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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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Neogenin and CDO form complexes. Lysates from the indicated cell lines were immunoprecipitated (IP) and Western blotted (WB) with the indicated antibodies. Immunoblotting of straight lysates with antibodies to nonimmunoprecipitated proteins are shown as a control. (A and B) C2C12 cells were cultured in GM (G) or DM (D). Immunoblotting of straight lysates to MHC in A indicates the degree of differentiation of the cells. (C) C2C12 cells were cultured on plastic dishes (−) or as a single cell suspension in the presence of EDTA (+). (D and E) 293T cells were transiently transfected with expression vectors for neogenin and/or CDO as indicated. (F) 293T cells were transiently transfected with expression vectors for neogenin and CDO and cultured on plastic dishes (−) or as a single cell suspension in the presence of EDTA (+). (G) 293T cells were transiently transfected with expression vectors for neogenin and either full-length CDO (CDO) or CDO deletion mutants as indicated.
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fig7: Neogenin and CDO form complexes. Lysates from the indicated cell lines were immunoprecipitated (IP) and Western blotted (WB) with the indicated antibodies. Immunoblotting of straight lysates with antibodies to nonimmunoprecipitated proteins are shown as a control. (A and B) C2C12 cells were cultured in GM (G) or DM (D). Immunoblotting of straight lysates to MHC in A indicates the degree of differentiation of the cells. (C) C2C12 cells were cultured on plastic dishes (−) or as a single cell suspension in the presence of EDTA (+). (D and E) 293T cells were transiently transfected with expression vectors for neogenin and/or CDO as indicated. (F) 293T cells were transiently transfected with expression vectors for neogenin and CDO and cultured on plastic dishes (−) or as a single cell suspension in the presence of EDTA (+). (G) 293T cells were transiently transfected with expression vectors for neogenin and either full-length CDO (CDO) or CDO deletion mutants as indicated.

Mentions: Ig/FNIII proteins can bind in a cis fashion to additional members of this family to form complexes that regulate their function. For example, Robo receptors bind to DCC to silence netrin-1–mediated attraction (Stein and Tessier-Lavigne, 2001), and BOC binds CDO to stimulate myogenesis (Kang et al., 2002). To test whether neogenin might also interact with CDO, lysates from C2C12 and F3 cells were immunoprecipitated with antibodies to CDO or neogenin and blotted with the reciprocal antibody. Neogenin was present in CDO immunoprecipitates, and CDO in neogenin immunoprecipitates, from both cell lines, suggesting that these two proteins do indeed form a complex (Fig. 7, A and B). More neogenin co-immunoprecipitated with CDO under DM than GM conditions, although this enhanced association in DM was not evident in the reciprocal co-immunoprecipitation (Fig. 7, A and B). Co-immunoprecipitation of neogenin and CDO was not diminished when C2C12 cells were collected as a single-cell suspension in the presence of EDTA (which blocks cadherin-mediated adhesion), suggesting that the interaction occurs in a cis fashion (Fig. 7 C). CDO associates with N- and M-cadherins in myoblasts (Kang et al., 2003), and cadherins also co-immunoprecipitated with neogenin (Fig. 7 B), suggesting these proteins may interact together in a higher order structure. Furthermore, consistent with netrin-3 functioning as a neogenin ligand in C2C12 cells, it too was observed in neogenin immunoprecipitates (Fig. 7 B).


Netrins and neogenin promote myotube formation.

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

Neogenin and CDO form complexes. Lysates from the indicated cell lines were immunoprecipitated (IP) and Western blotted (WB) with the indicated antibodies. Immunoblotting of straight lysates with antibodies to nonimmunoprecipitated proteins are shown as a control. (A and B) C2C12 cells were cultured in GM (G) or DM (D). Immunoblotting of straight lysates to MHC in A indicates the degree of differentiation of the cells. (C) C2C12 cells were cultured on plastic dishes (−) or as a single cell suspension in the presence of EDTA (+). (D and E) 293T cells were transiently transfected with expression vectors for neogenin and/or CDO as indicated. (F) 293T cells were transiently transfected with expression vectors for neogenin and CDO and cultured on plastic dishes (−) or as a single cell suspension in the presence of EDTA (+). (G) 293T cells were transiently transfected with expression vectors for neogenin and either full-length CDO (CDO) or CDO deletion mutants as indicated.
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fig7: Neogenin and CDO form complexes. Lysates from the indicated cell lines were immunoprecipitated (IP) and Western blotted (WB) with the indicated antibodies. Immunoblotting of straight lysates with antibodies to nonimmunoprecipitated proteins are shown as a control. (A and B) C2C12 cells were cultured in GM (G) or DM (D). Immunoblotting of straight lysates to MHC in A indicates the degree of differentiation of the cells. (C) C2C12 cells were cultured on plastic dishes (−) or as a single cell suspension in the presence of EDTA (+). (D and E) 293T cells were transiently transfected with expression vectors for neogenin and/or CDO as indicated. (F) 293T cells were transiently transfected with expression vectors for neogenin and CDO and cultured on plastic dishes (−) or as a single cell suspension in the presence of EDTA (+). (G) 293T cells were transiently transfected with expression vectors for neogenin and either full-length CDO (CDO) or CDO deletion mutants as indicated.
Mentions: Ig/FNIII proteins can bind in a cis fashion to additional members of this family to form complexes that regulate their function. For example, Robo receptors bind to DCC to silence netrin-1–mediated attraction (Stein and Tessier-Lavigne, 2001), and BOC binds CDO to stimulate myogenesis (Kang et al., 2002). To test whether neogenin might also interact with CDO, lysates from C2C12 and F3 cells were immunoprecipitated with antibodies to CDO or neogenin and blotted with the reciprocal antibody. Neogenin was present in CDO immunoprecipitates, and CDO in neogenin immunoprecipitates, from both cell lines, suggesting that these two proteins do indeed form a complex (Fig. 7, A and B). More neogenin co-immunoprecipitated with CDO under DM than GM conditions, although this enhanced association in DM was not evident in the reciprocal co-immunoprecipitation (Fig. 7, A and B). Co-immunoprecipitation of neogenin and CDO was not diminished when C2C12 cells were collected as a single-cell suspension in the presence of EDTA (which blocks cadherin-mediated adhesion), suggesting that the interaction occurs in a cis fashion (Fig. 7 C). CDO associates with N- and M-cadherins in myoblasts (Kang et al., 2003), and cadherins also co-immunoprecipitated with neogenin (Fig. 7 B), suggesting these proteins may interact together in a higher order structure. Furthermore, consistent with netrin-3 functioning as a neogenin ligand in C2C12 cells, it too was observed in neogenin immunoprecipitates (Fig. 7 B).

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.

Show MeSH
Related in: MedlinePlus