A disintegrin-like and metalloproteinase with thrombospondin motifs 18 (ADAMTS18) cleaves fibronectin and regulates its fibrillogenesis

Remodelling of the extracellular matrix plays a crucial role in the development, maintenance and repair of all tissues. Therefore, identifying the regulators of this process is essential. Among these, A disintegrin and metalloproteinase with a thrombospondin motif 18 (ADAMTS18) has been implicated in fibronectin (FN) matrix regulation. Knockout of ADAMTS18, either in mouse models or in vitro, was shown to lead to FN accumulation, mutation in epithelial branching and reduction in endothelial sprouting. However, the mechanisms by which ADAMTS18 influences endothelial specific functions and the extracellular matrix, particularly in the regulation of FN fibrils remains unclear. In this study, using both siRNA-mediated knockdown and overexpression of ADAMTS18 in primary endothelial cells, we delineated some of these mechanisms. Using global RNA sequencing of endothelial cells we demonstrated differential gene regulation of vessel-development and endothelial adhesion genes with ADAMTS18-siRNA knockdown, whereas cell matrix and cell cycle associated genes were affected by overexpression of ADAMTS18. Consistent with the later, we observed reduced endothelial cell proliferation and altered cell cycle with ADAMTS18 overexpression. Using mass spectrometry, we identified two sites in FN that are proteolytically cleaved by ADAMTS18, including a cleavage site in the linker FN-I5-6. Cleavage at this site generated FN molecules lacking the N-terminal FN-I1-5 (29kDa) fragment that is known to be essential for FN fibrillogenesis. Accordingly, ADAMTS18 overexpression greatly impaired FN fibrillogenesis in endothelial cultures and in co-culture with fibroblasts. Our results implicate ADAMTS18 in FN-associated extracellular matrix remodelling and suggest an important role for ADAMTS18 in endothelium biology. Overall design: To investigate the role of ADAMTS18 in endothelial cells, we over-expressed human ADAMTS18 in human umbilical vein endothelila cells (HUVEC) using a lentiviral vector (LV). In addition, to study the effect of knockdown of ADAMTS18 we reduced ADAMTS18 expression in HUVEC using siRNA. We then performed gene expression profiling based on data derinved from RNA-sequencing from 4 independent HUVEC cultures (pool of 3 donors) for each treatment. and harvested after 2 or 3 days. Comparitive gene expression profiling was performed for the over-expression of ADAMTS18 via comparison to control transduced cells over-expressing a reporter gene (green fluorescent protein; GFP) 3 days after transduction. For the knockdown study siADAMTS18 samples were compared to control siRNA (siCTRL) samples, generated 2 days following transfection.