Individual Timp deficiencies differentially impact pro-MMP-2 activation.
Membrane-type matrix metalloproteinases (MT-MMPs) have emerged as key enzymes in tumor cell biology. The importance of MT1-MMP, in particular, is highlighted by its ability to activate pro-MMP-2 at the cell surface through the formation of a trimolecular complex comprised of MT1-MMP/tissue inhibitor of metalloproteinase-2 (TIMP-2)/pro-MMP-2. TIMPs 1-4 are physiological MMP inhibitors with distinct roles in the regulation of pro-MMP-2 processing. Here, we have shown that individual Timp deficiencies differentially affect MMP-2 processing using primary mouse embryonic fibroblasts (MEFs). Timp-3 deficiency accelerated pro-MMP-2 activation in response to both cytochalasin D and concanavalin A. Exogenous TIMP-2 and N-TIMP-3 inhibited this activation, whereas TIMP-3 containing matrix from wild-type MEFs did not rescue the enhanced MMP-2 activation in Timp-3(-/-) cells. Increased processing of MMP-2 did not arise from increased expression of MT1-MMP, MT2-MMP, or MT3-MMP or altered expression of TIMP-2 and MMP-2. To test whether increased MMP-2 processing in Timp-3(-/-) MEFs is dependent on TIMP-2, double deficient Timp-2(-/-)/-3(-/-) MEFs were used. In these double deficient cells, the cleavage of pro-MMP-2 to its intermediate form was substantially increased, but the subsequent cleavage of intermediate-MMP-2 to fully active form, although absent in Timp-2(-/-) MEFs, was detectable with combined Timp-2(-/-)/-3(-/-) deficiency. TIMP-4 associates with MMP-2 and MT1-MMP in a manner similar to TIMP-3, but its deletion had no effect on pro-MMP-2 processing. Thus, TIMP-3 provides an inherent regulation over the kinetics of pro-MMP-2 processing, serving at a level distinct from that of TIMP-2 and TIMP-4.
Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada.
Jane L
JL
English
Zamaneh
Z
Kassiri
Ilpo
I
Koskivirta
Susan J
SJ
Atkinson
Marco
M
Di Grappa
Paul D
PD
Soloway
Hideaki
H
Nagase
Eero
E
Vuorio
Gillian
G
Murphy
Rama
R
Khokha
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003208:Concanavalin A / Q000494:pharmacology; D006224:Cricetinae; D003433:Crosses, Genetic; D015638:Cytochalasin D / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D053511:Matrix Metalloproteinase 14; D053517:Matrix Metalloproteinase 15; D053513:Matrix Metalloproteinase 16; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism / Q000502:physiology*; D020782:Matrix Metalloproteinases / Q000378:metabolism; D053504:Matrix Metalloproteinases, Membrane-Associated; D000090023:Metallothionein 3; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008954:Models, Biological; D011485:Protein Binding; D011506:Proteins / Q000235:genetics* / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000378:metabolism / Q000502:physiology*; D019714:Tissue Inhibitor of Metalloproteinases; D000097792:Tissue Inhibitor of Metalloproteinase-4
D003208:Concanavalin A; D015638:Cytochalasin D; D053511:Matrix Metalloproteinase 14; D053517:Matrix Metalloproteinase 15; D053513:Matrix Metalloproteinase 16; D020778:Matrix Metalloproteinase 2; D020782:Matrix Metalloproteinases; D053504:Matrix Metalloproteinases, Membrane-Associated; D000090023:Metallothionein 3; D011506:Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D019717:Tissue Inhibitor of Metalloproteinase-3; D019714:Tissue Inhibitor of Metalloproteinases; D000097792:Tissue Inhibitor of Metalloproteinase-4; C507098:MMP15 protein, human; C507102:MMP16 protein, human; C507096:Mmp14 protein, mouse; C507100:Mmp15 protein, mouse; C507104:Mmp16 protein, mouse; C000715430:Mt3 protein, mouse