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 -