Effect And Mechanism Of USP7 On Chondrocyte Proliferation,differentiation And Apoptosis

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USP7 regulates the proliferation and differentiation of chondrocytes through the Sox9-PTHr P-PTH1 R axis Objective Ubiquitination is a post-translational modification that maintains proteostasis and can be removed by deubiquitination under deubiquitinases.Ubiquitin-specific peptidase 7(USP7)is one of the most wildly studied deubiquitinases and regulates multiple cellular events and diseases.Endochondral ossification is an important healing method to repair bone fracture defects,especially huge bone defects,and it is a progress in which chondrocytes undergo proliferation,differentiation,apoptosis and finally are replaced by the bone tissue.Sex-determining region Y-box 9(Sox9)is necessary for chondrogenesis and regulates the various stages of endochondral ossification.In chondrocytes,Sox9 is degraded by ubiquitination but deubiquitinases inhibits its ubiquitination.Sox9 upregulates parathyroid hormone-related protein(PTHr P),and PTHr P/parathyroid hormone 1 receptor(PTH1R)and Indian hedgehog(Ihh)/Smoothened(Smo)form a negative feedback loop to regulate endochondral ossification.ATDC5 cells are commonly used to stimulate the proliferation and differentiation of chondrocytes in vitro,and are widely used to study the mechanisms of endochondral ossification.The aim of this study was to explore the regulatory effects of USP7 on the proliferation and differentiation of ATDC5 cells in vitro and in vivo,and the potential mechanisms involved.Materials and Methods1.q RT-PCR,Western Blot,and immunofluorescence staining were used to observe the expression of USP7 in ATDC5 cells after chondrogenic induction.Under USP7 knockdown and its inhibitor HBX41108,the expression of markers of chondrogenic and hypertrophic differentiation was assessed by q RT-PCR and Western Blot,the chondrocyte differentiation was assessed by alcian blue,toluidine blue and alkaline phosphatase(ALP)stainings,and chondrocyte proliferation speed was assessed by the CCK-8 assay and crystal violet staining.2.ATDC5 cells transfected with USP7-nc and USP7-sh2 lentivirus were seeded into the fibrin gel and transplanted subcutaneously into the dorsa of BALB/c nude male mice.Six weeks later,samples were harvested and observed.Histological stainings were used to observe cartilage-like tissue formation and immunohistochemistry stainings were used to observe the expression of markers of chondrocyte proliferation and differentiation.3.Under USP7 knockdown and its inhibitor HBX41108,the related markers of Ihh/Smo and PTHr P/PTH1 R signalings were assessed by q RT-PCR and Western Blot.Supplementation with the Ihh signaling antagonist cyclopamine,deletion of PTH1 R with si-PTH1 R,and supplementation with the PTHr P analogue abaloparatide were used to detect chondrocyte proliferation and differentiation by Western Blot and CCK-8 and verify the regulation of USP7 on Ihh/Smo and PTHr P/PTH1 R signalings.4.The interaction of USP7 protein and Sox9,PTHr P,or Ihh proteins were assessed by co-immunoprecipitation(Co-IP).The relationship between Sox9 and PTHr P was tested by chromatin immunoprecipitation and deletion of Sox9 with si-Sox9.Results1.USP7 was located in both the cytoplasm and nucleus of wild ATDC5 cells.After chondrogenic induction,the expression of USP7 was significant different.USP7 knockdown and its inhibitor HBX41108 suppressed chondrocyte proliferation and markers of chondrogenic differentiation but improved markers of hypertrophic differentiation.Histological stainings showed the same trend.2.The in vivo study of histological stainings showed that the USP7 knockdown group had less cartilage-like tissue formation.Immunohistochemistry stainings showed that chondrocyte proliferation and markers of chondrogenic differentiation decreased,but markers of hypertrophic differentiation increased in the USP7 knockdown group.The in vivo study obtained the same results as the in vitro study.3.USP7 knockdown and its inhibitor HBX41108 activated Ihh/Smo signaling,but inhibited PTHr P/PTH1 R signaling.Supplementation with cyclopamine suppressed Ihh/Smo and PTHr P/PTH1 R signalings,and inhibited ATDC5 cell proliferation and differentiation.Cyclopamine partly reduced the positive influence of USP7 knockdown in hypertrophic differentiation.Si-PTH1 R increased hypertrophy,but this was reversed by cyclopamine supplementation.Supplementation with abaloparatide activated PTH1 R to upregulate proliferation and chondrogenic differentiation but downregulated hypertrophic differentiation.Abaloparatide partly reversed the inhibition of chondrocyte proliferation and chondrogenic differentiation by USP7 knockdown.4.USP7 interacted with Sox9,but not PTHr P or Ihh protein.Si-Sox9 inhibited the expression of PTHr P and Sox9 protein bound to PTHr P gene promoter.Conclusion USP7 promotes ATDC5 cell proliferation and chondrogenic differentiation,but inhibits hypertrophic differentiation.Sox9 protein bounds to PTHr P gene promoter to activate PTHr P/PTH1 R signaling to exert these effects.Ihh/Smo promotes PTHr P/PTH1 R signaling to modulate chondrocyte proliferation and chondrogenic differentiation but Ihh/Smo enhances the hypertrophic differentiation independently of PTHr P/PTH1 R signaling.Part ? USP7 modulates TNF-? stimulated chondrocytes proliferation,apoptosis and inflammatory response through attenuating Bi P-e IF2?-ATF4-CHOP and NF-?B/p65 signaling Objective Inflammation has a dual effect on bone defect repair.Little and transient inflammatory stimulation promotes bone defect repair,but continuous and excessive inflammatory stimulation has the opposite effect.Osteoarthritis(OA)is the common chronic disease in articular cartilage,of which the main cell type is the chondrocyte.OA is characterized by extracellular cartilage matrix degradation,decreased number of chondrocytes,and increased chondrocyte apoptosis and inflammatory response.And in turn,chondrocyte apoptosis and inflammation accelerate the progression of OA.Studies of USP7 on chondrocytes under inflammation are still at the early stage.The abnormal activated binding protein(Bi P)-eukaryotic translation factor 2 alpha(e IF2?)-activating transcriptional factor 4(ATF4)-transcriptional factor C/EBP homologous protein(CHOP)signaling of endoplasmic reticulum stress and nuclear factor kappa-B(NF-B)/p65 signaling are positively related to the degree of cartilage destruction in OA.This study aimed to explore the effects of USP7 on ATDC5 cell proliferation,apoptosis and inflammatory response under TNF-?-stimulated inflammation,and investigate the underlying mechanisms.Materials and Methods1.Knee OA model of mice was constructed by the anterior cruciate ligament transection(ACLT).8 weeks after surgery,haematoxylin-eosin staining,Safranin O-Fast Green staining,and toluidine blue staining were used to verify the model construction.Immunohistochemistry staining was used to observe the expression of USP7.2.ATDC5 cells were cultured in chondrogenic medium for 48 hours with different concentrations of TNF-?.Alcian blue and toluidine blue stainings,and the CCK-8assay were used to detect chondrocyte proliferation.Western Blot,flow cytometry,and Caspase-3 activity were used to detect chondrocyte apoptosis.ELISA and q RT-PCR were used to detect inflammatory response.Proper concentration of TNF-?was selected to mimics inflammatory stimulation in vitro based on these results.3.Under USP7 knockdown and its inhibitor HBX41108 with proper concentration of TNF-? for 48 hours,chondrocyte proliferation was measured by histological stainings and the CCK-8 assay,apoptosis was measured by Western Blot,flow cytometry,Caspase-3 activity,and TUNEL staining,and inflammatory response was measured by q RT-PCR and ELISA.4.Under USP7 knockdown and its inhibitor HBX41108 with proper concentration of TNF-? for 48 hours,the related markers of Bi P-e IF2?-ATF4-CHOP and NF-?B/p65 signalings were measured by Western Blot and immunofluorescence staining.Under USP7 knockdown with proper concentration of TNF-?,supplementation with the endoplasmic reticulum stress inhibitor 4-phenylbutyric acid(4-PBA),deletion of CHOP with si-CHOP,and the p65 signaling inhibitor QNZ to detect chondrocyte proliferation,apoptosis and inflammatory response and to verify the regulation of USP7 on signalings under inflammation.Results1.Eight weeks after surgery,histological stainings showed that the OA group had less number of chondrocytes and decreased cartilage thickness,which indicated that knee OA model of mice was successfully constructed by ACLT.Immunohistochemistry staining showed that USP7 decreased in the knee articular cartilage of the OA group.2.ATDC5 cell proliferation decreased,but cell apoptosis and inflammation increased with increasing TNF-?.Considering that 20 ng/m L and 40 ng/m L TNF-? had no significant difference on chondrocyte proliferation and apoptosis,and the state of chondrocyte was poor under 40 ng/m L TNF-?,20 ng/m L TNF-? was selected in the following experiments to mimics inflammatory stimulation.3.Under inflammatory stimulation,USP7 knockdown and its inhibitor HBX41108 decreased ATDC5 cell proliferation and chondrocyte extracellular matrix formation,but upregulated apoptosis related proteins,apoptosis rate,Caspase-3 activity,TUNEL positive cells and inflammatory cytokines in chondrocytes and their supernatants.4.Under inflammatory stimulation,USP7 knockdown and its inhibitor HBX41108 activated Bi P-e IF2?-ATF4-CHOP and NF-?B/p65 signalings.Supplementary with4-PBA,si-CHOP,and QNZ partly reversed effects of USP7 knockdown on chondrocyte proliferation,apoptosis and inflammatory response.Conclusion Under TNF-?-stimulated inflammation,USP7 promotes ATDC5 cell proliferation,but suppresses apoptosis and inflammatory response.USP7 attenuates Bi P-e IF2?-ATF4-CHOP and NF-?B/p65 signaling to modulate chondrocytes under inflammation.