Effect And Mechanism Of DDIT3/CHOP On Autophagy In ATDC5 Chondrocytes

Objective: The fate and function of chondrocytes are crucial to endochondral ossification and cartilage development.Under physiological condition,chondrocytes synthesize abundant extracellular matrix and may be sensitive to ER stress.ER stress is also implicated in the pathology of cartilage and bone related diseases.Additionally,autophagy is an important metabolic pathway to maintain cellular homeostasis.Upon ER stress,autophagy can be activated to cope with protein-folding challenges.Autophagy has been considered to play a positive role in chondrocyte proliferation,differentiation as well as extracellular matrix metabolism.These suggest that there may be crosstalk between ER stress and autophagy in chondrocytes.DNA damage-inducible transcript 3(DDIT3)/ C/EBP homologous protein(CHOP)is a typical ER stress marker.Our study previously showed that DDIT3/CHOP was expressed in the mouse growth plate as well as ATDC5 chondrocytes.However,whether DDIT3/CHOP can regulate autophagy in chondrocytes remains unclear.In this study,the relationship between DDIT3/CHOP and autophagy was evaluated in the tibial growth plate and costal primary chondrocytes from wild-type and DDIT3 KO mice.Then the DDIT3 overexpression and knockdown chondrogenic ATDC5 cells were used to study the regulation mechanism of DDIT3/CHOP on autophagy,which may offer new strategies for skeletal disorders therapy.Materials and methods: 1.Purchased DDIT3+/-mice with the genetic background of C57BL/6J were mated with wild-type mice to breed more heterozygous mice and then generate DDIT3-/-(DDIT3 KO)mice by self-fertilization.The genotyping was identified by PCR analysis.The expression of autophagic markers in chondrocytes from tibial growth plate of DDIT3 KO and wild-type mice were detected by immunohistochemistry staining.Primary costal chondrocytes were isolated and Western blot was applied to evaluate the expression of autophagy-related proteins.2.The lentivirus vector system was applied to establish ATDC5 cells with DDIT3/CHOP overexpression and knockdown.Infection efficiency was analyzed by q RT-PCR and Western blot.The effect of DDIT3/CHOP overexpression and knockdown on the expression of autophagic markers were detected by Western blot.Immunofluorescence staining was applied to detect the LC3 B positive cells.TEM was applied to identify the formation of autophagosomes and autolysosomes.3.CHIP was applied to detect the interaction between DDIT3/CHOP and SIRT1 in wildtype ATDC5 cells treated with tunicamycin for 12 h.q RT-PCR and Western blot were used to detect the role of DDIT3/CHOP on SIRT1 expression.4.SIRT1 expression in ATDC5 cells after transiently transfection was evaluated by q RTPCR and Western blot.The expression of autophagic markers were detected by Western blot.Immunofluorescence staining was applied to detect the LC3 B positive cells.Cells were observed by TEM to identify the formation of autophagosomes and autolysosomes.5.DDIT3 overexpression ATDC5 cells were transfected with si RNAs that targeting SIRT1.p LVX and DDIT3 overexpression groups were treated with SIRT1 inhibitor ex-527 or AKT activator SC79 for 24 h.DDIT3 knockdown cells were transiently transfected with SIRT1 overexpression plasmids.The expression of autophagy-related proteins,p-AKT and AKT were detected by Western blot.Results: 1.WT and DDIT3-/-(DDIT3 KO)mice were successfully generated.IHC showed that the expression of autophagic markers were decreased in the tibial growth plate from DDIT3 KO mice.Western blot demonstrated that DDIT3/CHOP knockout inhibited the autophagic activity of chondrocytes.2.The DDIT3/CHOP overexpression and knockdown ATDC5 cells were established and the transfection efficiency was satisfactory.Western blot demonstrated that DDIT3 overexpression promoted autophagic activity of ATDC5 cells,however,the opposite trends were observed in DDIT3 knockdown cells.Immunofluorescence results showed that DDIT3 overexpression group presented significantly more LC3 B positive cells than the control group.An increased number of autophagosomes and autolysosomes were observed in DDIT3 overexpression cells.3.CHIP analysis revealed that DDIT3/CHOP directly bound to the SIRT1 gene promoter.q RT-PCR and Western blot confirmed the positive regulation of DDIT3/CHOP on SIRT1.4.Western blot indicated that overexpressing SIRT1 promoted autophagy in ATDC5 cells.And more LC3 B positive cells were observed in SIRT1 overexpression group by immunofluorescence staining.TEM showed that SIRT1 overexpression promoted the formation of autophagosomes and autolysosomes.5.The promoted autophagic activity due to DDIT3 overexpression was partly reversed after inhibiting SIRT1 expression or enzymatic activity.The inhibited autophagy of DDIT3 knockdown cells were reversed after overexpressing SIRT1.The AKT signaling was inhibited in DDIT3 overexpression cells,while the opposite trend was observed in DDIT3 knockdown cells.After the incubation of AKT agonist SC79,the promoted autophagic activity due to DDIT3 overexpression was partly reversed.Inhibition of SIRT1 activated AKT signaling in the DDIT3 overexpression cells.Conclusion: This study demonstrated that DDIT3/CHOP promoted autophagy in ATDC5 chondrocytes.The mechanism might be related to the inhibition of AKT signaling via DDIT3/CHOP directly binding to the SIRT1 gene promoter.These results offer a novel insight into the interaction between ER stress and autophagy in chondrocytes as well as contribute to existing knowledge of chondrocyte metabolism and new strategies for skeletal disorder therapy.