Identification of candidate angiogenic inhibitors processed by matrix metalloproteinase 2 (MMP-2) in cell-based proteomic screens: disruption of vascular endothelial growth factor (VEGF)/heparin affin regulatory peptide (pleiotrophin) and VEGF/Connective tissue growth factor angiogenic inhibitory complexes by MMP-2 proteolysis.
Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2-/- mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2-/- cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis.
Dean
Richard A
RA
University of British Columbia, Centre for Blood Research, 4.401 Life Sciences Institute, 2350 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3.
Butler
Georgina S
GS
Hamma-Kourbali
Yamina
Y
Delbé
Jean
J
Brigstock
David R
DR
Courty
José
J
Overall
Christopher M
CM
D020533:Angiogenesis Inhibitors / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002465:Cell Movement; D049109:Cell Proliferation; D055513:Connective Tissue Growth Factor; D017077:Culture Media, Conditioned; D016207:Cytokines / Q000378:metabolism*; D004789:Enzyme Activation; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D007553:Isotope Labeling; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D010455:Peptides / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D015203:Reproducibility of Results; D013379:Substrate Specificity; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D020533:Angiogenesis Inhibitors; C526317:CCN2 protein, human; C526318:CCN2 protein, mouse; D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D016207:Cytokines; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D042461:Vascular Endothelial Growth Factor A; C066845:pleiotrophin; D055513:Connective Tissue Growth Factor; D020778:Matrix Metalloproteinase 2