Role of cell surface metalloprotease MT1-MMP in epithelial cell migration over laminin-5 - PubMed (original) (raw)

Role of cell surface metalloprotease MT1-MMP in epithelial cell migration over laminin-5

N Koshikawa et al. J Cell Biol. 2000.

Erratum in

Abstract

Laminin-5 (Ln-5) is an extracellular matrix substrate for cell adhesion and migration, which is found in many epithelial basement membranes. Mechanisms eliciting migration on Ln-5 need to be elucidated because of their relevance to tissue remodeling and cancer metastasis. We showed that exogenous addition of activated matrix metalloprotease (MMP) 2 stimulates migration onto Ln-5 in breast epithelial cells via cleavage of the gamma2 subunit. To investigate the biological scope of this proteolytic mechanism, we tested a panel of cells, including colon and breast carcinomas, hepatomas, and immortalized hepatocytes, selected because they migrated or scattered constitutively in the presence of Ln-5. We found that constitutive migration was inhibited by BB94 or TIMPs, known inhibitors of MMPs. Limited profiling by gelatin zymography and Western blotting indicated that the ability to constitutively migrate on Ln-5 correlated with expression of plasma membrane bound MT1-MMP metalloprotease, rather than secretion of MMP2, since MMP2 was not produced by three cell lines (one breast and two colon carcinomas) that constitutively migrated on Ln-5. Moreover, migration on Ln-5 was reduced by MT1-MMP antisense oligonucleotides both in MMP2+ and MMP2- cell lines. MT1-MMP directly cleaved Ln-5, with a pattern similar to that of MMP2. The hemopexin-like domain of MMP2, which interferes with MMP2 activation, reduced Ln-5 migration in MT1-MMP+, MMP2+ cells, but not in MT1-MMP+, MMP2- cells. These results suggest a model whereby expression of MT1-MMP is the primary trigger for migration over Ln-5, whereas MMP2, which is activated by MT1-MMP, may play an ancillary role, perhaps by amplifying the MT1-MMP effects. Codistribution of MT1-MMP with Ln-5 in colon and breast cancer tissue specimens suggested a role for this mechanism in invasion. Thus, Ln-5 cleavage by MMPs may be a widespread mechanism that triggers migration in cells contacting epithelial basement membranes.

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Figures

Figure 1

Figure 1

Effects of the hydroxamate MMP inhibitor BB94 on constitutive cell migration over Ln-5. Indicated cell lines were incubated under optimized conditions (see Materials and Methods for experimental details) in the upper compartment of Transwell chambers, which contained purified Ln-5 (200 or 400 ng/ml) in the lower compartment. Constitutive migration occurred in all cases. Likewise, BB94 blocked migration in a dose-dependent fashion. Each bar represents the mean + SD of three wells. The same results were obtained by coating the lower surface of Transwell filters with Ln-5, rather than adding soluble Ln-5 to the lower chamber.

Figure 3

Figure 3

Inhibitory effects of mAb MIG-1 on Ln-5 BRL cell scattering and migration. (A) BRL cells were incubated in control medium (None) or Ln-5 (60 ng/ml) in the presence of mAbs to Ln5 TR-1 (control) or MIG-1 (20 μg/ml), which blocks migration induced by MMP2-cleaved Ln-5. Cell morphology was examined under a phase-contrast microscope after incubation for 16 h. MIG-1, not TR-1, inhibits scattering induced by Ln-5. (B) Transwell chamber migration of BRL cells, stimulated by Ln-5 (200 ng/ml), is blocked by MIG-1, not TR-1 control, in a dose-dependent fashion. Each bar represents the mean + SD of three wells.

Figure 3

Figure 3

Inhibitory effects of mAb MIG-1 on Ln-5 BRL cell scattering and migration. (A) BRL cells were incubated in control medium (None) or Ln-5 (60 ng/ml) in the presence of mAbs to Ln5 TR-1 (control) or MIG-1 (20 μg/ml), which blocks migration induced by MMP2-cleaved Ln-5. Cell morphology was examined under a phase-contrast microscope after incubation for 16 h. MIG-1, not TR-1, inhibits scattering induced by Ln-5. (B) Transwell chamber migration of BRL cells, stimulated by Ln-5 (200 ng/ml), is blocked by MIG-1, not TR-1 control, in a dose-dependent fashion. Each bar represents the mean + SD of three wells.

Figure 2

Figure 2

(A) Effect of TIMPs on BRL migration over Ln-5. BRL cells were incubated in Transwell chambers, in control medium or in the presence of Ln-5 (200 ng/ml) and the indicated concentrations of MMP inhibitors in the lower chamber. In the presence of Ln-5, BRL cells migrate constitutively. Both TIMP-1 and TIMP-2 inhibit migration, to levels comparable to BB94. Each bar represents the mean + SD for cell migration of two wells. (B) Lack of inhibition by BB94 of scattering induced by MMP2-cleaved Ln-5. BRL cells were incubated in 24-well culture plates in control medium (top), Ln-5 (80 ng/ml) (middle) or MMP2-cleaved Ln-5 (80 ng/ml) (bottom), in the presence of BB94, 500 nM or 2 μM. Cell morphology was examined under a phase-contrast microscope after incubation for 16 h. Scattering occurs with both intact Ln-5 and cleaved Ln-5. However, BB94 inhibits scattering induced by Ln-5, not by cleaved Ln-5, further supporting involvement of MMPs in the constitutive scattering of BRL cells.

Figure 2

Figure 2

(A) Effect of TIMPs on BRL migration over Ln-5. BRL cells were incubated in Transwell chambers, in control medium or in the presence of Ln-5 (200 ng/ml) and the indicated concentrations of MMP inhibitors in the lower chamber. In the presence of Ln-5, BRL cells migrate constitutively. Both TIMP-1 and TIMP-2 inhibit migration, to levels comparable to BB94. Each bar represents the mean + SD for cell migration of two wells. (B) Lack of inhibition by BB94 of scattering induced by MMP2-cleaved Ln-5. BRL cells were incubated in 24-well culture plates in control medium (top), Ln-5 (80 ng/ml) (middle) or MMP2-cleaved Ln-5 (80 ng/ml) (bottom), in the presence of BB94, 500 nM or 2 μM. Cell morphology was examined under a phase-contrast microscope after incubation for 16 h. Scattering occurs with both intact Ln-5 and cleaved Ln-5. However, BB94 inhibits scattering induced by Ln-5, not by cleaved Ln-5, further supporting involvement of MMPs in the constitutive scattering of BRL cells.

Figure 4

Figure 4

(A and B) Gelatin zymography of (A) conditioned media (CM) (60 μl/lane) or (B) crude plasma membrane fractions (2 μg/lane) from BRL, MCF-10A, and HT-1080 cells. The positions of pro-MMP9 (92 kD), pro-MMP2 (72 kD), and activated MMP2 (62 kD) are indicated. Note the presence of activated MMP2 in BRL, but not MCF-10A plasma membrane fractions. (C and D) Western blotting of BRL, MCF-10A, or HT-1080 conditioned media (CM) (300 μl/each lane) or crude plasma membrane fractions (2 μg/each lane) with antibodies to TIMP-2 (C) or MT1-MMP (D). In C, purified human TIMP-2 (50 ng) was run in the indicated lane. The respective positions of TIMP-2 and MT1-MMP are indicated to the right of gels. (asterisk) Processed form of MT1-MMP (see text).

Figure 4

Figure 4

(A and B) Gelatin zymography of (A) conditioned media (CM) (60 μl/lane) or (B) crude plasma membrane fractions (2 μg/lane) from BRL, MCF-10A, and HT-1080 cells. The positions of pro-MMP9 (92 kD), pro-MMP2 (72 kD), and activated MMP2 (62 kD) are indicated. Note the presence of activated MMP2 in BRL, but not MCF-10A plasma membrane fractions. (C and D) Western blotting of BRL, MCF-10A, or HT-1080 conditioned media (CM) (300 μl/each lane) or crude plasma membrane fractions (2 μg/each lane) with antibodies to TIMP-2 (C) or MT1-MMP (D). In C, purified human TIMP-2 (50 ng) was run in the indicated lane. The respective positions of TIMP-2 and MT1-MMP are indicated to the right of gels. (asterisk) Processed form of MT1-MMP (see text).

Figure 5

Figure 5

Cleavage of Ln-5 by MT1-MMP. (A) Ln-5 (1 μg) was treated with recombinant soluble MT1-MMP at the indicated concentrations, electrophoresed by 6% SDS-PAGE under reducing conditions, and then analyzed by Western blotting with a polyclonal antibody (1963) against rat Ln-5 γ2 chain. The positions of Ln-5 γ2 (140 kD), γ2′ (100 kD), and γ2x (80 kD) chains are indicated. In the presence of increasing concentrations of MT1-MMP, the γ2 appears to chase into the γ2′ and the γ2x chain. (B) Schematic depiction of the position of the MMP2 cleavage site (Giannelli et al. 1997) and the predicted position of the MT1-MMP sites. The γ2′ has been described (Vailly et al. 1994). (C) Scheme of the predicted size of MMP2 and MT1-MMP cleavage products of the Ln-5 γ2 chain.

Figure 7

Figure 7

Effect of MT1-MMP antisense oligonucleotides on Ln-5 migration by BRL or HT-29 cells. (A) Cells were pretreated for 2 d in culture with MT1-MMP antisense (AS) or control, scrambled (Cont.) oligonucleotides, and tested in migration assays. Ln-5 and oligonucleotides, at the same concentrations as treatment, or PBS were present throughout the assay in the lower Transwell chambers. Each bar represents the mean + SD for cell migration of two (BRL) or four (HT-29) wells. (B) BRL cells were pretreated with MT1-MMP AS or control oligonucleotides, and tested in the scattering assay as in Fig. 2, in the presence of either intact or MMP2-cleaved Ln-5. Addition of either intact or MMP2-cleaved Ln-5 causes a morphological change in BRL cells, which loosen cell–cell contacts, elongate, and scatter. MT1-MMP AS treatment inhibits these morphological changes when they are induced by intact Ln-5. In contrast, cleaved Ln-5 still causes scattering of AS-treated cells, supporting a link between reduction of MT1-MMP expression, Ln-5 cleavage and scattering response.

Figure 7

Figure 7

Effect of MT1-MMP antisense oligonucleotides on Ln-5 migration by BRL or HT-29 cells. (A) Cells were pretreated for 2 d in culture with MT1-MMP antisense (AS) or control, scrambled (Cont.) oligonucleotides, and tested in migration assays. Ln-5 and oligonucleotides, at the same concentrations as treatment, or PBS were present throughout the assay in the lower Transwell chambers. Each bar represents the mean + SD for cell migration of two (BRL) or four (HT-29) wells. (B) BRL cells were pretreated with MT1-MMP AS or control oligonucleotides, and tested in the scattering assay as in Fig. 2, in the presence of either intact or MMP2-cleaved Ln-5. Addition of either intact or MMP2-cleaved Ln-5 causes a morphological change in BRL cells, which loosen cell–cell contacts, elongate, and scatter. MT1-MMP AS treatment inhibits these morphological changes when they are induced by intact Ln-5. In contrast, cleaved Ln-5 still causes scattering of AS-treated cells, supporting a link between reduction of MT1-MMP expression, Ln-5 cleavage and scattering response.

Figure 6

Figure 6

Schematic model for mechanisms of MMP-dependent Ln-5 cell migration.

Figure 6

Figure 6

Schematic model for mechanisms of MMP-dependent Ln-5 cell migration.

Figure 8

Figure 8

Effect of MMP2 HLD on Ln-5 migration by BRL or HT-29 cell. Purified HLD was added to the lower Transwell chambers at 0, 1, or 3 μg/ml. Results shown are from two separate, representative experiments (1 and 2) performed on different occasions. Each bar represents the mean + SD for cell migration of four wells.

Figure 9

Figure 9

Detection of Ln-5, MT1-MMP, and MMP2 in human colon carcinoma tissue specimens by double immunofluorescence and confocal microscopy. Cryostat sections were double immunostained for either Ln-5 and MT1-MMP or Ln-5 and MMP2. Expression of Ln-5 is discrete, in some cases limited to the outside layer of cell nests. Colocalization with MT1-MMP and MMP2 is shown in the panels on the right (yellow) by combining the specific fluorophore spectra recorded for Ln-5 (green) and MT1-MMP or MMP2 (red). Examples of colocalization are indicated by arrowheads. A slight difference in the appearance of Ln-5–specific immunoreactivity in top and middle panels is due to the use of either mouse mAb D4B5 (combination with rabbit anti–MT1-MMP) or rabbit polyclonal 1963 (combination with mouse monoclonal to MMP2). Control sections incubated with normal mouse IgGs and rabbit IgGs were completely negative (not shown). Bar size, 43 μm.

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