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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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Induction of Mig6 by HGF/Met signaling in cultured cells and coexpression of Mig6 with Met in vivo. (A–C) Northern blot analysis showing mig6 mRNA up-regulation upon HGF, FGF, or PDGF stimulation for 4 h (A and B) or the indicated times (C) in MLP29 and C2C12 cells. 18S or glyceraldehyde 3–phosphate dehydrogenase (G3PDH) mRNA levels were used as internal control. Western blot (W.B.) analysis showing Mig6 protein induction upon HGF and EGF stimulation in MLP29 cells (D) and primary hepatocytes (E). Cells were grown in 10% FBS, then stimulated with empty media, 40 ng/ml HGF, or 10 ng/ml EGF for the indicated times, lysed, and analyzed by SDS-PAGE and immunoblotting using a specific anti-Mig6 antiserum. Immunoblots for α-tubulin and Met were used as internal controls. Immunocytochemistry analysis showing Mig6 protein induction upon HGF stimulation in MLP29 cells. Cells stimulated as above with empty media (F) or with HGF (G), fixed, and stained using a specific anti-Mig6 antiserum. NT, not treated. Bar, 50 μm. (H–N) In situ hybridization analyses for mig6 and met mRNA transcripts in selected organs of E13.5 wild-type (+/+) and Met signaling–deficient (metd/d) embryos. Co-expression of mig6 and met is observed in alveoli of the lungs (Lu), liver (Li), intercostal muscle (i.m.), and body wall muscle (b.w.) (I, J, M, and N). Mig6 transcript levels are reduced in metd/d embryo lungs and liver (K). Mig6 sense probe was used as negative control on adjacent sections (H, L). Ri, ribs. Bar, 300 μm.
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fig1: Induction of Mig6 by HGF/Met signaling in cultured cells and coexpression of Mig6 with Met in vivo. (A–C) Northern blot analysis showing mig6 mRNA up-regulation upon HGF, FGF, or PDGF stimulation for 4 h (A and B) or the indicated times (C) in MLP29 and C2C12 cells. 18S or glyceraldehyde 3–phosphate dehydrogenase (G3PDH) mRNA levels were used as internal control. Western blot (W.B.) analysis showing Mig6 protein induction upon HGF and EGF stimulation in MLP29 cells (D) and primary hepatocytes (E). Cells were grown in 10% FBS, then stimulated with empty media, 40 ng/ml HGF, or 10 ng/ml EGF for the indicated times, lysed, and analyzed by SDS-PAGE and immunoblotting using a specific anti-Mig6 antiserum. Immunoblots for α-tubulin and Met were used as internal controls. Immunocytochemistry analysis showing Mig6 protein induction upon HGF stimulation in MLP29 cells. Cells stimulated as above with empty media (F) or with HGF (G), fixed, and stained using a specific anti-Mig6 antiserum. NT, not treated. Bar, 50 μm. (H–N) In situ hybridization analyses for mig6 and met mRNA transcripts in selected organs of E13.5 wild-type (+/+) and Met signaling–deficient (metd/d) embryos. Co-expression of mig6 and met is observed in alveoli of the lungs (Lu), liver (Li), intercostal muscle (i.m.), and body wall muscle (b.w.) (I, J, M, and N). Mig6 transcript levels are reduced in metd/d embryo lungs and liver (K). Mig6 sense probe was used as negative control on adjacent sections (H, L). Ri, ribs. Bar, 300 μm.

Mentions: Mig6 was chosen for further analysis because its expression was induced more strongly by HGF—4.5- to 80-fold by 4 h of HGF stimulation—than by fibroblast growth factor 2 (FGF2) or PDGF (Fig. 1, A and B). Other transcripts did not show this preference for HGF (Fig. S1 B). The reduced response to FGF2 and PDGF was not due to lack of specific receptors or downstream transducers, because both growth factors induced robust phosphorylation of ERK/MAPKs in MLP29 cells (Fig. S2; available at http://www.jcb.org/cgi/content/full/jcb.200502013/DC1). Time courses of HGF stimulation revealed that Mig6 mRNA and protein were induced half maximally after 1 h and maintained for several hours (Fig. 1, C and D). The induction of Mig6 protein by EGF was more transient than that induced by HGF (Fig. 1 D). Induction of Mig6 protein by HGF in primary hepatocytes followed delayed kinetics (Fig. 1 E). We also confirmed the induction of endogenous Mig6 protein by immunostaining of MLP29 cells (Fig. 1, F and G). We next investigated the expression of Mig6 and Met in embryonic tissues. By in situ hybridization analysis, we found that both transcripts coexpressed in alveoli of embryonic (E13.5) lung, in liver parenchyma, and in intercostal and body wall muscles of wild-type embryos (Fig. 1, I, J, M, and N). Consistent with Met regulating mig6 transcript levels under physiologic conditions, we found reduced levels of mig6 mRNA in embryos that expressed a signaling-deficient Met receptor (metd/d) (Maina et al., 1996, 2001) (Fig. 1 K). Expression of Mig6 protein was confirmed in structures positive for mig6 mRNA, including intercostal and body wall muscles (Fig. S2).


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)

Induction of Mig6 by HGF/Met signaling in cultured cells and coexpression of Mig6 with Met in vivo. (A–C) Northern blot analysis showing mig6 mRNA up-regulation upon HGF, FGF, or PDGF stimulation for 4 h (A and B) or the indicated times (C) in MLP29 and C2C12 cells. 18S or glyceraldehyde 3–phosphate dehydrogenase (G3PDH) mRNA levels were used as internal control. Western blot (W.B.) analysis showing Mig6 protein induction upon HGF and EGF stimulation in MLP29 cells (D) and primary hepatocytes (E). Cells were grown in 10% FBS, then stimulated with empty media, 40 ng/ml HGF, or 10 ng/ml EGF for the indicated times, lysed, and analyzed by SDS-PAGE and immunoblotting using a specific anti-Mig6 antiserum. Immunoblots for α-tubulin and Met were used as internal controls. Immunocytochemistry analysis showing Mig6 protein induction upon HGF stimulation in MLP29 cells. Cells stimulated as above with empty media (F) or with HGF (G), fixed, and stained using a specific anti-Mig6 antiserum. NT, not treated. Bar, 50 μm. (H–N) In situ hybridization analyses for mig6 and met mRNA transcripts in selected organs of E13.5 wild-type (+/+) and Met signaling–deficient (metd/d) embryos. Co-expression of mig6 and met is observed in alveoli of the lungs (Lu), liver (Li), intercostal muscle (i.m.), and body wall muscle (b.w.) (I, J, M, and N). Mig6 transcript levels are reduced in metd/d embryo lungs and liver (K). Mig6 sense probe was used as negative control on adjacent sections (H, L). Ri, ribs. Bar, 300 μm.
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Related In: Results  -  Collection

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fig1: Induction of Mig6 by HGF/Met signaling in cultured cells and coexpression of Mig6 with Met in vivo. (A–C) Northern blot analysis showing mig6 mRNA up-regulation upon HGF, FGF, or PDGF stimulation for 4 h (A and B) or the indicated times (C) in MLP29 and C2C12 cells. 18S or glyceraldehyde 3–phosphate dehydrogenase (G3PDH) mRNA levels were used as internal control. Western blot (W.B.) analysis showing Mig6 protein induction upon HGF and EGF stimulation in MLP29 cells (D) and primary hepatocytes (E). Cells were grown in 10% FBS, then stimulated with empty media, 40 ng/ml HGF, or 10 ng/ml EGF for the indicated times, lysed, and analyzed by SDS-PAGE and immunoblotting using a specific anti-Mig6 antiserum. Immunoblots for α-tubulin and Met were used as internal controls. Immunocytochemistry analysis showing Mig6 protein induction upon HGF stimulation in MLP29 cells. Cells stimulated as above with empty media (F) or with HGF (G), fixed, and stained using a specific anti-Mig6 antiserum. NT, not treated. Bar, 50 μm. (H–N) In situ hybridization analyses for mig6 and met mRNA transcripts in selected organs of E13.5 wild-type (+/+) and Met signaling–deficient (metd/d) embryos. Co-expression of mig6 and met is observed in alveoli of the lungs (Lu), liver (Li), intercostal muscle (i.m.), and body wall muscle (b.w.) (I, J, M, and N). Mig6 transcript levels are reduced in metd/d embryo lungs and liver (K). Mig6 sense probe was used as negative control on adjacent sections (H, L). Ri, ribs. Bar, 300 μm.
Mentions: Mig6 was chosen for further analysis because its expression was induced more strongly by HGF—4.5- to 80-fold by 4 h of HGF stimulation—than by fibroblast growth factor 2 (FGF2) or PDGF (Fig. 1, A and B). Other transcripts did not show this preference for HGF (Fig. S1 B). The reduced response to FGF2 and PDGF was not due to lack of specific receptors or downstream transducers, because both growth factors induced robust phosphorylation of ERK/MAPKs in MLP29 cells (Fig. S2; available at http://www.jcb.org/cgi/content/full/jcb.200502013/DC1). Time courses of HGF stimulation revealed that Mig6 mRNA and protein were induced half maximally after 1 h and maintained for several hours (Fig. 1, C and D). The induction of Mig6 protein by EGF was more transient than that induced by HGF (Fig. 1 D). Induction of Mig6 protein by HGF in primary hepatocytes followed delayed kinetics (Fig. 1 E). We also confirmed the induction of endogenous Mig6 protein by immunostaining of MLP29 cells (Fig. 1, F and G). We next investigated the expression of Mig6 and Met in embryonic tissues. By in situ hybridization analysis, we found that both transcripts coexpressed in alveoli of embryonic (E13.5) lung, in liver parenchyma, and in intercostal and body wall muscles of wild-type embryos (Fig. 1, I, J, M, and N). Consistent with Met regulating mig6 transcript levels under physiologic conditions, we found reduced levels of mig6 mRNA in embryos that expressed a signaling-deficient Met receptor (metd/d) (Maina et al., 1996, 2001) (Fig. 1 K). Expression of Mig6 protein was confirmed in structures positive for mig6 mRNA, including intercostal and body wall muscles (Fig. S2).

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