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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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Induction of EGFR tyrosine phosphorylation by rDIII. (A) Incubation of MDA-MB-231 cells with 185 nM rDIII for 5 min (lane 4, top) stimulated phosphorylation of EGFR. There is no EGFR stimulation for the 2-min rDIII sample (lane 3), or the 5-min no ligand control (lane 1). To exclude nonspecific effects due to cross-linking, cells were exposed to BS3 in the absence of ligand (lane 2). To ensure that equal amounts of EGFR protein were loaded, blots were stripped and reprobed with EGFR pAb (bottom). (B) EGFR phosphorylation by 185 nM rDIII (lane 1, top) and 1.7 nM EGF (lane 2) for 5 min in the absence of BS3. For control, ligand was omitted (lane 3), and the loading controls are shown in the bottom panel.
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fig3: Induction of EGFR tyrosine phosphorylation by rDIII. (A) Incubation of MDA-MB-231 cells with 185 nM rDIII for 5 min (lane 4, top) stimulated phosphorylation of EGFR. There is no EGFR stimulation for the 2-min rDIII sample (lane 3), or the 5-min no ligand control (lane 1). To exclude nonspecific effects due to cross-linking, cells were exposed to BS3 in the absence of ligand (lane 2). To ensure that equal amounts of EGFR protein were loaded, blots were stripped and reprobed with EGFR pAb (bottom). (B) EGFR phosphorylation by 185 nM rDIII (lane 1, top) and 1.7 nM EGF (lane 2) for 5 min in the absence of BS3. For control, ligand was omitted (lane 3), and the loading controls are shown in the bottom panel.

Mentions: MDA-MB-231 cells treated with rDIII for 5 min in the presence of BS3 showed distinct EGFR phosphorylation (Fig. 3 A). BS3 alone did not induce EGFR phosphorylation. In control experiments without cross-linker, significant EGFR phosphorylation was detected in samples containing rDIII (Fig. 3 B) or EGF, whereas control samples devoid of ligand failed to show EGFR phosphorylation. These data show that rDIII stimulates EGFR phosphorylation.


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)

Induction of EGFR tyrosine phosphorylation by rDIII. (A) Incubation of MDA-MB-231 cells with 185 nM rDIII for 5 min (lane 4, top) stimulated phosphorylation of EGFR. There is no EGFR stimulation for the 2-min rDIII sample (lane 3), or the 5-min no ligand control (lane 1). To exclude nonspecific effects due to cross-linking, cells were exposed to BS3 in the absence of ligand (lane 2). To ensure that equal amounts of EGFR protein were loaded, blots were stripped and reprobed with EGFR pAb (bottom). (B) EGFR phosphorylation by 185 nM rDIII (lane 1, top) and 1.7 nM EGF (lane 2) for 5 min in the absence of BS3. For control, ligand was omitted (lane 3), and the loading controls are shown in the bottom panel.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172889&req=5

fig3: Induction of EGFR tyrosine phosphorylation by rDIII. (A) Incubation of MDA-MB-231 cells with 185 nM rDIII for 5 min (lane 4, top) stimulated phosphorylation of EGFR. There is no EGFR stimulation for the 2-min rDIII sample (lane 3), or the 5-min no ligand control (lane 1). To exclude nonspecific effects due to cross-linking, cells were exposed to BS3 in the absence of ligand (lane 2). To ensure that equal amounts of EGFR protein were loaded, blots were stripped and reprobed with EGFR pAb (bottom). (B) EGFR phosphorylation by 185 nM rDIII (lane 1, top) and 1.7 nM EGF (lane 2) for 5 min in the absence of BS3. For control, ligand was omitted (lane 3), and the loading controls are shown in the bottom panel.
Mentions: MDA-MB-231 cells treated with rDIII for 5 min in the presence of BS3 showed distinct EGFR phosphorylation (Fig. 3 A). BS3 alone did not induce EGFR phosphorylation. In control experiments without cross-linker, significant EGFR phosphorylation was detected in samples containing rDIII (Fig. 3 B) or EGF, whereas control samples devoid of ligand failed to show EGFR phosphorylation. These data show that rDIII stimulates EGFR phosphorylation.

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