MT1-MMP-dependent and -independent regulation of gelatinase A activation in long-term, ascorbate-treated fibroblast cultures: regulation by fibrillar collagen

TY - JOUR

T1 - MT1-MMP-dependent and -independent regulation of gelatinase A activation in long-term, ascorbate-treated fibroblast cultures

T2 - regulation by fibrillar collagen

AU - Ruangpanit, Neeracha

AU - Price, John T

AU - Holmbeck, Kenn

AU - Birkedal-Hansen, Henning

AU - Guenzler, Volkmar

AU - Huang, Xinfan

AU - Chan, Danny

AU - Bateman, John F

AU - Thompson, Erik W

N1 - (c)2001 Elsevier Science.

PY - 2002/1/15

Y1 - 2002/1/15

N2 - 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.

AB - 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.

KW - Ascorbic Acid/pharmacology

KW - Cells, Cultured

KW - Collagen/metabolism

KW - Collagen Type I/genetics

KW - Cross-Linking Reagents

KW - Enzyme Activation

KW - Fibrillar Collagens/metabolism

KW - Fibroblasts/cytology

KW - Humans

KW - Matrix Metalloproteinase 14

KW - Matrix Metalloproteinase 2/metabolism

KW - Matrix Metalloproteinases, Membrane-Associated

KW - Metalloendopeptidases/metabolism

KW - Procollagen-Proline Dioxygenase/antagonists & inhibitors

KW - Skin/cytology

KW - Time Factors

U2 - 10.1006/excr.2001.5403

DO - 10.1006/excr.2001.5403

M3 - Journal article

C2 - 11777335

SN - 0014-4827

VL - 272

SP - 109

EP - 118

JO - Experimental Cell Research

JF - Experimental Cell Research

IS - 2

ER -