Proteomic Investigation On The Molecular Mechanism Underlying Osteoarthritic Chondrocyte Mal-differentiation

BackgroundCartilage defect repair is one of the most difficult problems for the medical society to conquer in osteoarthritis(OA) treatment. Numerous studies indicated that the osteoarthritic chondrocytes undergo dedifferentiation and abnormal redifferentiation, characterized by chondroctye reduced synthesizing normal differention phenotypic proteins, the type II collagen and aggrecan, while began to synthesize the abnormal terminal differentiation phenotypic protein, the type X collagen, and other abnormal redifferentiation phenotypic proteins, the typeⅠandⅢcollagen. The mal-differentiation status of chondrocytes existing in the OA pathologic microenvironment is believed to be a reason for chondrocytes' functional inability to repair pericellular matrix and subsequential self-repair failure of defected cartilage.2-dimensional gel electrophoresis(2-DE), as an efficient technique used for differentially expressed proteins screening, was used to investigate the molecular mechanisms underlying OA chondrocyte mal-differentiation. However, there were many obstacles and challenges in applying the 2-DE technique. Interference of the proteoglycan and glycosaminoglycans(GAGs) in gel focusing and the dedifferentiation of cultured chondroytes are the two main issues associated with the 2-DE-technique.ObjectivesThe objective of this study is to optimize a feasible scheme capable of avoiding the interference of proteoglycan and GAGs in gel focusing, and avoiding the lost of pathologic phenotype and phenotype modulation molecules, so as to use the 2-DE technique to explore the molecular mechanism underlying osteoarthritic chondrocyte mal-differentiation. Based on the differentially expressed proteins isolated by the 2-DE technique, further studies were undertaken to validate the differential expression status and to understand the impacts of the downregulation of a newly isolated protein, REST corepressor 1(CoREST1) on chondrocyte phenotype modulation.MethodNormal articular cartilage(NC) was obtained from the femoral condyle or head of 20 patients that was diagnosis with uncurable severe leg trauma or femoral neck fracture that had performed amputation or half/total hip replacement. Osteoarthritic cartilage was obtained from 19 patients that was diagnosis with OA in the knee and had undergo a total knee replacement. Outbridge classification system was used to categorize cartilage samples from OA condyles into Outbridge classificationⅠ(OAⅠ) and Outbridge classificationⅡ(OAⅡ) and normal cartilage into Outbridge classification 0 control(NC). Chondrocytes were isolated by enzyme digestion in 0.1% hyaluronidase and 0.2% collagenaseⅡDMEM solution with constant agitation at 37℃for 4 hours. Chondrocyte number was counted and every 1×106 cells were centrifuged to form a pellet for liquid nitrogen frozen storage. Pellets of 1×107 chondrocytes from 10 patients were pooled and soluble protein fraction was extracted. Protein concentration was quantified by the modified Bradford's dye-binding assay and 100μg protein solution was loaded in a gel strip for 2-DE analysis.2-DE gels were silver stained to colorate the isolated protein spots. Gel images were scanned and input into the gel image analyzing software ImageMaster 2-D Platinum(V3.0) to compare protein expression difference and screen for the differentially expressed protein spots. The differential expressed protein spots were cut off and digested for mass spectrometry(MS) analysis to understand corresponding protein identities. Each protein were identified and confirmed by checking the biological information provided by MS and location of the corresponding protein spot in gel images.The differentially expression profile of one of the differentially expressed proteins, the REST corepressor 1(CoREST1) between normal and OA chondrocytes, was confirmed with real-time RT-PCR technique. SiRNA interference was subsequentially performed to knock-down the mRNA expression level of CoREST1 to mimic CoREST1 downregulation in osteoarthritic chondrocytes. The siRNA with the highest CoREST1 gene knock-down efficiency was selected and the optimal gene knock-down reaction parameters were explored. After CoREST1 knock-down, the expression level of normal differentiation phenotypic genes(the typeⅡcollagen and aggrecan), terminal differentiation phenotypic gene(the type X collagen) and the pathologic redifferentiation phenotypic gene towards fibroblast (the typeⅠcollagen) were compared with that before CoREST1 knock-down. RNA in situ hybridization was subsequentially carried out to observe the distribution difference of CoREST1 gene expression in normal and OA cartilage.ResutlsPerfect 2-D gel focusing was achieved and high-quality gel images were generated.135 protein spots were screened to have been differentially expressed and 31 protein spots were identified by MS. Aside from those reported to have pathologic phenotypic molecules such as collagenⅥ, Annexin A2 and neuropolypeptide h3 in OAⅡ, numerous molecules functionally correlated with chondrocyte phenotype modulation were detected. Among them, human protein disulfide isomerase (PDⅠ), procollagen-proline,2-oxoglutarate 4-dioxygenase(P4H2), TGF-β2 were upregulated in OAⅡ, while mitogen-activated protein kinase kinase kinase 7(MAPKKK7) and REST corepressor 1 (CoREST1) were downregulated.CoREST1 mRNA level was confirmed to be downregulated by 45.3%(Mann-Whitney U test, Z=-2.382, P=0.016) and 49.6%(Mann-Whitney U test, Z=-2.648, P=0.005) respectively in OAI and OAⅡcompared to that of normal cartilage with real-time RT-PCR, which showed significant difference between normal and OA chondrocytes. The CoREST1 gene expression level in the primary cultured chondrocytes was significantly knocked-down by meanly 95.7% compared to those without siRNA interference. After CoREST1 knock-down, the mRNA level of collagenⅡand aggrecan was downregulated by 83.0%(T test, P＜0.01) and 50.6%(T test, P<0.01) respectively. The fibroblast marker gene, collagen I was also suppressed by 91.0%(T test, P<0.01). However, the chondrocyte terminal differentiation marker gene, collagen X was upregulated by 43.7%, which showed no significant difference with the CoREST1 knock-down (T test, P=0.066).RNA in situ hybridization observations and analysis revealed that the CoREST1 gene expression level was reppressed in both OAI and OAⅡcartilage. However, CoREST1 was downregulated the most severely maily in the middle layer of Outerbridgel and deep layer of Outerbridge 2 cartilage.Conclusions(1) We successfully optimized the 2-DE scheme for investigating the molecular mechanism of osteoarthritic chondrocyte phenotype mal-differetiation by using soluble protein samples directly from freshly isolated chondroctyes. This scheme eliminated both the interferences of proteoglycan and GAGs on 2D gel focusing and cell culture induced phenotype loss.(2) Pathologic phenotypic molecules such as annexin A2, neuropolypeptide h3 and unregulated collagen VI expression in OAII chondrocytes verified previous reports on OA chondrocyte phenotype abnormality.(3) The expression patterns of osteoarthritic chondrocyte phenotype regulating factors demonstrated disordered mechanisms in osteoarthritic chondrocyte phenotype modulation.(4) We discovered that the downregulation of CoREST1 in OA chondroctyes and demonstrated that CoREST1 was involved in the modulation mechanism of osteoarthritic chondrocytes mal-differentiation by triggering articular chondrocyte dedifferentiation. This dedifferentiation effect driven by CoREST1 was not accompanied by a terminal differentiation or a redifferentiation towards fibroblast phenotype.(5) CoREST1 downexpression mainly existed in the middle layer of Outerbridgel and deep layer of Outerbridge 2 OA cartilage, which indicated that chondrocyte dedifferentiation level was inhomogenous in different cartilage regions.