Available online http://arthritis-research.com/content/8/4/R124 ResearchVol 8 No 4 article Open Access Expression profiling of metalloproteinases and their inhibitors in synovium and cartilage Rose K Davidson1, Jasmine G Waters1, Lara Kevorkian1, Clare Darrah2, Adele Cooper2, Simon T Donell2 and Ian M Clark1 1School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK 2Institute of Orthopaedics, Norfolk and Norwich University Hospital, Norwich NR4 7UY, UK Corresponding author: Ian M Clark, [email protected] Received: 24 May 2006 Revisions requested: 19 Jun 2006 Revisions received: 11 Jul 2006 Accepted: 14 Jul 2006 Published: 19 Jul 2006 Arthritis Research & Therapy 2006, 8:R124 (doi:10.1186/ar2013) This article is online at: http://arthritis-research.com/content/8/4/R124 © 2006 Davidson et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Cartilage destruction in osteoarthritis (OA) is thought to be The four most significantly upregulated genes (P < 0.0001) in mediated by two main enzyme families; the matrix OA synovium compared to the fractured NOF are MMP28, metalloproteinases (MMPs) are responsible for cartilage ADAMTS16, ADAMTS17 and TIMP2. For MMP9, MMP10, collagen breakdown, whereas enzymes from the 'a disintegrin MMP12, MMP17, MMP23, MMP28, ADAMTS4, and and metalloproteinase domain with thrombospondin motifs' ADAMTS9, there is a significant correlation between expression (ADAMTS) family mediate cartilage aggrecan loss. Tissue levels in the synovium and cartilage, suggesting similar inhibitors of metalloproteinases (TIMPs) regulate the activity of mechanisms of regulation. Additionally, we have shown that in these enzymes. Although cartilage destruction in OA might be cartilage the median level of steady-state mRNA for MMP13 is driven by the chondrocyte, low-grade synovitis is reported in approximately 20-fold higher than MMP28 and approximately patients with all grades of this disease. 1,500-fold higher than ADAMTS16, with expression of this latter gene approximately 150-fold higher in synovium than Our earlier work profiling these gene families in cartilage cartilage. identified a number of genes that are regulated in OA, which are hence implicated in the disease process. Because the synovium might contribute to cartilage-matrix destruction in OA, we have extended the screening in the current study. We have profiled MMP, ADAMTS and TIMP genes in both cartilage and This study is the most comprehensive analysis of the metzincin synovium from patients with either OA of the hip or a fracture to family of proteinases in the joint to date and has identified the neck of femur (NOF), giving a more complete picture of several proteinase genes not previously reported to be proteolysis in this disease. expressed or regulated in synovium. age is an important risk factor, will be an increasing health and economic burden on society. Introduction Osteoarthritis (OA) is a debilitating degenerative joint disease The molecular mechanisms underlying cartilage destruction in characterized by degradation of articular cartilage. Recent sta- OA are poorly understood (reviewed in [2]). Cartilage is made tistics show that approximately 5 million people in the UK suf- up of two main extracellular matrix (ECM) macromolecules: fer from moderate-to-severe OA. These patients are type II collagen and aggrecan, a large aggregating proteogly- predominantly older than 45 years of age, with the major mor- can [3,4]. The former endows the cartilage with its tensile bidity in patients over 60 years of age [1]. Given the current strength, whereas the latter enables cartilage to resist com- demographic trend towards an older population, OA, for which ADAMTS = a disintegrin and metalloproteinase domain with thrombospondin motifs; BLAST = basic local alignment search tool; CT = threshold cycle; ECM = extracellular matrix; Ki = inhibition constant; MMP = matrix metalloproteinase; MRI = magnetic resonance imaging; NOF = neck of femur; OA = osteoarthritis; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; RA = rheumatoid arthritis; TIMP = tissue inhibitor of metallo- proteinases. Page 1 of 10 (page number not for citation purposes) Arthritis Research & Therapy Vol 8 No 4 Davidson et al. pression. Quantitatively, more minor components (e.g. type IX, magnetic resonance imaging (MRI) reinforce the idea that syn- XI and VI collagens, biglycan, decorin and cartilage oligomeric ovitis is a frequent feature, even of early OA [25]. matrix protein) also have important roles in controlling the supramolecular organization of the matrix [3]. Normal cartilage We recently published a complete expression profile of all ECM is in a state of dynamic equilibrium, with a balance MMPs, the ADAMTS family and the four TIMPs in normal car- between synthesis and degradation. For the degradative proc- tilage compared with OA cartilage [26]. This identified a ess, there is a balance between proteinases that degrade the number of genes that are regulated in OA, which are hence ECM and their inhibitors. In OA, the dogma is that a disruption implicated in the disease process. Because the synovium of this balance, in favour of proteolysis, leads to pathological might contribute to cartilage-matrix destruction in OA, we have cartilage destruction. extended the screening in the current study. Hence, we have profiled MMP, ADAMTS and TIMP genes in both cartilage The matrix metalloproteinases (MMPs) are a family of 23 and synovium from normal and OA joints, giving a more com- enzymes in humans that facilitate ECM turnover and break- plete picture of proteolysis in this disease. down in physiology and pathology [5]. The MMP family con- tains the only mammalian proteinases that can specifically Materials and methods degrade triple helical collagens at neutral pH. These so-called Cartilage samples 'collagenases' specifically cleave a single locus in all three col- Human articular cartilage was obtained from femoral heads of lagen chains at a point three-quarters of the length from the N- patients undergoing total-hip-replacement surgery at the Nor- terminus of the molecule. The 'classical' collagenases (MMP- folk and Norwich University Hospital (Norwich, UK). Samples 1, MMP-8 and MMP-13) have differing substrate specificities from patients with OA (n = 16, of which 7 patients were female for collagens I, II and III, with MMP-13 showing a preference and 9 patients were male; age range, 61–84 years) were com- for type II collagen [6]. More recently, gelatinase A (MMP-2) pared with cartilage from patients undergoing hip replacement and MT1-MMP (MMP-14) have also been shown to make the following fracture to the neck of femur (NOF; n = 24, of which specific collagen cleavage, although with less catalytic effi- 19 patients were female and 5 patients were male; age range, ciency than the classical collagenases, at least in vitro [7,8]. A 52–92 years). OA was diagnosed using the clinical history second group of proteinases has been identified that affect and an examination, coupled with X-ray findings; confirmation ECM synthesis and degradation. The 'a disintegrin and metal- of gross pathology was made at time of joint removal. The frac- loproteinase domain with thrombospondin motifs' (ADAMTS) ture patients had no known history of joint disease and their family contains 19 members [9]; these include enzymes cartilage was free of lesions; 80% of these patients underwent involved in collagen biosynthesis as procollagen propepti- surgery within 36 hours of fracture. This study was performed dases (ADAMTS-2, ADAMTS-3 and ADAMTS-14) [10-13]. with Ethical Committee approval and all patients provided Other members of the family are so-called 'aggrecanases' informed consent. (ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-8, ADAMTS- 9 and ADAMTS-15) that degrade the interglobular domain Femoral head dissection separating G1 and G2 of aggrecan at a specific Glu373– Intact femoral heads were washed in sterile PBS. Cartilage Ala374 bond [14-18]. samples were removed from the femoral head using a razor blade, chopped into 2–5 mm pieces and snap frozen in liquid A family of four specific inhibitors, the tissue inhibitors of met- nitrogen within 15–30 minutes of surgery. Synovium was alloproteinases (TIMPs), has been described [19]. These are washed in sterile PBS, snap frozen and stored in liquid nitro- endogenous inhibitors of MMPs and potentially the ADAMTS gen. family. The ability of the TIMPs to block active MMPs is largely promiscuous. TIMP-3 seems to be the most potent inhibitor of RNA extraction from articular cartilage the ADAMTS family, with a subnanomolar Ki against ADAMTS- Extractions were performed on the same day or within 24 4 and ADAMTS-5 [20]. hours of dissection from the femoral head following the basic method of Price et al. [27]. Cartilage was weighed and ground Although the dogma has been that OA is a 'noninflammatory' under liquid nitrogen using a freezer mill (SPEX CertiPrep disease driven by the chondrocyte, there is certainly evidence 6750, Glen Creston, Stanmore, Middlesex, UK). TRIzol® rea- for synovial involvement. Low-grade synovitis with thickening gent (Invitrogen, Paisley, UK) was added immediately to of the lining layer, increased vascularity and inflammatory-cell ground cartilage (1 ml/0.2 g cartilage); the solution was mixed infiltration is reported in patients with all grades of OA [21]. thoroughly and incubated at room temperature for 5 minutes. This results in the expression of cytokines and proteinases that Ground cartilage was pelleted at 9,500 g for 10 minutes at could contribute to the pathogenesis of the disease [22]. 4°C and the supernatant recovered. To each 0.5 ml of TRIzol®, Arthroscopic studies have also shown that inflammatory 300 µl of chloroform was added; the resulting solution was changes in the OA synovium often occur where this tissue is vortexed for 15 seconds and incubated at room temperature in contact with cartilage [23,24].