ADAM17 co-purifies with TIMP-3 and modulates endothelial invasion responses in three-dimensional collagen matrices.
In this study, we investigated potential mechanisms through which the known anti-angiogenic factor, tissue inhibitor of metalloproteinase-3 (TIMP-3) blocks angiogenesis. As a strategy to identify TIMP-3 binding proteins, we used tandem affinity purification, employing recombinant adenoviruses constructed to deliver TIMP-3 fused to C-terminal S and His tags (TIMP-3-S-His) or TIMP-1-S-His control to endothelial cells prior to extraction. Western blotting of final eluates revealed robust binding of A Disintegrin and Metalloproteinase (ADAM) 17 and a slight association of ADAM15 to TIMP-3, but not TIMP-1 control. To confirm a functional requirement for ADAM15 and 17 in mediating angiogenic events, a model of endothelial cell invasion was utilized. Silencing of ADAM17, but not ADAM15, expression using small interfering RNA (siRNA) interfered with invasion, resulting in decreased density of invading cells and decreased invasion distance. Stable EC lines expressing short hairpin RNA directed to ADAM17 were similarly inhibited. To confirm these results, dominant negative mutants (DeltaMPs) of ADAM10, ADAM15 or ADAM17 were delivered using recombinant lentiviruses. Expression of ADAM17 DeltaMP, but not ADAM10 or ADAM15 DeltaMP, decreased invasion density and distance. Further, time-lapse analyses revealed ADAM17 DeltaMP cells exhibited far greater numbers of protruding sprouts compared to control, suggesting an inability of extended processes to retract properly. Immunofluorescence analyses revealed ADAM17 localized to bifurcations in invading sprouts. These data jointly indicate a role for ADAM17 in modulating endothelial sprouting events during angiogenesis.
Kwak
Hyeong-il
HI
Department of Molecular & Cellular Medicine, Texas A&M Health Science Center, College Station, TX 77843-1114, USA.
Mendoza
E Adriana
EA
Bayless
Kayla J
KJ
D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000072197:ADAM10 Protein; D000072198:ADAM17 Protein; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism; D022081:Cell Surface Extensions / Q000201:enzymology; D002478:Cells, Cultured; D003094:Collagen; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000502:physiology*; D025941:Protein Interaction Mapping; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000502:physiology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics / Q000378:metabolism*; D054457:Tissue Scaffolds; D014161:Transduction, Genetic
D008565:Membrane Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C088390:TIMP3 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; D019717:Tissue Inhibitor of Metalloproteinase-3; D003094:Collagen; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; C098660:ADAM15 protein, human; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human