KERATAN SULPHATE SMALL LEUCINE-RICH PROTEOGLYCANS AS POTENTIAL BIOMARKERS OF DEGENERATIVE JOINT AND DISC DISEASES.

Abstract

Introduction: Several small leucine-rich proteoglycans (SLRPs) are involved in the regulation of collagen fibril size(s) in a variety of different musculoskeletal tissues. In hyaline articular cartilage the major SLRPs involved in regulation of type II collagen fibrils are believed to be decorin and fibromodulin. These two SLRPs along with another family member, lumican, have also been identified in intervertebral disc tissues. In recent studies, we serendipitously discovered that, keratocan and lumican [two keratan sulphate (KS) substituted members of the SLRP family] were unusually expressed in extracts from degenerative joint and degenerative disc tissues. The object of this study has been to further investigate this finding with a view to examining the increased expression of keratocan and lumican using qualitative Western blot analysis and quantitative ELISA methods. Our working hypothesis is that the increased expression of these two SLRPs in degenerative joint and disc tissue results from a reparative deposition of a type I collagen fibrillar ¡®scar¡^-.

Methods: Monoclonal antibodies were produced to core protein epitopes in lumican and keratocan. Degenerate cartilage was obtained from patients undergoing routine joint replacement for either hip or knee joints, whilst normal articular cartilage tissue was obtained from surgical knee procedures. In addition, disc samples were obtained from patients undergoing a variety of spinal procedures and were Graded I-IV using a modified Thompson score. The tissue was diced and extracted in a 4M guanidine HCl buffer, pH6.8 containing an inhibitor cocktail for 48 h at 4¢ªC. Samples were then dialysed exhaustively against Milli Q water and assayed for glycosaminoglycan (GAG) content using the DMMB assay. Cartilage extracts containing equal amounts of GAG were then separated by SDS-PAGE and transferred to nitrocellulose for Western blotting using mMAbs to either keratocan or lumican. In addition, a competitive ELISA has been developed for quantifying keratocan and lumican.

Results: Western blot analysis of normal and degenerative articular cartilage revealed the presence of both keratocan and lumican. However, the presence of these SLRPs was substantially increased in the degenerate articular cartilge extracts. In addition, these proteins were also present in extracts of intervertabral disc with an increase being apparent in those disc samples with increased pathology. Preliminary data for the development of a quantitative ELISA for these two SLRPs shows promise.

Discussion: The unexpected increase in the detection of keratocan and lumican in degenerative articular cartilage and disc suggests their potential as biomarkers for the onset of degenerative joint and disc disease. However, this will involve the development of a quantitative assay and the investigation of the presence of these molecules in synovial fluid and serum.