P157 PROTEOMICDIFFERENTIAL EXPRESSION ANALYSIS OF OSTEOARTHRITIS-RELATEDPROTEINS IN HUMAN ARTICULAR CHONDROCYTES: ROLE OF HSP90 IN OSTEOARTHRITIS

Abstract

Purpose: To analyze, by a proteomic approach, the protein changes that are characteristic of OA chondrocytes, and identify new OA-related proteins. Methods: Chondrocytes were obtained from 10 OA patients undergoing joint replacement, and from 10 cartilages from autopsies without history of joint disease. Whole cell proteins were resolved by means of two-dimensional gel electrophoresis (2DE) and stained with SYPRORuby. Protein expression patterns of gels from OA and normal chondrocyte proteins were analyzed with PDQuest 7.3.1 software. OA-related proteins were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) or MALDI-TOF/TOF mass spectrometry. Western blot and immunohistochemistry analysis were performed with a polyclonal anti-Hsp90β antibody. Interleukin-1β (5 ng/ml) and Geldanamycin (1-1000 nM) were used to stimulate NO synthesis and inhibit Hsp90, respectively. Normal chondrocytes were treated with these compounds for up to 48 h prior to evaluation of NO production by the Griess method. Results: We examined an average of 700 protein spots that were present in the different 2-DE gels. Both qualitative and quantitative changes in protein expression patterns between normal and OA chondrocytes were studied. Fifty nine protein spots were found to be statistically increased or only present in OA cells compared to the pattern of normal chondrocytes (ratio OA:N ≥2.0, p<0.05), whereas 50 were decreased in OA chondrocytes or only present in normal cells (ratio OA:N ≤0.5, p<0.05). Many of these proteins were identified, and some of them are shown in Table 1. According to their cellular role, 30% of the over-expressed proteins were involved in cellular metabolism, 11% in cell signalling, 11% in protein targeting, 9% in protein synthesis or turnover, 6% in transport processes, and 23% of them were structural proteins. From the set of under-expressed proteins, 33% of them were implicated in metabolism, 20% in cell signalling, 17% in protein targeting, 8% in transport, 5% in protein synthesis or turnover and 20% were structural. Validation of the results was performed for the NOS positive modulator Hsp90β