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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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EGF released from autocrine cells does not stimulate migration of neighboring cells. WT HMECs were labeled with CellTracker green and cells expressing EGF-Ct were labeled with CellTracker orange. Cells were mixed overnight and followed for 4 h by two color fluorescence time-lapse microscopy using 4 min intervals. Shown are the initial images overlaid with trajectories marked at 40-min intervals. Left, cells in the absence of exogenous EGF. Right, cells in the presence of 2 nM exogenous EGF.
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fig6: EGF released from autocrine cells does not stimulate migration of neighboring cells. WT HMECs were labeled with CellTracker green and cells expressing EGF-Ct were labeled with CellTracker orange. Cells were mixed overnight and followed for 4 h by two color fluorescence time-lapse microscopy using 4 min intervals. Shown are the initial images overlaid with trajectories marked at 40-min intervals. Left, cells in the absence of exogenous EGF. Right, cells in the presence of 2 nM exogenous EGF.

Mentions: The above data suggests that spatial restriction in EGFR-mediated signaling is an important aspect of its role in cell migration. However, this implies that released EGF is restricted to the producing cell and does not affect all the cells in a population. To verify that the EGF-Ct autocrine loop indeed operates locally, we made visual observations of individual HMEC in mixed populations of EGF-Ct–expressing and WT cells. WT HMEC were labeled green and the EGF-Ct–expressing cells were labeled red using fluorescent vital dyes. Their paths were then tracked over a period of 4 h. As can be seen in the example photograph of Fig. 6, EGF-Ct–expressing cells exhibited strong locomotion behavior while WT cells, even those next to EGF-Ct-expressing cells, did not move significantly from their original locations. However, when exogenous EGF was added to the media, migration of WT HMEC was stimulated (in accord with data shown in Fig. 2), confirming that their motility apparatus was functional. The observation that autocrine EGF-stimulated migration of the EGF-Ct–expressing cells but not of neighboring WT cells supports the notion that the EGF autocrine loop is operating in spatially restricted, or “local,” fashion. This is consistent with the predictions of a recent theoretical study that concluded that for typical values displayed by the EGFR autocrine system, a major portion of released autocrine ligand should be captured within a micron of the release point (Shvartsman et al., 2001).


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

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

EGF released from autocrine cells does not stimulate migration of neighboring cells. WT HMECs were labeled with CellTracker green and cells expressing EGF-Ct were labeled with CellTracker orange. Cells were mixed overnight and followed for 4 h by two color fluorescence time-lapse microscopy using 4 min intervals. Shown are the initial images overlaid with trajectories marked at 40-min intervals. Left, cells in the absence of exogenous EGF. Right, cells in the presence of 2 nM exogenous EGF.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: EGF released from autocrine cells does not stimulate migration of neighboring cells. WT HMECs were labeled with CellTracker green and cells expressing EGF-Ct were labeled with CellTracker orange. Cells were mixed overnight and followed for 4 h by two color fluorescence time-lapse microscopy using 4 min intervals. Shown are the initial images overlaid with trajectories marked at 40-min intervals. Left, cells in the absence of exogenous EGF. Right, cells in the presence of 2 nM exogenous EGF.
Mentions: The above data suggests that spatial restriction in EGFR-mediated signaling is an important aspect of its role in cell migration. However, this implies that released EGF is restricted to the producing cell and does not affect all the cells in a population. To verify that the EGF-Ct autocrine loop indeed operates locally, we made visual observations of individual HMEC in mixed populations of EGF-Ct–expressing and WT cells. WT HMEC were labeled green and the EGF-Ct–expressing cells were labeled red using fluorescent vital dyes. Their paths were then tracked over a period of 4 h. As can be seen in the example photograph of Fig. 6, EGF-Ct–expressing cells exhibited strong locomotion behavior while WT cells, even those next to EGF-Ct-expressing cells, did not move significantly from their original locations. However, when exogenous EGF was added to the media, migration of WT HMEC was stimulated (in accord with data shown in Fig. 2), confirming that their motility apparatus was functional. The observation that autocrine EGF-stimulated migration of the EGF-Ct–expressing cells but not of neighboring WT cells supports the notion that the EGF autocrine loop is operating in spatially restricted, or “local,” fashion. This is consistent with the predictions of a recent theoretical study that concluded that for typical values displayed by the EGFR autocrine system, a major portion of released autocrine ligand should be captured within a micron of the release point (Shvartsman et al., 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.

Show MeSH
Related in: MedlinePlus