The role of ADAMTS-1, -4 and -5 in multiple sclerosis.

ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs)-1, -4 and -5 are secreted enzymes which are members of the glutamyl endopeptidases (GEPs) group of ADAMTSs. These enzymes break down chondroitin sulphate proteoglycans (CSPGs) which are key components of brain extracellular matrix (ECM). In multiple sclerosis (MS), CSPG breakdown by ADAMTSs may enable axonal regeneration or conversely it may lead to alterations of the ECM, allowing influx of inflammatory cells promoting tissue damage.ADAMTS-1, -4 and -5 mRNA expression was studied by quantitative real-time PCR (qRT-PCR) using the Taqman method in SHSY-5Y and SK-N-DZ human neuroblastoma cells, undifferentiated or differentiated to a more neuronal phenotype using retinoic acid (RetA). Modulation by pro-inflammatory cytokines ((interleukin-1 IL-1) or tumour necrosis factor (TNF)), which are involved in the pathogenesis of MS, was also studied. As ADAMTS-1 was the most abundant ADAMTS in the neuronal cell lines, it was investigated at its protein level in both cell lines by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) with western blotting. Furthermore, the presence of ADAMTS-1 at its mRNA and protein levels was confirmed by the small interfering RNA (siRNA) technique in SHSY-5Y cells. Cryostat sections of normal and MS central nervous system (CNS) tissue white matter, obtained from the UK Multiple Sclerosis Tissue Bank, were used to determine the localisation of V0/V2 neoepitopes of versican, derived by ADAMTS cleavage, using immunohistochemistry.SHSY-5Y and SK-N-DZ cells expressed mRNA for ADAMTS-1, -4 and -5. ADAMTS-1 expression was significantly increased on cellular differentiation with Ret A in SHSY-5Y cells. Its expression was confirmed at the mRNA and protein level. IL-1 B and TNF had no effect on ADAMTS mRNA expression in SHSY-5 Y cells. However, ADAMTS-1 mRNA expression was upregulated by IL-1 B in differentiated SK-N-DZ and there was also a significant increase in ADAMTS-4 mRNA expression with TNF treatment. ADAMTS-mediated versican breakdown, as determined immuohistochemically by versican (V0/V2) neoepitopes expression, was increased in MS brain tissue compared to normal brain tissue.In conclusion, ADAMTS-1, -4 and -5 were constitutively expressed in SHSY-5Y and SK-N-DZ neuronal cells. Modulation by the cytokines tested was seen in the SK-N-DZ cells. From these in vitro studies, neuronal ADAMTSs in the CNS may have a potential role in MS pathogenesis. However further investigation is needed on primary neuronal cells and CNS to elucidate the role of neuronal ADAMTSs and their contribution in MS.ADAMTSs do appear to be involved in increased proteolysis of versican at the known cleavage site in human brain tissue as indicated by (V0/V2) versican neoepitopes expression. Upregulation of versican (V0/V2) neoepitopes was observed in lesional MS sections on immunohistochemistry. These enzymes require further investigation, by immunohistochemical methods for co-localisation with versican (V0/V2) neoepitopes in MS, to determine which of the ADAMTSs generates these neoepitopes.