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. 2009 Jun 19;284(34):23001–23011. doi: 10.1074/jbc.M109.002808

FIGURE 5.

FIGURE 5.

Defects in Mmp14−/− dermal fibroblast function are confined to the collagen-invasive phenotype. A, Mmp14+/+, Mmp14−/−, or Mmp14−/− fibroblasts transduced with control or MT1-MMP, MT1-MMPE→A, MT1-MMPΔCT, MT1-MMPΔHPX, MT1-MMPMMP-1CAT, or MT1-MMPMMP-1CAT/ΔHPX retroviral vectors were seeded atop collagen gels as islands in the center of each well and migration monitored in the presence of 10% FCS for 72 h. After 72 h the cells were stained with toluidine blue, and the distance migrated by the advancing front of cells was measured in 5 randomly selected fields (bar = 5 mm). Results are shown for three experiments (mean ± S.E.). B, proliferative activity of Mmp14+/+ or Mmp14−/− skin fibroblasts was measured by BrdUrd uptake in the presence of 0.5% FCS (control), 0.5% FCS, and fibroblast growth factor-2 (10 ng/ml) or 10% FCS as described under “Experimental Procedures.” BrdUrd uptake (green) in cells double-stained with propidium iodide (red) are marked by white arrows (bar, 50 μm) and quantified as the mean ± S.E. in 5 randomly selected fields in a single representative experiment of 3 performed. C and D, invasive activity of Mmp14+/+ or Mmp14−/− skin fibroblasts in acid-extracted, cross-linked type I collagen versus pepsin-extracted, non-cross-linked type I collagen gels. Mmp14+/+, Mmp14−/−, or Mmp14−/− skin fibroblasts transduced with MT1-MMPE→A, MT1-MMPΔCT, MT1-MMPΔHPX, MT1-MMPMMP-1CAT, or MT1-MMPMMP-1CAT/ΔHPX retroviral vectors were seeded atop either acid- or pepsin-extracted collagen gels in the presence of 10% FCS and stimulated with PDGF-BB for a 5-day culture period. The number of invading cells was determined in 10 randomly selected fields in a single representative experiment of 3 performed (C). As shown in hematoxylin and eosin-stained cross-sections, the invasion-null phenotype of Mmp14−/− fibroblasts cultured atop acid-extracted collagen is reversed after transduction with MT1-MMP or MT1-MMPMMP-1CAT but not MT1-MMPE→A retroviral expression vectors (bar = 100 μm). The double-headed arrows mark the boundaries of the collagen gel, whereas the black arrow marks the overlying layer of fibroblasts applied to the surface of the matrix.