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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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Detection of DIII in lysates of mammary gland tissue. Mammary lysates were resolved on a 4–12% Bis-Tris gradient gel in MES buffer under reducing conditions. D4B5 detected a distinct band of ∼20 kD that very likely is DIII (top panel). In contrast to WT, DIII could be found at day 10 of lactation as well as at day 1 of involution in TIMP-3– (−/−) tissue. After day 1 of involution, DIII was detectable at all time points of involution in both the involuting and lactating mice (day 2–7 of involution). Stripping and reprobing the blot with 2778 revealed unscheduled fragmentation of Ln-5 γ2 chain (bottom panel). Fragmentation occurred in −/− 1 d earlier than in WT mammary glands (day 1 of involution, WT, −/−). The exact identity of the various fragments in the range of 30–100 kD is not known. The Ln-5 γ2 chain profile at day 10 of lactation as well as at day 7 of involution does not differ between WT and −/− tissue, when detected with 2778 (not depicted). To ensure equal loading in each lane, blots were stripped and reprobed with Actin mAb (MAB 1501, CHEMICON International; middle panel).
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fig9: Detection of DIII in lysates of mammary gland tissue. Mammary lysates were resolved on a 4–12% Bis-Tris gradient gel in MES buffer under reducing conditions. D4B5 detected a distinct band of ∼20 kD that very likely is DIII (top panel). In contrast to WT, DIII could be found at day 10 of lactation as well as at day 1 of involution in TIMP-3– (−/−) tissue. After day 1 of involution, DIII was detectable at all time points of involution in both the involuting and lactating mice (day 2–7 of involution). Stripping and reprobing the blot with 2778 revealed unscheduled fragmentation of Ln-5 γ2 chain (bottom panel). Fragmentation occurred in −/− 1 d earlier than in WT mammary glands (day 1 of involution, WT, −/−). The exact identity of the various fragments in the range of 30–100 kD is not known. The Ln-5 γ2 chain profile at day 10 of lactation as well as at day 7 of involution does not differ between WT and −/− tissue, when detected with 2778 (not depicted). To ensure equal loading in each lane, blots were stripped and reprobed with Actin mAb (MAB 1501, CHEMICON International; middle panel).

Mentions: Our in vitro data support the hypothesis that DIII is a cryptic migratory ECM cue released upon MMP-2 cleavage of Ln-5. To investigate the physiological relevance of these data, we tested for liberated DIII in the involuting mammary gland. The stages of mammary gland involution are well characterized with respect to MMP expression (Talhouk et al., 1992; Lund et al., 1996). We investigated the presence of DIII in wild-type (WT) as well as tissue inhibitor of metalloproteinase 3 (TIMP-3)– (−/−) mice because deficiency of MMP inhibitors should favor DIII liberation. TIMP-3 binds tightly to the ECM (Fata et al., 2001; Leco et al., 2001) and interferes strongly with localized proteolysis of ECM molecules such as Ln-5. To detect DIII, we used mAb D4B5, which recognizes an epitope present on mouse DIII only when it is liberated from Ln-5 (i.e., it is not reactive with intact mouse Ln-5 γ2-chain). Mammary gland tissue lysates from mice killed on day 10 of lactation as well as day 1, 2, 3, 4, and 7 of involution were subjected to SDS-PAGE and WB. In WT mice, DIII is detectable in involuting tissue starting at day 2 of involution, but it is not visible at day 1 of involution or day 10 of lactation (Fig. 9) . In contrast, in the −/− mice, the DIII fragment is clearly detectable at day 10 of lactation, as well as day 1 of involution. At day 2, 3, 4, and 7 of involution, approximately equal levels of DIII were apparent in both WT and −/− tissue.


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)

Detection of DIII in lysates of mammary gland tissue. Mammary lysates were resolved on a 4–12% Bis-Tris gradient gel in MES buffer under reducing conditions. D4B5 detected a distinct band of ∼20 kD that very likely is DIII (top panel). In contrast to WT, DIII could be found at day 10 of lactation as well as at day 1 of involution in TIMP-3– (−/−) tissue. After day 1 of involution, DIII was detectable at all time points of involution in both the involuting and lactating mice (day 2–7 of involution). Stripping and reprobing the blot with 2778 revealed unscheduled fragmentation of Ln-5 γ2 chain (bottom panel). Fragmentation occurred in −/− 1 d earlier than in WT mammary glands (day 1 of involution, WT, −/−). The exact identity of the various fragments in the range of 30–100 kD is not known. The Ln-5 γ2 chain profile at day 10 of lactation as well as at day 7 of involution does not differ between WT and −/− tissue, when detected with 2778 (not depicted). To ensure equal loading in each lane, blots were stripped and reprobed with Actin mAb (MAB 1501, CHEMICON International; middle panel).
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Related In: Results  -  Collection

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fig9: Detection of DIII in lysates of mammary gland tissue. Mammary lysates were resolved on a 4–12% Bis-Tris gradient gel in MES buffer under reducing conditions. D4B5 detected a distinct band of ∼20 kD that very likely is DIII (top panel). In contrast to WT, DIII could be found at day 10 of lactation as well as at day 1 of involution in TIMP-3– (−/−) tissue. After day 1 of involution, DIII was detectable at all time points of involution in both the involuting and lactating mice (day 2–7 of involution). Stripping and reprobing the blot with 2778 revealed unscheduled fragmentation of Ln-5 γ2 chain (bottom panel). Fragmentation occurred in −/− 1 d earlier than in WT mammary glands (day 1 of involution, WT, −/−). The exact identity of the various fragments in the range of 30–100 kD is not known. The Ln-5 γ2 chain profile at day 10 of lactation as well as at day 7 of involution does not differ between WT and −/− tissue, when detected with 2778 (not depicted). To ensure equal loading in each lane, blots were stripped and reprobed with Actin mAb (MAB 1501, CHEMICON International; middle panel).
Mentions: Our in vitro data support the hypothesis that DIII is a cryptic migratory ECM cue released upon MMP-2 cleavage of Ln-5. To investigate the physiological relevance of these data, we tested for liberated DIII in the involuting mammary gland. The stages of mammary gland involution are well characterized with respect to MMP expression (Talhouk et al., 1992; Lund et al., 1996). We investigated the presence of DIII in wild-type (WT) as well as tissue inhibitor of metalloproteinase 3 (TIMP-3)– (−/−) mice because deficiency of MMP inhibitors should favor DIII liberation. TIMP-3 binds tightly to the ECM (Fata et al., 2001; Leco et al., 2001) and interferes strongly with localized proteolysis of ECM molecules such as Ln-5. To detect DIII, we used mAb D4B5, which recognizes an epitope present on mouse DIII only when it is liberated from Ln-5 (i.e., it is not reactive with intact mouse Ln-5 γ2-chain). Mammary gland tissue lysates from mice killed on day 10 of lactation as well as day 1, 2, 3, 4, and 7 of involution were subjected to SDS-PAGE and WB. In WT mice, DIII is detectable in involuting tissue starting at day 2 of involution, but it is not visible at day 1 of involution or day 10 of lactation (Fig. 9) . In contrast, in the −/− mice, the DIII fragment is clearly detectable at day 10 of lactation, as well as day 1 of involution. At day 2, 3, 4, and 7 of involution, approximately equal levels of DIII were apparent in both WT and −/− tissue.

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