title: MT1-MMP-dependent and -independent regulation of gelatinase A activation in long-term, ascorbate-treated fibroblast cultures: regulation by fibrillar collagen.
abstract: Human skin fibroblasts were cultured long-term in the presence of ascorbic acid to allow formation of a three-dimensional collagen matrix, and the effects of this on activation of secreted matrix metalloproteinase-2 (MMP-2) were examined. Accumulation of collagen over time correlated with increased levels of both mature MMP-2 and cell-associated membrane type 1-MMP (MT1-MMP), and subsequently increased mRNA levels for MT1-MMP, providing temporal resolution of the "nontranscriptional" and "transcriptional" effects of collagen on MT-1MMP functionality. MMP-2 activation by these cultures was blocked by inhibitors of prolyl-4-hydroxylase, or when fibroblasts derived from the collagen alpha1(I) gene-deficient Mov-13 mouse were used. MMP-2 activation by the Mov-13 fibroblasts was rescued by transfection of a full-length alpha1(I) collagen cDNA, and to our surprise, also by transfection with an alpha1(I) collagen cDNA carrying a mutation at the C-proteinase cleavage, which almost abrogated fibrillogenesis. Although studies with ascorbate-cultured MT1-MMP-/- fibroblasts showed that MT1-MMP played a significant role in the collagen-induced MMP-2 activation, a residual MT1-MMP-independent activation of MMP-2 was seen which resembled the level of MMP-2 activation persisting when wild-type fibroblasts were cultured in the presence of both ascorbic acid and MMP inhibitors. We were also unable to block this residual activation with inhibitors specific for serinyl, aspartyl, or cysteinyl enzymes.
authors.affiliation: VBCRC Breast Cancer Invasion and Metastasis Unit, St. Vincent's Institute of Medical Research, Melbourne, Australia.
authors.forename: Neeracha
authors.initials: N
authors.lastname: Ruangpanit
authors.forename: John T
authors.initials: JT
authors.lastname: Price
authors.forename: Kenn
authors.initials: K
authors.lastname: Holmbeck
authors.forename: Henning
authors.initials: H
authors.lastname: Birkedal-Hansen
authors.forename: Volkmar
authors.initials: V
authors.lastname: Guenzler
authors.forename: Xinfan
authors.initials: X
authors.lastname: Huang
authors.forename: Danny
authors.initials: D
authors.lastname: Chan
authors.forename: John F
authors.initials: JF
authors.lastname: Bateman
authors.forename: Erik W
authors.initials: EW
authors.lastname: Thompson
mesh_terms: D001205:Ascorbic Acid / Q000494:pharmacology*; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D024042:Collagen Type I / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D024022:Fibrillar Collagens / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D053511:Matrix Metalloproteinase 14; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000378:metabolism* / Q000502:physiology; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors; D012867:Skin / Q000166:cytology; D013997:Time Factors
chemical_list: C000716128:Col1a1 protein, mouse; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D003432:Cross-Linking Reagents; D024022:Fibrillar Collagens; C507096:Mmp14 protein, mouse; D003094:Collagen; D011393:Procollagen-Proline Dioxygenase; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D053511:Matrix Metalloproteinase 14; D001205:Ascorbic Acid