Exogenously added GPI-anchored tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) displays enhanced and novel biological activities.
The family of tissue inhibitors of metalloproteinases (TIMPs) exhibits diverse physiological/biological functions including the inhibition of active matrix metalloproteinases, regulation of proMMP activation, cell growth, and the modulation of angiogenesis. TIMP-1 is a secreted protein that can be detected on the cell surface through its interaction with surface proteins. The diverse biological functions of TIMP-1 are thought to lie, in part, in the kinetics of TIMP-1/MMP/surface protein interactions. Proteins anchored by glycoinositol phospholipids (GPIs), when purified and added to cells in vitro, are incorporated into their surface membranes. A GPI anchor was fused to TIMP-1 to generate a reagent that could be added directly to cell membranes and thus focus defined concentrations of TIMP-1 protein on any cell surface independent of protein-protein interaction. Unlike native TIMP-1, exogenously added GPI-anchored TIMP-1 protein effectively blocked release of MMP-2 and MMP-9 from osteosarcoma cells. TIMP-1-GPI was a more effective modulator of migration and proliferation than TIMP-1. While control hTIMP-1 protein did not significantly affect migration of primary microvascular endothelial cells at the concentrations tested, the GPI-anchored TIMP-1 protein showed a pronounced suppression of endothelial cell migration in response to bFGF. In addition, TIMP-1-GPI was more effective at inducing microvascular endothelial proliferation. In contrast, fibroblast proliferation was suppressed by the agent. Reagents based on this method should assist in the dissection of the protease cascades and activities involved in TIMP biology. Membrane-fixed TIMP-1 may represent a more effective version of the protein for use in therapeutic expression.
Medizinische Poliklinik, Ludwig-Maximilians-Universität, Schillerstrasse 42, D-80336 München, Germany.
Roghieh
R
Djafarzadeh
Anke
A
Mojaat
Ana Belen
AB
Vicente
Irene
I
von Lüttichau
Peter J
PJ
Nelson
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology; D017261:Glycosylphosphatidylinositols / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D061965:Matrix Metalloproteinase Inhibitors; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D013499:Surface Properties; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism* / Q000494:pharmacology*
D017261:Glycosylphosphatidylinositols; D061965:Matrix Metalloproteinase Inhibitors; D011993:Recombinant Fusion Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D008666:Metalloendopeptidases