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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 and Cdo induce MyoD activities synergistically, and the Cdo-binding deficient Stx4 mutant failed to enhance myotube formation. a 10T1/2 cells were cotransfected with a MyoD-luciferase reporter and the expression vectors for MyoD and β-galactosidase as an internal control. In addition, control, Stx4, and/or Cdo expression vectors were cotransfected as indicated. Forty-eight hours later, the reporter activities were measured and normalized relative to the internal control. The experiment was performed as triplicates and repeated three times with similar results. *p < 0.01. b Lysates of C2C12 cells stably transfected with indicated Stx4 vectors were immunoblotted with antibodies to MHC and pan-Cadherin as a loading control. The relative signal intensities of MHC to pan-Cadherin were quantified and added under the blot. c C2C12 cells were transiently cotransfected with control (pcDNA), Stx4, or Stx4 mutants along with a GFP expression vector to mark the transfectant. Then, cells were induced to differentiate for 2 days, followed by immunostaining with an antibody to MHC and DAPI stain. Size bar = 100 μm. d Quantification of myotube formation of cell lines shown in panel c. Values represent means of triplicate determinations ± 1 SD. The experiment was repeated three times with similar results. Significant difference from control, *p < 0.01, **p < 0.005
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Fig4: Stx4 and Cdo induce MyoD activities synergistically, and the Cdo-binding deficient Stx4 mutant failed to enhance myotube formation. a 10T1/2 cells were cotransfected with a MyoD-luciferase reporter and the expression vectors for MyoD and β-galactosidase as an internal control. In addition, control, Stx4, and/or Cdo expression vectors were cotransfected as indicated. Forty-eight hours later, the reporter activities were measured and normalized relative to the internal control. The experiment was performed as triplicates and repeated three times with similar results. *p < 0.01. b Lysates of C2C12 cells stably transfected with indicated Stx4 vectors were immunoblotted with antibodies to MHC and pan-Cadherin as a loading control. The relative signal intensities of MHC to pan-Cadherin were quantified and added under the blot. c C2C12 cells were transiently cotransfected with control (pcDNA), Stx4, or Stx4 mutants along with a GFP expression vector to mark the transfectant. Then, cells were induced to differentiate for 2 days, followed by immunostaining with an antibody to MHC and DAPI stain. Size bar = 100 μm. d Quantification of myotube formation of cell lines shown in panel c. Values represent means of triplicate determinations ± 1 SD. The experiment was repeated three times with similar results. Significant difference from control, *p < 0.01, **p < 0.005

Mentions: The promyogenic function of Cdo involves the activation of MyoD via p38MAPK pathway [1]. Therefore, we assessed the effect of Stx4 or/and Cdo expression on MyoD activation by using a MyoD-responsive reporter. To do so, 10T1/2 fibroblasts were cotransfected with a MyoD-luciferase construct and a MyoD expression vector along with expression vectors for Stx4 and/or Cdo. Forty-eight hours later, lysates were subjected to a luciferase assay. The expression of Stx4 or Cdo singly with MyoD enhanced the luciferase activity approximately 2.5-fold and 2.7-fold, respectively, while coexpression of Stx4 and Cdo enhanced the MyoD-reporter activity to approximately 5.8-fold compared to control MyoD-expressing cells (Fig. 4a). These data suggest that Stx4 and Cdo can activate MyoD cooperatively.Fig. 4


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 and Cdo induce MyoD activities synergistically, and the Cdo-binding deficient Stx4 mutant failed to enhance myotube formation. a 10T1/2 cells were cotransfected with a MyoD-luciferase reporter and the expression vectors for MyoD and β-galactosidase as an internal control. In addition, control, Stx4, and/or Cdo expression vectors were cotransfected as indicated. Forty-eight hours later, the reporter activities were measured and normalized relative to the internal control. The experiment was performed as triplicates and repeated three times with similar results. *p < 0.01. b Lysates of C2C12 cells stably transfected with indicated Stx4 vectors were immunoblotted with antibodies to MHC and pan-Cadherin as a loading control. The relative signal intensities of MHC to pan-Cadherin were quantified and added under the blot. c C2C12 cells were transiently cotransfected with control (pcDNA), Stx4, or Stx4 mutants along with a GFP expression vector to mark the transfectant. Then, cells were induced to differentiate for 2 days, followed by immunostaining with an antibody to MHC and DAPI stain. Size bar = 100 μm. d Quantification of myotube formation of cell lines shown in panel c. Values represent means of triplicate determinations ± 1 SD. The experiment was repeated three times with similar results. Significant difference from control, *p < 0.01, **p < 0.005
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Fig4: Stx4 and Cdo induce MyoD activities synergistically, and the Cdo-binding deficient Stx4 mutant failed to enhance myotube formation. a 10T1/2 cells were cotransfected with a MyoD-luciferase reporter and the expression vectors for MyoD and β-galactosidase as an internal control. In addition, control, Stx4, and/or Cdo expression vectors were cotransfected as indicated. Forty-eight hours later, the reporter activities were measured and normalized relative to the internal control. The experiment was performed as triplicates and repeated three times with similar results. *p < 0.01. b Lysates of C2C12 cells stably transfected with indicated Stx4 vectors were immunoblotted with antibodies to MHC and pan-Cadherin as a loading control. The relative signal intensities of MHC to pan-Cadherin were quantified and added under the blot. c C2C12 cells were transiently cotransfected with control (pcDNA), Stx4, or Stx4 mutants along with a GFP expression vector to mark the transfectant. Then, cells were induced to differentiate for 2 days, followed by immunostaining with an antibody to MHC and DAPI stain. Size bar = 100 μm. d Quantification of myotube formation of cell lines shown in panel c. Values represent means of triplicate determinations ± 1 SD. The experiment was repeated three times with similar results. Significant difference from control, *p < 0.01, **p < 0.005
Mentions: The promyogenic function of Cdo involves the activation of MyoD via p38MAPK pathway [1]. Therefore, we assessed the effect of Stx4 or/and Cdo expression on MyoD activation by using a MyoD-responsive reporter. To do so, 10T1/2 fibroblasts were cotransfected with a MyoD-luciferase construct and a MyoD expression vector along with expression vectors for Stx4 and/or Cdo. Forty-eight hours later, lysates were subjected to a luciferase assay. The expression of Stx4 or Cdo singly with MyoD enhanced the luciferase activity approximately 2.5-fold and 2.7-fold, respectively, while coexpression of Stx4 and Cdo enhanced the MyoD-reporter activity to approximately 5.8-fold compared to control MyoD-expressing cells (Fig. 4a). These data suggest that Stx4 and Cdo can activate MyoD cooperatively.Fig. 4

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