Effect And Mechanism Of ERN1 Regulating Mouse Cartilage Growth And Development

Objective:The cartilage tissue conditional knockout mouse model of ERN1?endoplasmic reticulum to nucleus signaling 1?was propagated by Cre/LoxP system,the growth and development phenotypes of mice at different time points were observed and studied,the relationship between ERN1 gene and cartilage growth and development was further clarified,and the mechanism of ERN1 regulating cartilage growth and development in mice was preliminarily discussed,which is helpful to analyze the new regulation mechanism of ERN1 and cartilage development,and provide experimental basis for the subsequent development of biological agents involved in cartilage formation and repair.Methods:Using Cre/LoxP system,flox positive heterozygote micewithout Neo and Flp(ERN1^flox/+)were mated with Col2-Cre mice to obtain flox positive and Col2-Cre positive mice(Col2-Cre;ERN1^flox/+)and flox positive and Col2-Cre negative mice(ERN1^flox/+).Then Col2-Cre;ERN1^flox/+mice were self-bred or mated with ERN1^flox/+mice.Finally,ERN1 gene conditional knockout mice model(Col2-Cre;ERN1^flox/flox,Abbreviated as:ERN1CKO)was obtained and its genotypes were identified.The cartilage and other tissues of the experimental group?ERN1 CKO?and the control group(ERN1^flox/flox)were extracted from the same litter,and PCR and Western blot were used to verify whether the construction was successful.The body length and femur length of experimental mice and control mice at different stages were measured and paraffin sections were made for HE staining and SafraninO-Fast Green Staining to observe the developmental phenotype of mouse growth plate.Western blot and RT-PCR were used to detect the expression of chondrodevelopment-related marker genes in primary chondrocytes and cartilage tissue,immunohistochemistry?IHC?was used to verify the expression of hypertrophic chondrocyte marker genes in mouse growth plate.Packaging ERN1 and its different domain adenoviruses,RT-PCR was used to detect the effect of ERN1 on chondrocyte differentiation.Western blot was used to detect the expression of endoplasmic reticulum stress related marker genes in primary chondrocytes and C28I2 cells.The relationship between ERN1 and autophagy was detected by immunofluorescence in C28I2 overexpression and knockdown ERN1;ERN1 binding protein CHERP,in chondrocytes was screened by mass spectrometry.CHERP recombinant plasmid was constructed by LIC method,and the interaction between ERN1 and CHERP was verified by CO-IP.The relationship between ERN1 and CHERP and their combination in regulating autophagy was identified by overexpression and knockout of ERN1.Results:Genomic PCR and Westernblotting results indicated that ERN1 CKO mice had knocked out ERN1 in cartilage tissue,and ERN1 was normally expressed in other tissues,which proved that the conditional knock out model of ERN1 cartilage tissue was successfully constructed.At 4,8 and 12weeks,the body length and femur length of ERN1 CKO mice were longer than those of control mice?P<0.05?.HE and SafraninO-Fast Green Staining showed that the structure of cartilage growth plate of two kinds of mice was normal at embryonic stage?E15,E17,E19?.At 4 and 8 weeks,the total length of tibial growth plate,the length of hypertrophic area and the proportion of hypertrophic area in ERN1 CKO mice were higher than those in control mice.The results of primary chondrocyte mRNA level and cartilage tissue protein level showed that the expression of hypertrophic chondrocyte marker genes COL10A1 and MMP13 was up-regulated in ERN1 CKO mice.In addition,IHC suggested that the expression of COL2A1,Aggrecan and Ki67 was lower than that of control mice.Then,ERN1 and its different domain adenoviruses were successfully packaged,and the over-expression of ERN1 recombinant adenovirus inhibited chondrocyte differentiation.TM can induce endoplasmic reticulum stress in C28I2 human chondrocytes and normal mouse primary chondrocytes,but can not be activated in ERN1 CKO mouse primary chondrocytes.Overexpression of ERN1 recombinant plasmid in C28I2 cells can promote autophagy,while knocking down ERN1 can inhibit autophagy.The eukaryotic expression plasmid CHERP was successfully constructed and the interaction between ERN1 and CHERP was confirmed.To determine the regulatory relationship between ERN1 and CHERP and the effect of their combination on autophagy.Conclusions:?1?The Cre/Loxp system was used to successfully obtain ERN1 cartilage tissue-specific knockout?conditional knockout,cKO?mice.Phenotype analysis of the mice showed that ERN1 cKO mice resulted in abnormal growth and development of mouse cartilage;?2?Overexpression of ERN1 inhibits chondrocyte differentiation and promotes autophagy,ERN1 deficiency can not activate ER stress and autophagy normally in vitro;?3?ERN1 participates in chondrocyte differentiation and cartilage development by combining CHERP,regulating ER Stress and autophagy activation through the fusion of autophagic vesicles and lysosomes.