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Netrins and neogenin promote myotube formation.

Kang JS, Yi MJ, Zhang W, Feinleib JL, Cole F, Krauss RS - J. Cell Biol. (2004)

Bottom Line: These proteins stimulate myotube formation and enhance myogenic bHLH- and NFAT-dependent transcription.Furthermore, neogenin binds to CDO in a cis fashion, and myoblasts lacking CDO are defective in responding to recombinant netrin.It is proposed that netrin-3 and neogenin may promote myogenic differentiation by an autocrine mechanism as components of a higher order complex of several promyogenic cell surface proteins.

View Article: PubMed Central - PubMed

Affiliation: Brookdale Department of Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, New York, NY 10029, USA.

ABSTRACT
Differentiation of skeletal myoblasts into multinucleated myotubes is a multistep process orchestrated by several families of transcription factors, including myogenic bHLH and NFAT proteins. The activities of these factors and formation of myotubes are regulated by signal transduction pathways, but few extracellular factors that might initiate such signals have been identified. One exception is a cell surface complex containing promyogenic Ig superfamily members (CDO and BOC) and cadherins. Netrins and their receptors are established regulators of axon guidance, but little is known of their function outside the nervous system. We report here that myoblasts express the secreted factor netrin-3 and its receptor, neogenin. These proteins stimulate myotube formation and enhance myogenic bHLH- and NFAT-dependent transcription. Furthermore, neogenin binds to CDO in a cis fashion, and myoblasts lacking CDO are defective in responding to recombinant netrin. It is proposed that netrin-3 and neogenin may promote myogenic differentiation by an autocrine mechanism as components of a higher order complex of several promyogenic cell surface proteins.

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Netrin-2 promotes myotube formation. (A) Photomicrographs of C2C12 cells were treated with 5% FBS plus or minus recombinant chicken netrin-2 for 24 h, and fixed and stained with an antibody to MHC. Bars: (top) 0.5 mm; (bottom) 0.2 mm. (B) Quantification of myotube formation. Values represent means of triplicate determinations ± 1 SD. (C) Western blot analysis of muscle-specific proteins by C2C12 cells treated with 5% FBS plus or minus recombinant chicken netrin-2 for 24 h. Cell lysates were probed with the indicated antibodies.
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fig4: Netrin-2 promotes myotube formation. (A) Photomicrographs of C2C12 cells were treated with 5% FBS plus or minus recombinant chicken netrin-2 for 24 h, and fixed and stained with an antibody to MHC. Bars: (top) 0.5 mm; (bottom) 0.2 mm. (B) Quantification of myotube formation. Values represent means of triplicate determinations ± 1 SD. (C) Western blot analysis of muscle-specific proteins by C2C12 cells treated with 5% FBS plus or minus recombinant chicken netrin-2 for 24 h. Cell lysates were probed with the indicated antibodies.

Mentions: To assess the effects of netrin-3 on myoblast cell lines, we initially attempted to synthesize recombinant mouse protein in COS and 293T cells but, similar to the findings of Puschel (1999), were unable to produce sufficient quantities of secreted, soluble material. Mouse netrin-3 is orthologous to human netrin-2L and, although not orthologous to chicken netrin-2, is grouped with both these proteins by dendrogram analyses in a sub-category designated “netrin-2–like”, distinguishable from a “netrin-1–like” group (Puschel, 1999). Therefore, we chose to use recombinant chicken netrin-2 for this work as it is commercially available. C2C12 cells were cultured in GM, then transferred into 5% FBS plus or minus chicken netrin-2, and observed 24 h later. C2C12 cell proliferation was not altered by netrin-2 treatment (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200405039/DC1). However, although the control cultures displayed small MHC+ myotubes under these conditions, the netrin-2–treated cultures showed distinctly larger myotubes with increases in the total number of nuclei in MHC+ cells and in the average number of nuclei/myotube (Fig. 4, A and B). When analyzed for expression of muscle-specific proteins, the cells that received netrin-2 produced significantly more TnT than control cells, but the levels of MyoD, myogenin, MHC, and CDO were unchanged (Fig. 4 C). Thus, similar to overexpression of neogenin, treatment of C2C12 cells with a neogenin ligand resulted in enhanced myotube formation and alterations in expression of TnT, but not several other muscle markers.


Netrins and neogenin promote myotube formation.

Kang JS, Yi MJ, Zhang W, Feinleib JL, Cole F, Krauss RS - J. Cell Biol. (2004)

Netrin-2 promotes myotube formation. (A) Photomicrographs of C2C12 cells were treated with 5% FBS plus or minus recombinant chicken netrin-2 for 24 h, and fixed and stained with an antibody to MHC. Bars: (top) 0.5 mm; (bottom) 0.2 mm. (B) Quantification of myotube formation. Values represent means of triplicate determinations ± 1 SD. (C) Western blot analysis of muscle-specific proteins by C2C12 cells treated with 5% FBS plus or minus recombinant chicken netrin-2 for 24 h. Cell lysates were probed with the indicated antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172498&req=5

fig4: Netrin-2 promotes myotube formation. (A) Photomicrographs of C2C12 cells were treated with 5% FBS plus or minus recombinant chicken netrin-2 for 24 h, and fixed and stained with an antibody to MHC. Bars: (top) 0.5 mm; (bottom) 0.2 mm. (B) Quantification of myotube formation. Values represent means of triplicate determinations ± 1 SD. (C) Western blot analysis of muscle-specific proteins by C2C12 cells treated with 5% FBS plus or minus recombinant chicken netrin-2 for 24 h. Cell lysates were probed with the indicated antibodies.
Mentions: To assess the effects of netrin-3 on myoblast cell lines, we initially attempted to synthesize recombinant mouse protein in COS and 293T cells but, similar to the findings of Puschel (1999), were unable to produce sufficient quantities of secreted, soluble material. Mouse netrin-3 is orthologous to human netrin-2L and, although not orthologous to chicken netrin-2, is grouped with both these proteins by dendrogram analyses in a sub-category designated “netrin-2–like”, distinguishable from a “netrin-1–like” group (Puschel, 1999). Therefore, we chose to use recombinant chicken netrin-2 for this work as it is commercially available. C2C12 cells were cultured in GM, then transferred into 5% FBS plus or minus chicken netrin-2, and observed 24 h later. C2C12 cell proliferation was not altered by netrin-2 treatment (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200405039/DC1). However, although the control cultures displayed small MHC+ myotubes under these conditions, the netrin-2–treated cultures showed distinctly larger myotubes with increases in the total number of nuclei in MHC+ cells and in the average number of nuclei/myotube (Fig. 4, A and B). When analyzed for expression of muscle-specific proteins, the cells that received netrin-2 produced significantly more TnT than control cells, but the levels of MyoD, myogenin, MHC, and CDO were unchanged (Fig. 4 C). Thus, similar to overexpression of neogenin, treatment of C2C12 cells with a neogenin ligand resulted in enhanced myotube formation and alterations in expression of TnT, but not several other muscle markers.

Bottom Line: These proteins stimulate myotube formation and enhance myogenic bHLH- and NFAT-dependent transcription.Furthermore, neogenin binds to CDO in a cis fashion, and myoblasts lacking CDO are defective in responding to recombinant netrin.It is proposed that netrin-3 and neogenin may promote myogenic differentiation by an autocrine mechanism as components of a higher order complex of several promyogenic cell surface proteins.

View Article: PubMed Central - PubMed

Affiliation: Brookdale Department of Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, New York, NY 10029, USA.

ABSTRACT
Differentiation of skeletal myoblasts into multinucleated myotubes is a multistep process orchestrated by several families of transcription factors, including myogenic bHLH and NFAT proteins. The activities of these factors and formation of myotubes are regulated by signal transduction pathways, but few extracellular factors that might initiate such signals have been identified. One exception is a cell surface complex containing promyogenic Ig superfamily members (CDO and BOC) and cadherins. Netrins and their receptors are established regulators of axon guidance, but little is known of their function outside the nervous system. We report here that myoblasts express the secreted factor netrin-3 and its receptor, neogenin. These proteins stimulate myotube formation and enhance myogenic bHLH- and NFAT-dependent transcription. Furthermore, neogenin binds to CDO in a cis fashion, and myoblasts lacking CDO are defective in responding to recombinant netrin. It is proposed that netrin-3 and neogenin may promote myogenic differentiation by an autocrine mechanism as components of a higher order complex of several promyogenic cell surface proteins.

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