Degradation of fibronectin fibrils by matrilysin and characterization of the degradation products.
Matrilysin is a metalloproteinase expressed in a variety of tumors as well as in some types of normal tissue. In addition to regulating normal tissue remodelling, metalloproteinases are believed to play a role in tumor cell invasion and metastasis by degrading components of the extracellular matrix, for example the highly insoluble fibronectin fibrils found in the interstitial stroma. In this study we examined whether matrilysin can degrade fibronectin fibrils produced by human foreskin fibroblasts and characterized the degradation products of soluble fibronectin. Using indirect immunofluorescence microscopy, we demonstrate for the first time degradation of the fibronectin fibrils upon incubation with 15 nM active matrilysin. Removal of matrilysin resulted in regrowth of the fibrils, suggesting that matrilysin was not cytotoxic. Immunoblotting with specific monoclonal antibodies revealed initial degradation of soluble fibronectin within 1 h. Further degradation occurred over a period of 20 h. Degradation of soluble fibronectin resulted in one fragment of 58 kDa containing the gelatin-binding domain, two fragments of 37 and 38 kDa, which were part of the cell attachment domain, and three fragments of 36, 33, and 30 kDa recognized by an antibody raised against the C-terminal heparin-binding domain. In addition to most of these fragments, several intermediates and unique fragments of 31 and 34 kDa could be found in the conditioned medium of human foreskin fibroblasts treated with matrilysin. Isolation of these fragments may allow further studies to determine their influences on cell migration, attachment, and signal transduction, which are expected to be different from the effects of undegraded fibronectin.
von Bredow
D C
DC
Department of Pathology, University of Arizona, Tucson 85724, USA.
Nagle
R B
RB
Bowden
G T
GT
Cress
A E
AE
D000911:Antibodies, Monoclonal; D002478:Cells, Cultured / Q000187:drug effects; D000939:Epitopes / Q000276:immunology; D005347:Fibroblasts / Q000187:drug effects; D005353:Fibronectins / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D020783:Matrix Metalloproteinase 7; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*; D008970:Molecular Weight; D012995:Solubility; D013997:Time Factors
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005353:Fibronectins; D008666:Metalloendopeptidases; D020783:Matrix Metalloproteinase 7