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Autocrine epidermal growth factor signaling stimulates directionally persistent mammary epithelial cell migration.

Maheshwari G, Wiley HS, Lauffenburger DA - J. Cell Biol. (2001)

Bottom Line: The possibly diverse effect of presenting a growth factor in autocrine as opposed to exogenous (or paracrine) mode is an especially important issue in cell biology.Addition of exogenous EGF to these cells abrogates their enhanced directional persistence, reducing their directionality to a level similar to wild-type cells.These findings emphasize the functional importance of spatial restriction of EGFR signaling, and suggest critical implications for growth factor-based therapeutic treatments.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

ABSTRACT
Cell responses to soluble regulatory factors may be strongly influenced by the mode of presentation of the factor, as in matrix-bound versus diffusible modes. The possibly diverse effect of presenting a growth factor in autocrine as opposed to exogenous (or paracrine) mode is an especially important issue in cell biology. We demonstrate here that migration behavior of human mammary epithelial cells in response to stimulation by epidermal growth factor (EGF) is qualitatively different for EGF presented in exogenous (paracrine), autocrine, and intracrine modes. When EGF is added as an exogenous factor to the medium of cells that express EGF receptor (EGFR) but not EGF, cell migration speed increases while directional persistence decreases. When these EGFR-expressing cells are made to also express via retroviral transfection EGF in protease-cleaveable transmembrane form on the plasma membrane, migration speed similarly increases, but directional persistence increases as well. Addition of exogenous EGF to these cells abrogates their enhanced directional persistence, reducing their directionality to a level similar to wild-type cells. If the EGFR-expressing cells are instead transduced with a gene encoding EGF in a soluble form, migration speed and directional persistence were unaffected. Thus, autocrine presentation of EGF at the plasma membrane in a protease-cleavable form provides these cells with an enhanced ability to migrate persistently in a given direction, consistent with their increased capability for organizing into gland-like structures. In contrast, an exogenous/paracrine mode of EGF presentation generates a "scattering" response by the cells. These findings emphasize the functional importance of spatial restriction of EGFR signaling, and suggest critical implications for growth factor-based therapeutic treatments.

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Cell types used in this study. WT HMECs express EGFR endogenously, but lack the expression of EGF. EGF-Ct cells express the EGF-Ct construct. EGF-Ct lacks the NH2-terminal extracellular domain of the human EGF precursor. The sEGF construct is simply the 53 amino acid long mature EGF without the NH2-terminal, COOH-terminal, or the transmembrane domain of EGF precursor; it binds to the EGFR during receptor transport to the cell surface (Wiley et al., 1998).
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fig1: Cell types used in this study. WT HMECs express EGFR endogenously, but lack the expression of EGF. EGF-Ct cells express the EGF-Ct construct. EGF-Ct lacks the NH2-terminal extracellular domain of the human EGF precursor. The sEGF construct is simply the 53 amino acid long mature EGF without the NH2-terminal, COOH-terminal, or the transmembrane domain of EGF precursor; it binds to the EGFR during receptor transport to the cell surface (Wiley et al., 1998).

Mentions: To test the hypothesis that autocrine presentation of EGF family ligands provides for a distinct manner of cell regulation, we have generated an experimental cell system in which the presentation mode of EGF in a MEC line can be altered. Wiley et al. (1998) used retroviral transfection to individually express each of two forms of EGF ligands in the EGFR-expressing human MEC (HMEC) line (Stampfer and Yaswen, 1993) (Fig. 1). One is the mature EGF 53 amino acid peptide retaining its cytoplasmic and transmembrane domain (EGF-Ct); this version is protealytically cleaved at the cell plasma membrane, diffuses into the extracellular environment and is recaptured by cell-surface receptors, thus operating in classical autocrine mode. The second is simply the mature EGF 53 amino acid peptide containing a signal sequence (sEGF). This version is generated in soluble form in the lumen of the protein synthesis pathway where it can bind newly synthesized (and perhaps recycling) intracellular EGFR, thus operating in a presumably pathological intracrine mode. The parental HMEC cells (subtype 184A1) produce low levels of endogenous EGFR ligands and thus require exogenous EGF for optimum growth in culture. This allows them to be used to compare autocrine and intracrine presentation modes of EGF with that of typical paracrine mode, where the ligand is provided exogenously in the cell culture medium.


Autocrine epidermal growth factor signaling stimulates directionally persistent mammary epithelial cell migration.

Maheshwari G, Wiley HS, Lauffenburger DA - J. Cell Biol. (2001)

Cell types used in this study. WT HMECs express EGFR endogenously, but lack the expression of EGF. EGF-Ct cells express the EGF-Ct construct. EGF-Ct lacks the NH2-terminal extracellular domain of the human EGF precursor. The sEGF construct is simply the 53 amino acid long mature EGF without the NH2-terminal, COOH-terminal, or the transmembrane domain of EGF precursor; it binds to the EGFR during receptor transport to the cell surface (Wiley et al., 1998).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199328&req=5

fig1: Cell types used in this study. WT HMECs express EGFR endogenously, but lack the expression of EGF. EGF-Ct cells express the EGF-Ct construct. EGF-Ct lacks the NH2-terminal extracellular domain of the human EGF precursor. The sEGF construct is simply the 53 amino acid long mature EGF without the NH2-terminal, COOH-terminal, or the transmembrane domain of EGF precursor; it binds to the EGFR during receptor transport to the cell surface (Wiley et al., 1998).
Mentions: To test the hypothesis that autocrine presentation of EGF family ligands provides for a distinct manner of cell regulation, we have generated an experimental cell system in which the presentation mode of EGF in a MEC line can be altered. Wiley et al. (1998) used retroviral transfection to individually express each of two forms of EGF ligands in the EGFR-expressing human MEC (HMEC) line (Stampfer and Yaswen, 1993) (Fig. 1). One is the mature EGF 53 amino acid peptide retaining its cytoplasmic and transmembrane domain (EGF-Ct); this version is protealytically cleaved at the cell plasma membrane, diffuses into the extracellular environment and is recaptured by cell-surface receptors, thus operating in classical autocrine mode. The second is simply the mature EGF 53 amino acid peptide containing a signal sequence (sEGF). This version is generated in soluble form in the lumen of the protein synthesis pathway where it can bind newly synthesized (and perhaps recycling) intracellular EGFR, thus operating in a presumably pathological intracrine mode. The parental HMEC cells (subtype 184A1) produce low levels of endogenous EGFR ligands and thus require exogenous EGF for optimum growth in culture. This allows them to be used to compare autocrine and intracrine presentation modes of EGF with that of typical paracrine mode, where the ligand is provided exogenously in the cell culture medium.

Bottom Line: The possibly diverse effect of presenting a growth factor in autocrine as opposed to exogenous (or paracrine) mode is an especially important issue in cell biology.Addition of exogenous EGF to these cells abrogates their enhanced directional persistence, reducing their directionality to a level similar to wild-type cells.These findings emphasize the functional importance of spatial restriction of EGFR signaling, and suggest critical implications for growth factor-based therapeutic treatments.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

ABSTRACT
Cell responses to soluble regulatory factors may be strongly influenced by the mode of presentation of the factor, as in matrix-bound versus diffusible modes. The possibly diverse effect of presenting a growth factor in autocrine as opposed to exogenous (or paracrine) mode is an especially important issue in cell biology. We demonstrate here that migration behavior of human mammary epithelial cells in response to stimulation by epidermal growth factor (EGF) is qualitatively different for EGF presented in exogenous (paracrine), autocrine, and intracrine modes. When EGF is added as an exogenous factor to the medium of cells that express EGF receptor (EGFR) but not EGF, cell migration speed increases while directional persistence decreases. When these EGFR-expressing cells are made to also express via retroviral transfection EGF in protease-cleaveable transmembrane form on the plasma membrane, migration speed similarly increases, but directional persistence increases as well. Addition of exogenous EGF to these cells abrogates their enhanced directional persistence, reducing their directionality to a level similar to wild-type cells. If the EGFR-expressing cells are instead transduced with a gene encoding EGF in a soluble form, migration speed and directional persistence were unaffected. Thus, autocrine presentation of EGF at the plasma membrane in a protease-cleavable form provides these cells with an enhanced ability to migrate persistently in a given direction, consistent with their increased capability for organizing into gland-like structures. In contrast, an exogenous/paracrine mode of EGF presentation generates a "scattering" response by the cells. These findings emphasize the functional importance of spatial restriction of EGFR signaling, and suggest critical implications for growth factor-based therapeutic treatments.

Show MeSH