title: Tissue inhibitor of metalloproteinases-4 inhibits but does not support the activation of gelatinase A via efficient inhibition of membrane type 1-matrix metalloproteinase.
abstract: The tissue inhibitors of metalloproteinases 1-4 (TIMPs) have discrete regulatory roles in the activation of matrix metalloproteinase (MMP)-2 (gelatinase A), an important basement membrane-degrading MMP pivotal to tumor metastasis and angiogenesis. TIMP-2 binds to both the hemopexin C domain of progelatinase A and the active site of membrane type-1 (MT1) MMP. This trimeric complex presents the cell surface-bound gelatinase A zymogen to a free MT1-MMP molecule for activation. To investigate the role of TIMP-4 in the activation process, we developed a new procedure for the expression and purification of recombinant human TIMP-4 from baby hamster kidney cells. The recombinant TIMP-4 was a potent inhibitor of gelatinase A (apparent K(i) [Ki(app.)] < or = 9 pM; k(on) (association rate constant), 4.57 +/- 0.13 x 10(6) M(-1)s(-1)) and was less dependent upon hemopexin C domain interactions than TIMP-2 in its mode of binding and inhibition. Unlike TIMP-1, TIMP-4 strongly inhibited MT1-MMP (Ki(app.) < or = 100 pM; k(on), 3.49 +/- 0.34 x 10(6) M(-1)s(-1)) and blocked the concanavalin A-induced cellular activation of progelatinase A. In concanavalin A-stimulated homozygous Timp2 -/- fibroblasts or unstimulated MT1-MMP-transfected Timp2 -/- cells, which cannot activate progelatinase A, activation was restored by the addition of 0.3-5 nM TIMP-2 but not by TIMP-4, unequivocally showing the TIMP-2 dependency of MT1-MMP-induced activation of gelatinase A and the fact that TIMP-4 cannot support activation. The dominance of TIMP-2 in the activation process was further supported by the preferential binding of TIMP-2 compared with TIMP-4 to the hemopexin C domain of progelatinase A in inhibitor mixtures and by the ability of TIMP-2 to displace TIMP-4 from the hemopexin C domain. Hence, TIMP-4 regulates gelatinase A activity by efficient inhibition of MT1-MMP-mediated activation and by inhibiting the activated enzyme and, thus, is a tumor resistance factor in the peritumor stroma.
authors.affiliation: Faculty of Dentistry and Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: H F
authors.initials: HF
authors.lastname: Bigg
authors.forename: C J
authors.initials: CJ
authors.lastname: Morrison
authors.forename: G S
authors.initials: GS
authors.lastname: Butler
authors.forename: M A
authors.initials: MA
authors.lastname: Bogoyevitch
authors.forename: Z
authors.initials: Z
authors.lastname: Wang
authors.forename: P D
authors.initials: PD
authors.lastname: Soloway
authors.forename: C M
authors.initials: CM
authors.lastname: Overall
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D005347:Fibroblasts; D018093:Gelatinases / Q000378:metabolism; D007700:Kinetics; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019714:Tissue Inhibitor of Metalloproteinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D000097792:Tissue Inhibitor of Metalloproteinase-4
chemical_list: D004792:Enzyme Precursors; D018093:Gelatinases; D020778:Matrix Metalloproteinase 2; D061965:Matrix Metalloproteinase Inhibitors; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D011994:Recombinant Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D019714:Tissue Inhibitor of Metalloproteinases; D000097792:Tissue Inhibitor of Metalloproteinase-4; C069860:progelatinase