ABSTRACT
ADAMTS-13 is the Von Willebrand factor (vWF) cleaving protease, responsible for the cleavage and down-regulation of the pro-thrombotic properties of ultra large VWF multimers. It is expressed predominantly by the hepatic stellate cells of the liver, but is also found to be expressed in other tissues, including brain. Reduced ADAMTS-13 is associated with a variety of thrombotic microangiopathies. Since the cellular origin and regulation of ADAMTS-13 expression in the brain is unknown, we aimed to investigate this in four different central nervous system (CNS)-derived cell lines, SHSY-5Y (human neuroblastoma), U373 (human astroglioma), CHME-3 (human foetal microglia) and hCMEC/D3 (adult human brain endothelial cells). All cell lines expressed ADAMTS-13 mRNA constitutively with neuroblastoma cells showing the highest expression. Interleukin (IL)-1ß down-regulated ADAMTS-13 mRNA expression in astroglioma cells and microglial cells whereas TNF and IL-6 treatment showed no significant differences in ADAMTS-13 mRNA expression in any cell line tested. ADAMTS-13 protein expression was reduced in a dose-dependent manner only in astroglioma cells following stimulation by IL-1ß. The ability of IL-1ß to significantly reduce ADAMTS-13 mRNA expression in human microglia and astroglioma cells suggests a role in the haemostasis of the local microenvironment under inflammatory conditions. This is the first report of ADAMTS-13 expression in cells of the CNS; however, its function remains to be determined.
Subject(s)
ADAM Proteins/genetics , Astrocytes/drug effects , Down-Regulation/drug effects , Interleukin-1beta/pharmacology , Microglia/drug effects , Nerve Tissue Proteins/genetics , Neurons/drug effects , ADAM Proteins/biosynthesis , ADAMTS13 Protein , Adult , Astrocytes/enzymology , Astrocytes/pathology , Astrocytoma/enzymology , Astrocytoma/pathology , Brain/cytology , Brain/embryology , Brain/growth & development , Cell Line/enzymology , Cell Line, Tumor/enzymology , Enzyme Induction/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Hemostasis/physiology , Humans , In Vitro Techniques , Interleukin-6/pharmacology , Microglia/enzymology , Microglia/pathology , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Nerve Tissue Proteins/biosynthesis , Neuroblastoma/enzymology , Neuroblastoma/pathology , Neurons/enzymology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/genetics , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
In this study we examined the ability of tissue factor (TF) alone, or in conjunction with factor VIIa, factor Xa and TFPI in activating a number of key signalling pathways associated with cellular growth, stress and differentiation responses in human endothelial cells. We used luciferase reporter systems to demonstrate the activation of p42/44 MAPK by the TF-FVIIa complex, mediated via the PAR1 receptor. TF alone was capable of interacting with the cell surface and was sufficient to activate the JNK-SAPK pathway and subsequently AP-1, but the level of activation was enhanced by the activity of FXa on PAR1 and 2. Furthermore, the phosphorylated form of the transmembrane-cytoplasmic domain of TF was directly responsible for activation of these pathways. CREB activation occurred in response to TF-FVIIa in a non-protease dependent manner but was lowered on addition of FXa. Finally, NFkappaB activation occurred in response to FVIIa or FXa, with the latter exhibiting higher levels of activation. In conclusion, we have shown that TF is capable of activating differing signalling pathways, via more than one mechanism. The differential influence of TF is modified depending on the presence of other coagulation factors and ultimately acts as a deciding factor in the determination of cellular fate.