A22 WHOLE DEGRADOME EXPRESSION PROFILING IN NORMAL AND OSTEOARTHRITIC CARTILAGE

Purpose: Cartilage destruction in osteoarthritis (OA) is thought to be mediated by extracellular proteases. The main focus of research to date has been on the role of two enzyme families; the matrix metalloproteases (MMPs) with respect to cartilage collagen breakdown, and enzymes from the ‘a disintegrin and metalloprotease domain with thrombospondin motifs’ (ADAMTS) family with respect to cartilage aggrecan loss. It is now known that there are around 561 proteases in the human genome (the ‘degradome’): 21 aspartyl, 148 cysteine, 186 metallo-, 178 serine and 28 threonine proteases. The expression of the vast majority of these genes in cartilage is unknown. We have therefore profiled the complete degradome in cartilage from patients with either OA or fracture to the neck of femur (NOF), giving a more complete picture of proteolysis in this tissue and disease. Methods: Cartilage was collected at total hip replacement surgery and snap frozen in liquid nitrogen. Cartilage was ground under liquid nitrogen in a freezer mill and RNA purified by a combination of TRIzol (Invitrogen) and RNeasy (Qiagen). RNA was reverse transcribed and gene expression assessed by quantitative real-time PCR using Taqman low density arrays on the ABI Prism 7900. Results: Expression of 560 proteases and inhibitor genes was detectable in cartilage using this methodology. Preliminary analysis shows that approximately 197 proteases are differentially expressed in OA cartilage compared to NOF at a statistical significance of p< 0.05 (79 metallo-, 50 serine-, 41 cysteine-, 6 aspartyl-, 2 threonine proteases and 19 protease inhibitors). At a significance of p< 0.0001, there were 3 aspartyl, 14 cysteine, 15 metallo-, 12 serine proteases and 5 inhibitors which are differentially regulated in OA. Conclusions: This is the most comprehensive analysis of protease gene expression in cartilage to date. It has identified several protease genes not previously reported to be expressed or regulated in cartilage and provides the foundation on which to build functional studies.