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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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Effect of ligand presentation on cell speed. Filled, hatched, and blank bars represent the cell speeds of EGF-Ct– expressing cells, sEGF-expressing cells, and WT HMECs, respectively. Cell speeds of the three cell types were monitored in the absence of exogenous EGF, in the presence of 2 nM exogenously added EGF, and in the presence of 10 μg/ml 225 mAb EGFR-blocking antibody. Errors represent ± SEM. Numbers above the bars are the number of individual cell tracks used in the analysis.
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fig2: Effect of ligand presentation on cell speed. Filled, hatched, and blank bars represent the cell speeds of EGF-Ct– expressing cells, sEGF-expressing cells, and WT HMECs, respectively. Cell speeds of the three cell types were monitored in the absence of exogenous EGF, in the presence of 2 nM exogenously added EGF, and in the presence of 10 μg/ml 225 mAb EGFR-blocking antibody. Errors represent ± SEM. Numbers above the bars are the number of individual cell tracks used in the analysis.

Mentions: Wild-type (WT) HMEC as well as cells expressing either sEGF or EGF-Ct were used in these studies. The structure of the artificial sEGF and EGF-Ct genes are shown in Fig. 1. Migration speeds of HMEC for the three different EGF presentation modes were quantified using time-lapse video microscopy of individual cells (Fig. 2). In the absence of exogenous EGF, WT HMEC exhibited a baseline mean migration speed of ∼50 μm/h. EGF-Ct–expressing cells (autocrine presentation) exhibited substantially greater mean speed at ∼120 μm/h, whereas sEGF-expressing cells (intracrine presentation) showed only a small increase in mean speed to roughly 65 μm/h. Addition of 2 nM exogenous EGF increased the mean migration speeds of both WT and sEGF-expressing cells to 90 μm/h, whereas the mean speeds of EGF-Ct–expressing cells remained at 120 μm/h. The differences among mean speeds of the three cell types in the presence of 2 nM exogenous EGF are not statistically significant. Addition of 225 mAb, which blocks the binding of extracellular, but not intracellular EGF to its receptor (Wiley et al., 1998), decreased the mean migration speed of each of the cell types to the range of 25–35 μm/h. The reduction from the original WT and sEGF cell baselines of 50–65 μm/h is likely due to blocking of the low levels of endogenous EGF family autocrine ligands in these cells (Wiley et al., 1998). Together, these findings indicate that the migration speed of the HMEC is essentially governed by signaling from cell surface EGF–EGFR complexes; this is consistent with recent evidence that key pathways of EGFR-mediated induction of enhanced cell migration reside at the plasma membrane (Haugh et al., 1999; Glading et al., 2001). Signaling by EGF–EGFR complexes at the cell surface in EGF-Ct–expressing HMEC is relatively unaffected by additional exogenous EGF. In contrast, WT and sEGF-expressing cells migrate at maximal speeds only when signaling by surface EGF–EGFR complexes is increased by addition of exogenous EGF.


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

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

Effect of ligand presentation on cell speed. Filled, hatched, and blank bars represent the cell speeds of EGF-Ct– expressing cells, sEGF-expressing cells, and WT HMECs, respectively. Cell speeds of the three cell types were monitored in the absence of exogenous EGF, in the presence of 2 nM exogenously added EGF, and in the presence of 10 μg/ml 225 mAb EGFR-blocking antibody. Errors represent ± SEM. Numbers above the bars are the number of individual cell tracks used in the analysis.
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Related In: Results  -  Collection

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

fig2: Effect of ligand presentation on cell speed. Filled, hatched, and blank bars represent the cell speeds of EGF-Ct– expressing cells, sEGF-expressing cells, and WT HMECs, respectively. Cell speeds of the three cell types were monitored in the absence of exogenous EGF, in the presence of 2 nM exogenously added EGF, and in the presence of 10 μg/ml 225 mAb EGFR-blocking antibody. Errors represent ± SEM. Numbers above the bars are the number of individual cell tracks used in the analysis.
Mentions: Wild-type (WT) HMEC as well as cells expressing either sEGF or EGF-Ct were used in these studies. The structure of the artificial sEGF and EGF-Ct genes are shown in Fig. 1. Migration speeds of HMEC for the three different EGF presentation modes were quantified using time-lapse video microscopy of individual cells (Fig. 2). In the absence of exogenous EGF, WT HMEC exhibited a baseline mean migration speed of ∼50 μm/h. EGF-Ct–expressing cells (autocrine presentation) exhibited substantially greater mean speed at ∼120 μm/h, whereas sEGF-expressing cells (intracrine presentation) showed only a small increase in mean speed to roughly 65 μm/h. Addition of 2 nM exogenous EGF increased the mean migration speeds of both WT and sEGF-expressing cells to 90 μm/h, whereas the mean speeds of EGF-Ct–expressing cells remained at 120 μm/h. The differences among mean speeds of the three cell types in the presence of 2 nM exogenous EGF are not statistically significant. Addition of 225 mAb, which blocks the binding of extracellular, but not intracellular EGF to its receptor (Wiley et al., 1998), decreased the mean migration speed of each of the cell types to the range of 25–35 μm/h. The reduction from the original WT and sEGF cell baselines of 50–65 μm/h is likely due to blocking of the low levels of endogenous EGF family autocrine ligands in these cells (Wiley et al., 1998). Together, these findings indicate that the migration speed of the HMEC is essentially governed by signaling from cell surface EGF–EGFR complexes; this is consistent with recent evidence that key pathways of EGFR-mediated induction of enhanced cell migration reside at the plasma membrane (Haugh et al., 1999; Glading et al., 2001). Signaling by EGF–EGFR complexes at the cell surface in EGF-Ct–expressing HMEC is relatively unaffected by additional exogenous EGF. In contrast, WT and sEGF-expressing cells migrate at maximal speeds only when signaling by surface EGF–EGFR complexes is increased by addition of exogenous EGF.

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
Related in: MedlinePlus