Proteomics Analysis Of Human Articular Cartilage Differential Expression Protein In Knee Osteoarthritis

Objective. Osteoarthritis (OA) is one of the most common cause ofmusculoskeletal disability related to aging and is characterized by progressivedegeneration of articular cartilage. Articular cartilage is mainly composed ofextracellular matrix (ECM) and a single type of cells called chondrocytes.Chondrocytes are important in control of cartilage integrity through productionand secretion of collagen, proteogiycans and enzymes for cartilage metabolism.ECM is a narrow tissue region surrounding chondrocytes in articular cartilage.This region has been shown to be rich in collagen (typeⅡ,Ⅵ,ⅨandⅪcollagen), proteoglycans (including aggrecan, fibromodulin, lumican anddecorin) and noncollagenous proteins(e.g, cartilage oligomeric matrixprotein(COMP), cartilage matrix protein(CMP), cartilage intermediate layerprotein(CILP), chondroadherin and fibronectin). Many of these componentsplay a structural role, while others represent degradation products which maybe involved in regulating chondrocyte function. There is a clear relationshipbetween biochemical composition of the cartilage ECM and susceptibility toOA.Nevertheless, little is known about the molecular mechanism that controlmatrix turnover in articular cartilage in OA. Recently, changes in thetranscriptome of OA cartilage were studied using DNA microarray analysis. However, alterations in level of mRNAs are not always well correlated withthe level of proteins due to post-transcriptional regulation, post-translationalmodification and differential stability of the proteins.A proteomics approach, in which entire proteins in tissue or cells areidentified and quantified directly, has been shown to be a valuable way to bringinsight into the molecular basis of disease etiology. More recently, theproteome of human normal articular chondrocytes was characterized bytwo-dimensional polyacrylamide gel electrophoresis (2-DE) and tandem massspectrometry (MS/MS) of chondrocytes isolated from normal cartilage. Indeed,these studies have improved our knowledge of the proteome of normalchondrocytes, and lay the ground work for investigation of pathologies ofcartilage diseases. A similar approach was used to study the secretory profile ofexplanted OA cartilage in culture medium. This study showed that synthesis oftypeⅡcollagen was strongly up-regulated in OA cartilage and furthermoreidentified potential cartilage-specific regulatory molecules. These findings,however, may be limited by the fact that tissues or cells in culture do notalways maintain in vivo characteristics. For example, dissection andexplantation can activate extracellularly regulated kinase pathways whichotherwise are sequestered.To better understand the mechanisms involved in the progressive destructionof articular cartilage in OA and to obtain insight into the molecular basis of theetiology of OA, here we studied the profile of proteins extracted from non-cultured human articular cartilage samples of normal or OAjoints by 2-DEand MS/MS.Method. Proteins extracted from either normal or OA knee joint cartilageswere separated by two-dimensional gel electrophoresis (2-DE). Thedifferentially expressed proteins in OA cartilages were chosen to be furtheridentified by linear iron trap tandem mass spectrometry (LTQ-FT/MS).Results. A total of 1436±49 or 1472±7 protein spots were resolved by 2-DEof normal or OA cartilage extractions, respectively. 16 spots from OA cartilagesamples were found to have statistically significant change amount of proteinscompared with normal samples. Of 16 spots, the identities of 14 proteins wereunambiguously determined by LTQ-FT/MS. These OA associated proteins fellinto 5 groups, including glycolysis and energy production (ADH, ADK, ENOA,KPYM and FR), signaling (ANNX-Ⅰ, PEBP and TUB), Redox (PRDX3 andSODM), and cartilage matrix (COLL-Ⅰand COLL-Ⅵ). Interestingly, two novelRING (Really Interesting New Gene) domain-containing proteins, RF, Zn-RF,were identified, suggesting novel pathways of cartilage protein regulation.Conclusion. This study shows that 2-DE followed by LTQ-FT/MS can besuccessfully used to characterize the proteome of cartilage without in vitroculturing which could obfuscate physiological differences. The definition ofunique OA-associated proteins described here provide significant mechanisticinsights into OA by corroborating previously suggested mechanisms and bydefining unique players with roles yet to be defined in disease pathogenesis.