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Mitogen-inducible gene 6 is an endogenous inhibitor of HGF/Met-induced cell migration and neurite growth.

Pante G, Thompson J, Lamballe F, Iwata T, Ferby I, Barr FA, Davies AM, Maina F, Klein R - J. Cell Biol. (2005)

Bottom Line: Here we report a mechanism by which mitogen-inducible gene 6 (Mig6; also called Gene 33 and receptor-associated late transducer) negatively regulates HGF/Met-induced cell migration.The effect is observed by Mig6 overexpression and is reversed by Mig6 small interfering RNA knock-down experiments; this indicates that endogenous Mig6 is part of a mechanism that inhibits Met signaling.Because Mig6 also is induced by HGF stimulation, our results suggest that Mig6 is part of a negative feedback loop that attenuates Met functions in different contexts and cell types.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, 82152 Munich-Martinsried, Germany.

ABSTRACT
Hepatocyte growth factor (HGF)/Met signaling controls cell migration, growth and differentiation in several embryonic organs and is implicated in human cancer. The physiologic mechanisms that attenuate Met signaling are not well understood. Here we report a mechanism by which mitogen-inducible gene 6 (Mig6; also called Gene 33 and receptor-associated late transducer) negatively regulates HGF/Met-induced cell migration. The effect is observed by Mig6 overexpression and is reversed by Mig6 small interfering RNA knock-down experiments; this indicates that endogenous Mig6 is part of a mechanism that inhibits Met signaling. Mig6 functions in cells of hepatic origin and in neurons, which suggests a role for Mig6 in different cell lineages. Mechanistically, Mig6 requires an intact Cdc42/Rac interactive binding site to exert its inhibitory action, which suggests that Mig6 acts, at least in part, distally from Met, possibly by inhibiting Rho-like GTPases. Because Mig6 also is induced by HGF stimulation, our results suggest that Mig6 is part of a negative feedback loop that attenuates Met functions in different contexts and cell types.

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Mig6 overexpression in cortical neurons inhibits HGF-induced migration. Cortical neurons were dissected from E15.5 mouse embryos and electroporated with expression plasmids encoding GFP (A) or Mig6FL-V5. Electroporated cells were plated for 24 h (A, D–F) or 3 d (B and C) onto coverslips for immunocytochemical analysis. (A) GFP fluorescent cells (green) among untransfected Hoechst dye–labeled cells. (B and C) After transfection with Mig6FL-V5, cells were cultured for 3 d and immunostained with α-microtubule-associated protein 2 (α-MAP2) (B) or α-Mig6 (α-Mig) antibodies (C). Mig6 did not affect neuronal differentiation. (D–G) Cells were fixed and doubly labeled using α-V5 (D) and α-Mig6 (E) antibodies. Nearly all V5-labeled cells also express Mig6. Similar results were obtained with a HIS-epitope–tagged Mig6 expression plasmid (not depicted). (H) Quantification of Hoechst dye–labeled cortical neurons that have migrated onto the lower face of the Boyden chamber membrane. Cells were left nonelectroporated (−) or were electroporated with expression plasmids encoding GFP or with Mig6FL-HIS. Neurons were seeded onto the upper compartment of the Boyden chamber in the absence (black bars) or presence (gray bars) of 50 ng/ml HGF. The cells were fixed and counted after 24 h. The expression of Mig6FL-HIS completely prevented HGF-mediated cell migration. (I) Quantification of migrating cortical neurons expressed as fold of induction over unstimulated cells. Cells were electroporated with LacZV5 control or Mig6FL-V5 expression plasmids in the presence of 50 ng/ml HGF or 100 ng/ml SDF-1. Mig6FL-V5 inhibited HGF-induced (P < 0.0001, t test), but not SDF-1–induced, cell migration (P > 0.28, t test). Bar, 50 μm.
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fig5: Mig6 overexpression in cortical neurons inhibits HGF-induced migration. Cortical neurons were dissected from E15.5 mouse embryos and electroporated with expression plasmids encoding GFP (A) or Mig6FL-V5. Electroporated cells were plated for 24 h (A, D–F) or 3 d (B and C) onto coverslips for immunocytochemical analysis. (A) GFP fluorescent cells (green) among untransfected Hoechst dye–labeled cells. (B and C) After transfection with Mig6FL-V5, cells were cultured for 3 d and immunostained with α-microtubule-associated protein 2 (α-MAP2) (B) or α-Mig6 (α-Mig) antibodies (C). Mig6 did not affect neuronal differentiation. (D–G) Cells were fixed and doubly labeled using α-V5 (D) and α-Mig6 (E) antibodies. Nearly all V5-labeled cells also express Mig6. Similar results were obtained with a HIS-epitope–tagged Mig6 expression plasmid (not depicted). (H) Quantification of Hoechst dye–labeled cortical neurons that have migrated onto the lower face of the Boyden chamber membrane. Cells were left nonelectroporated (−) or were electroporated with expression plasmids encoding GFP or with Mig6FL-HIS. Neurons were seeded onto the upper compartment of the Boyden chamber in the absence (black bars) or presence (gray bars) of 50 ng/ml HGF. The cells were fixed and counted after 24 h. The expression of Mig6FL-HIS completely prevented HGF-mediated cell migration. (I) Quantification of migrating cortical neurons expressed as fold of induction over unstimulated cells. Cells were electroporated with LacZV5 control or Mig6FL-V5 expression plasmids in the presence of 50 ng/ml HGF or 100 ng/ml SDF-1. Mig6FL-V5 inhibited HGF-induced (P < 0.0001, t test), but not SDF-1–induced, cell migration (P > 0.28, t test). Bar, 50 μm.

Mentions: We next asked whether the functions of Mig6 were specific to cells of hepatic origin, or whether Mig6 had a more general role in controlling cell migration across different cell lineages, including neurons from the neocortex (Powell et al., 2001). To test the consequences of Mig6 overexpression, embryonic cortical neurons were transfected with a Mig6FL-V5 construct, and Mig6 expression was monitored by immunofluorescence microscopy (Fig. 5, D–G). Our modified electroporation protocol (see Materials and methods) led to transfection efficiencies of 40–70%, as judged by GFP fluorescence (Fig. 5 A). Mig6 overexpression did not affect survival (unpublished data) or differentiation of cortical neurons as judged by the expression of microtubule-associate protein 2 (Fig. 5 B,C). HGF-induced cell migration was assayed 36 h after transfection of expression plasmids that encoded GFP or Mig6FL-HIS. Quantification of all (transfected and untransfected) cells on the lower face of the porous membrane revealed a three- to fourfold increase of migrated cells in the presence of HGF, and a seven- to eightfold stimulation among the GFP transfectants (Fig. 5 H). In contrast, overexpression of Mig6 completely prevented HGF from inducing cell migration (Fig. 5 H). In a separate set of experiments, we compared cells that were transfected with plasmids that encoded Mig6FL-V5 or LacZV5 in the absence or presence of HGF. 24 h after transfection, LacZV5 control cells responded to HGF with a six- to sevenfold higher migration rate (Fig. 5 I). In contrast, Mig6FL-V5-expressing cells failed to migrate (Fig. 5 I; P < 0.0001, t test). The effect of Mig6 on HGF-induced cell migration was specific, because Mig6 was unable to block cell migration in response to the chemokine stromal cell–derived factor-1 (SDF-1) (Fig. 5 I; P > 0.28, t test).


Mitogen-inducible gene 6 is an endogenous inhibitor of HGF/Met-induced cell migration and neurite growth.

Pante G, Thompson J, Lamballe F, Iwata T, Ferby I, Barr FA, Davies AM, Maina F, Klein R - J. Cell Biol. (2005)

Mig6 overexpression in cortical neurons inhibits HGF-induced migration. Cortical neurons were dissected from E15.5 mouse embryos and electroporated with expression plasmids encoding GFP (A) or Mig6FL-V5. Electroporated cells were plated for 24 h (A, D–F) or 3 d (B and C) onto coverslips for immunocytochemical analysis. (A) GFP fluorescent cells (green) among untransfected Hoechst dye–labeled cells. (B and C) After transfection with Mig6FL-V5, cells were cultured for 3 d and immunostained with α-microtubule-associated protein 2 (α-MAP2) (B) or α-Mig6 (α-Mig) antibodies (C). Mig6 did not affect neuronal differentiation. (D–G) Cells were fixed and doubly labeled using α-V5 (D) and α-Mig6 (E) antibodies. Nearly all V5-labeled cells also express Mig6. Similar results were obtained with a HIS-epitope–tagged Mig6 expression plasmid (not depicted). (H) Quantification of Hoechst dye–labeled cortical neurons that have migrated onto the lower face of the Boyden chamber membrane. Cells were left nonelectroporated (−) or were electroporated with expression plasmids encoding GFP or with Mig6FL-HIS. Neurons were seeded onto the upper compartment of the Boyden chamber in the absence (black bars) or presence (gray bars) of 50 ng/ml HGF. The cells were fixed and counted after 24 h. The expression of Mig6FL-HIS completely prevented HGF-mediated cell migration. (I) Quantification of migrating cortical neurons expressed as fold of induction over unstimulated cells. Cells were electroporated with LacZV5 control or Mig6FL-V5 expression plasmids in the presence of 50 ng/ml HGF or 100 ng/ml SDF-1. Mig6FL-V5 inhibited HGF-induced (P < 0.0001, t test), but not SDF-1–induced, cell migration (P > 0.28, t test). Bar, 50 μm.
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fig5: Mig6 overexpression in cortical neurons inhibits HGF-induced migration. Cortical neurons were dissected from E15.5 mouse embryos and electroporated with expression plasmids encoding GFP (A) or Mig6FL-V5. Electroporated cells were plated for 24 h (A, D–F) or 3 d (B and C) onto coverslips for immunocytochemical analysis. (A) GFP fluorescent cells (green) among untransfected Hoechst dye–labeled cells. (B and C) After transfection with Mig6FL-V5, cells were cultured for 3 d and immunostained with α-microtubule-associated protein 2 (α-MAP2) (B) or α-Mig6 (α-Mig) antibodies (C). Mig6 did not affect neuronal differentiation. (D–G) Cells were fixed and doubly labeled using α-V5 (D) and α-Mig6 (E) antibodies. Nearly all V5-labeled cells also express Mig6. Similar results were obtained with a HIS-epitope–tagged Mig6 expression plasmid (not depicted). (H) Quantification of Hoechst dye–labeled cortical neurons that have migrated onto the lower face of the Boyden chamber membrane. Cells were left nonelectroporated (−) or were electroporated with expression plasmids encoding GFP or with Mig6FL-HIS. Neurons were seeded onto the upper compartment of the Boyden chamber in the absence (black bars) or presence (gray bars) of 50 ng/ml HGF. The cells were fixed and counted after 24 h. The expression of Mig6FL-HIS completely prevented HGF-mediated cell migration. (I) Quantification of migrating cortical neurons expressed as fold of induction over unstimulated cells. Cells were electroporated with LacZV5 control or Mig6FL-V5 expression plasmids in the presence of 50 ng/ml HGF or 100 ng/ml SDF-1. Mig6FL-V5 inhibited HGF-induced (P < 0.0001, t test), but not SDF-1–induced, cell migration (P > 0.28, t test). Bar, 50 μm.
Mentions: We next asked whether the functions of Mig6 were specific to cells of hepatic origin, or whether Mig6 had a more general role in controlling cell migration across different cell lineages, including neurons from the neocortex (Powell et al., 2001). To test the consequences of Mig6 overexpression, embryonic cortical neurons were transfected with a Mig6FL-V5 construct, and Mig6 expression was monitored by immunofluorescence microscopy (Fig. 5, D–G). Our modified electroporation protocol (see Materials and methods) led to transfection efficiencies of 40–70%, as judged by GFP fluorescence (Fig. 5 A). Mig6 overexpression did not affect survival (unpublished data) or differentiation of cortical neurons as judged by the expression of microtubule-associate protein 2 (Fig. 5 B,C). HGF-induced cell migration was assayed 36 h after transfection of expression plasmids that encoded GFP or Mig6FL-HIS. Quantification of all (transfected and untransfected) cells on the lower face of the porous membrane revealed a three- to fourfold increase of migrated cells in the presence of HGF, and a seven- to eightfold stimulation among the GFP transfectants (Fig. 5 H). In contrast, overexpression of Mig6 completely prevented HGF from inducing cell migration (Fig. 5 H). In a separate set of experiments, we compared cells that were transfected with plasmids that encoded Mig6FL-V5 or LacZV5 in the absence or presence of HGF. 24 h after transfection, LacZV5 control cells responded to HGF with a six- to sevenfold higher migration rate (Fig. 5 I). In contrast, Mig6FL-V5-expressing cells failed to migrate (Fig. 5 I; P < 0.0001, t test). The effect of Mig6 on HGF-induced cell migration was specific, because Mig6 was unable to block cell migration in response to the chemokine stromal cell–derived factor-1 (SDF-1) (Fig. 5 I; P > 0.28, t test).

Bottom Line: Here we report a mechanism by which mitogen-inducible gene 6 (Mig6; also called Gene 33 and receptor-associated late transducer) negatively regulates HGF/Met-induced cell migration.The effect is observed by Mig6 overexpression and is reversed by Mig6 small interfering RNA knock-down experiments; this indicates that endogenous Mig6 is part of a mechanism that inhibits Met signaling.Because Mig6 also is induced by HGF stimulation, our results suggest that Mig6 is part of a negative feedback loop that attenuates Met functions in different contexts and cell types.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, 82152 Munich-Martinsried, Germany.

ABSTRACT
Hepatocyte growth factor (HGF)/Met signaling controls cell migration, growth and differentiation in several embryonic organs and is implicated in human cancer. The physiologic mechanisms that attenuate Met signaling are not well understood. Here we report a mechanism by which mitogen-inducible gene 6 (Mig6; also called Gene 33 and receptor-associated late transducer) negatively regulates HGF/Met-induced cell migration. The effect is observed by Mig6 overexpression and is reversed by Mig6 small interfering RNA knock-down experiments; this indicates that endogenous Mig6 is part of a mechanism that inhibits Met signaling. Mig6 functions in cells of hepatic origin and in neurons, which suggests a role for Mig6 in different cell lineages. Mechanistically, Mig6 requires an intact Cdc42/Rac interactive binding site to exert its inhibitory action, which suggests that Mig6 acts, at least in part, distally from Met, possibly by inhibiting Rho-like GTPases. Because Mig6 also is induced by HGF stimulation, our results suggest that Mig6 is part of a negative feedback loop that attenuates Met functions in different contexts and cell types.

Show MeSH
Related in: MedlinePlus