Processing of syndecan-2 by matrix metalloproteinase-14 and effect of its cleavage on VEGF-induced tube formation of HUVECs

Abstract

Syndecans (SDCs) are transmembrane proteoglycans that are involved in cell adhesion and cell communication. Specifically, SDC2 plays a key role in tumorigenesis, metastasis, and angiogenesis. Previously, we found that rat SDC2 is shed by matrix metalloproteinase-7 (MMP-7) in colon cancer cells. Here, we analyzed the susceptibility of rat SDC2 to various MMPs. We found that the rat SDC2 ectodomain (ECD) fused to the C-terminal Fc region, which was expressed in mammalian cells, was cleaved more efficiently by MMP-14 than MMP-7. Likewise, when anchored on the surface of HeLa cells, rat SDC2 was cleaved more efficiently by the treatment of MMP-14 than MMP-7 and was shed more readily by membrane-anchored MMP-14 than soluble MMP-14. Furthermore, MMP-14 cleaved recombinant SDC2-ECD expressed in into multiple fragments. Using N-terminal amino acid sequencing and the top-down proteomics approach, we determined that the major cleavage sites were S↓L, T↓M, T↓L, D↓P, and N↓L for rat SDC2-ECD and S↓G, S↓P, P↓K, N↓I D↓P, and S↓L for human SDC2-ECD. Finally, the rat and human SDC2-ECD lost the ability to suppress vascular endothelial growth factor-induced formation of capillary-like tubes by human umbilical vein endothelial cells following cleavage by MMP-14, but its major cleavage-site mutant of rat SDC2-ECD did not. These results suggest that MMP-14 is a novel enzyme responsible for degrading SDC2 and impairing its physiological roles including angiogenesis.

Role of Extracellular Matrix in Gastrointestinal Cancer-Associated Angiogenesis

“…This was in line with the results conducted on 119 CRC patients where lower levels of circulating sEndoglin associated with a higher angiogenic activity [252]. More recently, Lee and co-authors, using a rat colonic cell line as a model, demonstrated that MT1-MMP was responsible for the degradation of the proteoglycan syndecan-2 [256]. Through its N-terminal domain, syndecan-2 selectively promotes a VEGFA-dependent neovascularization enhancing 6-O heparan sulfate chains' sulfation [257].…”

ADAMTS-1 and syndecan-4 intersect in the regulation of cell migration and angiogenesis

“…Syndecan ectodomain shedding is an important regulatory mechanism allowing for rapid changes in cell surface receptor dynamics; shedding occurs juxtamembrane and can be carried out by a number of matrix proteinases (Manon-Jensen et al, 2010). MMP7 and MMP14 are both able to shed the extracellular domain of syndecan-2, and MMP9 has been shown to shed syndecan-4 from the cell surface in response to TNFα (Kwon et al, 2014;Lee et al, 2017;Ramnath et al, 2014). There was a significant increase in MMP9 expression in ADAMTS-1 siRNA-treated cells, and upon loss of MMP9, via either an inhibitor or siRNA depletion, syndecan-4 was no longer lost from the cell surface.…”

Thrombin-cleaved syndecan-3/-4 ectodomain fragments mediate endothelial barrier dysfunction

“…A number of sheddases expressed on cell membranes, including MMPs and ADAM (a disintegrin and metalloprotease) metalloendopeptidases, are believed to cleave transmembrane molecules such as SDC and release their ectodomains [3,4]. Other proteases in the circulation, including secreted MMPs, plasmin and thrombin, have been shown to be able to further degrade the ectodomains into fragments [2,3,5].…”

“…Recently, S2ED was also shown to be cleaved by MMP14 to produce multiple fragments. Moreover, the authors found that in cleaving S2ED, MMP14 impaired the ability of S2ED to decrease endothelial capillary-tube formation in vitro , which has implications in angiogenesis [4].…”