Investigation of ADAMTS-9 expression in models of central nervous system inflammation.

The ADAMTSs (a disintegrin and metalloproteinase with thrombospondin type 1-like motifs) are a group of peptidases with important roles in normal physiology and pathology. ADAMTS-9 is expressed in the central nervous system (CNS) and has been reported to cleave aggrecan and versican, which are chondroitin sulphate proteoglycans (CSPGs) with critical roles in the extracellular matrix (ECM) of the brain. CSPGs are structural components of the glial scar, which forms in response to destructive CNS inflammation, inhibiting axonal outgrowth but protecting neurones against inflammatory infiltrates. Therefore, ADAMTS-9 has the potential to be involved in normal processing of the brain ECM as well as contributing to or protecting against pathology. In this study the expression of ADAMTS-9 was analysed in CNS-derived cells in vitro under basal conditions and following treatment with factors pertinent to CNS inflammatory disorders. ADAMTS-9 expression was also analysed in rat brains following transient middle cerebral artery occlusion (tMCAo), a model of focal cerebral ischaemia.Real-time RT-PCR data demonstrated that ADAMTS-9 mRNA was constitutively expressed by human astrocytic (U373-MG, U87-MG and B327-01), microglial (CHME3) and neuronal (SHSY-5Y) cell lines in vitro, under basal conditions. In U373-MG cells, ADAMTS-9 mRNA expression was increased 25 and 8-fold following treatment with 1 ng/mL interleukin-1 beta (IL-1beta) and tumour necrosis factor (TNF) respectively. In B327-01 cells, IL-1beta (100 ng/mL) modulated a 6-fold increase in ADAMTS-9 mRNA expression. In contrast, treatment with 1 ng/mL interferon-gamma (IFN-gamma) led to a decrease in ADAMTS-9 expression in B327-01 cells. The effect of IFN-gamma in CHME3 cells differed to that observed in B327-01 in that 10 ng/mL increased expression of ADAMTS-9 mRNA 2-fold in the astrocytic cells. The study also demonstrated that ADAMTS-9 mRNA expression was modulated by retinoic acid in SHSY-5Y cells. Immunocytochemical analysis of U373-MG cells indicated that ADAMTS-9 protein is localised in the nucleus under basal conditions.Real-time RT-PCR analysis of ADAMTS-9 mRNA expression demonstrated it was up-regulated in tMCAo tissue compared to sham-operated at 6, 24 and 120 h post-procedure. In addition ADAMTS-9 mRNA levels were higher in ipsilateral (occluded) hemispheres when compared to contralateral (non-occluded) hemispheres in tMCAobrains. Western blotting indicated that the mature form of the ADAMTS-9 protein was only detected in brains subjected to tMCAo at 24 h. An ADAMTS-9-specific riboprobe was generated by subcloning and expression of a plasmid containing ADAMTS-9 cDNA in the XL-1 blue strain of E. coli followed by in vitro transcription and labelling with digoxigenin (DIG). The probe was utilised to perform in situ hybridisation, which showed that neurones were the predominant cell-type expressing ADAMTS-9 mRNA in tMCAo tissue.Preliminary bioinformatical analysis of the putative human and rat ADAMTS-9 promoter regions demonstrated that consensus binding sites for transcription factors (nuclear factor kappa B [NFkappaB], activator protein 1 [AP-1], hypoxia inducible factor-1 [HIF-1] and interferon regulatory factor 1 [IRF-1]) pertinent to CNS inflammation were present in the sequence. Such transcription factors are potentially involved in activating the expression of ADAMTS-9 expression. Consequently, this study confirms that ADAMTS-9 is expressed by endogenous CNS cells and its expression is modulated by CNS inflammatory conditions both in vitro and in vivo.