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Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution.

Schenk S, Hintermann E, Bilban M, Koshikawa N, Hojilla C, Khokha R, Quaranta V - J. Cell Biol. (2003)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. sschenk@scripps

ABSTRACT
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.

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Stimulation of ERK1/2 phosphorylation by rDIII. Time course of ERK1/2 activation after exposure to rDIII. Before lysate preparation, MCF-7 (A) or MDA-MB-231 (B) cells were treated with rDIII for the indicated time periods at 37°C. The ratio of phosphorylated ERK1/2 bands (P-ERK1/2, top panels) to total ERK1/2 protein bands (ERK1/2, bottom panels) was quantified. The control signal (no ligand) was set to 1 and the relative ERK phosphorylation intensity calculated and depicted as bar graphs (bottom panels). (A) ERK phosphorylation was performed with two distinct, purified preparations of rDIII protein (Prep. A and Prep. B). (B) One representative experiment and the mean ± SD (n = 3) of relative ERK1/2 phosphorylation intensity is depicted. ERK1/2 activation induced by EGF (C) but not by control protein rDIII-V (D). MCF-7 cells were stimulated with EGF (C) for up to 30 min or with rDIII, rDIII-V or EGF for 5 min and phosphorylated ERK1/2 were detected as described in the legend to A. As compared with rDIII (lane 3, D) and EGF (lane 4, D,) no phosphorylation signal above control level (lane 1, D) was seen with rDIII-V (lane 2, D). (E) Dependency of ERK1/2 activation on EGFR. Before stimulation for 5 min with either rDIII (lane 5) or EGF (lane 2), MCF-7 cells were preincubated with either AG1478 or 528. Both EGFR inhibitors diminish phosphorylation of ERK1/2 (top panel) by rDIII (lanes 6 and 7) or EGF (lanes 3 and 4). The top bands (∼25 kD) in lane 4 (EGF + 528) and 7 (rDIII + 528) originates from the IgG light chain of 528. The total amount of loaded ERK1/2 protein is shown in the bottom panel.
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fig5: Stimulation of ERK1/2 phosphorylation by rDIII. Time course of ERK1/2 activation after exposure to rDIII. Before lysate preparation, MCF-7 (A) or MDA-MB-231 (B) cells were treated with rDIII for the indicated time periods at 37°C. The ratio of phosphorylated ERK1/2 bands (P-ERK1/2, top panels) to total ERK1/2 protein bands (ERK1/2, bottom panels) was quantified. The control signal (no ligand) was set to 1 and the relative ERK phosphorylation intensity calculated and depicted as bar graphs (bottom panels). (A) ERK phosphorylation was performed with two distinct, purified preparations of rDIII protein (Prep. A and Prep. B). (B) One representative experiment and the mean ± SD (n = 3) of relative ERK1/2 phosphorylation intensity is depicted. ERK1/2 activation induced by EGF (C) but not by control protein rDIII-V (D). MCF-7 cells were stimulated with EGF (C) for up to 30 min or with rDIII, rDIII-V or EGF for 5 min and phosphorylated ERK1/2 were detected as described in the legend to A. As compared with rDIII (lane 3, D) and EGF (lane 4, D,) no phosphorylation signal above control level (lane 1, D) was seen with rDIII-V (lane 2, D). (E) Dependency of ERK1/2 activation on EGFR. Before stimulation for 5 min with either rDIII (lane 5) or EGF (lane 2), MCF-7 cells were preincubated with either AG1478 or 528. Both EGFR inhibitors diminish phosphorylation of ERK1/2 (top panel) by rDIII (lanes 6 and 7) or EGF (lanes 3 and 4). The top bands (∼25 kD) in lane 4 (EGF + 528) and 7 (rDIII + 528) originates from the IgG light chain of 528. The total amount of loaded ERK1/2 protein is shown in the bottom panel.

Mentions: MAPK activation is a well characterized downstream signaling event that follows stimulation of the growth factor receptor EGFR (Chen et al., 2001). In MCF-7 breast cancer cells (Fig. 5 A), extracellular signal–regulated kinase 1 (ERK1; p44) and ERK2 (p42) were rapidly phosphorylated upon rDIII treatment. Phosphorylation levels peaked 5 min after stimulation, were maintained for 10–20 min, and returned to background levels after 30 min. Stimulation of ERK1/2 by EGF demonstrated similar kinetics, with a pronounced peak at 5 min after stimulation (Fig. 5 C). In contrast to rDIII and EGF, rDIII-V did not stimulate ERK1/2 (Fig. 5 D). In MDA-MB-231 cells (Fig. 5 B), phosphorylated forms of ERK1/2 were also detected on rDIII or EGF stimulation, but with the following distinctive features: there was a more prominent phospho-ERK2 band consistent with a higher expression of total ERK2 compared with ERK1. Constitutive levels of phosphorylated ERK1/2 in nonstimulated MDA-MB-231 cells were higher than in MCF-7 cells, as reported previously (Seddighzadeh et al., 1999). rDIII-induced ERK1/2 phosphorylation was completely blocked by AG1478, a selective inhibitor of EGFR tyrosine kinase, and by 528, an EGFR-blocking antibody (Fig. 5 E). These data strongly support our hypothesis that rDIII induces ERK1/2 activation via binding to EGFR.


Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution.

Schenk S, Hintermann E, Bilban M, Koshikawa N, Hojilla C, Khokha R, Quaranta V - J. Cell Biol. (2003)

Stimulation of ERK1/2 phosphorylation by rDIII. Time course of ERK1/2 activation after exposure to rDIII. Before lysate preparation, MCF-7 (A) or MDA-MB-231 (B) cells were treated with rDIII for the indicated time periods at 37°C. The ratio of phosphorylated ERK1/2 bands (P-ERK1/2, top panels) to total ERK1/2 protein bands (ERK1/2, bottom panels) was quantified. The control signal (no ligand) was set to 1 and the relative ERK phosphorylation intensity calculated and depicted as bar graphs (bottom panels). (A) ERK phosphorylation was performed with two distinct, purified preparations of rDIII protein (Prep. A and Prep. B). (B) One representative experiment and the mean ± SD (n = 3) of relative ERK1/2 phosphorylation intensity is depicted. ERK1/2 activation induced by EGF (C) but not by control protein rDIII-V (D). MCF-7 cells were stimulated with EGF (C) for up to 30 min or with rDIII, rDIII-V or EGF for 5 min and phosphorylated ERK1/2 were detected as described in the legend to A. As compared with rDIII (lane 3, D) and EGF (lane 4, D,) no phosphorylation signal above control level (lane 1, D) was seen with rDIII-V (lane 2, D). (E) Dependency of ERK1/2 activation on EGFR. Before stimulation for 5 min with either rDIII (lane 5) or EGF (lane 2), MCF-7 cells were preincubated with either AG1478 or 528. Both EGFR inhibitors diminish phosphorylation of ERK1/2 (top panel) by rDIII (lanes 6 and 7) or EGF (lanes 3 and 4). The top bands (∼25 kD) in lane 4 (EGF + 528) and 7 (rDIII + 528) originates from the IgG light chain of 528. The total amount of loaded ERK1/2 protein is shown in the bottom panel.
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fig5: Stimulation of ERK1/2 phosphorylation by rDIII. Time course of ERK1/2 activation after exposure to rDIII. Before lysate preparation, MCF-7 (A) or MDA-MB-231 (B) cells were treated with rDIII for the indicated time periods at 37°C. The ratio of phosphorylated ERK1/2 bands (P-ERK1/2, top panels) to total ERK1/2 protein bands (ERK1/2, bottom panels) was quantified. The control signal (no ligand) was set to 1 and the relative ERK phosphorylation intensity calculated and depicted as bar graphs (bottom panels). (A) ERK phosphorylation was performed with two distinct, purified preparations of rDIII protein (Prep. A and Prep. B). (B) One representative experiment and the mean ± SD (n = 3) of relative ERK1/2 phosphorylation intensity is depicted. ERK1/2 activation induced by EGF (C) but not by control protein rDIII-V (D). MCF-7 cells were stimulated with EGF (C) for up to 30 min or with rDIII, rDIII-V or EGF for 5 min and phosphorylated ERK1/2 were detected as described in the legend to A. As compared with rDIII (lane 3, D) and EGF (lane 4, D,) no phosphorylation signal above control level (lane 1, D) was seen with rDIII-V (lane 2, D). (E) Dependency of ERK1/2 activation on EGFR. Before stimulation for 5 min with either rDIII (lane 5) or EGF (lane 2), MCF-7 cells were preincubated with either AG1478 or 528. Both EGFR inhibitors diminish phosphorylation of ERK1/2 (top panel) by rDIII (lanes 6 and 7) or EGF (lanes 3 and 4). The top bands (∼25 kD) in lane 4 (EGF + 528) and 7 (rDIII + 528) originates from the IgG light chain of 528. The total amount of loaded ERK1/2 protein is shown in the bottom panel.
Mentions: MAPK activation is a well characterized downstream signaling event that follows stimulation of the growth factor receptor EGFR (Chen et al., 2001). In MCF-7 breast cancer cells (Fig. 5 A), extracellular signal–regulated kinase 1 (ERK1; p44) and ERK2 (p42) were rapidly phosphorylated upon rDIII treatment. Phosphorylation levels peaked 5 min after stimulation, were maintained for 10–20 min, and returned to background levels after 30 min. Stimulation of ERK1/2 by EGF demonstrated similar kinetics, with a pronounced peak at 5 min after stimulation (Fig. 5 C). In contrast to rDIII and EGF, rDIII-V did not stimulate ERK1/2 (Fig. 5 D). In MDA-MB-231 cells (Fig. 5 B), phosphorylated forms of ERK1/2 were also detected on rDIII or EGF stimulation, but with the following distinctive features: there was a more prominent phospho-ERK2 band consistent with a higher expression of total ERK2 compared with ERK1. Constitutive levels of phosphorylated ERK1/2 in nonstimulated MDA-MB-231 cells were higher than in MCF-7 cells, as reported previously (Seddighzadeh et al., 1999). rDIII-induced ERK1/2 phosphorylation was completely blocked by AG1478, a selective inhibitor of EGFR tyrosine kinase, and by 528, an EGFR-blocking antibody (Fig. 5 E). These data strongly support our hypothesis that rDIII induces ERK1/2 activation via binding to EGFR.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. sschenk@scripps

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus