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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 enhances myogenic bHLH factor– and NFAT-dependent transcription. (A) C2C12 cells were transiently transfected with 4Rtk-luc and either a control or neogenin expression vector and analyzed for luciferase reporter activity. (B) 10T1/2 cells were transiently transfected with 4Rtk-luc, expression vectors for MyoD and E12, and either a control or neogenin expression vector and analyzed for luciferase reporter activity. (C) C2C12 cells were transiently transfected with NFAT-luc and a control, neogenin or CDO expression vector and analyzed for luciferase reporter activity. (D) 10T1/2 cells were transiently transfected with NFAT-luc and either a control or neogenin expression vector and analyzed for luciferase reporter activity. (A–D) Values are means ± 1 SEM from three experiments, each performed in triplicate. (E) Western blot analysis of NFATc3 in C2C12 and 10T1/2 cells treated with 5% FBS plus or minus recombinant chicken netrin-2 for 6 or 30 h. Cell lysates were probed with the indicated antibodies. The top and bottom arrows indicate the presumptive phosphorylated and dephosphorylated forms of NFATc3, respectively. (F and G) Netrin-2 requires neogenin to stimulate NFATc3 levels. (F) Western blot analysis of NFATc3 in C2C12 cells transiently transfected with control (−) or neogenin RNAi (+) vectors that were sorted and treated with 5% FBS plus or minus netrin-2. (G) Similar to F except that the cells were the stable neogenin RNAi transfectants shown in Fig. 5.
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fig6: Neogenin enhances myogenic bHLH factor– and NFAT-dependent transcription. (A) C2C12 cells were transiently transfected with 4Rtk-luc and either a control or neogenin expression vector and analyzed for luciferase reporter activity. (B) 10T1/2 cells were transiently transfected with 4Rtk-luc, expression vectors for MyoD and E12, and either a control or neogenin expression vector and analyzed for luciferase reporter activity. (C) C2C12 cells were transiently transfected with NFAT-luc and a control, neogenin or CDO expression vector and analyzed for luciferase reporter activity. (D) 10T1/2 cells were transiently transfected with NFAT-luc and either a control or neogenin expression vector and analyzed for luciferase reporter activity. (A–D) Values are means ± 1 SEM from three experiments, each performed in triplicate. (E) Western blot analysis of NFATc3 in C2C12 and 10T1/2 cells treated with 5% FBS plus or minus recombinant chicken netrin-2 for 6 or 30 h. Cell lysates were probed with the indicated antibodies. The top and bottom arrows indicate the presumptive phosphorylated and dephosphorylated forms of NFATc3, respectively. (F and G) Netrin-2 requires neogenin to stimulate NFATc3 levels. (F) Western blot analysis of NFATc3 in C2C12 cells transiently transfected with control (−) or neogenin RNAi (+) vectors that were sorted and treated with 5% FBS plus or minus netrin-2. (G) Similar to F except that the cells were the stable neogenin RNAi transfectants shown in Fig. 5.

Mentions: CDO signals to enhance myogenic bHLH factor–dependent transcription (Cole et al., 2004), and netrin-1 signals through DCC to stimulate NFAT-mediated transcription (Graef et al., 2003). Therefore, we assessed whether neogenin could stimulate reporter constructs specific for myogenic bHLH factors and NFAT. In transient assays of C2C12 cells, cotransfection of the neogenin expression vector enhanced activity of a reporter construct driven by four reiterated myogenic E-boxes (4Rtk-luc), approximately threefold above that seen with a control vector lacking a cDNA insert (Fig. 6 A). Cotransfection of 10T1/2 fibroblasts with expression vectors for MyoD and its heterodimeric partner E12 led to activation of 4Rtk-luc, and this activity was also enhanced approximately threefold by coexpression of neogenin (Fig. 6 B). However, neogenin could not activate the reporter in the absence of MyoD (unpublished data).


Netrins and neogenin promote myotube formation.

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

Neogenin enhances myogenic bHLH factor– and NFAT-dependent transcription. (A) C2C12 cells were transiently transfected with 4Rtk-luc and either a control or neogenin expression vector and analyzed for luciferase reporter activity. (B) 10T1/2 cells were transiently transfected with 4Rtk-luc, expression vectors for MyoD and E12, and either a control or neogenin expression vector and analyzed for luciferase reporter activity. (C) C2C12 cells were transiently transfected with NFAT-luc and a control, neogenin or CDO expression vector and analyzed for luciferase reporter activity. (D) 10T1/2 cells were transiently transfected with NFAT-luc and either a control or neogenin expression vector and analyzed for luciferase reporter activity. (A–D) Values are means ± 1 SEM from three experiments, each performed in triplicate. (E) Western blot analysis of NFATc3 in C2C12 and 10T1/2 cells treated with 5% FBS plus or minus recombinant chicken netrin-2 for 6 or 30 h. Cell lysates were probed with the indicated antibodies. The top and bottom arrows indicate the presumptive phosphorylated and dephosphorylated forms of NFATc3, respectively. (F and G) Netrin-2 requires neogenin to stimulate NFATc3 levels. (F) Western blot analysis of NFATc3 in C2C12 cells transiently transfected with control (−) or neogenin RNAi (+) vectors that were sorted and treated with 5% FBS plus or minus netrin-2. (G) Similar to F except that the cells were the stable neogenin RNAi transfectants shown in Fig. 5.
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fig6: Neogenin enhances myogenic bHLH factor– and NFAT-dependent transcription. (A) C2C12 cells were transiently transfected with 4Rtk-luc and either a control or neogenin expression vector and analyzed for luciferase reporter activity. (B) 10T1/2 cells were transiently transfected with 4Rtk-luc, expression vectors for MyoD and E12, and either a control or neogenin expression vector and analyzed for luciferase reporter activity. (C) C2C12 cells were transiently transfected with NFAT-luc and a control, neogenin or CDO expression vector and analyzed for luciferase reporter activity. (D) 10T1/2 cells were transiently transfected with NFAT-luc and either a control or neogenin expression vector and analyzed for luciferase reporter activity. (A–D) Values are means ± 1 SEM from three experiments, each performed in triplicate. (E) Western blot analysis of NFATc3 in C2C12 and 10T1/2 cells treated with 5% FBS plus or minus recombinant chicken netrin-2 for 6 or 30 h. Cell lysates were probed with the indicated antibodies. The top and bottom arrows indicate the presumptive phosphorylated and dephosphorylated forms of NFATc3, respectively. (F and G) Netrin-2 requires neogenin to stimulate NFATc3 levels. (F) Western blot analysis of NFATc3 in C2C12 cells transiently transfected with control (−) or neogenin RNAi (+) vectors that were sorted and treated with 5% FBS plus or minus netrin-2. (G) Similar to F except that the cells were the stable neogenin RNAi transfectants shown in Fig. 5.
Mentions: CDO signals to enhance myogenic bHLH factor–dependent transcription (Cole et al., 2004), and netrin-1 signals through DCC to stimulate NFAT-mediated transcription (Graef et al., 2003). Therefore, we assessed whether neogenin could stimulate reporter constructs specific for myogenic bHLH factors and NFAT. In transient assays of C2C12 cells, cotransfection of the neogenin expression vector enhanced activity of a reporter construct driven by four reiterated myogenic E-boxes (4Rtk-luc), approximately threefold above that seen with a control vector lacking a cDNA insert (Fig. 6 A). Cotransfection of 10T1/2 fibroblasts with expression vectors for MyoD and its heterodimeric partner E12 led to activation of 4Rtk-luc, and this activity was also enhanced approximately threefold by coexpression of neogenin (Fig. 6 B). However, neogenin could not activate the reporter in the absence of MyoD (unpublished data).

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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