| BackgroundOsteoarthritis(OA)is a degenerative joint disease associated with aging,and related to the destruction of articular cartilage.Once OA occurs,treatment methods are very limited,and the commonly used treatment measures are mainly to relieve the pain symptoms.At present,there is no drug that can interfere with the progression of OA.Therefore,it is particularly important to clarify the pathogenesis of OA,and then targeted early intervention.Some specific causes,such as trauma or neoplasms,can cause secondary OA.Primary OA may be related to age,gender,obesity,and other factors,but the mechanism is not clear,which brings difficulties for early intervention.Epidemiological studies show that OA mainly affects the elderly people;the incidence of premenopausal OA in women is similar to that in men.From the beginning of perimenopause,the incidence of OA in women is significantly higher than that in men.Traditionally,it is believed that the main cause of OA in postmenopausal women is the decrease of estrogen(E2)level.However,some clinical phenomena cannot be explained by the decrease of E2 level alone,suggesting that other factors may play roles.Studies found that in perimenopausal women,follicle-stimulating hormone(FSH)levels rise earlier than E2 levels fall.When E2 level was still at a relatively normal level,but FSH has been rises,the prevalence of OA began to increase significantly.In addition,our previous study have found that FSH is related to the severity of knee osteoarthritis in postmenopausal women and is an independent risk factor for OA.However,the effects of FSH on articular cartilage or osteoarthritis are poorly studied and need to be clarified.Based on this phenomenon,this study explores the effect of FSH on chondrocytes and seeks for the pathogenesis of postmenopausal OA.FSH is a glycoprotein hormone synthesized by pituitary,and its receptor FSHR is a seven-fold transmembrane G protein coupled receptor(GPCR).In general,when ligands bind to receptors on the cell membrane,they can activate the downstream second messenger through different G? proteins(G?s,G?i,G?q,and G? 12),acting on different reaction elements to perform functions.Traditionally,it was believed that FSH mainly plays a role in the gonadal axis and regulates follicle development and estrogen secretion by binding to FSHR on the ovary.According to our previous study,FSHR is expressed in human and mouse chondrocytes.We hypothesized that FSH affects specific Ga proteins by binding to FSHR and activates downstream signaling pathways,thus influencing chondrocytes.OA is a disease that affects the entire joint.Articular cartilage damage is existed through throughout OA.The only cell type is chondrocyte.They can produce extracellular matrix(ECM)components.The main component of ECM is collagen,which accounts for 60%of the dry weight.The main collagen component of articular cartilage is type ? collagen(Coll-?)?accounting for 90-95%of all collagens.Studies have shown that chondrocytes appear abnormal differentiation during OA.Chondrocytes can undergo dedifferentiation under some pathological conditions(such as inflammation and long-term culture in vitro).Since dedifferentiation of chondrocytes plays an important role in the occurrence and development of OA,it has gradually attracted public attention.The dedifferentiated chondrocytes are considered as naive chondrocytes.The dedifferentiated chondrocytes showed the characteristics of mesenchymal stem cells,such as the cell morphology changed from round to fibroblast phenotype.The dedifferentiation of chondrocytes was mainly characterized by decreased expression of Coll-?.Therefore,chondrocyte dedifferentiation can lead to the disappearance of chondrocyte phenotype,ECM remodeling,mechanical properties damaging and the occurrence of OA.Therefore,this study explored the effect of FSH on chondrocyte dedifferentiation by detecting the effects of FSH on Coll-? and cytoskeleton of chondrocytes.ERK1/2 MAPK and P38 MAPK signaling pathways have been shown involving in chondrocyte dedifferentiation and OA.Inhibition of p38 signaling pathway can prevent the hypertrophic differentiation of chondrocytes.Inhibition of ERK signaling pathway can increase the expression of Coll-?,and promote chondrocyte differentiation.Thus,ERK1/2 and p38 signaling pathways play opposite roles in regulating chondrocyte differentiation.This study aims to investigate the effect of FSH on chondrocyte differentiation by observing the effects of FSH on ERX1/2 and p38 signaling pathways.The roles of ERK1/2 MAPK and p38 MAPK signaling pathways in dedifferentiation were further clarified by using ERK 1/2 and p38 signaling inhibitors.The FSH antibody blocking mouse model is a classic model for investigating the loss of FSH effects.It has been adopted for studying osteoporosis and obesity after menopause.Studies have shown that blocking FSH by FSH antibody improves osteoporosis;improve the occurrence of obesity and hypercholesterolemia and in mice.In this study,we used FSH antibody to block FSH to observe whether blocking FSH could prevent knee cartilage degeneration and chondrocyte dedifferentiation in mice.Studies have shown that the superficial zone protein(SZP)or proteoglycan 4(Prg4)is decreased in the early stage of OA and is an early marker of OA.Early growth response protein 1(Egrl)is significantly increased in OA cartilage and regulates OA progression,which is considered as a marker of OA.We examined these two indicators to find evidence that FSH regulates postmenopausal OA.Based on the phenomenon that OA is increased in postmenopausal women,this study explored the effect of FSH on chondrocytes through basic studies in vivo and in vitro.In vitro studies using primary chondrocytes and ATDC5 chondrocytes showed that FSH promoted the dedifferentiation of chondrocytes.In vivo,FSH antibody was used to block FSH in the mouse model,and it was found that blocking FSH can prevent cartilage degeneration and inhibit chondrocytes dedifferentiation.Exploring its mechanism,it was found that FSH,by binding FSHR and coupling G?i protein.On the one hand FSH activates ERK1/2 and inhibits the p38 signaling pathway,on the other hand inhibits CREB-SOX9 signaling,eventually leads to the decrease of Coll-II expression and ECM remodeling.Our study reveals the effect of FSH on chondrocyte dedifferentiation,and established the signaling pathway of FSH regulating dedifferentiation,thus providing a supporting basis and therapeutic target for FSH affecting postmenopausal OA.Objectives:1.Using primary chondrocytes and ATDC5 chondrocyte lines to observe whether high FSH can cause chondrocyte dedifferentiation2.Using ERK1/2 and p38 pathway inhibitors,G?i protein inhibitors and FSHR small interfering RNAs to clarify the signaling pathway that FSH regulates chondrocyte dedifferentiation3.Tissue localization of FSHR was performed by detecting the expression of FSHR in articular cartilage.To investigate the role of blocking FSH in preventing articular cartilage degeneration,Coll-II expression,number of chondrocytes,cartilage thickness,OA score and OA marker expression of knee cartilage in FSHAb mice were detected.Methods:1.Cell culture:mouse primary cells and ATDC5 chondrocyte lines were cultured according to the culture conditions.Cells were starved to remove the serum hormones.2.Mouse model:9-week-old female C57BL/6J mice were divided into two groups:one group is FSH antibody(FSHAb)group and the other is IgG control group.Mice were intraperitoneally injected FSHAb and IgG antibody,respectively,for 8 weeks.3.CCK8 experiment was performed on primary chondrocytes:time gradients(24h,48h,72h)and concentration gradients(Ong/mL,10ng/mL,100ng/mL)were set to observe the effect of FSH on chondrocyte viability.4.Real-time quantitative PCR(qRT-PCR)assay:after the primary chondrocytes were stimulated by FSH(10ng),differentiation related genes were detected:Col2a1,acan,sox9,sox5,Mmp3,Mmp13,Coll0a1,Ihh,Pthlh.The effect on cell differentiation was observed.The effect of FSH on Prg4 and Egr1 of primary chondrocytes was detected to determine the effect of FSH on OA markers(Prg4,Egr1).5.Chondrocyte differentiation markers were then detected by Western blot(WB)assay6.Cellular immunofluorescence assay:to detect the expression of FSHR.The effect of FSH(30ng/mL)on Egrl and Prg4 was detected by cellular immunofluorescence in ATDC5 cells.7.Rhodamine phalloidin staining.After stimulating primary chondrocytes with FSH(0ng/mL,3ng/mL,10ng/mL,30ng/mlL).The expression of F-actin in cartilage cytoskeleton of ATDC5 cells was detected by Rhodamine phalloidin staining to determine the effect of FSH on cell morphology.8.Fshr siRNA interference experiment:3 pairs of siRNA(#1,#2,#3)were designed.After siRNA was transfected,the expression of Fshr was detected by qRT-PCR,and the most efficient one(#2)was selected for the subsequent experiment.Primary chondrocytes were transfected with#2 Fshr siRNA.9.cAMP assay:The expression of cAMP was detected after FSH stimulation of primary chondrocytes.The pertussis toxin(PTX)can interfere with the expression of G?i,and was used to determine the role of Gai.Forskolin can stimulate cAMP and is used as a positive control.Cayman's cAMP kit was used for detection.10.Immunohistochemical(IHC)assay.The expression of FSHR was detected by immunohistochemical(IHC)assay to determine the location of FSHR in articular cartilage.The expression of Coll-II and Coll-X was detected to determine the effect of FSH blockade on chondrocyte differentiation.Ki67 staining was used to determine the effect of blocking FSH on cell proliferation.Egr1 staining was used to determine the effect of blocking FSH on Egr1.11.Safranin O/fast green staining.The assay was used to distinguish cartilage and bone tissue,and to observe the damage of cartilage tissue.12.Mankin score and OARSI score were performed on the knee tissue sections of mice to analyze the injury and degeneration of articular cartilage.13.MicroCT was used to detect bone structure of mice.14.Statistical analysis:data were analyzed by GraphPad.The Mann-Whitney test after nonparametric test was used for comparison between two groups,and the Kruskal-Wallis test after nonparametric test was used for comparison between multiple groups.A p value less than 0.05 was considered statistically significant.Results:1.FSH did not affect chondrocyte apoptosis and proliferation CCK-8 kit was used to detect FSH at 0,10,100 ng at 24h,48h and 72h.Results shows that FSH did not affect chondrocyte proliferation and apoptosis.2.FSH promotes chondrocyte dedifferentiationqRT-PCR results showed that FSH affected the indexes related to chondrocyte differentiation.FSH mainly down-regulates chondrocyte differentiation indexes,such as Col2a1,Acan,Sox9 and Sox5 genes related to cartilage matrix synthesis.Mmp3,Mmp13,Coll0a1 genes related to hypertrophy;Ihh was down-regulated and pthlh was up-regulated,indicating FSH inhibits chondrocyte differentiation.Western blot results showed that FSH inhibited the synthesis of Coll-? and its transcription factor SOX9 in a dose-dependent manner.Besides,FSH inhibits phosphor-CREB,indicating that CREB/SOX9/Coll-II was affected.Rhodamine phalloidin staining shows that FSH effects cell morphology,chondrocytes changes from a round phenotype to fibroblast phenotype.3.FSHR expressed on ATDC5 chondrocyte cell lineWe have previously observed the expression of FSHR on primary chondrocytes,but whether FSHR expresses on chondrocyte lines has never been studied.Cell immunofluorescence showed that FSHR receptor was expressed on the cell surface after induction.4.FSH regulates chondrocyte dedifferentiation by binding FSHRInterference with Fshr by siRNAs was performed.After Fshr siRNA interfering,FSH downregulated Coll-? was reversed.5.G?i protein is involved in FSH regulated chondrocyte dedifferentiationThe cAMP kit was used to detect whether FSH affected the expression of the second messenger cAMP.The results showed that FSH inhibited the synthesis of cAMP,and the inhibition of G?i by PTX reversed the FSH-down-regulated cAMP and Coll-?,suggesting that G?i was involved in the FSH-regulated dedifferentiation of chondrocytes.6.ERK1/2 and p38 signaling pathways are involved in chondrocyte dedifferentiationFSH can up-regulate ERK1/2 but down-regulate the phosphorylation of p38.PD98059,an ERK1/2 inhibitor,can reverse FSH downregulation of Coll-?.FSH down-regulated Coll-? was promoted with p38 inhibitors.These results suggest that ERK1/2 and p38 have opposite regulatory effects on Coll-? and chondrocyte dedifferentiation.7.FSHR is located in the superficial zone of mouse knee cartilageImmunohistochemistry of tissue sections showed that FSHR is mainly located at the superficial zone(SZ)of articular cartilage.8.Mouse FSH antibody(FSHAb)blockade can improve trabecular parameters,prevent chondrocyte dedifferentiation and articular cartilage injuryFSH antibody blockade improves trabecular parameters,increases Coll-?expression,and prevents chondrocyte dedifferentiation.FSHAb improves chondrocyte loss,cartilage articular surface damage,and OA related scores such as Mankin Score and OARSI Score.Ki67-positive cells was increased after blocking FSH,suggesting that blocking FSH might be beneficial to the improvement of cartilage degeneration.9.FSH affects OA markersRecent studies have shown that Prg4 and Egr1 are markers of OA.We show that FSH can up-regulate the expression of Egrl and Egr1.Blocking FSH can decrease the expression of Egr1.Above results suggest that FSH may contribute to OA by affecting OA markers.Conclusions:1.FSH decrease cartilage anabolism and promotes chondrocyte dedifferentiation.2.FSH is involved in the reduction of Coll-? synthesis and promotion of chondrocyte dedifferentiation through binding of FSHR,which then couples G?i protein.On one hand FSH activates of ERK1/2 signaling and inhibits p38 signaling.On the other hand inhibits Sox9-CREB-Coll-?.3.Blockage of FSH can improve bone structure and improve a series of cartilage degeneration,such as chondrocyte dedifferentiation,chondrocyte injury and chondrocyte loss.4.Prg4 and Egr1 are important molecular mechanisms and targets of FSHAb in improving cartilage degeneration. |
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