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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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Effects of rDIII on gene expression. (A) Microarray hybridization. Amplified RNA prepared from MCF-7 cells was cultured in the presence (cy3) or absence (cy5) of rDIII and cohybridized to the microarray containing 83 human cDNAs related to cancer cell metastasis (filled histogram). The open histogram represents RNA isolates where the cy3 and cy5 labels were switched. Ratios below 1.0 were inverted and multiplied by −1 to aid in their interpretation. The MMP-2 gene expression signal exceeded a twofold signal intensity change threshold, independent of the dye label orientation. Results are expressed as the mean ± SD from six fluorescence signals. (B) Semi-quantitative RT-PCR. Changes in MMP-2 expression were assessed on the influence of 185 nM rDIII, 2.5 nM Ln-5, or 0.17 nM EGF, with or without AG1478 or LA1. DMSO was added to the controls. As a control for normalization, GAPDH was amplified similarly to MMP-2. Amplified cDNAs of MMP-2 (504 bp) and GAPDH (516 bp) were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining.
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fig7: Effects of rDIII on gene expression. (A) Microarray hybridization. Amplified RNA prepared from MCF-7 cells was cultured in the presence (cy3) or absence (cy5) of rDIII and cohybridized to the microarray containing 83 human cDNAs related to cancer cell metastasis (filled histogram). The open histogram represents RNA isolates where the cy3 and cy5 labels were switched. Ratios below 1.0 were inverted and multiplied by −1 to aid in their interpretation. The MMP-2 gene expression signal exceeded a twofold signal intensity change threshold, independent of the dye label orientation. Results are expressed as the mean ± SD from six fluorescence signals. (B) Semi-quantitative RT-PCR. Changes in MMP-2 expression were assessed on the influence of 185 nM rDIII, 2.5 nM Ln-5, or 0.17 nM EGF, with or without AG1478 or LA1. DMSO was added to the controls. As a control for normalization, GAPDH was amplified similarly to MMP-2. Amplified cDNAs of MMP-2 (504 bp) and GAPDH (516 bp) were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining.

Mentions: Activated ERKs phosphorylate downstream transcription factors that regulate gene expression (Hazzalin and Mahadevan, 2002). We investigated a possible effect of rDIII on gene expression using cDNA microarrays, which carried probes for members of the integrin, protein tyrosine kinase and MMP families, MMP inhibitors, and ECM molecules (Seftor et al., 2001). Spiking experiments with exogenous control RNAs allowed a 2.0-fold change in signal intensity to be considered statistically significant (Bilban et al., 2002). In two independent experiments (two chips), we found that MMP-2, MMP-9, and phosphoinositide 3-kinase gene expression were up-regulated on rDIII treatment, whereas urokinase plasminogen activator expression was down-regulated (Fig. 7 A, only MMP-2 data are shown). Enhanced expression of MMP-2 by rDIII treatment was confirmed by semi-quantitative RT-PCR (Fig. 7 B). Induction of MMP-2 gene expression was also observed in cells stimulated with EGF, but not with intact Ln-5. To investigate whether rDIII-induced MMP-2 gene expression is EGFR dependent, cells were incubated with AG1478 and LA1 before treatment with rDIII. Blockage of EGFR function by these compounds inhibited induction of MMP-2 gene expression by rDIII. These results provide independent evidence that rDIII interacts with EGFR and triggers downstream signaling, resulting in altered gene expression.


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)

Effects of rDIII on gene expression. (A) Microarray hybridization. Amplified RNA prepared from MCF-7 cells was cultured in the presence (cy3) or absence (cy5) of rDIII and cohybridized to the microarray containing 83 human cDNAs related to cancer cell metastasis (filled histogram). The open histogram represents RNA isolates where the cy3 and cy5 labels were switched. Ratios below 1.0 were inverted and multiplied by −1 to aid in their interpretation. The MMP-2 gene expression signal exceeded a twofold signal intensity change threshold, independent of the dye label orientation. Results are expressed as the mean ± SD from six fluorescence signals. (B) Semi-quantitative RT-PCR. Changes in MMP-2 expression were assessed on the influence of 185 nM rDIII, 2.5 nM Ln-5, or 0.17 nM EGF, with or without AG1478 or LA1. DMSO was added to the controls. As a control for normalization, GAPDH was amplified similarly to MMP-2. Amplified cDNAs of MMP-2 (504 bp) and GAPDH (516 bp) were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining.
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Related In: Results  -  Collection

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fig7: Effects of rDIII on gene expression. (A) Microarray hybridization. Amplified RNA prepared from MCF-7 cells was cultured in the presence (cy3) or absence (cy5) of rDIII and cohybridized to the microarray containing 83 human cDNAs related to cancer cell metastasis (filled histogram). The open histogram represents RNA isolates where the cy3 and cy5 labels were switched. Ratios below 1.0 were inverted and multiplied by −1 to aid in their interpretation. The MMP-2 gene expression signal exceeded a twofold signal intensity change threshold, independent of the dye label orientation. Results are expressed as the mean ± SD from six fluorescence signals. (B) Semi-quantitative RT-PCR. Changes in MMP-2 expression were assessed on the influence of 185 nM rDIII, 2.5 nM Ln-5, or 0.17 nM EGF, with or without AG1478 or LA1. DMSO was added to the controls. As a control for normalization, GAPDH was amplified similarly to MMP-2. Amplified cDNAs of MMP-2 (504 bp) and GAPDH (516 bp) were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining.
Mentions: Activated ERKs phosphorylate downstream transcription factors that regulate gene expression (Hazzalin and Mahadevan, 2002). We investigated a possible effect of rDIII on gene expression using cDNA microarrays, which carried probes for members of the integrin, protein tyrosine kinase and MMP families, MMP inhibitors, and ECM molecules (Seftor et al., 2001). Spiking experiments with exogenous control RNAs allowed a 2.0-fold change in signal intensity to be considered statistically significant (Bilban et al., 2002). In two independent experiments (two chips), we found that MMP-2, MMP-9, and phosphoinositide 3-kinase gene expression were up-regulated on rDIII treatment, whereas urokinase plasminogen activator expression was down-regulated (Fig. 7 A, only MMP-2 data are shown). Enhanced expression of MMP-2 by rDIII treatment was confirmed by semi-quantitative RT-PCR (Fig. 7 B). Induction of MMP-2 gene expression was also observed in cells stimulated with EGF, but not with intact Ln-5. To investigate whether rDIII-induced MMP-2 gene expression is EGFR dependent, cells were incubated with AG1478 and LA1 before treatment with rDIII. Blockage of EGFR function by these compounds inhibited induction of MMP-2 gene expression by rDIII. These results provide independent evidence that rDIII interacts with EGFR and triggers downstream signaling, resulting in altered gene expression.

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