title: Cellular activation of MMP-2 (gelatinase A) by MT2-MMP occurs via a TIMP-2-independent pathway.
abstract: The role of membrane-type (MT) 2-matrix metalloproteinase (MMP) in the cellular activation of MMP-2 and the tissue inhibitor of matrix metalloproteinase (TIMP) requirements for this process have not been clearly established. To address these issues a TIMP-2-free cell line derived from a Timp2-/- mouse was transfected for stable cell surface expression of hMT2-MMP. Untransfected cells did not activate endogenous or exogenous TIMP-2-free MMP-2 unless both TIMP-2 and concanavalin A (ConA) were added. Transfected cells expressing hMT2-MMP efficiently activated both endogenous and exogenous MMP-2 (within 4 h) via the 68-kDa intermediate in the absence of TIMP-2 and ConA. In contrast, activation of MMP-2 by Timp2-/- cells expressing recombinant hMT1-MMP occurred more slowly (12 h) and required the addition of 0.3-27 nm TIMP-2. Addition of TIMP-2 or TIMP-4 did not enhance MMP-2 activation by MT2-MMP at any concentration tested; furthermore, activation was inhibited by both TIMPs at concentrations >9 nm, consistent with the similar association rate constants (k(on)) calculated for the binding of TIMP-4 and TIMP-2 to MT2-MMP (3.56 x 10(5) m(-1) s(-1) and 6.52 x 10(5) m(-1) s(-1), respectively). MT2-MMP-mediated activation involved cell surface association of the MMP-2 in a hemopexin carboxyl-terminal domain (C domain)-dependent manner: Exogenous MMP-2 hemopexin C domain blocked activation, and cells expressing hMT2-MMP did not bind or activate a truncated form of MMP-2 lacking the hemopexin C domain. These studies demonstrate the existence of an alternative TIMP-2-independent pathway for MMP-2 activation involving MT2-MMP, which may be important in mediating MMP-2 activation in specific tissues or pathologies where MT2-MMP is expressed.
authors.affiliation: Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: C J
authors.initials: CJ
authors.lastname: Morrison
authors.forename: G S
authors.initials: GS
authors.lastname: Butler
authors.forename: H F
authors.initials: HF
authors.lastname: Bigg
authors.forename: C R
authors.initials: CR
authors.lastname: Roberts
authors.forename: P D
authors.initials: PD
authors.lastname: Soloway
authors.forename: C M
authors.initials: CM
authors.lastname: Overall
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002478:Cells, Cultured; D003208:Concanavalin A / Q000494:pharmacology; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D017353:Gene Deletion; D005822:Genetic Vectors / Q000378:metabolism; D006466:Hemopexin / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D053517:Matrix Metalloproteinase 15; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism*; D014162:Transfection
chemical_list: D018076:DNA, Complementary; C507098:MMP15 protein, human; C507100:Mmp15 protein, mouse; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003208:Concanavalin A; D019716:Tissue Inhibitor of Metalloproteinase-2; D006466:Hemopexin; D053517:Matrix Metalloproteinase 15; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2