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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 inhibits HGF-mediated cell migration. (A–C) MLP29 cells were transfected with a mixture of YFP and Mig6FL-V5 expression plasmids in a ratio of 1:5. 24 h later, cells were starved in 0.1% FBS for an additional 24 h, harvested, and seeded onto coverslips for immunocytochemical analysis. Cells were fixed and Mig6 overexpression was detected using a Mig6-specific antibody (B). YFP-expressing cells (A) are strongly positive for Mig6 (C). Bar, 50 μm. (D–I) MLP29 cells were transfected with Mig6FL-V5 or LacZV5 expression plasmids and stained with anti-Mig6 and anti-V5 antibodies as above. LacZV5 transfected cells were weakly positive for (endogenous) Mig6 and strongly positive for the V5 epitope (D–F). Mig6FL-V5 transfected cells showed strong costaining for Mig6 and V5 (G–I) indicating efficient overexpression of Mig6. (J) Western blot (W.B.) using V5 antibodies to detect Mig6FL-V5 or LacZV5 expression. (K–N) Photographs of Hoechst dye–labeled cells after migration through the Boyden chamber membrane. Cells were transfected with the indicated expression plasmids as described above, and a portion was seeded onto the upper face of the membrane. Cells were exposed to 10% FBS (K and L) or 40 ng/ml HGF (M and N). Reduced migration due to Mig6 overexpression was observed in the YFP-positive (see quantification below) and in the total Hoechst dye–positive cell population (compare M with N). Bar, 100 μm. Quantification of migration of YFP-expressing cells indicated as fold of induction over unstimulated cells, in the absence (O) or presence (P) of the DNA polymerase inhibitor, aphidicolin. Mig6 overexpression reduced HGF-stimulated cell migration by approximately threefold in the absence (O; P < 0.001, t test) or presence of aphidicolin (P; P < 0.001, t test). In contrast, cell migration stimulated (Stim.) with 10% FBS in the absence or presence of aphidicolin was unaffected by Mig6 overexpression (P = 0.45 and P = 0.121, respectively; t test).
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fig2: Mig6 overexpression inhibits HGF-mediated cell migration. (A–C) MLP29 cells were transfected with a mixture of YFP and Mig6FL-V5 expression plasmids in a ratio of 1:5. 24 h later, cells were starved in 0.1% FBS for an additional 24 h, harvested, and seeded onto coverslips for immunocytochemical analysis. Cells were fixed and Mig6 overexpression was detected using a Mig6-specific antibody (B). YFP-expressing cells (A) are strongly positive for Mig6 (C). Bar, 50 μm. (D–I) MLP29 cells were transfected with Mig6FL-V5 or LacZV5 expression plasmids and stained with anti-Mig6 and anti-V5 antibodies as above. LacZV5 transfected cells were weakly positive for (endogenous) Mig6 and strongly positive for the V5 epitope (D–F). Mig6FL-V5 transfected cells showed strong costaining for Mig6 and V5 (G–I) indicating efficient overexpression of Mig6. (J) Western blot (W.B.) using V5 antibodies to detect Mig6FL-V5 or LacZV5 expression. (K–N) Photographs of Hoechst dye–labeled cells after migration through the Boyden chamber membrane. Cells were transfected with the indicated expression plasmids as described above, and a portion was seeded onto the upper face of the membrane. Cells were exposed to 10% FBS (K and L) or 40 ng/ml HGF (M and N). Reduced migration due to Mig6 overexpression was observed in the YFP-positive (see quantification below) and in the total Hoechst dye–positive cell population (compare M with N). Bar, 100 μm. Quantification of migration of YFP-expressing cells indicated as fold of induction over unstimulated cells, in the absence (O) or presence (P) of the DNA polymerase inhibitor, aphidicolin. Mig6 overexpression reduced HGF-stimulated cell migration by approximately threefold in the absence (O; P < 0.001, t test) or presence of aphidicolin (P; P < 0.001, t test). In contrast, cell migration stimulated (Stim.) with 10% FBS in the absence or presence of aphidicolin was unaffected by Mig6 overexpression (P = 0.45 and P = 0.121, respectively; t test).

Mentions: The only cellular context in which Mig6 has been implicated is cell division. However, because HGF/Met signaling is critical for cell migration, we tested the effects of Mig6 overexpression on HGF/Met-mediated migration in the Boyden chamber migration assay. This assay tests the capacity of cells to migrate through a porous membrane that separates upper and lower compartments. MLP29 cells were transfected with a plasmid that encodes YFP, or cotransfected with plasmids that encode full-length Mig6 (Mig6FL) and YFP (Fig. 2, A–C). We confirmed the expression of exogenous Mig6 by immunostaining of cells and immunoblotting of lysates from cells expressing V5 epitope–tagged versions of Mig6 (Mig6FL-V5) in comparison with V5 epitope–tagged β-galactosidase (LacZV5) (Fig. 2, D–J). For each experimental condition, equal numbers of cells were seeded onto coverslips for immunocytochemical analysis, or onto the upper compartment of the Boyden chamber and were exposed to varying concentrations of HGF or 10% FBS in the lower compartment. Cell migration through the membrane into the lower compartment was stimulated by HGF in a dose-dependent manner (unpublished data). At 40 ng/ml HGF, migration of transfected, YFP-positive cells was enhanced 30- to 40-fold over unstimulated cells (PFig. 2, O and P). Representative images of Hoechst dye–labeled cells, which migrated into the lower compartment of the Boyden chamber, are shown in Fig. 2 (K–N). The presence of Mig6FL reduced HGF-stimulated cell migration by approximately threefold (Fig. 2 O, P < 0.001, t test). In contrast, cell migration that was stimulated with 10% FBS was unaffected by Mig6 overexpression (P = 0.121, 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 inhibits HGF-mediated cell migration. (A–C) MLP29 cells were transfected with a mixture of YFP and Mig6FL-V5 expression plasmids in a ratio of 1:5. 24 h later, cells were starved in 0.1% FBS for an additional 24 h, harvested, and seeded onto coverslips for immunocytochemical analysis. Cells were fixed and Mig6 overexpression was detected using a Mig6-specific antibody (B). YFP-expressing cells (A) are strongly positive for Mig6 (C). Bar, 50 μm. (D–I) MLP29 cells were transfected with Mig6FL-V5 or LacZV5 expression plasmids and stained with anti-Mig6 and anti-V5 antibodies as above. LacZV5 transfected cells were weakly positive for (endogenous) Mig6 and strongly positive for the V5 epitope (D–F). Mig6FL-V5 transfected cells showed strong costaining for Mig6 and V5 (G–I) indicating efficient overexpression of Mig6. (J) Western blot (W.B.) using V5 antibodies to detect Mig6FL-V5 or LacZV5 expression. (K–N) Photographs of Hoechst dye–labeled cells after migration through the Boyden chamber membrane. Cells were transfected with the indicated expression plasmids as described above, and a portion was seeded onto the upper face of the membrane. Cells were exposed to 10% FBS (K and L) or 40 ng/ml HGF (M and N). Reduced migration due to Mig6 overexpression was observed in the YFP-positive (see quantification below) and in the total Hoechst dye–positive cell population (compare M with N). Bar, 100 μm. Quantification of migration of YFP-expressing cells indicated as fold of induction over unstimulated cells, in the absence (O) or presence (P) of the DNA polymerase inhibitor, aphidicolin. Mig6 overexpression reduced HGF-stimulated cell migration by approximately threefold in the absence (O; P < 0.001, t test) or presence of aphidicolin (P; P < 0.001, t test). In contrast, cell migration stimulated (Stim.) with 10% FBS in the absence or presence of aphidicolin was unaffected by Mig6 overexpression (P = 0.45 and P = 0.121, respectively; t test).
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fig2: Mig6 overexpression inhibits HGF-mediated cell migration. (A–C) MLP29 cells were transfected with a mixture of YFP and Mig6FL-V5 expression plasmids in a ratio of 1:5. 24 h later, cells were starved in 0.1% FBS for an additional 24 h, harvested, and seeded onto coverslips for immunocytochemical analysis. Cells were fixed and Mig6 overexpression was detected using a Mig6-specific antibody (B). YFP-expressing cells (A) are strongly positive for Mig6 (C). Bar, 50 μm. (D–I) MLP29 cells were transfected with Mig6FL-V5 or LacZV5 expression plasmids and stained with anti-Mig6 and anti-V5 antibodies as above. LacZV5 transfected cells were weakly positive for (endogenous) Mig6 and strongly positive for the V5 epitope (D–F). Mig6FL-V5 transfected cells showed strong costaining for Mig6 and V5 (G–I) indicating efficient overexpression of Mig6. (J) Western blot (W.B.) using V5 antibodies to detect Mig6FL-V5 or LacZV5 expression. (K–N) Photographs of Hoechst dye–labeled cells after migration through the Boyden chamber membrane. Cells were transfected with the indicated expression plasmids as described above, and a portion was seeded onto the upper face of the membrane. Cells were exposed to 10% FBS (K and L) or 40 ng/ml HGF (M and N). Reduced migration due to Mig6 overexpression was observed in the YFP-positive (see quantification below) and in the total Hoechst dye–positive cell population (compare M with N). Bar, 100 μm. Quantification of migration of YFP-expressing cells indicated as fold of induction over unstimulated cells, in the absence (O) or presence (P) of the DNA polymerase inhibitor, aphidicolin. Mig6 overexpression reduced HGF-stimulated cell migration by approximately threefold in the absence (O; P < 0.001, t test) or presence of aphidicolin (P; P < 0.001, t test). In contrast, cell migration stimulated (Stim.) with 10% FBS in the absence or presence of aphidicolin was unaffected by Mig6 overexpression (P = 0.45 and P = 0.121, respectively; t test).
Mentions: The only cellular context in which Mig6 has been implicated is cell division. However, because HGF/Met signaling is critical for cell migration, we tested the effects of Mig6 overexpression on HGF/Met-mediated migration in the Boyden chamber migration assay. This assay tests the capacity of cells to migrate through a porous membrane that separates upper and lower compartments. MLP29 cells were transfected with a plasmid that encodes YFP, or cotransfected with plasmids that encode full-length Mig6 (Mig6FL) and YFP (Fig. 2, A–C). We confirmed the expression of exogenous Mig6 by immunostaining of cells and immunoblotting of lysates from cells expressing V5 epitope–tagged versions of Mig6 (Mig6FL-V5) in comparison with V5 epitope–tagged β-galactosidase (LacZV5) (Fig. 2, D–J). For each experimental condition, equal numbers of cells were seeded onto coverslips for immunocytochemical analysis, or onto the upper compartment of the Boyden chamber and were exposed to varying concentrations of HGF or 10% FBS in the lower compartment. Cell migration through the membrane into the lower compartment was stimulated by HGF in a dose-dependent manner (unpublished data). At 40 ng/ml HGF, migration of transfected, YFP-positive cells was enhanced 30- to 40-fold over unstimulated cells (PFig. 2, O and P). Representative images of Hoechst dye–labeled cells, which migrated into the lower compartment of the Boyden chamber, are shown in Fig. 2 (K–N). The presence of Mig6FL reduced HGF-stimulated cell migration by approximately threefold (Fig. 2 O, P < 0.001, t test). In contrast, cell migration that was stimulated with 10% FBS was unaffected by Mig6 overexpression (P = 0.121, 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