ABSTRACT
Fractalkine shedding is believed to occur constitutively and following induction via the activity of two membrane-bound enzymes, ADAM-10 and ADAM-17. However, our previous work suggested that ADAM-17 is not involved in the proteolytic release of fractalkine under TNF treatment of a human adult brain endothelial cell line, hCMEC/D3. The pro-inflammatory cytokine, TNF, has previously been shown to be expressed in the perivascular cuffs in multiple sclerosis. Here we sought to identify, using siRNAs to silence the expression of ADAM-10 and ADAM-17, whether ADAM-10 is responsible for TNF-induced shedding of fractalkine from the cell membrane in hCMEC/D3. Our findings suggest that ADAM-10, and not ADAM-17, is the major protease involved in fractalkine release under pro-inflammatory conditions in this human adult brain endothelial cell model.
Subject(s)
ADAM Proteins/genetics , Amyloid Precursor Protein Secretases/genetics , Brain/blood supply , Chemokine CX3CL1/metabolism , Endothelial Cells/metabolism , Membrane Proteins/genetics , RNA, Small Interfering/genetics , Tumor Necrosis Factor-alpha/pharmacology , ADAM10 Protein , ADAM17 Protein , Adult , Cell Line , Endothelial Cells/drug effects , Gene Knockdown Techniques , Gene Silencing , Humans , Inflammation/metabolismABSTRACT
ADAM-17 expression is localised to endothelial cells in the human central nervous system (CNS) and is increased in multiple sclerosis (MS) white matter, suggesting a role in MS pathogenesis. Expression of ADAM-17, TIMP-3, and fractalkine were investigated in a human brain endothelial cell line (hCMEC/D3) after pro-inflammatory cytokine treatment. Tumour necrosis factor (TNF) significantly increased fractalkine mRNA (>100 fold) and protein expression, which was associated with increased shedding of fractalkine from the cell. Fractalkine shedding may regulate immune cell trafficking into the CNS, however, this does not appear to be directly controlled by ADAM-17 activity.
Subject(s)
ADAM Proteins/metabolism , Chemokine CX3CL1/metabolism , Cytokines/metabolism , Cytokines/pharmacology , Endothelial Cells/metabolism , ADAM Proteins/genetics , ADAM17 Protein , Cell Line , Chemokine CX3CL1/genetics , Chemotaxis, Leukocyte/immunology , Encephalitis/immunology , Encephalitis/metabolism , Encephalitis/physiopathology , Endothelial Cells/drug effects , Endothelial Cells/immunology , Gene Expression/drug effects , Gene Expression/physiology , Humans , RNA, Messenger/analysis , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Tissue Inhibitor of Metalloproteinase-3/genetics , Tissue Inhibitor of Metalloproteinase-3/metabolism , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Up-Regulation/drug effects , Up-Regulation/immunologyABSTRACT
PURPOSE: Injury to the adult optic nerve, caused mechanically or by diseases, is still not reparable because the retinal ganglion cells (RGCs) are not allowed to regrow their axons and die retrogradely, although they possess the intrinsic propensity to regenerate axons in experimental conditions. METHODS: In vitro propagated embryonic stem cells derived from the early chicken neural tube (NTSCs) were used to examine whether transplanted NTSCs produce growth-promoting factors and pave the microenvironment, thus facilitating axonal regeneration within the rat optic nerve. RESULTS: NTSCs survived within the site where the optic nerve had been cut and continued to be nestin-positive, thus preserving their undifferentiated cell phenotype. Transplanted NTSCs activated the matrix metalloproteases (MMP)-2 and -14 in glial fibrillary acidic protein (GFAP)-positive optic nerve astrocytes. MMP2 production correlated with immunohistochemically visible degradation of inhibitory chondroitin sulfate proteoglycans (CSPGs). In addition, NTSCs produced a panoply of neurite-promoting factors including oncomodulin, ciliary neurotrophic factor, brain-derived neurotrophic factor and crystallins beta and gamma. Cut axons intermingled with NTSCs and passed through the zone of injury to enter the distal optic nerve over long distances, arriving at the thalamus and midbrain. CONCLUSIONS: This study showed evidence that paving of the distal optic nerve microenvironment with proteolytically active MMPs and providing stem-cell-derived growth factors is a suitable method for facilitating regenerative repair of the optic nerve. Understanding the molecular mechanisms of this repair has fundamental implications for development of NTSC-based subsidiary therapy after neural injuries.
