Articular cartilage proteoglycan biosynthesis and sulfation

Sammanfattning: Glucosamine (GlcN) and glucosamine sulfate (GS) have been used to treat the patients with osteoarthritis(OA) as a disease-modifying agent. Previousin vitro studies have focused on the effects of GlcN or GSon cartilage metabolism, whereas in vivo studies have investigated their potential for the treatment of OA. Although these results have raised promises of the disease-modifying effects of GlcN or GS, the cellular mechanisms behind these proposed effects are not clear. In general, the effectiveness of GS in thetreatment of OA as a symptomatic and as a disease-modifying agent is a matter of debate. Loss of proteoglycans (PGs) in OA could be partly due to deficient water binding e.g., by undersulfation of glycosaminoglycans (GAGs). In this study, the molar ratios of chondroitin sulfate (CS)disaccharide isoforms were analyzed with fluorophore-assisted carbohydrate electrophoresis to investigate the hypothesis that sulfate deficiency is involved with the development of bovine and human OA. Our present results indicate that the molar ratio of non-sulfated CS disaccharide in human samples was much lower than that detected in bovine samples, and it did not increase in human OA samples. Conversely, this ratio significantly decreased in bovine OA samples. Furthermore, the steady-state levels of aggrecan mRNA expression and sulfated GAG synthesis were analyzed by using Northern blot assay, quantitative real time reverse transcription polymerase chain reaction and[35S]sulfate incorporation analyses in bovine primary chondrocyte cultures. Aggrecan which is a large CS-PG of cartilage provides osmotic resistance for the cartilage helping it to absorb the compressive loads. Loss of PGs is a major cause of joint dysfunction and disability in OA. However, our results from 25 individual animals showed that none of the different forms of hexosamines, nor the GS salt, could stimulate aggrecan mRNA expression or GAG synthesis in bovine primary chondrocytes. Glucosamine is produced intracellularly from endogenous glucose, and is one of the basic sugar structures required for CS synthesis. It is converted to UDP-glucuronic acid (GlcA) and UDP-N-acetylgalactosamine (UDP-GalNAc) before use for the synthesis of CS polysaccharide chain. If exogenous GS is made available to the cultured cells, it can be directly incorporated into the CS synthesis by UDP-GalNAc via GlcN-6-phosphate bypassing fructose-6-phosphate. Thus, the levels of intracellular UDP-N-acetylhexosamines and UDP-GlcA were explored with reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry in bovine primary chondrocytes to analyze whether a physiologically attainable level of GS could stimulate CS synthesis by increasing intracellular UDP-sugar levels. Our present results with the cells from nine individual animals did not support this hypothesis. In conclusion, bovine and human articular cartilage PGs were not undersulfated in the early stage of OA. Exogenous GS did not increase steady state levels of aggrecan mRNA expression, GAG synthesis or intracellular levels of nucleotide-activated precursors of GAG synthesis in bovine primary chondrocytes.