EXPRESSION OF ADAM-TS4 BY CHONDROCYTES IN OSTEOARTHRITIS IN RELATION TO CHANGES IN DNA METHYLATION STATUS

Abstract

In osteoarthritis (OA) there is a loss of matrix components, especially aggrecan, which is a major structural component important for the integrity and function of articular cartilage. The breakdown of aggrecan is mediated by enzymes from the ADAM-TS (a disintegrin and metalloproteinase with thrombospondin motifs) family and recent studies have suggested that, in humans, ADAM-TS4 (aggrecanase-1) plays a major role. Articular chondrocytes do not express ADAM-TS4 in contrast to clonal OA chondrocytes. Since in any somatic cell non-expressed genes are thought to be silenced by DNA methylation in the promoter region, the aims of the project were twofold:

1. to localize enzyme expression for ADAM-TS4 by immunocytochemistry and

2. to determine whether ‘unsilencing’ (i.e. DNA de-methylation) in the promoter of ADAM-TS4 was associated with the abnormal enzyme synthesis.

Using immunocytochemistry, we confirmed that there is an increased expression of ADAM-TS4 in OA chondrocytes, which initially occurs in chondrocytes of the superficial zone. As the Mankin score increases, ADAM-TS4 positive chondrocytes were found in duplets, then quadruplets until, at Mankin score > 10, all the cells in a typical OA clone were immunopositive for ADAM-TS4, suggesting that abnormal enzyme expression was inherited by daughter cells. DNA was extracted from femoral head cartilage of 24 patients, who had undergone hip replacement surgery for either symptomatic OA or following a fracture of neck of femur (#NOF). The latter was used as control due to the inverse relationship between OA and osteoporosis. For OA samples, it was important to sample only those regions for which immunocytochemistry had shown the presence of ADAM-TS4 synthesizing cells, i.e. the superficial zones near the weight-bearing region. DNA methylation only occurs at cytosines of the sequence 5′...CG...3′, the so-called CpG sites. To determine methylation status of specific CpG sites, methylation sensitive restriction enzymes were used, which will only cut DNA in the absence of methylation. By designing PCR primers that bracketed these sites, presence or absence of PCR bands could distinguish between methylated and non-methylated CpGs respectively. The ADAM-TS4 promoter contains a total of 13 CpG sites. Using restriction enzyme/primers combinations, it was possible to analyze 7 of these sites for methylation status. In the control group, all 7 CpG sites were methylated, while there was an overall 49% decrease of methylation in the OA group (p=< 0.0001). Some of the CpG sites were more consistently demethylated then others, one site at −753bp upstream from the transcription start site, showed a 86% decrease in methylation in OA compared to the control group (p=0.0005), while at other sites the decrease in methylation ranged from 36–50%. Conclusions. This study confirmed by immunocytochemistry that ADAM-TS4 is produced by OA chondrocytes, contributing to the degradation of their matrix. This abnormal enzyme expression is associated with DNA methylation. If a causal relationship could be proven in the future, then DNA de-methylation might play an important role in the pathogenesis of osteoarthritis and future therapies might be directed at influencing the methylation status.