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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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MT1-MMP Western blot and remodeling of the CNS  inhibitory substrate. (a) Western blots of cell lysates and salt-washed plasma membranes using a monoclonal antibody against  MT1-MMP. The protein fractions were electrophoresed on a  10% SDS polyacrylamide gel, blotted onto a nitrocellulose membrane, and probed with MT1-MMP antibody. Bound antibody  was visualized by peroxidase-conjugated second antibody and  chemiluminescence. Lane 1, C6 cell homogenate (CH C6); lane  2, C6 cell plasma membranes (PM C6); lane 3, plasma membranes of MT1-MMP–transfected fibroblasts (PM MT); lane 4,  plasma membranes of mock-transfected fibroblasts (PM Mo).  Molecular mass markers are indicated at the left side, proMT1-MMP and processed MT1-MMP (arrows) on the right. Two nonspecific bands are seen between the pro- and mature MT1-MMP  forms since they are visualized also in the absence of primary antibody. (b) Culture dishes coated with inhibitory CNS membrane  proteins were pretreated in different ways and their inhibitory  activity assayed for 3T3 fibroblast spreading. Treatment with C6  homogenate (50 μg) or plasma membranes (30 μg) of C6 glioma  cells or MT1-MMP–transfected fibroblasts reduced the inhibitory  property significantly, whereas plasma membranes of mock-transfected fibroblasts did not. Pretreatment of the CNS substrate with gelatinase A/MMP-2 (0.1, 1, and 10 μM) did not influence its nonpermissive substrate property. Values are means ±  SEM of three experiments performed in triplicate. Significance  with the two-tailed Student's t test: P < 0.01 (**).
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Figure 2: MT1-MMP Western blot and remodeling of the CNS inhibitory substrate. (a) Western blots of cell lysates and salt-washed plasma membranes using a monoclonal antibody against MT1-MMP. The protein fractions were electrophoresed on a 10% SDS polyacrylamide gel, blotted onto a nitrocellulose membrane, and probed with MT1-MMP antibody. Bound antibody was visualized by peroxidase-conjugated second antibody and chemiluminescence. Lane 1, C6 cell homogenate (CH C6); lane 2, C6 cell plasma membranes (PM C6); lane 3, plasma membranes of MT1-MMP–transfected fibroblasts (PM MT); lane 4, plasma membranes of mock-transfected fibroblasts (PM Mo). Molecular mass markers are indicated at the left side, proMT1-MMP and processed MT1-MMP (arrows) on the right. Two nonspecific bands are seen between the pro- and mature MT1-MMP forms since they are visualized also in the absence of primary antibody. (b) Culture dishes coated with inhibitory CNS membrane proteins were pretreated in different ways and their inhibitory activity assayed for 3T3 fibroblast spreading. Treatment with C6 homogenate (50 μg) or plasma membranes (30 μg) of C6 glioma cells or MT1-MMP–transfected fibroblasts reduced the inhibitory property significantly, whereas plasma membranes of mock-transfected fibroblasts did not. Pretreatment of the CNS substrate with gelatinase A/MMP-2 (0.1, 1, and 10 μM) did not influence its nonpermissive substrate property. Values are means ± SEM of three experiments performed in triplicate. Significance with the two-tailed Student's t test: P < 0.01 (**).

Mentions: To biochemically evaluate the localization of MT1-MMP, a C6 total cell homogenate and a salt-washed C6 plasma membrane fraction were prepared. These fractions were separated on 10% SDS-PAGE and blotted. Incubation of the blots with anti–MT1-MMP antibody revealed two specific bands: one at the molecular weight of proMT1-MMP (63 kD) and another band at 58 kD, corresponding to the molecular weight of active MT1-MMP (Fig. 2 a) (Ohuchi et al., 1997). The 58-kD band was clearly more intense in the plasma membrane fraction than in the cell homogenate fraction.


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)

MT1-MMP Western blot and remodeling of the CNS  inhibitory substrate. (a) Western blots of cell lysates and salt-washed plasma membranes using a monoclonal antibody against  MT1-MMP. The protein fractions were electrophoresed on a  10% SDS polyacrylamide gel, blotted onto a nitrocellulose membrane, and probed with MT1-MMP antibody. Bound antibody  was visualized by peroxidase-conjugated second antibody and  chemiluminescence. Lane 1, C6 cell homogenate (CH C6); lane  2, C6 cell plasma membranes (PM C6); lane 3, plasma membranes of MT1-MMP–transfected fibroblasts (PM MT); lane 4,  plasma membranes of mock-transfected fibroblasts (PM Mo).  Molecular mass markers are indicated at the left side, proMT1-MMP and processed MT1-MMP (arrows) on the right. Two nonspecific bands are seen between the pro- and mature MT1-MMP  forms since they are visualized also in the absence of primary antibody. (b) Culture dishes coated with inhibitory CNS membrane  proteins were pretreated in different ways and their inhibitory  activity assayed for 3T3 fibroblast spreading. Treatment with C6  homogenate (50 μg) or plasma membranes (30 μg) of C6 glioma  cells or MT1-MMP–transfected fibroblasts reduced the inhibitory  property significantly, whereas plasma membranes of mock-transfected fibroblasts did not. Pretreatment of the CNS substrate with gelatinase A/MMP-2 (0.1, 1, and 10 μM) did not influence its nonpermissive substrate property. Values are means ±  SEM of three experiments performed in triplicate. Significance  with the two-tailed Student's t test: P < 0.01 (**).
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Related In: Results  -  Collection

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Figure 2: MT1-MMP Western blot and remodeling of the CNS inhibitory substrate. (a) Western blots of cell lysates and salt-washed plasma membranes using a monoclonal antibody against MT1-MMP. The protein fractions were electrophoresed on a 10% SDS polyacrylamide gel, blotted onto a nitrocellulose membrane, and probed with MT1-MMP antibody. Bound antibody was visualized by peroxidase-conjugated second antibody and chemiluminescence. Lane 1, C6 cell homogenate (CH C6); lane 2, C6 cell plasma membranes (PM C6); lane 3, plasma membranes of MT1-MMP–transfected fibroblasts (PM MT); lane 4, plasma membranes of mock-transfected fibroblasts (PM Mo). Molecular mass markers are indicated at the left side, proMT1-MMP and processed MT1-MMP (arrows) on the right. Two nonspecific bands are seen between the pro- and mature MT1-MMP forms since they are visualized also in the absence of primary antibody. (b) Culture dishes coated with inhibitory CNS membrane proteins were pretreated in different ways and their inhibitory activity assayed for 3T3 fibroblast spreading. Treatment with C6 homogenate (50 μg) or plasma membranes (30 μg) of C6 glioma cells or MT1-MMP–transfected fibroblasts reduced the inhibitory property significantly, whereas plasma membranes of mock-transfected fibroblasts did not. Pretreatment of the CNS substrate with gelatinase A/MMP-2 (0.1, 1, and 10 μM) did not influence its nonpermissive substrate property. Values are means ± SEM of three experiments performed in triplicate. Significance with the two-tailed Student's t test: P < 0.01 (**).
Mentions: To biochemically evaluate the localization of MT1-MMP, a C6 total cell homogenate and a salt-washed C6 plasma membrane fraction were prepared. These fractions were separated on 10% SDS-PAGE and blotted. Incubation of the blots with anti–MT1-MMP antibody revealed two specific bands: one at the molecular weight of proMT1-MMP (63 kD) and another band at 58 kD, corresponding to the molecular weight of active MT1-MMP (Fig. 2 a) (Ohuchi et al., 1997). The 58-kD band was clearly more intense in the plasma membrane fraction than in the cell homogenate fraction.

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