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Membrane-type 1 matrix metalloprotease (MT1-MMP) enables invasive migration of glioma cells in central nervous system white matter.

Beliën AT, Paganetti PA, Schwab ME - J. Cell Biol. (1999)

Bottom Line: Plasma membranes of both MT1-MMP-transfected fibroblasts and C6 glioma cells inactivated inhibitory myelin extracts, and this activity was sensitive to the same protease inhibitors.Interestingly, pretreatment of CNS myelin with gelatinase A/MMP-2 could not inactivate its inhibitory property.These data imply an important role of MT1-MMP in spreading and migration of glioma cells on white matter constituents in vitro and point to a function of MT1-MMP in the invasive behavior of malignant gliomas in the CNS in vivo.

View Article: PubMed Central - PubMed

Affiliation: Brain Research Institute, University of Zurich and Swiss Federal Institute of Technology, 8057 Zurich, Switzerland. belien@hifo.unizh.ch

ABSTRACT
Invasive glioma cells migrate preferentially along central nervous system (CNS) white matter fiber tracts irrespective of the fact that CNS myelin contains proteins that inhibit cell migration and neurite outgrowth. Previous work has demonstrated that to migrate on a myelin substrate and to overcome its inhibitory effect, rat C6 and human glioblastoma cells require a membrane-bound metalloproteolytic activity (C6-MP) which shares several biochemical and pharmacological characteristics with MT1-MMP. We show now that MT1-MMP is expressed on the surface of rat C6 glioblastoma cells and is coenriched with C6-MP activity. Immunodepletion of C6-MP activity is achieved with an anti-MT1-MMP antibody. These data suggest that MT1-MMP and the C6-MP are closely related or identical. When mouse 3T3 fibroblasts were transfected with MT1-MMP they acquired the ability to spread and migrate on the nonpermissive myelin substrate and to infiltrate into adult rat optic nerve explants. MT1-MMP-transfected fibroblasts and C6 glioma cells were able to digest bNI-220, one of the most potent CNS myelin inhibitory proteins. Plasma membranes of both MT1-MMP-transfected fibroblasts and C6 glioma cells inactivated inhibitory myelin extracts, and this activity was sensitive to the same protease inhibitors. Interestingly, pretreatment of CNS myelin with gelatinase A/MMP-2 could not inactivate its inhibitory property. These data imply an important role of MT1-MMP in spreading and migration of glioma cells on white matter constituents in vitro and point to a function of MT1-MMP in the invasive behavior of malignant gliomas in the CNS in vivo.

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Western blot of bNI-220–enriched CNS protein extract using a polyclonal rabbit antibody against bNI-220 (Huber  et al., 1998). CNS protein extract enriched for bNI-220 was preincubated for 1 h at 37°C in the presence of plasma membranes of  C6 cells and transfected fibroblasts. The protein fractions then  were electrophoresed on a 5% SDS polyacrylamide gel, blotted  onto a nitrocellulose membrane, and then probed with purified  anti–bNI-220 polyclonal rabbit antibody. Bound antibody was visualized with an peroxidase-conjugated secondary antibody followed by chemiluminescent detection. Plasma membranes of C6  glioma cells (PM C6, lane 1) and MT1-MMP–transfected fibroblasts (PM MT, lane 2) could degrade the bNI-220 completely  (C6 glioma cells) or partially (MT1-MMP–transfected fibroblasts), whereas mock-transfected fibroblast plasma membranes  could not (PM Mo, lane 3). Digestion of bNI-220 in the presence  of PM MT was blocked by 100 nM TIMP2 (lane 4), but not by  100 nM TIMP1 (lane 5) or 1 μM BB94 (lane 6). Arrow indicates  the bNI-220 level, arrowheads point to a nonspecific band, indicating that in all lanes equal amounts of protein were loaded.
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Figure 11: Western blot of bNI-220–enriched CNS protein extract using a polyclonal rabbit antibody against bNI-220 (Huber et al., 1998). CNS protein extract enriched for bNI-220 was preincubated for 1 h at 37°C in the presence of plasma membranes of C6 cells and transfected fibroblasts. The protein fractions then were electrophoresed on a 5% SDS polyacrylamide gel, blotted onto a nitrocellulose membrane, and then probed with purified anti–bNI-220 polyclonal rabbit antibody. Bound antibody was visualized with an peroxidase-conjugated secondary antibody followed by chemiluminescent detection. Plasma membranes of C6 glioma cells (PM C6, lane 1) and MT1-MMP–transfected fibroblasts (PM MT, lane 2) could degrade the bNI-220 completely (C6 glioma cells) or partially (MT1-MMP–transfected fibroblasts), whereas mock-transfected fibroblast plasma membranes could not (PM Mo, lane 3). Digestion of bNI-220 in the presence of PM MT was blocked by 100 nM TIMP2 (lane 4), but not by 100 nM TIMP1 (lane 5) or 1 μM BB94 (lane 6). Arrow indicates the bNI-220 level, arrowheads point to a nonspecific band, indicating that in all lanes equal amounts of protein were loaded.

Mentions: A bovine spinal cord detergent extract enriched for bNI-220 over an ion-exchange column was preincubated with plasma membranes of mock- and MT1-MMP–transfected fibroblasts. Western blots with an affinity-purified rabbit polyclonal antibody against a bNI-220 peptide sequence (Ab 472; Huber et al., 1998) revealed disappearance or diminution of the bNI-220 band after treatment with plasma membranes of MT1-MMP–transfected fibroblasts or of C6 glioma cells, but not after treatment with membranes of mock-transfected fibroblasts (Fig. 11). This degradation could be inhibited by the addition of 100 nM TIMP2, but not by the addition of 1 μM BB94 or 100 nM TIMP1 (Fig. 11). We conclude that NI-220 can be degraded specifically by MT1-MMP–enriched plasma membranes.


Membrane-type 1 matrix metalloprotease (MT1-MMP) enables invasive migration of glioma cells in central nervous system white matter.

Beliën AT, Paganetti PA, Schwab ME - J. Cell Biol. (1999)

Western blot of bNI-220–enriched CNS protein extract using a polyclonal rabbit antibody against bNI-220 (Huber  et al., 1998). CNS protein extract enriched for bNI-220 was preincubated for 1 h at 37°C in the presence of plasma membranes of  C6 cells and transfected fibroblasts. The protein fractions then  were electrophoresed on a 5% SDS polyacrylamide gel, blotted  onto a nitrocellulose membrane, and then probed with purified  anti–bNI-220 polyclonal rabbit antibody. Bound antibody was visualized with an peroxidase-conjugated secondary antibody followed by chemiluminescent detection. Plasma membranes of C6  glioma cells (PM C6, lane 1) and MT1-MMP–transfected fibroblasts (PM MT, lane 2) could degrade the bNI-220 completely  (C6 glioma cells) or partially (MT1-MMP–transfected fibroblasts), whereas mock-transfected fibroblast plasma membranes  could not (PM Mo, lane 3). Digestion of bNI-220 in the presence  of PM MT was blocked by 100 nM TIMP2 (lane 4), but not by  100 nM TIMP1 (lane 5) or 1 μM BB94 (lane 6). Arrow indicates  the bNI-220 level, arrowheads point to a nonspecific band, indicating that in all lanes equal amounts of protein were loaded.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132902&req=5

Figure 11: Western blot of bNI-220–enriched CNS protein extract using a polyclonal rabbit antibody against bNI-220 (Huber et al., 1998). CNS protein extract enriched for bNI-220 was preincubated for 1 h at 37°C in the presence of plasma membranes of C6 cells and transfected fibroblasts. The protein fractions then were electrophoresed on a 5% SDS polyacrylamide gel, blotted onto a nitrocellulose membrane, and then probed with purified anti–bNI-220 polyclonal rabbit antibody. Bound antibody was visualized with an peroxidase-conjugated secondary antibody followed by chemiluminescent detection. Plasma membranes of C6 glioma cells (PM C6, lane 1) and MT1-MMP–transfected fibroblasts (PM MT, lane 2) could degrade the bNI-220 completely (C6 glioma cells) or partially (MT1-MMP–transfected fibroblasts), whereas mock-transfected fibroblast plasma membranes could not (PM Mo, lane 3). Digestion of bNI-220 in the presence of PM MT was blocked by 100 nM TIMP2 (lane 4), but not by 100 nM TIMP1 (lane 5) or 1 μM BB94 (lane 6). Arrow indicates the bNI-220 level, arrowheads point to a nonspecific band, indicating that in all lanes equal amounts of protein were loaded.
Mentions: A bovine spinal cord detergent extract enriched for bNI-220 over an ion-exchange column was preincubated with plasma membranes of mock- and MT1-MMP–transfected fibroblasts. Western blots with an affinity-purified rabbit polyclonal antibody against a bNI-220 peptide sequence (Ab 472; Huber et al., 1998) revealed disappearance or diminution of the bNI-220 band after treatment with plasma membranes of MT1-MMP–transfected fibroblasts or of C6 glioma cells, but not after treatment with membranes of mock-transfected fibroblasts (Fig. 11). This degradation could be inhibited by the addition of 100 nM TIMP2, but not by the addition of 1 μM BB94 or 100 nM TIMP1 (Fig. 11). We conclude that NI-220 can be degraded specifically by MT1-MMP–enriched plasma membranes.

Bottom Line: Plasma membranes of both MT1-MMP-transfected fibroblasts and C6 glioma cells inactivated inhibitory myelin extracts, and this activity was sensitive to the same protease inhibitors.Interestingly, pretreatment of CNS myelin with gelatinase A/MMP-2 could not inactivate its inhibitory property.These data imply an important role of MT1-MMP in spreading and migration of glioma cells on white matter constituents in vitro and point to a function of MT1-MMP in the invasive behavior of malignant gliomas in the CNS in vivo.

View Article: PubMed Central - PubMed

Affiliation: Brain Research Institute, University of Zurich and Swiss Federal Institute of Technology, 8057 Zurich, Switzerland. belien@hifo.unizh.ch

ABSTRACT
Invasive glioma cells migrate preferentially along central nervous system (CNS) white matter fiber tracts irrespective of the fact that CNS myelin contains proteins that inhibit cell migration and neurite outgrowth. Previous work has demonstrated that to migrate on a myelin substrate and to overcome its inhibitory effect, rat C6 and human glioblastoma cells require a membrane-bound metalloproteolytic activity (C6-MP) which shares several biochemical and pharmacological characteristics with MT1-MMP. We show now that MT1-MMP is expressed on the surface of rat C6 glioblastoma cells and is coenriched with C6-MP activity. Immunodepletion of C6-MP activity is achieved with an anti-MT1-MMP antibody. These data suggest that MT1-MMP and the C6-MP are closely related or identical. When mouse 3T3 fibroblasts were transfected with MT1-MMP they acquired the ability to spread and migrate on the nonpermissive myelin substrate and to infiltrate into adult rat optic nerve explants. MT1-MMP-transfected fibroblasts and C6 glioma cells were able to digest bNI-220, one of the most potent CNS myelin inhibitory proteins. Plasma membranes of both MT1-MMP-transfected fibroblasts and C6 glioma cells inactivated inhibitory myelin extracts, and this activity was sensitive to the same protease inhibitors. Interestingly, pretreatment of CNS myelin with gelatinase A/MMP-2 could not inactivate its inhibitory property. These data imply an important role of MT1-MMP in spreading and migration of glioma cells on white matter constituents in vitro and point to a function of MT1-MMP in the invasive behavior of malignant gliomas in the CNS in vivo.

Show MeSH
Related in: MedlinePlus