The Mechanism About The Mutated HDAC4 Inhibiting Chondrocytes Hypertrophy And Promoting Proliferation

Objective:Osteoarthritis(OA)is one of the major diseases that cause functional disability and economic burden in middle-aged and elderly people.In recent years,the incidence of OA has increased year by year,and the age of onset has gradually become younger.However,the clinical treatment of OA is still limited to symptomatic treatment,such as relieving joint pain,joint swelling and other symptoms.In severe cases,only joint replacement surgery can be performed.There is no effective method for repairing cartilage damage.Cartilage tissue engineering is one of the key research areas in the research of articular cartilage injury repair.It can be cultured and expanded in vitro by a fewer chondrocytes,and then attached to a certain scaffold material to be transplanted into the Cartilage defect to form a new vital cartilage tissue.Cartilage damage is repaired.However,it was found that in the process of multiple subcultures,the chondrocytes gradually became hypertrophy and lost their phenotype,and the proliferative ability was also significantly reduced,which could not produce normal cartilage matrix and lose the ability to form cartilage.Therefore,effectively inhibiting chondrocytes hypertrophy and chondrocytes promoting proliferation in the process of in vitro expansion is one of the key problems to be solved in cartilage tissue engineering.Histone deacetylase 4(HDAC4)is a class II histone deacetylase(HDACs)that is mainly distributed in brain,muscle and cartilage tissues.The studies found that HDAC4 can inhibit chondrocytes hypertrophy by inhibiting the transcriptional activity of Runx-2(a key gene for chondrocyte hypertrophy),which plays a central role in chondrocyte hypertrophy,skeletal development and OA.It is worth noting that our previous results showed that HDAC4 not only inhibits chondrocyte hypertrophy,but also promotes chondrocyte proliferation.However,only HDAC4 can enter the nucleus to function effectively.Studies have confirmed that Caspase-3 can induce the degradation of HDAC4,and14-3-3 protein can bind to HDAC4 in the cytoplasm,which is the main reason for reducing HDAC4 to enter into the chondrocyte nucleus.Therefore,we hypothesized that the mutant Caspase-3 degradation site and the 14-3-3 binding site can increase HDAC4 in the chondrocyte nucleus,which will play a key role in inhibiting chondrocyte hypertrophy and promoting chondrocyte proliferation,in turn optimizes the seed cells(chondrocytes)of cartilage tissue engineering.The main objective of this study was to investigate whether HDAC4 mutated with Caspase-3 cleavage site and 14-3-3 protein binding sites can inhibit chondrocyte hypertrophy,maintain normal function of chondrocytes and promote chondrocyte proliferation more effectively,in turn optimize cartilage tissue engineering seed cells(chondrocytes),At the same time the research preliminaryly study the mechanism of HDAC4 promoting chondrocytes proliferation.Methods:1.Constructed RFP-HDAC4-GFP and RFP-AD-S246/467/632A/A289E-HDAC4-GFP adenovirus,and transfected them in human chondrocytes cultured in vitro with Caspase-3 inhibitor.48 hours after transfection,detected whether the mutation of HDAC4 Caspase-3 cleavage site 298 in the mutant group was successful by Western blot.2.Detected whether the HDAC4 14-3-3 protein binding site 246/467/632 was successfully mutated by immunoprecipitation.3.The human chondrocytes were divided into : empty virus group,wild-type HDAC4(WT-HDAC4)group,WT-HDAC4+Caspase inhibitor and mutant HDAC4(M-HDAC4)group.At 48 hours after transfection,the HDAC4 subcellular localization was observed by fluorescence microscopy and western blot.4.The chondrocyte hypertrophy markers were detected by western blot and realtime-PCR: RUNT-related transcription factor 2(Runx-2),type X collagen and metalloproteinase-13(MMP-13)and chondrocyte function markers: type II collagen,Sox-9 and aggrecan.CCK-8 and western blot for detection of chondrocyte proliferation,observed the function of mutated HDAC4 in inhibiting chondrocyte hypertrophy and promoting chondrocyte proliferation.5.The interaction between HDAC4 and PCNA protein in each group was detected by co-immunoprecipitation.ATDC5 chondrocyte line was cultured with different lengths of HDAC4 fragment plasmids with GFP(1-289,1-326,1-669,629-1040 and full-HDAC4),and the interaction site of HDAC4 with PCNA was detected by co-immunoprecipitation.Results:1.The Caspase-3 cleavage fragment(N-HDAC4)was significantly reduced in the M-HDAC4 group,the mutation in Caspase-3 cleavage site(289)of HDAC4 can reduce significantly the cleavage of HDAC4.2.The mutation of 14-3-3 protein binding site 246,467,632 in M-HDAC4 can effectively repress the interaction between HDAC4 and 14-3-3 protein.3.Compared with the WT-HDAC4 group,at 48 h after transfection,the expression of HDAC4 in the nuclear in M-HDAC4 group was increased significantly(***P<0.001,n=3).4.Compared with the WT-HDAC4 group,the m RNA level of chondrocyte hypertrophy markers Runx-2,Type-X-collagen and MMP-13(*P<0.05,n=6)were significantly decreased in M-HDAC4 group.5.Compared with the WT-HDAC4 group,the protein level of chondrocyte hypertrophy markers Runx-2(***P<0.001,n=3),Type-X-collagen(*P<0.05,n=3)and MMP-13(**P<0.01,n=3)were significantly reduced in M-HDAC4 group.6.Compared with the WT-HDAC4 group,the m RNA level of chondrocyte function markers Type ? collagen,Aggrecan and Sox-9(*P<0.05,n=6)were significantly increased in M-HDAC4 group.7.Compared with the WT-HDAC4 group,the protein level of chondrocyte function markers Type ? collagen(***P<0.001,n=3),Aggrecan(*P<0.05,n=3)and Sox-9(**P<0.01,n=3)were significantly increased in M-HDAC4 group.8.The proliferation assy of CCK-8 showed that compared with the WT-HDAC4 group,the proliferation of chondrocytes in the M-HDAC4 group were significantly increased at 24 h(**P <0.01,n=3),48 h(*P <0.05,n= 3),72h(***P<0.001,n=3),5d(***P <0.001,n=3)after transfection.Western blot results showed that compared with the WT-HDAC4 group,PCNA protein protein was significantly increased in M-HDAC4 group(**P <0.01,n=3).9.The co-immunoprecipitation results showed that there are direct interaction between HDAC4 protein and PCNA.And the binding ability of M-HDAC4 is stronger than WT-HDAC4..10.Binding site detection results show that endogenous PCNA in chondrocytes can bind to 1-669,629-1040 and full-HDAC4,but not to 1-289,1-326 HDAC4 fragment,suggesting that the binding site of HDAC4 to PCNA minght be between 326 and 669.Conclusion:1.Mutation of the Caspase-3 cleavage site and the 14-3-3 binding sites of HDAC4 can inhibits effectively chondrocyte hypertrophy,maintains the normal function of chondrocytes and promotes chondrocyte protliferation.2.The binding between HDAC4 and PCNA might be related to the mechanism of HDAC4 promoting chondrocytes proliferation;the binding site might be between HDAC4 326 and 669 in HDAC4.