Figure 3.

Characterization of MT1-MMP–overexpressing MDCK cells. A, Proliferative response of wild-type (▪), vector control-transfected (•), full-length MT1-MMP (□), cytosolic tail-deleted MT1-MMP (MT1-MMP_ct; ▵), and soluble MT1-MMP (ΔMT1-MMP; ○) during a 7-d culture period. Cell number at each time point was measured in triplicate and the error bars represent the SD of a single representative experiment of three performed. The inset shows Western blots of MT1-MMP expression in either control or MT-MMP transfectants as indicated. Asterisks (*) mark the position of active MT1-MMP species whereas the arrowheads mark the position of a membrane-anchored, catalytically inactive fragment of MT1-MMP (M_r ∼43 kD; Lehti et al. 1998). MT1-MMP stable transfectants did not express detectable levels of MT2-MMP or MT3-MMP, as assessed by Western or Northern blot analysis, respectively. B, Epithelial morphology, E-cadherin staining, and SF/HGF-induced scattering responses of control or MT1-MMP–transfected cells cultured as described in Materials and Methods in either the absence or presence of SF/HGF. C, In the presence of SF/HGF, control vector-transfected MDCK cells cultured atop type I collagen gels for 3 d display widespread, but shallow foci of invasion (arrows) as viewed en face (top; bar, 100 μm), in cross-section (middle; bar, 100 μm), or by TEM (bottom; bar, 5 μm). In contrast, MDCK cells overexpressing MT1-MMP generated numerous large pits in the collagen matrix (arrows) extending well below the surface monolayer as viewed en face (top), in cross-section by light microscopy (middle) or by TEM (bottom). D, After 12 d, MT1-MMP overexpressing cells cultured in the absence of SF/HGF formed pits in the underlying collagen gel whereas SF/HGF-stimulated cells generated interconnecting cyst-like structures extending to the surface of the underlying polycarbonate membrane (compare to Fig. 1, A–D). Invasion by MT1-MMP transfectants was inhibited completely by TIMP-2, but not TIMP-1. Bar, 100 μm.