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Syntaxin 4 regulates the surface localization of a promyogenic receptor Cdo thereby promoting myogenic differentiation.

Yoo M, Kim BG, Lee SJ, Jeong HJ, Park JW, Seo DW, Kim YK, Lee HY, Han JW, Kang JS, Bae GU - Skelet Muscle (2015)

Bottom Line: Stx4 depletion decreases specifically the cell surface localization of Cdo without changes in surface N-Cadherin levels.Interestingly, Cdo depletion reduces the level of GLUT4 and Stx4 at cell surface.Consistently, overexpression of Cdo in C2C12 myoblasts generally increases glucose uptake, while Cdo depletion reduces it.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women's University, Seoul, 140-742 Republic of Korea.

ABSTRACT

Background: Syntaxins are a family of membrane proteins involved in vesicle trafficking, such as synaptic vesicle exocytosis. Syntaxin 4 (Stx4) is expressed highly in skeletal muscle and plays a critical role in insulin-stimulated glucose uptake by promoting translocation of glucose transporter 4 (GLUT4) to the cell surface. A cell surface receptor cell adhesion molecule-related, down-regulated by oncogenes (Cdo) is a component of cell adhesion complexes and promotes myoblast differentiation via activation of key signalings, including p38MAPK and AKT. In this study, we investigate the function of Stx4 in myoblast differentiation and the crosstalk between Stx4 and Cdo in myoblast differentiation.

Methods: The effects of overexpression or shRNA-based depletion of Stx4 and Cdo genes on C2C12 myoblast differentiation are assessed by Western blotting and immunofluorescence approaches. The interaction between Cdo and Stx4 and the responsible domain mapping are assessed by coimmunoprecipitation or pulldown assays. The effect of Stx4 depletion on cell surface localization of Cdo and GLUT4 in C2C12 myoblasts is assessed by surface biotinylation and Western blotting.

Results: Overexpression or knockdown of Stx4 enhances or inhibits myogenic differentiation, respectively. Stx4 binds to the cytoplasmic tail of Cdo, and this interaction seems to be critical for induction of p38MAPK activation and myotube formation. Stx4 depletion decreases specifically the cell surface localization of Cdo without changes in surface N-Cadherin levels. Interestingly, Cdo depletion reduces the level of GLUT4 and Stx4 at cell surface. Consistently, overexpression of Cdo in C2C12 myoblasts generally increases glucose uptake, while Cdo depletion reduces it.

Conclusions: Stx4 promotes myoblast differentiation through interaction with Cdo and stimulation of its surface translocation. Both Cdo and Stx4 are required for GLUT4 translocation to cell surface and glucose uptake in myoblast differentiation.

No MeSH data available.


Related in: MedlinePlus

Stx4 is expressed in skeletal muscles and induced in myoblast differentiation. a RT-PCR analysis of hindlimb muscles from E15.5 embryos and P1, P5, P7, P14, and P30 mice for the expression of Stx4, Cdo, MyoD, Myogenin, and 18S rRNA serves as a loading control. b Immunoblot analysis of C2C12 cells from various differentiation days (D) for the expression of Stx4, Cdo, Myogenin, MHC, and pan-Cadherin serves as a loading control. c Immunoblot analysis for Stx4 protein expression in Cdo+/+ and Cdo−/− primary myoblasts during differentiation, and pan-Cadherin serves as a loading control. d qRT-PCR analysis for Stx4 mRNA expression in Cdo+/+ and Cdo−/− primary myoblasts during differentiation
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Fig1: Stx4 is expressed in skeletal muscles and induced in myoblast differentiation. a RT-PCR analysis of hindlimb muscles from E15.5 embryos and P1, P5, P7, P14, and P30 mice for the expression of Stx4, Cdo, MyoD, Myogenin, and 18S rRNA serves as a loading control. b Immunoblot analysis of C2C12 cells from various differentiation days (D) for the expression of Stx4, Cdo, Myogenin, MHC, and pan-Cadherin serves as a loading control. c Immunoblot analysis for Stx4 protein expression in Cdo+/+ and Cdo−/− primary myoblasts during differentiation, and pan-Cadherin serves as a loading control. d qRT-PCR analysis for Stx4 mRNA expression in Cdo+/+ and Cdo−/− primary myoblasts during differentiation

Mentions: In the previous study, we performed a yeast two-hybrid screening to identify interacting proteins for Cdo, and JLP and Bnip2 are two such proteins implicated in Cdo-mediated myogenesis [13, 14]. In the same screen, Syntaxin (Stx) 1 was identified as an interacting protein for Cdo. Stx1 and Stx4 share high homology and have similar domain structures consisting of Stx, t-SNARE, and transmembrane domain (TD) [28]. While Stx1 is expressed predominantly in neural cell types, Stx4 is the major form in skeletal muscles [29, 30]. Therefore, we examined whether Stx4 plays a role in myogenesis, especially in association with Cdo. First, we have assessed the expression pattern of Stx4 and Cdo in mouse hindlimb muscles from various developmental stages. The expression of Stx4 was detected throughout the examined stages; however, the level of Cdo, MyoD, and Myogenin decreased after the postnatal day 7 which may reflect the fast muscle growth during early postnatal life (Fig. 1a). Next, we have examined the expression pattern of Stx4 protein during myoblast differentiation. C2C12 cells were grown to near-confluency (D0) and induced to differentiate by switching to the differentiation medium for a total of 3 days (D3), followed by immunoblotting. As shown in Fig. 1b, the level of Stx4 is enhanced progressively during myoblast differentiation, while the Cdo protein is expressed throughout the differentiation time course, and the expression of Myogenin and myosin heavy chain (MHC) was dramatically enhanced at D2 or D3, respectively. These data suggest that Stx4 might be important for myoblast differentiation. Since Cdo and Stx4 were coexpressed in developing skeletal muscles, we examined the relationship between Cdo and Stx4 by using primary myoblasts isolated from Cdo+/+ or Cdo−/− mice. Previously, we have shown that Cdo-deficient primary myoblasts display defects in myoblast differentiation and p38MAPK activation [26]. Cdo+/+ or Cdo−/− myoblasts at high cell density (D0) were induced to differentiate by removal of basic fibroblast growth factor (bFGF) for 2 days. The expression of Stx4 in Cdo−/− myoblasts was substantially increased at D2 compared to that of Cdo+/+ myoblasts, whereas there was only slight or no difference at D0 and D1 (Fig. 1c). In addition, the qRT-PCR analysis showed that Stx4 transcript levels were increased at D1 in Cdo-deficient myoblasts, but no difference in cells at D0 or D2 (Fig. 1d). These data suggest that the Stx4 expression level alone may not be sufficient to induce myoblast differentiation when Cdo is deficient.Fig. 1


Syntaxin 4 regulates the surface localization of a promyogenic receptor Cdo thereby promoting myogenic differentiation.

Yoo M, Kim BG, Lee SJ, Jeong HJ, Park JW, Seo DW, Kim YK, Lee HY, Han JW, Kang JS, Bae GU - Skelet Muscle (2015)

Stx4 is expressed in skeletal muscles and induced in myoblast differentiation. a RT-PCR analysis of hindlimb muscles from E15.5 embryos and P1, P5, P7, P14, and P30 mice for the expression of Stx4, Cdo, MyoD, Myogenin, and 18S rRNA serves as a loading control. b Immunoblot analysis of C2C12 cells from various differentiation days (D) for the expression of Stx4, Cdo, Myogenin, MHC, and pan-Cadherin serves as a loading control. c Immunoblot analysis for Stx4 protein expression in Cdo+/+ and Cdo−/− primary myoblasts during differentiation, and pan-Cadherin serves as a loading control. d qRT-PCR analysis for Stx4 mRNA expression in Cdo+/+ and Cdo−/− primary myoblasts during differentiation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4561423&req=5

Fig1: Stx4 is expressed in skeletal muscles and induced in myoblast differentiation. a RT-PCR analysis of hindlimb muscles from E15.5 embryos and P1, P5, P7, P14, and P30 mice for the expression of Stx4, Cdo, MyoD, Myogenin, and 18S rRNA serves as a loading control. b Immunoblot analysis of C2C12 cells from various differentiation days (D) for the expression of Stx4, Cdo, Myogenin, MHC, and pan-Cadherin serves as a loading control. c Immunoblot analysis for Stx4 protein expression in Cdo+/+ and Cdo−/− primary myoblasts during differentiation, and pan-Cadherin serves as a loading control. d qRT-PCR analysis for Stx4 mRNA expression in Cdo+/+ and Cdo−/− primary myoblasts during differentiation
Mentions: In the previous study, we performed a yeast two-hybrid screening to identify interacting proteins for Cdo, and JLP and Bnip2 are two such proteins implicated in Cdo-mediated myogenesis [13, 14]. In the same screen, Syntaxin (Stx) 1 was identified as an interacting protein for Cdo. Stx1 and Stx4 share high homology and have similar domain structures consisting of Stx, t-SNARE, and transmembrane domain (TD) [28]. While Stx1 is expressed predominantly in neural cell types, Stx4 is the major form in skeletal muscles [29, 30]. Therefore, we examined whether Stx4 plays a role in myogenesis, especially in association with Cdo. First, we have assessed the expression pattern of Stx4 and Cdo in mouse hindlimb muscles from various developmental stages. The expression of Stx4 was detected throughout the examined stages; however, the level of Cdo, MyoD, and Myogenin decreased after the postnatal day 7 which may reflect the fast muscle growth during early postnatal life (Fig. 1a). Next, we have examined the expression pattern of Stx4 protein during myoblast differentiation. C2C12 cells were grown to near-confluency (D0) and induced to differentiate by switching to the differentiation medium for a total of 3 days (D3), followed by immunoblotting. As shown in Fig. 1b, the level of Stx4 is enhanced progressively during myoblast differentiation, while the Cdo protein is expressed throughout the differentiation time course, and the expression of Myogenin and myosin heavy chain (MHC) was dramatically enhanced at D2 or D3, respectively. These data suggest that Stx4 might be important for myoblast differentiation. Since Cdo and Stx4 were coexpressed in developing skeletal muscles, we examined the relationship between Cdo and Stx4 by using primary myoblasts isolated from Cdo+/+ or Cdo−/− mice. Previously, we have shown that Cdo-deficient primary myoblasts display defects in myoblast differentiation and p38MAPK activation [26]. Cdo+/+ or Cdo−/− myoblasts at high cell density (D0) were induced to differentiate by removal of basic fibroblast growth factor (bFGF) for 2 days. The expression of Stx4 in Cdo−/− myoblasts was substantially increased at D2 compared to that of Cdo+/+ myoblasts, whereas there was only slight or no difference at D0 and D1 (Fig. 1c). In addition, the qRT-PCR analysis showed that Stx4 transcript levels were increased at D1 in Cdo-deficient myoblasts, but no difference in cells at D0 or D2 (Fig. 1d). These data suggest that the Stx4 expression level alone may not be sufficient to induce myoblast differentiation when Cdo is deficient.Fig. 1

Bottom Line: Stx4 depletion decreases specifically the cell surface localization of Cdo without changes in surface N-Cadherin levels.Interestingly, Cdo depletion reduces the level of GLUT4 and Stx4 at cell surface.Consistently, overexpression of Cdo in C2C12 myoblasts generally increases glucose uptake, while Cdo depletion reduces it.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women's University, Seoul, 140-742 Republic of Korea.

ABSTRACT

Background: Syntaxins are a family of membrane proteins involved in vesicle trafficking, such as synaptic vesicle exocytosis. Syntaxin 4 (Stx4) is expressed highly in skeletal muscle and plays a critical role in insulin-stimulated glucose uptake by promoting translocation of glucose transporter 4 (GLUT4) to the cell surface. A cell surface receptor cell adhesion molecule-related, down-regulated by oncogenes (Cdo) is a component of cell adhesion complexes and promotes myoblast differentiation via activation of key signalings, including p38MAPK and AKT. In this study, we investigate the function of Stx4 in myoblast differentiation and the crosstalk between Stx4 and Cdo in myoblast differentiation.

Methods: The effects of overexpression or shRNA-based depletion of Stx4 and Cdo genes on C2C12 myoblast differentiation are assessed by Western blotting and immunofluorescence approaches. The interaction between Cdo and Stx4 and the responsible domain mapping are assessed by coimmunoprecipitation or pulldown assays. The effect of Stx4 depletion on cell surface localization of Cdo and GLUT4 in C2C12 myoblasts is assessed by surface biotinylation and Western blotting.

Results: Overexpression or knockdown of Stx4 enhances or inhibits myogenic differentiation, respectively. Stx4 binds to the cytoplasmic tail of Cdo, and this interaction seems to be critical for induction of p38MAPK activation and myotube formation. Stx4 depletion decreases specifically the cell surface localization of Cdo without changes in surface N-Cadherin levels. Interestingly, Cdo depletion reduces the level of GLUT4 and Stx4 at cell surface. Consistently, overexpression of Cdo in C2C12 myoblasts generally increases glucose uptake, while Cdo depletion reduces it.

Conclusions: Stx4 promotes myoblast differentiation through interaction with Cdo and stimulation of its surface translocation. Both Cdo and Stx4 are required for GLUT4 translocation to cell surface and glucose uptake in myoblast differentiation.

No MeSH data available.


Related in: MedlinePlus