorderly.(3)Western blot: Compared with non-damaged cartilage,FBXO21 and MMP13 in damaged cartilage were significantly up-regulated at the protein level.On the contrary,COL2A1 was significantly down-regulated.(4)Baseline data sheet of 24 OA patients: 10 cases of knee OA patients had relatively low expression of FBXO21 and 14 cases of relatively high expression of FBXO21 in injured cartilage.(5)Correlation of FBXO21 expression value with baseline data of 24 knee OA patients: The relatively high expression of FBXO21 is closely related to KellgrenLawrence grade.(6)Spearman correlation analysis of FBXO21 expression value and baseline data of 24 knee OA patients: The relatively high expression of FBXO21 is closely related to body mass index(BMI),obesity grade and Kellgren-Lawrence grade.2.The expression of FBXO21 in the knee articular cartilage of OA rats is elevated and is positively correlated with the severity of OA(1)Evaluation of OA model modeling: In the OA model induced by aging and MIA,the success of OA model modeling is confirmed by gross imaging,plain radiograph,MRI and gross imaging scores.The cartilage surface of the normal knee joint is smooth and complete,there is only a small amount of clear and non-sticky knee joint fluid in the joint cavity,the bone structure of the knee joint is intact,the knee cartilage surface is flat,and the knee joint space is normal.With the aggravation of OA,the cartilage surface is increasingly rough,fibrosis,cracks,and erosion to the subchondral bone,the joint fluid increases and becomes extremely viscous,and the joint space becomes narrow.Compared with the control group,the four groups of 12 M,18 M,MIA 2 week and MIA 3 week have significant statistical significance.(2)Western blot: The expression of FBXO21 and MMP13 gradually increased in the OA model induced by aging and MIA.On the contrary,the expression of COL2A1 decreased.FBXO21 was statistically significant in the four groups of 12 M,18 M,MIA2 week and MIA 3 week.(3)Immunohistochemistry: The expression of FBXO21 gradually increased in the aging OA model,on the contrary,the expression of COL2A1 decreased.Compared with the 1M control group,the expression changes of COL2A1 in the 6M,12 M and 18 M groups and FBXO21 in the 12 M and 18 M groups were statistically significant.3.The expression of FBXO21 in OA chondrocytes is elevated and is positively correlated with the severity of OA(1)Identification of rat primary chondrocytes: The identification was performed by alcian blue,safranin O,toluidine blue and COL2A1 immunohistochemical(IHC)staining,which proved that the cells were indeed rat primary chondrocytes.(2)The protein expression of FBXO21 is increased in the IL-1? treated OA model of rat primary chondrocytes,and FBXO21 is expressed in the cytoplasm and nucleus.(3)Western blot: FBXO21 expression gradually increased in three different OA models caused by IL-1?,TNF-? and LPS treatment.Compared with the control group,rat primary chondrocytes treated with 20ng/ml IL-1?,20ng/ml TNF-?,2?g/ml LPS and those treated with 10,20ng/ml TNF-?,1?g/ml LPS In SW1353 cells,the level of FBXO21 protein increased statistically.4.Knockdown of FBXO21 can delay the OA cartilage degeneration of rat cartilage treated with MIA(1)q PCR and Western blot: The expression of FBXO21 was significantly reduced,proving that the knockdown of FBXO21 was successful;The protective markers including COL2A1,Aggrecan,LC3 ?/?,Beclin1 and Bcl2/Bax were all significantly upregulated.The destructive markers,including MMP13,were significantly downregulated,except for the changes in MMP3 protein that were not statistically significant.(2)Gross imaging,plain X-ray,MRI and gross imaging scores: After knocking down FBXO21,the cartilage surface of the knee joint is rough,fibrosis,cracks and erosion until the subchondral bone is reduced,the viscous joint fluid is reduced,and the joint space narrowness is relieved.Moreover,compared with the control group,it has statistical significance in both the KD-01 and KD-02 groups.(3)Histological staining score: Safranin,Toluidine blue,Mankin and OARSI scores showed that after knocking down FBXO21,cartilage degradation was reduced,cartilage thickness increased,and disordered chondrocytes decreased,Mankin and OARSI scores were significantly reduced,with statistics significance.5.Overexpression of FBXO21 can promote OA cartilage degeneration in rat cartilage treated with MIA(1)q PCR and Western blot: The expression of FBXO21 was significantly increased,proving that the overexpression of FBXO21 was successful;protective markers including COL2A1,LC3 ?/?,Beclin1 and Bcl2/Bax were all significantly down-regulated,except for COL2A1 m RNA and Aggrecan in vivo protein.The destructive markers including MMP3 and MMP13 protein levels were significantly upregulated,except for changes in MMP13 m RNA in the body that were not statistically significant.(2)Gross imaging,plain X-ray,MRI and gross imaging scores: After overexpression of FBXO21,the surface of knee cartilage is rough,fibrosis,cracks and erosion until the subchondral bone increases significantly,viscous joint fluid increases,and joint space stenosis increases.Moreover,compared with the control group,the OEFBXO21 group had statistical significance.(3)Histological staining score: Safranin,Toluidine blue,Mankin and OARSI scores showed that after overexpression of FBXO21,cartilage degradation increased,cartilage thickness decreased,chondrocytes in disorder increased,Mankin and OARSI scores increased,with statistics significance.6.Knockdown of FBXO21 can delay the OA cartilage degeneration of rat chondrocytes treated by IL-1?(1)q PCR and Western blot: The expression of FBXO21 was significantly reduced,proving that the knockdown of FBXO21 was successful;protective markers including COL2A1,Aggrecan,LC3 ?/?,Beclin1 and Bcl2/Bax were all significantly upregulated,except for Bcl2/Bax in vitro protein.The destructive markers,including MMP3 and MMP13,were significantly down-regulated,with statistical significance.(2)Flow cytometry analysis: After knocking down FBXO21,the apoptosis rate of IL-1?-treated chondrocytes was significantly reduced,which was statistically significant.(3)m RFP-GFP-LC3 adenovirus double labeling and transmission electron microscopy: After knocking down FBXO21,autophagolysosomes(ALs)and autophagosomes(APs)with double membrane characteristics of IL-1?-treated chondrocytes were significantly up-regulated,with statistical significance.7.Overexpression of FBXO21 can promote OA cartilage degeneration in rat chondrocytes treated by IL-1?(1)q PCR and Western blot: The expression of FBXO21 was significantly increased,proving that the overexpression of FBXO21 was successful;protective markers including COL2A1,Aggrecan,LC3 ?/?,Beclin1 and Bcl2/Bax were all significantly down-regulated.The destructive markers including MMP3 and MMP13 were significantly up-regulated.(2)Flow cytometric analysis: After overexpression of FBXO21,the apoptosis rate of IL-1?-treated chondrocytes was significantly increased,which was statistically significant.(3)m RFP-GFP-LC3 adenovirus double labeling and transmission electron microscopy: After overexpression of FBXO21,autophagolysosomes(ALs)and autophagosomes(APs)with double membrane characteristics of IL-1?-treated chondrocytes were significantly reduced,which was statistically significant.8.FBXO21 interacts with ERK and phosphorylates it(1)Direct mechanism: Through proteomics analysis(LC-MS/MS),ERK peptide sequence was found in the IP complex,showing that FBXO21 may interact with ERK.The interaction between ERK and FBXO21 was confirmed by two-way immunoprecipitation.(2)Western blot: Overexpression of FBXO21 significantly increased the expression of phosphorylated ERK in IL-1?-treated rat chondrocytes.On the contrary,knocking down FBXO21 significantly reduced the expression of phosphorylated ERK,which was statistically significant.9.FBXO21 activates ERK through phosphorylation to inhibit IL-1? treatment of autophagy in rat chondrocytes(1)Western blotting and m RFP-GFP-LC3 adenovirus double labeling: The upregulation of LC3 II/I,Beclin1,APs and ALs mediated by FBXO21 knock down were both reversed after SD rat OA chondrocytes(20ng/ml IL-1? treatment)treated with ERK activator Honokiol,which was statistical significance.10.FBXO21 enhances the OA cartilage degeneration of rat chondrocytes treated by IL-1? by inhibiting autophagy(1)Western blot and flow cytometric analysis: The decreased of apoptosis,and the up-regulation of COL2A1 and Aggrecan expression mediated by FBXO21 knock down were both reversed after SD rat OA chondrocytes(20ng/ml IL-1? treatment)treated with autophagy inhibitor 3-methyladenine(3-MA),which was statistical significance.11.JUNB can bind to the promoter of FBXO21(1)FBXO21 transcription factor prediction: Use JASPAR database to predict that JUNB is a potential transcription factor of FBXO21(Homo sapiens).(2)Chromatin immunoprecipitation: On the IL-1?-treated SW1353 cell line,JUNB and FBXO21 promoter have direct target binding at the predictive binding site1,PCR2(96.43 times),which is statistically significant.12.JUNB promotes OA cartilage degeneration by activating the expression of FBXO21(1)Western blot: JUNB not only significantly increased the expression of damaged cartilage in patients with knee OA,but also was positively correlated with the expression of FBXO21 in damaged cartilage.(2)Western blot,m RFP-GFP-LC3 adenovirus double-labeling and flow cytometry experiment: On the IL-1?-treated SW1353 cell line,we observed that after overexpression of FBXO21,KD-JUNB-mediated MMP3 decreased,and the increase in autophagy level was significantly reversed,With statistical significance.In primary rat chondrocytes,we observed that after overexpression of FBXO21,the KD-JUNBmediated decrease in MMP3,and the increase in COL2A1,aggrecan and autophagy levels were significantly reversed,with statistical significance.Conclusion:1.The expression of FBXO21 in the articular cartilage of knee OA patients is significantly up-regulated,and is related to clinicopathological characteristics including Kellgren-Lawrence grade,BMI and obesity grade.2.The expression of FBXO21 in the articular cartilage of SD rats in the two OA models of MIA and aging is increased,and is positively correlated with the severity of OA,which is dependent on the treatment time gradient.3.The expression of FBXO21 in the chondrocytes of IL-1?,TNF-? and LPS three OA models is increased,and it is positively correlated with the severity of OA,which is treatment concentration gradient dependent.4.Injecting FBXO21 knockdown adenovirus into the knee joint cavity can delay the OA cartilage degeneration of rat cartilage treated by MIA,including increased cartilage matrix anabolism,decreased catabolism,increased autophagy,decreased apoptosis,and decreased cartilage destruction.5.Injecting FBXO21 over-expressing adenovirus into the knee joint cavity can promote the OA cartilage degeneration of rat cartilage treated by MIA.6.Knockdown of FBXO21 can delay the OA cartilage degeneration of rat primary chondrocytes treated with IL-1?,including increased cartilage matrix anabolism,decreased catabolism,increased autophagy and decreased apoptosis.7.Overexpression of FBXO21 can promote OA cartilage degeneration in rat chondrocytes treated by IL-1?.8.In IL-1?-treated rat primary chondrocytes,FBXO21 spatially interactive with ERK and activates ERK by phosphorylation.9.FBXO21 activates ERK through phosphorylation,thereby inhibiting the autophagy of IL-1?-treated rat primary chondrocytes.10.FBXO21 enhances the OA cartilage degeneration of rat primary chondrocytes treated by IL-1? by inhibiting autophagy.11.On the IL-1?-treated SW1353 cell line,JUNB and the FBXO21 promoter have a direct target binding at the predicted binding site 1(96.43 times).12.JUNB not only up-regulates in damaged cartilage of patients with knee OA,but also positively correlates with the expression of FBXO21 in damaged cartilage.12.JUNB accelerates the OA degeneration by activating the expression of FBXO21 in SW1353 cells and rat chondrocytes,including decreased cartilage matrix anabolism,increased catabolism,and decreased autophagy.14.In summary,the JUNB-FBXO21-ERK axis promotes cartilage degeneration by inhibiting autophagy in OA. |
PDF Full Text Request