Limits...
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.

Show MeSH

Related in: MedlinePlus

Endogenous Mig6 inhibits HGF-induced cell migration in MLP29 cells. MLP29 cells were transfected with GFP-specific siRNAs as a negative control (A) or with mig6-specific siRNAs (B) for 4 d, then fixed, and Mig6 protein levels were detected by immunofluorescence using a Mig6-specific antibody (green label). (B) Cells were visualized using Hoechst dye. Note the efficient knock-down of Mig6 protein levels upon transfection of mig6 siRNAs. Bar, 50 μm. (C) Western blot (W.B.) analysis of MLP29 cells transfected with GFP siRNAs (−) or mig6-specific siRNAs (+) for 4 d. After transfection, the cells were stimulated with 40 ng/ml HGF for the indicated times and the cell lysates were analyzed using SDS-PAGE and immunoblotting using α-Mig6– or α-tubulin–specific (α-Tub) antibodies. The specific reduction of Mig6 levels is seen most clearly at the 4-h time point. (D–G) Representative fields of GFP siRNA– and mig6 siRNA–transfected MLP29 cells (labeled with Hoechst dye) that migrated into the lower compartment of the Boyden chamber in the presence or the absence of HGF. MLP29 cells were treated as described above, then transfected a second time with siRNA oligonucleotides, incubated for 24h, harvested, plated into the upper compartment of the Boyden chamber, and allowed to attach onto the membrane. Different concentrations of HGF were added to the media of the lower compartment of the chamber and the cells were allowed to migrate for 16 h. Note the increase of cell migration of mig6 knock-down cells after HGF stimulation (compare panels F and G). Bar, 100 μm. (H) Quantification of migration expressed as fold of induction over unstimulated cells. Increasing concentrations of HGF were added to the lower chamber after transfection of control GFP siRNAs (black bars) or mig6 siRNAs (gray bars). 5 ng/ml HGF, P = 0.02; 10 ng/ml HGF, P < 0.0002; 40 ng/ml HGF, P < 0.01, t test.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171179&req=5

fig3: Endogenous Mig6 inhibits HGF-induced cell migration in MLP29 cells. MLP29 cells were transfected with GFP-specific siRNAs as a negative control (A) or with mig6-specific siRNAs (B) for 4 d, then fixed, and Mig6 protein levels were detected by immunofluorescence using a Mig6-specific antibody (green label). (B) Cells were visualized using Hoechst dye. Note the efficient knock-down of Mig6 protein levels upon transfection of mig6 siRNAs. Bar, 50 μm. (C) Western blot (W.B.) analysis of MLP29 cells transfected with GFP siRNAs (−) or mig6-specific siRNAs (+) for 4 d. After transfection, the cells were stimulated with 40 ng/ml HGF for the indicated times and the cell lysates were analyzed using SDS-PAGE and immunoblotting using α-Mig6– or α-tubulin–specific (α-Tub) antibodies. The specific reduction of Mig6 levels is seen most clearly at the 4-h time point. (D–G) Representative fields of GFP siRNA– and mig6 siRNA–transfected MLP29 cells (labeled with Hoechst dye) that migrated into the lower compartment of the Boyden chamber in the presence or the absence of HGF. MLP29 cells were treated as described above, then transfected a second time with siRNA oligonucleotides, incubated for 24h, harvested, plated into the upper compartment of the Boyden chamber, and allowed to attach onto the membrane. Different concentrations of HGF were added to the media of the lower compartment of the chamber and the cells were allowed to migrate for 16 h. Note the increase of cell migration of mig6 knock-down cells after HGF stimulation (compare panels F and G). Bar, 100 μm. (H) Quantification of migration expressed as fold of induction over unstimulated cells. Increasing concentrations of HGF were added to the lower chamber after transfection of control GFP siRNAs (black bars) or mig6 siRNAs (gray bars). 5 ng/ml HGF, P = 0.02; 10 ng/ml HGF, P < 0.0002; 40 ng/ml HGF, P < 0.01, t test.

Mentions: We next investigated, using RNA interference, whether endogenous Mig6 suppressed HGF/Met-mediated cell migration by knocking down Mig6 protein levels (Elbashir et al., 2001). MLP29 cells were transfected with siRNAs specific for GFP or mig6, and the levels of Mig6 protein were analyzed by immunostaining and immunoblotting. Mig6 siRNA, but not control GFP siRNA, specifically knocked down endogenous Mig6 immunoreactivity 96 h after transfection (Fig. 3, A and B). Mig6 siRNA also suppressed HGF-stimulated induction of Mig6 (Fig. 3 C; compare 4-h time point in the presence and absence of mig6 siRNA). The reduction of protein levels was specific for mig6, because endogenous α-tubulin and Met levels were unaffected (Fig. 3 C and not depicted). To investigate the role of Mig6 protein in cell migration, cells were transfected with mig6 siRNA oligonucleotides, and subjected to the Boyden chamber assay with different concentrations of HGF (Fig. 3). Representative images of Hoechst dye–labeled cells that migrated into the lower compartment are shown in Fig. 3 (D–G). The induction of cell migration by HGF under these conditions was less strong, yet was still dose dependent (Fig. 3 H). Quantification of migrating cells revealed that under conditions of optimal HGF concentrations, knock down of Mig6 enhanced cell migration by approximately twofold (Fig. 3 H). Similar results were obtained with a separate set of siRNA oligonucleotides (unpublished data). These findings demonstrated that Mig6 is a physiologic inhibitor of HGF/Met-mediated cell migration of MLP29 cells.


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)

Endogenous Mig6 inhibits HGF-induced cell migration in MLP29 cells. MLP29 cells were transfected with GFP-specific siRNAs as a negative control (A) or with mig6-specific siRNAs (B) for 4 d, then fixed, and Mig6 protein levels were detected by immunofluorescence using a Mig6-specific antibody (green label). (B) Cells were visualized using Hoechst dye. Note the efficient knock-down of Mig6 protein levels upon transfection of mig6 siRNAs. Bar, 50 μm. (C) Western blot (W.B.) analysis of MLP29 cells transfected with GFP siRNAs (−) or mig6-specific siRNAs (+) for 4 d. After transfection, the cells were stimulated with 40 ng/ml HGF for the indicated times and the cell lysates were analyzed using SDS-PAGE and immunoblotting using α-Mig6– or α-tubulin–specific (α-Tub) antibodies. The specific reduction of Mig6 levels is seen most clearly at the 4-h time point. (D–G) Representative fields of GFP siRNA– and mig6 siRNA–transfected MLP29 cells (labeled with Hoechst dye) that migrated into the lower compartment of the Boyden chamber in the presence or the absence of HGF. MLP29 cells were treated as described above, then transfected a second time with siRNA oligonucleotides, incubated for 24h, harvested, plated into the upper compartment of the Boyden chamber, and allowed to attach onto the membrane. Different concentrations of HGF were added to the media of the lower compartment of the chamber and the cells were allowed to migrate for 16 h. Note the increase of cell migration of mig6 knock-down cells after HGF stimulation (compare panels F and G). Bar, 100 μm. (H) Quantification of migration expressed as fold of induction over unstimulated cells. Increasing concentrations of HGF were added to the lower chamber after transfection of control GFP siRNAs (black bars) or mig6 siRNAs (gray bars). 5 ng/ml HGF, P = 0.02; 10 ng/ml HGF, P < 0.0002; 40 ng/ml HGF, P < 0.01, t test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171179&req=5

fig3: Endogenous Mig6 inhibits HGF-induced cell migration in MLP29 cells. MLP29 cells were transfected with GFP-specific siRNAs as a negative control (A) or with mig6-specific siRNAs (B) for 4 d, then fixed, and Mig6 protein levels were detected by immunofluorescence using a Mig6-specific antibody (green label). (B) Cells were visualized using Hoechst dye. Note the efficient knock-down of Mig6 protein levels upon transfection of mig6 siRNAs. Bar, 50 μm. (C) Western blot (W.B.) analysis of MLP29 cells transfected with GFP siRNAs (−) or mig6-specific siRNAs (+) for 4 d. After transfection, the cells were stimulated with 40 ng/ml HGF for the indicated times and the cell lysates were analyzed using SDS-PAGE and immunoblotting using α-Mig6– or α-tubulin–specific (α-Tub) antibodies. The specific reduction of Mig6 levels is seen most clearly at the 4-h time point. (D–G) Representative fields of GFP siRNA– and mig6 siRNA–transfected MLP29 cells (labeled with Hoechst dye) that migrated into the lower compartment of the Boyden chamber in the presence or the absence of HGF. MLP29 cells were treated as described above, then transfected a second time with siRNA oligonucleotides, incubated for 24h, harvested, plated into the upper compartment of the Boyden chamber, and allowed to attach onto the membrane. Different concentrations of HGF were added to the media of the lower compartment of the chamber and the cells were allowed to migrate for 16 h. Note the increase of cell migration of mig6 knock-down cells after HGF stimulation (compare panels F and G). Bar, 100 μm. (H) Quantification of migration expressed as fold of induction over unstimulated cells. Increasing concentrations of HGF were added to the lower chamber after transfection of control GFP siRNAs (black bars) or mig6 siRNAs (gray bars). 5 ng/ml HGF, P = 0.02; 10 ng/ml HGF, P < 0.0002; 40 ng/ml HGF, P < 0.01, t test.
Mentions: We next investigated, using RNA interference, whether endogenous Mig6 suppressed HGF/Met-mediated cell migration by knocking down Mig6 protein levels (Elbashir et al., 2001). MLP29 cells were transfected with siRNAs specific for GFP or mig6, and the levels of Mig6 protein were analyzed by immunostaining and immunoblotting. Mig6 siRNA, but not control GFP siRNA, specifically knocked down endogenous Mig6 immunoreactivity 96 h after transfection (Fig. 3, A and B). Mig6 siRNA also suppressed HGF-stimulated induction of Mig6 (Fig. 3 C; compare 4-h time point in the presence and absence of mig6 siRNA). The reduction of protein levels was specific for mig6, because endogenous α-tubulin and Met levels were unaffected (Fig. 3 C and not depicted). To investigate the role of Mig6 protein in cell migration, cells were transfected with mig6 siRNA oligonucleotides, and subjected to the Boyden chamber assay with different concentrations of HGF (Fig. 3). Representative images of Hoechst dye–labeled cells that migrated into the lower compartment are shown in Fig. 3 (D–G). The induction of cell migration by HGF under these conditions was less strong, yet was still dose dependent (Fig. 3 H). Quantification of migrating cells revealed that under conditions of optimal HGF concentrations, knock down of Mig6 enhanced cell migration by approximately twofold (Fig. 3 H). Similar results were obtained with a separate set of siRNA oligonucleotides (unpublished data). These findings demonstrated that Mig6 is a physiologic inhibitor of HGF/Met-mediated cell migration of MLP29 cells.

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