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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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Overexpression of neogenin promotes myotube formation by C2C12 cells. (A) C2C12 cells were stably transfected with a control expression vector lacking a cDNA (−) or with an expression vector harboring a neogenin cDNA (+). Western blots of the indicated cells were probed with neogenin or, as a control, pan-cadherin antibodies. The exposure of this film was for a shorter duration than the neogenin blot shown in Fig.1, so as to more clearly highlight the quantitative difference between the transfectants. (B) Photomicrographs of C2C12/puro and C2C12/neogenin cells were cultured in 5% FBS for 24 h, and fixed and stained with an antibody to MHC. Bar, 0.2 mm. (C) Quantification of myotube formation. Values represent means of triplicate determinations ± 1 SD. (D) Western blot analysis of muscle-specific proteins by C2C12 cell transfectants over a 2-d time course of differentiation.
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fig2: Overexpression of neogenin promotes myotube formation by C2C12 cells. (A) C2C12 cells were stably transfected with a control expression vector lacking a cDNA (−) or with an expression vector harboring a neogenin cDNA (+). Western blots of the indicated cells were probed with neogenin or, as a control, pan-cadherin antibodies. The exposure of this film was for a shorter duration than the neogenin blot shown in Fig.1, so as to more clearly highlight the quantitative difference between the transfectants. (B) Photomicrographs of C2C12/puro and C2C12/neogenin cells were cultured in 5% FBS for 24 h, and fixed and stained with an antibody to MHC. Bar, 0.2 mm. (C) Quantification of myotube formation. Values represent means of triplicate determinations ± 1 SD. (D) Western blot analysis of muscle-specific proteins by C2C12 cell transfectants over a 2-d time course of differentiation.

Mentions: To assess a role for neogenin in myogenesis, we transfected different “strains” of C2C12 cells and F3 cells with a vector engineered to drive expression of a human neogenin cDNA and a puromycin resistance gene. Transfected cultures were selected and analyzed for neogenin expression. Stable overexpression of neogenin was achieved only in one strain of C2C12 cells. These cells, previously described by us and designated as C2C12(E) cells (Kang et al., 1998), are highly differentiation-proficient in that they: (a) expressed detectable myogenin before a shift into DM; (b) were less sensitive to inhibition of differentiation by moderate levels of serum (e.g., 5% FBS); and (c) completed the differentiation process over a 2-d time period. These cells were used for all subsequent experiments and are simply referred to as C2C12 cells. Neogenin vector transfectants (C2C12/neogenin cells) produced two- to threefold more neogenin than control vector transfectants (C2C12/puro cells; Fig. 2 A). Overproduction of neogenin modestly decreased proliferation of C2C12 cells in GM, relative to vector controls (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200405039/DC1), and did not alter their morphology in GM (not depicted). When observed 24 h after shifting the cultures to medium containing 5% FBS (a time at which the differentiation process was in mid-progression), C2C12/neogenin cells had formed larger myotubes with more nuclei than did C2C12/puro cells (Fig. 2 B). C2C12/neogenin cultures displayed an increase in the total number of nuclei present in myosin heavy chain (MHC)–positive cells and in the average number of nuclei/myotube (Fig. 2 C). When analyzed for expression of muscle-specific proteins, including MyoD, myogenin, MHC, and troponin T (TnT), C2C12/neogenin cells showed accelerated expression of TnT, but otherwise were similar to C2C12/puro cells (Fig. 2 D). Overexpression of neogenin therefore resulted in enhanced formation of myotubes without dramatic changes in several biochemical markers of differentiation.


Netrins and neogenin promote myotube formation.

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

Overexpression of neogenin promotes myotube formation by C2C12 cells. (A) C2C12 cells were stably transfected with a control expression vector lacking a cDNA (−) or with an expression vector harboring a neogenin cDNA (+). Western blots of the indicated cells were probed with neogenin or, as a control, pan-cadherin antibodies. The exposure of this film was for a shorter duration than the neogenin blot shown in Fig.1, so as to more clearly highlight the quantitative difference between the transfectants. (B) Photomicrographs of C2C12/puro and C2C12/neogenin cells were cultured in 5% FBS for 24 h, and fixed and stained with an antibody to MHC. Bar, 0.2 mm. (C) Quantification of myotube formation. Values represent means of triplicate determinations ± 1 SD. (D) Western blot analysis of muscle-specific proteins by C2C12 cell transfectants over a 2-d time course of differentiation.
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Related In: Results  -  Collection

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fig2: Overexpression of neogenin promotes myotube formation by C2C12 cells. (A) C2C12 cells were stably transfected with a control expression vector lacking a cDNA (−) or with an expression vector harboring a neogenin cDNA (+). Western blots of the indicated cells were probed with neogenin or, as a control, pan-cadherin antibodies. The exposure of this film was for a shorter duration than the neogenin blot shown in Fig.1, so as to more clearly highlight the quantitative difference between the transfectants. (B) Photomicrographs of C2C12/puro and C2C12/neogenin cells were cultured in 5% FBS for 24 h, and fixed and stained with an antibody to MHC. Bar, 0.2 mm. (C) Quantification of myotube formation. Values represent means of triplicate determinations ± 1 SD. (D) Western blot analysis of muscle-specific proteins by C2C12 cell transfectants over a 2-d time course of differentiation.
Mentions: To assess a role for neogenin in myogenesis, we transfected different “strains” of C2C12 cells and F3 cells with a vector engineered to drive expression of a human neogenin cDNA and a puromycin resistance gene. Transfected cultures were selected and analyzed for neogenin expression. Stable overexpression of neogenin was achieved only in one strain of C2C12 cells. These cells, previously described by us and designated as C2C12(E) cells (Kang et al., 1998), are highly differentiation-proficient in that they: (a) expressed detectable myogenin before a shift into DM; (b) were less sensitive to inhibition of differentiation by moderate levels of serum (e.g., 5% FBS); and (c) completed the differentiation process over a 2-d time period. These cells were used for all subsequent experiments and are simply referred to as C2C12 cells. Neogenin vector transfectants (C2C12/neogenin cells) produced two- to threefold more neogenin than control vector transfectants (C2C12/puro cells; Fig. 2 A). Overproduction of neogenin modestly decreased proliferation of C2C12 cells in GM, relative to vector controls (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200405039/DC1), and did not alter their morphology in GM (not depicted). When observed 24 h after shifting the cultures to medium containing 5% FBS (a time at which the differentiation process was in mid-progression), C2C12/neogenin cells had formed larger myotubes with more nuclei than did C2C12/puro cells (Fig. 2 B). C2C12/neogenin cultures displayed an increase in the total number of nuclei present in myosin heavy chain (MHC)–positive cells and in the average number of nuclei/myotube (Fig. 2 C). When analyzed for expression of muscle-specific proteins, including MyoD, myogenin, MHC, and troponin T (TnT), C2C12/neogenin cells showed accelerated expression of TnT, but otherwise were similar to C2C12/puro cells (Fig. 2 D). Overexpression of neogenin therefore resulted in enhanced formation of myotubes without dramatic changes in several biochemical markers of differentiation.

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