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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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Expression of neogenin, netrin-3, CDO, and muscle-specific proteins in C2C12, F3, and 10T1/2 cells. (A) Western blot analysis of C2C12 cell differentiation. Cultures were brought to ∼90% confluence (day 0) and shifted into DM for the indicated period of time and harvested. Western blots were probed with antibodies against the indicated proteins. Immunoblotting for myogenin and MHC indicates the progression of cell differentiation; immunoblotting with a pan-cadherin antibody serves as a loading control (Kang et al., 2003). (B) Western blot analysis of F3 myoblasts cultured in GM (G) or DM (D) for 2 d. Blots were probed with antibodies against the indicated proteins. (C) Western blot analysis of 10T1/2 cell derivatives. 10T1/2 cells stably transfected with a control expression vector (−) or with a MyoD expression vector (+) were cultured in DM for 24 h. Blots were probed with antibodies against the indicated proteins. (D) Northern blot analysis of netrin-3 mRNA expression during C2C12 cell differentiation. The ethidium bromide-stained gel is shown as a loading control.
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fig1: Expression of neogenin, netrin-3, CDO, and muscle-specific proteins in C2C12, F3, and 10T1/2 cells. (A) Western blot analysis of C2C12 cell differentiation. Cultures were brought to ∼90% confluence (day 0) and shifted into DM for the indicated period of time and harvested. Western blots were probed with antibodies against the indicated proteins. Immunoblotting for myogenin and MHC indicates the progression of cell differentiation; immunoblotting with a pan-cadherin antibody serves as a loading control (Kang et al., 2003). (B) Western blot analysis of F3 myoblasts cultured in GM (G) or DM (D) for 2 d. Blots were probed with antibodies against the indicated proteins. (C) Western blot analysis of 10T1/2 cell derivatives. 10T1/2 cells stably transfected with a control expression vector (−) or with a MyoD expression vector (+) were cultured in DM for 24 h. Blots were probed with antibodies against the indicated proteins. (D) Northern blot analysis of netrin-3 mRNA expression during C2C12 cell differentiation. The ethidium bromide-stained gel is shown as a loading control.

Mentions: Our interest in Ig/FNIII repeat proteins led us to examine expression of netrin receptors of this class during myogenic differentiation in vitro. Western blot analyses of C2C12 myoblasts revealed a uniform level of neogenin in cells cultured in growth medium (GM) and cells transferred into DM over a period of 4 d (Fig. 1 A). This contrasts with expression of CDO, which was transiently up-regulated during the differentiation time course (Fig. 1 A; Kang et al., 1998). Duplicate blots probed with an antibody against DCC revealed only trace levels of this protein (unpublished data). F3, a myoblast line derived by treatment of 10T1/2 fibroblasts with 5-azacytidine, also expressed neogenin under both GM and DM conditions (Fig. 1 B). 10T1/2 fibroblasts and 10T1/2 cells converted to myoblasts by stable expression of MyoD expressed indistinguishable levels of neogenin (Fig. 1 C); again, this contrasts with CDO expression, which was induced by MyoD (Fig. 1 C; Kang et al., 1998). Finally, expression of oncogenic Ras in C2C12 cells, which results in reduction of MyoD and CDO and blocks differentiation (Kang et al., 1998), had no effect on neogenin levels (unpublished data). Together, these results indicate that neogenin is expressed in the myogenic lineage, but its expression is not dependent on myogenic factors.


Netrins and neogenin promote myotube formation.

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

Expression of neogenin, netrin-3, CDO, and muscle-specific proteins in C2C12, F3, and 10T1/2 cells. (A) Western blot analysis of C2C12 cell differentiation. Cultures were brought to ∼90% confluence (day 0) and shifted into DM for the indicated period of time and harvested. Western blots were probed with antibodies against the indicated proteins. Immunoblotting for myogenin and MHC indicates the progression of cell differentiation; immunoblotting with a pan-cadherin antibody serves as a loading control (Kang et al., 2003). (B) Western blot analysis of F3 myoblasts cultured in GM (G) or DM (D) for 2 d. Blots were probed with antibodies against the indicated proteins. (C) Western blot analysis of 10T1/2 cell derivatives. 10T1/2 cells stably transfected with a control expression vector (−) or with a MyoD expression vector (+) were cultured in DM for 24 h. Blots were probed with antibodies against the indicated proteins. (D) Northern blot analysis of netrin-3 mRNA expression during C2C12 cell differentiation. The ethidium bromide-stained gel is shown as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172498&req=5

fig1: Expression of neogenin, netrin-3, CDO, and muscle-specific proteins in C2C12, F3, and 10T1/2 cells. (A) Western blot analysis of C2C12 cell differentiation. Cultures were brought to ∼90% confluence (day 0) and shifted into DM for the indicated period of time and harvested. Western blots were probed with antibodies against the indicated proteins. Immunoblotting for myogenin and MHC indicates the progression of cell differentiation; immunoblotting with a pan-cadherin antibody serves as a loading control (Kang et al., 2003). (B) Western blot analysis of F3 myoblasts cultured in GM (G) or DM (D) for 2 d. Blots were probed with antibodies against the indicated proteins. (C) Western blot analysis of 10T1/2 cell derivatives. 10T1/2 cells stably transfected with a control expression vector (−) or with a MyoD expression vector (+) were cultured in DM for 24 h. Blots were probed with antibodies against the indicated proteins. (D) Northern blot analysis of netrin-3 mRNA expression during C2C12 cell differentiation. The ethidium bromide-stained gel is shown as a loading control.
Mentions: Our interest in Ig/FNIII repeat proteins led us to examine expression of netrin receptors of this class during myogenic differentiation in vitro. Western blot analyses of C2C12 myoblasts revealed a uniform level of neogenin in cells cultured in growth medium (GM) and cells transferred into DM over a period of 4 d (Fig. 1 A). This contrasts with expression of CDO, which was transiently up-regulated during the differentiation time course (Fig. 1 A; Kang et al., 1998). Duplicate blots probed with an antibody against DCC revealed only trace levels of this protein (unpublished data). F3, a myoblast line derived by treatment of 10T1/2 fibroblasts with 5-azacytidine, also expressed neogenin under both GM and DM conditions (Fig. 1 B). 10T1/2 fibroblasts and 10T1/2 cells converted to myoblasts by stable expression of MyoD expressed indistinguishable levels of neogenin (Fig. 1 C); again, this contrasts with CDO expression, which was induced by MyoD (Fig. 1 C; Kang et al., 1998). Finally, expression of oncogenic Ras in C2C12 cells, which results in reduction of MyoD and CDO and blocks differentiation (Kang et al., 1998), had no effect on neogenin levels (unpublished data). Together, these results indicate that neogenin is expressed in the myogenic lineage, but its expression is not dependent on myogenic factors.

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