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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

The cell-surface-resident Cdo was specifically decreased in Stx4-depleted C2C12 cells. a C2C12 cells were transfected with pSuper or shStx4 expression vectors, and cells at the indicated differentiation time points were subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were also analyzed as control. b C2C12/pSuper or C2C12/shStx4 cells were transfected with Cdo-GFP expression vectors and subjected to immunostaining with N-Cadherin antibody, followed by confocal microscopy. The boxed area is shown as an enlarged view. The white arrows mark the area where the localization of Cdo-GFP under the N-Cadherin-resident cell surface is located. Size bar = 10 μm. c C2C12/pSuper or C2C12/shCdo cells were transfected with pcDNA or Stx4 expression vectors and subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were analyzed as control. d Control or shCdo-transfected C2C12 cells at D1 were subjected to surface biotinylation followed by streptavidin-bead pulldown and immunoblotting with indicated antibodies. e Control or shCdo expression vector transfected C2C12 cells were immunoprecipitated with antibody to GLUT4 and immunoblotted with antibodies to GLUT4, Stx4, and Cdo. f Stable C2C12 cells transfected with control, Cdo, or shCdo expression vector were incubated with or without 10 μg/ml insulin for 1 h, followed by 2-NBDG incubation for a further 1 h. Glucose uptake was measured by the relative fluorescence intensity. The experiment was repeated for three independent assays with similar results. Significant difference from insulin-incubated cells, *p < 0.05, **p < 0.01
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Fig6: The cell-surface-resident Cdo was specifically decreased in Stx4-depleted C2C12 cells. a C2C12 cells were transfected with pSuper or shStx4 expression vectors, and cells at the indicated differentiation time points were subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were also analyzed as control. b C2C12/pSuper or C2C12/shStx4 cells were transfected with Cdo-GFP expression vectors and subjected to immunostaining with N-Cadherin antibody, followed by confocal microscopy. The boxed area is shown as an enlarged view. The white arrows mark the area where the localization of Cdo-GFP under the N-Cadherin-resident cell surface is located. Size bar = 10 μm. c C2C12/pSuper or C2C12/shCdo cells were transfected with pcDNA or Stx4 expression vectors and subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were analyzed as control. d Control or shCdo-transfected C2C12 cells at D1 were subjected to surface biotinylation followed by streptavidin-bead pulldown and immunoblotting with indicated antibodies. e Control or shCdo expression vector transfected C2C12 cells were immunoprecipitated with antibody to GLUT4 and immunoblotted with antibodies to GLUT4, Stx4, and Cdo. f Stable C2C12 cells transfected with control, Cdo, or shCdo expression vector were incubated with or without 10 μg/ml insulin for 1 h, followed by 2-NBDG incubation for a further 1 h. Glucose uptake was measured by the relative fluorescence intensity. The experiment was repeated for three independent assays with similar results. Significant difference from insulin-incubated cells, *p < 0.05, **p < 0.01

Mentions: Next, we examined whether Stx4 regulated Cdo translocation to cell surface. To do so, we have assessed whether Stx4 depletion altered the level of Cdo at the cell surface by surface biotinylation. Stx4 knockdowned C2C12 cells displayed decreased Cdo protein levels at the cell surface as well as total Cdo proteins in lysates (Fig. 6a). Furthermore, this effect on Cdo levels appears to be specific since N-Cadherin levels did not alter in these cells. To further examine, C2C12/pSuper or C2C12/shStx4 cells were transfected with a Cdo-GFP vector and subjected to immunostaining with a Cadherin antibody to label the membrane and confocal microscopy. Cdo-GFP proteins were found at the cell membrane and intracellular compartments in both cell types. However, the signals of Cdo-GFP and Cadherin were partially superimposed at the membrane in control cells, whereas Cdo-GFP and Cadherin signals did not largely overlap at the membrane in Stx4-depleted cells (Fig. 6a). Next, we assessed whether the amount of Cdo at the cell surface is rescued by Stx4 in Cdo-depleted C2C12 cells. C2C12/shCdo cells were transfected with the Stx4 expression vector, and 24 h later, cells were analyzed by surface biotinylation. At this condition, Cdo was decreased in both pcDNA and Stx4-transfected C2C12/shCdo cells, compared to the control C2C12/pSuper cells (Fig. 6c). Consistently, the biotinylated Cdo levels were decreased in control C2C12/shCdo cells while Stx4 overexpression restored the biotinylated Cdo levels in C2C12/shCdo cells to the control C2C12/pSuper cells. These data suggest that Stx4 enhances Cdo translocation to the cell surface thereby stimulating Cdo-mediated p38 activation and myoblast differentiation.Fig. 6


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)

The cell-surface-resident Cdo was specifically decreased in Stx4-depleted C2C12 cells. a C2C12 cells were transfected with pSuper or shStx4 expression vectors, and cells at the indicated differentiation time points were subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were also analyzed as control. b C2C12/pSuper or C2C12/shStx4 cells were transfected with Cdo-GFP expression vectors and subjected to immunostaining with N-Cadherin antibody, followed by confocal microscopy. The boxed area is shown as an enlarged view. The white arrows mark the area where the localization of Cdo-GFP under the N-Cadherin-resident cell surface is located. Size bar = 10 μm. c C2C12/pSuper or C2C12/shCdo cells were transfected with pcDNA or Stx4 expression vectors and subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were analyzed as control. d Control or shCdo-transfected C2C12 cells at D1 were subjected to surface biotinylation followed by streptavidin-bead pulldown and immunoblotting with indicated antibodies. e Control or shCdo expression vector transfected C2C12 cells were immunoprecipitated with antibody to GLUT4 and immunoblotted with antibodies to GLUT4, Stx4, and Cdo. f Stable C2C12 cells transfected with control, Cdo, or shCdo expression vector were incubated with or without 10 μg/ml insulin for 1 h, followed by 2-NBDG incubation for a further 1 h. Glucose uptake was measured by the relative fluorescence intensity. The experiment was repeated for three independent assays with similar results. Significant difference from insulin-incubated cells, *p < 0.05, **p < 0.01
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Fig6: The cell-surface-resident Cdo was specifically decreased in Stx4-depleted C2C12 cells. a C2C12 cells were transfected with pSuper or shStx4 expression vectors, and cells at the indicated differentiation time points were subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were also analyzed as control. b C2C12/pSuper or C2C12/shStx4 cells were transfected with Cdo-GFP expression vectors and subjected to immunostaining with N-Cadherin antibody, followed by confocal microscopy. The boxed area is shown as an enlarged view. The white arrows mark the area where the localization of Cdo-GFP under the N-Cadherin-resident cell surface is located. Size bar = 10 μm. c C2C12/pSuper or C2C12/shCdo cells were transfected with pcDNA or Stx4 expression vectors and subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were analyzed as control. d Control or shCdo-transfected C2C12 cells at D1 were subjected to surface biotinylation followed by streptavidin-bead pulldown and immunoblotting with indicated antibodies. e Control or shCdo expression vector transfected C2C12 cells were immunoprecipitated with antibody to GLUT4 and immunoblotted with antibodies to GLUT4, Stx4, and Cdo. f Stable C2C12 cells transfected with control, Cdo, or shCdo expression vector were incubated with or without 10 μg/ml insulin for 1 h, followed by 2-NBDG incubation for a further 1 h. Glucose uptake was measured by the relative fluorescence intensity. The experiment was repeated for three independent assays with similar results. Significant difference from insulin-incubated cells, *p < 0.05, **p < 0.01
Mentions: Next, we examined whether Stx4 regulated Cdo translocation to cell surface. To do so, we have assessed whether Stx4 depletion altered the level of Cdo at the cell surface by surface biotinylation. Stx4 knockdowned C2C12 cells displayed decreased Cdo protein levels at the cell surface as well as total Cdo proteins in lysates (Fig. 6a). Furthermore, this effect on Cdo levels appears to be specific since N-Cadherin levels did not alter in these cells. To further examine, C2C12/pSuper or C2C12/shStx4 cells were transfected with a Cdo-GFP vector and subjected to immunostaining with a Cadherin antibody to label the membrane and confocal microscopy. Cdo-GFP proteins were found at the cell membrane and intracellular compartments in both cell types. However, the signals of Cdo-GFP and Cadherin were partially superimposed at the membrane in control cells, whereas Cdo-GFP and Cadherin signals did not largely overlap at the membrane in Stx4-depleted cells (Fig. 6a). Next, we assessed whether the amount of Cdo at the cell surface is rescued by Stx4 in Cdo-depleted C2C12 cells. C2C12/shCdo cells were transfected with the Stx4 expression vector, and 24 h later, cells were analyzed by surface biotinylation. At this condition, Cdo was decreased in both pcDNA and Stx4-transfected C2C12/shCdo cells, compared to the control C2C12/pSuper cells (Fig. 6c). Consistently, the biotinylated Cdo levels were decreased in control C2C12/shCdo cells while Stx4 overexpression restored the biotinylated Cdo levels in C2C12/shCdo cells to the control C2C12/pSuper cells. These data suggest that Stx4 enhances Cdo translocation to the cell surface thereby stimulating Cdo-mediated p38 activation and myoblast differentiation.Fig. 6

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