Latent MMP-9 is bound to TIMP-1 before secretion.
Expression patterns of matrix metalloproteinase-9 (MMP-9) and its specific inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1), are closely correlated with physiological and pathological processes characterized by the degradation and accumulation of the extracellular matrix (ECM). Both, activated MMP-9 and pro-MMP-9 can bind to TIMP-1, and most cell types secrete MMP-9 in complex with TIMP-1. Utilizing immunofluorescence, we observed intracellular co-localization of MMP-9 and TIMP-1 in stimulated human fibrosarcoma cells. In the present study we searched for the origin of the complex formation between the latent enzyme and its specific inhibitor on a subcellular level. Fluorescence resonance energy transfer (FRET) between the fluorescently labeled enzyme and its inhibitor in co-transfected cells were measured. MMP-9 and TIMP-1 were fused to cyan (CFP) and yellow (YFP) variants of the green fluorescent protein and transiently expressed in human hepatoma cells. The intracellular distribution of fluorescently labeled TIMP-1 and MMP-9 was analyzed by confocal laser scanning microscopy. Intracellular complex formation in the Golgi apparatus was verified, demonstrating FRET between MMP-9-CFP and TIMP-1-YFP. Our data provide evidence that the proMMP-9-TIMP-1 complex is already present in the Golgi apparatus. This may be of significance for a number of intracellular and extracellular biochemical processes involving proMMP-9. However, the magnitude and functional relevance of this finding remain unknown.
Roderfeld
Martin
M
Department of Medicine II, Gastroenterology, University Hospital Giessen & Marburg GmbH, Justus Liebig University Giessen, D-35392, Giessen, Germany.
Graf
Jürgen
J
Giese
Bernd
B
Salguero-Palacios
Rebeca
R
Tschuschner
Annette
A
Müller-Newen
Gerhard
G
Roeb
Elke
E
D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D031541:Fluorescence Resonance Energy Transfer; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D011485:Protein Binding; D013347:Subcellular Fractions / Q000201:enzymology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism*
D019715:Tissue Inhibitor of Metalloproteinase-1; D020780:Matrix Metalloproteinase 9