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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 inhibits HGF-induced neurite outgrowth and branching of sympathetic neurons. (A) Western blotting (W.B.) using an α-Mig6 antibody showing the time course of endogenous Mig6 protein induction upon HGF stimulation in postnatal day 40 (P40) superior cervical ganglion (SCG) neurons (top panel). The α-MAPK antibody was used as internal control. Representative P40 SCG neurons transfected with YFP (B) or with a mixture of YFP plus Mig6FL-V5 expression plasmids (C). Cells were grown in medium supplemented with 10 ng/ml HGF for 48 h. Note the significant reduction in neurite complexity of the Mig6FL-V5-expressing cell compared with YFP control. (D) Sholl analysis (see Materials and methods) of P40 SCG neurons transfected with YFP alone and exposed for 48 h to control media (CON/YFP) or 10 ng/ml HGF (HGF/YFP). (E) Sholl analysis of P40 SCG neurons stimulated with 10 ng/ml HGF after being transfected with YFP (black bars) or YFP plus Mig6FL-V5 (gray bars). Mig6FL-V5 expression was able to block HGF-induced neurite outgrowth completely (compare panels D and E, gray bars). (F) Numbers of neurite branch points per neuron in different experimental conditions. P40 SCG neurons were treated as described above, and the numbers of branch points per neuron were counted in the presence (HGF) or absence (CON) of 10 ng/ml HGF. (G) Percentage of surviving P40 SCG neurons in different experimental conditions. After 48 h of HGF exposure, the neurons were transfected with the control LACZV5 or the Mig6FL-V5 expression plasmids. Upon stimulation with HGF, the expression of Mig6FL-V5 did not affect P40 SCG neuron survival. (H) Sholl analysis of P40 SCG neurons transfected with Mig6-specific or GFP-specific siRNA oligonucleotides together with an expression plasmid encoding YFP in the presence of 10 ng/ml HGF. Endogenous Mig6 knock-down by siRNA induced a modest increase in neurite branching. Distances in D, E, and H measured in μm.
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fig6: Mig6 inhibits HGF-induced neurite outgrowth and branching of sympathetic neurons. (A) Western blotting (W.B.) using an α-Mig6 antibody showing the time course of endogenous Mig6 protein induction upon HGF stimulation in postnatal day 40 (P40) superior cervical ganglion (SCG) neurons (top panel). The α-MAPK antibody was used as internal control. Representative P40 SCG neurons transfected with YFP (B) or with a mixture of YFP plus Mig6FL-V5 expression plasmids (C). Cells were grown in medium supplemented with 10 ng/ml HGF for 48 h. Note the significant reduction in neurite complexity of the Mig6FL-V5-expressing cell compared with YFP control. (D) Sholl analysis (see Materials and methods) of P40 SCG neurons transfected with YFP alone and exposed for 48 h to control media (CON/YFP) or 10 ng/ml HGF (HGF/YFP). (E) Sholl analysis of P40 SCG neurons stimulated with 10 ng/ml HGF after being transfected with YFP (black bars) or YFP plus Mig6FL-V5 (gray bars). Mig6FL-V5 expression was able to block HGF-induced neurite outgrowth completely (compare panels D and E, gray bars). (F) Numbers of neurite branch points per neuron in different experimental conditions. P40 SCG neurons were treated as described above, and the numbers of branch points per neuron were counted in the presence (HGF) or absence (CON) of 10 ng/ml HGF. (G) Percentage of surviving P40 SCG neurons in different experimental conditions. After 48 h of HGF exposure, the neurons were transfected with the control LACZV5 or the Mig6FL-V5 expression plasmids. Upon stimulation with HGF, the expression of Mig6FL-V5 did not affect P40 SCG neuron survival. (H) Sholl analysis of P40 SCG neurons transfected with Mig6-specific or GFP-specific siRNA oligonucleotides together with an expression plasmid encoding YFP in the presence of 10 ng/ml HGF. Endogenous Mig6 knock-down by siRNA induced a modest increase in neurite branching. Distances in D, E, and H measured in μm.

Mentions: Besides regulating cell migration, HGF is a chemoattractant and neurite growth-promoting factor for subsets of neurons, including sympathetic neurons (Maina and Klein, 1999; Thompson et al., 2004). We next tested the effects of Mig6 on neurite growth of paravertebral sympathetic neurons. Most postnatal day 40 sympathetic neurons of the superior cervical ganglion survive in culture without addition of neurotrophic factors (unpublished data). Exogenous HGF induced Mig6 expression (Fig. 6 A), and stimulated outgrowth and branching of neurites. To quantify this effect, we transfected the cells with an expression plasmid that encoded YFP by way of gene gun, and used Sholl analysis (see Materials and methods) to determine neurite complexity. HGF (10 ng/ml) significantly increased neurite complexity and branching as compared with nontreated control cultures (Fig. 6, D and F). The effect was most pronounced close to the soma, whereas there was no significant increase in complexity in the longest neurites (Fig. 6 D). To investigate neurite complexity, we compared the neurite arbors of cells that were transfected with expression vectors encoding Mig6 plus YFP with cells expressing YFP alone (Fig. 6, B and C). Overexpression of Mig6 greatly reduced neurite outgrowth and branching of HGF-stimulated cells (Fig. 6 E). The effect was most pronounced close to the cell soma, where HGF had its strongest effects in comparison with control cDNAs, such as YFP (Fig. 6 E). This effect was specific for HGF-treated neurons, because in untreated neurons, expression of Mig6 did not cause a reduction of branch points (Fig. 6 F). Exogenous Mig6 also did not affect survival of HGF-treated neurons, which suggests that Mig6 specifically inhibited Met signaling toward neurite growth (Fig. 6 G). We next performed Mig6 knock-down experiments and found a modest increase in neurite length in Mig6 siRNA-treated, as compared with GFP siRNA-treated, cells. The increase in Mig6 knock-down cells was significant in neurites that extended furthest from the soma. Because the induction of endogenous Mig6 protein is delayed (see Fig. 6 A), the effect of knocking down mig6 mRNA may be visible only in the longest neurons that took the most time to grow. These results suggest that Mig6 plays a role in suppressing neurite complexity that is induced by HGF/Met signaling.


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 inhibits HGF-induced neurite outgrowth and branching of sympathetic neurons. (A) Western blotting (W.B.) using an α-Mig6 antibody showing the time course of endogenous Mig6 protein induction upon HGF stimulation in postnatal day 40 (P40) superior cervical ganglion (SCG) neurons (top panel). The α-MAPK antibody was used as internal control. Representative P40 SCG neurons transfected with YFP (B) or with a mixture of YFP plus Mig6FL-V5 expression plasmids (C). Cells were grown in medium supplemented with 10 ng/ml HGF for 48 h. Note the significant reduction in neurite complexity of the Mig6FL-V5-expressing cell compared with YFP control. (D) Sholl analysis (see Materials and methods) of P40 SCG neurons transfected with YFP alone and exposed for 48 h to control media (CON/YFP) or 10 ng/ml HGF (HGF/YFP). (E) Sholl analysis of P40 SCG neurons stimulated with 10 ng/ml HGF after being transfected with YFP (black bars) or YFP plus Mig6FL-V5 (gray bars). Mig6FL-V5 expression was able to block HGF-induced neurite outgrowth completely (compare panels D and E, gray bars). (F) Numbers of neurite branch points per neuron in different experimental conditions. P40 SCG neurons were treated as described above, and the numbers of branch points per neuron were counted in the presence (HGF) or absence (CON) of 10 ng/ml HGF. (G) Percentage of surviving P40 SCG neurons in different experimental conditions. After 48 h of HGF exposure, the neurons were transfected with the control LACZV5 or the Mig6FL-V5 expression plasmids. Upon stimulation with HGF, the expression of Mig6FL-V5 did not affect P40 SCG neuron survival. (H) Sholl analysis of P40 SCG neurons transfected with Mig6-specific or GFP-specific siRNA oligonucleotides together with an expression plasmid encoding YFP in the presence of 10 ng/ml HGF. Endogenous Mig6 knock-down by siRNA induced a modest increase in neurite branching. Distances in D, E, and H measured in μm.
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fig6: Mig6 inhibits HGF-induced neurite outgrowth and branching of sympathetic neurons. (A) Western blotting (W.B.) using an α-Mig6 antibody showing the time course of endogenous Mig6 protein induction upon HGF stimulation in postnatal day 40 (P40) superior cervical ganglion (SCG) neurons (top panel). The α-MAPK antibody was used as internal control. Representative P40 SCG neurons transfected with YFP (B) or with a mixture of YFP plus Mig6FL-V5 expression plasmids (C). Cells were grown in medium supplemented with 10 ng/ml HGF for 48 h. Note the significant reduction in neurite complexity of the Mig6FL-V5-expressing cell compared with YFP control. (D) Sholl analysis (see Materials and methods) of P40 SCG neurons transfected with YFP alone and exposed for 48 h to control media (CON/YFP) or 10 ng/ml HGF (HGF/YFP). (E) Sholl analysis of P40 SCG neurons stimulated with 10 ng/ml HGF after being transfected with YFP (black bars) or YFP plus Mig6FL-V5 (gray bars). Mig6FL-V5 expression was able to block HGF-induced neurite outgrowth completely (compare panels D and E, gray bars). (F) Numbers of neurite branch points per neuron in different experimental conditions. P40 SCG neurons were treated as described above, and the numbers of branch points per neuron were counted in the presence (HGF) or absence (CON) of 10 ng/ml HGF. (G) Percentage of surviving P40 SCG neurons in different experimental conditions. After 48 h of HGF exposure, the neurons were transfected with the control LACZV5 or the Mig6FL-V5 expression plasmids. Upon stimulation with HGF, the expression of Mig6FL-V5 did not affect P40 SCG neuron survival. (H) Sholl analysis of P40 SCG neurons transfected with Mig6-specific or GFP-specific siRNA oligonucleotides together with an expression plasmid encoding YFP in the presence of 10 ng/ml HGF. Endogenous Mig6 knock-down by siRNA induced a modest increase in neurite branching. Distances in D, E, and H measured in μm.
Mentions: Besides regulating cell migration, HGF is a chemoattractant and neurite growth-promoting factor for subsets of neurons, including sympathetic neurons (Maina and Klein, 1999; Thompson et al., 2004). We next tested the effects of Mig6 on neurite growth of paravertebral sympathetic neurons. Most postnatal day 40 sympathetic neurons of the superior cervical ganglion survive in culture without addition of neurotrophic factors (unpublished data). Exogenous HGF induced Mig6 expression (Fig. 6 A), and stimulated outgrowth and branching of neurites. To quantify this effect, we transfected the cells with an expression plasmid that encoded YFP by way of gene gun, and used Sholl analysis (see Materials and methods) to determine neurite complexity. HGF (10 ng/ml) significantly increased neurite complexity and branching as compared with nontreated control cultures (Fig. 6, D and F). The effect was most pronounced close to the soma, whereas there was no significant increase in complexity in the longest neurites (Fig. 6 D). To investigate neurite complexity, we compared the neurite arbors of cells that were transfected with expression vectors encoding Mig6 plus YFP with cells expressing YFP alone (Fig. 6, B and C). Overexpression of Mig6 greatly reduced neurite outgrowth and branching of HGF-stimulated cells (Fig. 6 E). The effect was most pronounced close to the cell soma, where HGF had its strongest effects in comparison with control cDNAs, such as YFP (Fig. 6 E). This effect was specific for HGF-treated neurons, because in untreated neurons, expression of Mig6 did not cause a reduction of branch points (Fig. 6 F). Exogenous Mig6 also did not affect survival of HGF-treated neurons, which suggests that Mig6 specifically inhibited Met signaling toward neurite growth (Fig. 6 G). We next performed Mig6 knock-down experiments and found a modest increase in neurite length in Mig6 siRNA-treated, as compared with GFP siRNA-treated, cells. The increase in Mig6 knock-down cells was significant in neurites that extended furthest from the soma. Because the induction of endogenous Mig6 protein is delayed (see Fig. 6 A), the effect of knocking down mig6 mRNA may be visible only in the longest neurons that took the most time to grow. These results suggest that Mig6 plays a role in suppressing neurite complexity that is induced by HGF/Met signaling.

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