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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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RNAi-mediated reduction of neogenin levels blocks myotube formation. (A) Western blot analysis of RNAi-mediated “knockdown” of neogenin. C2C12 cells were transiently transfected with a GFP expression vector and either pSilencer (−) or pSilencer containing neogenin RNAi sequences (+). GFP-positive cells were sorted and cell lysates probed with antibodies to neogenin or, as a control, a pan-cadherin antibody. (B) C2C12 cells were transiently transfected with pSilencer (control) or pSilencer containing RNAi sequences targeted against neogenin, plus an expression vector for nlacZ (nuclear-targeted β-gal). Cultures were fixed and double stained for MHC and β-gal activity. Bars: (top) 0.2 mm; (bottom): a higher magnification micrograph, 0.1 mm. (C) Quantification of myotube formation. Values represent means of triplicate determinations ± 1 SD. (D) C2C12 cells were transiently transfected with pSilencer and pBabePuro (pSil/pBP); pSilencer containing neogenin RNAi sequences and pBabePuro (RNAi/pBP); or pSilencer containing neogenin RNAi sequences and pBabePuro harboring a human neogenin cDNA (RNAi/hNeogenin), in each case plus an expression vector for nlacZ. Cultures were fixed and double stained for MHC and β-gal activity. Bar, 0.2 mm. (E) Quantification of myotube formation. Values represent means of triplicate determinations ± 1 SD. The transfection efficiencies for these experiments were ∼10%, a value chosen to minimize fusion of independent β-gal+ transfectants.
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fig3: RNAi-mediated reduction of neogenin levels blocks myotube formation. (A) Western blot analysis of RNAi-mediated “knockdown” of neogenin. C2C12 cells were transiently transfected with a GFP expression vector and either pSilencer (−) or pSilencer containing neogenin RNAi sequences (+). GFP-positive cells were sorted and cell lysates probed with antibodies to neogenin or, as a control, a pan-cadherin antibody. (B) C2C12 cells were transiently transfected with pSilencer (control) or pSilencer containing RNAi sequences targeted against neogenin, plus an expression vector for nlacZ (nuclear-targeted β-gal). Cultures were fixed and double stained for MHC and β-gal activity. Bars: (top) 0.2 mm; (bottom): a higher magnification micrograph, 0.1 mm. (C) Quantification of myotube formation. Values represent means of triplicate determinations ± 1 SD. (D) C2C12 cells were transiently transfected with pSilencer and pBabePuro (pSil/pBP); pSilencer containing neogenin RNAi sequences and pBabePuro (RNAi/pBP); or pSilencer containing neogenin RNAi sequences and pBabePuro harboring a human neogenin cDNA (RNAi/hNeogenin), in each case plus an expression vector for nlacZ. Cultures were fixed and double stained for MHC and β-gal activity. Bar, 0.2 mm. (E) Quantification of myotube formation. Values represent means of triplicate determinations ± 1 SD. The transfection efficiencies for these experiments were ∼10%, a value chosen to minimize fusion of independent β-gal+ transfectants.

Mentions: To assess the effect of reducing neogenin levels on myotube formation, an RNAi approach was taken. A sequence from the mouse neogenin coding region was inserted into the pSilencer vector, and was cotransfected with a GFP expression vector into C2C12 cells; the pSilencer vector without an insert was used as a control. Transfected cultures were sorted for the presence of GFP and assessed for reduction of neogenin levels. A representative Western blot is shown in Fig. 3 A. GFP-sorted cells that received the neogenin RNAi vector produced fewer and smaller myotubes than sorted control transfectants (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200405039/DC1). To more precisely quantify this effect, C2C12 cells were cotransfected with the neogenin RNAi vector or the vector lacking an insert, plus a plasmid directing expression of nlacZ (encoding nuclear-localized β-galactosidase; β-gal). 2 d later, the cultures were transferred to 5% FBS for 24 h, and then fixed and double stained for MHC and β-gal activity. When control vector-transfectants fused with nontransfected cells, many (often most) of the nuclei in the myotube became positive for β-gal activity, presumably because the cytoplasmically translated protein diffused within the myotube (Fig. 3 B). When the number of nuclei in β-gal+ cells was scored, ∼56% had more than five nuclei, ∼21% had two to four nuclei and ∼23% had a single nucleus (Fig. 3 C). In contrast, the distribution of β-gal+ cells that received the neogenin RNAi vector was strongly skewed toward single nucleus-containing cells, with only about ∼5% displaying more than five nuclei (Fig. 3 C). Cells that received an RNAi vector containing a neogenin sequence that was ineffective at reducing neogenin protein levels behaved equivalently to those that received the vector lacking an insert (unpublished data).


Netrins and neogenin promote myotube formation.

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

RNAi-mediated reduction of neogenin levels blocks myotube formation. (A) Western blot analysis of RNAi-mediated “knockdown” of neogenin. C2C12 cells were transiently transfected with a GFP expression vector and either pSilencer (−) or pSilencer containing neogenin RNAi sequences (+). GFP-positive cells were sorted and cell lysates probed with antibodies to neogenin or, as a control, a pan-cadherin antibody. (B) C2C12 cells were transiently transfected with pSilencer (control) or pSilencer containing RNAi sequences targeted against neogenin, plus an expression vector for nlacZ (nuclear-targeted β-gal). Cultures were fixed and double stained for MHC and β-gal activity. Bars: (top) 0.2 mm; (bottom): a higher magnification micrograph, 0.1 mm. (C) Quantification of myotube formation. Values represent means of triplicate determinations ± 1 SD. (D) C2C12 cells were transiently transfected with pSilencer and pBabePuro (pSil/pBP); pSilencer containing neogenin RNAi sequences and pBabePuro (RNAi/pBP); or pSilencer containing neogenin RNAi sequences and pBabePuro harboring a human neogenin cDNA (RNAi/hNeogenin), in each case plus an expression vector for nlacZ. Cultures were fixed and double stained for MHC and β-gal activity. Bar, 0.2 mm. (E) Quantification of myotube formation. Values represent means of triplicate determinations ± 1 SD. The transfection efficiencies for these experiments were ∼10%, a value chosen to minimize fusion of independent β-gal+ transfectants.
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Related In: Results  -  Collection

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fig3: RNAi-mediated reduction of neogenin levels blocks myotube formation. (A) Western blot analysis of RNAi-mediated “knockdown” of neogenin. C2C12 cells were transiently transfected with a GFP expression vector and either pSilencer (−) or pSilencer containing neogenin RNAi sequences (+). GFP-positive cells were sorted and cell lysates probed with antibodies to neogenin or, as a control, a pan-cadherin antibody. (B) C2C12 cells were transiently transfected with pSilencer (control) or pSilencer containing RNAi sequences targeted against neogenin, plus an expression vector for nlacZ (nuclear-targeted β-gal). Cultures were fixed and double stained for MHC and β-gal activity. Bars: (top) 0.2 mm; (bottom): a higher magnification micrograph, 0.1 mm. (C) Quantification of myotube formation. Values represent means of triplicate determinations ± 1 SD. (D) C2C12 cells were transiently transfected with pSilencer and pBabePuro (pSil/pBP); pSilencer containing neogenin RNAi sequences and pBabePuro (RNAi/pBP); or pSilencer containing neogenin RNAi sequences and pBabePuro harboring a human neogenin cDNA (RNAi/hNeogenin), in each case plus an expression vector for nlacZ. Cultures were fixed and double stained for MHC and β-gal activity. Bar, 0.2 mm. (E) Quantification of myotube formation. Values represent means of triplicate determinations ± 1 SD. The transfection efficiencies for these experiments were ∼10%, a value chosen to minimize fusion of independent β-gal+ transfectants.
Mentions: To assess the effect of reducing neogenin levels on myotube formation, an RNAi approach was taken. A sequence from the mouse neogenin coding region was inserted into the pSilencer vector, and was cotransfected with a GFP expression vector into C2C12 cells; the pSilencer vector without an insert was used as a control. Transfected cultures were sorted for the presence of GFP and assessed for reduction of neogenin levels. A representative Western blot is shown in Fig. 3 A. GFP-sorted cells that received the neogenin RNAi vector produced fewer and smaller myotubes than sorted control transfectants (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200405039/DC1). To more precisely quantify this effect, C2C12 cells were cotransfected with the neogenin RNAi vector or the vector lacking an insert, plus a plasmid directing expression of nlacZ (encoding nuclear-localized β-galactosidase; β-gal). 2 d later, the cultures were transferred to 5% FBS for 24 h, and then fixed and double stained for MHC and β-gal activity. When control vector-transfectants fused with nontransfected cells, many (often most) of the nuclei in the myotube became positive for β-gal activity, presumably because the cytoplasmically translated protein diffused within the myotube (Fig. 3 B). When the number of nuclei in β-gal+ cells was scored, ∼56% had more than five nuclei, ∼21% had two to four nuclei and ∼23% had a single nucleus (Fig. 3 C). In contrast, the distribution of β-gal+ cells that received the neogenin RNAi vector was strongly skewed toward single nucleus-containing cells, with only about ∼5% displaying more than five nuclei (Fig. 3 C). Cells that received an RNAi vector containing a neogenin sequence that was ineffective at reducing neogenin protein levels behaved equivalently to those that received the vector lacking an insert (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.

Show MeSH