Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution.
Therefore, the elucidation of their identities and functions is of great interest.Here, we show that matrix metalloproteinases (MMPs) generate a domain (DIII) from the ECM macromolecule laminin-5.These findings indicate that ECM cues may operate via direct stimulation of receptor tyrosine kinases in tissue remodeling, and possibly cancer invasion.
Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. sschenk@scripps
Extracellular matrix (ECM) fragments or cryptic sites unmasked by proteinases have been postulated to affect tissue remodeling and cancer progression. Therefore, the elucidation of their identities and functions is of great interest. Here, we show that matrix metalloproteinases (MMPs) generate a domain (DIII) from the ECM macromolecule laminin-5. Binding of a recombinant DIII fragment to epidermal growth factor receptor stimulates downstream signaling (mitogen-activated protein kinase), MMP-2 gene expression, and cell migration. Appearance of this cryptic ECM ligand in remodeling mammary gland coincides with MMP-mediated involution in wild-type mice, but not in tissue inhibitor of metalloproteinase 3 (TIMP-3)-deficient mice, supporting physiological regulation of DIII liberation. These findings indicate that ECM cues may operate via direct stimulation of receptor tyrosine kinases in tissue remodeling, and possibly cancer invasion.
- Breast/cytology/drug effects/enzymology*
- Cell Adhesion Molecules/metabolism*
- Epidermal Growth Factor/metabolism*
- Epithelial Cells/cytology/drug effects/enzymology*
- Extracellular Matrix Proteins/genetics/metabolism*
- Matrix Metalloproteinases/metabolism*
- Receptor, Epidermal Growth Factor/metabolism*
- Basement Membrane/metabolism
- Breast Neoplasms/genetics/metabolism
- Cell Movement/genetics
- Cells, Cultured
- Matrix Metalloproteinase 2/metabolism
- Mice, Knockout
- Mitogen-Activated Protein Kinases/metabolism
- Neoplasm Invasiveness/genetics
- Peptide Fragments/metabolism
- Protein Binding/genetics
- Protein Structure, Tertiary/genetics
- Receptor Protein-Tyrosine Kinases/metabolism
- Tissue Inhibitor of Metalloproteinase-3/deficiency/genetics
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fig8: Breast cell migration stimulated by rDIII. (A) Micrographs of the lower surfaces of Transwell filters after migration of MDA-MB-231 (a–c), MCF-7 (d–f), or MCF10A (g–i) cells on coated Ln-5. The micrographs a, d, and g show migration on coated Ln-5 only. rDIII was added at increasing concentrations to the bottom (MDA-MB-231, 6 nM, b and 12 nM, c) or top chambers (MCF-7, 70 nM, e 185 nM, f and MCF10A, 70 nM, h and 460 nM, i). The magnification used to count and photograph migrated cells may differ between cell lines and assays depending on how heavily cells migrated. The results are summarized in the corresponding bar graphs. MDA-MB-231: *p (b and a) = 0.0044, **p (c and a) = 2.18E-08, ANOVA p = 6.38E-08; MCF-7: *p (e and d) = 2.96E-07, **p (f and d) = 3.07E-11, ANOVA p = 2.36E-16; MCF10A: *p (h and g) = 6.94E-07, **p (i and g) = 1.41E-07, ANOVA p = 3.43E-11. Micrographs shown are representative for several independent experiments. (B) Migration of MDA-MB-231 cells on coated Ln-5 challenged with rDIII. The effect of rDIII or EGF on MDA-MB-231 cell migration in the absence and presence of LA1, as well as constitutive migration on Ln-5 (in the absence of any stimuli) and its dependency on EGFR is depicted. Note that if membranes were not coated with Ln-5, MDA-MB-231 cells did not migrate at all (not depicted). To normalize values, each data point (cell number migrated per well) was divided by the average cell number migrated per well that was determined for Ln-5 (relative cell migration). Average cell number of Ln-5 was set to 1. Values in bar graphs represent the mean ± SD of at least three independent experiments. No change detected in 5 out of 5 assays with *p (b and a) = 0.97 - 0.16 (- hypothesis confirmed). Statistically significant changes were found in ** 7 out of 8, *** 3 out of 3, **** 3 out of 3 and ***** 3 out of 4 assays with **p (c and a) = 1.9E-06 – 0.0147; ***p (d and c) = 8.8E-07 – 3.6E-06; ****p (e and a) = 1.37E-06 – 1.6E-05; *****p (f and e) = 8.4E-06 – 2.2E-05. (C) DIII is a cleavage product of Ln-5 and is detectable in conditioned medium of human MCF10A cells. WB of MMP-2 cleaved Ln-5 and concentrated conditioned medium were detected with 2778 (lanes 1–6), or D4B5 (lane 7). The bottom and top WBs are identical, except that the bottom panels are overexposed, depicting DIII more clearly. Cleavage of Ln-5 with MMP-2 for 2 h (lane 3), 17 h (lane 4), and 24 h (lane 5) results in the appearance of the γ80 chain, DIII-V, and DIII. In conditioned medium from MCF10A cells DIII is detectable using both 2778 (lane 6) and D4B5 (lane 7). For comparison, purified, non-MMP treated Ln-5, which is mainly composed of the γ140 and γ100 chains, was loaded in lane 2 and rDIII in lane 1. (D) Stimulated migration of MMP-2 cleaved Ln-5 depends on EGFR. MCF10A cells were allowed to migrate on Transwell membranes, which were coated with either uncleaved (top panels; −MMP-2) or MMP-2-cleaved Ln-5 (bottom panels; +MMP-2). Cells remained untreated (left panels, Ln-5), or were treated with LA1 for 30 min before seeding. LA1 was added where indicated (right panels, +LA1). The corresponding bar graph is shown with *p (b and a) = 0.0030; **p (c and a) = 7.27E-05; ***p (d and a) = 0.1333 and ***p (d and c) = 1.39E-05.
Because EGFR and ERK1/2 have motogenic properties (Klemke et al., 1997; Xie et al., 1998), we tested rDIII for its ability to affect cell motility. In Transwell migration assays with MDA-MB-231 cells on Ln-5, addition of rDIII resulted in a 1.5–2-fold increase in migratory activity. Though the increase in migration was modest, it was highly reproducible in multiple independent experiments. More pronounced stimulation of migration on Ln-5 was observed in rDIII-treated MCF-7 and MCF10A cells (Fig. 8 A).