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Contribution of MT1-MMP and of human laminin-5 2 chain degradation to mammary epithelial cell migration

Christine Gilles^1,*,, Myriam Polette^2,*, Christelle Coraux², Jean-Marie Tournier², Guerrino Meneguzzi³, Carine Munaut¹, Laure Volders¹, Patricia Rousselle⁴, Philippe Birembaut² and Jean-Michel Foidart¹

¹ Laboratory of Tumor and Developmental Biology, University of Liège, C.H.U. Sart-Tilman, B23, Liège, Belgium
² Unité INSERM U.514, Laboratoire Pol Bouin, IFR 53, C.H.U. Maison Blanche, Reims, France
³ Unité INSERM U.385, Faculty of Medicine, Nice, France
⁴ Institut de Biologie et de Chimie des Protéines, CNRS, U.P.R. 412, Lyon, France
^* These authors contributed equally to this research
Author for correspondence (e-mail: cgilles{at}ulg.ac.be )

View larger version (63K): [in a new window]   Fig. 1. In vitro migration assay. MCF10A cells were plated inside a glass ring and were allowed to migrate as an outgrowth after the ring removal. (A) Sequential video images of a migratory culture of MCF10A cells, taken immediately, 24, 48 or 72 hours after the removal of the ring and showing the expansion of the outgrowth. (B) Images of nuclei stained with Hoechst dye and visualized under epifluorescence illumination (a, at the edge of the outgrowth; c, distant from the outgrowth periphery). Trajectories of 20 randomly selected nuclei (indicated with a white dots in a and c) were quantified in each area (b, at the edge of the outgrowth; d, distant from the outgrowth periphery). Scale bars: 2.35 mm in A; 80 µm in B.

View larger version (16K): [in a new window]   Fig. 2. Differential expression of MMPs mRNA in migratory cultures of MCF10A cells. RT-PCR analyses for MT1-MMP, MMP-2, MMP-9, MMP-1, MMP-3 and MMP-11 were performed on total RNA extracted from migratory cultures of MCF10A cells.

View larger version (78K): [in a new window]   Fig. 3. Specific overexpression and subcellular organization of MT1-MMP in migratory MCF10A cells at the periphery of the outgrowth. (A) In situ hybridization on a migratory culture of MCF10A cells performed with a MT1-MMP antisense probe 72 hours after the removal of the ring. (B) MT1-MMP immunolabelling on a migratory culture of MCF10A cells. MT1-MMP labelling is in red and DAPI staining is in blue. (C) MT1-MMP immunolabelling (in red) on a migratory culture of MCF10A cells (a). Phase-contrast microscopy analysis of the corresponding area (b). (D) Confocal microscopy analyses of MT1-MMP in migratory MCF10A cells 72 hours after the removal of the ring. Twenty-four successive optical sections were taken from the apical (section 2) to the basal (section 22) surface of the cells (sections 2, 12, 15, 16, 20 and 22 are shown). MT1-MMP labelling was mainly found in the lamellipodia at the leading edge of migratory cells (arrows) as well as at the basal surface of the cells in contact with the substrate (section 16 and 20). Scale bars: 60 µm in A,B; 20µm in C; 10 µm in D.

View larger version (21K): [in a new window]   Fig. 4. Inhibitory effect of BB94, TIMP-2 and MT1-MMP antisense oligonucleotides on MCF10A cell migration. 72 hours after the removal of the ring, BB-94 (at 5x10-6M) and TIMP-2 (1 µg/ml) were added for 1 hour before the quantification of migration speed of cells at the periphery of the outgrowth. MT1-MMP oligonucleotides antisense (+) or scrambled control oligonucleotides (-) were added to the medium immediately after the ring removal for 144 hours before the quantification of the cell migration speed at the periphery of the outgrowth. *P<0.05.

View larger version (67K): [in a new window]   Fig. 5. Ln-5 deposition in migratory cultures and contribution to MCF10A cell migration. (A) Ln-5 immunolabelling of migratory cultures of MCF10A cells 72 hours after the removal of the ring. Ln-5 was detected mainly in migratory cells at the periphery of the wound (a) but was also found deposited in the matrix as clearly shown by traces left behind cells that have migrated out of the outgrowth (b, arrow). (B) Seventy-two hours after the removal of the ring, Ln-5 blocking antibodies (clone P3H9-2) were added for 4 hours before the quantification of cell migration speed at the periphery of the outgrowth. *P<0.05. (C) 3 and 6 integrins immunolabelling of migratory cultures of MCF10A cells. (a,c, periphery of the outgrowth; b,d, distant from the periphery of the outgrowth). Scale bars: 60 µm in Aa; 80 µm in Ab; 20 µm in C.

View larger version (41K): [in a new window]   Fig. 6. Ln-5 2 chain degradation in migratory cultures of MCF10A cells and cleavage of Ln-5 2 by MT1-MMP. (A) Western blot analyses of the Ln-5 2 subunit in migratory cultures of MCF10A cells incubated in complete growth medium (MCF10A/FCS) 72 hours after the removal of the ring, in purified Ln-5 from SCC25 cells used as a control and in purified Ln-5 incubated with increasing amounts of the catalytic domain of human MT1-MMP (100 ng and 500 ng). (B) Western blot analyses of the Ln-5 2 subunit 72 hours after the removal of the ring in migratory cultures of MCF10A cells incubated in complete growth medium (MCF10A/FCS), in EGF-induced migratory cultures (MCF10A/EGF) and in stationary cultures incubated in EGF/FCS-free medium (Control). (C) Western blot analyses of MT1-MMP in migratory cultures of MCF10A cells incubated in complete growth medium (MCF10A/FCS), in EGF-induced migratory cultures (MCF10A/EGF) and in stationary cultures incubated in EGF/FCS-free medium (Control). HT1080 cells are used as a control showing the three main forms of MT1-MMP at 63, 60 and 43 kDa.